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Hemoglobin Estimation

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46 views2 pages

Hemoglobin Estimation

Uploaded by

chamlingraikasish
Copyright
© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
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ESTIMATION OF HEMOGLOBIN BY SAHLI’s METHOD a.k.a.

ACID HEMATIN METHOD


PRINCIPLE OF SAHLI’s METHOD / ACID HEMATIN METHOD:
When the blood is added to N/10 Hydrochloric acid (HCl), the hemoglobin present in RBCs is
converted to acid hematin which is a dark brown colored compound. The color of the formed
acid hematin complex corresponds to the Hemoglobin concentration in the blood and is matched
with the standard which is a reference brown glass given in the Sahli’s apparatus by diluting with
N/10 hydrochloric acid or distilled water until the color of acid hematin complex match with the
color of the standard.

REAGENTS REQUIRED FOR SAHLI’s METHOD / ACID HEMATIN METHOD


 N/10 hydrochloric acid (It is prepared by diluting concentrated hydrochloric acid 0.98 ml
in distilled water and volume is made up 100 ml).
 Distilled water

APPARATUS & EQUIPMENTS REQUIRED FOR SAHLI’s METHOD / ACID


HEMATIN METHOD
 Sahli’s Apparatus
o Hemoglobin pipette (0.02 ml or 20 µl capacity)
o Sahli’s graduated Hemoglobin tube
o Thin glass rod Stirrer for Hemoglobin Tube
o Sahli’s Comparator box with brown glass standard
o Spirit swab
o Blood Lancet
o Dry cotton swab
o Pasteur pipette
PROCEDURE OF SAHLI’s METHOD / ACID HEMATIN METHOD:
 Place N/10 Hydrochloric acid is taken in Hemoglobin tube (has two graduations – one
side gm/dl, and other side shows the Hb %age), up to the mark 2 gm% – the lowest
marking (yellow marking).
 Take blood sample in Sahli’s pipette exactly up to 20 µl mark.
 Wipe out the surface of the pipette with the help of tissue paper/ cotton so that excess
blood may not be added to the Hb tube.
 Add the blood into N/10 hydrochloric acid taken in the hemoglobin tube, rinse the pipette
with the same solution and mix properly with the help of stirrer.
 Place the tube at room temperature for 10 minutes for complete conversion of
hemoglobin into acid hematin.
 After the reaction completes, place the Hb tube in the column in Sahli’s Comparator box
and start diluting the dark brown coloured compound (Acid Hematin) formed in the Hb
tube using the N/10 HCl or distilled water by adding drop by drop of it into the solution
and mix with the help of stirrer after each addition.
 This process is done until the endpoint comes matching the color of standard with the
color of the test.
 Once the color is matched with the standard brown glass, lift the stirrer up and note down
the reading in Sahli’s Hb tube by taking the lower meniscus in consideration.

Note:

 Although the graduated tube is marked in both grams and percent figures, result should
always be reported in grams. This is because :
a) No single hemoglobin value can be considered as 100% since it varies with the
age and sex of the individual and altitude.
b) Hemoglobinometers of different manufacturers have different values as 100% so
that same sample of blood will yield different results on different instruments.

ADVANTAGES OF SAHLI’s METHOD / ACID HEMATIN METHOD


⇒ It is the simple and easy method and may be done at any place because apparatus can be
picked up anywhere.

DISADVANTAGES OF SAHLI’s METHOD / ACID HEMATIN METHOD


 Perfect matching with the brown glass standard is not possible.
 Carboxyhemoglobin, methemoglobin and sulfhemoglobin are not converted to acid
hematin.
 HbF is also not converted to acid hematin and therefore this method is not suitable in
small infants.
 Development of colour is slow and acid hematin is not stable.
 Source of light will influence the visual comparision of colors.
 Personal error in matching brown glass standard with test solution.
 Color of brown glass standard fades with time.

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