PMLS 1

Lesson 8 Nature of the Clinical

Laboratory
 Intended Learning Outcomes
At the end of the lesson, the student should be able
to:
1. Discuss the different sections of the laboratory
and the tests done in each
2. Compare and contrast the different types of
clinical laboratories
3. Identify the salient points of the laws governing
the establishment, operation and maintenance
of clinical lab in the Phil.
4. Discuss the importance of quality assurance in
the clinical lab
 The Clinical Laboratory
• The clinical lab is an essential component of health
institutions. Its main task is to provide accurate and reliable
information to medical doctors for the diagnosis, prognosis,
treatment, and management of diseases.
• Seventy percent of all decisions performed by medical
doctors are based on laboratory test results., thus the need
for accurate and reliable test results.
• The clinical laboratory is the place where specimens (e.g.,
blood and other body fluids, tissues, feces, hair, nails)
collected from patients are processed, analyzed,preserved,
and properly disposed.
• Clin lab vary acc.to size, function,and complexicity of the
tests performed.
 Classification of Clinical Laboratories
According to function:
1. Clinical Pathology is a clinical lab that focuses on the
areas of clinical chemistry, immunohematology, and
blood banking, medical microbiology, immunology and
serology, hematology, parasitology, clinical microscopy,
toxicology, therapeutic drug monitoring, and
endocrinology.
2. Anatomic Pathology is a clin lab that focuses on the
areas of histopathology, immunohistopathology, cytology,
autopsy, and forensic pathology among others.it is
connected with the diagnosis of diseases through
microscopic exam of tissues and organs.
 Classification of clinical laboratories
According to Institutional characteristics
1. An institution- based is a clin lab that operates
within the premises or part of an institution
such as a hospital, school, medical clinic,
medical facility for overseas workers and
seafarers, birthing homes, psychiatric facility,
drug rehabilitation center, and others.
2. Free-standing clin lab is not part of an
established institution. The most common
example is a free-standing out-patient clinical
lab
 Classification of clinical laboratories
According to ownership
1. Government –owned clin lab are owned , wholly or
partially, by national or local govt units.Ex are the clin and
anatomical lab of DOH- run govt hospitals like San Lazaro
Hospital,Jose R.Reyes Memorial Medical Center, UP-Phil
General Hospital, and local-govt – run hospital-based
clinical lab of Ospital ng Maynila Medical Center, Sta. Ana
Hosp, and Bulacan Medical Center.
2. Privately-owned clinical lab are owned, established, and
operated by an individual, corporation, institution,
association, or organization. Ex. Are St. Luke’s Medical
Center, Makati Medical Center, and MCU-FDTMF Hosp.
 Classification of clinical laboratories
According to Service Capability
1. Primary category are licensed to perform basic,
routine lab testing, namely routine urinalysis,
routine stool exam, routine hematology, or CBC
that includes hemoglobin,hematocrit, WBC, and
RBC count, WBC differential count, and
qualitative platelet count, blood typing, and
Gram staining (if hospital-based). Equipt
requirements are, but not limited
to,microscopes centrifuge, hematocrit
centrifuge.
2. Secondary category (hospital and non-hospital-
based) are licensed to perform lab tests being done
by primary category clin lan along with routine
clinical chemistry tests like blood glucose
concentration, blood urea nitrogen, blood uric acid,
blood creatinine, cholesterol, determination
qualitative platelet count, and if hospital-based,
Gram stain, KOH mount, and crossmatching.
A minimum requirement of 20 sq. m. is needed
for the floor area . Personnel requirement depends
on the workload.
Minimum equipts are microscopes, centrifuge,
Hematocrrit centrifuge, semi-automated chemistry
analyzers, autoclave, incubator, and oven.
3.Clinical Lab under the tertiary category (hospital
and non-hospital-based) are licensed to perform all
the lab tests performed in the secondary category
lab plus :
(1)Immunology and serology (e.g., NSA-Ag for
dengue, rapid plasma reagin, (2)Treponema
pallidum particle agglutination tests);
(2) microbiology, bacteriology and mycology (e.g.,
differential staining techniques,culture and I.D. of
bacteria and fungi from specimens antimicrobial
susceptibility testing;
(3) special clinical chemistry (e.g., clinical
enzymology, therapeutic drug monitoring, markers
for certain diseases)
(4) Special hematology (e.g., bone marrow
studies, special staining for abnormal blood
cells, red cell morphology); and
(5) Immunohematology and blood banking (e.g.,
blood donation program, antibody screening
and I.D., preparation of blood components.
Tertiary lab have a minimum floor area
requirement of at least 60 sq.m.
Equipment requirements include those seen in
2ndary category lab along with automated
chemistry analyzer, BSC class II, serofuge, among
others
4. National Reference Lab is a lab in a govt
hospital designated by DOH to provide special
diagnostic functions and services for certain
diseases.
These functions include referral services,
provision of confirmatory testing, assistance for
research activities, implementation of External
Quality Assurance Program (EQAP) of the govt,
resolution of conflicts regarding test results of
different lab, and training of medical
technologist on certain specialized procedures
that require standardization.
Laws on the Operation, Maintenance,
and Registration of Clinical Lab in the
Philippines.
• READ ON THESE.
 Sections of the Clinical Laboratory
• A clin lab is made up of different sections
cohesively and comprehensively performing
different activities and procedures for each
specimen collected from patients to produce
reliable test results.
• At the forefront of these activities are the clinical
lab personnel, namely the pathologist, medical
technologists/clinical lab scientists, medical
technicians, phlebotomists, and other lab
personnel.
 Introduction to Microbiology

