HEMATOLOGY CELL PROCEDURES

Manual count

- Manual counts are performed using a HEMACYTOMETER or counting

chamber

- Manua dilutions are made calibrated automated pipettes and diluents.

- Principle for cell counts is the same for some blood components; WBC, RBC,

platelets

- Principle: the blood is diluted with appropriate known volume of diluting

fluid and then counting is done by using hemacytometer

- Dilution, diluting fluid and area counted vary for RBC, WBC and platelets

- Any particle e.g sperm can be counted using this system

- Equipment used:

1. Hemacytometer: consists of

i. Counting chamber

Types of counting chambers

a. Improved neubauer

 Triple lined dividing central large square are closer

 Central square has different divisions

  Central area is divided into 25 large squares

 25 squares are subdivided into 16 squares

 By dividing central squares by 25 the RBC and Platelet

count become easy to do.

b. Fuschs Rosenthal

c. Spear levys

d. Turks

e. Bass-jones

ii. Coverglass

iii. Pipette

iv. Rubber tube

2. Thoma pipet

3. Suction device

4. Thick cover slip

5. Cell counter

6. Diluting fluids
Manual Cell counts

1. RBC count

- Rarley performed due to inaccuracy

- WBCs need not be destroyed when RBCs are counted

- Highest in morning

- Diluting fluid: isotonic saline

OTHER DILUTING SOLUTIONS

i. Dacies fluid(formol citrate)

ii. Hayem’s diluting fluid

iii. Gower solution

iv. Toissons fluid

v. 3.8% sodium citrate

vi. Bethel’s fluid

- Dilution = 1: 200

- Objective: 40X

- Area counted: 0.2mm2(5 tertiary squares of center space)

- Counted on both sides of the chamber

- RBC count calculated using hemacytometer formula

- Calculation

Average total of RBCs in 5 squares x dilution correction factor x

volume/depth correction factor

OR

Average total of RBC x 5 x 200 x 10

- High RBC COUNT: Polycythemia or leukemia

- Normal values:

i. Female: 3.8-5.2 million/cumm

ii. Male: 4.5-5.5 million/cumm

iii. At birth: 6-6 million/cumm

- Low RBC COUNT: Anemia, acute leukemia

2. WBC count

- Number of WBCs in 1 liter or 1 microliter

- Diluting fluid: should be hypotonic

a. 1% ammonium oxalate

b. Weak acid: 3% acetic acid

 c. Weak acid: 1% hydrochloric acid with 1 drop of methylviolet or crystal

violet

d. Turks solution

- Diluting fluids lyse RBC so as to not intefer

- Dilution= 1:20

- Area counted: 4mm2(4 large corner cells)

- Objective: 10X

- Helps detect infections

- Calculation

Total WBC COUNT= Average total WBC x dilution factor x volume

correction factor

OR

= Average number of cells counted x dilution factor

Area counted(mm2) x depth factor

- Normal values

i. Females:4.5-11.0 x 109/L
 ii. Males: same as females

iii. At birth: 10.0-30.0 x 109/L

3. Corrected WBC count

- Nucleated RBC—percursors of to normal red blood cells

- Must be correctd if >5 N-RBCs

- FALSELY counted as WBC—appear as WBCs

- NOT LYSED by WBC diluting fluid

- When conducting WBC count , total includes both WBCs and nucleated red

blood cells

- To correct this formula used:

a. Uncorrected WBC x 100

b. Add 100 to total NRBCs observed

c. Divide second total from first total.

