This document discusses various methods for determining hemoglobin levels and performing complete blood counts. It describes colorimetric methods, including direct visual and photoelectric techniques. Specific gravity and gasometric methods are also covered. Procedures are provided for reticulocyte counting using wet and dry methods. The hemocytometry method for red blood cell counting is explained in detail, including use of diluting fluids, Thoma pipettes, and counting chambers.

1. HEMOGLOBIN
DETERMINATION
BOND KING
NITISH
RAHUL DEV

2. I. COLORIMETRIC
METHOD
A. Direct visual colorimetric Method
 Tall quist method
 Dare’s Hemoglobinometer
 Acid Hematin method
 Alkaline Hematin method

3. B. Photoelectric colorimetric method
1. Oxyhemoglobin method
 Measures normal hemoglobin
 Used 0.007 N NH4OH or 0.1% Na2Co3
 Read with the wavelength at 540 nm

4. 2. Cyanmethemoglobin (HiCN)
 Also known as hemiglobin cyanide or
ferrihemoglobin cyanide
 All forms of hemoglobin are measured
except sulfohemoglobin
 Uses Drabkin’s solution
 Potassium ferricyanide
 Potassium cyanide
 Dihydrogen potassium phospate
 Distilled water
 PH = 7.0-7.4 ( blood capacity )
 Used sahli pipet= (0.02ml or 20 micro liter)

5. PROCEDURE
 Place 5ml of Drabkin’s reagent into a
testube
 Get 0.02 ml of whole blood using sahli pipet
 Place the 0.02 ml of blood in to drabkin's
reagent through rinsing it.
 Mix and let it stand for 10 minutes
 Read in a spectrophotometer at 540 nm.

6. II. Specific gravity method/Gravitational
method CUSO4 method
 Specific gravity of copper sulfate = 1.053
with an hemoglobin equivalent of 12.5 gm%
 Mass blood
Procedure
 Collect blood sample
 Drop a blood into the solution
 Observe the activity of the blood
 Within 12 seconds, describe how the drop
of blood behaves.

7. Interpretation :-
 Maintain = equal to 1.053 = 12.5 gm%
 Sink = > 1.053 = >12.5 gm%
 Float = < 1.053 = <12.5 gm%

8. III. Gasometric Method
 Indirect method
 Based on the assumption that 1gm Hb
can carry approximately 1.34 ml O2.
IV. Chemical Method
 Indirect method
 Based on the assumption that 1gm Hb
contains approximately 3.47 mg iron.

9. RETICULOCYTE COUNTING
I. Wet method
 New methylene blue method
 Cook, meyer and tureen
 seiverd’s method
Procedure
 Get blood sample
 Secure equal proportion of blood and stain.
 Mix it and letit stand for 10 minutes
 Make a smear.
 Dry the smear
 Examine under microscope using OIO
 Count reticulocytes in relation to 1,000 RBC.

10. II. Dry method
 Schiling’s rapid method =(BCB method).
 Sabin’s method = (janus green /neutral
red)
 Seiverd’s method =(BCB method)
 Osogood- wilhelm method = (new
methylene blue method)
BCB = Brilliant crystal Blue

11. PROCEDURE
 Spread stain thinly on a glass slide and air
dry.
 Place a small drop of blood upon the layer
of the dried stain.
 Place a cover slip on the drop of blood.
 Allow to stand for 10 minutes
 Examine under the microscope under OIO
 Count reticulocytes in relation to 1,000
RBCs.

12. COMPUTATION
% Reticulocytes count = no.of retics.counted X 100
1000 RBCs
Example :-
12/1000X 100 = 1.2% normal in adult

13. RBC COUNT
A. Hemocytometry method (microscopic
method) (used hemoglobinometer)
 Diluting fluids
 Thoma pipets
 Counting chambers / Hemocytometer

14. 1.Diluting fluids
 Hayerm’s
 Gower’s
 Toisson’s
 Bethel’s
 Formol-citrate/Dacies solutn
 NSS
 3.8 % sodium citrate
- easy to prepare
- must have preservative method
- must be safe
- no corrosive, non-caustic
- should be isotonic

15. 2.Thoma pipet
 Bead - identification of type of pipet
- used for mixing
- seperating color
 Upper calibration of RBC pipet = 101
 Capacity of bulb is 100 times capacity of
stem
 Constant volume of RBC pipet = 100[ 101-1]
RBC thoma- red bead
WBC thoma – white bead

16. Thoma pipet
Bulb/ mixing chamber
Short stem
Bead
Long stem

17. 3. Counting chamber
Raised platform
Drawing ruled area
Counting chamber
H-shaped
moat

18. Counting chamber
 Improved neubaber- commonly used
 Cover slip= depth of the counting chamber
(0.1mm)
 1 ruled area = 1mm2
 1 large square width and length 1mm
 Center of large square have 25 small
squares and each 25 small square has 16
small square which is used in RBC count.
 Total 400 small square are found in center
of large square.

19. WBC
WBC
R R
R
R R
WBC
WBC

21. Procedure
 Collect blood
 Suck blood to 0.5 mark of the pipet.
 Suck diluting fluid to 101 mark.
 Shake pipet for 2 minutes.
 Discard first few drops.
 Charge the counting chamber at an angle from
30- 35 degree.
 Count the RBC under HPO using 5 RBC squares
of central large square
 Compute.

22. Computation
 RBC count = RBC counted X DCF X VCF
 DCF = Volume of blood / amt of blood
sucked
 VCF = volume desired / area x depth of
the counting chamber x nos of squares
used.
DCF= diluting correction factor
VCF = volume correction factor
 For RBC pipet DCF = 200 and VCF is 50
 VCF = 1/ 0.04x 0.1x 5 =50

23. Errors
Technical error
 Pipetting
 Shaking the pipet
 Charging the counting chamber
 Application of cover slip
 Counting of the cells
 Computation
 Reporting of results.

24. Never leave that till tomorrow which you can do
today.
Thank you.