Hematology Technique 1

Moderate leucocytosis and Thrmbocytosis is seen post prandially

Inc in RBC ,haptoglobin following exercise loss of plasma + re entry from marginal capillaries to circulation

Exercise – brief ,severe – mild lymphocytosis Prolonged – neutrophilia – release from marginal pool

into circulation Stress fibrinolysis ,leucocytosis Hb and PCV is 5% less in recumbant than in upright

position PCV – hands horizontal -2-4% lower than in vertical Smoking --. Inc erythropoiesis ,plt hyper

aggregability,dec platelet survival Serum Iron – maximum at morning and lowest at

evening – 30 % variation seen

Capillary blood collection

Adult – Middle or ring finger Infants – Great toe / side of heel 70% spirit Wipe away the first one or two drops and

use subsequent drops Puncture – just off centre Perpendicular to the finger print ridges

Tests done using capillary blood

1. Hb

2. PS

3. BG

4. WBC count

5. Osmotic fragility

6. Reticulocyte count

Excessive anticoagulant – false low PCV False leucopenia Mix the tubes with anticoagulant additives

gently 5 – 10 times Blood storage – 4C

Anticoagulants1. Double oxalate

2. EDTA

3. Tri Sodium Citrate

4. Heparin

5. Acid Citrate Dextrose and Citrate Phosphate Dextrose

6. Defibrinated Blood

Double oxalate Wintrobe’s mixture Ammonium Oxalate + Potassium Oxalate Used in1. PCV2. ESR3. Hb4. WBC Count5. PT K – shrink the RBC Ammonium – swells the RBC Not suitable for Peripheral smear

EDTA Most frequently used Anticoagulant Can be used for Platelet count Action –Combines with ionized Calcium Unsuitable for Calcium and Iron analysis Na3 –EDTA – high PH not used K2 –EDTA – MC used more soluble

,better chelating agent Inhibits Platelet Function and factor 8- not

used for routine studies of homeostasis

Tri Sodium Citrate

Usual anticoagulant for Coagulation investigation

Used for PT and Plasma Coagulation studies

Heparin

For determining Carboxy Hb Osmotic fragility Red cell enzyme determinants Li salt of Heparin is used Causes Clumping of Platelets & Leucocytes Not

useful for Blood cell counting Inactivates 10a and 2 a Platelets and WBC show clumping and blood film

stain with a bluish tinge if heparin is used as anticoagulant

ACD & CPD

Anti coagulants in Blood Banking Mostly used now is CPD with Adenine – CPDA The storage life of RBC in ACD =21 days CPDA = 35 days Adenine will hastens the loss of 2,3 DPG from

the Red cells Citrate removes Calcium by forming a loose

ionic complex

Defibrinated blood

Blood agitated continuously using glass beads or paper slips Fibrin clots around these bodies removed from the sample together with platelets leaving RBC and WBC suspended in serum

When anticoagulant is contra indicated LE cell preperation

Specimen Stability

At room temperature with EDTA –for Hb , PCV ,red cell indices ,counts ---stable up to 8 hours

Long term storage

- 70 C Lyophilization & storage at -20C Containers – for coagulation and platelet

studies – siliconized containers to prevent Contact activation

Preparation of Blood1. Diluted Blood – counts ,Hb estimation- not stable –

platelet count (30 min) …others ( 2 hrs)2. Blood Smear – shud be stained 2 hrs after it is

stained…after staining keep indefinitly3. Oxalated Blood – ESR –within 2hrs …PCV – one

day Blood grouping and R h -2 days4. Blood Plasma – PT ,Prothrombin Consumption

test , Thromboplastin Generation test,FibrinogenDefficiency Test

5. Blood Serum – no fibrinogen – Coomb’s test ,Rh titre ,Heterophile Antibody test

6. Cell suspension- grouping , Rh typing ,titre , Coomb’s test

Hb estimation-Sahli’s acid haematin method Other methods1. Cyanmethaemoglobin method –best available

method2. Measurement of Iron content – Wong’s Method3. Estimation of free protoporphyrin in RBC4. Oxy Hb method5. Alkali haemetin method6. Acid hematin method 7. Autmated / semi automated method

Sahli’s method

N/10 HCl converts Hb to acid hematin N/10 HCl upto the lowest mark Blood is sucked upto 20 mm3 mark in pipette Allow for 10 minutes 95% of adult Hb acid Hematin in 10 min 1 hr – for 100 % conversion Acid hematin is a colloid suspension so it cannot

be read by colourimetrically Only adult H forms acid hematin

Cyan metHb method

Hb cyan met Hb by Potassium ferricyanide

All derivatives except sulphHb Drabkin’s solution – diluting solution1. Potassium ferricyanide2. Potassium cyanide3. Distilled water