•Microbiology is the study of living

organisms (bacteria, virus, fungi, parasite) of
microscopic size. Anton van Leeuwenhoek
was the one who first observed microbes.
•Clinical microbiology deals with the
isolation and identification of bacterial
pathogens causing disease, so physicians can
treat the patients.
 Microbiology
• This section is subdivided into four sections:
bacteriology, mycobacteriology, mycology, and
virology.
• At present, the work in this section is more
focused on the I.D. of bacteria and fungi on
specimen received.
• Specimens usually submitted are blood and
other body fluids, stool, tissues, and swabs
from different sites of the body.
 Parasites

Leishmania species

Plasmodium falciparum
Fungi
 Identification of microbes
• Morphology
• Gram stain reaction
• Results of certain biochemical reactions
• Grow bacteria in appropriate culture media
• serology
Gram’s Stain
Morphology
Culture Media
 Specimens for microbiological exam
When a patient has a particular disease
symptoms and a microbiological infection is a
suspect, proper specimen should be
submitted to the lab.
• Ex. Sore throat – throat swab is done
• Possible kidney or urinary tract infection-
urinalysis is done and also urine culture
• Gastroenteritis –will require stool specimens
 Specimens to collect
• Most frequently cultured sites
-Throat using sterile cotton swab or
polyester-tipped swab
-genito-urinary tract -clean catch urine
• Next frequently performed culture
- wounds
- sputum
- culture suspected of fungal infection
 Throat Cultures
• Done primarily to differentiate the Lancefield group A
beta-hemolytic streptococcal ( Streptococcus
pyogenes) sore throat pharyngitis from a viral throat
infection.
• Sore throat caused by group A must identified and
treated because, sequelae can occur in some patients
and can lead to scarlet fever or acute rheumatic fever ,
followed by a chronic rheumatic disease.
• Acute glumerulonephritis can also follow an untreated
group A streptococcal throat infection in some patients
 Throat
• Gently swabbing the throat and surfaces
• Usual culture medium used- blood agar plate
• Material on the swab should be cultured right
away but if not possible, culturette is used-
sterile swab in a plastic tube with an ampule
of modified Stuart’s transport medium.
• Usual cause of strep throat- Strep pyogenes
Throat swab

Blood agar plate

Stuart’s transport medium

 Culture Medium
• Nutritive substance where organisms are
grown
• Inoculating loop is used
• Multiple interrupted streak is used to get
isolated colonies
Dilution streak technique
 Antibiotic Sensitivity Testing
• 0nce the organism causing the infection has
been identified, antibiotic sensitivity must be
determined.
• Two principal methods are employed to
determine antimicrobial susceptibility testing:
- agar-disk diffusion or Kirby-Bauer Method
- dilution testing
 Disk-diffusion method
• Turbidity of the bacterial inoculum should be
compared with a standard that represents a
known no. of bacterial suspension.
• The standard is the 0.5 McFarland which
contains 99.5 ml of 1% v/v sulfuric acid and o.5
mL 0f 1.175% barium chloride to obtain a barium
sulfate suspension with a specific optical density.
• This provides a turbidity comparable to that of a
bacterial suspension containing approx. 1.5 x
10⁸CFU/mL.
 Microdilution method
• This method permits a quantitative result to
be reported (MIC) indicating the amount of
drug needed to inhibit the microorganism
being tested.
• MIC or minimum inhibitory concentration is
the lowest concentration of an antibiotic that
will visibly inhibit the growth of the organism
being tested.
 Antibiotic Sensitivity Test