- Calculation

= uncorrected WBC count x 100

No.of corrected N-RBCs/100WBC + 100

4. Platelet count

- Number of platelets in 1 liter or 1 microliter

- Platelets adhere to foreign objects and to each other

- Difficult to count

- Diluting fluid: 1% ammonium oxalate—lyses nRBCs

- Dilution= 1:200

- Objective: 40X phase contrast microscope

- Area covered: 1mm2(25 small squares in large center)

- Diagnose bleeding diorders

- Sources of error:

i. Age of specimen

ii. Clumping of platelets

iii. Debris in diluting fluid

iv. Platelet adherence to glass

v. Incorrect dilution of specimen

- In case of thrombocytopenia: dilution has to be increased. And added to

calculation
- In case of thrombocytosis: fewer squares counted

- Normal ranges:150,000-400,000 per microliter or 150 to 400 x 10 9/L

i. Female: 157,000-371,000/microliter

ii. Male: 135,000-317,000/microliter

- Increased platelets: postsplenectomy, polycythemia vera, chroninc

myelogenous leukemia,

- Decreased platelets; aplastic anemia, idiopathic thrombocytopenia and

acute leukemia

Platelet estimate of Report estimate as

0-49,000/uL Markedly decreased
50,000-99,000/uL Moderately decreased
100,000-149,000/uL Slowly decreased
150,000-199,000/uL Low decreased
200,000-400,00/uL Normal
401,000-599,000/uL Slowly increased
600,000-800,000/uL Moderately increased
Above 800,000/uL Markedly increased

5. Hemoglobin count

- Principle:
 o In the cyanmethemoglobin method, also known as

hemiglobincyanide method/HiCN method

o Blood is diluted in a solution of potassium cyanide and potassium

fericyanidde.

o Hemoglobin is oxidized to methemoglobin by potassium

ferricyanide

o Potassium cyanide converts methemoglobin to

cyanemethemoglobin

o Absorbance: 540nm

- Reagent: modified Drabkins reagent. Pale and yellow with a ph value of

7.2

- Contents of reagent shorten conversion time to 3 mins and has noionic

detergent that lyses RBCs

- When mixed with blood

- Stable HiCN is formed

- Higher in morning

- Screen for anemia and RBC breakdown

- High in HIGHER ALTITUDES, dependent on age and sex

- Increase in strenuous muscular activity

- Sources of error:

i. Drabkins reagent is sensitive to light---stored in brown bottle in dark

ii. Inceased WBC count and platelet count causes turbidity and false

high results------centrifuge and measure supernatant

iii. Lipemic sample can interfere and give false result--------add 00.1ml

of patients blood to drabkins reagent and us solution as a reagent

blank

iv. HbS and HbC may be resistant to hemolysis causing turbdity------

dilution with distilled water (1:2)

v. Abnormal globulin found in multiple myeloma or Waldestrom

macroglobulinemia may precipitate------add 0.1g of potassium

carbonate to drabkins reagent

- Normal ranges:

i. Female: 12-16g/L

ii. Male: 13-18g/L

iii. At birth: 15-20g/L

- Low: critical valueless than 5.0g/L

i. anemia caused by RBCs dying earlier than normal known as

hemolytic anemia,: transfusion of blood e.g reaction to chemical or

 drugs and reaction to infectious agents, various systemic blood e.g

leukemia, SLE, hodgkins disease

ii. bleeding,

iii. Thalassemia

iv. Pernicious anemia

v. Iron deficiency

vi. poor nutrition( low iron level, folate, vitamin B12, or B6

- High=hyperchromia : caused by hypoxia

i. COPD(chronic obstructive pulmonary disease)

ii. Sickle cell anemia

iii. Thalassemia

iv. Transfusion reaction

v. Hemolysis

vi. Dehydration

vii. Polycythemia vera

viii. High altitude(mountaneous places) to low altitudes

Oligochromia: decreased hemoglobin levels found in anemias.

