SECTION 1

Clinical Laboratory
Practice Review

Credentialing
Process Definition
Accreditation A voluntary process of external peer review
in which a private, nongovernmental agency
i grants puhlic recognition to an institution
that meets certain standards
Certification A voluntary process by which a nongovern-
mental agency grants recognition to an
individual who has met certain educational
requirements and demonstrated entry level
competency by examination
Licensure A mandatory process by which a state grants
permission to an individual or organization
to engage in a given occupation or business
Kegistration A process by which qualified individuals are
listed on an official roster maintained by a
governmental or nongovernmental agency
2 CLINICAL LABORATORY PRACTICE REVIEW
--
Exumples
American Association of Blood Banks
College of American Pathologists
Joint Cornmission on Accreditation of Health
Care Organizations
National Accrediting Agency for Clinical
Laboratory Sciences
American Medical Technologists
American Society of Clinical Pathologists
International Society for Clinical Laboratory
Technology
National Credentialing Agency for Laboratory
Personnel
Licensure of laboratory personnel is required in
California, Florida, Georgia, Hawaii, Louisiana,
Montana, Nevada, North Dakota, Rhode Island,
Tennessee, and West Virginia. Many states
require licensure of clinical laboratories.
 Regulatory Agencies
Agency Authority
Department of Transportation Federal agency that regulates packaging, labeling, and transportation of
(DOT) biological products
Envirorlmental Protection Federal agency that regulates disposal of toxic chemical and biohazardous
Agency (EPA) wastes
- - - - ~ - - - - - - - - - - - ~~~ ~ - ~ ~

Food and Drug Administration
(FDA)
Health Care Financing
Administration
(HCFA)
Department of Health and
Human Services (HHS)
Department of Public Health
Nuclear Regulatory Commission
(NRC)
Occupational Safety and
Health Administration (OSHA)
Substance Abuse and Mental
Health Services Administration
(SAMHSA)
Federal agency that regulates market entry of instruments and reagents,
regulates production of donor blood and components, and licenses blood banks
Federal agency that administers Medicare and federal portion of Medicaid
program. Writes regulations for and enforces Clinical Laboratory Amendments
of 1988 (CLIA '88).
Federal agency that interprets and implements federal regulations related to
healthcare. Includes HCFA, FDA, and CDC.
State agency that develops rules and regulations to implement a state's public
health laws
Federal agency that licenses laboratories that use radionucleotides
Federal agency that regulates employee safety in the workplace
Federal agency that issues mandatory guidelines for laboratories performing
forensic toxicology. Formerly National Institute on Drug Abuse (NIDA).

CLINICAL LABORATORY PRACTICE REVIEW 3

4
C - - --- - -
 Agencies That Issue Guidelines/Standards
Agency
Amer-ican Association of Blood Batiks (AABK)
Centers for Disease Control and Prevention (CDC)
- -
International Organization for Standardization (ISO)
Sational Committee for Clinical Lal~oratory
Sta~ldards(KCCLS)
4 CLINICAL 1,ABOKATORY PRACTICE REVIEW
Guidelines/Stnndards
Technical standards for blood banks
Standards and guidelines for I~ospitalsand laboratories,
primarily related to infei.tion control and safe work practices
Standards to facilitate the international exchange oi goods
and srrvices. Developed through a volurltary worldwide con-
sensus process (e.g., IS0 9000 provides a framrwork for
quality management and quality assurarlce).
S t a ~ ~ d a r on
d s all aspects of laboratory practlce developed
through a volulltary consensus pi-ocess
 Clinical Laboratory Improvement Amendments
of 1988 (CLIA '88)
All laboratories that examine human specimens for diagnosis, prevention, or treatment
Applies to of disease
Exernptioils Work done for lcgal or cmployment eligibility purposes; research not used for patient care;
VA, armed forces, Substance Abuse and Mental Health Services Admirlistration
Levels of co~l~plexity Waived, provider-performed microscopy, moderate complexity, high complexity
Administered by Health Care Finance Administration (HCFA)
Areas for compliance Personnel qualifications/competency assessment, patient test management, quality
control, quality assurance, proficiency tesling

CLINICAL LABORATORY PRACTICE REVIEW 5 1

 OSHA Standards
Yeur Standard Explanation I
I
I
1984 Respiratory Protection Stdndard Requires a respiratory protection plan !
I
1987 Hazdrd Communication Standard Requires elnplovers to inform employees about hazardous
"Right-to-Know Law" substances ill the workplace and to educate and train them I
in safe and proper liandling I1
1991 Occupational Exposures to Requires a chemical hygiene plan to minimize pel.soilnel
Hazardous Chemicals in exposures to hazardous chemicals
Laboratories-"Ldboratory Standard"
1991 Bloodburne Pathogens Standard hlandates strirlgent work practices and procedures to I
minimize worker exposure to bloodborile pathogens 1I
1992 (revised) For~naldehydeStandard Requires monitoring of forinaldehyde exposure, engineering I
controls, persolla1 protective equipment, training, emergency 1
action plan
1

6 CLINICAL LABORATORY PRACTICE REVIEW

 Bloodborne Pathogens
I

Specimens That Are Usi~allyNot Infectious I

Specimens Tlrat Are Potentially Infectious [Unless Visibly Bloody) II

I

Blood Feces I
I
Pus and purulent fluids Nasal secretions I
Semen Sputurn I
Vagirlal secretions Sweat I
Cerebrospinal fluid
Pleural fluid
Peritorleal fluid
Pericardial fluid
A~nnioticfluid
Tears
Urine
Vonlitus 1
Breast milk I
I

CLINICAL LABORATORY PRACTICE REVIEW 7

 Hazard Categories of Chemicals
Classification Example Effect Comments
Corrosives Glacial acetic acid, Visible destructio~lof human Chemicdis with pH <2 or >12. Separate
hydrochloric acid, tissue upon contact. Can produce inorganic acids from organic acids.
sodium hydroxide injury upon inhalation or contact. Concentrated acids and bases can
generate large amounts of beat when
mixed with water.
-

Toxic substances Cy'mides, sulfides I~lterierewith metabolic pro- Threshold limit values [TtWs) indicate
cesses when ingested, inhaled, the amount an individual can be
01-absorbed through the skin exposed to without adverse effect.
Carcinogens Renzidine, Capable of causing cancer OSHA requires ir~onitoringof exposuri.
fornialdehyde to formaldehyde.
Mutagens a n d Benzene, lead, Mutagells induce genetic Special precautions during pregnancy
teratogens mercury, ~.adio- mutatiolls. Teratogens cause
active material, physical defects in developing
toluene embryo.
lg~litahles Acetone, alcohols, Fire Flamrnahles have a flashpoint below
ether, xylene 100°F. Combustibles have a flashpoint at
or above I0O"F. The flasl~point1s the low-
est temperature that produces sufficient
vapor lo form an ignitable mixture at the
surface of the liquid. A hear source, such
as an electrical spark or open flame,
must be present to ignite tlie vapor.
Corltinued
8 CLINICAL LABORATORY PRACTICE REL'IEW
 Hazard Categories of Chemicals Continued
Classification Example Effect Comments
Reactives Ether, perchloric Explosion Ethyl and isopropyl ether form explosive peroxides on
acid, picric acid, exposure to air or light; must be stored in an explosion-
sodium azide proof refrigerator. Perchloric acid should be separated
from other acids; niay react explosively with organic com-
pounds, including acetic acid. Picric acid becomes shock-
sensitive when dehydrated and is a more powerful explo-
sive than TNT. Pouring sodium azide solutions down the
drain can cause explosive lead or copper azides to form
from plumbing.

CLINICAL LABORATORY PRACTICE REVIEW 9

 Hazard Identification System
National Fire Protection Association (NFPA)
Hazard Svrnbol 0 I 2 3 4
Health Blue diamond No unusual Caution: May Warning: May Warning: Danger: May be
hazard cause irritation be harmful if Corrosive or fatal on short ex-
inhaled or toxic. Avoid posure. Special-
absorbed skin contact ized protective
or inhalation. equipment
required.
Flamn~ability Red diamond Not Combustible Caution: Warning: Danger:
combustible if heated Combustible Flammable Flammable
liquid. Flash liquid. Flash gas or extremely
point of point below flammable
100-200°F. 100°F. liquid
Reactivity/ Yellow Stable: Not Caution: May Warning: Danger: May Danger:
stability dianiond reactive react if heated, Unstable, or be explosive Explosive
when mixed or mixed with may react if if spark occurs, material at
with water water mixed with if heated room
water under confine- temperature
ment, or mixed
with water
Special White
hazard diamond
information

10 CLINICAL LABORATORY PRACTICE REVIEW

- - - - - --
 Storage of Chemicals
Cherniml
Category Examples Storage Guidelines
Acids Organic: formic, glacial acetic, citric Store below counter level or in acid cabinets.
Inorganic: hydrochloric, nitric, sulfuric Separate from flammable and combustible material.
Oxidizing: chromic, nitric, perchloric, Separate organic acids from inorganic acids.
sulfuric Separate oxidizing acids from organic acids.
Separate from bases and active metals (e.g., sodium,
potassium, magnesium) .
Bases Ammonium hydroxide, potassium Separate from acids.
hydroxide, sodium hydroxide Store inorganic hydroxides in polyethylene containers.
- - - - - - -

Flammables Acetone, alcohols, xylene Store in approved safety cans or cabinets.

No more than 60 gallons permitted in a single flammable
storage cabinet.
Keep away from oxidizing acids and oxidizers.
pp -- - - - - - - - - - -

Oxidizers Nitric acid, perchloric acid, sulfuric Keep away from reducing agents (e.g., zinc, alkaline
acid, acetic acid, potassium chloride, metals, formic acid).
hydrogen peroxide Keep away from flammable and combustible materials.
- - -

Water- Sodium, potassium Keep away from water.

reactive Store in a dry, cool place.
chemicals

CLINICAL LABORATORY PRACTICE REVIEW 11


Class of Fire Combustible Material
A Cloth, wood, paper
B Flammable liquids
and gases
C Operating electrical
equipment
D Combustible/
reactive metals
Fire Safety
Extinguishers to Use Comments
Pressurized water (A) Do not use pressurized water on electrical fires
Dry chemical (ABC) or burning liquids.
Dry chemical (ABC)
Carbon dioxide (BC)
Dry chemical (ABC) Dry chemical may cause electrical equipment
Carbon dioxide (BC) damage.
Halon Halon is first choice for computer and electrical
equipment fires. Carbon dioxide is second
choice.
Leave to professional
firefighters.

12 CLINICAL LABORATORY PRACTICE REVIEW

--
 Commonly Used Anticoagulants
Stopper
Anticoagulant Color Mode of Action Examples of Use Comments
EDTA (Na, or K,) Lavender Chelates Ca2+ CBCs, differentials, Prevents platelets from clumping.
erythrocyte sedi- Causes minirnal morphologic changes
mentation rate (ESR) in WBCs. ntbe should be at least half
full.
Heparin (Na, Li, Green Neutralizes Many chemistries, Best anticoagulant for prevention of
or NH,) thrombin osmotic fragility, hemolysis. Not satisfactory for
plasma hemoglobin, differentials; causes a blue background.
blood gases
Sodium citrate Light Blue Binds Ca2+ Most coagulation 3.2% or 3.8%. Preserves labile clotting
tests factors. Tube must be filled completely
to achieve 9:l blood-to-anticoagulant
ratio. Reduce anticoagulant for hemat-
ocrits over 55%.
Sodium fluoride/ Gray Oxalate binds Glucose, lactic acid Preserves glucose for 24 hours at room
potassium oxalate Ca2+.Fluoride temperature and 48 hours at 4OC. Ox-
inhibits alate distorts cellular morphology;
glycolysis. don't use for differentials.
- - - -

Lithium Gray Iodoacetate Glucose, lactic acid Used to preserve glucose and stabilize
iodoacetate/ inhibits lactic acid levels.
lithium heparin glycolysis.

CLINICAL LABORATORY PRACTICE REVIEW 13

 Tests Requiring a Fasting Specimen
Fasting blood sugar
Glucose tolerance test
Riglycerides
Lipid panel
Gastrin
Insulin

14 CLINICAL LABORATORY PRACTICE REVIEW

-- -- -- - - - - --
 Recommended Order of Drawing/Filling Tubes IB
Procedure Order
Drawing evacuated tubes Sterile, red, light blue, green, lavender, gray
Filling evacuated tubes from syringe Sterile, light blue, lavender, green, gray, red
Filling microcontainers from finger-/heel-stick Lavender, green, red

CLINICAL LABORATORY PRACTICE REVIEW 15

- -- --- - ---
 Situations in Phlebotomy
- -
Situation Action
IV or blood transfusion running Never draw above an IV site. Use opposite arm or perform fingerstick, if possi-
ble. Otherwise, turn off IV for at least 2 minutes, apply tourniquet below IV
site, select vein other than the one with the IV, draw and discard first 5 mL.
Note that blood was taken from IV arm. Don't draw from an IV site for 1 hour
after IV is discontinued.
Fistula Draw from opposite arm.
Indwelling lines and catheters, Usually not drawn by lab. The first 5 mL of blood drawn should be discarded.
heparin locks, cannulas Lab may draw below heparin lock if nothing is being infused.
Sclerosed veins Select another site.
Hematoma Draw below.
Streptokinase/Tissue plasminogen Minimize venipunctures. Hold pressure until bleeding has stopped.
activator (TPA)
Edema Select another site.
Scars, burns, tatoos Select another site.
- ~

Mastectomy Draw from opposite arm.

Patient refuses Try to persuade. If unsuccessful, respect wishes and notify nurse. Never draw
without consent. Charges of assault and battery could result.
Unidentified patient Ask nurse to properly identify patient before drawing.

16 CLINICAL LABORATORY PRACTICE REVIEW

 -- - -
Specimens Requiring Special Handling
Requirement Tests Comments
Chilling Arterial blood gases, adrenocorticotropic Place in crushed ice or a mixture of ice and
hormone (ACTH), ammonia, gastrin, water. Do not use ice cubes alone because
glucagon, lactic acid, parathyroid RBCs may lyse.
hormone (PTH) , renin
Warming Cold agglutinins, cryoglobulins Use 37°C heat block, heel warmer, or hold in
hand.
Protection from light Bilirubin, carotene, vitamin A, Wrap in aluminum foil.
vitamin B,,
Chain of custody Any test used as evidence in legal Chain of custody form; lock boxes may be
proceedings; e.g., blood alcohol, drug required.
screens, DNA analysis

.
, CLINICAL LABORATORY PRACTICE REVIEW 17

--
 .--. ---4'-

Specimen Processing
Serum or plasma should be separated from cells within 2 hours of collection (unless collected in a gel separator
tube).
Allow red top tubes to clot sufficiently (20-30 minutes) before centrifugation to avoid fibrin strands.
Centrifuge 1025 minutes at 1000-1200 X g.
Keep tubes capped during centrifugation to avoid loss of CO,, change of pH, evaporation, or aerosol formation.
Lipemic specimens can be ultracentrifuged at 105gto remove chylomicrons (triglycerides).
If analysis will be delayed more than 5 hours, serum or plasma for most tests can be capped and stored at 4°C.
Some analytes should be frozen. (Do not freeze whole blood.) Avoid repeated freezing and thawing.
Specimens for lactate dehydrogenase (LD) should be kept at room temperature.
For acid phosphatase, add citrate (10 mg/mL) and freeze.
Glucose is stable in serum separator tubes for 24 hours and in sodium fluoride tubes for 24 hours at room
temperature or 48 hours at 4°C.
 Examples of Criteria for Specimen Rejection
Missing or inadequate label
Collected at wrong time
Collected in wrong tube
Insufficient specimen
Inadequate volume of blood in anticoagulant tube
Exposure to temperature extremes
Prolonged transit
Clots in CBC tube
Hemolysis (depending on test ordered)
Lipemia (depending on test ordered)

CLINICAL LABORATORY PRACTICE REVIEW 19

I
L

Centrifuge Fast Facts

Relative centrifugal force Force acting on sample being centrifuged. gs (gravities). Function of rpm and radius.
RPM Revolutions per minute. Speed of centrifugation. Determined by tachometer.
Radius Distance from center of rotation to bottom of tube when rotating.
- -

Fixed-angle centrifuge Tubes are at fixed angle when rotating. Capable of higher speeds. Produces a slanted
sediment surface.
- - - - -

Horizontal centrifuge Tubes are in horizontal position when rotating. Produces a flat sediment surface.
Ultra centrifuge High-speed. Capable of 100,000 rpm.
Usual gs and time for 1000-1200 x g for 10 5 5 minutes
centrifuging blood
Safety Make sure centrifuge is balanced. Don't open while spinning. Keep tubes capped.
Clean daily with disinfectant.
Quality control Check speed every 3 months. Should be within 5 % .
Check timer with stopwatch every 3 months. Should not be more than 10% off.
Check temperatures of refrigerated centrifuges monthly. Should be within fZ°C.
Check brushes every 2 months.

20 CLINICAL LABORATORY PRACTICE REVIEW

 Packaging of Biologics for Shipment in Mail
Primary container Test tube, vial, etc. containing etiologic agent. Must be securely closed and watertight.
Must be placed in a secondary container.
Secondary container Must be durable and watertight. Can contain more than one primary container, but not
more than 50 mL total. Must contain enough absorbent material, such as paper towels, to
absorb contents in case of breakage. Must be placed in an approved mailing container.
Mailing container Must be made of corrugated fiberboard, cardboard, wood, or other material of equivalent
strength with an external dimension of no less than 3.9 inches
Labeling Etiologic agent label must be placed on mailing container. Must have biohazard symbol
and phone number for CDC for notification in case of leakage.
(Regulations published in July 21, 1980 Federal Register under Interstate Shipment of Etiologic Agents)

CLINICAL LABORATORY PRACTICE REVIEW 21

v p e s of Glass
Aluminosilicate glass (Corex) Six times stronger than borosilicate and better able to resist clouding due to alkali
and scratching.
Boron-free Alkali resistant. Poor heat resistance. Used for highly alkaline solutions.
Borosilicate glass (Kimax High resistance to thermal shock and chemical attack. Heavy walls to minimize
and Pyrex) mechanical breakage. Most beakers, flasks, and pipets are made of this. Minimal
contamination of liquids by elements in the glass. Can be heated and autoclaved.
Flint glass Soda-lime glass containing oxides of sodium, silicon, and calcium. Least expen-
sive but poor resistance to high temperatures and sudden changes of temperature.
Resistance to chemical attack is only fair. Can release alkali and affect some deter-
minations. Used for some disposable laboratory glassware.
High silica Heat, chemical, and electrical tolerance. Excellent optical properties. Used for
high-precision analytic work, optical reflectors, mirrors.
Low actinic Amber or red. Used to reduce exposure to light.

22 CLINICAL LABORATORY PRACTICE REVIEW

b
 Glassware Inscriptions
A Class A. Meets high standards for accuracy.
-

20°C Temperature of calibration. This is the temperature the glassware and solution should be for maximum
accul acy.
TC To contain. The vessel is calibrated to hold a specified volume (e.g., volumetric flasks).
TD To deliver. The vessel is calibrated to deliver a specified volume (e.g., graduated cylinders).
 Volumetric Glassware
Beaker Wide-mouthed, straight-sided jar with a pouring spout. Not accurate enough for critical
measurements.
Erlenmeyer flask Sloping sides. Graduated markings. May be used to hold liquids, mix solutions, or measure
noncritical volumes.
Florence flask Spherical base with a long cylindrical neck. Single calibration mark. Only for noncritical
measurements.
Volumetric flask Pear-shaped. Long neck with a single calibration mark. Manufactured to strict standards.
Glassware and solutions should be at room temperature. Used to accurately prepare standards
and reagents. Should not be used to store solutions.
Graduated cylinder Upright, straight-sided tube with a flared base to provide stability. Used to make noncritical
(graduates) measurements. Most are marked "TD." Should not be used for measuring volumes less than 5
mL or volumes less than 10% of capacity. Use the graduate closest in size to the volume to be
measured.

24 CLINICAL LABORATORY PRACTICE REVIEW

- -. --
 -

Glasd Pipets
Vblumetric pipet Ransfer pipet. Single calibration mark. Calibrated to accurately deliver a fixed volume of
aqueous solutions such as nonviscous samples and standards. The last drop is touched off
against the wall of the receiving vessel.
Ostwald-Folin pipet Transfer pipet. Similar to volumetric pipet but bulb is closer to the delivery tip, reducing the
surface area in contact with the liquid. Etched ring near the upper end signifies that it is a
blow-out pipet. Used for accurate measurement of viscous fluids such as whole blood or
serum. Not widely used in laboratories today.
Serologic pipet Graduated or measuring pipet. Graduation marks down to the tip. Etched ring near the up-
per end signifies that it is a blow-out pipet. Used for preparing serial dilutions and measur-
ing reagents. Generally not considered accurate enough for measuring samples or standards.
Mohr pipet Graduated or measuring pipet. Does not have graduation marks all the way to the tip and
does not have a frosted band near the upper end. Delivery is made "point to point." Not
commonly used.
-

Micropipets Disposable pipets for volumes ranging from 1-1000 ILL.Single calibration mark. Filled by
r.
capillary action. TC. Must be rinsed out with diluent to deliver exact amount.

CLINICAL LABORATORY PRACTICE REVIEW 25

-
-
*d1*

'Ifrpes of Plastic
Polycarbonate Stronger than polypropylene and better temperature tolerance, but chemical resistance is not
as good. Clear. Resistant to shattering. Used for centrifuge tubes.
Polyolefins Relatively inert chemically. Resistant to most acids, alkalis, and salts. (Concentrated sulfuric
(polyethylene, acid attacks polyethylene). Polyethylene used in most disposable plasticware; can't be
polypropylene) sterilized. Polypropylene can be sterilized.
Polyvinyl chloride Soft and flexible, but porous. Frequently used as tubing.
Teflon Extremely inert. Excellent temperature tolerance and chemical resistance. Nonwettable sur-
face. Antiadhesive properties. Used for stir bars, stopcocks, tubing.

26 CLINICAL LABORATORY PRACTICE REVIEW

a
 Grades of Chemicals
Reagent or analytical reagent grade Meets specifications of the American Chemical Society. Very high purity.
Recommended for both qualitative and quantitative analysis.
Ultra pure reagents Also called spectrograde, nanograde, or high-performance liquid chromatog-
raphy (HPLC) pure. Used for gas chromatography, HPLC, fluorometry, and
trace metal determinations.
Chemically pure Not of sufficient purity to use as analytic reagents
Purified, practical, technical, or Not of sufficient purity to use as analytic reagents
commercial nrade
USP or NF Grade Meet specifications of U.S. Pharmacopeia or National Formulary. Not injuri-
ous to health. Not necessarily of sufficient purity to use as analytic
reagents.

CLINICAL LABORATORY PRACTICE REVIEW 27

Reagent Grade Water

Type I Used in all quantitative and most qualitative lab procedures, for electrophoretic analyses, toxicology
screening procedures, and in preparation of buffers, standards, and controls. Should not be stored.
'Ifrpe I1 Used in reagents with preservatives or reagents that are sterilized, and in most stains. Can be stored for
short periods.
'Ifrpe I11 Used for washing and preliminary rinsing of glassware or as a source of water for further purification.

28 CLINICAL LABORATORY PRACTICE REVIEW

 Water Purification
Process
Distillation Water is boiled and steam is
condensed.
Deionization Water is passed through a
bed of mixed cation and
anion exchange resins.
Reverse osmosis Water is forced under
pressure through a semiper-
meable membrane.
Activated Water is passed through a
carbon filtration bed of activated carbon.
Ultrafiltration Water is passed through
semipermeable membranes
of pore size 10.22 pm.
UItraviolet Exposure to 185 nm for
oxidation and oxidation; 254 nm for
sterilization sterilization
Removes
Bacteria, pyrogens, particulate
matter, dissolved ionized
solids, some dissolved
organic contaminants
Dissolved ionized gases and
solids
Dissolved organic contaminants,
ionic solids, bacteria
Dissolved organic compounds
and chlorine
Particulate matter, bacteria,
pyrogens
Organic contaminants
(oxidation) and bacteria
(sterilization)
Comments
Does not meet conductivity
requirement of type I water
Resin must be frequently
replaced or regenerated.
Inadequate alone for produc-
tion of reagent grade water.
Frequently used to pretreat
water before deionization.
Used with deionization.
Used after other purification
processes.

CLINICAL LABORATORY PRACTICE REVIEW 29

Brightfield Microscopy Fast Facts

Achromatic objective Least expensive objective. Partially corrects for chromatic and spherical aberrations.
Aperture diaphragm Controls angle and amount of light sent to objective
Binocular microscope One with two oculars
Blue filter Used to eliminate yellow color emitted by tungsten
Brightfield microscope Uses transmitted light and lenses. Objects appear dark against white background. Used
for most routine clinical work.
Compound microscope One with two lens systems-objectives and oculars
Condenser Focuses light on specimen
Depth of focus Distance throughout which all parts of specimen are clearly in focus simultaneously
Field diaphragm Limits area of illumination to image field
Field of view Area of specimen that can be seen
Immersion oil Used to help objective gather light from a wide numerical aperture. Provides high reso-
lution. Type B (high viscosity) is commonly used.
Kohler illumination Method of focusing and centering light path and spreading light uniformly. Ensures opti-
mum contrast and resolution.
Magnification, total Magnification of the ocular x magnification of the objective. lOOOX is the highest mag-
nification achievable with a brightfield microscope.
Continued

30 CLINICAL LABORATORY PRACTICE REVIEW

 >
r
---.

Brightfield Microscopy Fast Facts

Numerical aperture (NA) Mathematical expression of light admitted by lens. The higher the NA, the greater the
resolution.
Objectives Lenses attached to the revolving nosepiece. Most commonly used are low power (10x1,
high power (40X), and oil immersion (50 or 1 0 0 ~ ) .
Ocular Eye piece. Usually lox.
Parcentric Property whereby an object in the center of a field at one magnification remains in the
center of the field at other magnifications
Parfocal Property whereby an object remains in focus from one magnification to another
Planachromatic objective More expensive objective that corrects for curvature of the field, resulting in a flat field
with uniform focus
Resolution Ability to reveal fine detail and distinguish between two close points
- -

Rheostat Light control knob. Light intensity should not be regulated by the condenser or di-
aphragms.
Tbngsten-halogen bulb Type of bulb used for brightfield microscopy
- - -

Virtual image Image seen through the microscope. It is upside down and reversed.
Working distance Distance between the slide and the objective. Decreases with higher magnification ob-
jectives.

CLINICAL LABORATORY PRACTICE REVIEW 31

L

Other 'Ifrpes of Microscopy

Explanation Application in Clinical Laboratory
Light Microscopes
Darkfield Brightfield microscope with special condenser. Objects Identification of live Deponema
appear white against a black background. pallidum and other microorganisms
Fluorescent Brightfield microscope with two special filters. Excitation filter Direct and indirect fluorescent
selects wavelength of light presented to specimen. Barrier or antibody stains in microbiology
emission filter permits specific wavelength of fluorescent light and immunology
from specimen to pass to eyepiece. Fluorescent dyes absorb
light of one wavelength and emit light of a longer wavelength.
Objects appear green, yellow, or orange against a black
background.
Interference Brightfield microscope with special slit aperture below Wet mounts
contrast condenser, polarizer, and special amplitude filter (modulator)
in back of each objective. Gives a 3-D effect to unstained
specimens.
Phase Brightfield microscope with phase condenser and phase Manual platelet counts, urine
contrast objectives. Subtle differences in refractive index are converted sediments (good for hyaline casts)
into clear-cut variations of light intensity and contrast.
Good for living cells, unstained specimens.
Continued

1 32 CLINICAL LABORATORY PRACTICE REVIEW

i
 Other IIfrpes of Microscopy
Explanation Application in Clinical Laboratory
Polarizing Brightfield microscope with two crossing filters-polarizing Identification of crystals in urine and
filter located below condenser, analyzer between objective synovial fluid. Confirmation of fat or
and eyepiece. Objects that can refract light (birefringent) oval fat bodies in urine sediment.
appear white against a black background.
Electron Microscopes
Transmission Beam of electrons passes through specimen and is focused Virology, cells (organelles)
onto a fluorescent screen or photographic plate.
Magnification >100,00OX.
Scanning Ream of electrons strikes surface of specimen and is focused Virology, cells (surface)
onto photographic film or cathode ray tube. Forms 3-D image.
Magnification >1,00OX.