MIC- minimum inhibitory concentration

 Urine
• Collection of urine requires the cooperation of
the patient
• morning urine before taking any fluid is collected
• For routine urinalysis – urine must be collected in
a clean, dry container, midstream sample is
collected
• If urine culture is requested a clean-catch
midstream sample is collected in a sterile
container
• Volume of urine should at least be 12 mL.
 Urine Culture
• Done to diagnose bacterial infections of the urinary tract (
bladder, ureter, kidney, and urethra)
• UTIs are of two types:
- lower UTIs of bladder or urethra such as cystitis,
infection of the bladder
- upper UTIs of the ureters and kidneys, such as
pylonephritis, an infection of the renal parenchyma or
kidney
• Urine culture is done by using one selective culture
medium and one nonselective culture medium
• BAP- non selective
• MacConkey Agar = selective
 Urine Culture
• Before collection, proper cleaning of the collection site
especially for females, is very important.
• Clean-catch midstream urine is necessary.
• If a patient cannot urinate, a catheterized specimen
can be obtained. Urine from the tubing (not the bag) is
sent to the lab and not the whole catheter.
• Urine is sent immediately to the lab or if not cultured
immediately, must be refrigerated to prevent bacterial
growth.
• Urine is normally sterile within the urinary bladder but
is usually contaminated during the process of
collection.
 Urine Culture
• Quantitative urine culture is required to
differentiate true infections from
contamination.
• Presence of bacteria in a clean-catch culture
does not necessarily mean a UTI unless the
no. of organisms is significant.
• In many UTIs , laboratory findings show
presence of 100,000 (10₅) or more colony
forming units (CFU)per mL of urine
Method of streaking
 Blood Culture
• Venipuncture site must be cleaned before
puncture to avoid possible contamination.
• One process is to apply 70% alcohol solution
to remove dirt and lipids.
• A circular motion moving from the site out . A
1% to 10% povidone –iodine is used followed
by an alcohol rinse.
• Blood is usually cultured on BHI with SPS
 Blood Culture
• Blood is usually sterile and no microorganisms
should be present.
• Infections involving the blood stream are serious.
• Bacteremia (bacteria in the blood) can have
serious consequences for the patient.
• Septicemia or sepsis indicates a situation in
which the bacteria in the blood or a toxin
produced by the bacteria is causing harm to the
host.
• Fungemia , the presence of fungi in the blood ,
can be found in immunosupressed patients.
• When a patient is septic, the bacteria can release
exotoxins or endotoxins. Particularly in the case
of endotoxins, septic shock can occur
• Symptoms of septic shock: chills, lowered blood
pressure (hypotension), respiratory distress, and
disseminated intravascular coagulation (DIC)
• DIC is a serious complication of septic shock in
which clotting factors are a high rate.
• Once these clotting factors are depleted,
excessive bleeding can occur.
Portals of entry for organisms that can
cause bacteremia
• Genitourinary tract – 25%
• Respiratory tract ------20%
• Abscesses --------------10%
• Surgical awound infections- 5%
• Biliary tract ------------5%
• Miscellaneous sites—5%
• Uncertain sites --------25%
 Culture media for blood
• Usually liquid broth like trypticase soy broth,
brain heart infusion broth, peptone supplement
or thioglycolate broth
• In addition, an anticoagulant, 0.025% to 0.05%
sodium polyanethol sulfonate (SPS) is added.
• A blood/medium ratio of 1:5 or 1:10 is adequate
for most laboratories
• After 6-18 hours of incubation at 35◦C, blind
cultures are done on (CHOC. SBA, MAC)
• Blood cultures are examined visually at least once
a day for 7 days.
Anaerobic glass jar
 Automated blood culture
Advantages:
• More rapid detection time for many pathogens
• Monitoring of growth without visual inspection
or subculture of the subcultured bottles
• Ability to use the report in conjunction with a
computerized laboratory management
information system.
• Ex. BD diagnostic System
 BD Diagnostic System
• Bottles are incubated in the incubator chamber
where constant rocking occurs to enhance
growth.
• In the chamber is a detector of CO₂ produced by
bacterial metabolism.
• Gas-permeable sensors use fluorescence to
detect CO₂.
• The system is alarmed to make personnel aware
of positive samples
• Blood culture bottles in then are subcultured and
gram stained.
BD diagnostic system
 Stool Specimen
• Contains large numbers of bacteria (normal flora)
and a stool specimen is usually cultured only to
isolate certain types of pathogenic enteric
organisms.
• Stool specimens should be cultured within 2
hours. If this cannot be done transport media
special for stool samples can be used.
• Stool can also be used to identify certain
parasites.
• Stool specimens are usually cultured to detect
enteric pathogens.
 Cerebrospinal fluid
• A physician collects CSF through a lumbar
puncture.
• Rapid handling of CSF in the lab is extremely
impt. because of the serious nature of meningitis
and organisms recovered in meningitis are
sensitive to temperature change , so refrigeration
is not done.
• CSF is typically placed in sterile container.
• CSF is sent to the lab immediately for testing
including culture and gram stain
 Respiratory
• Sputum is usually collected in the morning
• Deep coughing will usually bring a good
sputum specimen.
• A good specimen has lower than 10 squamous
epithelial cells when gram stained.
• Other respiratory specimens includes
bronchial washings, lavages, and brushings,
which are collected by physicians in a
procedure called bronchoscopy.
 Swabs
• Are used to collect cultures from various
openings of the body, such as the nose,
throat, mouth, vagina, anus, and wounds.
• These swabs must be collected carefully and
placed in the proper transport media before
they are taken to the lab.
• If swabs are properly handled, the
microorganisms may dry out or their numbers
may be insufficient for culture.
 Genito-urinary cultures
• Done primarily to determine the cause of
urethritis, vaginitis, and cervicitis.
• Orgnisms recovered from these sources are often
sexually transmitted: Neisseria gonorrheae and
Chlamydia trachomatis.
• These infections can cause pelvic inflammatory
disease (PID) leading to infertility
• Chlamydial infections have surpassed gonorrheal
infections as the most prevalent STD in the US.
 Gram stain for N. gonorrhea
• Gram-stained smears from urethral discharge