Deteremination
- Done by automated instrument

 Colorimetric

i. Direct visual

 Acid hematin: using Comparator block

 Procedure:

o Dispense 0.1 N HCl up to 2mark in Sahil grad tube

o Aspirate blood using sahil pipette upto 0.02 cc mark

o Dispense into grad tube

o Gentle draw up and down to mix

o Stand for 5 mins(to convert Hgb to Acid hematinti)

o Added diluted water by block

o Compare using comprator block

o Value obtained when sample has same color as block

 Sources of error

a. High WBC count & platelet count = turbidity (false

increase

b. Hemoglobin S&C

c. Lipemic blood

 Alkaline hematin: for neonates since Hgb-F is alkali resistant

ii. Photoelectric method/indirect

 Cyanmethoglobin(HiCN)

 Also known as hemiglobincyanide

 Reference method for Hgb determination

 Standard approved by CLSI

 All forms of Hgb are measured except Sulf-Hgb

 Uses drabkins solutionlight absorbance at 540 nm

 Drabkins contains:

i. potassium cyanide: converts Met-Hgb to Cyanmmet-

Hgb

ii. Potassium ferricyanide: converts Hgb to Met-Hgb

iii. Dihydrogen potassium phosphate: allows to be read

spectotrophetrically

iv. Non-ionic detergent: improves lysis

 Conversion of hemoglobin to cyanmethemoglobin

 Procedure;

 5ml of drabkins rgt is added to 0.02 cc mark of Sahil pipette

 Mix well and stand for 10 mins

 Set spectrometer @ 540nm

  Read absorbance with value of Hgb

Sources of error

 Hemoglobin conc. Falsely elevated by lipemia(cloudy plasma)

 Extreme high WBC count

 High RBC count with hemoglobin C or S

 Specimen conditions

 Gasometric method/oxygen capacity method

i. Indirect method

ii. Also known as Van slyke oxygen capacity

iii. 1g hemoglobin = 1.34ml oxygen

iv. Also known as Van slyke oxygen capacity

 Chemical method

i. Indirect method

ii. 1g Hbg = 3.47mg iron

iii. Known as Keneddys or Wongs

  Specific gravity/copper sulfate

i. Specific gravity of copper sulfate = 1.053

ii. Also known as copper sulfate method

iii. hemoglobin equivalent of 12.5g/dl

iv. If blood sinks. Greater sp.gravity and more than 12.5g/dl

v. If floats. Less sp. Gravity and less than 12.5g/dl

vi. Changed daily

6. Hematocrit

 Known as packed cell volume(PCV)

 Determinants: RBC and plasma

 Volume of packed RBC that occupies a given volume of whole blood.

 Principle: anticoagulant whole blood is centrifuged and the total

volume of red cell mass is expressed as a percentage or a decimal.

 Macroscopic observation of volume of packed RBCs

 High levels:

 Indicate in increase in number of erythrocytes or decrease in

plasma volume.

 Indicates: erythrocytosis, polycythemia vera, shock

 Conditions: macrocytic anemia, spherocytosis, thalassemia,

hypochromic anemia, sickle cell anemia

 Low levels:

 indicate low number of red blood cells also indicates low oxygen

carrying capacity.

