Course: 2nd year Nursing Faculty: Pathology Kanachur Institute of Medical Sciences

1. ● Hemoglobin estimation
● RBC, WBC and platelet
counts
Dr. Salman Ansari
Dept. of Pathology
Kanachur Institute of Medical Sciences

2. Contents
● Hemoglobin estimation
● RBC count
● WBC count
● Platelet count

3. HEMOGLOBIN ESTIMATION

4. Intro
- Hb carries oxygen from lungs to tissues
- Carbon dioxide from tissues to lungs

7. Causes of low hemoglobin
● Decreased intake
● Menstrual bleeding
● Bleeding from any cause
● Cancer
● Chemotherapy

8. Causes of increased hemoglobin
● Polycythemia vera
● lung/heart disorders
● Renal cancer
● Smoking
● hypoxemia

9. When to check hemoglobin?
Indications for Hb estimation:
- if anemia is suspected(low hemoglobin)
- if polycythemia is suspected(high hemoglobin)
- to monitor anemia treatment
- to estimate red cell indices
- for selecting blood donors

10. Methods of estimation
- Colorimetric estimation - by comparing colour of
sample. E.g: Sahli’s method
- Physical method: specific gravity
- Chemical method: iron content of Hb
- Gasometric method: O2-carrying capacity of Hb

12. Sahli’s acid hematin method
Principle:
Hb is converted into acid hematin by N/10 HCl, which
produces brown colour which is matched with reference
tube.
Intensity of brown colour depends on amount of acid
hematin and amount of Hb in blood sample.

13. Equipment needed
1. Sahli hemoglobinometer
2. Sahli pipette
3. Glass rod stirrer
4. Dropping pipette
5. Reagents: N/10 hydrochloric acid, distilled water

15. Procedure
1. Take N/10 HCl with a dropper into the hemoglobinometer
tube up to the lowest mark.
2. Draw blood up to 20 cu mm (0.02 mL) mark of Hb pipette
and wipe off the excess blood
3. Entire blood in the Hb pipette is transferred into the tube of
hemoglobinometer.
4. Mix well and leave to stand for 10 minutes, which allows
formation of acid hematin(brown colour)

16. 9. If the solution in the tube is darker, add distilled water
with a dropper and stir the solution with a glass rod.
Repeat till the color of the solution exactly matches
the color of the comparator.
10.Take the tube out and remove the glass rod. Take
the reading from the lower meniscus.
11.Hemoglobin is recorded as gram/dL (or gm%).

18. Disadvantages
- Reading is very subjective, so results may vary
- High chance of technical errors due to improper mixing of
blood, pipetting error
- Carboxy Hb, meth Hb and sulph Hb cannot be converted with
this method
- Colour of comparator may fade with time

19. RBC count

20. Normal range
Adult male: 4.5 - 5.5 million/mm3
Adult female: 3.8 - 5.2 million/mm3
At birth: 4.0 - 6.0 million/mm3

21. RBC count
Equipment:
● Hemocytometer(Counting chamber)
● RBC pipette
● Coverslip
● Diluting fluid

25. Principle:
Blood is mixed with an isotonic solution and the RBCs
in a portion of diluting fluid are counted in the chamber
under microscope

26. Procedure
1. Take blood up to 0.5 mark in the RBC pipette and wipe
off excess blood on the outside of the pipette
2. Place the tip of the pipette in the diluting fluid and with
suction draw fluid till it reaches the 101 mark above the
bulb
3. Mix the contents by holding the pipette in horizontal
position

27. 4. Discard 3-4 drops of fluid from the pipette
5. Put a coverslip on the ruled area of the chamber
and touch the free end of the pipette with the
coverslip and pinch the aspiration tube. Fluid will
move into it via capillary action

28. 4. After 5 minutes, count the RBCs in the small squares in
each corner of the ruled area and at the central
square(16 x 5 = 80 squares in total)
5. Multiply the total number by 10,000 and express results
as:
Total RBC count = ___/mm3 of blood

31. WBC Count

32. WBC count
- Number of WBCs in 1 microlitre of blood
- It is done to look for increase or decrease in total
number of white cells in blood
- Leukocytosis: increase in total number of WBCs
- Leukopenia: decrease in total number of WBCs
- Done in any patient with fever or blood disorders

33. Normal range

34. Causes of high WBC count(leukocytosis)
● Bone marrow
disorders. E.g: CML
● Infections: bacterial
● Autoimmune
disorders: E.g:
Rheumatoid arthritis

35. Causes of low WBC count(leukopenia)
● Viral infection
● Autoimmune disease
● Cancers
● Hypersplenism
● Drug-induced: chemotherapy

36. WBC estimation - Manual method
Blood sample mixed with diluent which breaks down RBCs
and stains nucleus of WBCs
WBCs are then counted in a hemocytometer counting
chamber(Neubauer chamber)
Equipment:
- Neubauer chamber
- WBC pipette
- Test tube, coverslip, diluting fluid

41. Procedure
1. Aspirate blood upto 0.5 mark and wipe off excess blood
2. Place tip of pipette in diluting fluid and draw up to 11 mark
above bulb
3. Mix contents by holding pipette in horizontal position
4. Discard 3-4 drops
5. Put a coverslip on ruled area of the hemocytometer
6. Touch the free end of the coverslip and pinch the
aspiration tube. Fluid flows under the coverslip due to
capillary action

42. 7. After 5 minutes, count the total number of WBCs in the 4
big corner squares, and multiply by 50 to obtain WBC
count. Express results as:
Total WBC count: ____ / mm3 of blood

44. PLATELET COUNT

45. Platelet Count
- Can be counted manually under a microscope or with
machine
- Difficult to count(small size)
- Normal count: 150,000-400,000/mm3
- >400,000: high platelet count - thrombocytosis
- <150,000: low platelet count - thrombocytopenia
- Platelet count <20,000: spontaneous, heavy bleeding

46. Causes of high platelet count
Thrombocytosis:
- Bleeding
- Cancers
- Hemolytic anemia

47. Causes of low platelet count
Thrombocytopenia:
- Leukemia
- Idiopathic thrombocytopenia purpura(ITP)
- medications(like heparin)
- Heavy alcohol consumption

48. When to check platelet count?
- Suspected bleeding disorder

49. Manual method
- Using RBC pipette, Neubauer’s chamber - central
square(similar to RBC count)
- Count in the central square after 15 minutes
- Calculation: multiply the number of platelets counted by 2000

52. References:
● Dr. Purnima S. Rao - Textbook of Pathology & Genetics For
Nursing
Questions:
salman.s.ansari92@gmail.com