CLINICAL LABORATORY PRACTICE REVIEW 33

!
i
Computers
-

Hardware The physical parts of a combuter

- - - -- - --
--
CPU Central processing unit. The electronic portion of a computer.
Input devices Mechanisms whereby the computer receives data; e.g., keyboards, bar-code readers, com-
puter links (interfaces)
Output devices Means by which computer delivers information; e.g., printers, CRT screens
RAM Random access memory. "Volatile memory." Working memory used for temporary storage
of programs and data. Content is lost each time the computer is turned off.
ROM Read only memory. Part of the memory that js permanently protected from being modified,
erased, or written over. Not affected by loss of power. Used for boot-level and other system
instructions.
Hard drive Magnetic-coated metal plate inside the CPU for storing data
Floppy drive Removable flexible plastic disk with a magnetic coating, used to store information external
to the CPU
Optical disk Laser-read compact disk
Disk drive Unit that allows data to be extracted and entered on disks
Software Programs that tell the computer what to do
Systems software Programs that control and run the operating system of the computer
Application software Programs designed to meet specific needs of the users
Continued
34 CLINICAL 1,ABORATORY PRACTICE REVIEW
 Computers
Word processor Application program that allows for the manipulation of text. Used to write letters, reports.
Spreadsheet Application program used to manipulate numbers and perform mathematical calculations.
Used to prepare financial statements, budgets, cash projections.
Database Application program used to organize, store, sort, and retrieve data (words or numbers).

CLINICAL LABORATORY PRACTICE REVIEW 35

.- ', "
_-a

Local area network (LAN)
Wide area network (WAN)
Laboratory information system (LIS)
Client server
Computer Networks
A computer network that connects computers and their equipment in close
geographic proximity (e.g., building, campus)
A computer network that connects computers and their equipment over a
large geographic area (e.g., Internet)
Network of computer components designed to incorporate all aspects of
the informational needs of the laboratory and its customers from the intake
of requests for services to the delivery of results. Can provide patient infor-
mation, test information, work lists, test results, financial functions, pro-
ductivity and workload monitoring, quality management, interface with
other computer systems.
A system that allows users to tap into the LIS and extract the information
they want.

i1 36 CLINICAL LABORATORY PRACTICE REVIEW

 Phases of QualityAssurance
Phase Definition Examples
- - - - - - - - - - -

Preanalytical Everything that precedes actual test performance Test ordering by physician, test ordering by
nursing/clerical staff, patient preparation, pa-
tient identification, specimen collection, spec-
imen transport, specimen processing
Analytical Everything related to the actual testing process Test analysis, quality control, reagents, cali.
bration, preventive maintenance
Postanalytical Everything that comes after test analysis Verification of calculations and reference
ranges, review of results, procedures for noti-
fication of critical values, result reporting,
test interpretation by physician, follow-up pa-
tient care

CLINICAL LABORATORY PRACTICE REVIEW 37

t d
 Quality Assurance
Quality assurance Process by which a laboratory ensures quality results by closely monitoring the preanalyti-
cal, analytical, and postanalytical stages of testing.
Quality control Part of the analytical stage of quality assurance; process of monitoring results from control
samples to verify quality of patient results.
Sensitivity Ability of the method to detect slight differences in concentration. High sensitivity means
few false negatives and is desirable in screening tests.
Specificity The ability of the method to determine solely the compound it is supposed to measure. High
specificity means few false positives and is desirable in confirmatory tests.
Accuracy How close a measurement is to the true value. Implies freedom from error.
Precision Reproducibility. Indicates how close test measurements are to each other when the same
test is run on the same sample time after time. Usually expressed in terms of standard devi-
ation (SD) or coefficient of variation. Precision does not imply accuracy. Precision implies
freedom from variation.
Imprecision A large amount of scatter about the mean. Most often caused by technique errors such as
variability in pipetting or inattention to detail by the operator. Results in an increase in the
SD or a broadening of the distribution about the mean.
Standard A solution that contains a known amount of an analyte. Used to calibrate an assay method.
Standard, primary A substance that can be accurately weighed or measured to produce a solution of an exactly
known concentration. Must be 99.98% pure. This level of purification is impossible to attain
with most biologic materials and is not necessary for routine testing.
Continued

38 CLINICAL LABORATORY PRACTICE REVIEW

 Quality Assurance Continued
Coefficient of Expresses standard deviation as a percentage. CV % = [SD + mean) X 100. Allows
variation (CV) comparison of the precision of tests having different concentration ranges or units. The
lower the CV, the higher the precision.
Normal distribution The Gaussian bell-shaped curve.
Mean + 1 SD = 68.2 % of observations
Mean + 2 SD = 95.45 % of observations (referred to as 95 % confidence interval] 1
*
Mean 3 SD = 99.73% of observations
Levey-Jennings chart Normal distribution curve lying on its side, marked with the mean and *1, 2, and 3 SD.
Thirty sequential control results are plotted on x axis.
Control limits Numerical limits within which the values of controls must fall for the assay to be considered
valid. Many labs use the mean ?2 SD. One determination in 20 will fall outside 2 2 SD. This
is an anticipated part of normal variation.
Outlier A control result outside of the established limits. May be due to chance or may indicate a
problem in the test system. If it occurs more frequently than once in 20 successive runs,
then patient results cannot be reported. Investigation must be carried out.
Shift Six consecutive control values fall on the same side of the mean.
Trend Control values increasing or decreasing for six consecutive runs.
Error, random Error affecting a particular determination that does not recur in a regular pattern (e.g., error
due to use of dirty glassware or the wrong pipet, voltage fluctuation, sampling error, antico-
agulant or drug interference]. Indicated by a control value that is significantly different from
the other values on a Levey-Jennings chart, or violation of the I,, and R4, Westgard rules.
Usually a one-time error and controls and samples can be rerun with success.
Continued
40 CLINICAL LABORATORY PRACTICE REVIEW
 Quality Assurance Continued
Procedure manual A set of instructions for methods used in the laboratory. Must be approved, signed, and
dated by the director of the laboratory.
Proficiency testing Testing of unknowns submitted by an outside agency. Unknowns are not to receive prefer-
ential treatment. Results are reported to the agency, which compares them to results from
other laboratories. One commonly used provider of proficiency testing is the College of
American Pathologists. CLIA '88 requires that all laboratories performing moderately or
highly complex tests participate in proficiency testing.
Correlation study A study to verify the accuracy of a new method, where split patient samples are analyzed by
the existing method and the new method. Requires a minimum of 40 patient samples repre-
senting a wide range of concentrations. Reference values (existing method) are plotted on
the x axis and new values on the y axis. Perfect correlation is indicated by a straight line
passing through zero at a 45" angle. The correlation coefficient (r) can be derived mathemat-
ically. Values range from - 1 to + 1. A value of 0 indicates no correlation between the two
methods. A value of + 1 indicates a perfect direct relationship between the two methods. A
value of 0.95 or higher is considered excellent agreement.

42 CLINICAL LABORATORY PRACTICE REVIEW

 Westgard Multirules
Rule Explanation m e of Error Comments
l,, One control exceeds the mean by more than Initiates testing of other rules. If there
2 SD, but less than 3 SD. A warning flag that are no violations of the remaining
indicates a possible change in accuracy or rules, the control values are considered
precision. acceptable.
l,, One control exceeds the mean by more Random Rejection rule
than 3 SD.
2,s l b o consecutive controls exceed the mean Systematic Rejection rule
by more than 2 SD, but less than 3 SD and
in the same direction.
R,, The difference between two consecutive Random Rejection rule
controls is greater than 4 SD.
4,s Four consecutive controls exceed the mean Systematic Rejection rule
by more than 1 SD and in the same direction.
10, Ten consecutive controls fall on one side of Systematic Rejection rule
the mean.

CLINICAL LABORATORY PRACTICE REVIEW 43

b 2
 A Generic Approach to an Out-Of-Range Control
Step
Repeat the same control.
Run a new vial of the same control.
Run a new control from a different lot.
Perform maintenance, check reagents,
and rerun the control.
Recalibrate and rerun the control.
Get assistance.
Document corrective action.
Rationale
Check for human error. V a h rn-Qht have been due to expected random
error. (1 in 20 results will be outside ?2 SD.)
First vial may have been outdated, improperly stored, contaminated.
First lot may have been outdated, improperly stored.
Problem might be due to inadequate preventive maintenance. Reagents
might have been outdated, improperly stored, contaminated.
Calibration may have shifted.
Supervisor or service rep might be able to locate source of problem.
Provides a record for future reference. Will point out repetitive problems.

44 CLINICAL LABORATORY PRACTICE REVIEW

 Miscellaneous Quality Control
Balances Verify accuracy monthly using class S weights.
Diluters Verify accuracy and precision on receipt, after service or repair, and on a regular sched-
ule using spectrophotometric procedure with potassium dichromate.
Dispensers Verify accuracy and precision on receipt, after service or repair, and on a regular sched-
ule using gravimetric method.
Micropipets Verify accuracy and precision on receipt, after service or repair, and on a regular sched-
ule. Primary method for calibration is gravimetric method. Secondary method is spec-
trophotometric procedure with potassium dichromate (unacceptable for volumes <10
$1 -
Temperature-controlled Check daily with a National Institute for Standards and Technology (N1ST)-certified
equipment: water thermometer or one calibrated against an NIST-certified thermometer. Most should be
baths, heat blocks, within t 1°C.
refrigerators, freezers
Thermometers Verify every 6-12 months with an NIST-certified thermometer. Should be within 21°C.
Water Monitoring of resistivity (ionic content), microbiologic content, pH, soluble silica.

CLINICAL LABORATORY PRACTICE REVIEW 45

 Procedure Manual Format (NCCLS and CLIA '88)
1. Requirements for specimen collection and processing and criteria for specimen rejection
2. Procedures for microscopic examinations, including the detection of inadequately prepared slides
3. Step-by-step performance of the procedure, including test calculations and interpretation of results
4. Preparation of slides, solutions, calibrators, controls, reagents, stains, and other materials used in testing
5. Calibration and calibration-verification procedures
6. Reportable range for patient test results
7. Control procedures
8. Remedial action to be taken when calibration or control results fail to meet the laboratory's criteria for acceptability
9. Limitations in methodologies, including interfering substances
10. Reference range
11. Imminent life-threatening laboratory results (panic or critical vaIues)
12. Pertinent literature references
13. Appropriate criteria for specimen storage and preservation to ensure specimen integrity until testing is completed
14. The laboratory's system for reporting patient results, including the protocol for reporting critical values
15. Description of the course of action to be taken in the event a test system becomes inoperable
16. Criteria for the referral of specimens, including procedures for specimen submission and handling
Manufacturer's package inserts may be used for items 1-13. Items not provided by manufacturer must be provided
by the laboratory.
Procedures and changes must be approved, signed, and dated by the director. Procedures must be retained for 2
years after date of discontinuation.
46 CLINICAL LABORATORY PRACTICE REVIEW
Col.lege of American Pathologists (CAP) Guidelines
for Retention of Specimens
Specimen Retention Period
- - - - - - -

Blood bank specimens 7 days post-transfusion or 10 days post-crossmatch

- -- -
Blood smears 7 days
Bone marrow smears 10 years
-
Microbiology slides, stained 7 days
Serum/CSF/body fluids 24 hours

CLlNICAL LABORATORY PRACTICE REVIEW 47

 CAP Guidelines for Retention of Records
Record n p e Retention Period
Accession records 2 years
Donor and recipient records Indefinitely
Instrument maintenance records 2 years
Patient test records 2 years
-- --
Quality control records 2 years (exception: 5 years for blood bank)

48 CLINICAL LABORATORY PRACTICE REVIEW

 -

Quality Assurance
Quality assurance Process by which a laboratory ensures quality results by closely monitoring the preanalyti-
cal, analytical, and postanalytical stages of testing.
Quality control Part of the analytical stage of quality assurance; process of monitoring results from control
samples to verify quality of patient results.
Sensitivity Ability of the method to detect slight differences in concentration. High sensitivity means
few false negatives and is desirable in screening tests.
-

Specificity The ability of the method to determine solely the compound it is supposed to measure. High
specificity means few false positives and is desirable in confirmatory tests.
Accuracy How close a measurement is to the true value. Implies freedom from error.
-- - -

Precision Reproducibility. Indicates how close test measurements are to each other when the same
test is run on the same sample time after time. Usually expressed in terms of standard devi-
ation (SD) or coefficient of variation. Precision does not imply accuracy. Precision implies
freedom from variation.
Imprecision A large amount of scatter about the mean. Most often caused by technique errors such as
variability in pipetting or inattention to detail by the operator. Results in an increase in the
SD or a broadening of the distribution about the mean.
I
Standard A solution that contains a known amount of an analyte. Used to calibrate an assay method.
Standard, primary A substance that can be accurately weighed or measured to produce a solution of an exactly
known concentration. Must be 99.98% pure. This level of purification is impossible to attain
with most biologic materials and is not necessary for routine testing.
- - - - -
Continued

38 CLINICAL LABORATORY PRACTICE REVIEW

 Quality Assurance Continued
- - - - - - - - - -

Coefficient of Expresses standard deviation as a percentage. CV % = (SD + mean) X 100. Allows

variation (CV) comparison of the precision of tests having different concentration ranges or units. The
lower the CV, the higher the precision.
Normal distribution The Gaussian bell-shaped curve.
Mean 5 1 SD = 68.2 % of observations
Mean 2 2 SD = 95.45% of observations (referred to as 95% confidence interval)
Mean 2 3 SD = 99.73% of observations
Levey-Jennings chart Normal distribution curve lying on its side, marked with the mean and ?1, 2, and 3 SD.
Thirty sequential control results are plotted on x axis.
Control limits Numerical limits within which the values of controls must fall for the assay to be considered
valid. Many labs use the mean 5 2 SD. One determination in 20 will fall outside 2 2 SD. This
is an anticipated part of normal variation.
Outlier A control result outside of the established limits. May be due to chance or may indicate a
problem in the test system. If it occurs more frequently than once in 20 successive runs,
then patient results cannot be reported. Investigation must be carried out.
Shift Six consecutive control values fall on the same side of the mean.
Trend Control values increasing or decreasing for six consecutive runs.
Error, random Error affecting a particular determination that does not recur in a regular pattern (e.g., error
due to use of dirty glassware or the wrong pipet, voltage fluctuation, sampling error, antico-
agulant or drug interference). Indicated by a control value that is significantly different from
the other values on a Levey-Jennings chart, or violation of the l,, and R,, Westgard rules.
Usually a one-time error and controls and samples can be rerun with success.
Continued
40 CLINICAL LABORATORY PRACTICE REVIEW
 Quality Assurance Continued
Standard, secondary A substance of lower purity whose concentration is determined by assay and comparison
with a primary standard.
Calibrator Substance with an assigned value that the manufacturer establishes by using a reference
method or by using reference materials traceable to primary standards. Used to set the value
reported by the laboratory's method or instrument.
Control A sample that is chemically and physically similar to the unknown specimen and is tested
in exactly the same manner. Monitors precision of the test system. Controls should be run at
all levels of clinical importance. For quantitative tests, CLIA '88 requires that at least two
control samples of different concentrations (normal and abnormal) be assayed at least every
24 hours. Some states mandate three pools (low abnormal, normal, high abnormal) for
some tests. [For qualitative tests, positive and negative controls must be included with each
run of patient specimens.)
- -

Measures of Statistical parameters describing the spread of data about the mean; e.g., standard
dispersion deviation, coefficient of variation, and range. Measurements of precision.
- --- - - - - - - - -- - - - -

Range Difference between the highest and lowest values in a data set. An indication of the disper-
sion of data points.
-

Mean Sum of all observations divided by the number of observations. The average of all observa-
tions.
Standard deviation Statistical expression of the dispersion of values around the mean. Equal to the square root
(SD) of the sum of the squared differences from the mean divided by the number of values minus
one. A minimum of 20 analyses are needed for the calculation.
Continued

CLINICAL LABORATORY PRACTlCE REVIEW 39

 Quality Assurance Continued
Error, systematic Recurring, measurable error that is inherent in a test procedure. Is continuous and affects all
results. Indicated by a trend or a shift on a Levey-Jennings chart, or violation of the 2,,, 4,,
or 10, Westgard rules. Requires investigation to determine cause. Examples: dirty photome-
ter, faulty ISE, evaporation or contamination of standards or reagents.
False rejection Rejection of an analytical run because quality control results indicate arlalytical problems
that are not really present. Use of Westgard rules minimizes false rejections.
Linearity Reportable range.
Reportable range The range of concentrations that may be reported without the need for further treatments
such as dilution. Also known as linearity.
Reference range Formerly called normal values. Each laboratory should set its own reference ranges for all
analytes. 100-150 data points should be gathered from a representative healthy client popu-
lation. Data are arranged in sequential order and the data points that occupy the 2.5 % posi-
tion at the low end and the 97.5% position at the high end define the range. (95% confi-
dence interval. One normal person in 20 will fall outside the reference range.)
Significant change A change greater than three times the quality control SD. Indicates a real change in the pa-
tient.
Delta check Comparison of patient data with previous results. Delta limits are set to detect specimen
mix-up or other errors. When limits are exceeded, one must determine if a major medical
change has occurred in the patient or if a laboratory error occurred.
Preventive A schedule of daily, weekly, and monthly maintenance of equipment designed to keep it in
maintenance peak operating condition. Must be documented.
Continued

CLINICAL LABORATORY PRACTICE REVIEW 41

 Commonly Used Prefixes in the Metric System
Deci = 10-1
Centi = 10-2
Milli = 10--3
Micro = 10-6
Nano = 10-9
Pico = 10-12
Femto = 10-15

50 CLINICAL LABORATORY PRACTICE REVIEW

d
 General Laboratory Calculations
If the temperature of a refrigerator is 4"C, what is the temperature
in OF?
O F = (1.8 x C) + 32 = 39.2

If the room temperature is 73"F, what is the temperature in "C?

73 = 1.8C + 32

Standard Deviation (SD) =

J Z 6-x ) ~
- Z = sum, x = individual value, K = mean, n = number of values

Coefficient of variation (%) = SD x 100 What is the CV for a procedure whose mean is 100 and whose
Mean standard deviation is 3?
cv = 3
100
x 100 = 3%

Dilution = What is the dilution if 0.1 mL of serum is diluted with 0.4 mL of saline?
Vol. of specimen 0.1 - 0.1 1
-
Vol. of specimen + vol. of dilluent 0.1 + 0.4 0.5 5
How would you prepare a 1:10 dilution of urine?
1 part of urine + 9 parts of diluent
Correcting for a dilution: Value obtained A specimen for a glucose is diluted 1:s. The value of the diluted
for diluted specimen x reciprocal of specimen is 100 mg/dL. What value should be reported?
dilution 100 mg/dL x 5 = 500 mg/dL

CLINICAL LABORATORY PRACTICE REVIEW 51

C
 Laboratory Tests/Medical Records tI
Laboratory tests can only be ordered by physicians.
Requests for laboratory tests must be made in writing or by electronic means.
Test results are not to be released to anyone except the treating physician without the patient's consent.
Unauthorized release of test results can lead to charges of breach of confidentiality or invasion of privacy.
The most common cause of erroneous lab reports is clerical error.
To correct an erroneous report, draw a line through the incorrect value so that it is still legible. Do not erase or
white-out. Record the corrected value and date the entry.
Laboratory records are the property of the laboratory or of the hospital if the laboratory is owned by the hospital.
Patients have a right to their own healthcare information, including laboratory test results.

CLINICAL LABORATORY PRACTICE REVIEW 49

i
I
 SECTION 2

Clinical Chemistry
Review
 Conventional Units vs. SI Units
Comparison of Selected Reference Ranges
Amlyte Conventional Units sz Units
Bilirubin, total 0.2-1.0 mg/dL 3-1 7 pmol/L
BUN 7- 18 mg/dL 2.5-6.4 mmol/L
Calcium, total 8.7-10.2 mg/dL 2.18-2.25 mmol/L
Chloride 98-106 mEq/L 98-106 rnmol/L
-- ~ - - - --

Creatinine Male: 0.6-1.2 mg/dL Male: 53-106 mmol/L

Female: 0.5-1.1 mg/dL Female: 44-97 mmol/L
Glucose, fasting 70-110 mg/dL 3.9-6.0 mmol/L
Magnesium 1.6-2.4 mEq/L 0.65-1.0 mmol/L
Potassium 3.4-5.0 mEq/L 3.4-5.0 mmol/L
Sodium
Total protein 6.5-8.3 g/dL 65-83 g/L
Uric acid Male: 3.5-7.2 mg/dL Male: 208-428 pmol/L
Female: 2.6-6.0 mg/dL Female: 155-357 pmol/L

54 CLINICAL CHEMISTRY REVIEW

 Patient Variables That Affect Chemistry Values
Factor Examples of Amlytes Affected
Age Albumin, alkaline phosphatase, phosphorus, cholesterol
Gender Albumin, alkaline phosphatase, creatinine, uric acid
Diurnal variation 1'in AM: adrenocorticotropic hormone (ACTH), cortisol, iron
?' in PM:acid phosphatase, growth hormone, parathyroid hormone (PTH), thyroid-
stimulating hormone (TSH)
Day-to-day variation 20% or more for alanine aminotransferase (ALT), bilirubin, iron, TSH, triglycerides
Recent food ingestion 1'glucose, insulin, triglycerides, gastrin
.1chloride, phosphorus, potassium
Foods and beverages Avocados, bananas, eggplant, pineapple, plums, and walnuts cause false-positive
5-hydroxyindole acetic acid.
Bananas, vanilla cause false-positive vanillylmandelic acid (VMA].
Caffeine increases cortisol.
Alcohol increases triglycerides and gamma glutamyl transaminase (GGT).
Posture '? when standing: albumin, cholesterol
- - -

Activity '? in ambulatory patients: creatinine kinase (CK)

'? with exercise: lactic acid, creatinine, protein, CK, aspartate aminotransferase (AST],
lactate dehydrogenase (LD)
.1with exercise: cholesterol, triglycerides
Stress 1'ACTH, cortisol, catecholamines

CLINICAL CHEMISTRY REVIEW 55

 Effect of Anticoagulant Contamination
on Chemistry Analytes
Anticoagulant Amlytes Affected
Ethylenediaminetetra- calcium, magnesium
acetic acid (EDTA) Inhibits alkaline phosphatase, CK, amylase
? potassium with K, EDTA, ? sodium with Na, EDTA.
Sodium fluoride/ .1calcium, magnesium
potassium oxalate Fluoride inhibits enzymes; interferes with BUN by urease and uric acid by uricase
Sodium citrate .1calcium, magnesium
Inhibits alkaline phosphatase, CK, amylase
Heparin Inhibits alkaline phosphatase
? ammonia with ammonium heparin, ? lithium with lithium heparin, ? sodium with
sodium heparin if tube is not completely filled
Lithium iodoacetate Inhibits CK
? lithium

56 CLINICAL CHEMISTRY REVIEW

 Collection, Processing, and Storage Factors That
Affect Chemistry Results
Factor Examples of A ~ l y t e Affected
s
Squeezing site of capillary puncture I' potassium
Pumping fist during venipuncture '? potassium, lactic acid, calcium, phosphorus
-1 PH
Tourniquet >1 minute ? potassium, total protein, cholesterol, iron
IV fluid contamination Common cause of I' glucose, potassium, or drugs, depending on IV
Serum separator tubes -1 tricyclic antidepressants, some antiarrhythmics
Hemolysis ? potassium, magnesium, phosphorus, LD, acid phosphatase, AST, iron,
total protein, ammonia
Exposure to light -1 bilirubin, carotene
Delay in separating serum/plasma ? ammonia, lactic acid, potassium, magnesium, LD
-1 glucose
Temperature of storage -1 at room temperature: glucose, acid phosphatase
I' at room temperature: lactic acid, ammonia
-1 at 4°C: LD

CLINICAL CHEMISTRY REVIEW 57

 Factors That Affect Enzyme Reactions
Factor Effect
Substrate First-order kinetics: [enzyme] > [substrate].
concentration Reaction rate proportional to [substrate].
Zero-order kinetics: [substrate] > [enzyme].
Reaction rate proportional to [enzyme].
Enzyme Velocity of reaction is proportional to [enzyme]
concentration as long as [substrate] > [enzyme].
Comments
Use zero-order reaction [excess substrate).
Report in U/L. [U = International Unit =
amount of enzyme that will catalyze the
reaction of 1 micromole of substrate per
minute under specified conditions.)

PH Extremes of pH may denature enzymes.
Temperature An increase of 10°C doubles the rate of reaction
until around 40-50°C when denaturation of
enzyme may occur.
Cofactors Activators = inorganic cofactors (metallic and
nonmetallic ions)
Coenzymes = organic cofactors (nucleotide
phosphates and vitamins)
The higher the concentration of cofactors,
the greater the rate of reaction.
Inhibitors Interfere with the reaction.
Most reactions occur at pH 7-8. Use buffers.
37°C is most commonly used in U.S.
Must be present in excess.
Common cofactors: nicotinamide adenine
dinucleotide (NAD) o nicotinamide adenine
dinucleotide, reduced form (NADH). NADH
has absorbance at 340 nm; NAD does not.
Must be lacking.

58 CLINICAL CHEMISTRY REVIEW

L
 ----.*..- &

Differences in Analyte Concentrations /

1
Higher in Higher in Higher in Higher in
Plasma Serum Plasma Higher in Capillary Venom Blood
than in than in than in Blood than in than in
Serum Plasma Whole Blood Venom Blood Capillary Blood
Total protein Potassium Glucose Glucose (in postprandial Calcium
LD Phosphorus specimen) Total protein
Calcium Glucose Potassium
CK
Bicarbonate
Alkaline phosphatase
(ALP)
Albumin
Aspartate aminotransferase
(ASTI
Triglycerides

CLINICAL CHEMISTRY REVIEW 59

L
 Differences in Electrolyte Concentrations
Higher Concentration in RBCs Higher Concentration in Plasma
Potassium Sodium
Phosphorus Chloride
Magnesium

60 CLINICAL CHEMISTRY REVIEW


Panel
Basic metabolic panel
Comprehensive metabolic panel
Electrolyte panel
Hepatic function panel
Lipid panel
Thyroid panel
Chemistry Panels
Tesl
Sodium, potassium, chloride, CO,, glucose, creatinine, BUN
Sodium, potassium, chloride, CO,, glucose, creatinine, BUN, albumin, total
protein, ALP, AST, bilirubin, calcium
Sodium, potassium, chloride, CO,
Albumin, alanine aminotransferase (ALT), AST, ALP, bilirubin (total and direct)
- -
Cholesterol (total), HDL cholesterol, triglycerides
Thyroxine (T,), thyroid hormone binding ratio (THBR or T3 uptake), thyroid-
stimulating hormone (TSH)
CLINICAL CHEMISTRY REVIEW 61
 ..~.".~_Tlln..-sr
Chemistry Tests
Analyte Reference Range Clinical Significance
Sodium 138-146 mmol/L T (hypernatremia): hyperaldosteronism,
or mEq/L ingestion or administration of large
amounts of sodium, excessive sweating,
burns, hyperventilation, diabetes
insipidus, osmotic diuresis
.1(hyponatremia): heart failure, liver
disease, nephrotic syndrome, renal
failure, inappropriate antidiuretic
hormone (ADH) secretion, excessive
fluid intake, vomiting, diarrhea, burns,
diuretic therapy, hypoaldosteronism
Potassium 3.7-5.3 mmol/L '? (hyperkalemia): acidosis, crush injuries,
or mEq/L tissue hypoxia, insulin deficiency, digitalis
overdose, IV administration, transfusion
of aged blood, renal failure, hypoaldo-
steronism, diuretics, defects in renal
tubular secretion
.1(hypokalemia): alkalosis, increased
insulin, diuretics, decreased intake,
increased GI or urinary loss
62 CLINICAL CHEMISTRY REVIEW
Other
Major extracellular cation. Contri-
butes almost half to plasma
osmolality. Central role in maintain-
ing normal distribution of water and
osmotic pressure. Levels regulated
by aldosterone. Ion-selective electrode
is most common method. Artifac-
tually low levels in samples with
elevated lipids or protein with some
methods. Normal Na+:K+ ratio in
serum approximately 30:l. Critical:
< 120 and > 160. If sodium heparin is
used, tubes must be completely filled.
Major intracellular cation. Critical:
<2.8 and X . 2 . Hyper- and hypo-
kalemia cause cardiac arrhyth-
mias. Pseudohyperkalemia due to
squeezing site of capillary puncture,
prolonged tourniquet time, pumping
of fist during venipuncture, contam-
ination with IV fluid, hemolysis,
prolonged contact with RBCs, icing
specimen before separation, leuko-
cytosis, thrombocytosis. Serum
Continued
F - w
Chemistry Tests Continued
Analyte Reference Range Clinical Significance Other
values 0.1-0.2 mmol/L higher than
plasma due to release from platelets
during clotting. Most common
method is ISE with membrane in-
corporating valinomycin.
Chloride 98-109 mmol/L T prolonged diarrhea, renal tubular Major extracellular ion. Maintains
disease, hyperparathyroidism, hydration, osmotic pressure, ionic
dehydration balance. Changes usually parallel
J prolonged vomiting, burns, salt-losing changes in sodium. Most common
renal diseases, overhydration method is ISE. Other methods:
colorimetric and coulometric
titration.
- -

CO,, total 23-30 mmol/L ? metabolic alkalosis, compensated Primarily bicarbonate. Keep sample
respiratory acidosis capped to prevent loss of CO,.
J metabolic acidosis, compensated
respiratory alkalosis
Glucose, 70-110 mg/dL ? (hyperglycemia): diabetes mellitus, Major source of cellular energy.
fasting (3.9-6.1 mmol/L) pancreatitis, hepatic failure, renal disease Levels decrease at room tempera-
J (hypoglycemia): insulinoma, insulin- ture. Use sodium fluoride or iodo-
induced hypoglycemia, acetate to preserve. Hexokinase is
hypopituitarism most specific and widely used
method. Critical: <40 mg/dL
Continued

CLINICAL CHEMISTRY REVIEW 63

 p
- -?Zb.--
-
---
Chemistry Tests Continued
Analyte Reference Range Clinical Significance Other
(coma, seizures, death) and >SO0
mg/dL (acidosis, coma). > 126
mg/dL on more than one occasion
= diabetes mellitus.