are examined for the presence of
polymorphonuclear cells (PMNs) and gram-
neg. intracellular diplococci within the PMNs
in men only.
• Vaginal flora contaminates smears in women,
and if done, the positive smear is only
presumptive evidence of gonorrhea.
Neisseria gonorrheae
 N. Gonorrheae Culture
• Modified Thayer Medium a selective culture
medium incubated in a CO₂ atmosphere are
required for optimal recovery from clinical
specimens
• Antibiotics like vancomycin –against gram pos
• Colistin- against gram neg.
• Nystatin – against fungal contaminants
 Chlamydial Infections
• Nucleic acid testing is used
• For females, swab is inserted in endocervical
canal and rotated for 10 to 30 secs. and placed
in the transport tube
• For males, the swab is inserted into the
urethra 2 to 4 cm. using a rotating motion,
then placed in the transport tubes.
• NA amplification method is more sensitive
than culture method.
 Genito-urinary cultures
• Other organisms that can cause vaginitis are
Gardnerella vaginitis and Trichomonas vaginalis
• T. vaginalis an STD can be seen using wet mount
of vaginal secretions
• G. vaginalis are short, gram neg. bacilli or
coccobacilli. When the exfoliated vaginal
epithelial cells are covered with tiny gram-
variable bacilli and coccobacilli, they arre known
as clue cells
T. vaginalis and G. vaginalis
 Test for fungi (mycology)
• The study of fungi (yeast and mold)
• Patients who are immunocompressed like
persons with HIV/AIDS, transplantation (solid
organ or bone marrow) patients requiring
immunosuppression, are prone to develop
opportunistic fungal infections.
 Fungi as a source of infection
• Fungi normally live a nonpathogenic existence in
nature, enriched by decaying nitrogenous material.
• Humans become infected through accidental exposure
by inhalation of spores or by introduction into tissue
through trauma.
• Any alteration in the immunologic status of the host
can result in infection by fungi that are normally
nonpathogenic; most yeast infections are opportunistic
• Most frequently isolated yeast is Candida albicans
which can be part of the normal flora of the GIT and
mouth in healthy persons.
 Fungal infections or mycoses
• Superficial mycoses – confined to the
outermost layer of skin and hair with
symptoms of discoloration, scaling or
abnormal skin pigmentation.
• Cutaneous mycoses – affect the keratinized
layer of the skin, hair, or nails. Symptoms of
these infections include itching, scaling or
ringlike patches (ringworm) of the skin; brittle
or broken hairs; and thick discolored nails.
 Fungal infections or mycoses
• Subcutaneous infections affect the deeper layers
of the skin, including muscle and connective
tissue; these infections do not usually
disseminate to the blood to the different organs.
• Systemic mycoses affect the lungs and can
disseminate to internal organs of the deep tissues
of the body.
• Opportunistic mycoses- ordinarily, opportunistic
fungi do not cause any disease, but given a
chance, they can infect people.
Superficial mycoses
Cutaneous mycoses
Subcutaneous mycoses
 Methods of detection of fungi
DIRECT MICROSCOPIC
Grams stain- yeast will appear gram pos.
(purple or blue) and will often show KOH mount- Recommended
budding Preparation for detecting fungal
Elements in skin, hair, nails and
tissues.
India ink prep. Acid fast stain
Acid fast stain
Methods of detection of fungi
 Collection of specimens
• Stool
• Urogenital tract – as vaginal or urethral
discharge, or as prostatic secretion
• Sputum
• CSF
• Biopsy material from other tissues
• blood
 Stool specimen
• A single stool specimen may not be sufficient
because parasites shed eggs or cysts on a
variable schedule
• Recommended protocol is to collect three stool
samples 1 or 2 days apart, but all within a 10 day
period
• Collection of stool samples should always be
done before a radiologic study using barium
sulfate
• Certain medications can also affect the detection
of parasites.
 Stool collection
• A clean, dry, water proof container with a
tight-fitting lid
• Contaminating the stool with urine should be
avoided
• Sample should be sent to the lab as soon as
possible
 Blood specimen
• Thick and thin blood smears are made to allow
for better detection of parasites found in the
blood, such as Plasmodium spp. (malaria).
• Thick smear is used to screen a larger volume of
blood; thin smear is used to identify the parasite
because the parasites are not as distorted as with
the thick smear.
• Giemsa hematologic stain is used to detect blood
parasites
Methods for detection of parasites
Wet mount, direct smear
• A smear is prepared by mixing a small amount
of sample with a drop of physiologic saline on
a glass slide with a coverslip.
• This is repeated using iodine, and both slides
are examined trophozoites, helminth ova
(eggs) larvae, and cysts. Motility is also
observed
 Common parasites identified
• T. vaginalis – motile trophozoites can be
identified only on fresh urine specimen or
fresh genital secretions from both sexes.
• It appears pear shaped, elongated form and
moves in jerky, undulating motion
• T. vaginalis infection is considered an STD .
• Cause of vaginitis, urethritis. and prostatitis
•
 Common parasites identified
• Many parasitic infections are diagnosed
through identification of their eggs or larvae in
a fecal sample
• Stool specimens for ova and parasitic studies
are preserved in formalin or polyvinyl alcohol
(PVA)
 Macroscopic exam
• Observe for consistency of the specimen:
- soft or liquid specimen trophozoite stages are
more likely to be found
- formed stool will more likely yield cysts
- occasionally, adult helminths can be seen on the
surface of the stool
• Presence of blood should be noted; dark feces
may indicate bleeding high in the GIT, whereas
bright red feces indicates bleeding at a lower
level
 Microscopic exam
• Direct wet mounts of the specimen are
observed to detect helminth eggs and motile
trophozoite stages of protozoan
• Identification of of intestinal protozoa and
helminth egg is based on recognition of
specific characteristics.
Pictures of some parasitic ova
 Blood and tissue parasites
• Malaria is one of the most common infectious
disease WW.
• Diagnosis depends upon symptoms
(headache, fever, chills, sweats, nausea) and
identification of the Plasmodium spp.in the RBC.
• Lung tissue or secretions can be infected with
Pneumocystis jiroveci (formerly P. carinii), which
is symptomatic only in immunosuppressed
individuals like in patients with HIV .
Plasmodium falciparum in RBC
Blood bank/Immunohematology