 Low anemias and >50 Years old

 INDICATE: anemia, leukemia, lymphomas, adrenal insufficiency,

chronic disease, acute and chronic blood loss

 Simple and reliable

 Used to evaluate and classify anemia according to red cell indices

 When blood is centrifuged heavy particles fall to bottom

 Light particles settle on top of heavier particles

 Hematocrit: percentage of RBCs in a volume of whole blood

 Expressed as ratio or %

 Reagent: anticoagulant oxalate

 Centrifuge: 3000rpm for 30 mins

DETERMINATION

1. Macrohematocrit:

i. Method of collection: venipuncture

ii. Amount of blood: large

iii. Centrifuge @ 2,00-2,300 rpm

iv. For 30 mins

v. Separation of buffy coat: completely

Principle same as wintrobe method

1. Wintrobe method

- Anticoagulant: double oxalate

- Length: 115mm

- Diameter: 3.0mm

- HCT marking: below upwards

- ESR marking: below upwards

Procedure

 Fill the wintrobe tube with blood up to the 10 mark

  Place inside a test tube with cotton in between to make it

stable

 Centrifuge @ 2,000-3,000 RPM for 30 mins

 Remove tubes and carefully place on a wintrobe stand

 Measure height of RBC layer in mm

 It is expressed in %

2. Hadens modification method

- Anticoagulant: 1.1% sodium oxalate

3. Van Allens method

- Anticoagulant: 1.6% sodium oxalate

4. Sanford-Magath

- 1.3% sodium oxalate

5. Brays method

- Anticoagulant: heparin

2. Microhematocrit

 Capillary puncture

i. 3 layers: plasma, buffy coat, packed RBCs

 ii. Height of Capillary tubes: 75mm

iii. Bore: 1.155mm

iv. Pricking: 2-3mm

v. Clay: 4-6mm

 Procedure

o Perform skin puncture

o fill 2 capillary tubes

o apply clay seal

o centrifuge @ 10,000-15,000 RPM for 5 mins

o remove the tube

o measure RBCs layer on micro-hct reader

 Centrifuge @ 15,000 rcf for 5 mins

 Normal range

i. Female: 37-47%

ii. Male: 40-54%

iii. At birth: 45-60%

 Less: critical valueless than 15%

 Hematocrit value obtained from automated instrumnts is slightly lower

than centrifugation method

Sources of error

 Increases

- Insufficient centrifugation

- Inclusion of buffy coat

- Dehydration

- Disorder such as: macrocytic anemia, hypochromic anemia

and sickle-cell anemia

 Decreases

- Improper sealing of capillary tube

- Increased conc of anticoagulants

- Prolonged centrifugation

- Acute blood loss

Inteferring factors in Hct determination

 High altitude

 Normal value vary with age and sex

 Lower value in men and women older than 60 years

 Severe dehydration from falsely rises Hct

 LAYERS OF BLOOD AFTER CENTRIFUGATION

o TRAPPED PLASMA:amount of plasma that still remains in the

RBC portion after the microhematocrit has been spun

RULE OF 3

i. Value of hematocrit is 3x value of hemoglobin

Apples to specimen with normocytic normochromic erhythrocytes

6. Differential count

 Relative proportion of different leukocytes expressed as a percentage

 ROUTINE: count 100 WBC

 LEUKOPENIA: count 50 WBC

 LEUKOCYTOSIS: count 200 WBC

WBC Reference range

segmenters R: 47-77%

A: 1.8-7.8 x 10^9/L
Band R: 0-7%
 A: 0.07 x 10^9/L
Lymphocytes R: 20-40%

A: 1.0-4.8 x 10^9/L
Monocytes R: 2-10%

A: 0.01-0.8 x 10^9/L
Eosinophils R: 0-6%

A: 0-0.6 X 10^9/L
Basophils R:0-1%

A: 0-0.2 x 10^9/L

CLASSIFCATION NEUTROPHIL COUNT

Mild 1.0—2.0 x 10^9/L
Moderate 0—1.0 x 10^9/L
Severe <0—1.0 x 10^9/L

 Granuolocytes: neutrophils, basophils, eosinophils

 Agranulocytes: lymphocytes and monocytes

 Specimen:

i. peripheral blood, bone marrow, body fluid sediments e.g spinal fluid

ii. Also whole blood smears from EDTA or prepared free flowing

capillary blood

iii. Smears made within 1 hr and stored at rtp

 ABSOLUTE COUNT: more accurate

 Peripheral blood smear preparation

 Blood smear methods:

1. Two slide

o Also known as wedge method, push smear, spreader slide

o Simple and most used

o Uses 2 slides one for smear and another as spreader or pusher

o Diameter of blood: 2-3mm

o 0.25 inch from the slide

o Angle of spreader: 30-40’ or 30-45’

o Fixative: methanol

o Have thick and thin portions

o Smooth , even, free, from ridges and waves or holes

Characterisitic of good smear

o Should have a feathery area

o Visible transition from thick to thin

o Smear covers ¾ of slides length

o Smooth surfaces
 o Leukocytes should not be bunched on top or bottomof edge.