BUN 8-26 mg/dL T kidney disease Synthesized by liver from ammonia.

(3.0-9.2 mmol/L) -1 overhydration or liver disease Excreted by kidneys. Urease reagent.
Do not use sodium fluoride, EDTA,
citrate, or ammonium heparin. Test is
not sensitive. Urine-dilute 1:20 or
150 and refrigerate or acidify.
Creatinine 0.7-1.5 mg/dL T kidney disease Waste product formed by dehydra-
160-130 pmol/L) tion of creatine (mainly in muscles).
Jaffe's reaction (alkaline picrate) is
nonspecific. Enzymatic methods are
more specific. Tests are not sensitive.
Normal BUN:creatinine ratio = 12-20.
Urine-dilute 1:loo.
Uric acid 2.5-7.0 mg/dL T gout, renal failure, ketoacidosis, Increased = risk of renal calculi and
(0.15-0.40 lactate excess, high nucleoprotein diet, joint tophi.. Uricase method. EDTA
mmol/L) leukemia, lymphoma, polycythemia and fluoride interfere. Urine-adjust
-1 administration of ACTH, renal pH to 7.5-8 to prevent precipitation.
tubular defects
Continued
64 CLINICAL CHEMISTRY REVIEW
 - P.
4
'

Chemistry Tests Continued

Amlyte Reference Range Clinical Significance Other
Cholesterol Desirable T hypothyroidism, nephrosis, diabetes, Direct correlation with risk of coronary
<200 mg/dL liver disease artery disease.
severe liver insufficiency
Mglycerides Desirable T risk factor for atherosclerotic cardio- Main form of lipid storage. Requires
<200 mg/dL vascular disease a fasting specimen.
k malnutrition
Total protein 6.0-8.0 g/dL T dehydration, chronic inflammation, <4.5 g/dL associated with peripheral
(60-80 g/L) multiple myeloma edema. Biuret method. Alkaline
-1 nephrotic syndrome, malabsorption, copper reagent reacts with peptide
overhydration, hepatic insufficiency, bonds.
malnutrition, agammaglobulinemia
Albumin 3.5-5.0 g/dL T dehydration Largest fraction of plasma proteins.
(35-50 g/L) -1 malnutrition, liver disease, Synthesized by liver. Regulates
nephrotic syndrome, osmotic pressure. Measure by dye
chronic inflammation binding; e.g., bromcresol green(BCG),
bromcresol purple (BCP) .
Bilirubin, 0.1-1.2 mg/dL T liver disease, hemolysis, hemolytic From breakdown of RBCs. Metabolized
total (1.7-20.5 pmol/L) disease of the newborn in liver. In infants, >20 mg/dL associated
with brain damage (kernicterus) and
may require exchange transfusion.
Protect from light. Diazo reagent +
methyl alcohol or caffeine.
Continued
CLINICAL CHEMISTRY REVIEW 65
 Contents
SECTION 1: CLINICAL
. . LABORATORY PRACTICE REVIEW . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
1. Credential~ng. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
2 . Regulatory Agencies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
3 . Agencies that Issue Guidelines/Standards . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
4 . Clinical Laboratory Improvement Amendments of 1988 (CLIA '88) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
5. OSHAStandards . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
6 . Bloodborile Pathogens . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
7 . Hazard Categories of Chemicals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
8 . Hazard Identification System-National Fire Protection Association (NFPA) . . . . . . . . . . . . . . . . . . . . . . . 10
9 . Storage of Chemicals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
10. Firesafety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
11. Commonly Used Anticoagulants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
12. Tests Requiring a Fasting Specimen . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14
13. Recommended Order of Drawing/Filling Tubes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
14. Situations in Phlebotomy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .16
15. Specimens Requiring Special Handling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
16. SpecimenProcessing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .18
17. Exan~plesof Criteria for Specimen Rejection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19
18. Centrifuge Fast Facts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .20
19. Packaging of Biologics for Shipment in Mail . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21
20 . TypesofGlass . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22
21 . Glassware Inscriptions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23
22 . Volumetric Glassware . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .24
23 . GlassPipets . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .25
24 . Types of Plastic . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .26
25 . GradesofChemicals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27
26 . Reagent Grade Water . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .28
27 . Water Purification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29
 Contents
SECTION 1: CLINICAL LABORATORY PRACTICE REVIEW ...................................... 1
1. Credentialing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
2 . Regulatory Agencies. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
3 . Agencies that Issue Guidelines/Standards . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
4 . Clinical Laboratory Improvement Amendments of 1988 (CLIA '88) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
5 . OSHAStandards . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
6 . Bloodborne Pathogens . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
7. Hazard Categories of Chemicals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
8 . Hazard Identification System-National Fire Protection Association (NFPA) . . . . . . . . . . . . . . . . . . . . . . . 10
9 . Storage of Chemicals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
10. Firesafety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
11. Commonly Used Anticoagulants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
12. Tests Requiring a Fasting Specimen . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14
13. Recommended Order of Drawing/Filling Thbes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
14. Situations in Phlebotomy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16
15. Specimens Requiring Special Handling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
16. Specimen Processing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18
17. Examples of Criteria for Specimen Rejection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19
18. Centrifuge Fast Facts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
19. Packaging of Biologics for Shipment in Mail . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21
20 . TypesofGIass . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22
21 . Glassware Inscriptions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23
22 . Volumetric Glassware . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24
23 . Glass Pipets . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25
24 . Types of Plastic . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26
25 . Grades of Chemicals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27
26 . Reagent Grade Water . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28
27 . Waterpurification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29
 28 . Brightfield Microscopy Fast Facts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
29 . Other v p e s of Microscopy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
30 . Computers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
31. ComputerNetworks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ?
32 . Phases of Quality Assurance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
33. Quality Assurance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
34 . Westgard Multirules . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
35 . A Generic Approach to an Out-of-Range Control . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
36 . Miscellaneous Quality Control . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
37. Procedure Manual Format (NCCLS and CLIA '88) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
38 College of American Pathologists (CAP) Guidelines for Retention of Specimens . . . . . . . . . . . . . . . . . . . . . 4
39 . CAP Guidelines for Retention of Records . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
40 . Laboratory TestsIMedical Records. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
41. Commonly Used Prefixes in the Metric System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
42 . General Laboratory Calculations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . !

SECTION 2: CLINICAL CHEMISTRY REVIEW ................................................ 5

43 . Conventional Units vs . SI Units-Comparison of Selected Reference Ranges . . . . . . . . . . . . . . . . . . . . . . . 5
44 . Patient Variables that Affect Chemistry Values . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
45 . Effect of Anticoagulant Contamination on Chemistry Analytes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
46 . Collection, Processing. and Storage Factors that Affect Chemistry Results . . . . . . . . . . . . . . . . . . . . . . . . . 5
47 . Factors that Affect Enzyme Reactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
48 . Differences in Analyte Concentrations. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
49 . Differences in Electrolyte Concentrations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
50 . ChemistryPanels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . t
51. ChemistryTests . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
52 . Enzymes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
53. Summary of Diagnostic Enzymology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . i
54.CKandLDIsoenzymes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
55. Cardiac Markers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7

xii CONTENTS

56 . Regulation of Glucose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 74
57. Diabetes Mellitus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 75
58. Tests for Diabetes Mellitus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 76
59. 'Ifrpical Laboratory Findings in Uncontrolled Diabetes Mellitus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 79
60 . Lipoproteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 80
61. Characteristics of Lipoproteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 81
62 . Lipid Levels (National Cholesterol Education Program Adult Treatment Panel) . . . . . . . . . . . . . . . . . . . . . 82
63. Protein Electrophoresis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 83
64. Common Serum Protein Electrophoresis Patterns . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 84
65 . Inborn Errors of Metabolism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 85
66. Bilirubin Metabolism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 87
67. Bilirubin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 88
68. Differential Diagnosis of Jaundice . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 89
69 . Iron and Related Tests in Selected Diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 90
70 . Structural Classes of Hormones . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 91
71. Hormones . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 92
72.ThyroidTests . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 97
73. Thyroid Test Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 98
74 . Acid-Base Balance Terminology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 99
75. Acid-Base Imbalances . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 101
76. Arterial Blood Gases Terminology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 102
77 . Blood Gas Parameters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 103
78. Blood Gas Instrumentation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 104
79 . Sources of Error in Arterial Blood Gases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 105
80 . Therapeutic Drug Monitoring . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 106
81 . Therapeutic Drugs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 107
82.Toxicology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 108
83 . Visible Light . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 110
84.Spectrophotometry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111
85. Chemistry Instrumentation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 112

CONTENTS xiii
 86. Automated Chemistry Analyzers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 115
87. Calculated Chemistry Values . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 116
88. Chemistry Calculations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 120

SECTION 3: CLINICAL MICROBIOLOGY REVIEW ............................................. 123

89. Biosafety Levels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 124 .
90. Biologic Safety Cabinets . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 125
91. Sterilization and Disinfection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 126
92 . Bacterial Toxins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 128
93. Specimen Collection Guidelines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 129
94. Specimen Preservation and Storage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 130
95.FragileOrganisms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 131
96. Examples of Criteria for Rejection of Specimens in Microbiology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 132
97. TheGramStain . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 133
98. Staining Properties of Gram-Positives and Gram-Negatives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 134
9 9 . v p e s o f M e d i a . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .135
100. Routine Primary Media for Aerobes and Facultative Anaerobes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 136
101. Media for Culture of Anaerobes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 140
102. Special Bateriologic Media . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 141
103. Aerotolerance Test . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 144
104. Organisms Requiring lncubation in lncreased CO, . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 145
105. Gram-Positive Cocci . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 146 .
106. Summary of Tests for Identification of Gram-Positive Cocci . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 151
107. Gram-Positive Bacilli . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 155
108. Neisseria and Related Organisms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 158
109. Enterobacteriaceae . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 161
110. Biochemical Tests for Identification of Enterobacteriareae. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 162
111. Antigens of Enterobacteriaceae . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .166
112. Commonly Isolated Enterobacteriareae (Usual Reactions) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 167
113. Summary of Key Reactions for Enterobacteriaceae . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 171
114. Appearance of Enterobactenaceae on Selected Media . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 172
xiv CONTENTS
146. Trematodes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 225
147. Blood and Tissue Protozoa . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 227
148.Plasmodium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .229
149. Blood and Tissue Helminths . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 230
150. Parasites of the Urogenital Tract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 231
151. Stains Used in Mycology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 232
152. Media Used in Mycology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 233
153.Dermatophytes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 234
154.DimorphicFungi . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .235
155.Yeasts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .237
156. Contaminants/Opportunistic Fungi . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 239
157. Fungal Pathogens by Site . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 242
158. Viral Structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 243
159. Viral Replication . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 244
160. Human DNA Viruses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .245
:
161. Human RNA Viruses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 246
162. Common Viral Pathogens . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 248
163. Specimens for Viral Culture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 249 .
164. Viruses that May Be Cultured from Clinical Specimens . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .250
165. Methods Used to Diagnose Viral Infections . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 251
166. Tissue Cultures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 253
167. Comparison of Microorganisms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 254

SECTION 4: HEMATOLOGY REVIEW ...................................................... 255

168. Adult-Reference Ranges: Comparison of Conventional and SI Units . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 256
169. Pediatric Hematologic Findings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 257
170. Bloodcells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .258
171. Hematopoiesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 259
172. Changes During Cell Maturation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 261
173. Erythrocytic Developmental Series . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 262
174. Asynchronous Erythropoiesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 263
xvi CONTENTS
 .
175.Hemoglobin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 264
176. Hemoglobin Electrophoresis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 263
177. Hemoglobin Derivatives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .266
178. Troubleshooting the Wright's-Stained Blood Smear . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 267
179. RBCMorphology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .270
180.RBCInclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 273
181. Summary of RBC Inclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 275
182. Erythrocyte Indices . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 276
183. Hemoglobinopathy vs . Thalassemia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 278
184.Anemias . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 279
185. Differentiation of Microcytic Hypochromic Anemias . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 284
186. Anemia Due to Blood Loss . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .285
187. Highlights of Granulocytic Maturation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 286
188. Normal Leukocytes of the Peripheral Blood . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 287
189. Leukocyte Abnormalities . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .288
190. Quantitative Abnormalities of Leukocytes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 289
191. Acute vs . Chronic Leukemia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 290
192. Acute Nonlymphocytic Leukemia (ANLL) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 291
193. Acute Lymphoblastic Leukemia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 292
194. Chronic Myeloproliferative Disorders (CMPD) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .293
195. Leukemoid Reaction vs . Chronic Myelogenous Leukemia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 294
196. Chronic Lymphoproliferative Disorders . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .295
197. Plasma Cell Disorders . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 296
198. Hematology Special Stains . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 297
199. Erythrocyte Sedimentation Rate (ESR) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 299
200 . Other Manual Hematology Procedures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 301
201 . Sources of Error in Selected Manual Hematology Tests . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 303
202 . Changes in Blood at Room Temperature . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 305
203 . Interfering Factors on Most Hematology Analyzers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 306
1 204 . Hematology Calculations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 308
: 205 . Overview of Hernostasis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 312
CONTENTS xvii
235 . Labeled Immunoassay Terminology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 357
236 . Comparison of Radioimmunoassay and Enzyme Immunoassay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 358
237 . ImmunoassayLabels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 359
238 . Principles of Labeled Immunoassays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 360
239 . Labeled Immunoassays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 363
240 . Nontreponemal Tests for Syphilis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 364
241 . 'I'reponemal Tests for Syphilis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 365
242 . Tests for the Diagnosis of Infectious Mononucleosis (IM) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 366
243 . Weil-Felix Reaction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 367 .
244 . Antinuclear Antibodies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 368
245.HepatitisTests . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .370
246 . Hepatitis Profiles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 371
247. HIVTesting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .372
248 . Other Serological Tests . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 374
249 . Interpretation of Serological Tests . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 376
250 . Serology Calculations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 377

SECTION 6: IMMUNOHEMATOLOGY REVIEW ............................................... 379

251 . Primary vs . Secondary Response . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .380
252 . 1gGvs.IgM . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .381
253 . Signs of Antigen-Antibody Reactions in Blood Bank Testing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 382
254 . Factors that Affect Agglutination . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 383
255 . Antigen-Antibody Enhancement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .384
256 . Antihuman Globulin Serum . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .385
257 . Antiglobulin Testing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 386
258.AandBAntigens . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 388
259 . ABO Genotypes and Phenotypes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 389
260 . Using Punnett Square to Predict ABO ?frpe . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 390
261 . Frequency of ABO Types . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 391
262. ABOSystem . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 392
263.ABOTyping . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 393
CONTENTS xix
295. Blood Donor Deferrals (AABB) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 430
296.BloodCollection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .432 .
297 . Anticoagulants/Additives/Rejuvenating Solutions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .433
298 . CPDA-1 Ingredients . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .435
299 . Donor Serological Testing Required by AABB and/or FDA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .436
300. Autologous Transfusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 437
301. BloodComponents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 438
302 . Opensystem . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 442
303. Changes in Stored Blood . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 443
304 . Blood Bank Quality Control . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 444
305. FDA Required Reports . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 446

SECTION 7: URINALYSIS AND BODY FLUIDS REVIEW ......................................... 447

306 . Changes in Urine at Room Temperature . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 448
307. UrineVolume . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .449
308. Urine Color and Clarity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .450
309. Chemical Urinalysis by Reagent Strip . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 451
310 . Confirmatory/Supplemental Urine Chemistry Tests . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .455
311. Effect of High Levels of Ascorbic Acid on Urinalysis Tests . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 457
312. Cells in the Urine Sediment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 458
313 . Crystals Found in Acid or Neutral Urine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 460
314. Crystals Found in Alkaline Urine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 462
315. Casts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .463
316. Miscellaneous Urine Sediment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 465
317 . Renal Disorders . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .466
318 . Cerebrospinal Fluid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 467
319. Meningitis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 469
320 . BodyFluids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 470
321. Transudates and Exudates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 471
322. CellsinBodyFluids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 472
323.BodyFluidCells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .473
CONTENTS xxi
353. Laboratory Operating Costs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 509
354 . Payroll Hours . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 510
355 . Break-Even Points . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .511
356 . Reimbursement for Services . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .512
357 . Legal Terminology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 514
358. Competency-Based Instruction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .517
359 . The ABCs of Writing Behavioral Objectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 518
360 . Learning Styles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .520
361. What Students Remember . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .521 .
362 . Domains of Learning . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .522
363 . Bloom's Cognitive Taxonomy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 523
364. The Seven Principles of Good Teaching . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .524
365. Planning for Instruction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .525
366. TheLessonPlan . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 526
367. Instructional Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .527
368.Testing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 528
.
369 . Norm-Referenced vs . Criterion-Referenced Tests . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 529
370. m e s of Test Questions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .530
371. Testing at Different Cognitive Levels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .532
372 . Changing Educational Paradigms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .533
373. What Students Say About Their Best Teachers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .534
374 . What Students Say About Their Worst Teachers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 536
375. Legal Issues for Educational Programs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .537
376. Test-Taking Tips . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .538

CONTENTS xxiii
324.SynovialFluid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 474 .
325. Synovial Fluid Crystals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 475
326 . SemenAnalysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 476
327. Amniotic Fluid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 478

SECTION 8: MANAGEMENT AND EDUCATION REVIEW ........................................ 479

328.ManagementSkills . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .480
329. The Foundations of Management . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 481
330. The Management Process . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 482
331. Laboratory Management . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 483
332 . McGregor's Theory X and Theory Y . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 484
333. Management Styles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 485
334 . Maslow's Hierarchy of Needs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 486
335. Principles of Total Quality Management/Continuous Quality Improvement . . . . . . . . . . . . . . . . . . . . . . . . 487
336. Team Problem-Solving Method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .488
337. Federal Legislation Governing Hiring Practices . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .489
338. Federal Legislation Governing Employee Compensation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .490
339. Interview Questions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 491
340. Components of a Criteria-Based Job Description . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 493
341. Components of an Employee Performance Appraisal System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 494
342. Performance Standards . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 495
343. Evaluation Errors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 496
344. Testing Personnel Competency Assessment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 497
345. Documents to be Included in Personnel File . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .498
346 . Requirements of OSHA's Bloodborne Pathogen Standard . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 499
347. Requirements for a Chemical Hygiene Plan . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 500
348. Requirements of OSHA's Formaldehyde Standard . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 501
349 . Clinical Laboratory Improvement Amendments of 1988 (CLIA '88) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 502
350 . Test Complexity under CLIA '88 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .503
351. Personnel Required in High-Complexity Laboratories under CLIA '88 . . . . . . . . . . . . . . . . . . . . . . . . . . . . 506
352 . Laboratory Finances . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .507
xxii CONTENTS
r . 1
264. ABO Discrepancies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .394
265. Non-Group Specific Transfusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 9 6 1
266 . Rh Genotypes and Phenotypes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .3 9 7 1
267 . Using Punnett Square to Predict Rh V p e . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 9 8 ;
268. RhAntigens . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .399
269. Frequency of Rh Antigens . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 400
270. FrequencyofRhGenes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 401
271. Breaking the Rh Secret Code . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 402
272.RhTyping . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .403
273. Positive Rh Controls . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .404
274.RhTypingSera . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 0 S i
275 . Selection of Rh v p e . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .4 0 7 1
276 . Frequency of Other Selected Blood Group Antigens . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 0 8 ;
277. ISystem . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 410
278. Antibody Characteristics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 411
279 . Antibody Identification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 413
280. lnterpretation of Antibody Panels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 1 4 i
281 . Cold Antibodies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .4 1 5 ;
282 . Compatibility Testing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .416
283.Crossmatches . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 1 7 '
284. The Major Crossmatch . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 418 i
285. The lncompatible Crossmatch . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .419
286. Emergency Transfusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 420
287 . NewbornTesting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 421
288. Conditions for Reissue of Blood . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 2 2 ~
289. Transfusion Reactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 423
290 . Transfusion Reaction Investigation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 425
291 . Transfusion-Associated Diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 426
292 . Hemolytic Disease of the Newborn . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 427
293. Rh Immune Globulin (RhIG) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 428
294 . Allogeneic Blood Donor Criteria (AABB) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 429
xx CONTENTS
206. Coagulation Factors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 313
207 . Summary of Coagulation Factors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 315
208. Inherited Factor Deficiencies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 316
209 . Acquired Factor Deficiencies. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 318
210. Coagulation Tests. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .319
211. Effect of Factor Deficiencies on PT and APTT . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 321
212 . Substitution Studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .322
213 . Laboratory Findings in Selected Coagulation Abnormalities . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 324
214 . Disseminated Intravascular Coagulation vs . Primary Fibrinolysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 326
215 . Anticoagulant Therapy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 327 .
216 . Thrombolytic Therapy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .328
217. Sources of Error in Coagulation Testing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 329
218. Causes for Rejection of Specimens for Coagulation Testing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 331

SECTION 5: IMMUNOLOGY REVIEW ...................................................... 333

219. ImmunoIogyTerms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .334
220. Branches of the Immune System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .337
221 . Types of Immunity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 338
222.AdaptiveImmunity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .339
223. Cells of the Immune System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 340
224.LymphoidOrgans . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 342
225 . Laboratory Identification of Lymphocytes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 343
226. Common Lymphocyte Markers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .344
227. Immunoglobulin Structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 345 .
228 . Immunoglobulins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .346
229 . Complement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .348
230. ImmunePhenomena . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .349
231. Hypersensitivity Reactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 350
232 . Agglutination Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 351
233 . Precipitation Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 353
234 . Other Serological Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 355
xviii CONTENTS
 RBC Morphology
Abnormality Description
Anisocytosis Variation i n size
Macrocytes RBCs greater than 9 p,m
Microcytes RBCs less than 6 p,m
Poikilocytosis Variation in shape
Elliptocytes/0valocytes Oval or pencil-shaped
Crenated RBCs Round cell with knobby,
uniform projections
Burr cells (echinocytes) Round cell with spiny,
unevenly spaced projections.
Variable number in different
fields.
Acanthocytes Shrunken cells with irregular,
spiny projections
270 HEMATOLOGY REVIEW
- -- -- - -
Significance
Seen in many anemias
Vitamin B,, deficiency, folic acid deficiency, pernicious
anemia, hemolytic anemia, cirrhosis, alcoholism. Nor-
mal in newborns.
Iron-deficiency anemia, thalassemia
Seen in many anemias
Hereditary ovalocytosis, various anemias
- - - - -
Osmotic imbalance. If seen in most cells in thin part of
smear, don't report. Probably artifact due to excess an-
ticoagulant or slow drying.
Damage to RBC membrane, uremia, carcinoma of
stomach, bleeding peptic/gastric ulcers
Damage to RBC membrane, alcoholic cirrhosis, post-
splenectomy, abetalipoproteinemia
Continued
 RBC Morphology Continued
Abnormality Description Significance
Polychromasia Bluish-gray color Young RBCs. Reticulocytes (retics) with supravital
stain. Sign of active erythropoiesis. 1-2 % in normal
adult. Increased with acute blood loss, hemolytic ane-
mia, following treatment for iron deficiency or perni-
cious anemia.
Target cells (codocytesl Bull's-eye, "Mexican hat cell" Hemoglobinopathies, thalassemia, alcoholism, post-
splenectomy. May be artifact if observed in only one
part of smear.
Stornatocytes RBC with slitlike central pallor Hereditary stomatocytosis, hereditary spherocytosis,
thalassemia, alcoholic cirrhosis, Rh null disease. May be
artifact in parts of smear that are too thin or too thick.
Spherocytes Small, dark-staining RBCs Damaged RBC membrane. Hereditary spherocytosis,
without central pallor autoantibodies, burns, hemoglobinopathies, hemoly-
sis, ABO hemolytic disease of the newborn, incompati-
ble blood transfusion, transfusion of stored blood. A
few are normal due to aging of RBCs.
~-~ - -

Rouleaux RBCs resemble stack of coins Serum protein abnormality; e.g., increased globulins
or fibrinogen. Seen in multiple myeloma and
macroglobulinemia. May be artifact due to delay in
spreading drop of blood or smear that is too thick.
Agglutination RBCs in irregular clumps Autoantibodies, cold autoagglutinin

272 HEMATOLOGY REVIEW

- -- --- --- - --- - -- -- --
 RBC Inclusions
inclusion Stain Descrintion Explanation Significance Conditions
Basophilic Wright's and Multiple, irregular Aggregation of Coarse: exposure Exposure to lead, acceler-
stippling new methy- purple inclusions RNA (ribosomes) to lead. Fine: ated or abnormal hemoglo-
lene blue evenly distributed young RBC. bin synthesis, thalassemia
throughout cell
Howell-Jolly Wright's and Round, purple, Nuclear rem- Usually pitted Postsplenectomy,
bodies new methy- 1-2 k m in dia- nants (DNA) by spleen. Seen thalassemia, hemolytic and
lene blue meter. Usually with accelerated megaloblastic anemias,
only one per cell. or abnormal sickle cell anemia
erythropoiesis.
Cabot rings Wright's Reddish purple May be part of Rapid blood Pernicious anemia,
rings or figure mitotic spindle, regeneration, thalassemia, postsplenec-
eights remnant of abnormal tomy, lead poisoning
microtubules, or erythropoiesis
fragment of nu-
clear membrane
Pappen- Wright's Small purplish- Iron particles Faulty iron Sideroblastic anemias, post-
heimer (siderotic blue granules. Vary utilization splenectomy, thalassemia,
bodies granules in size, shape, sickle cell anemia,
with Prussian number. Usually at hemochromatosis
blue stain.) periphery in clusters.
Continued

HEMATOLOGY REVIEW 273

 --

Summary of RBC Inclusions

Inclusion Wright's Stain New Methylene Blue Stain Prussian Blue Stain
Reticulum Cell appears polychromatophilic Yes No
Howell-Jolly bodies Yes Yes No
Pappenheimer bodies Yes Yes Yes
Siderotic granules Yes, but called Pappenheimer bodies Yes Yes
- -

Heinz bodies No Yes No

HEMATOLOGY REVIEW 275

 Erythrocyte Indices
Index Definition Formula Reference Ranges Comments
Mean cor- Average volume HCT (%) Male = 80-94 fL Useful in classification of
puscular of the RBC MCV = Female = 81-99 fL anemias. RBCs of normal
volume RBCs (X 1012/L) MCV are described as
(MCV) normocytic. RBCs with MCV
>lo0 appear macrocytic on the
blood smear. RBCs with MCV
<80 appear microcytic on the
blood smear. MCV is an aver-
age. A combination of micro-
cytes and macrocytes may be
observed with a normal MCV.
Mean cor- Average weight Hgb (g/dL) X 10 27-31 pg Varies in proportion to MCV.
puscular of hemoglobin MCH = Least useful of the indices.
hemoglobin in the average RBC (X 1012/L)
(MCH) RBC
Continued

276 HEMATOLOGY REVIEW

-- -
 --

Hemoglobinopathy vs. Thalassemia

Hemoglobinopathy Thalassemia
Abnormality Qualitative abnormality. Abnormality in amino Quantitative abnormality. Amino acid
acid sequence of globin chain, but not in amount sequence of globill chains is normal, but
of globin produced. there is underproduction of one or more
globin chains.
Examples Sickle cell anemia and trait, hemoglobin C Beta thalassemia major and minor
disease and trait

