Hemoglobin Test; How to Perform:

Apparatus:

Hemoglobinometer, pricking needle (blood lancet) and spirit swab.

Table of Contents

Sahli’s method:

Hemoglobinometer:

It is a box which contains:

Stirrer: Simple glass rod used to mix the blood with acid or distilled water.
Hemoglobin pipette: it is a simple pipette having capillary tube in it. This is connected to the teat (mouth piece) through a rubber tube. There is ‘a marking on the pipette indicating 20 cu mm (0.02 ml) volume.
Sahli’s tube (Hemoglobin tube): It is a graduated tube having markings on two sides. On one side in gm°/o (gm per 100ml) from 0 to 24 and other side 0-1700/0. Gram % means Hb in gm per 100 ml ‘of blood. Simple percent means percentage of hemoglobin in relation to normal value of Hb which is considered as 100%.
Comparator: It consisted of a box having one slot for Sahli’s (hemoglobin) tube and two non-fading standard brown tinted glass pieces for matching the color of diluted blood. A translucent white glass is Fitted at the back to provide uniform illumination for matching the color.
Simple rubber dropper: To add acid or water drop by drop.

PRINCIPLE:

The haemoglobin is synthesized by maturing nucleated RBCs in the bone marrow and remains intracellular. The red cell membranes is destroyed (hemolysis) by acid and Hb pigment is released into the plasma and forms acid hematin. So the known quantity of blood (Hb) is mixed with N/ 10 HO leading to formation of acid haematin which gives brown color. Now mixture containing acid haematin is diluted with distilled water till its color matches standard color in the glass fitted in the comparator. So the color matching is done macrosc0pically. Value of Hb is noted down from the Sahli’s tube.

PROCEDURE:

With the help of a dropper, take N/ 10 HCl in Sahli’s tube up to mark 20%.
Under all aseptic condition give a bold prick on the tip of the finger with the help of pricking needle.
Wipe off the first drop of blood and suck the blood from the second drop in Hb pipette 0.2 uL up to mark 20 cu mm. Fill the Hb pipette by capillary action.
Wipe the tip of the pipette with the help of cotton to remove sticked blood around the tip. Extra blood is removed by filter paper/tissue paper.
Blow out the blood from the pipette into acid in Sahli’s tube and rinse the pipette with the same.
Mix the blood in acid with stirrer and wait for 10 minutes.
Dilute the mixture by adding distilled water drop by drop till the color of mixture matches with standard color in comparator.
Each time you have to mix it with help of stirrer after adding the distilled water
Once the colour is matched, lift the stirrer up and note down the reading in Sahli’s tube by taking the lower meniscus in consideration.
Usually in coloured solution upper meniscus is considered for taking the reading but it is a transparent color solution and lower meniscus can be recorded.
Now add one more drop of distilled water and mix it properly. If color is still matching note down the reading, if it is lighter it shows reading taken before dilution was correct. If not add a drop of distilled water again and match the color.
Reading is expressed in Hb gm/lOO ml of blood.

Normal value of Hemoglobin:

At birth: _13, 19. s gm.
Neonatal Hb: 14 5-22.2 gm/dl or ng/%.
Child: (6-12 yrs) ’ 11.5 -1S 5 gm/dl or ng/%.
Adult male(18-49 yrs) 13.5 17.5 gm/dl or ng/% .
Adult female(18-49 yrs) 12.0 16.0 gm/dl or ng/%.

Old Methods of Hb estimation:

CuSO4 specific gravity method .
Haldane’s carboxy haemoglobin method.

New Method of Hb estimation:

Cyanmet haemoglobin method:

In this method spectrophotometer is used. The blood is treated with potassium cyanide and potassium ferri cynide, which converts ferrous state of the iron into ferric state (methaemoglobin) and form scyanmet haemoglobin. In this method the anticoagulant used is EDTA. The capillary blood can also be used. The spectrophotometer should be properly standardized at 540 nm cuvette. The dirty, scratched or unmatched cuvette and deteriorated reagents’ should not be used. Cyanmet Hb reagent is unstable in light so it should be kept in dark cabinet and in a dark glass bottle. The cap of the bottle should be tightly capped. Cyanmet Hb reagent contains cyanide which is dangerous so it standling should be very careful.

Other Methods Used for Hb Estimation:

Alkali hematin method: Alkali is used to form alkali hematin and color is matched with standard.
lron estimation method: It is based on the principle that 100 mg Hb contains 347 mg of iron. Value of iron is estimated in blood which gives value of Hb.
Vanslyke’s method: The amount of oxygen combined with Hb is measured. One gm of Hb can carry 1.34 ml of oxygen. 50 quantity of oxygen is measured in 100 ml of blood.
Tallquist scale method: In this method a drop of blood is absorbed on an absorbent paper and color of this paper is matched with standard.
Haldane’s carbox hemoglobin method.
CuSO4 specific gravity method.
Cyanmethaemoglobin method.

RESULT:

The age and sex of the subject are noted. The result is written in the following pattern:

Average =-15.2 gm/dl
Slightlylighterthan the standard = 14.2 gm/dl 4.
Exact reading = 15 gm/dl
Slightly darker than standard = 15.2 gm/dl
% Hb =-15.1×100/15 =103%
% Hb = 15.1×100

PRECAUTIONS:

Hemoglobin pipettee and Sahli’s tube should be clean and dry before use.
Suck the blood exactly up to the mark of 20 cu mm.
There should not be any air bubble in the pipette with blood.
Wait for 8-10 minutes after adding the blood in acid.
Add distilled water drop by drop done avoid over dilution.
The matching of color should be done against natural source of light or electrical tube light (white light).
Blood sample and acid should be taken in accurate and precise amount.

Objectives:

Draw backes of Sahli’s method:

This method estimates only oxy Hemoglobin. Carboxy and met-hemoglobin can not be estimated.
Acid hematin is not a true solution, some degree of turbidity interferes with colour matching.

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