2. Ehrlichs

a. One ½ coverglass of 22mm square are used

b. Cover slip technique

c. Prepared for bone marrow preparation

d. Disadvantages:

o Cover glass is easily broken

o Require chemically clean coverglass

o Difficult to prepare

o Difficult to label, stain and transport

3. Beacons cover glass and slide method

a. There is even distribution of cells but yields limited blood smears

4. Spunners methodautomated slide making

 0.2 ml of blood from skin puncture

 Can be used to create a good smear

 Disadvantage; requires fresh blood sample

 5. Buffy coat smear for WBCs <1.0 x 10^9/L--LEISHMANIA

6. Thick blood smears for blood parasites(malaria): Chagas disease,

babesiosis, ehrilichiosis, relapsing fever

7. Wet preparation

 For determination of RBC abnormalities

 To detect sickle cell anemia and spherocytes

 Blood is diluted with Isotonic NaCl beneath the coverslip

P= PASS

A= ANGLE

S= SPEED

S=SAMPLE

Thick and thin smears and buffy coat smears

Thick smear:

 Blood is spread out to cover an area about 4 times its original area

 Criteria for thickness: slide placed on piece of newspaper : SMALL PRINT

IS VISIBLE

 Used for: diagnoses: parasitic diagnoses

Thin smear:

 Used for: diff count, platelet count, retic count, malarial parasite,

stained red cell, siderocyte count

i. Requirement for proper blood film

 Use a chemically clean glass slide and cover glass

 Not too large or too small blood drop

 Proper angling and pressure of spreader

ii. Methods of drying blood smears:

 Air drying

 Heating in an oven over low fire

 Chemical drying in ethyl alcohol

iii. Fixatives for blood films

 Methanol

 Absolute ethyl alcohol

 Absolute ethyl alcohol and ether

 1% solution mercury chloride and 1% formalin

iv. Factors affecting thickness/thinness of blood smear

  Size of the drop

I. Large drop=thick smear

II. Small drop= thin smear

 Angle of the spreader

I. Proper angle 34-45’degrees=good smear

II. Increased=thick smear

III. Decreased= thin smear

 Pressure exerted when pushing the spreader

I. Heavy pressure=thin smear

II. Light pressure= thin smear

 Speed of spreader slide

I. Too fast = thick smear

II. Too slow = thin smear

Buffy coat smear

 For WBCs

 Less than 1.0 x 109/L

STAINING OF BLOOD SMEARS

 Most important part of routine hematological examination.

 Essential for identification of hematopoetic cells

 Most used stains are polychromic stains: ROMANOWSKY GROUP

 Stained: 2-3 hours after collection

 Ph LEVEL: 6.8

1. WRIGHTS STAIN

i. Methylene bluebasic dye. Stains acidic cellular components

ii. Eosinacidic dye. Stains basic(eosinophilic) cellular components.

o Sodium phosphate –added to the stain and has Ph of 6.4

2. MAY-GRUNWALDS –GIEMSA STAIN

I. Both giemsa and may-grunwalds are added to smear

3. GIEMSA STAIN

4. LEISHMANS STAIN

5. JENNERS STAIN

6. FIELD STAIN

7. PANOPTIC STAIN

i. Combination of romanowsky stain and another stain

8. SUPRAVITAL STAIN
 i. Used to stain and inspect living cells which have been removed

from the body.

Criteria of a good stain

1. Macroscopically: Appear redbrownish (WRIGHTS STAIN) stain

2. Microscopically: are pink

a. Neutrophil granules: LILAC/PURPLE

b. RBC: PINK

c. Esoniphil graanules: RED

d. Nuclei of WBC: PURPLISH BLUE

e. BASOPHILS: DARK PURPLE

f. PLATELETS: PURPLE/DARK LILAC

g. MONOCYTES:GREY GROUND GLASS

APPEARANCE

o Causes of overstained smears

1. Too thick smears

2. Insufficient washing

3. Too prolonged staining time

4. Execessive alkalinity of the stain, buffer or water

o Solution

1. Check ph not if its alkaline or acidic

2. Shorten stain time

3. Prolong buffering time

o Causes of understained smear:

1. Too thin smears

2. Insufficient staining time

3. Old stain

4. Excessive washing

5. Excessive acidity of the stain, buffer or water

o Correction:

1. Lengthen staining

2. Check stain and buffer ph

3. Shorten buffering

4. Shorten washing time

Methods Of counting DLC

1. LONGITUDINAL Strip diff countTAIL TO HEAD of smear

2. Exaggerated battlement methodpattern of consecutive fields beginning

near tail on a horizontal edge. MOST COMMON METHOD

3. ZIG-ZAG: FROM SIDE to SIDE. Cross-sectional

Absolute count vs relative count

 Refer to white cell differential

 Relative count: percentage of a particular cell counted from 100WBC

DIFFERENTIAL

 Absolute count:

i. Count derived from total white count multiplied by

percentage of any particular white cell

ii. Clinically significant

iii. More accurate

 Normal range:

a. Neutrophils: 2.0--7.0 x 10^9/l (40-80%)

b. Band: 0.0—0.7% x 10^9/l (0-7%)

c. Lymphocytes: 1.0--3.0 x 10^9/l (20-40%)

d. Monocytes: 0.2--1.0 x 10^9/l (2-10%)

e. Eosinophils: 0.02—0.5 x 10^9/l (1-6%)

f. Basophils: 0.02—0.1 x 10^9/l (<1—2%)

6. Blood indices

 Test used to diagnose cause and types of anemia

 Describe size,shape and oxygen carrying protein content in red blood

cells

 Indices include following measurements:

1. Mean corpsular volume

 Average volume of RBC

 In Fl or 10 -15

 Formula

Hematocrit (%) x 10

RBC count(1012)/L

 Normal cell volume= 80-100 fl(normocytic cells)

 Smaller cells: <80 fl(microcytic cells)

 Conditions: iron deficiency anemia, lead poisoning,

sideroblastic anemia, thalassemia

 Large cells:>100fl(macrocytic cells)

Conditions: Vitamin B12 deficiency, liver disease,

reticulocytosis, megaloblastic anemia—hypersegmented,

alcoholism, competitive parasite, hemolytic anemia with

reticulocytosis, normal newborn

2. Mean corpsular hemoglobin(MCH)

 This is the average weight of hemoglobin in a RBC

 Expressed in pictograms(pg) or 1012g

 Directly proportional to size of cell and concentration of

hemoglobin

 Formula

Hemoglobin (g/dl) x 10

RBC cont (1012)/L

 Reference range for adults= 28—32pg

 INCREASE: macrocytic anemia

 DECREASE: microcytic anemia, hpochromic anemia

  Interfering factors: hyperlipedemia falsely elevates the MCH.

High heparin concentration also elevates MCH

 NOT CONSIDERED in classification of anemia

3. Mean corpsular hemoglobin concentration(MCHC)

 It is the average concentration of hemoglobin and hematocrit

levels in each individual erythrocyte

 Valuable in monitoring therapy for anemia

 Units used are gram per deciliter

Hemoglobin (g/dl) x 100

Hematocrit

 Nonchromic cells range from 32—37 g/dl

 Hypochromic cells <32g/dl

 IDA

 Thalassemia

 Microcytic anemia

 Chronic blood loss anemia

 Hyperchromic cells >37g/dl: error in RBC or presence

ofspherocytes, Lipemia, active cold agglutination disease

 4. Red cell distribution width

 Determined from RBC histogram

 RDW is used because MCV is less reliable in describing

erythrocyte population when considerable variationin erythrocyte

size occurs

 RDW is coefficient of variation of size distribution of RBCs

 RDW is essentially a response to the degree of anistocytosis

(unequal RBCs) and coefficient of the variation of the MCV

 Formula

Standard deviation of MCV x 100

MCV

 Normal value: 11.5—14.5%

 Increased values indicates: ANISOCYTOSIS(unequal RBC size);