278 HEMATOLOGY REVIEW

- - - -- - - -
 -

Anemias
Hemoglobin
Anemia Etiology Classification Blood Smear Electrophoresis Other
Iron deficiency Inadequate iron Microcytic, Aniso, poik, Normal .1RBC, ? red-cell distri-
for hemoglobin hypochromic hypochromic bution width (RDW), &
, i synthesis^ microcytes serum iron, '? total iron-
binding capacity (TIBC) ,
serum ferritin
Sideroblastic ~nzymatic Microcytic, Dimorphic Normal Siderocytes, ? serum
anemia defect in heme hypochromic RBCs, Pappen- iron, 4 TIBC, '? serum
synthesis heimer bodies, ferritin. retics.
basophilic ? ringed sideroblasts in
stippling marrow.
- - - - -

Anemia of Defective iron Usually normo- Normal or Normal -1 serum iron, normal
chronic utilization cytic, normo- hypochromic, TIBC, ? serum ferritin,
diseases chromic. One- microcytic and free erythroc te
fourth to one- RBCs protoporphyrin. Yretics
third may be
microcytic,
hypochromic
Beta thalas- Decreased pro- Microcytic, Aniso, poik, >90% A, ? RBC, normal RDW,
semia minor duction of hypochromic hypochromic 3.5-7% A,, normal serum iron and
chains (hetero- microcytes, F may be slightly TIBC
zygous) target cells, increased
Continued
HEMATOLOGY REVIEW 279
 ---

Anemias Continued
Hemoglobin
Anemia Etiology Classification Blood Smear Electrophoresis Other
Sickle cell Inheritance of Normocytic, Aniso, poik, >80% S, Hgb S polymerizes
anemia (SS) sickle cell gene normochromic sickle cells, frag- 1-20% F, under decreased 0, and
from both parents. mented cells, 2-4.5 % A,, decreased blood pH.
Substitution of target cells, nu- no A Disease not evident in
valine for gluta- cleated RBCs, newborn because of ?
mic acid in sixth spherocytes, Hgb F. Positive solubility
position of Howell-Jolly test. Retics 5-20%. May
B chain. bodies, basophilic have leukocytosis with
stippling, sidero- shift to left and
tic granules, poly- thrombocytosis.
chromasia.
Sickle cell Inheritance of Normocytic, Occasional target 50-65 % A, Positive solubility test
trait (AS) sickle cell gene normochromic cell. No sickle 35-45 % S,
from one parent cells unless normal F,
hypoxic. normal to slightly
increased A,
Continued

HEMATOLOGY REVIEW 281

 --- --

Anemias Continued
Hemoglobin
Anemia Etiology Classification Blood Smear Electrophoresis Other
and polychro- ? indirect bilirubin,
masia. Spherocytes. ? osmotic fragility
Autoimmune Autoantibodies Normocytic, Polychromasia, Normal ? retics, ? indirect
hemolytic normochromic spherocytes, bilirubin,
anemia nucleated RBCs haptoglobin,
positive direct
antiglobulin test

HEMATOLOGY REVIEW 283

-
 --

Differentiation of Microcytic Hypochromic Anemias

Free
Percent Erythrocyte Retics
Serum satura- Serum Protopor- (Before
Iron TZBC tion Ferritin phyrin (FEP) RDW lkeatment) Other
Iron 1 t t -1 or Normal
deficiency Normal hemoglobin
electrophoresis
Beta Normal Normal Normal Normal Normal Normal 1 or Increased Hgb A,
thalassemia Normal
minor
Anemia of 1 Normal Normal ? t Normal
chronic
disease
Sideroblastic T 1 7 t Variable t 1 Dimorphic RBC
anemia population,
siderotic gran-
ules, bas0 hilic
S
stippling,
ringed sidero-
blasts in marrow

284 HEMATOLOGY REVIEW

 RBC Morphology Continued
A bnonnality Description Significance
Schistocytes RBC fragments, helmets, Fragmentation of RBCs. Seen with disseminated
triangular cells intravascular coagulation (DIC), hemolysis, artificial
heart valves, burns, microangiopathic hemolytic
anemia
- - ~

Blister cells RBCs with one or more
vacuoles
Sickle cells Crescent, S- or C-shaped, boat-
(drepanocytes) shaped, oat-shaped
Hemoglobin C crystals Blunt, six-sided, dark staining
projection. "Bar of gold."
"Washington monument."
Hemoglobin SC crystals Glovelike intracellular crystals
Teardrops (dacryocytes) Teardrop-shaped
Hypochromia Central pallor greater than
one-third cell diameter
Anisochromia Mixture of normochromic and
hypochromic RBCs
Microangiopathic hemolytic anemia, pulmonary
emboli in sickle cell anemia
Sickle cell anemia
Hemoglobin C disease
Hemoglobin SC disease
Myelofibrosis, various anemias
Iron-deficiency anemia, thalassemia
Dimorphic anemia, post-transfusion
Continued

HEMATOLOGY REVIEW 271

 Anemia Due to Blood Loss
Acute Blood Loss Chronic Blood Loss
RBCs Normocytic, normochromic Microcytic, hypochromic (due to iron deficiency)
WBCs Increased with shift to left for about 2-4 days Normal
Reticulocytes Increased within 3-5 days. Peak around 10 days. Normal
- - - - - - - ----- - - - - - - - -

Hematocrit Steady during first few hours due to vaso- Decreased

and constriction and other compensatory
hemoglobin mechanisms. Full extent of hemorrhage evident
within 48-72 hours.
Other Platelets increase within 1 hour. Nucleated Decreased serum iron and ferritin
RBCs may be released.

HEMATOLOGY REVIEW 285

 -

RBC Inclusions Continued

Inclusion Stain Description Explanation Significance Conditions
Siderotic Prussian Blue granules of Aggregates of Faulty iron utili- Sideroblastic anemias, post-
granules blue varying size and iron particles zation in hemo- splenectorny, thalassemia,
shape globin sy~ithesis sickle cell anemia,
he~riochromatosis
Reticulocytes New methy- Blue-staining Residual RNA >2% increased Hemolytic anemia, blood
lene blue network (ribosomes) erythropoiesis. loss, following treatment
(polychro- <0.1% decreased for iron deficiency or
masia on erythropoiesis. megaloblastic anemia
Wright's stain)
Heinz bodies Supravital Round blue Precipitated, Normal during Glucose-6-phosphate
stain; e.g., inclusions, varying oxidized, aging but pitted dehydrogenase (G6PD)
crystal violet, sizes, close to cell denatured by spleen deficiencies, unstable
brilliant membrane. May hemoglobin hemoglobins, chemical
cresyl blue, be more than one. injury to RRCs, drug-
methylene induced hemolytic anemia
blue

274 HEMATOLOGY REVIEW

 Highlights of Granulocytic Maturation
Stage Key Characteristics
Myeloblast 15-20 km. Small amount of dark blue cytoplasm. Usually no granules. Nucleus has del-
icate chromatin with nucleoli. QgSAKsJ kc =( ~b,?
Promyelocyte Similar to myeloblast but has primary (nonspecific) granules
-

Myelocyte Presence of secondary (specific) granules (eosinophilic, basophilic, or neutrophilic).

Last stage to divide.
Metamyelocyte Nucleus begins to indent.
Band Nuclear indentation is more than half.
Segmented Neutrophil ?tYo to five nuclear lobes connected by thin strands of chromatin.

286 HEMATOLOGY REVIEW

-- - - - -

-- -
 -

Erythrocyte Indices Continued

Index Definition Formula Reference Ranges Comments
Mean cor- Average Hgb (g/dL) X 100 32-36 g/dL RBCs with normal MCHC are
puscular concentration of MCHC = described as normochromic.
hemoglobin hemoglobin per HCT ( % ) They have an area of central
concentration unit volume of pallor approximately one-third
(MCHC) RBCs the diameter of the cell. The
MCHC is decreased in hypo-
chromic cells and the area of
central pallor is increased.
RBCs cannot accommodate
more than 37 g/dL of hemo-
globin. Higher levels are in-
dicative of problems with the
specimen (hyperlipidemia,
cold agglutinins) or the instru-
ment.

HEMATOLOGY REVIEW 277

- -- - -
 Normal Leukocytes of the Peripheral Blood
Adult Reference Adult Reference
Range (Relative Range (Absolute
Cell Size Nucleus Cytoplasm Count) Count)
Neutrophil 10-15 p m Segmented. Two to five Pinkish-tan with 50-70% 2.5-7.0 x 109/L
lobes connected by thread- neutrophilic
like filament of chromatin. granules
Eosinophil 12-16pm Band-shaped or seg- Large red 1-3 % 0.05-0.30 X 1OY/L
mented into two lobes granules
Basophil 10-14km Usually difficult to see Dark purple 0-1 %
because of overlying granules
granules
Monocyte 12-22 wm Round, horseshoe-shaped, Gray-blue with 24%
or lobulated. Convoluted. indistinct uink
Loose strands of granules.
chromatin. Vacuoles.
Occasional
pseudopods.
Lymphocyte 6-8 pm or Round or oval. Dense Sparse to 20-40 % 1-4 x l o 9 / ~
8-15pm blocks of chromatin. abundant. Sky
Indistinct chromatin/ blue. May contain
parachromatin separation a few azurophilic
granules.

HEMATOLOGY REVIEW 287

 Anemias Continued
Hemoglobin
Anemia Etiology Classification Blood Smear Electrophoresis Other
basophilic
stippling
Beta thalas- Decreased pro- Microcytic, Marked aniso Little or no A, MCV 50-60 fL, retics
semia major duction of p hypochromic and poik, hypo- up to 98 % F. 2-8 % . Hgb F distribu-
(Cooley's chains (homo- chromic micro- tion is heterogeneous
anemia) zygous) cytes, target among RBCs.
cells, ovalo-
cytes, nucleated
RBCs, basophilic
stippling
Pernicious Reduced intrinsic Macrocytic Macro-ovalocytes, Normal Achlorhydria,
factor secondary teardrops, hyper- .L serum B,,,
to gastric atrophy. segmentation, .1intrinsic factor,
Deficiency of d, WBCs, abnormal Schilling test,
vitamin B,, irn- .1platelets t lactate dehydrogenase
pairs DNA (LD)
synthesis.
Continued

280 HEMATOLOGY REVIEW

- - --
 Leukocyte Abnormalities
Abnormality Description Significance
Shift to the left Presence of immature granulocytes in peripheral blood Bacterial infection, inflammation
--- - -- - - ~-~

Toxic granulation Dark staining granules in cytoplasm of neutrophils Infection, inflammation

Dohle bodies Light blue patches in cytoplasm of neutrophils Infection, burns
Vacuolization Phagocytic vacuoles in cytoplasm of neutrophils Septicemia, drugs, toxins, radia-
tion
Hypersegmentation More than 5 % of segmented neutrophils with five- One of first signs of pernicious
lobed nuclei or any with more than five lobes anemia
Pelger-Huet anomaly Most neutrophils have round or bilobed nuclei. Inherited disorder. No clinical ef-
fect. May be misinterpreted as a
shift to the left.
Auer rods Red needles in cytoplasm of leukemic myeloblasts and Nonlymphocytic leukemia
monoblasts
- - - - - - - ---- - - - - - - -

Variant lymphocytes One or more of the following: large size, elongated or Viral infections (e.g., infectious
(atypical or reactive) indented nucleus, immature chromatin, increased mononucleosis, cytomegalovirus)
parachromatin, nucleoli, increased cytoplasm, dark
blue or very pale cytoplasm, peripheral basophilia,
scalloped edges due to cytoplasm being indented by
adjacent RBCs, frothy appearance, many azurophilic
granules

288 HEMATOLOGY REVIEW

- -- - ---
 Anemias Continued
Hemoglobin
Anemia Etiology Classification Blood Smear Electrophoresis Other
Hemoglobin Inheritance of Normocytic, Aniso, poik, >90 C,
C disease gene for Hgb C normochromic slight hypochro- <7% F, no A
(CC) from both masia, many
parents. Lysine target cells,
is substituted fragmented cells,
for glutamic acid folded cells
in the sixth posi- ("pocketbook
tion of p chain. cells "), occasional
Hgb C crystals,
occasional micro-
spherocytes
Hemoglobin Inheritance of Normocytic, Many target cells 50-60% A,
C trait (AC) gene for Hgb C normochromic 30-40% C
from one parent.
SC disease Inheritance of Normocytic, Target cells, Equal amounts Positive solubility test
(sc) one sickle cell normochromic pocketbook cells, of C and S,
gene and one occasional SC normal to 7 % F,
Hgb C gene crystals ("gloves") no A.
Hereditary Defect of cell Normocytic, Varying degrees Normal T MCHC, ? retics,
spherocytosis membrane normochromic of aniso, poik, k haptoglobin,
Continued

282 HEMATOLOGY REVIEW

- -- --- - - - - -
- -- --
 Quantitative Abnormalities of Leukocytes
Neutrophilia Neutropenia Lymphocytosis Monocytosis Eosinophilia Basophilia
Bacterial Acute infection Infectious Convalescence from Allergies Chronic myelo-
infection Antibodies mononucleosis viral infections Skin diseases genous leukemia
Inflammation Drugs Cytomegalovirus Chronic infections Parasitic Polycythemia vera
Hemorrhage Chemicals Whooping cough TB infections
Hemolysis Radiation Acute infectious Subacute bacterial
Stress lymphocytosis endocarditis
Parasitic infections
Rickettsia1 infections

HEMATOLOGY REVIEW 289

 Acute vs. Chronic Leukemia
Acute Chronic
Age Mostly children and young adults Mostly middle-aged and elderly
Onset Sudden Insidious
- ~

Median survival 3 months 2-6 years

time if untreated
White count Usually increased, but may be normal Increased
or decreased
Platelets Low to markedly decreased Normal or increased at first. Decreased in late stages.
Leukocytes Many blasts More mature cells
Anemia Normocytic, normochromic None until late in disease
lleatment Chemotherapy, bone marrow transplant Chemotherapy or none
Cause of death Infection, hemorrhage Infection, hemorrhage
Diagnosis Peripheral blood smear, bone marrow Peripheral blood smear
examination, cytochemical stains,
surface markers, chromosome analysis,
electron microscopy
Other Majority go into blast crisis

290 HEMATOLOGY REVIEW

- - - ---
 Acute Nonlymphocytic Leukemia (ANLL)
I)rpe Name Principle Featurels)
AML (MO) Acute myelogenous Undifferentiated blasts
without maturation
AML (Ml) Acute myelogenous with Predominance of myeloblasts with minimal maturation. Some promyelo-
minimal maturation cytes may be present. Auer rods may be present. Pseudo Pelger-Huet
anomaly may be present.
AML (M2) Acute myelogenous Myeloblasts predominate. Some maturation to or beyond promyelocyte
with maturation stage. Auer rods are common. Most comrnon type of acute myelogenous
leukemia.
APL (M3) Acute promyelocytic The promyelocyte is the predominant cell. Myeloblasts and myelocytes
may be present. Auer rods are common. High incidence of DlC. Uncom-
mon type.
AMML (M4) Acute myelomonocytic Early myelogenous cells predominate with approximately 20% monocytic
cells. Blast may have indented and convoluted nuclei. Auer rods may be
present. Pseudo Pelger-Huet anomaly may be present. Second most corn-
mon type.
AMoL (M5) Acute monocytic M5a has a predominance of monoblasts. M5b has promonocytes and
monocytes as well. Uncommon types\ p
I+; rc
;;4-c &Sbase
I
EL (M6) Acute erythroleukemia Erythroblasts with megaloblastoid changes and other d z a s t i c features,
(Di Guglielmo syndrome) myeloblasts, and promyelocytes. Auer rods may be present.
MegL (M7) Acute megakaryoblastic Proliferation of megakaryoblasts and atypical megakaryocytes. Megakary-
ocytic fragments may be seen in the peripheral blood. Uncommon type.
HEMATOLOGY KEVIEW 291
- . - -- -- - -
- - - -- -- - - - - - --
 Acute Lymphoblastic Leukemia
French-American-British
(FAB) Morphologic Classification
L1 (Small cell homogeneous) Uniform population of small lymphoblasts with scanty cytoplasm, round nu-
clei with occasional clefting, homogeneous chromatin, inconspicuous nucle-
oli. Children.
~ - -

L2 (Large cell heterogeneous) Heterogeneous population of large, pleomorphic lymphoblasts with nuclear
clefting and indentation. Older children and adults.
'\(~urkitt type) Uniform population of relatively large lymphoblasts with deeply basophilic
cytoplasm, vacuoles, and round to oval nuclei without indentations. Sec-
6 ' J ~ ~ i n rLs\ j m ~ \ * n e ondary to Burkitt's lymphoma.
Immunologic Marker Classification
B-cell ALL 1 5 % of ALL. Corresponds to FAB type L3.
Pre-B cell ALL 10-15% of ALL. Most are FAB type L1.
-- - - - - - pp - - - - - - - -

Pro-B cell ALL 85% of childhood ALL and 75 % of adult ALL. FAB type L1 or LZ. Highest
remission rate.
T-cell ALL 15% of ALL. FAB type L1 or L2. Poor prognosis.

292 HEMATOLOGY REVIEW

 Chronic Myeloproliferative Disorders (CMPD)
Disorder Other Names Principle Feature
- - - - - - - --

Chronic myelogenous Chronic granulocytic Marked leukocytosis with an increase in mature and
leukemia (CML) leukemia immature cells of the granulocytic series. Thrombocytosis is
common. Philadelphia chromosome may be found in malig-
nant cells. Anemia.
Agnogenic rnyeloid Idiopathic myelofibrosis. Bone marrow fibrosis. Extramedullary hematopoiesis. All
metaplasia (AMM) Myelofibrosis with myeloid stages of myeloid maturation in peripheral blood, including
metaplasia. immature eosinophils and basophils. Nucleated RBCs.
Teardrop RBCs. Defective platelets.
Polycythemia Primary polycythemia. Increase in all cellular bone marrow elements. Increased
Vera (PV) Erythremia. hemoglobin and hernatocrit. Increased leukocyte alkaline
phosphatase (LAP).
Essential thrombo- Platelets >lo00 X 109/L. Marked platelet anisocytosis.
cythernia (ET) Occasional megakaryocyte fragments.

HEMATOLOGY REVIEW 293

 - -

Leukemoid Reaction vs.

Chronic Myelogenous Leukemia
Lenkemoid Reaction CML

Peripheral blood smear Shift to the left (blasts rare), toxic granulation, Shift to the left with blasts,
Dohle bodies eosinophilia, basophilia
LAP score High Low
Philadelphia chromosome Negative Usually positive

294 HEMATOLOGY REVIEW

- -- -- -- .
 -

Chronic Lymphoproliferative Disorders

Leukemia Key Characteristics
Chronic lymphocytic Most common leukemia in adults. B-CLL is most common. Lymphocytosis with relatively
leukemia (CLL) mature lymphocytes. Smudge cells. May be accompanied by anemia, thrombocytopenia,
and production of autoantibodies (autoimmune hemolytic anemia or idiopathic thrombocy-
topenia purpura).
Hairy cell leukemia Hairy cells: scant to abundant, agranular, light grayish-blue cytoplasm with irregular hair-
like projections. Round to oval nuclei with loose chromatin. Pancytopenia.

HEMATOLOGY REVIEW 295

- -
 - - -- --

Plasma Cell Disorders

Disorder Key Characteristics
Multiple myeloma Malignant plasma cells in marrow. Rare abnormal plasma cell on peripheral smear. Normo-
cytic, normochromic anemia. Rouleaux on blood smear. Elevated erythrocyte sedimenta-
tion rate due to increased globulins. M spike on serum protein electrophoresis (monoclonal
gammopathy). May have Bence Jones proteinuria. Lytic bone disease.
- - - ---- -

Plasma cell leukemia Abnormal plasma cells in peripheral blood (> 2 X lo9/ L). Pancytopenia. Rouleaux. Mono-
clonal gammopathy.
Waldenstrom's Monoclonal gammopathy due to increased IgM. Rare plasmacytoid lymphocytes or plasma
macroglobulinemia cells on peripheral smear. Rouleaux. May have Bence Jones proteinuria and cryoglobulins.

296 HEMATOLOGY REVIEW

-- -
--

Hematology Special Stains

Stain Detects Significance
-

New methylene blue RNA Used for reticulocyte counts

Prussian blue Iron
Peroxidase (myelo- Peroxidase found in primary
peroxidase) granules and Auer rods
Sudan black Lipids in primary and secondary
granules of granulocytes
Napthol ASD chloro- Esterase i n primary granules
acetate esterase of myeloid cells
a-naphthyl acetate Nonspecific esterase in monocyte
esterase or a-naphthyl precursors
butyrate esterase
Combined esterase Esterase enzymes in myeloid and
monocy tic cells
TdT Primitive lymphoid cells
Periodic acid-Schiff (PAS) Glycogen
Detects siderocytes
Differentiates AML M1, M2, and M3 (positive)
from ALL (negative)
Differentiates AML M1, M2, and M3 (positive)
from ALL (negative)
Differentiates AML M1, M2, and M3 (positive)
from ALL (negative)
Strong positive in rnonocytic cells. Inhibited by
addition of sodium fluoride. Positive in M4 and
MS.Negative in ALL.
Used to evaluate the ratio of myelocytic and
nlonocytic cells on a smear based on different
color of staining. Differentiates M4 and MS.
Activity is elevated in L1 and L2.
RBC precursors in erythroleukernia stain in-
tensely. Normal RRCs and normal RBC precur-
sors are negative. Large coarse clumps in lym-
phoblasts in ALL.
Continued
HEMATOLOGY REVIEW 297
 Hematology Special Stains Continued
Stain Detects Significance
Tartrate resistant acid Tartrate resistant acid phosphatase Hairy cell leukemia
phosphatase (TRAP)
Leukocyte alkaline Alkaline phosphatase in bands and Increased in leukemoid reaction and poly-
phosphatase (LAP) segmented neutrophils cythemia vera. Decreased in CML. Score 100
segmented neutrophils and bands from 0-4 +
and total. Normal: 20-100.

298 HEMATOLOGY REVIEW

--
 Erythrocyte Sedimentation Rate (ESR)
Measures
Clinical significance
Methods
Sources of error
Excess anticoagulant
Hemolysis
Specimen at room temperature
more than 4 hours
Room temperature below 22"-27°C
Room temperature above 22"-27°C
Tilted tube
Inclusion of buffy coat in reading
Inaccurate reading of scale
Bubbles in column
Amount of settling of RBCs in a column of anticoagulated whole blood
Nonspecific indicator of inflammation. Increased in acute and chronic
infections, rheumatic fever, rheumatoid arthritis, myocardial infarction,
TB, subacute bacterial endocarditis
Wintrobe and Westergren. Westergren is preferred. Longer column in-
creases sensitivity.
RBCs shrink. ESR J.
RBC column is decreased. ESR J.
RBCs swell. ESR J.
Cells settle more slowly. ESR -1.
Cells settle faster. ESR T.
ESR T. A 3" angle from vertical can accelerate ESR by 30%.
ESR 4
ESR could be increased or decreased.
ESR 4
Continued
HEMATOLOGY REVIEW 299
- -- -- - - - p-p
 Erythrocyte Sedimentation Rate (ESR) Continued
Inaccurate timing ESR could be increased or decreased.
Anemia Change in RBC:plasma ratio favors rouleaux formation. ESR 1'.
Polycythemia Increased viscosity. ESR J.
Anisocytosis Cells don't rouleaux as readily. ESR J.
Poi kilocy tosis Cells don't rouleaux a s readily. ESR J.
Agglutinated RBCs Agglutinates settle faster. ESR '?.
Macrocytosis Large cells settle faster. ESR ?.
Microcytosis Small cells settle more slowly. ESR J.
Increased fibrinogen or globulins Favors rouleaux formation. ESR ?.

300 HEMATOLOGY REVIEW

- - - - -
 - --

Other Manual Hematology Procedures

Manual white blood cell Acetic acid is used as diluent to lyse RBCs. WBCs are counted in a Neubauer
(WBC) count hemacytometer. Nucleated RBCs can't be distinguished from WBCs.
Eosinophil count Phloxine stains eosinophils red. Cells are counted in a special hemacytometer with
a larger volume than a Neubauer chamber (Fuchs-Rosenthal or Speirs-Levy).
Hematocrit Packed cell volume (PCV). Microhematocrit tubes are centrifuged at 10,000 rpm for
5 minutes. The ratio of the volume occupied by the RBCs to the volume of whole
blood is determined. Values may be slightly higher than automated hematocrit
(HCT) due to trapped plasma. (Automated HCT is a calculated value).
Hemoglobin Cyanmethemoglobin method. Potassium cyanide and potassium ferricyanide
reagent (Drabkin's). Potassium ferricyanide oxidizes ferrous ions to ferric ions,
forming methemoglobin. Methemoglobin reacts with cyanide ions to form cyan-
methemoglobin. Read at 540 nm. A commercially prepared standard is used to pre-
pare a standard curve. Measures all forms of hemoglobin except sulfhemoglobin.
Sugar water test (sucrose Screening test for paroxysmal nocturnal hemoglobinuria (PNH). Sucrose provides a
hemolysis) medium of low ionic strength that promotes the binding of complement. PNH RBCs
are complement-sensitive and lyse. Normal RBCs are unaffected.
Ham test (acid-serum test) For confirmation of PNH. Serum is acidified, which activates complement. PNH
RBCs lyse. Normal RBCs are unaffected.
Hemoglobin A, by Screening test for beta thalassemia minor (? Hgb A,)
microcolumn
Continued

HEMATOLOGY REVlEW 301

 Other Manual Hematology Procedures Continued
Hemoglobin F Alkali denaturation: Hgb A is denatured; Hgb F is not. Acid elution: Hgb A is eluted
and RBCs appear as ghost cells. Hgb F resists acid and KBCs with a high concentra-
tion stain pink. Hgb F is increased in hereditary persistence of fetal hemoglobin
(HPFH), sickle cell anemia, thalassemia. In thalassemia there is a heterocellular dis-
tribution of Hgb F (some cells contain Hgb F and some don't). HPFH can be pan-
cellular (uniform distribution of Hgb F among the RBCs) or heterocellular.
Solubility test for sickling Sickling hemoglobins produce a turbid solution when mixed with sodium dithion-
hemoglobin ite. Screening test only. Not specific for Hgb S. Does not differentiate SS from AS.
Confirm with hemoglobin electrophoresis.
Osmotic fragility Measures the ability of RRCs to take up water without lysing. RBCs are incubated
with various concentrations of NaC1. The amount of hemolysis is determined by
reading the absorbance of the supernatant from each tube. Normal = hemolysis be-
ginning at 0.45% and complete at 0.30%. Increased in hereditary spherocytosis.
Decreased with target cells, sickle cell anemia, iron deficiency anemia, thalassemia.
LE prep Outdated test for diagnosis of lupus erythematosus. Antinuclear antibody in pa-
tient's serum digests nucleus of damaged WBC, which is then phagocytized by a
neutrophil or monocyte.
Malaria smears Examine thin and thick smears. Thick smear concentrates parasites and is used to
screen. It is not fixed, so RBCs lyse, making detection easier. Thin smear must be
examined to speciate. Refer to Section 3 for speciation.

