HEMOGLOBIN DETERMINATION

BOND KING NITISH RAHUL DEV

I. COLORIMETRIC METHOD

A. Direct visual colorimetric Method Tall quist method Dare’s Hemoglobinometer Acid Hematin method Alkaline Hematin method

B. Photoelectric colorimetric method1. Oxyhemoglobin method Measures normal hemoglobin Used 0.007 N NH4OH or 0.1%

Na2Co3 Read with the wavelength at 540 nm

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)

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.

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.

Interpretation :- Maintain = equal to 1.053 = 12.5 gm

% Sink = > 1.053 = >12.5 gm% Float = < 1.053 = <12.5 gm%

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.

RETICULOCYTE COUNTING

I. Wet method New methylene blue method Cook, meyer and tureen seiverd’s methodProcedure 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.

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

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.

COMPUTATION

% Reticulocytes count = no.of retics.counted X 100

1000 RBCs

Example :-

12/1000X 100 = 1.2% normal in adult

RBC COUNT

A. Hemocytometry method (microscopic method) (used hemoglobinometer)

Diluting fluids Thoma pipets Counting chambers /

Hemocytometer

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

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

Thoma pipet

Bead

Bulb/ mixing chamber

Long stem

Short stem

3. Counting chamber

H-shaped moat

Raised platform

Counting chamber

Drawing ruled area

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.

WBC

WBCWBC

WBC

R

RR

R R

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.

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

Errors

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

Never leave that till tomorrow which you can do today.

Thank you.