Post transfusion, post-treatment,idiopathic sideroblastic anemia

Conditions

Normal RDW---DECREASED OR INCREASED MCV= macrocytic or micorcytic

Increaed RDW---NORMAL MCV= Anistocytes size inaverage size and normal

range

Abnormal RDW----ABNORMAL RDW= anistocyte size in average size and above

or below normal range

 Critical value WBC count: <2,500 per microliter and > 30,000 per

microliter

 Critical values platelet count: <30,000 microliters

OTHER HEMATOLOGIC TESTS

1. RETIC COUNT

 Last immature red blood cell development stage

 Remains in bone marrow 2-3 days and 1 day in peripheral blood

 Reticulocyte contains: RNA, mitochondria, ribosomes

 Retic count: assess erythropetic activity of the bone marrow

 SUPRAVITAL STAIN—very important

 New methylene blue is used or brilliant cresyl blue or pure azore

  Any non-nucleated red blood cell with 2 or more particles of blue

stained granulofilaments after new methylene blue stain is a

RETICULOCYTE

 Precipitates RNA to form deep blue mesh-like network

 Stress reticulocytes exhibit much dense like network

 Expressed in %

 Formula

Number of reticulocytes x 100

100 RBC s counted

 Norml range:

Adults: 0.5--1.5%

New born: 2.5--6.5%

2 week to 1 year: 1--3%

 Formula With Millers disk

Number of reticulocytes in LARGE SQUARE x 100

Number of Red blood cells in SMALL SQUARE x 9

Absolute reticulocyte count

 Actual number of reticulocytes in 1 liter or 1 microliter of blood

  Calculated result should be converted from 1012 to 109

 Formula

%reticulocyte count x total rbc count x 1012 /100

 Normal referenc range

25—75 x 109/L

o If less t= patient has low erythropoetic response to their anemia

 Aplastic anemia

 Acute benzol poisoning

 Chronic infections

 Anaplastic crisis of HA

o If more=

 Pregnancy

 At birth

 Menstruation

Corrected reticulocyte count

 Dependent on degree of anemia

 Low hematocrit specimen

 Percentage of retics may be FALSELY ELEVTAED

  Due to whole blood with few red blood cells

 Formula

Reticulocyte count x hematocrit

Normal hematocrit value based on age and gender

 Normal value: same as stated on normal reticulocyte values

Reticulocyte production Index

 Reticulocytes released from marrow prematurely called SHIFT

RETICULOCYTES

 Shifted from bone marrow to peripheral blood earlier than usual

 These cells take 2-3 days to lose reticula

 When erythropoeisi is elevated a correction is made for presence of

shifrt reticulocytes

 Maturation index

a. 36-45%= 1 day

b. 26-35%= 1.5 days

c. 16-25%= 2 days

d. <15 days= 2.5 days

 CALCUATION OF RPI

= CORRECTED RETIC COUNT IN %

MATURATION IN DAYS

2. ESR

 It is an established parameter of inflammation

 Non-specific indicator of disease

 Not recommended as screening test to detect inflammatory conditions

asymptomatic patients

 C-reactive protein level more predictable and reliable

 When blood allowed to stand at rtp undisturbed for a period of time red

blood cells settle at bottom

 ESR is distance in millimeters that red cells fall in an hour.

 Increased ESR: rouleaux, increased fibrinogen levels, relative increase

of plasma globulins and absolute incrase of plasma globuluns

 ESR is directly propotional to RED BLOOD CELL MASS

 ESR is inversely proportional to plasma visocosity

 Stages of sedimentation:
 1. AGGREGATION(10 mins)—intial rouleaux formation

2. RAPID/FAST SETTLING(40 mins)

3. SLOW/FINAL DEDIMEMTATION(10 mins)

 Clinical conditions associated with increased ESR

1. Anemia

2. Infections

3. Inflammation

4. Tissue necrosiss(myocardial infarction)

5. Hemolytic anemia

Fctors that affect ESR

 Red blood cells have a negative charge

 Plasma cells have a positive charge

 Red cells repel

 Incrased plasma counteracts this

 Red blood cells settle more rapidly as a result of formation of rouleaux

Methods of measuring ESR

1. Wintrobe method
  Smaller than westergren(115mm)

 Anticoagulant; Oxalate

 Length: 115mm

 Bore: 3mm

 Normal range for Wintrobe method

Male—>0—9mm/hr

Female0—20mm/hr

Children0—13mm/hr

2. Westergren method

 Most sensitive because of the tube used(longer)