302 HEMATOLOGY REVIEW

-- - - - -- --
 Sources of Error in
Selected Manual Hematology Tests
Manual Hemoglobin
Lipemia Value is increased because of turbidity (higher absorbance). Use patient plasma in
reagent to blank.
WBC >30 X 109/L Value is increased because of turbidity (higher absorbance). Centrifuge reaction tube
and read absorbance of supernatant.
Hgb S or C Cells are resistant to lysis. Value is increased because of turbidity (higher absorbance).
Dilute mixture 1:2 with distilled water. Reread. Multiply result X 2.
Manual Hematocrit
Tourniquet left on too long Increased due to hemoconcentration
Excess anticoagulant Decreased due to shrinkage of RBCs
Hemolysis Decreased due to destruction of RBCs
Insufficient mixing Could b e higher or lower, depending on portion sampled
Inadequate time or speed Decreased values due to insufficient packing of cells
of centrifugation
Inclusion of buffy coat Increases value
in reading
Parallax error Failure to look straight down when reading result. Could increase or decrease value.
Continued
HEMATOLOGY REVIEW 303

Selected Manual Hematology Tests Continued
Incorrect use of
hematocrit reader
Poikilocytosis
Manual Reticulocyte
Refractive artifacts
Other inclusions
' h b e Solubility Test for Hgb S
Lipemia
Leukocytosis
Polycy themia
Increased globulins
Blood from infant less
than 6 months old
Recent transfusion
Hemoglobin less
than 7 g/dL
304 HEMATOLOGY REVIEW
Sources of Error in
Could be higher or lower, depending on error
- - - -- - -
Increased, due to trapped plasma
Increased values. (RNA disappears when out of focus.)
Pappenheimer bodies, Howell-Jolly bodies, Heinz bodies are seen with supravital
stain. Differentiate by other stains.
False-positive due to turbidity
False-positive due to turbidity
False-positive
False-positive due to turbidity
False-negative due to predominance of Hgb F
False-negative
False-negative
 Changes in Blood at Room Temperature
MCV t (due to RBCs swelling)
Hematocrit ? (due to t MCV)
MCHC L (due to t hematocrit)
ESR L (swollen RBCs don't rouleaux)
Osmotic fragility t
WBC count L
WBC morphology Necrobiotic cells, karyorrhexis (nuclear disintegration), degranulation, vacuolization

HEMATOLOGY REVIEW 305

- -- - - -- -
 Interfering Factors on Most Hematology Analyzers
- ~ - -

Condition Effect Parameters Affected Resolution

WBC over Turbidity may Increases RBC. May Dilute blood and rerun to get accurate
50,000 increase Hgb. increase Hgb. WBC. Subtract WBC from RBC. Follow manufacturer's
WBCs are counted HCT and indices instructions for obtaining accurate Hgb. Perform spun
and sized as RBCs. inaccurate. HCT. Recalculate indices.
Cold Agglutinates are Decreases RBC. Prewarm blood to 37°C. and rerun.
agglutinins counted and sized Increases MCV,
as RBCs. MCH, MCHC.
HCT inaccurate.
Nucleated Some analyzers Increases WBC. Uncorrected WBC X 100
RBCs count as WBCs. Corrected WBC =
100 + NRBCs per 100 WBCs
Lipemia lbrbidity may affect May increase Hgb, Follow manufacturer's instructions for obtaining
Hgb reading. MCH, MCHC. accurate Hgb. Recalculate indices.

Hemolysis RBCs are lysed. Decreases RBC and If hemolysis is in vitro, recollect.
HCT. Increases MCH
and MCHC.
Giant plate- Platelet clumps are Decreases platelets Examine blood film. For platelet clumps or satellitism,
lets, plate- counted as WBCs. (PLT). recollect in sodium citrate. Multiply by 1.1 dilution fac-
let clumps, Increases WBC. tor. Manual counts or film estimates may be required.
or satellitism
Continued
306 HEMATOLOGY REVIEW

-- - - -- --- - - - - -
 Interfering Factors on Most
Hematology Analyzers Continued
Condition Effect Parameters Affected Resolution
Old RBCs swell. Increases MCV. Recollect.
specimen Platelets and Decreases PLT. Automated
WBCs degenerate. differential count (auto
diff) may be inaccurate.
- - -

Schistocytes, Not counted as RBCs. Decreases RBC. HCT and Manual counts. Spun HCT.
microcytes May be counted a s PLT. indices may be inaccurate. Recalculate indices.
Resistant RBCs don't lyse. Increases WBC. Manual Hgb, adding water to lyse RBCs.
RBCs (e.g., Counted as .WBCs. Hgb inaccurate. Manual WBC. Recalculate indices.
Hgb S or C)
Fragile WBCs WBCs are damaged in Decreases WBC. May Manual counts.
(e.g., chemo- analyzer. May be increase PLT.
therapy) counted as PLT.

HEMATOLOGY REVIEW 307

-
 -- -

Hematology Calculations
Formula Example Calculation

retics Der 1000 RBCs What is the reticulocyte count if reticulum is observed in
Reticulocyte 15 of 1000 RBC?
15
Retic % = -= 1.5
10
Reticuloc~te% retics in square A x 100 What is the reticulocyte count if 60 reticulocytes are
using Miller Disc =
RBCs in square counted in square A and 300 RBCs are counted in
square B?
60 X 100
Retic % = 300 = 2.2

Absolute What is the absolute reticulocyte count if the reticulocyte

Reticulocyte count is 2% and the RBC is 5.2 X 1012/L?
Count (ARC) retic % x RBC count (1012/L) X 1000 2 X 5.2 X 1000
( x 109/~)= ARC =
100 100 = 104 x 1 0 9 / ~

Corrected What is the corrected reticulocyte count if the uncor-

Reticulocyte HCT(%) rected reticulocyte count is 5 % and the hematocrit is 36 % ?
Count (CRC) = retic % X 36
4s CRC=5X - = 4 %
45

Continued

308 HEMATOLOGY REVIEW

 -

Hematology Calculations Continued

Formula Example Calculation
Reticulocvte What is the RPI if the corrected retic is 5% and the HCT
~roductidn
Index (RPI) =
corrected retic count
maturation time correction factor*
is 35%; (Maturation time correction factor for HCT of
35% is 1.5).

RPI =
5
1.5 = 3.3%
I
HCT ( % ) x 10 Calculate the MCV if the RBC is 3.0 X 10I2/L, the Hgb is
MCV = 6 gm/dL, and the HCT is 20%.
RBC (1012/L)
20 X 10
MCV = -------
3 = 66.7 fL

Hgb (g/dL) X 10 Calculate the MCH if the RBC is 3.0 X 1012/L, the Hgb is
MCH = RBC (1012/L) 6 gm/dL, and the HCT is 20 % .
6 X 10
MCH = -
3
= 20 pg.

Hgb (g/dL) X 100 Calculate the MCHC if the RBC is 3.0 X 1012/L, the Hgb is
MCHC =
HCT (%) 6 g~n/dL,and the HCT is 20 % .
6 X 100
MCHC = -
- 30%

-
*The maturation time correction factor is based on the patient's hematocrit and is obtained from a maturation timetable.
Continued

HEMATOLOGY REVIEW 309

 Hematology Calculations Continued
Formula Example Calculation
Rules of Three: What should the hemoglobin be if the RBC is 4.1 x 1012/L?
RBC X 3 = Hgb Z 0.5 Hgb = 4.1 X 3 = 12.3 % 0.5 = 11.8-12.8 g/dL
Hgb X 3 = H C T ? 3 % What should the hematocrit be if the hemoglobin is 12.3 g/dL?
HCT = 12.3 X 3 = 36.9 % 3 = 33.9-39.9%

Cells per mm3 (or pL) = No. of cells counted X Calculate the WBC count if blood is drawn to the 0.5 mark
depth factor (always 10) x reciprocal of and diluent to the 11 mark in a WBC diluting pipet and
dilution X reciprocal of area counted (mm2) 100 WBCs are counted in the 4 "W" squares of a Neubauer
hemacytometer.
Cells per pL = 100 X 10 x 20 X '/4 = 5000