• ABO blood Group - discovered by

Landsteiner and his students.
- consists of four phenotypes: A, B, AB,
and O. These phenotype can be explained
on the presence of two antigens on the
surface of the red blood cells.
• Isoantibodies of ABO System- These
isoantibodies are not manifested in infants
until three to six months of age.
• Rh Blood Group Cell System- This is more
complicated than the ABO blood group system
because it consists of six related red blood cell
antigens (C.D, E, c, d, e). CDE is the most
common and often used.
• Antihuman Globulin Reaction- This reaction
takes place when the antiglobulin antibody reacts
with any antibody coated on the RBC.
Because it is an IgM type,it will react with any
antibody coating adjacent to the RBC, If this
antibody is finally detected, it will cause severe
transfusion reaction.
• Compatibility testing and Cross Matching. The
effective way to ensure safe blood transfusion is
to perform blood typing including Rh typing and
cross matching. Major cross matching is involved
in testing the donor’s red cells and the patient’s
serum
Cross matching
• Blood Transfusion- Method of therapeutic
replacement of blood or one of its components which
outweighs the risk of contracting an infectious
disease.Blood transfusion reactions are characterized
as immune ( hemolytic,febrile,or graft versus host
transfusion injury) and none immune (circulatory
overload, iron overload, and shock
• Blood components and Derivatives of blood
transfusion. Whole blood is not used in blood
transfusion. Depending on the condition of the patient,
only components, (products obtained from whole
blood through centrifugation), blood derivatives or
fractions (products derived by complex separation
from whole blood) are transfused.
 Blood components
• Packed red cells - (by centrifugation). Used when
the oxygen-carrying capacity of hemoglobin is
diminished or lost such as in anemia;
• Plasma which is obtained by removing the clotted
blood is appropriately used to replace
coagulation factors
• Platelets are usually used in case of deceasing
platelet counts or bleeding tendency due to
abnormal platelet functions.
BLOOD COMPONENTS
 Hematology and Coagulation Studies
• This section deals with the enumeration of cells
in the blood and other body fluids (e.g., CSF,
pleural fluid, etc.)
• Exams done in this section include complete
blood count, (CBC), hemoglobin, hematocrit,
WBC differential count, blood smear preparation,
and staining for other body fluids.
• Coagulation studies focus on blood testing for the
determination of various coagulation factors.
 Introduction to Hematology
• Hematology is the branch of medicine
concerned in the study of formed elements of
blood and blood-forming tissues
• Composed of two sections:
a. routine examination
b. special examination
• Routine exam includes complete blood count,
hemoglobin count, and hematocrit
determinations
• Special exam includes reticulocyte, platelet
counts, blood indexes, and coagulation tests.
 Importance of the hematology lab
• Performs test to detect and monitor treatment of
anemias, leukemias, and inherited blood disorders such
as hemophilia
• The effects of certain treatments such as cancer therapy
can be determined by hematological tests
• Hematological tests can give information about a
patient’s general well-being
• Also aids in the dx and management of diseases that
originate from other systems
 Introduction to Hematology
• Blood is composed of formed elements
suspended in a fluid called plasma
• Formed elements : rbc, leukocytes (wbc)
thrombocytes (platelets), produced and matured
in the bone marrow
• Released in the blood stream to:
- transport oxygen
- blood clotting
- providing immunity
 Introduction to Hematology
• Plasma-consists of
- 90% water
- the remainder is dissolved solids such
as proteins (including antibodies,
albumin, and coagulation factors)
 Introduction to Hematology
• How to study the formed elements
- counting the cells as in RBC count,
WBC count
- stained blood smear to determine % of
cell types, structures and form
 Formed elements
• Erythrocytes
- most numerous
- gives blood its color
- live an average of 120 days
- function- transport oxygen from the lungs to
the tissues and carbon
dioxide from the tissues to the lungs (Hb) , the
major component of red blood cells
 Formed elements
• Leukocytes
- least numerous of the blood cells
- each microliter of blood contains
5,000 to 10,000 leukocytes
- composed of granulocytes:
neutrophils, basophils, eosinophils
- agranulocytes:
lymphocytes and monocytes
 The Leukocytes
• (WBCs) and platelets
(thrombocytes) (~1%)
Leukocytes are involved in the
body’s defense against the invasion
of foreign antigens.
 Formed elements
• Platelets
- not actually whole cells but fragments of
cytoplasm that have been released into the
circulating blood from large cells in the bone
marrow
- they act on the vessel walls to form a plug to
stop bleeding
- they also chemicals that are important in other
stages of hemostasis
- present in hundreds of thousands per
microliter of blood
PACKED CELL
VOLUME OR
HEMATOCRIT
 How to collect blood sample
• By puncture exposing the capillaries
- middle or ring finger
- lateral or side portion of the heel -
• Venipuncture-
• Anticoagulated blood is used for most
hematological tests
Most frequently requested tests
• CBC ( complete blood count)
- red blood cell count (RBC count)
- white blood cell count (WBC count)
- hemoglobin (Hb)
- Hematocrit (Hct)
- differential count
- platelet count
- observation of blood cell morphology
 Special hematological tests
• Many tests other than CBC are performed such
as reticulocyte count, sedimentation rate, and
basic coagulation tests.
• Hematological diseases :
1. leukemia- out of control production of WBC
but WBC have not matured enough and cannot
provide immunity. The patient may be highly
susceptible to infection even though the
leukocyte is high.
 Hematological diseases :
2. Anemia – decreased red cell production
due to iron deficiency. In this condition, red
cells do not function properly because they
do not contain enough hemoglobin . This
causes symptoms such as fatigue, pallor,
and shortness of breath due to decreased
oxygen to tissues.
 Inherited diseases
• Hemophilia – one of the factors in blood
clotting is missing or defective
• Abnormal hemoglobin function – Ex.
Sickle cell anemia due to the abnormal
structure of the hemoglobin
Inherited diseases
 Secondary or acquired hematological
diseases
• Abnormal- appearing red cells in patient
with severe or renal failure because cells
may be damaged as they circulate through
small blood vessels
• Diabetes may have lazy leukocytes
causing slow healing of wounds or
infections because some of the WBC
function improperly
• Lymphocytes develop an atypical appearance
in infectious mononucleosis
• Blood cells may also be affected by medication.
Ex. Aspirin may cause abnormal platelet
function. But this is temporary and will be
corrected if the drug is stopped
• Cancer chemotherapy treatments are
designed to stop the growth of cancer cells.
However, these drugs are not selective and may
also stop production of blood cells
RED BLOOD CELL COUNT