At birth – 13.5 – 19.5 g% Infants 11 – 13 g% 10- 12 yrs – 11.5 – 14.5 g% Men 14 – 18 g% Females -11.5 – 16.5 g% Raised Hb polycythemia ,smoking ,

Hypoxia and some renal conditions excess erythropoietin

Lowered anemia Clinical symptoms appear only below 7 – 9

g%

Hb A1 c

Glucose level of 4 – 6 weeks RBC insulin independent glucose intake Glu attaches to Hb A glycosylation /

Glycation HbA1c Remains for the life time of the RBC Normal range -3-6.5% of Normal Hb

Variation in HbA1c

Increase

1. Elevated level of Fetal Hb

2. < 2 yrs

3. Some hemoglobinopathies Decreased value

1. Blood containing Hb S ,D ,G ,C,E

Hematocrit

Percentage of packed red cells in 100 ml of blood

Wintrobe’s haematocrit tube Anti coagulant EDTA Fill upto mark 10 Centrifuge at 3000 rpm at 30 min 1mm =0.34 g Hb =1 lakh RBC / mm3

Layers

1. Plasma

2. Platelet

3. WBC

4. Nucleated RBC

5. Reticulocyte

6. Mature RBC

Buffy coat

WBC +Plt 1mm = 10 000 WBC /mm3

Normal PCV value

infants 42 – 54% Children 35 – 38 % Male 40 – 54% Females 35 – 45%

Inc –

1. Chronic hypoxia associated with pulmonary disease and congenital heart disease

2. PRV

3. Lowered plasma volume Lowered PCV

1. Acute blood loss

2. Anemia

PCV > 45% associated with thromboembolic disorders

PCV > 52 % - always investigated 15% of PRV leukemia which is resistant to

chemo therapy

Red cell indices

Mean corpuscular volume / MCV =85+_8 fl

MeanCorpuscularHb/MCH =29.5 +_ 2.5pg MCHC = 32.5 +_ 2.5 g/dL

MCV /RBC <12 T minor MCV / RBC > 14 Iron deficiency /

lead poisoning High MCHC1. Heriditary spherocytosis2. ABO incompatability3. AIHA

Erythrocyte Sedimentation Rate

When Anti coagulated blood is allowed to stand undisturbed the red cells sediment

Discrete RBC sediment slowly Aggregates of RBC settles more quickly Rate of sedimentation is directly proportional to

the square of the radius of the aggregate – Stoke’s law

ESR is the falling of a red cell coloum in 1 hour in mm

Factors influencing ESR

Internal factors

1. Effect of the plasma proteins

2. Influence of the plasma viscosity

3. Effect of the red cell factors

External factors affecting ESR

More dilution with anticoagulant more rapid falling Taller the column – more rapid falling Temperature above 27C and below 22C require

correction Time of blood collection –after 2 hrs ESR will be low Diameter of the tube –larger the diameter faster the

rate Position of the tube 3degree inclination 30% inc in

ESR

Stages of ESR

Formation of Rouleaux – 10 min Rapid settling – 40 min Final packing – 10 min

Modified Esrite method

Venous blood is mixed with 3.8% sodium citrate in the ratio 4:1

Push upto 0 level Take reading after 1 hour Nl – 1-7mm in first hour males 3 – 12 mm / one hour in females

Variations

Inc – pregnancy ,menstruation ,Inflammation ,TB , RA , Syphilis , MM

Dec – New born , PV , SS , CCF

Zeta Sedimentation rate

Effect of plasma macro molecules in decreasing the zeta potential of adjescent red cells

Measures the closeness with which the red cells aproach one another under a standard stress

Zetacrit – height of the red cells to the total height of the blood column

ZSR – expressed in % 40 – 51 %

Plasma viscosity

Blood cell counting

Diluting Fluid

1. Turk’s fluid – WBC count

2. Hymes fluid and Gower’s fluid for RBC count

3. Rees and Ecker solution for Platelet count

Counting chambers

1. Improved Neubaur

2. Fuch Rosenthal hemocytometer

WBC counting

To determine the no. of WBC per mm3 Turk’s fluid1. Glacial acetic acid – mordant for nuclear

stain ,dissolves granules out of WBC and Hb2. Gentian violet – stains nuclei3. Distilled water – lyses RBC Blood upto 0. TF upto 11 Difference in WBC in two big squares shud not

exceed 12

Calculation

Dilution correction 20 One big square = w Total 4w volue = 1*1*0.1 *4 = 0.4 mm3 So for 1mm3 *2.5 Totally =w* 20 * 2.5 =w*50 /mm3

Correction for nucleated RBC – in Hemolytic anemia and Erythroblastosis foetalis

Eosinophil count – diluting fluid – Actone and Eosin