(In a WBC diluting pipet, 0.5 to 11 is a dilution of 1:20 be-

cause the diluent up to the 1 mark on the stem does not
mix with the blood.)
(Area counted is 4 mm2.)
Calculate the RBC count if 10 pL of whole blood are diluted in
a Unopette containing 1.99 mL of diluent and 250 RBCs are
counted in four corner and one center "R" squares of the mid-
dle square millimeter (subdivided into 25 squares) of a
Neubauer hemacytometer.
Continued
 Hematology Calculations Continued
Formula Examvle Calculation
~~~~~~ ~ p ~ ~ -

Cells per pL = 250 X 10 X 200 X 5 = 2,500,000

(Dilution is 0.01:2 or 1:200.)
(Area counted is 1/5 mmz.)
Absolute WBC = Total WBC x relative count Calculate the absolute lymphocyte count if the total WBC is
(% from differential) 10 x 10'/L and there are 70% lymphocytes.
Absolute count = 10 X 0.70 = 7 X 109/L
The automated hematology analyzer reports a WBC of 30 X
Corrected uncorrected WBC x 100 lO9/L. The technologist counts 115 nucleated RBCs per 100
WBCs while performing the differential. What is the corrected
WBC = 100 + nucleated RBCs per 100 WBCs WBC.
30 X 100
Corrected WBC = +115 = 14.0 X 109/L

HEMATOLOGY REVIEW 311

- - - -
 -

Overview of Hemostasis
- - - - - - -

Primary hemostasis Vasoconstriction.

Platelet adhesion to exposed subendothelial connective tissue.
Platelet aggregation to form initial plug at injury site.
Secondary hemostasis Coagulation factors interact on platelet surface to produce fibrin.
Fibrin stabilization by factor XIII.
Fibrinolysis Release of tissue plasminoge~lactivator.
Conversion of plasminogen to plasmin.
Conversion of fibrin to fibrin degradation products (fibrin split products).

312 HEMATOLOGY REVIEW

 -

Coagulation Factors Continued

- ---- -

Present in
Vit K Present in Adsorbed Present in
Name Required? Plasma? Plasma? Serum? Pathway Other
IX Christmas Yes Yes No Yes Intrinsic Deficiency =
factor hemophilia B
X Stuart-Prower Yes Yes NO Yes Common
factor
XI Plasma throm- No Yes Yes Yes Intrinsic Deficiency =
boplastin hemophilia C
antecedent
XI1 Hageman factor No Yes Yes Yes Intrinsic Glass activation fac-
tor. Not required in
vivo. No clinical
signs of deficiency.
XI11 Fibrin stabilizing Stabilizes fibrin clot.
factor Deficiency leads to
poor wound heal-
ing. Assay by urea
solubility test.

314 HEMATOLOGY REVIEW

 Coagulation Factors
Present in
Vit K Present in Adsorbed Present in
Name Required? Plasma? Plasma? Serum? Pathway Other
\ Fibrinogen No Yes Yes No Common Converted to fibrin by
thrombin. Normal =
200-400 mg/dL.
I\ Prothrombin Yes Yes No No Common Precursor of thrombin
I11 Tissue throm- High molecular weight
boplastin Extrinsic lipoprotein present in all
tissues. Liberated by
trauma.
IV Ca2+ Intrinsic, Round by sodium citrate.
extrinsic, Must be replaced by
and common reagents.
V LabiIe factor No Yes Yes No Common Deteriorates at room
temperature
VII Stable factor Yes Yes No Yes Extrinsic
VIII Antihemo- No Yes Yes No Intrinsic Deficiency = hemophilia
philic factor A. Most common inher-
ited bleeding abnormal-
ity. Labile.

Continued
HEMATOLOGYREVIEW 313
 --

Summary of Coagulation Factors

Produced in liver
Require vitamin K for
synthesis
Affected by Cournadin
Consumed during clotting
Labile factors
Contact group
Factors in extrinsic pathway
Factors in intrinsic pathway
Factors in common pathway
All. (Von Willebrand's portion of VIII also produced in endothelial cells and
megakaryocytes.)
11, VII, IX, X (prothrombin group)
11, VII, IX, X (prothrombin group)
I, 11, V, VIII, XI11
V, VIII
Prekallikrein [Fletcher factor), high-molecular-weight kininogen (Fitzgerald factor),
XII, XI
111, VII
Prekallikrein (Fletcher factor), high-molecular-weight kininogen (Fitzgerald factor),
XII, XI, IX, VIII
X, V, 11, I

HEMATOLOGY REVIEW 315

- - -- - - - - - -
 - -

Inherited Factor Deficiencies

Factor Name of Disease Pearment Comments
I Afibrinogenemia, hypo- Cryoprecipitate Rare
fibrinogenemia, dys-
fibrinogenemia
11 Prothrombin deficiency Fresh frozen plasma Rare
V Labile factor deficiency Fresh frozen plasma Autosomal recessive
(Owen's disease)
VI I Factor VII deficiency Plasma or prothrombin complex Rare
VIII Hemophilia A Cryoprecipitate, factor VIII One of most common inherited coagula-
concentrate, l-desamino-8- tion disorders. Sex-linked recessive.
D-arginine vasopressin (DDAVP) Occurs primarily i11 males. Mothers are
carriers.
Von Willebrand's disease Cryoprecipitate, DDAVP One of most common inherited coagula-
tion disorders. Deficiency of VW factor
@art of factor VIII complex). Autosomal
dominant. Both sexes are affected.
IX Hemophilia B Plasma or commercial Sex-linked recessive
(Christmas disease) concentrates

Continued

316 HEMATOLOGY KEVIEW

 Inherited Factor Deficiencies Continued
Factor Name of Disease meahnent Comments
X Stuart-Prower factor Fresh frozen plasma or Rare
deficiency prothrombin complex
XI Hemophilia C Fresh frozen plasma Rare
XI1 Hageman trait None No bleeding disorders
XI11 Fibrin-stabilizing Plasma or lyophilized Rare
factor deficiency placental factor XI11

HEMATOLOGYREVIEW 317
 Acquired Factor Deficiencies
Condition Factors Affected
Liver disease Mild: 11, VII, IX, X
Moderate: also V, VIII
Severe: also I
Vitamin K deficiency 11, VII, IX, X
Coumadin therapy 11, VII, IX, X
-

Disseminated intravascular coagulation I, 11, V, VIII, and platelets

Primary fibrinolysis I, V, VIII

318 HEMATOLOGY REVIEW

 - -

Coagulation Tests
Test Significance
Bleeding time Prolonged with platelet abnormalities, vascular disease. Prolonged by aspirin.
Clot retraction Decreased with thrombocytopenia and qualitative platelet defects. Infrequently per-
formed.
Platelet aggregation Test of platelet adhesion, aggregation, and secretion. Detects qualitative platelet defects.
Prothrombin time (PT) Detects deficiencies in extrinsic and common pathways. Used to monitor Coumadin
therapy. Report in INR (International Normalized Ratio).
Activated partial throm- Detects deficiencies in intrinsic and common pathways. Most common test to monitor
boplastin time (APTT) heparin therapy.
- - - - -

Activated coagulation Used for bedside monitoring of heparin therapy

time (ACT)
Thrombin clotting Little used test to monitor heparin therapy
time (TCT)
Fibrinogen Estimation of fibrinogen level
Urea solubility Factor XI11 screening test
D-Dimer Positive in DIC, deep vein thrombosis, pulmonary embolism, and after lytic therapy.
Negative in primary fibrinolysis.
-

Continued

HEMATOLOGY REVIEW 3 19

-- - ---- ---- - -
 Coagulation Tests Continued
Test Significance
Fibrin(ogen) degradation Present in DIC, primary fibrinolysis, deep vein thrombosis, pulmonary embolism, after
(split) products lytic therapy
(FDP, FSP)
Plasminogen Precursor of plasmin. Decreased following lytic therapy, DIC, primary fibrinolysis.
Antithrombin 111 (AT-111) Heparin cofactor. Deficiencies associated with thrombosis.
Protein C and protein S Inhibitors of coagulation. Deficiencies associated with thromboembolic disorders.

320 HEMATOLOGY REVIEW

-- p- - -- - - -- - --- ------ --
 Effect of Factor Deficiencies on PT and APTT
Factor Deficiency PT APTT
I Prolonged Prolonged
11 Prolonged Prolonged
V Prolonged Prolonged
VII Prolonged Normal
VIII Normal Prolonged
IX Normal Prolonged
X Prolonged Prolonged
XI Normal Prolonged
XI1 Normal Prolonged

HEMATOLOGY REVIEW 321

-- - - --
 - -

Substitution Studies
PT APTT Deficiency Corrected By Not Corrected By
Abnormal Normal VII Fresh normal plasma Adsorbed plasma
Serum
Normal Abnormal VIII Fresh normal plasma Serum
Adsorbed plasma
IX Fresh normal plasma Adsorbed plasma
Serum
XI Fresh normal plasma
Adsorbed plasma
Serum
Fresh normal plasma
Adsorbed plasma
Serum

Continued

322 HEMATOLOGY REVIEW

-- - --
 Substitution Studies Continued
-- ~- ~

PT APTT Deficiency Corrected By Not Corrected By

Abnormal Abnormal I Fresh normal plasma
Adsorbed plasma Serum
Fresh normal plasma Adsorbed plasma
Serum
Fresh normal plasma
Adsorbed plasma Serum
X Fresh normal plasma Adsorbed plasma
Serum
To identify a factor deficiency, patient's plasma is mixed with plasma deficient in a single clotting factor. Failure of the factor-
deficient plasma to correct the clotting time of the patient's plasma indicates that the patient's plasma is missing the same factor
as the factor-deficient plasma.

HEMATOLOGY REVIEW 323

-. -- - - -- -
 Laboratory Findings in
Selected Coagulation Abnormalities
- -

Disease Laboratory Findings

Hemophilia A (classic hemophilia)
Hemophilia B
Von Willebrand's disease
Glanzmann's thrombasthenia
Decreased factor VIII.
APTT prolonged.
PT normal.
Bleeding time normal.
Platelet count normal.
APTT prolonged.
PT normal.
Bleeding time normal.
Platelet count normal.
Bleeding time prolonged.
Platelet aggregation abnormal with ristocetin.
Factor VIII may be decreased.
APTT may be prolonged.
PT normal.
Platelet count normal.
Clot retraction abnormal.
Bleeding time prolonged.
Platelet count normal.
Platelet aggregation abnormal with ADP,
collagen, thrombin, epinephrine.

Continued

324 HEMATOLOGY REVIEW

i -- -- - -- - -- - -
 Laboratory Findings in
Selected Coagulation Abnormalities Continued
Disease Laboratory Findings
Bernard-Soulier sy itdrome Bleeding time prolonged.
Giant platelets.
Platelet count decreased.
Clot retraction normal.
 Disseminated Intravascular Coagulation
vs. Primary Fibrinolysis
DZC Primary Fibrinolysis
PT Prolonged Prolonged
APTT Prolonged Prolonged
Fibrinogen Decreased Decreased
Platelets Decreased Normal
FSP Present Present
D-Dimer Positive Negative
AT-111 Decreased Normal
RBC morphology Schistocytes Normal

326 HEMATOLOGY REVIEW

-- - - -- - - - -
- - - - -

Anticoagulant Therapy
Heuarin Coumadin
Administration Subcutaneous or IV Oral
Action Antithrombin effect Vitamin K antagonist
Effect Immediate Slow-acting
Duration Short Long
Test for monitoring APTT PT
Reversed by Protamine sulfate Vitamin K
Other Requires AT-I11 to be effective Decreases production of 11, VII, IX, X

HEMATOLOGY REVIEW 327

I
 - - -.

Thrombolytic Therapy
Purpose To activate fibrinolytic system. Used to treat acute myocardial infarction, deep vein
thrombosis, pulmonary emboli.
Drugs used Streptokinase, urokinase, tissue plasminogen activator (TPA), pro-urokinase
(Pro-UK), acylated plasminogen streptokinase activated complex (APSAC)
Baseline studies PT, APTT, CBC, fibrinogen
Changes induced by therapy in fibrinogen, plasminogen, a-2-antiplasmin, factors V, VIII, IX,XI, XII.
I' in plasmin, fibrin(ogen) degradation products, D-dimer, APTT, PT, thrombin time.
Precautions Limit venipunctures. Apply pressure following venipuncture to stop bleeding.

328 HEMATOLOGY REVIEW

- -

Sources of Error in Coagulation Testing
Error
Incorrect anticoagulant
Drawing coagulation tube
before other anticoagulant
tubes
Probing to find vein
Incorrect ratio of blood
to anticoagulant
Failure to mix anticoagulant
with blood
Polycythemia
Heparin contamination from
catheter or heparin lock
Hemolysis
Lipemia
-
Comment
3.2 % or 3.8 % sodium citrate should be used. Labile factors are preserved better.
3.2% is preferred. Less subject to effects of polycythemia.
Contamination with other anticoagulants can interfere.
Tissue thromboplastin activates coagulation and shortens times.
Need nine parts of blood to one part of anticoagulant. Tubes less than 90% full
will have longer times.
Blood will clot.
Hematocrits above 55% lead to longer times. Anticoagulant must be reduced.
Will prolong times. Lines must be flushed with saline and the first 5 mL of blood
drawn discarded.
Hemolyzed RBCs may activate clotting factors. Hemolysis may interfere with pho-
toinetric reading.
May interfere with photo-optical methods. May be tested with electromechanical
instrument.
Continued
HEMATOLOGY REVIEW 329
 Sources of Error in Coagulation Testing Continued
Error
Improper storage of specimen
Improper storage or
reconstitution of reagents
Equipment malfunction,
e.g., temperature, timer,
detector, volumes dispensed
Comment
Should remain capped to prevent change in pH. Test within 2 hours if stored at
room temperature or 4 hours if stored at 4°C. Loss of labile factors will prolong
times.
Run controls once each shift to verify system performance.
Run controls once each shift to verify system performance.

330 HEMATOLOGY REVIEW

- - - A -
 Causes for Rejection of
Specimens for Coagulation Testing
More than 2 hours at room temperature
Exposure to extremes of temperature
Tube less than 90% full
Specimen clotted
Specimen hemolyzed

HEMATOLOGY REVIEW 331

- - - --- - -- -
 - - -

Immunology Terms
Acute phase reactant Protein that increases due to infection, injury, or trauma (e.g., C-reactive protein, alpha-1
antitrypsin, haptoglobin, fibrinogen, ceruloplasmin, alpha-1 acid glycoprotein, comple-
ment)
Alloantibody Antibody formed in response to antigens from individuals of the same species
Antigen A foreign substance that stimulates antibody production. Large, complex molecules (MW
>10,000), usually protein or polysaccharide.
Antibody Immunoglobulin produced by plasma cells in response to an antigen
Autoantibody Antibody against self
Avidity Strength of bond between antigen and antibody
Chemotaxin Chemical messenger that causes migration of cells in a particular direction
Clusters of Antigenic features of leukocytes
differentiation (CD)
- - - - -

Cytokine Chemical messenger produced by stimulated cells that affect the function of other cells
Epitope Determinant site on an antigen
Hapten A low molecular weight substance that can bind to an antibody once it is formed, but
that is incapable of stimulating antibody production unless it is bound to a larger carrier
molecule
Continued

334 IMMUNOLOGY REVIEW

- -- -- -- A
 -

Immunology Terms Continued

Histamine A vasoactive arnine that is released from mast cells and basophils during an allergic reac-
tion
Hypersensitivity A heightened state of immune responsiveness that causes tissue damage in the host
Immunity Resistance to infection
Immunogen Any substance that is capable of inducing an immune response
Immunoglobulin Antibody
Immunology The study of the reactions of a host when exposed to foreign substances
Inflammation Cellular and humoral mechanisms involved in the reaction of the body to injury or infec-
tion
Interferons Cytokines produced by T cells and other cells that inhibit viral synthesis or act as immune
regulators
Interleukins Cytokines produced by T cells or macrophages that stimulate a number of cell types
Ligand A molecule that binds to another molecule of a complementary configuration. The sub-
stance being measured in an immunoassay.
Lymphokines Polypeptides given off by antigen-stimulated T cells, which regulate the function of other
cells
Lysozyme Enzyme found in tears and saliva that attacks cell walls of microorganisms
Continued

IMMUNOLOGY REVIEW 335

--
 - --. -

Immunology Terms Continued

- - p p p p - -

MHC Major histocompatibility complex. Genes that control the expression of proteins found on
all nucleated cells.
Monoclonal antibody An antibody derived from a single B-cell clone
- - -

Opsonin Serum proteins that attach to a foreign substance and enhance phagocytosis
Phagocytosis The engulfment of cells or particulate matter by neutrophils and macrophages
Plasma cells Transformed B cell that secretes antibody
Polyclonal antibody Antibody produced by many B-cell clones
Postzone False-negative reactions in a serological test due to antigen excess
Prozone False-negative reactions in a serological test due to antibody excess
Seroconversion The change of a serological test from negative to positive due to development of detectable
antibody
Serum sickness A type 111 hypersensitivity reaction that results from buildup of antibodies to animal serum
used in some passive immunizations
Thymus A small, flat bilobed organ found in the thorax. Site of T-lymphocyte development.
Titer A means of expressing the concentration of an antibody. The reciprocal of the highest dilu-
tion in which a positive reaction occurs.
Vaccination Injection of immunogenic material in order to induce immunity

336 IMMUNOLOGY REVIEW

 Branches of the Immune System
Branch Definition Defense Against Cells Involved Examples
Cellular Cell mediated Viruses, fungi, myco- T lymphocytes Graft rejection,
bacteria, other intra- hypersensitivity
cellular pathogens, reaction, elimination of
tumor cells tumor cells
- - - - - - -

Humoral Antibody mediated Bacteria (extracellular) B lymphocytes, plasma cells Antibody production

IMMUNOLOGY REVIEW 337

 'Ifrpes of Immunity
n'pe Explanation Components Memory?
Natural or innate Nonspecific defenses with Intact skin, mucous membranes, stomach No
which one is born acidity, cilia, mucus, skin secretions,
lysozyme, IgA, phagocytes, complement,
interferon, inflammation
Acquired or adaptive Specific responses to T cells, B cells, plasma cells, antibodies, Yes
eliminate a microorganism cytokines

338 IMMUNOLOGY REVIEW

--- - -
 Adaptive Immunity

VPe Explanation Example Specific? Immediate? Long-Term?

Naturally acquired An individual infected Clinical or subclinical Yes No Yes
active immunity with a microorganism infection
produces an antibody.
Artificially acquired An individual exposed DPT, MMR, polio, Yes No Yes
active immunity to an antigen through a tetanus, Haemophilus
vaccine develops influenzae type b
immunity without having vaccine
the infection.
Naturally acquired An individual is pro- Babies are protected by Yes Yes No
passive immunity tected by antibodies pro- maternal antibodies that
duced in another cross the placenta and
individual. are present in breast
milk.
Artificially acquired An individual receives Rh immune globulin, Yes Yes No
passive immunity an immune globulin HBIG, anti-toxins
containing antibodies
produced in another
individual.

IMMUNOLOGY REVIEW 339

- -- -- - -.-
 --- -- --

Cells of the Immune System

Cell Function Comments
---- ~~ ~ ~ ---------- - - - - - - --

T lymphocytes Cell-mediated immunity Derived from cells in bone marrow. Develop T-cell
specific surface antigens in thymus (e.g., CD2,
CD3, CD4, CD8). 60-80% of peripheral lympho-
cytes.
Helper/ Orchestrates cell-mediated immunity. CD4+ (T4). 55-70% of peripheral T cells. Normal
inducer T cells Activates B cells, cytotoxic cells, and CD4 = 1,00O/pL. 111 AIDS, less than 200/bL.
natural killer cells
Suppressor/ Suppressor cells inhibit helper T cells. CD8+ (T8). 2 5 4 0 % of peripheral T cells.
cytotoxic T cells Cytotoxic cells kill other cells. (Normal CD4:CD8 ratio is 2:l. In AIDS, less than
0.5:l).
B lymphocytes Synthesize and secrete immunoglobulins Develop in bone marrow. Surface immunoglobu-
after antigenic challenge lins and CD19, CD20, CD21. Less than 15% of
lymphs.
Natural killer Recognize and destroy virally infected cells Non-T, non-B. Lack CD3, CD4, and CD8.
(NK) cells and tumor cells
(null cells)
Lymphokine- Use IL-2 to help lyse tumor cells Non-T, non-B
activated killer
cells (LAK)
- ~ ~ ~ ~ ------

Continued

340 IMMUNOLOGY REVIEW

 Cells of the Immune System Continued
Cell Function Comments
Eosinophils Suppress inflammatory reaction. Kill some Some phagocytic ability
parasites.
Basophils Granules contain histamine and heparin. Some phagocytic ability. Precursor of mast cell.
Role in hypersensitivity reactions.
Neutrophils Principal leukocyte associated with phago-
cytosis and localized inflammatory response.
Granules contain bactericidal enzymes.
Respond to chemotaxins.
Monocytes Phagocytes. Respond to chemotaxins. Pro- Precursor of macrophage
cess antigens and present to lymphocytes.
Produce interleukin-1 and other cytokines.

1MMUNOLOGY REVIEW 341

- - -

Laboratory Identification of Lymphocytes
To Obtain Lymphocytes
Density gradient centrifugation with
Ficoll-Hypaque
To Identify Lymphocytes
Direct or indirect immunofluorescence
Flow cytometry (FACS: fluorescence-
activated cell sorter)
Surface immunoglobulins
Rosette technique
-- - -
,
Layers from top to bottom: plasma, mononuclear cells, Ficoll-Hypaque,
RBCs and granulocytes
Use of labeled monoclonal antibodies against specific surface antigens.
Slides are read with a fluorescent microscope.
Use of labeled monoclonal antibodies against specific surface antigens.
Light scattering is measured as cells flow through a laser beam.
Immunoenzymatic method to demonstrate immunoglobulins on B cells
T lymphocytes form rosettes with sheep RBCs. Old method.
IMMUNOLOGY REVIEW 343
 -- - -

Common Lymphocyte Markers

T Cells B Cells

- - - -

Surface immunoglobulins

344 IMMUNOLOGY REVIEW

 Immunoglobulin Structure
Basic structure A four-chain polypeptide unit that consists of two heavy chains and two light chains held to-
gether by disulfide bonds
Heavy chains y, a,p, 6, and E. The two heavy chains in the immunoglobulin molecule are always of the same
type. They determine the immunoglobulin class (IgG, IgA, IgM, IgD, and IgE).
Light chains K or A. Both K and X light chains are found in all classes of immunoglobulins, but only one type
is present in a given molecule.
Fab fragment Fragment antigen-binding. Consists of one light chain and one-half of a heavy chain. The intact
immunoglobulin has two Fab fragments, each representing one antigen-binding site.
Fc fragment Fragment crystalline. The carboxy-terminal halves of the two heavy chains. This portion of the
molecule has no antigen binding ability.
Constant region The carboxy-terminal end of the immunoglobulin molecule, where the amino acid sequence is
the same for all chains of that type.
Variable region The amino-terminal end of the immunoglobulin molecule, where the amino acid sequence varies.
This part of the molecule is responsible for the specificity of a particular immunoglobulin. Also
known as the antigen-recognition unit.
Hypervariable Regions within the variable region that actually form the antigen-binding site. Through changes
region in the hypervariable region, an immense diversity of antigen-binding sites can be created.
Hinge region The flexible portion of the heavy chain, located between the first and second constant regions.
This allows the molecule to bend to let the two antigen-binding sites operate independently.
~ - - - - - - - - - ~~~~ ~-~ - - ~ - - - - ~

Joining chain A glycoprotein that serves to link immunoglobulin monomers together. Only found in IgM and se-
cretory IgA molecules.

IMMUNOLOGY REVIEW 345

---- -- - - - -- --- - -- - --
 Immunoglobulins

Form Monomer Pentamer Monomer and dirner Monomer Monomer

Molecular weight 150,000 900,000 160,000 or 400,000 180,000 190,000
(daltons)
Heavy chain Gamma (y) Mu (14 Alpha (4 Delta (6) Epsilon ( E )
Light chain Kappa or K O ~ X K or h K or h K or A
lambda
(Kor X)
-

% of total immuno- 75-80%

globulin
Serum concentration 800-1600 120-1 50 70-350 mg/dL 1-3 mg/dL 0.005 mg/dL
mg/dL mg/dL
Antigen-binding sites 2 10 2 or 4 2 2
Complement fixation Yes Yes No No No
- ~ p p

Crosses placenta? Yes

Continued

346 IMMUNOLOGY REVIEW

- - - --
 Immunoglobulins Continued
IgG IgM I@' IsE
Other Defense against bacteria First Ig produced In tears, sweat, Function Role in allergic reactions.
and viruses. Neutralizes in an immune saliva, respiratory unknown Binds to mast cells and
toxins. Opsonin. More response. Only and GI mucosa. triggers degranulation
efficient at precipitation Ig produced by First line of and release of histamine
than agglutination. newborn. Neu- defense. and heparin.
tralizes toxins.
Opsonin. Most
efficient Ig at
initiating coln-
plement cascade.
More efficient
at agglutination
than IgG.
Destroyed by
sulfhydryl com-
pounds.

IMMUNOLOGY REVIEW 347

- --
 Complement
Definition A series of more than 25 serum proteins that interact to enhance host defense re-
actions. Most are inactive enzyme precursors that are converted to active enzymes
in a precise order (cascade).
Functions Opsonization, chemotaxis, cell lysis
Classical pathway Triggered by antigen-antibody reactions. IgM is most efficient activator. A single
molecule attached to two adjacent antigenic determinants can initiate the cascade.
IgG1, 2, and 3 can activate complement but at least two molecules are required.
Recognition unit: C1 (first to bind).
Activation unit: C4, C2, C3.
Membrane attack complex: C5, C6, C7, C8, C9 (drills holes in cell membrane).
Alternative pathway Activated by bacteria, fungi, viruses, tumor cells, some parasites
Present in highest C3
concentration in plasma
Key component of both C3
pathways
Ions required Calcium, magnesium
Inactivation 5G°C for 30 minutes
Clinical manifestations Increased susceptibility to infection or accumulation of immune complexes with
of deficiency autoimmune manifestations
Significance of decreased levels Immune complex disease

348 IMMUNOLOGY REVIEW

 Immune Phenomena
- - - -

Explanation Application
Primary phenomena Combination of antibody with antigen Not easily detectable. Can be measured indi-
rectly by radioimmunoassay (RIA), enzyme
immunoassay (EIA) , immunofluorescence.
Secondary phenomena Precipitation, agglutination, Measured more readily. Basis for many
complement fixation serological tests.
Tertiary phenomena In viva reactions; e.g., inflammation, Some are useful diagnostically, e.g., skin
phagocytosis, deposition of immune testing.
complexes, immune adherence,
chemotaxis

IMMUNOLOGY REVIEW 349

-
 Hypersensitivity Reactions
m e 111: I)rpe IV:
Q p e I: Anaphylactic I)rpe 11: Cytotoxic Immune Complex Cell Mediated
Key reactant(s) IgE IgC, JgM, complement, IgG, IgM, complement T cells and
antigens found on macrophages
cell surfaces
Result Release of mediators Cytolysis due to anti- Deposits of antigen- Release of
from mast cells and body and complement antibody complexes lymphokines from
basophils antigen-stimulated
T cells
Symptoms Immediate Immediate Immediate Delayed
Example Anaphylaxis, hay fever, Bansfusion reactions, Serum sickness, Contact dermatitis,
asthma, food allergies hemolytic disease of systemic lupus tuberculin test
the newborn, auto- erythematosus (SLE),
immune hemolytic rheumatoid
anemia, idiopathic arthritis (RA)
thrombocytopenic
purpura (ITP), Good-
pasture's syndrome

350 lMMUNOLOGY REVIEW

-- --- - -
 Agglutination Methods
Method Principle Examples
Direct agglutination The process by which particulate antigens, such as Febrile agglutinins, Salmonella
cells, aggregate to form large complexes when and Shigella serotyping
specific antibody is present
Hemagglutination An antigen-antibody reaction that results in the ABO slide typing
clumping of RBCs
Passive agglutination A reaction in which soluble antigens are bound to Rheumatoid factor
latex beads, bentonite, or charcoal. The particles are
agglutinated by the corresponding antibody.
- - -

Passive hemagglutination A reaction in which soluble antigens are adsorbed Cold agglutinins
onto RBCs. The RBCs are agglutinated by the
corresponding antibody.
Reverse passive A reaction in which carrier particles coated with Rapid tests for identification of
agglutination antibody clump together due to combination with bacteria
antigen -
Agglutination inhibition An agglutination reaction based on competition Slide tests for human chorionic
between particulate antigen (reagent) and soluble gonadotrophin (HCG)
antigen (specimen) for limited sites on a reagent
antibody. Lack of agglutination is a positive result.
Continued

IMMUNOLOGY REVIEW 351

-- - -
 -- -

Agglutination Methods Continued

Method Principle Examples
Hemagglutination A test for detecting antibodies to certain viruses that Rubella antibody
inhibition agglutinate RBCs. In the presence of antibody, the
virus is neutralized and hemagglutination does not occur.
Coagglutination An agglutination reaction in which bacteria are used as Rapid tests for identification
the carrier for the antibody. (Protein A on the surface of of bacteria
S. aureus adsorbs the Fc portion of the antibody molecule.)
Rheumatoid factor can cause false-positive reactions in agglutination tests because it reacts with any 1gG. Heterophile antibodies
can cause false-positive reactions in hemagglutination tests.

352 IMMUNOLOGY REVIEW

- - - --
 Precipitation Methods
- - - -

Method Principle Application

Precipitation Soluble antigen combines with soluble antibody to pro- See following examples.
duce visible insoluble complexes. Precipitation methods
are less sensitive than agglutination methods because
more antibody is needed to produce a visible reaction.
Flocculation Soluble antigens react with specific antibody to form a VDRL, rapid plasma reagin (RPR)
precipitate of fine particles.
Ouchterlony Antigens and antibodies diffuse out from wells cut in gel Fungal antigens, extractable
technique and form precipitin lines where they meet. Double nuclear antigens
immunodiffusion.
Counter immuno- Antigens and antibodies are placed in wells that are Bacterial antigens
electrophoresis directly opposite one another in a gel. An electrophoretic
(CIE) charge is applied to drive the reactants toward each other.
A precipitin band forms where they meet.
Immunoelectro- Proteins are separated by electrophoresis, then subjected Identification of immunoglobu-
phoresis (IEP) to double diffusion with reagent antibodies placed in a lins in monoclonal
trough cut in the agar. The shape, intensity, and location gammopathies
of the precipitin arcs that develop are compared with
those of a normal control.
Continued

IMMUNOLOGY REVIEW 353

- --- - - - pp - . - -
 -

Precipitation Methods Continued

Method Principle Application
Immunofixation Proteins are separated by electrophoresis. A cellulose Identification of immunoglobu-
electrophoresis (IFE) acetate strip impregnated with antiserum is placed on the lins in monoclonal
separated proteins. The antiserum diffuses into the gel gammopathies
and antigen-antibody complexes precipitate.
-

Radial immuno- Antigen is measured based on the diameter of a precipitin Quantitation of immunoglobulins
diffusion (RID) ring that forms when it diffuses out of a well in a gel- and complement
containing antibody.
- - - - - - - - - - - - - -

Rocket electro- An electrical charge is applied to an RID assay. The height Immunoglobulins, complement
phoresis of the rocket-shaped precipitin band obtained is propor-
tional to the concentration of the antigen.
Nephelometry Light scattering by immune complexes is measured. Immunoglobulins, complement,
C-reactive protein

354 IMMUNOLOGY REVIEW

- --- - --
 Other Serological Methods
Method Pn'ncivle
Complement fixation Patient serum is incubated
with antigen and complement.
If the corresponding antihody
is present in the serum, it
forms a complex with the anti-
gen and complement. When
sensitized RBCs are added,
there is no free complement to
lyse them. No hemolysis is a
positive reaction.
Direct fluorescent The specimen is placed on a
antibody glass slide and overlaid with
fluorescein-labeled antibody.
If the corresponding antigen
is present, the labeled-
antibody binds and fluores-
cence will be seen with a
fluorescent microscope.
Comments Examvle
Patient serum must he inacti- Viral, fungal, rickettsia]
vated. Best for demonstration antibodies
of IgM antibodies. More sensi-
tive than agglutination and pre-
cipitation, but less sensitive
than labeled immunoassays.
Not widely used.
Detects antigens. Fluorescent Bacterial antigens
compounds absorb energy from
light source and convert it into
light of a longer wavelength
(lower energy) than that
absorbed. Fluorescent labels:
fluorescein isothiocyanate or
rhodamine B isothiocyanate.
Continued
IMMUNOLOGY REVIEW 355
 Other Serological Methods Continued
- - -

Method Principle Comments Example

Indirect fluorescent Reagent antigen on a glass "Sandwich technique." Detects Fluorescent antinuclear
antibody slide is overlaid with patient antibodies in serum. antibody (FANA),
serum. If the corresponding fluorescent trepone~nal
antibody is present in the antibody (FTA)
serum, it attaches to the
antigen. When fluorescein-
labeled antihuman globulin
is added, it attaches to the
antibody. Fluorescence will
be seen with a fluorescent
microscope.

356 IMMUNOLOGY REVIEW

 Labeled Immunoassay Terminology
Ligand The substa~lcebeing measured in an imlnunoassay
Isotopic Ail immunoassay that uses a radioisotope as the label
- - - - - - - -

Nonisotopic An imniunoassay that uses something other than a radioisotope as the label; e.g., enzyme, fluo-
rochrorne, chemiluminescent molecule
-

Competitive An irnniu~loassayin which the patient ligand and the labeled reagent ligand compete for a lirn-
ited number o f binding sites on a reagent antibody
-

Noncompetitive An irnmunoassay in which the reaction does not involve competition for binding sites. Noncom-
petitive assays Jre more sensitive than coiiipetitive assays.
-

Heterogenous A11 ~ ~ n n i i ~ n o a s in
s a which
y a separation step is requlred to remove free reactant from bound reac-
tant. Heterogeneous assays are inore sensitive than liomogeneous assays.
Ho~nogeneous An i~nmunoassayin which a separation step is not required. Hon~ogenousassays are easier to
automate.

IMMUNOLOGY REVIEW 357

 Comparison of Radioimmunoassay
and Enzyme Immunoassay
-
-

RIA EIA

Label Radioisotope, usually 1251 Enzyme

Measurement Counts per minute in sci~ltillationcounter Color develop~nentafter addition of substrate
Advdiitages Sensitivity Sensitivity
Specificity Specificity
Long sheli-life of reagents
No radiation hazard
No special disposal requirements
No need for scin~illationcounter
Disadvailtages Short shelf life of leagents
Radiation hazard
Regulations governing disposal of radioisotopes

358 IMMUNOLOGY REVIEW