HEMACYTOMETER
 Pipettes used in counting

The red cell diluting

The white cell
pipette. The 0.5 mark on
diluting fluid. The
the long stem is the mark
0.5 mark is the mark
to which blood is drawn.
to which blood is
The 101 mark on the short
drawn. The 11 mark
stem is the mark to which
is the mark to which
the diluting fluid is drawn.
diluting fluid mixed
The red bead identifies an
with blood. The white
or clear bead is for the RBC pipet
WBC count

WBC RBC
 Computation of RBC
• Cells/cu.mm = Avg.x D(mm) x DF
» A (mm2)
» = compute the ave
» Ex. Side 1= 480 (5 squares)
» Side 2= 520 ( 5 squares)
1000/2= 500
» RBC = 500 x 10mm x 200
» 0.2 mm2
» RBC = 500 x10,000 or 5,000,000/cu.mm.
 Computation of WBC
• Cells/cu.mm.= Avg. x D (mm) xDF
A (mm2)
= Ave. # cells x 10 x 20
4mm2
= ave. # of cells x 50
Compute the ave.
Side 1= 115 (4 big squares)
Side 2 = 125 (4 big squares)
Ave. 240/2 = 120
WBC/cu. mm. = 120 x 50 mm2
= 6,000,000/cu.mm.
ONE SIDE OF THE COUNTING CHAMBER
RED CELL COUNT
Hematocrit(Packed cell Volume)
• A macroscopic observation of volume of
the packed RBC in a sample of blood
• Hct is used to evaluate and classify the
various types of anemias according to cell
inidices
• When whole blood is centrifuged , the
heavier particles fall to the bottom of the
tube and the lighter particles settle on top
 Microhematocrit (EDTA)
• Separation of plasma and cellular
elements
• Reflects the red cell volume relative to a
given volume of blood
• Low hematocrit can indicate anemia or
presence of bleeding in the patient.
 Test for hemoglobin

• Specific gravity technique- a drop of blood is

placed on CuSo4 solution with a particular
density.
• Cyanmethemoglobin – blood + Drabkin’s
Reagent (with ion, cyanide and sod.
Bicarbonate) internationally test of choice.
• Manual method - add 0.02 ml of blood in a
Sahli-Helige pipette + 4.98 ml of Dabkin’s
Reagent. Absorbance is read in a spectrometer
• Automated
 Differential Count
• May be used to help diagnose the cause
of high or low WBC.
• Used to help diagnose and/or monitor
other diseases and conditions that affect 1
or more different types of WBC
• Information is useful to help diagnose
specific cause of an illness such as:
• Infection caused by bacteria, virus, fungi,
parasite.
• Inflammation
• Allergies, asthma
• Immune disorder
• Leukemia
• Myelodisplastic syndrome
• Myelopreliferative neoplasm
Preparation of blood smear
 Differential Count
• Neutrophil (seg) – 50-65%
• (Band) - 0-7%
• Eosinophil - 1-3%
• Basophil - 0-1%
• Monocyte - 3-9%
• Lymphocyte - 25-40%
 Erythrocyte sedimentation rate
• ESR is a simple frequently performed
hematology test. It is not specific for a
particular disease but is used as an
indicator of inflammation or tissue
injury.
• It may also be used to evaluate treatment
and follow the course of certain
inflammatory diseases.
 How to perform ESR
• A sample of anticoagulated blood is placed
in a calibrated tube of standard dimension,
and incubated in a vertical position for
exactly one hour. At the end of one hour,
the distance the RBC have fallen from the
plasma meniscus (a the zero mark) is
measured in millimeters
 Conditions in which ESR may be
increased
• Pregnancy increased plasma fibrinogen
• Anemia increased plasma globulins
• Macrocytosis
• Inflammatory diseases
• Cancer
• Acute and chronic infections
• Multiple myeloma
 Conditions in w/c ESR may be decreased

• Presence of sickle cells

• Spherocytosis
• Increased plasma viscosity
• microcytosis
Hemostasis and Coagulation lab.
• Usually included in the hematology section
• Asssess bleeding and clotting problems
• Coagulation lab tests prothrombin (PT)
and activated partial thrombin time (APTT)
to determine potential bleeding disorders
and to monitor anticoagulant therapy.
 Clinical Microscopy
• There are two major areas in this section of the
lab.
• The first area is alloted to routine and other
special examinations of the urine such as
macroscopic exam to determine color,
transparency, specific gravity, and pH level, and
microscopic exams to detect presence of
abnormal cells and/or parasites as well as to
quantify red cells and WBC and other chemicals
found in urine
 Clinical Microscopy
• The second area is assigned to the exam of
stool or routine fecalysis.
• Detection and identification of parasitic
worms and ova are the primary activities in
this area.
 Clinical Microscopy
Urinalysis- study of urine
• Historically, the routine urinalysis was one of the earliest
lab test performed, valuable to detect disease related to
kidney and urinary tract.
•By evaluating the three component parts of urinalysis:
•physical- color clarity, sp gravity
• chemical- pH, glucose, ketone bodies, protein, blood,
bilirubin, urobilirubin, nitrates, leukocytes, esterase,
•Microscopic sediments – cells, crystals, amorphous
deposits -
 Urinalysis

