hematology

1. Hematology Technique 1

2.  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

3. 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

4. Tests done using capillary blood
1. Hb
2. PS
3. BG
4. WBC count
5. Osmotic fragility
6. Reticulocyte count

5.  Excessive anticoagulant – false low PCV
 False leucopenia
 Mix the tubes with anticoagulant additives
gently 5 – 10 times
 Blood storage – 4C

6. Anticoagulants
1. Double oxalate
2. EDTA
3. Tri Sodium Citrate
4. Heparin
5. Acid Citrate Dextrose and Citrate Phosphate
Dextrose
6. Defibrinated Blood

7. Double oxalate
 Wintrobe’s mixture
 Ammonium Oxalate + Potassium Oxalate
 Used in
1. PCV
2. ESR
3. Hb
4. WBC Count
5. PT
 K – shrink the RBC
 Ammonium – swells the RBC
 Not suitable for Peripheral smear

8. 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

9. Tri Sodium Citrate
 Usual anticoagulant for Coagulation
investigation
 Used for PT and Plasma Coagulation
studies

10. 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

11. 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

12. 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

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

14. Long term storage
 - 70 C
 Lyophilization & storage at -20C
 Containers – for coagulation and platelet
studies – siliconized containers to prevent
Contact activation

15. Preparation of Blood
1. 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 indefinitly
3. Oxalated Blood – ESR –within 2hrs …PCV – one
day Blood grouping and R h -2 days
4. 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

16. Hb estimation-Sahli’s acid
haematin method
 Other methods
1. Cyanmethaemoglobin method –best available
method
2. Measurement of Iron content – Wong’s Method
3. Estimation of free protoporphyrin in RBC
4. Oxy Hb method
5. Alkali haemetin method
6. Acid hematin method
7. Autmated / semi automated method

17. 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

18. Cyan metHb method
 Hb  cyan met Hb by Potassium
ferricyanide
 All derivatives except sulphHb
 Drabkin’s solution – diluting solution
1. Potassium ferricyanide
2. Potassium cyanide
3. Distilled water

19.  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%

20. 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

21. 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

22. 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

24. Layers
1. Plasma
2. Platelet
3. WBC
4. Nucleated RBC
5. Reticulocyte
6. Mature RBC

25. Buffy coat
 WBC +Plt
 1mm = 10 000 WBC /mm3

26. Normal PCV value
 infants 42 – 54%
 Children 35 – 38 %
 Male 40 – 54%
 Females 35 – 45%

27.  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

28.  PCV > 45% associated with thromboembolic
disorders
 PCV > 52 % - always investigated
 15% of PRV  leukemia which is resistant to
chemo therapy

29. Red cell indices
 Mean corpuscular volume / MCV =85+_8
fl
 MeanCorpuscularHb/MCH =29.5 +_ 2.5pg
 MCHC = 32.5 +_ 2.5 g/dL

32.  MCV /RBC <12  T minor
 MCV / RBC > 14  Iron deficiency /
lead poisoning
 High MCHC
1. Heriditary spherocytosis
2. ABO incompatability
3. AIHA

34. 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

36. Factors influencing ESR
 Internal factors
1. Effect of the plasma proteins
2. Influence of the plasma viscosity
3. Effect of the red cell factors

37. 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

38. Stages of ESR
 Formation of Rouleaux – 10 min
 Rapid settling – 40 min
 Final packing – 10 min

41. 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

42. Variations
 Inc – pregnancy ,menstruation
,Inflammation ,TB , RA , Syphilis , MM
 Dec – New born , PV , SS , CCF

43. 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 %

44. Plasma viscosity

45. 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

46. Counting chambers
1. Improved Neubaur
2. Fuch Rosenthal hemocytometer

47. WBC counting
 To determine the no. of WBC per mm3
 Turk’s fluid
1. Glacial acetic acid – mordant for nuclear
stain ,dissolves granules out of WBC and Hb
2. Gentian violet – stains nuclei
3. Distilled water – lyses RBC
 Blood upto 0.
 TF upto 11
 Difference in WBC in two big squares shud not
exceed 12

49. 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

50.  Correction for nucleated RBC – in
Hemolytic anemia and Erythroblastosis
foetalis
 Eosinophil count – diluting fluid – Actone
and Eosin