 Principles of Labeled Immunoassays
Method V p e of Assay Principle
RIA Competitive Radiolabeled ligand and unlabeled ligand in the specimen compete for
Isotopic binding sites on reagent antibody. The more unlabeled ligand in the
Heteroge~irous specimen, the less labeled ligand binds. Free labeled ligand is separated
from bound labeled ligand. Solid-phase attachment is the most frequent
separation method. For example, antibody is adsorbed to the inner wall
of the assay tube and separation is achieved by aspiration or decanta-
tion. The amount of labeled ligand bound is determined by the counts
per minute (CPM) 011 a scintillation counter. CPM are inversely propor-
tional to the concentration of the ligand in the specimen.
Imniu~loradiometric The specimen is incubated with a labeled antibody. Solid-phase ligand
!
Noncompetitive
assay (IRMA) Isotopic is added to remove unbound labeled antibody. After centl-ilugation, the
Heterogeneous radioactivity in the supernatant is determined. CPM are directly propor- 1I
tional to the concentration of ligand in the specimen.
Enzyme-linked Competitive Enzyme-labeled ligand and unlabeled patient ligand compete for hind-
im~nunosorbent Nonisotopic ing sites on antibody niolecules attached to a solid phase. Free labeled
assay (ELISA) Heterogeneous ligand is removed by washing and a substrate is added. The enzyme ac-
tivity is inversely proportional to the concentration of the ligand in the
specimen.
Irldirect or non-
competitive ELlSA
Nonco~npefitive Antibody in the specimen attaches to a solid-phase antigen. After incrl
Nonisotopic batiori and washing to remove u ~ ~ h o u nantibody,
d an enzyme-labeled
1I
Heterogeneous antiglobulin is added. This second antihody reacts with the Fc portion I
of the patient antibody bound to the solid phase. Following another I
I
Continued I
I
360 IMMUNOLOGY REVIEW
 Principles of Labeled Immunoassays Contirlued
Method Q p e o f Assay Principle
Substrate-labeled Col~lpetitive Unlabeled ligand in the specimen ' ~ n dfluorogenic Iigand compete for
fluorescent Nonisotopic sites on reagent antibody. Free Idbeled ligand produces tluorescence.
immunoassay Homogeneous Bound labeled ligand does not produce fluorescence. Fluorescence is
(SLFLA) directly proportional to the concentration of the ligand in the specimen.
This is an automated neth hod.
Fluorescence Competitive Unlabeled hgand in the specimen and fluorogenic ligand compete for
polarization Nonisotopic sites on reagent antibody. Free labeled ligd~idrotates rapidly and emits
imrnunoassay (FPIA) Homogeneous little polarized tluorescence. Bound labeled ligand rotates more slowly
and emits more polarized fluorescence. The higher the concentration of
bound labeled ligand, the Illore poldrized fluorescence. The amount of
polarized fluorescence is iilversely proportional to the concentration of
the ligand in the specimen. This is an automated method.

362 IMMUNOLOGY REVIEW

 Nontreponemal Tests for Syphilis
VDRL RPR
Method Flocculation Flocculation
Detects Reagin Reagin
Reagent (s) Cardiolipin Cardiolipin with charcoal added
Positive reaction Microscopic clumps Macroscopic agglutination
Specimen(s) Inactivated serurn, CSF Serum (inactivation not required), plasma
Reactivity during May be negative in primary stage. Titers Sarne as VDRL
disease usualIy peak during secondary or early
late stages. Titers decline in late stage,
even when untreated. More rapid decline
with treatment. Becomes nonreactive
following successful treatment.
False-positives 10-30% may be biologic take positives; Sarne as VDRL
e.g., infectious niononucleosis (IM),
infectious hepatitis, malaria, leprosy.
lupus erythematosus, rheumatoid arthritis,
advanced age, pregnancy. Positive in other
treponemal infections such as yaws and pinta.
Other Replaced by RPR for serum. Still performed Good tor screening and treatment monitoring 1
on CSF.

364 IMMUNOLOGY REVIEW I

 Treponemal Tests for Syphilis
FTA-ABS Microhemagglutination Assay
Metllod lndirect fluorescent antibody Hemagglutination
Detects Ailtibody to T pallidurn Antibody to T pallidrlrr~
Reagent (s) Sorbent (nonpathogenic treponemes-Reiter Sheep RBCs coated with antigens from
strain), slides with Nichols strain of T pulli- Nichols strain of T pallidiirn, sorbent
dnm, fluorescein-labeled antihu~nanglobulin
Positive reaction Fluorescence Rough or jagged pattern of cells in microtiter
wells
Specimen(s) Serum, CSF Serum
Reactivity during Usually positive before reagin tests. Some Not as sensitive in primary syphilis as
disease false-negatives in primary syphilis. Usually FTA. Sensitivity close to 100% in sec-
positive for life. ondary syphilis. Usually positive in late stages.
False-positives Fewer than nontreponemal tests. Reactivity is Fewer than nontreponemai tests.
seen with other trepone~naldiseases, notably
yaws and pinta.
Other Sorbent rernoves nonspec~ficantibodies. Sorbent reliloves nonspecific antibodies. Used
Used to confirm reactive nontreponemal test. to confirm reactive nontreponemal test. Not
Not good for treatment monitoring. good for treatment monitoring.

IMMUNOLOGY REVIEW 365 ~

 Tests for the Diagnosis of
Infectious Mononucleosis (IM)
Antibodies Interpretation I
I
Heterophile antibodies* I

1M antiboclies: Agglutinate sheep, beef, ox, and horse
RRCs. Adsorbed by beef RBCs but not by guinea pig
kidney cells (GPK).
Serum sickness antibodies: Agglutinate sheep, beef,
and horse RBCs.
Adsorbed by beef RBCs and guinea pig kidney cells.
Forssman antibodies: Agglutinate sheep and horse
RBCs. Adsorbed by guinea pig kidney cells but not
by beef RBCs.
-
Agglutinalion of sheep or horse cells following adsorption
with GPK but riot with beef RBCs = IM.
No agglutination ot sheep or horse cells following
adsorption with GPK or beef KRCs = serum sickness.
Agglutination of sheep or horse cells following adsorption
with beef RBCs but riot with GPK = Forssman.

Specific viral antibodies

IgM-VCA (viral capsid antigen): Peaks 3-4 weeks after IgM anti-VCA, anti-EA, or IgG anti-VCA without anti-
infection. Undetectahlr after 12 weeks. EBNA = recent or current infection.
IgG-VCA: Appears in late acute phase and persists Anti-EBNA or IgG anti-VCA without IgM anti-VCA = past
for life. infection.
Anti-EA (early antigen): Appears early and persists
for years.
Anti-EBNA (EB nuclear antigen): Appears 2-3 months
after infection and persists indefinitely.
*No~ispecit'ic
antibodies that cross-react with antigcns other than those originally responsible for (heir prutluction

366 IMMUNOLOGY REVIEW

 Weil-Felix Reaction
!
Weil-Felix Renction I
I
Disease Organism TPansmission OX-2 OX-19 OX-K
Rocky Mountain Rickettsia rirkettsii Dog or wood tick + ++++ 0
spotted fever*
Epidemic typhus R. prouluzekii Body or head louse + ++l+ 0
Eilde~nictyphus R. typhi Rat flea + t++t 0
Scrub typhus R. tsutsugarr~ushi Mite 0 0 ++++
Q fever CoxieLLo burnetii Inhalation of barnyard dust or 0 0 0
ingestion of infected meat or milk
*Most conilliolr rickettsia1 disease in U.S.

IMMUNOLOGY REVIEW 367 1

 Antinuclear Antibodies
I
Indirect Fluo- I
rescent Antibody I
Antibody Against Specificity Sensitivity (IFA) Pattern Contrnents I
Anti-ds-DNA Double- Specific for SLE Found in only 50- Peripheral or Considered 1
stranded DNA 80% of patients ho~nngeneous diagnostic for
with SLE SLE, especially
when C3 is low.
Anti-Sm Extractable nu- Specific for SLE. Onlv found in 30% Speckled
clear antigen Not found in of patients with
associated with other disorders. SLE
RNA
Allti-DNP Deoxyribonu- Present iu SLE Present in 70-90% Peripheral or LE factor
i
cleoprotein and drug-induced of patients with homogeneous 1
SLE SLE
Anti-SS-A/Ro Extractable Present in SLE, Present in 25-40% Speckled Most often in pa-
l~uclearantigen. scleroderrna and of S1.E patients tients with cutan-
RNA protein Sjogren's eous manifesta-
conjugate. syndrome tions (photosensi- 1
tivity dermatitis) I
!
Anti-SS-R/La Ribonucleo- Present in SLE, Present in 10-15 0io Speckled
protein scleroderma and of SLE patients I
I
Sjogren's syndrome I

368 IMMUNOLOGY REVIEW

Continued ~
I
 Hepatitis Tests
Test Indication
Hepatitis A
Anti-HAV (total) Past infection and i~lilrlunity
Anti-HAV (1gM) Acute hepatitis A
Hepatitis B
I-IBsAg Acute or chronic infection HRV (carrier), infectivity
HBeAg Acute or chronic HBV (carrier), high degree of infect~vity
Anti-IIBc (total) Current or previous infectiun or a carrier
Anti-HBc (IgM) Recent acute infection
Anti-HBe Disease resolution and reduced infectivity
Anti-HBs Recovery and imlllunity
Hepatitis C
Anti-HCV Acute, chronic, or previous hepatitis C infection
Hepatitis D
Anti-HD Past or c u r r e n ~hepatitis D infection
Hepatitis E
None

370 IMMUNOLOGY REVIEW

 HIV Testing
Test Detects ripe of Test Principle Corrtments
ELISA Antibody to f1IV Screening Viral ailtigel1 IS coated oil s Most widely used screening
solid support. Patient serum procedure Pos~tivebmust be
added After il~cubationand confimled. Anti-HIV-1 and
washing, enzyme-labeled anti-HIV-2 (or c o m b ~ n a t i o ~ l
c ~ n t i l ~ u ~ globulin
nan (AHG) test) required on blood
is added followed by substrate. donors.
-- -
- --
Slide agglu- Alltibody to HIV Screcni~ig f IlV antigen is adsorbed onto Used when instrurrlentnrion is
tination tests carrier particles. The particles not avallahle.
agglutinate in the presence
of antibody.
Western blot Antibcrdy to HIV Confirrrlatory A lysate of HIV dntigen is Most cornnio~~ly used
sepdrated into conlponents by confirnmatory test. IIIV inkc-
electrophoresis and transferred tion is indicated by reactivity
to nitrocrllulose paper. The in twu oi the tollowi~~g bands:
resulting blot is cut into stl-ips p24, gp41, or gpI2U/lhO.
and reacted with serum.
Ldbefed a n t i h i ~ n ~ dglobulin
n
is ddded.

372 IMMUNOLOGY REVIEW

 Other Serological Tests
Test Common Method Reagents interpretation Comments
Rheumatoid Agglutinatioil IgG attached to Positive in 70-80% Rheumatoid factor is an autoantibody
arthritis (RA) latex or RBCs of patients with RA (usually IgM) against IgG. Not spe-
cific for RA. Also found in SLE, scle-
roderma, Sjogren's syndrome, B-cell
lymphoproliferative disorders.
Cold Hemagglutination Patient or Positive in primary PAP is caused by Mycoplusrna prreu-
agglutinins Group 0 RBCs atypical pneumonia moniur. Illcreased titer of Anti-I. Test
(PAP) is incubated in refrigerator. Specime~r
must be kept warm prior to testing to
prevent fdlse-negatives or decreased
riters. l-'ositives must be confirmed by
reversal of agglutination at 37°C.
Anti-strep- Neutralization Streptolysiii 0 , Titer is highest Streptolysin 0 lyses RBCs. Anti-strep-
tolysin group O KBCs dilution showing tolysin 0 in patient's s e n ~ nneutral-
~
0 (ASOJ no hernolysis. High izes streptolysin 0 reagent so RBCs
titers with strep are not lysed. Streptolysin 0 is oxy-
infecrions, rheu- gen Idbile. RBC control (RRCs +
matic fever, glo- buffer) should show no hemolysis.
~rierulonephritis. Streptolysin control (streptolysi~~ 0+
buffer + RBCs) should show hrmoly-
sis. Reported in Todd units (reciprocal
of highest dilution showing no he-
molysis). Norrnal is less than 166.
Continued
374 TMMUNOLOGY REVIEW
 SECTION 6

Immunohematology Review
 Primary vs. Secondary Response
Primary Secondary (Anamnestic)
Stirnulus First exposure to antigen Subsequent exposure to antigen
Lag Phase Days to months Hours
Type of antibody IgM at first. May switch to IgG after 2-3 weeks IgG
(isotype switching).
Titer Rises slowly. Peaks then declines. Rises faster and higher. Stays elevated longer.

380 IMMUNOHEMATOLOGY REVIEW

 IgG vs. IgM

Structure Monomer Pcntarner

Number of antigen-binding sites 2 10
'&pe of antibody Itnn~une Naturally occurring
O p t i ~ n ~ utemperature
n of reactivity 37°C 25°C or lower
Reacts in saline No Yes
-

Reacts best by indirect antiglobulin test (IAT) Yes No

Complement fixation Moderare Strong
Causes tr~nsfusuonreactions Ties hot usually, except
ABO
-- - -
Crosses placenta Yes No
Causes helnolytic disease of the ~iewborn Yes No
Destroyed by sl~lfhyclrylcompounds (dithiothrcitol [DTT], No Yes
2-n~ercaptoethanol[2-ME])
-

IAllMUNOI-IGMATOLOGY REVIEW 381

 Signs of Antigen-Antibody Reactions
in Blood Bank Testing

Explanation Union of antibody with antigen activates Antibodies attach to antigens on adjacent
complement which destrvys RBC membrane. RBCs, forming bridges between cells.
Requires CaL+.
!
!
Immunoglobulins One IgM n~uleculeor two IgG molecules can IgM is more effective in inducing agglutina-
activdte complement. (Kequires two Fc portions tion because of its size and number of anti-
of immunvglnhulin in close proximity on the gen-binding sites. I
RBC surface.) I
Grading PH = partial hemolvsis; some RBCs remain mf = rnixed field (some agglutinated RBCs
H = complete hemolysis, no RBCs remain in sea of free RRCs) !
wi = tiny agglutinates, turbid background
I
1+ = small agglutinates, turbid hackground
2+ = niediurn-sized agglutinates, clear
background
3+ = several large agglutinates
4+ = orie solid agglutinate

Comments False-negative reaction if overlooked. Most common reaction seen.

 Factors that Affect Agglutination
Factor Conlrrlents
Sensitization Stage Attachment of antibody to antigen
Temperature Cli~iicallysignificant antibodies react best at 37°C.
PH Most antibodies react at plI 5.5-8.5
Ionic strength Reducing the ionic strength of the medium facilitates interaction of antibody
with antigen (e.g., low ionic strength solutiori [LISS]).
A1ltigen:antibody ratio Too r~luchantibody can cause prozone (ialse-negative). Optirnul~lserum to cell
ratio is 8O:l. Usually two drops of serum to one drop of 2-5% RRCs. Follow
the nianufacturer's directions.
Incubation t ~ m e Depends on nledium. Usually 10-30 minutes. Follow the ma~~ufacturer's
directions.
Agglutil~ationStage Formation of antigen-antibody bridges between RBCs
Type oi antibody molecule 1gM is larger and can span the distance betweell RBCs more easily
Density of antigens and Affects ease of attach~nentof antibodies
location or] RBC surface
Zeta potential The difierence in charge between the riegatively charged RBC surface and the
cloud of positive ions that surround the RBCs. Reducing the zeta potential allows
the RBCs to move closer together (e.g., enzyme treatment of test cells).

IMMUNOHEMATOLOGY REI'IEW 383

 Antigen-Antibody Enhancement
Albumin 22% or 30% bovine serum alhui~iin.Reduces net negative charge of RBCs, allowilig thern to
come closer toget her.
Low ionic Lowers ionic strength of st~spendingmedium, allowing antigens and antibodies to move closer
strength together more rapidly. Reduces the incubation time in the indirect antiglobulin test to 10
solution (LISS) rninutes.
Polythylenegiycol Increases antibody uptake. Used for detection and identification oi weak IgG antibodies.
(PEG)
Enzymes Exarnples include ficin. bromelin, papain. Reduce RRC surface charge by cleaving sialic acid
molecules. M, N, S, Fyd, and Fyh antigens are destroyed.

384 IMMUNOHEMATOLOGY REVIEW


QP~
Polyspecific (broad spectrum)
Monospecific
Anti-IgG
Anti-C3d and antiLC3b
'Complement proteins.
Antihuman Globulin Serum
Detects Comments
IgG and C3d Used for direct antiglobulin test (DAT) and, in some
labs, for routine compatibility tests and antibody
detection. If positive, monospecific sera may be used
for follow-up testing. Disadvantage: reactions due to
complenlent binding by clinically insignificant cold
antibodies.
Can be used for routine compatibility tests and anti-
body identification. Detects clinically significant anti-
bodies.
C3d and C3b* Helpful in investigation of immune hemolytic anemia
IMMUNOHEMATOLOGY REVIEW 385
 Antiglobulin Testing
Direct (DAT) Indirect (lAT]
Detects In-viva sensitization of RBCs by In vitro sensitization of RBCs by IgG antibody
IgG antibody
Specimer~ EDTA (ethvlenediarninetetra-acetic acid) Serum, plasma, RBCs
red cells preterred
Incubation None required Serum or plasma with reagent RRCs or KBCs with
reagent antiserum
Application Ilemolytic disease o i the newborn. Antibody screen, crossmatch, RBC phenotyping,
transiusion reactions. autoin~mune weak D testing (D2)
hemoiytic anemia, dr-11g-induced
hemolytic anemia
False-positives Complement binding in vitro if RBCs are Cells wit11 positive DAT.
t<lkenfrom 3' red-top tube and broad- Ovel-centrif~~gation.
spectrum antihum,m globulin ( A X )
is ~rsed.
Septicemia.
Contarnination of specimen.
\Vharton's jelly in cord blood.
Overreading.
Overcentrifi~gation.
Continu~rf

386 IMMUNOHEMATOLOGY REVIEW

 Frequency of ABO Types
mve Whites (%I Blacks (%) Hbvanics (%IAsians (%)

IMMUNOHEMATOLOGY REVIEW 391

 ABO System

Group Antigen(s) on RBC Serum

0 Neither Anti-A, Anti-B

AB A and B Neither anti-A

n o r anti-B

392 IMMUNOIIEMATOLOGY REVIEW

 ABO Discrepancies

Anti-A Anti-B A , cells A, cells B cells 0 cells Auto Possible Cause* Resolution
0 0 0 0 0 0 0 Missing isosgglutinins Repeat reverse grouping
in group 0 using four drops of
serum. Iricubate at room
temperature for 30 min-
utes or at 4OC. (Run auto
control and screening
cells.)
4+ 0 1+ 0 4+ 0 0 A, with anti-A, Type cells with Anti-A,
Test serum with addi-
tional A, and A2 cells.
4+ 4+ 2+ 2+ 2+ 2+ 2+ Rouleaux Use washed RBCs sus-
pended in saline for for-
ward grouping. Perform
saline replacement tech-
nique in reverse grouping.
3+ 4+ 1+ 0 0 0 0 A,B with allti-A, Type cells with Anti-A,
Test serum with addi-
tional A, and Al cells.
"Othei explanations m a y be possible.
Continued

394 IMMUNOHEMATOLOGY REVIEW

 Non-Group Specific Transfusions
Patient RBC v p e Patient Plasma v p e Patient
vpe Can Receive Can Receive
0 0 only 0 , A, B, AB

AB AB, A, B, 0 AB

396 IMMUNOHEMATOLOGY REVIEW

 Using Punnett Square to Predict Rh Q p e
Mother's Genotype

Father's
Gerrotype

398 IMMUNOHEMATOLOGY REVIEW

 Frequency of Rh Antigens
Antim Whites (%) Blacks (%)

Note: Students do 11ot need to memorize all

frequeilcres but should know f~.cquencyof D
and which antigens are most and least
com1n011.

400 IMMUNOHEMATOLOGY REVIEW

 1 Positive Rh Controls
I
Possible Cause Resolution
Cells with positive DAT Type with monoclonal anti-D, chemically modified anti-D, or I
saline anti-D. !
I
Kouleaux or higll-titered cold agglutinin Repeat using washed, saline suspended RBCs. I
1
Bacterial contamination of cells or reagents Repeat with new specimen or reagent.

404 IMMUNOHEMATOLOGY REVIEW

 Rh Typing Sera
High-Protein Chemically Modified Anti-D Monoclonal/
Aati-D Anti-D PolycIonal Blend Saline Anti-D
Description Polyclonal IgG anti-El IgG allti-D converted to Blend of IgM monoclorial IgM ariti-D susprrided
in 20-2491 protein direct agglutinin. In 6 % anti-D and IgG polyclonal in saline
protein concentra!ion. anti-D. Low protein
content.
-- --
Protein ~ o r ~High
- Low l.onr Low
centration
-
Use Routir~etyping When Rh control is When Rh i:oritrol is When Rh control is
positive with high- positive with high- positive with high-
protein reagent protein reagent protrin reagenl
-
. -
Cor~trol Sarne ingredients a s ABO forward grouping ABO forward grouping 6 % bovine albumir~
reagent, except no serves as control for all serves as control for all
anti-D. Sho~rldbe except type AB. For type except type AB. For type
purchased fro111 AB, dn autocol~trol,6% AD, a n auto-control,
same manufacturer albumin, or saline may 6% albumin, or saline
as Anti-D. be recommended. may be recommended.
Follow the manu- Follow the manu-
facturer's instructions. facturer's instructions.
--
Used for l'e s Yes Yes No
weak L)
testing!
-
Cr~ntinued

1MMUNOHEMATOI.OGY REVIEW 405

 Selection of Rh Type
Recipient 'Ijpe Rh Q p e Patient Can Receive
Rh positive Rh positive or Rh negative
Weak D Rh positive or Rh negative
R h negative Rh negative or~ly,especially woinen of childbearing age. (If Kh positive must be given in an
emergency, Rh immune globulin can be given to prevent immunization.)

IMMUNOHEMAI'O1,OGY REVIEW 407

 Z antigen i antigen
Adult cells Much Trace
Cord cells Trace Much
i adult cells Trace Much

410 IMMUNOHEMATOLOGY REVIEW

 Antibody Identification
Reactions Possible Explanation
Same strength and in onc phasr onlv Suggestive of s~ngleantibody
Varying strength Multiple antibodies, antibody exhibiting dosage, anti-
gens of differing strength
In diiferent phases Combination of warm and cold antibodies, antibody
with wide thermal range
-.

All cells in AHG, ~utocontrolnegative Multiple antil~odies,antibody to high frequency antigen

I
All cells in AHG, autocontrol positive Warm autoantibody !

All cells at 37"C, negative in AIIG. autocontrol positive Kouleaux

IbIMUNOHEMATOLOGY REVIEW 413

 Cold Antibodies
Anti-A, Only fou~ldin subgroups of A. Agglutinates A, and A,B cells, but not A2 or 0 .
Anti-I Agglutinates all adult cells, except i adult. Does not agglutinate cord cells
Anti-i Agglutinates cord cells more strongly than adult cells.
Anti-H Most comrnon in A, and AIB. Agglutinates O cells most strongly, followed by A, and B; then A,
and A,B.
Anti-IH Most conlrllon in A, and AIB. Agglutinates cells that possess both I and H. Agglutirlates adult 0
cells most st~ongly.Wealtcr reactions with A, cells. Does not agglutinate cord cells.

IMMUNOHEMATOLOGY REVIEW 415

 Compatibility Testing
Specimen collected within 3 days of transfusion if the patient has been pregnant or transfused during the preceding
3 months.
Confirmation of identifying information on request fnrm and specimen.
Check of blood bank records
Repeat ABO type on donor.
Repeat Rh type on donor if unit 1s labeled R h negative (weak D not required)
ABO type on recipient.
Rh type on recipient (weak D not required).
Antibody screen on recipient.
Crossmatch.
Retain patient specimen and unit segment at I-6°C for 7 days follouring transfusion.

416 1MMUNOHEMATOLOGY REVIEW

 Crossmatches
Explanation When Performed Comnients
Antiglobulin crossmatch Recipient serum and Required when recipient
donor RBCs are tested has, or previously had, a
through the indirect clinically significant
antiglobulin phase. antibody
Abbreviated crossmatch Recipient serum and Per~nissibleif recipient Test of ABO incompatibility. An
donor RBCs are tested in does not have, and has antibody screen carried through
immediate spin only. never had, clinically signih- the AHG phase must be
cant antibodies performed and must he
negative.
Computer crossmatch Computer check of Permissible if recipient ABO typing of recipient must be
donor ABO and R h type does not have, and has done twice. Computer system
and recipient ABO and never had, clinically signifi- must be validated to prevent
Rh type cant antibodies release of ABO-incompatible
blood and must alert user to
discrepa~lciesand incompatibil-
ities.

IMMUNOHEMATOLOGY REVIEW 417

 The Major Crossmatch
Will Will Not
Detect ABO i~lcompatibility Detect all ABO typing errors
Detect most antibodies against Detect most Rh typing errors
donor cells
Detect all antibodies
Detect platelet or leukocyte antibodies
Preve~itirnmullizatio~l
Ensure ~iormalsurvival of RBCs

418 IMMUNOHEMATOLOGY REVIEW

 The Incompatible Crossmatch
Reactions Likely Cause Resolution
One antibody screening cell and one Alloantibody Identify antibody. Crossinatch units negative
donor positive in AHG. Negative for the corresponding antlgen.
autocuntrol.
Antibody screening cells. all donors Positive DAT on donor Keturn unit to collecting facility.
except one, and autocontrol negative
at 37'C and in AHG. One donor
positive in AHG only.
Antibody screening cells, donors, Lirarni autoantibody Best not to transfuse. If unavoidahle, find
and autocontrol positive in AHG. "least incompatible" unit.
- - -
Antibody screening cells, donors. and Rouleaux Saline replacement technique
autoco~itrolpositive at 37'C and
negative in AHG.

IMMUNOHEMATOLOGY REVIEW 419

 Emergency Transfusions
Give ABO and Rh compatible if time allows for typing, otherwise, give 0 negative RBCs.
Label must indicate that crossmatch was not completed.
Physician must sign emergency release.
Routine testing must b e completed and physician notified immediately of any incompatibility.

420 IMMUNOHEMATOLOGY REVIEW

 Newborn Testing*
Specimen Comments
ABO and Rh typing Cord blood, capillary, or venous blood ABO forward grouping only. Only required
once per admission.
Antibody screen Serum or plasma of mother or baby Only required once per admission
Crossmatch Serum or plasma of mother or baby If the antibody screen is positive, perform an
antiglobulin crossmatch on units negative
for corresponding antigen.
If the antibody screen is negative, crosslnatch
is not required.
'Younger than 4 months.

IMMUNOHEMATOLOGY KEVIEW 421 1

 Conditions for Reissue of Blood
RBCs were maintained between 1-10°C. (Red cells at room temperature reach 10°C in 313 minutes.)
Closure was not I~roken.
At least one segment remains attached to unit.
Unit is ~rlspectedpriol to release.
Records indicate that blood has been reissued.

422 IMMUNOHEMATOLOGY REVIEW


-.
5 ~ e Clinical Signs
Hen~olytic, Fevcr; clrills; shock;
i~~trdvascul.tr rerlal failure: DIC;
p i n ill chest, back,
or flank
He~nolvtic, F e ~ ~ ecr 3. n e ~ n inrild
~,
extr~vasrular jaundice 2 or more
days after
transfusion
Febrile An illcrease in Allti-leukocyte anti-
trnipcraturr 2 1 ° C bodies
tvitliin 24 hours of
transiusion, with
rio other explanation
Transfusion Reactions
Cause Laboratory Findings Other
Immediate destruction 111post-transiusio~l Most serious reaction.
of donor RBCs by specimens: hemoglobin May be fatal. Usually due
recipient antibody in urine and serum, to ad mini strati or^ of ABO-
positive n~ixedfield DAT inco~npatibleblood.
(unless donor cells arc
all tlrstroyed), decrease
ill haptoglobin, de-
creasetl hernoglohin and
lierrlatocrit [Hand 13)
--
Donor IiBCs are sensi- Increased bilirubirr. May he d u r to a n a ~ n ~ r e s -
tized by recipient IgG positive mixed-field LIAT. tic response. Kidd allti
antibocly and renloved decreased haptoglobii~, bodirs most common
fro111circulation. decreased I 1 and H at 2 cause. Usuallv not liie-
or mol-e days after threatening.
tr,r~lsfusion
None Common. Most often
seen in multipiv tsa~is-
fusrd patients or women
with multiple prrgrlan-
cies. Future trar~sfusions
should be with leuko-
poor RBCs. Antipyretics
Continrieptl
IMMUNOHEMATOLOGY KEVIEW 423
 Transfusion Reaction Investigation
Specimens needed
Immediate steps
Signs of a he~nolyticreaction
Further steps if ~ h e s eare signs
of a hemolytic reaction
Additional procedures
Pre-transfusion blood
Post-transfusion blood
Post-transfusion urine
Segment from unit
Blood bag with administration set and attdched IV solutions
Check all IDS and labels.
Check post-transfusion specimen for hernolysis.
Perform a DAT on post-transfusion specimen.
Hemolysis in post-transfusion s p e c i ~ ~ ~ ebut
r l , not in prc-transfusion specimen. I
Mixed field agglutination in DAT on post-transfusion specimen, but not on pre- I
transfusion specimen. I
Check hernoglohin in first voided urine after transfusion. I
Repeat ABO and Rh on pre- and post-transfusion specimens and unit. I
Repeat antibody screen on pre- and post-transfusion specimens and unit.
Repeat crossmatch on pre- and post-transfusion specimens.
I
Haptoglobin (decreased with hemolysis). I
I
Gram stain and culture of unit.
Bilirubin 5-7 hours after transfusion (sign of extravascular hernolysis).
BUN and creatinine (sign of renal involvement).
IMMUNOHEMATOLOGY REVIEW 425
 Transfusion-Associated Diseases
Infection Prevention Comments
HIV Donor screening Estimated risk: 1 in 676,000 transfusions
I
I
Hepatitis B Donor screening Estimated risk: 1 in 66,000 transfusions.
Hepatitis C Donor screening Estimated risk: <1 in 100,000
HTLV-I and -11 Donor screeiling Rare in United States
Syphilis Donor screening Limited risk in refrigerated or frozen components since spirochetes
can't survive in cold. Highest risk from platelets.
Malaria
Babesiosis
Donor history
Donor history
Plasrrlodiurrz is transmitted in RBCs.
Babesia is transmitted in RBCs. i
Chagas' disease

Cytomegalovirus
Donor history

Selected screening
Typanosorna crlui is transmitted in blood. Potential risk in donors
from Central and South America.
CMV is transmitted in WBCs. Risk to premature infants, bone mar-
l
row recipients, other iinmunocon~proinisedrecipients.
Graft-versus-host Irradiation of cellular Viable T lymphocytes in donor blood attack recipient. Usually
disease components fatal. Blood components should be irradiated for in~munocornpro-
mised recipients, for patients with leukemia and lymphoma, and
for recipients of blood from a first-degree relative, intrauterine or
exchange transfusion, or bone marrow transplant.
1
a

I
426 IMMUNOHEMATOLOGY REVIEW !
 Hemolytic Disease of the Newborn
ABO Rh
Mothers at risk Usually group 0 Rh negative
First child affected? Yes Not usually
Frequency Common Uncommon
- - -

Severity Mild Severe

DAT Weak positive or negative Strong positive
Best elution method Heat or freeze-thaw Acid or orgmic solvents
Spherocytes? Yes No
Predictable? Yes (antibody screen)
Preventable! No Yes (Rh immune globulin)

IMMUNOHEMATOLOGY REVIEW 427 1

 Rh Immune Globulin (RhIG)
Composition Anti-D der~vedfrom pools of human plasma
Use Prevent immunization to D
Administration Antepartum: To Rh-negative woman at 28 weeks of gestation
Postpartum: Within 72 hours of delivery when Rh-negative womari delivers Rh-positive
baby
Other obstetric events: To Rh-negative woman after sponta~leousor therapeutic abortion,
ectopic pregnancy, anlniocentesis, chorionic villus sampling, anteparturn hemorrhage.
or fetal death
Other: May also be administered to Rh-negative recipients of Rh-positive blood or
components
Prenatal evaluation ABO and Rh, including weak D. If Rh positive, womari is not a candidate for RhlG.
Antibody screen. If positive, identify antibody. If anti-D is present, woman is not a
candidate for RhIG.
Postpartum evaluation ABO and Rh, including weak D, on baby. If baby is Rh negative, mother is not a
candidate for RhIG.
If baby is Rh positive, draw mother's blood within 1 hour after delivery and perform
rosette test to screen for large fetal bleed. If positive, perform acid-elution test (Klei-
hauer-Betke) to quantitate fetal bleed. (Fetal cells resist acid elution and stain pink.
Adult cells lose hemoglobin and appear as "ghosts.")
Dose One dose per 15 mL of D-positive fetal RBCs (30 mL of fetal whole blood)
Note Residual anti-D from anteparturn RhIG may be detected following delivery and does not
disqualify woman from receiving postpartum RhIG.

428 IMMUNOHEMATOLOGY REVIEW 1

 Allogeneic Blood Donor Criteria (AABB)

Age At least 17
Weight At least 110 lb to donate 525 mL
Donation interval 8 weeks
Blood pressure Systolic 5 1 8 0 , diastolic 5100
Pulse 50-100 with no pathological cardiac irregularities
Hemoglobin 212.5 g/dL
Temperature 537.S°C

IMMUNOHEMATOLOGY REVIEW 429

 Blood Donor Deferrals (AABB)
Deferral Condition
3 days Aspirin-containing medications if donor is sole source of platelets
2 weeks Measles, mumps, polio, or yellow fever vaccines
4 weeks Rubella vaccine
G weeks
12 months Syphilis
Gonorrhea
Animal bite
HBIG
Tattoo
Mucous membrane exposure to blood
Skin penetration with sharp contaminated with blood or body fluids
Household or sexual contact with individual with hepatitis
Sexual contact with individual with HIV or at high risk
Travel to area endemic for malaria
3 years Malaria, or from an area endemic for malaria
Contirrned

430 IMMIJNOHEMATOLOGY REVIEW

 Anticoagulants/Additives/RejuvenatingSolutions
Name Abbreviation CLassification Shelf-Life Comments 1
I
Acid-citrnte-dextrose ACD Anticoagulant/ 21 days I
preservative I
Citrate-phosphate- CPD Anticoagulant/ 21 days Higher pH preserves 2,3-diphospho- I
dextrose preservative glycerate (2,3-DPC) better. Better 0,
delivery.
Citr~te-phosphate- CPDA-1 Anticoagulant/ 35 days Adenine incre~sesadenosine diphos-
dextrose with preservative phate (ADP).
adenine
Adsol AS- 1 Additive 42 days Additive is in satellite bag. After
plasma is removed, additive is added
to RBCs. Provides nutrients for irn-
proved viability.
Nutricel AS-2 Additive 35 days Additive is in satellite bag. After
plasma is rernuved, additive is added
to R6Cs. Provides nutrients for im-
proved viability.
Nutricel AS-3 Additive 42 days Additive is in satellite bag. After 1
plasma is removed, additive is added
to RGCs. Provides nutrients for irn-
proved viability. I
Contiiiued 1
IMMUNOHEMATOLOGY REVIEW 433
 Donor Serological Testing Required
by AABB and/or FDA
ABO
Rh (including weak D)
Antibody screen
RPR
HBsAg
Anti-HBc
Anti-HCV
Anti-HIV 1 a n d 2
HIV- I - Ag
Anti-HTLV-I and 11

436 IMMUNOHEMATOLOGY REVIEW

 Autologous Transfusions
Advantage No risk of disease transmission, incompatibility, or immunization
Age limit None
Hemoglobin Not less than 11 g/dL
Frequency of donation Every 3 days, but not within 72 hours of surgery
Contraindications Bacteremia
Testing ABO a n d Rh. If the unit will be transfused outside the collecting facility, HBsAg, HIV-1-
Ag, Anti-HIV-l and 2, anti-HCV, anti-HRc, and rapid plasma reagin (RPR) are also re-
quired.
Labeling "Autologous Donor," "For Autologous Use Only." Biohazard label if any hepatitis or
HIV test is confirmed to be positive.
Pretransfusion testing ABO and Rh of recipient and unit

IMMUNOHEMATOLOGY REVIEW 437 1

 Blood Components
Storage
Component Preparation Ten~perature Shelf-Life Indicatiorls Other
~ - ~ ~ - - ~~ --------- ~ ~~~ ~~~~ ~

Red blood Separated from 1-6°C 35 days in Anemia Test at least 4 units per
cells (RBCs) whole blood CPDA-1,42 ~ n o n t h 90Y1
. sllc~uld