• Composed of 3 parts:
- physical
- chemical
- microscopic
• The presence of disease can cause changes in:
- amount of urine formed or excreted
- Color of urine
- Appearance
-odor of urine
- Cells present in the urine
- Chemical constituents in urine
Function of the Urinary System
 Function of the Kidney
• hormones produced Function
- erythropoetin stimulates red cell synthesis
- rennin indirectly influences blood
pressure
- active Vit.D influences mobilization of
calcium
• Hormones that affect kidney function
- parathyroid hormone, aldosterone, and
antidiuretic hormone (ADH)
 Processes in the Formation of Urine
1. filtration of wastes products, salts, and
excess fluid from the blood
2. Reabsorption of water and solutes from the
filtrate
3. Secretion of certain ions and certain drugs
and chemicals
 Components of urine
• Major: Water, urea, and sodium chloride.
• Some solutes that are also found:
- electrolytes, - peptides
- vitamins - bile pigments
- carbohydrates - uric acid
- amino sugars- creatinine
- amino acids
 Renal threshold
• Almost all constituents in urine are also
present in the blood but in different amounts.
• Some substances like glucose have a high
renal threshold. This means that these
substances must reach a certain high level in
the blood before they are detected in the
urine.
 Diseases affecting urinalysis
Infections of the urinary tract
Urine stored in the urinary bladder is normally
sterile. Urinary tract infections (UTI) most
commonly occur in the urethra. If left
untreated , the infection may ascend to the
bladder, causing cystitis. In severe cases the
kidney may be involved. Pyelonephritis is an
inflammation of the kidney pelvis usually
caused by bacterial infection
 Diseases affecting urinalysis
Diseases of the kidney- results from the build
up of toxic products in the blood such as urea,
uric acid, creatinine. If kidney function is
suddenly lost, death will occur within few days
if treatment is not administered. One form of
treatment is dialysis. In hemodialysis, blood is
circulated outside the body passed over a
membrane to remove toxic waste products
and then the blood is returned to the body.
 Diseases affecting urinalysis
Diseases of the kidney
• Glumerulonephritis – infection of the
glumerulus. There is protein and RBC in the urine
• Tubular necrosis – a condition where in the blood
supply in the kidney is diminished, or upon
exposure to substances that are nephrotic (toxic
to the kidney cells)
• Polycystic kidney disease- inherited condition in
which glumerular function is lost due to cyst
formation in the kidneys.
 Other diseases with abnormal
urinalysis results
• Diabetes mellitus, hypertension, atherosclerosis, and
autoimmune diseases like lupus erythematosus.
• The kidneys are important in the regulation of blood
pressure. Prolonged high blood pressure can cause
kidney damage.
• Nephrotic syndrome, a condition characterized by
tissue edema and protein in the urine, is usually
associated with circulatory disorders.
• Malignancies arising in the urinary tract may cause
obstruction leading to abnormal urinalysis results.
 Types of urine specimens
• Preferred specimen is the first morning specimen
• urine may be collected by the midstream or
clean-catch procedure.
• Midstream is one in which the urine is collected
in the middle portion of the urine stream
• Clean-catch- required for culture to determine
presence of bacteria.
• The physician may order a 24-hour urine
specimen for protein, creatinine, urobilinogen,
and calcium
 Handling and preserving of urine
specimens
• Urine should be examined within one hour of
voiding.
• If not possible, urine may be refrigerated at 4-
6◦C for up to 8 h.
• If it cannot be refrigerated, some
preservatives like toluene, formalin or thymol
may be used.
 Urine volume
• Excessive production of urine – polyuria
• Excessive urination at night – nocturia
• Oliguria – insufficient production of urine
• Anuria – absence of urine production
 Routine urinalysis
• Physical characteristics- color, odor,
transparency, specific gravity
Color –normal color of urine is yellow
- color caused by food or medication
red- beets, rhubarb(in alkaline urine)
yellow-orange –carrots, some antibiotics
brown-black – melanin, homogentisic acid,Hb
yellow –brown
or green-brown – bilirubin or bile pigments
 Routine urinalysis
• Physical characteristics- odor
odor- freshly voided urine has a characteristic aromatic
odor. Changes in the odor may be due to diet, disease,
or the presence of organisms. The odor of urine of a
patient with diabetes is fruity due to the presence of
ketones which are products of fat metabolism.
phenyl ketonuria (PKU)- an inherited condition in
which amino acid phenylalanine is not metabolized ,
causes urine to have a musty or mousy odor. It is impt
that all newborns be tested for PKU to avoid mental
retardation if allowed to go untreated.
 Routine urinalysis
• Physical characteristics- appearance of the urine
- fresh normal urine is usually clear immediately
upon voiding. As the urine reaches room
temperature, or after refrigeration, it may
become turbid or cloudy depending on the pH of
the urine.
acidic pH- cloudiness is due to urates
basic pH- due to phosphates
- turbidity or cloudiness in a freshly voided urine
may be an indication of a disease. Four causes of
turbid urine: WBC, RBC, epithelial cells, bacteria
 Routine urinalysis
• Physical characteristics- specific gravity
-is the ratio of the weight of a given volume of
the solution (urine) to the weight of an equal
volume of water.
- the sp. gr. of urine indicates the concentration
of solids such as urea, phosphates, chlorides,
proteins, and sugars that are dissolved in urine.
- normal urine has sp. gr. of 1.005-1.030.
- sp.gr. can be measured with a
urinometer,refractometer, or reagent strip.
 Routine urinalysis
• chemical characteristics-
- reagent strips are the most widely used technique in
detecting chemical in urine and are available in many
types:
- test for pH, protein, glucose, ketones, bilirubin, blood
- some strips may test for urobilinogen, specific
gravity, leukocytes, bacteria
• Presence of these chemicals in the urine provide
information on the patient’s CHO metabolism. Kidney,
liver function, and acid-base balance.
• Performing the chemical test using reagent strip sh, be
done within 1 hour of urine collection.
 Principles of chemical tests
• Ph- measure of alkalinity or acidity or urine
- normal pH 5.5-8.0
- yellow to orange – acidic
- green to blue – basic
• Protein- indicates renal disease or UTI
• Glucose – glusoria, blood glucose level has exceeded
the renal threshold
• Ketone – incomplete metabolism of fats, ketones are
dev. and excreted in the urine
– present In diabetes, fasting or starvation
• Bilirubin – breakdown product of Hb.
- indication of liver disease, bile duct
obstruction or hepatitis
• Blood - presence of blood indicates infection
or presence of trauma of urinary tract
or bleeding in the kidney
• Urobilinogen- degradation product of bilirubin
which is formed by intestinal bacteria.
• Nitrite – gram neg. bacteria can convert nitrate
to nitrite
• Leukocyte - indicates infection
 Normal values
• Ph – 5.5 – 8
• Protein – neg. to trace
• Glucose – neg.
• Ketone – neg
• Bilirubin – neg
• Blood – neg
• Urobilinogen – 0.1-1.0 E.U./dL (Erhlich Unit)
• Bacteria (nitrite) – neg.
• Leukocyte – neg.
 Routine urinalysis
• Microscopic – sediments like cells, crystals, amorphous deposits
Cells – blood cells seen in disease or trauma
- leukocytes – increased in UTI
- epithelial cells – squamous epithelial cells result from
sloughed off from lining of the urinary tract
smaller bladder or renal tubular cells are found in renal disease
- bacteria – cocci or rod shape
- yeast
- protozoa like T. vaginalis
- spermatozoa
 Routine urinalysis
• Microscopic
Casts – when protein accumulates and
precipitates in the kidney tubules and washed
into the urine (hyaline, granular, cellular)
Crystals – acidic- normal - amorphous urates,
calcium oxalates
basic- normal-
amorphous phosphates, calcium
carbonate
 RENAL EPITHELIAL CAST GRANULAR CAST