 Length:300 mm

 Bore: 2.65mm

 Anticoagulant: sodium citrate

 Normal range for westergnern method; dependent on age

Males <50yrs---0-10mm/hr

Females <50 years---0-20mm/hr

Males >50yrs---0-20mm/hr

Females>50years—0-30mm/hr
 Children--0-10mm/hr

 Procedure:

 Draw the blood into the tube up to 0mm mark

 Wipe blood from bottom of tube with cotton

 Set tube upright

 Leave tube undisturbed for 1 hr

 At end read results

Factors affecting ESR

ERYTHROCYTES PLASMA TECHNICAL ERRORS

COMPOSITION
 Incr anemia  Inc. fibrinogen  3’tilt

 Incr. macrocyte  Inc. alpha-1-  Inc. vibration

 Dec. microcyte globulin  Inc.

 Dec. poikilocyte  Inc. alpha-2- temperature

 Dec. globulin  Inc. long tube

polycythemia  Dec. albumin  Inc. large bore

 Dec. lectin  Dec. cold

temperature

 Dec. short tube

  Dec. small bore


HIGH ESR LOW ESR

1. Pregnancy 1. polycythemia

2. Menstauration 2. poikilocytosis

3. Nephrosis 3. spherocyte

4. Tuberculosis 4. sickle cell

5. Hepatits 5. Hgb CC

6. Inflammation 6. Hypofibrinogenemia

7. Acute/chromic infection 7. Congestive heart failure

8. Hypo/hyperthyroidism

9. Myocardial infarction

10.Bacterial endocarditis

11.Rheumatic fever

12.Cryoglobulinemia

13.Waldenstrom

14.macroglobulinemia

 ZETA SEDIMENTATION RATIO

a. Centrifuge used: zeta centrifuge

b. Use spin capillary tubes

c. Reading is called ZETACRIT

d. Ratio

True hematocrit X 100

Zetacrit%

e. Normal value : 40-51% male and female

SPECIAL HEMATOLOGIC TESTS

Procedure to count eosinophils

1. Eosinophil count

a. Phloxine: Phloxine B, propylene glycol and sodium carbonate

Stains eosnophils red and lyses other cells

b. Dunger solution: Eosin stain

 OTHER Diluting fluids

1. PILOTS

2. MANNERS

3. RANDOLPHS
 4. HINKLEMAN

5. TANNENS

 Method of eosinophilic count

1. Thorns method

o Counting chamber used: fuchs—rosenthal chamber

o No. of squares counted: 16 big squares

o Area in sq.mm= 16 sq.mm

o Test adrenal cortical function

o Eosinophil high before fasting

o Eosinophil count 2-4 hours after giving ACTH

o Hypoadrenalism equal before fasting and after giving ACTH

2. Osmotic fragilty test

o Used to diagnose hereditary spherocytosis

o Increased OFT: decreased resistance

 Hemolytic anemia

 Hereditary spherocytosis

o Decreased OFT:
  SPLENECTOMY

 In liver disease

 Sickle cell anemia

 Iron-deficiency anemia

 Thalassemia

QUALITY CONTROL

1. Commercial control

 3 levels

a. Low

b. Normal

c. High

 Values stored in instrument computer

 Levey-jennings graph stored for each parameter

2. Mode to mode

 Most automated hematology instrument

 Primary and secondary mode

  Controls run on BOTH

 PRIMARY: automated or closed

 SECONDARY: manual or open

3. Delta checks

 LIS and instrument are interfaced

 Delta checks are conducted by LIS on select parameters

Automation

 2 general principles

1. Electronic impedance: measures volume ONLY

2. Light scattering: measures volume and internal characteristics of cells.

Known as coulture

a. Orthongal reflects structures inside cells

b. Forward angle reflects size and volume