by ceutrlfugation days in AS-1 have hernatocrit of 80%
or sedimentation or less. Hematocrit >
any time hefore 80%: inadequate I
the expiration preservative to support
date of the storage. One nit
whole blooct should increase hernat-
ocrit 3 % . I

RBCs froze11 Frozcn in glycerol 40% glycerol: 10 years from Anemia Check final wash for
withirl 6 days of -65°C. phlebotoniy, free hemoglobin to ,
I

collrction 24 hours after ensure adequate glyc-

20%) glycerol: cleglyceroli- erol removal. Virtually I
5-120°C. zation all plasma, anticoagu- I
1
lant, WBCs, and i
I-6°C after de- platelets are removed. ,
glycerolization. Safe for IgA deficient
pdtients. Used tu store I
rare cells.
-
Washed RBCs RBCs washed 1-6°C 24 hours after Anelliic patients One way of producing
with saline washing with paroxysnlal leuko-reduced KBCs.
nocturnal
-- I
Continued
$38 IMMUNOHEMATOLOGY REVIEW
 Blood Components Continued
Storage
Component Preparation Temperature Shelf-Life Indications Other
whichever recipients, donor T cells
occurs first recipients of blood inl~nunoincompetent.
horn a relative,
bone rnarrow
transplant
patients
Fresh fro7en Plasnla separated 5-18°C. After 12 months. Deficiency of Check for evidence of
plasma from whole blood thawing, After thawing, coagulation thawir~gand refreezing.
and frozen within 1-6°C. t~ansfusewithin factors Thawed at 30-37°C or
8 hours of 24 hor~rs. hy an FDA-approved
collection microwave.
Cryopre- Prepared by I-18°C. After 12 months. After Hemophilia 11, Check for evidence of
cipitate thawing FFP thawing, thawing, tlans- von Willehrand's thawing and refreezing.
between I -G°C, room fuse within 6 disease, Iiypo- Thaw at 30-37" C. Test
removing plasma, temperature. flours if unit is fibrinogenemia, at least 4 units/month.
and refreezing not entered, Factor XI11 Should contain at least
within 1 hour. within 4 hours deficiency 80 IU of Factor VlII and
if pooled. 150 rng of fibrinogen.
Contirrucd

440 1MMUNOHEMATOI.OGY REVIEW

 Changes in Stored Blood
Increased Decreased
Plasma K+ PI 1
Pldsmd NH, ATP
Plasma hemog1oL)in 2,3-DPG
Mirroaggregdtes Vlable RBCs, WRCs, and platelets
J,dbile coagulat~onfactors

IMMUNOHEMATOLOGY REVIEW 443

 Blood Bank Quality Control
Centrifuges Check rpm with tachometer quarterly.
Check timers with stopwatch quarterly.
Uetermine optinium speed and time for different procedures upon receipt, after repairs, and
semiannually.
Cell washers Check volume of saline and AHG in each tube. Verify time and speed of centrifugation peri-
odically.
Waterbaths Check temperature daily.
Heat blocks Check temperature daily. Periodically check each well.
Refrigerators System to monitor temperaturr continuously and to record temperature at least every 4
hours. Alarm system with audible signal. Must be 1-6°C.
Freezers Syslem to monitor temperature continuously and to record temperature at least every 4
hours. Alarm system with audible signal.
Alarms Check high and low temperatures of activation quartrrly.
Platelet incubators System to monitor temperature continuously and to ~ecordtemperature at least every 4
hours. Should be 20-24°C. Check rpm periodically.
Pipettes and droppers Determine average delivery volume. Calculate number of drops that will give 8O:l serum-
to-cell ratio.
Continued

444 IMMUNOHEMATOLOGY REVIEW

 Changes in Urine at Room Temperature
Bacteria multiply and may cause turbid~tyand a positive protein reactloll.
Bacteria convert urea to ammonia, which increases pH.
Bacteria metaholi~eglucose.
RBCs lyse in dilute or alkalilie urine.
Casts lyse in dlkaline urine.
WBCs disintegrate.
Bilirubin/urobilii1oge11 are lost through exposure to light and/or oxidation.
Ketones are lost through evaporation.

448 URINALYSIS AND BODY FLUIDS REVIEW

 Urine Volume
Normal dailv voli~me 1200-1500 mL
Nor~ilald ~ y - n i g hratio
t 211-3:l
Diuresis lncreaseci urine production
Polyuria B2000 mL/day (e.g., diabetes mellitus, diabetes insipidus)
Oligi~ria 1.500 mL/day (e.g.. dehydration, renal disease, obstructio~iof urinary tract)
Anuria No urine production
- I

URINALYSIS AND BODY FI.UIUS REVIEW 449

 Urine Color and Clarity
Urochrome Normal yellow color
Dilute urine Colorless 1
Concentrated urine Dark yellow, amber
Bilirubin Yellow-brown or olive-green. Yellow foam on shaking.
Homogentisic acid Normal on voiding. Brown or black on standing, beginning at surface.
Melanin Brow11 or black on standing
Methemoglobin Brown or black due to oxidation of hemoglobirl in acid urine
Myoglobin "Cola" on standing
Blood/hemoglobin Pink or red when fresh. "Col,~"or "smoky" on standing. Cloudy with RBCs. Clear with
hemoglobin.
Porphyrin Port-wine
Drugs, medications, food Green, blue, orange
Pst~udomonasinfection Green, blue-green
Urobilinogen Colorless when excreted. Oxidized to orange-red 01. orange-brown urobilin.
i
Crystals. WBCs, RBCs, Cloudy
epithelial cells, bacteria

450 URINALYSIS AND BODY FLUIDS REVIEW I

 Chemical Urinalysis by Reagent Strip
Test Normal Principle Significance Sources Of Error* Comments I

PH First AM: 5-6 Double buffer Useful in evalua- Decreased: Acid- Acid with proteinjmeat I
Random: 4.5-8 system tion of acid-base nlnover from protein diet. Alkaline with
bala~lce,manage- square. Increased: vegetarian diet.
ment of UTI and Specimen left at
renal calculi room temperature
i
Protein Negative-trace Protein-error
of indicator
Renal disease
too long.
False-positive: Highly
buffered or alkaline
Buffered to maintain
pH 3. Most sensitive to
ii
urine, prolonged dip- albumin. Blood, WBCs,
ping. False-negative: bacteria can cause posi-
Proteins other than tive reaction. Orthostatic
albumin proteinuria: a benign
condition in which pro-
I
tein is negative in the
first AM specimen and
positive after standing.
Glucose Negative Glucose oxi- Diabetes False-positive: Conta- Specific for glucose.
dasejperoxi- mellitus nlination with peroxide More sensitive and
dase or oxidizing detergents specific than copper
(bleach). False-negative: reduction test. For
High levels of ascorbic diabetic monitoring.
acid, glycolysis. specimen collected 2
Continued
 Chemical Urinalysis by Reagent Strip Continued
Test Normal Principle Significance Sources Of Error* Comments
hours after eating is pre-
ferred. Normal renal
threshold = 160-180
mg/dL.
Ketones Negative Sodium nitro- Increased fat Decreased in improperly Most sensitive to
prusside metabolism, e.g., stored specimens acetoacetic acid
reaction diabetes mellitus,
vomiting, star-
vation, low carbo-
hydrate diet
Blood Negative Peroxidase- Renal calculi, glo- Decreased: High levels Detects RBCs, hemoglo-
like activity merular disease, of ascorbic acid, nitrites, bin, and myoglobin
of hemoglobin tumors, trauma, protein, specific gravity. [muscle destruction)
pyelonephritis, Failure to mix specimen.
hemolytic anemia, False-positive: Men-
hemolytic trans- struation, oxidizing
fusion reaction, detergents, bacterial
burns, infections, peroxidase.
strenuous exercise
Bilirubin Negative Diazo reaction Liver disease, False-negative: Exposure Only conjugated
biliary obstruction to light, oxidation to bili- bilirubin is excreted in
verdin, hydrolysis of bili- urine.
~ - - -

'Sources of error may vary with brand of reagent strip. Refer to manufacturer's package insert.
Continued

452 URINALYSIS AND BODY FLUIDS REVIEW

 Blood Donor Deferrals (AABB) Continued

Deferral Condition
Permanent Parenteral drug use
Family history of Creutzfeldt-Jakob disease
a e a t e d with growth hormone
Viral hepatitis after 11th birthday
Positive HBsAg
Repeatedly reactive anti-HBc, anti-HCV, anti-HTLV, or anti-HIV
High-risk behavior for HIV
Sole donor to patient who developed post-transfusion hepatitis, HIV,
or HTLV
Babesiosis
Chagas' disease

IMMUNOHEMATOLOGY REVIEW 431

-- -- -- - - -
 Principles of Labeled Immunoassays Continued
Method I)tpe of Assay Principle
wash, substrate is added. The amount of enzyme label detected is di-
rectly proportional to the amount of antibody in the serum. Indirect
ELISAs are more sensitive than direct ELISAs.
Immunoenzymo- Noncompetitive The specimen is incubated with an enzyme-labeled antibody. A solid-
metric assay (IEMA) Nonisotopic phase ligand (usually on glass beads) is added and the tubes are
Heterogeneous centrifuged to remove unbound labeled antibody. The amount of la-
beled antibody in the supernatant is directly proportional to the concen-
tration of the ligand in the specimen. This method is more sensitive
than indirect ELISAs.
Sandwich enzyme- Noncompetitive The antigen in the specimen is sandwiched between antibody attached
multiplied Nonisotopic to a solid phase and enzyme-labeled antibody. Enzymatic activity is
immunoassay Heterogeneous directly proportional to the amount of antigen in the test sample.
Enzyme-multiplied Competitive The antigen in the specimen and an enzyme-labeled antigen compete
immunoassay Nonisotopic for binding sites on reagent antibody. When enzyme-labeled antigen
technique (EMIT) Homogeneous binds to antibody, enzyme activity is inhibited. Enzyme activity is
directly proportional to the concentration of the antigen in the speci-
men. This is an automated method that is frequently used for drug as-
says.
Continued

IMMUNOLOGY REVIEW 361

 Antinuclear Antibodies Continued
Antibody Against Specificity Sensitivity ZFA Pattern Comments
Anti-RNP Proteins com- Present in SLE, Speckled
plexed with nu- mixed connective
clear RNA tissue disease and
other autoimmune
diseases
Anti-histone Histone (nucleo- Present in SLE, Present in almost Honiogeneous Diagnostic of
protein that is a RA, primary all patients with drug-induced
major constituent biliary cirrhosis drug-induced lupus. High levels
of chromatin) lupus associated with
more active and
severe forms of
SLE.
Multiple ANAs can be present in a specimen. More than one pattern will be observed.
Fluorescence of nucleoli is due to antibody to RNA. Seen in scleroderma and polymyositis.

IMMUNOLOGY REVIEW 369

- - -
 HIV Testing Continued
Test Detects V p e of Test Principle Comments
Indirect Virus or antibody Confirmatory Antibody test: Serum is incu- Sensitivity and specificity
immuno- bated with virally infected comparable to Western blot.
fluorescence cells on a glass slide. Simple and rapid to perform.
assays Fluorescein-labeled antihuman
globulin is added. Antigen test:
Patient cells are fixed to a slide
and incubated with HlV-specific
antiserum.
P24 antigen HIV antigen Screening Anti-HIV-1 bound to a solid P24 antigen precedes antibody
test support is incubated with by several weeks. Positives
(HIV-1-Ag) serum. After washing, enzyme- must be confirmed by a neu-
laheled anti-HIV-1 is tralization assay. Required on
added followed by substrate. blood donors.
Polymerase Viral genome Adjunct to Viral DNA from infected cells Extremely sensitive technique.
chain reaction standard is amplified, then identified Will detect infections during
(PCR) testing using labeled probes. "window period." Not suitable
for routine screening.
Viral culture Virus Adjunct to Virus grown on cell culture Expensive, time-consuming,
standard and hazardous. Not routinely
testing performed.
Antibody tests indicate exposure to HIV.

IMMUNOLOGY REVIEW 373

 Other Serological Tests Continued
Test Common Method Reagents Interpretation Comments
C-reactive Latex agglutination Latex particles Agglutination = Sensitive, but nonspecific, indicator
protein coated with inflammation of inflammation. Originally named
anti-CRP because it was thought to be an anti-
body to the C polysaccharide of S.
pneumoniae.
Rubella Hemagglutination Viral preparation, Titer is highest Rubella = German measles = 3-day
Inhibition RBCs dilution showing measles. In-utero infections cause
no agglutination. fetal death or birth defects. Greatest
A titer of 8 or risk in first month of pregnancy. IgM
higher = immunity antibodies in a neonate are diagnostic
to rubella. of congenital rubella.
Febrile Bacterial agglu- Salmonella 0 and Positive reaction is Screening test for fever of unknown
agglutinins tination H, Brucella agglutination. origin. Weil-Felix reaction: rickettsia1
abortus, antibodies cross react with OX-2, OX-
Proteus OX 19. 19, and OX-K strains of Pmteus.
Widal's test: detects antibodies in ty-
phoid fever, tularemia, brucellosis.
Antibodies to 0 = recent infection.
Antibodies to H = past infection. Best
indicator of active infection is four-
fold increase in titer. Nonspecific test.
Infrequently ordered today.

IMMUNOLOGY REVIEW 375

- --
 ABO Discrepancies Continued
Anti-A Anti-B A , cells A, cells B cells 0 cells Auto Possible Cause* Resolution
4+ 2+ 0 0 4+ 0 0 Acquired B antigen Check medical history for
GI problem or sep-
ticemia. Type with hu-
man anti-B acidified to
pH 6.0 or with mono-
clonal anti-B.
4 + 4+ 2+ 0 0 2+ 0 AB with cold Perform antibody panel.
alloantibody Repeat forward grouping
with B cells lacking the
corresponding antigen.
*Other explanations may be possible.

IMMUNOHEMATOLOGY REVIEW 395

-- -
 -. -

Rh IIfrping Sera Continued

- - - -- - - -

High-Protein Chemically Modified Anti-D Monoclonal/

Anti-D Anti-D Pvlyclonal Blend Saline Anti-D
Comments High protein content Reacts in immediate IgM anti-D reacts with Lower incidence of
enables reagent to spin without high pro- D antigen in immediate false-positives with
react in immediate tein concentration. spin. IgG anti-D enables antibody coated RBCs
spin. False-positive Lower incidence of reagent to be used to
results if RBCs have false-positives with detect weak D. Lower
a positive DAT. antibody-coated RBCs. incidence of false-positives
with antibody-coated RBCs.

406 IMMUNOHEMATOLOGY REVIEW

- -- -- -
Frequency of Other Selected Blood
Group Antigens Continued
Antigen Whites (%) Blacks (%)

Note: Students do not need to memorize frequencies but should

know which antigens are common, which are uncommon, and
which show marked racial differences.

IMMUNOHEMATOLOGY REVIEW 409

- - -- . -
-
 - -

Antibody Characteristics
Naturally occurring ABO, Lewis, P,, MN, Lua
Clinically significant ABO, Rh, Kell, Duffy, Kidd, SsU
Warm antibodies Rh, Kell, Duffy, Kidd
Cold antibodies M, N, P,
Usually only react in AHG Kell, Duffy, Kidd
- - - - - - ~ ~

Can react in any phase of testing Lewis

Detection enhanced by enzyme treatment of test cells Rh, Lewis, Kidd, P,
Not detected with enzyme treatment of test cells M, N, Duffy
Enhanced by acidification M
Show dosage Rh other than D, MNS, Duffy, Kidd
Bind complement I, Kidd, Lewis
Cause in vitro hemolysis ABO, Lewis, Kidd, Vell, and some P,
Labile in vivo and in vitro Kidd
Common cause of anamnestic response (delayed transfusion reaction) Kidd
Associated with paroxysmal nocturnal hemoglobinuria Anti-P
Continued
 -- - -

Transfusion Reactions Continued

5ve Clinical Signs Cause Laboratow Findings Other
(aspirin, acetaminophen)
can be used to premed-
icate.
Allergic Hives (urticaria) Foreign plasma None Common. Treat with
proteins antihistamines. Transfu-
sion reaction investiga-
tion not required. Future
transfusions should be
with washed RBCs.
Anaphylactic Bronchospasms Anti-IgA in an IgA- None Rare. Transfuse with
reaction deficient recipient washed RBCs or blood
from an IgA-deficient
donor.
Circulatory Coughing, cyanosis, 'Itansfusion of too None Problem in children,
overload difficulty breathing large a volume cardiac and pulmonary
patients, elderly
Septicemia Fever, cramps, Contaminated blood Positive culture on unit Can be fatal
diarrhea, vomiting,
muscle pain, DIC,
shock, renal failure

424 IMMUNOHEMATOLOGY REVIEW

- - -
 Anticoagulants/Additives/
Rejuvenating Solutions Continued

Name Abbreviation Classification Shelf-Life Comments

Phosphate-inosine- PIPA Rejuvenating Used to salvage rare or type 0 units
pyruvate-adenine solution up to 3 days beyond expiration. Cells
are incubated with solution, then
washed. (Inosine might be toxic to re-
cipient.) Must be transfused within 24
hours or frozen.

434 IMMUNOHEMATOLOGY REVIEW

- -
- ___ _ _ - - --
 Blood Bank Quality Control Continued
Antisera Test with positive and negative controls each day of use. Use heterozygous cells for positive
controls.
~ - -

Reagent cells Check for hemolysis. Test daily with positive and negative controls.
Antihuman globulin Check anti-IgG activity by testing Rh-positive cells sensitized with anti-D.
QC records Retain 5 years or longer.

IMMUNOHEMATOLOGY REVIEW 445

-
 - -

Blood Components Continued

Storage
Component Preparation Tempemture Shelf-Life Indications Other
Platelets Centrifugation of 20-24°C 5 days with Prevent or stop Test at least 4 units/
whole blood agitation bleeding in month. 75% should
maintained at patient with contain 5.5 x 101°
room temperature thrombocytopenia platelets and pH 26.0.
within 8 hours or abnormal One unit should raise
of phlebotomy. platelet function. platelet count by
Enough plasma to Contraindicated 5,000-10,00O/pL in a
maintain pH at in thrombotic 75 kg recipient.
6.0 or higher. thrombocytopenic Shouldn't be sole
35 mL is mini- purpura (TTP), source of platelets
mum; 50-70 mL idiopathic if donor had aspirin
preferred. Can also thrombocytopenic within 3 days of dona-
be prepared by purpura (ITP). tion. Should not be
apheresis. used if visible aggre-
gates are present.
Granulocytes Cytapheresis 20-24°C 24 hours Neutropenia with 'Ransfuse as soon as
infection possible after collection.
Must be crossmatched
because RBCs are pres-
ent.

IMMUNOHEMATOLOGY REVIEW 441

 Blood Components Continued
Storage
Component Preparation Tempemture Shelf-Life indications Other
hernoglobinuria,
antibodies to
IgA, or history of
febrile reactions.
(Deglycerolized
RBCs are
preferred.)
---- - - -- -- - - - - - ~ -- --- - - - - - - - - - - - - - - - -

Leukocyte Best prepared by 1-6°C Closed system: Anemia with Must retain 80% of
reduced RBCs filtration same as RBCs. history of RBCs. To prevent febrile
Open system: febrile reactions reaction, no more than
24 hours. 5 x 106 leukocytes.
Rejuvenated Treated to restore 1-b°C 24 hours after Anemia Rejuvenating solution is
RBCs 2,3-DPG and ATP rejuvenation added 3 days after col-
up to 3 days after if not frozen lection to 3 days after
outdate expiration. Not for use
with cells in additive
solution.
RBCs, 1500-5000 rads 1-6°C Original outdate Intrauterine trans- For prevention of
irradiated prior to adrninis- or 28 days from fusions, immuno- graft-versus-host
tration irradiation, compromised disease. Renders
Continued

IMMUNOHEMATOLOGY REVIEW 439

.- - -
 -

Chemical Urinalysis by Reagent Strip Continued

Test Normal Principle Significance Sources O f E m r * Comments
rubin diglucuronide, high
levels of ascorbic acid or
nitrites, drugs causing
atypical colors. False-
positive: Urine pigments.
Urobili- 1 mg/dL or 1 Ehrlich's Liver disease, False-positive: Porphobili- Reagent strips do not
nogen Ehrlich unit reaction hemolysis nogen (with some brands detect absence of
@-dimethyl- of reagent strips) urobilinogen, only
aminobenzal- increase.
dehyde)
Nitrites Negative Greiss Urinary tract False-negative: Non- Test first AM
reaction infection nitrate-reducing bacteria, specimen.
insufficient dietary nitrate,
high levels of ascorbic acid,
some antibiotics, reduction
of nitrites to nitrogen, insuf-
ficient bladder incubation.
False-positive: Bacterial
contamination, medications
that color urine red.
*Sourcesof error may vary with brand of reagent strip. Refer to manufacturer's package insert.
Continued

URINALYSIS AND BODY FLUIDS REVIEW 453

 Confirmatory/Supplemental Urine Chemistry Tests
Substance[s)
Test Detected Principle Sources of Error Comments
- - ~ ~ ~ ~ - - - - -~- - -
~ ~~~~ ~~ ~ - - - ---

Suliosalicylic Protein Acid precipita- False-positive: Radiographic Detects all proteins, including
acid tion dyes, tolbutamide, some Bence Jones proteins
antibiotics, turbid urine.
False-negative: Highly
buffered alkaline urine.
Clinitest Reducing Copper False-positive: High levels of Nonspecific. Reacts with glu-
substances reduction ascorbic acid. False-negative: cose, galactose, fructose, pen-
Glycolysis, pass through. tose, maltose, lactose. (Sucrose
(Color goes through orange is not a reducing sugar.) Test
and returns to blue or blue- all infants to diagnose galac-
green. Repeat using two-drop tosemia. Not as sensitive for
method and two-drop color glucose as reagent strip. Self-
chart.) heating method. Perform in
rack to avoid burning.
Acetest Ketones Sodium nitro- False-negative: Improperly Most sensitive to acetoacetic
prusside reaction stored specimen acid
Ictotest Bilirubin Diazo reaction Decreased: Exposure to light, More sensitive than reagent
improperly stored specimen, strip. Less affected by
high levels of ascorbic acid, interfering substances.
nitrites. False-positive: Urine
pigments.
- - ~

Continued
URINALYSIS AND BODY FLUIDS REVIEW 455
Effect of High Levels of Ascorbic Acid
on Urinalysis Tests
False-Positive False-Negative or Decrease*
Clinitest Glucose
Blood
Bilirubin
Nitrite
Leukocyte esterase
'May vary with brand of reagent strip. Refer to manufacturer's
package insert.

URINALYSIS AND BODY FLUIDS REVIEW 457

- - - --- -- - --
 -

Cells in the Urine Sediment

Cell Description Origin Clinical Significance Comments
Squamous 40-50 pm. Flat. Lower urethra, Usually none. Improperly
epithelial cell Prominent round vagina collected clean-catch
nucleus. specimen.
Transitional 20-30 pm. Renal pelvis, ureters, Seldom significant May form syncytia
epithelial cell Spherical, pear- bladder, upper
shaped, or poly- urethra
hedral. Round
central nucleus.
Renal tubular Slightly larger than Renal tubules n b u l a r necrosis, toxins, Differentiate from polys by
epithelial cell a WBC. Round. viral infections, renal adding 2% acetic acid to
Eccentric round rejection visualize nucleus
nucleus.
Oval fat body Renal tubular Renal tubules Same as renal tubular Maltese crosses with polar-
epithelial cell con- epithelial cells ized light
taining fat droplets
White blood Usually polymor- Kidney, bladder, or Cystitis, pyelonephritis, 0-5/high-power field (HPF)
cell (WBC) phonuclear. Approxi- urethra tumors, renal calculi are normal. Clumps of
WBCs are associated
mately 12 pm. with acute infection.
Granular appearance.
Continued

458 URINALYSIS AND BODY FLUIDS REVIEW

- -- -
m e Description
Hyaline Homogenous with parallel
sides and rounded ends
Granular Same as hyaline, but
contain granules
RBC RBCs in cast matrix.
Yellowish to orange color.
Blood Contain hemoglobin.
Yellowish to orange color.
WBC Leukocytes incorporated
into cast matrix. Irregular
in shape.
Epithelial Renal tubular epithelial cells Renal tubular damage
cell incorporated into cast matrix
Significance Comments
0-2/low-power field Most common type. Least significant.
(LPF) are normal. Contain Tamm-Horsfall protein only. Dissolve
Increased with stress, in alkaline urine. Same refractive index as
fever, trauma, exercise, urine, so may be overlooked if light is too
renal disease. bright.
0-l/LPF is normal. May originate from disintegration of cellular
Increased with stress, casts
exercise, glomerulo-
nephritis, pyelonephritis.
Acute glomerulonephritis, Pinpoints source of bleeding in kidney. Most
strenuous exercise fragile of casts. Often in fragments.
Same as RBC cast From disintegration of RBC casts
Pyelonephritis Pinpoints kidneys as the site of infection
Transitional and squamous epithelial cell
casts do not exist. These cells are found dis-
tal to renal tubules and collecting ducts
where casts are formed.
Continued
URINALYSIS AND BODY FLUIDS REVIEW 463
- -
 Crystals Found in Acid or Neutral Urine
Crystal Description Significance Comments
Amorphous urates Irregular granules None Form pink precipitate in bottom of tube. May
obscure significant sediment. Dissolved by
warming to 60°C.
Uric acid Very pleomorphic. Four- Usually normal Birefringent. Polarizes light.
sided, six-sided, star-
shaped, rosettes, spears,
plates. Colorless, red-
brown, or yellow.
Calcium oxalate Octahedral (eight-sided) Normal. From Occasionally found in slightly alkaline urine.
envelope form is most oxalate-rich Monohydrate form may be mistaken for RBCs.
common. Also dumbbell foods. Most common constituent of renal calculi.
and ovoid forms.
Leucine Yellow, oily-looking Severe liver Often accompanied by tyrosine
spheres with radial and disease
concentric striations
Qrosine Fine yellow needles in Severe liver Often accompanied by leucine
sheaves or rosettes disease
Cystine Hexagonal (six-sided) Cystinuria Must be differentiated from uric acid. Does
not polarize light. Confirm by cyanide-nitro-
prusside test.
Continued

460 URINALYSIS AND BODY FLUIDS REVIEW

- - --- - -
 - -- - - - --

Crystals Found in Alkaline Urine

Crystal
Amorphous phosphates
Triple phosphate
Ammonium biurate
Calcium phosphate
- -
Description Significance Comments
Irregular granules None Form white precipitate in bottom of tube.
Dissolve with 2% acetic acid.
"Coffin-lid" crystal None
Yellow-brown "thorn None Seen in old specimens
apples" and spheres
Needles, rosettes, None Only needle form seen in alkaline urine
"pointing finger"
- - - -- - - - - - ---

Calcium carbonate Colorless dumbbells

or aggregates None

462 URINALYSIS AND BODY FLUIDS REVIEW

-
 - - - -

Miscellaneous Urine Sediment

Structure Description Significance Cornrnents
Bacteria Rods, cocci Urinary tract infection or Usually accompanied by WBCs
contaminant when clinically significant, unless
patient is neutropenic
Yeast 5-7 pm. Ovoid, colorless, Usually due to vaginal or fecal Differentiate from RBCs by adding
smooth and refractile. May contamination. May be due to 2% acetic acid. RBCs are lysed;
be budding. kidney infection. May be seen yeast are not. Presence of pseudo-
in urine of diabetic patients. hyphae indicates kidney infection.
Sperm 4-6 pm head with Usually not significant in an
40-60 pm tail adult. May be a sign of sexual
abuse in a child.
- - -

Trichomonas Resembles WBC. Rapid, Contaminant from genital tract Should not be reported unless
jerky, nondirectional motility. infection motile
Mucus Transparent, long, thin, Large amount seen with chronic May be mistaken for hyaline casts
ribbonlike structure with inflammation of urethra or
tapering ends bladder

URINALYSIS AND BODY FLUIDS REVIEW 465

L- -- -- -
 Renal Disorders
Disorder Cause Reagent Strip Sediment Other
Acute glomer- Inflammation and Protein, blood RBCs (some dysmorphic), Frequently follows a
ulonephritis damage to glomeruli WBCs, RBCs, and/or group A strep infection
hemoglobin casts
Nephrotic Increased glomerular Protein (large Casts (all kinds), free fat Hypoproteinemia,
syndrome permeability amount) and oval fat bodies hyperlipidemia
Pyelonephritis Infection of upper Protein, leukocyte WBCs, WBC casts,
urinary tract involving esterase, nitrite bacteria
interstitial tissue of
kidney
Cystitis Bladder infection Leukocyte esterase, WBCs, bacteria, possibly
nitrite RBCs. No casts.

466 URINALYSIS AND BODY FLUIDS REVIEW

- - - -
 Cerebrospinal Fluid
Normal Abnormalities Comments
Color Colorless Xanthochromia = slight pink, Examine for xanthochromia within 1 hour
orange, or yellow supernatant. of collection to avoid false-positives due to
Due to oxyhemoglobin or bili- lysis of RBCs. Centrifuge specimen and
rubin. Seen with subarachnoid compare to water.
hemorrhage. Red or pink, uni-
formly distributed in all tubes =
subarachnoid hemorrhage.
Red or pink, diminishing from
tube 1 through 3 = blood from
traumatic tap.
Clarity Clear Cloudy with infection or bleeding
WBC Adult: 0-5 lymphs/pL. Increased in meningitis Perform cell count in a hemacytometer
Newborn: 0-30 within 30 minutes of collection
monos/pL.
RBC 0 Increased with subarachnoid Uniform distribution with subarachnoid
hemorrhage or traumatic tap hemorrhage. Uneven distribution with
traumatic tap. (More in tube 1.)
Glucose 60-70% of blood Decreased in bacterial Blood glucose method
glucose meningitis
Continued

URINALYSIS AND BODY FLUIDS REVIEW 467

- -- -- - -- - - - --
 Meningitis
Bacterial Viral Mycobacterial Fungal
WBC Increased Increased Increased Increased
Differential Polys Lymphs Lymphs Lymphs
Protein Increased Increased Increased Increased
Glucose Decreased Normal Decreased Normal or decreased
Lactate Increased Normal Increased Increased
Other Weblike clot or pellicle

URINALYSIS AND BODY FLUIDS REVIEW 469

- - - - - - -- - -
 Body Fluids
Definition Other Terms
Effusion Abnormal accumulation of fluid in a body cavity.
Classified as transudate and exudate.
Serous fluid Fluid contained in pericardial, peritoneal, and
pleural cavities
Pericardial fluid Fluid surrounding heart Pericardiocentesis fluid
Peritoneal fluid Fluid in abdominal cavity Abdominal fluid, ascitic fluid
Pleural fluid Fluid surrounding lungs Chest fluid, thoracentesis fluid,
empyema fluid
Synovial fluid Fluid in joints Joint fluid

470 URINALYSIS AND BODY FLUIDS REVIEW

- --
 Transudates and Exudates
Ransudate Exudate
Etiology Systemic disorder that affects fluid Condition involving membranes within
filtration and reabsorption; e.g., con- the body cavity; e.g., infection, malig-
gestive heart failure, hypoalbuminemia, nancy, inflammation, hemorrhage.
cirrhosis. Problem originating outside
of the body cavity.
v p e of process Noninflammatory Inflammatory
Color Colorless Yellow, brown, red, green
Clarity Clear Cloudy
Specific gravity (1.015 >1.015
Protein t 3 g/dL > 3 g/dL
Fluidserum protein t0.5 >0.5
Spontaneous clotting No Yes
LD (lactate dehydrogenase) (200 U/L >ZOO U/L
F1uid:serum LD <O.G >0.6
WBC <1,OOO/pL >l,OOO/~L
Differential Predominantly mononuclears Predominantly polys

URINALYSIS AND BODY FLUIDS REVIEW 471

- -- --- --- -- - -
 Chemical Urinalysis by Reagent Strip Continued
Test Normal Principle Significance Sources Of Error* Comments
Leuko- Negative Granulocytic Urinary tract False-positive: Oxidizing Will detect intact and
esterase infection agents. Decreased reaction: lysed polys.
esterase reaction High glucose, protein, Lymphs do not
specific gravity, or react.
ascorbic acid.
Specific Random PKa change of Indication of kid- Increased: Protein. Measures ionizable
gravity specimen: polyelectrolyte ney's concentrat- Decreased: Alkaline urine. substances only. Not
1.003-1.030 ing ability and always the same as
state of hydration specific gravity by re-
fractometer.
'Sources of error may vary with brand of reagent strip. Refer to manufacturer's package insert.

454 URINALYSIS AND BODY FLUIDS REVIEW

 - -

Cells in Body Fluids

- -

Fluid Normal Cells Abnormal Cells

Cerebrospinal fluid Lymphocytes RBCs
Monocytes Neutrophils
Ependymal cells Eosinophils
Choroid plexus cells Basophils
Siderophages
Blasts/malignant cells
(Nucleated RBCs and cartilage cells may be present from tap)
Serous fluid (peritoneal, Lymphocytes RBCs
pericardial, pleural) Monocytes/histiocytes Neutrophils
Mesothelial cells Eosinophils
Basophils
Lupus erythematosus (LE) cells
Malignant cells
Synovial fluid Lymphs RBCs
Monocytes/histiocytes Neutrophils
Synovial cells LE cells
RA cells (ragocytes)
Malignant cells (rare)

472 URINALYSIS AND BODY FLUIDS REVIEW

-- - -- -- - - -
 Confirmatory/Supplemental Urine
Chemistry Tests Continued
Substance(s)
=st Detected Principle Sources o f E m r Comments
Watson- Urobilinogen, Ehrlich's alde- Decreased: Exposure to light, Collect specimen from 2-4 PM.
Schwartz porphobilinogen hyde reaction more than 1 hour at room Store in dark. Urobilinogen is
Test temperature. False-positive: soluble in chloroform and
Warm aldehyde reaction. butanol. Porphobilinogen is
(Urine should be at room not soluble in either.
temperature.)
Hoesch Test Porphobilinogen Ehrlich's alde- Similar to Watson-Schwartz Urobilinogen doesn't react
hyde reaction unless very high.

456 URINALYSIS AND BODY FLUIDS REVIEW

 Body Fluid Cells
Cell Significance Size Cytoplasm Nucleus Comments
Mesothelial Normal in serous Variable. Abundant. Oval. About one-half "Fried egg" appear-
fluids. From lining 12-20 pm Basophilic. cell diameter. May be ance. May be in
of pericardial, May have eccentric. Dark purple. singles, pairs, or
pleural, and peri- vacuoles Stippled. May have sheets. Homogenous
toneal cavities. and frayed nucleoli and peri- cells of uniform size.
Synovial Normal in syno- borders. nuclear clear zone. Each retains its
vial fluid. From May be multinu- individuality. Slitlike
synovial membrane. cleated (similar size opening (window)
and shape). between cells.
Histiocyte Found in normal 15-30 pm Abundant. Round or irregular. Abnormal forms:
(Macrophage) fluids Colorless. Eccentric. Delicate erythrophage (contains
May chromatin. RBCs), siderophage
have many (contains siderotic
vacuoles granules), signet ring
and ingested (large lipid vacuole
matter. that pushes nucleus
to the edge)
Malignant Wmor Great Scant Often multiple with Bizarre appearance.
cells variation variation in size. Irregular in shape.
Hyperchromatism. Tendency to fuse.
Nucleoli. May have Cells lose individuality.
mitotic figures. No window between
cells. Nuclear molding.
3D appearance.

URINALYSIS AND BODY FLUIDS REVIEW 473

 Cells in the Urine Sediment Continued
Cell Description Origin Clinical Significance Comments
Glitter cell WBC with Brownian Same as WBC Same as WBC Seen in hypotonic urine
movement of
granules. Stain
faintly or not at all.
Red blood Biconcave disk, Kidney, bladder, Infection, trauma, Crenated in hypertonic
cell (RBC) approximately 7 km. or urethra tumors, renal calculi. urine. Lyse in hypotonic
Smooth. Non- Dysmorphic RBCs urine and with 2 %
nucleated. indicate glomerular acetic acid.
bleeding.

URINALYSlS AND BODY FLUIDS REVIEW 459