RED CELL CASTS WHITE CELL CASTS

URINE STRIP
 Anatomic Pathology
Section of Histopathology/Cytology
• Activities performed in this section include
tissue (removed surgically as in biopsy and
autopsy), processing, cutting into sections,
staining, and preparation for microscopic
exam by a pathologist
 Specialized Sections of the Lab
Immunohistochemistry-a specialized section of the
lab that combines anatomical, clinical, and
biochemical techniques where antibodies
(monoclonal and polyclonal) bounded to enzymes
and fluorescent dyes are used to detect presences
of antigens in tissues. This is used in the diagnosis
of some types of cancers by detecting the presence
of tumor-specific antigens, oncogenes, and tumor
suppressor genes. It can also be used to assess the
responses of patients to cancer therapy as well as
diagnosis of certain neurodegenerative disorders.
 Specialized Sections of the Lab
Molecular Biology and Biotechnology- using
different reagents and enzymes, DNA and RNA
are identified and sequenced to detect any
pathologic conditions/disease processes.
• The most current technique is the use of
polymerase chain reaction, (PCR) . This
technique can screen genetic indicators of
disease and diagnosis of cancer and infectious
diseases.
 Laboratory Testing Cycle
• The lab testing cycle encompasses all activities
starting from a medical doctor writing a lab
request up to the time (called turnaround
time (TAT) the results are generated and
become useful info for the treatment and
management of the patient.
• The cycle has three phases, namely,
preanalytic, analytic, and post-analytic
 Quality Assurance in the Clinical Lab
• QA encompasses all activities performed by the
lab personnel to ensure reliability of tests results.
• It is organized, systematic, well-planned and
regularly done with results properly documented
and consistently reviewed.
• QA in the clinical lab has two major
components:Internal Quality Assurance System
(IQAS) and External Quality Assurance System (E
QAS)
 Quality Assurance in the Clinical Lab
• IQAS includes day-to-day activities that are
undertaken in order to control factors or
variables that may affect test results.
• Regular review and audit of results are done in
order to identify weaknesses and consequently
perform corrective actions.
• EQAS is a system for checking performance
among clinical lab and is facilitated by designated
external agencies. The National Reference Lab
(NRL) is the DOH designated EQAS.
• An unknown sample with known test results is
regularly sent to a clinical lab for testing.
• Results are then returned to the external
facility and are compared with the known
result.
• This procedure determines the performance
of the lab
• A certificate of performance is given to the
participating clinical lab.
 NRL-EQAS
• National Kidney and Transplant Institute (NKTI)-
Hematology and Coagulation
• Research Institute of Tropical Medicine (RITM)-
Microbiology (identification and antibiotic
susceptibility testing and Parasitology (I.D. of ova
and quantitation of malaria)
• Lung Center of the Phil (LCP).-clinical chemistry
(for testing 10 analytes, namely glucose,
creatinine, total protein, albumin, blood urea
nitrogen, uric acid, cholesterol, sodium,
potassium, and chloride)
• East Avenue Medical Center (EAMC)- drugs of
abuse (methamphetamine and cannabinoids)
• San Lazaro Hospital STD-AIDS Cooperative
Center Laboratory (SACCL)- infectious
immunology hepatitis B surface antigen
(HBsAg), human immunodeficiency virus
(HIV), hepatitis C virus (HCV)