Haemoglobin estimation practical is for students who are doing DMLT or PGDMLT. Methods 1. Sahli acid haematin method 2. Cyanmethhaemoglobin method

1. DR. RAJASHREE MUTHA
MBBS,DPB
(Pathology &Bacteriology)

2.  Defn: It is conjugated protein, synthesized in RBC, It gives red colour to blood. It
consists of 2 components, Haem & Globin.
 Functions:
 To carry oxygen from lungs to tissues and
 To carry carbon dioxide away from tissues to lungs.
 Acid Base balance
 Normal Range
 Males-14-18 gm/dl
 Females 11.5-14.5 gm/dl
 Clinical Significance
 Hb- Low- Haemodilution-Anemia,Pregnancy
 Hb-High: Haemoconcentration
 -Dehydration-Diarrhoea, Vomiting
 -Polycythemia
 -Congenital heart disease
 -Chronic lung disease

3. Methods of Haemoglobin estimation:
1. Sahli method (Acid haematin method)
2. Colorimetric (Cyanmethhaemoglobin method)
3. Gasometric-Hb calculated by O2 carrying capacity
4. Chemical method – Acid hematin or alkaline
haematin method
5. Specific gravity method – CuSo4 (Blood bank)

4. DR. RAJASHREE MUTHA
MBBS,DPB
(Path&Bact)

5.  It consists of
 1. Haemoglobin pipette
2. Haemoglobin tube
 3. Comparator- brown glass
 Use :
 1. Haemoglobin estimation

6.  When blood is added to 0.1N hydrochloric acid,
haemoglobin is converted to brown colored
acid haematin.
 This acid haematin after dilutation, compared
with standard brown glass of a sahli’s
haemoglobinometer.

7.  Sample Capillary blood or thoroughly mixed
anticoagulated (E.D.T.A.)
 • With the help of Pasture pipette add 0.1 N
hydrochloric acid in the haemoglobin tube up to the
lowest (20%)mark.
 • Draw blood up to 20μl in the Hb-pipette in such way
that it does not contain air bubble. Wipe excess
blood with dry cotton.
 • Transfer the blood to the haemoglobin tube, mix
well and allow the tube to stand for 10 minutes.
 • Dilute the solution by adding distilled water drop by
drop and mix carefully, until the color of the solution
gets mixed with brown color plates in comparator.
 • The level of the solution is noted in gm/dl.

9.  1. When blood is mixed with Drabkin’s reagent
containing potassium ferricyanide and potassium
cyanide, haemoglobin reacts with potassium
ferricyanide, and converted to
cyanamethamoglobin (HCN) by the cyanide.
 2. The intensity of color is proportional to
haemoglobin concentration and it is compared
with a known cyanamethamoglobin standard at
540 nm (green filter) using colorimeter.

10.  Sample Capillary blood or anticoagulated (E.D.T.A.)
blood.
 Pipette in the tube labeled as follows:
 Additions Test tube Blank tube
 Drabkin’s reagent, ml 5.0 5.0
 Blood, ml 0.02 ---
 • Mix well and allow the tube to stand for 5 minutes.
 • Read the absorbance of Test by setting blank to
zero at 540nm (green filter).
 • Read the absorbance of standard. 15 g/dl.
Calculate the concentration of test using following
formula.

11. Calculation:
O.D.TEST
Haemoglobin g/dl = ————— × Conc.of Std.(15)
O.D. STD
Result
The Haemoglobin concentration of the given
blood sample by Drabkin’s method
is ————g/dl

12. 1. Essentials of Medical Microbiology. Shastry A.
India : Jaypee publication;2020
2. Textbook of microbiology, Anantnarayan and
panikar.
3. Text book of Medical Parasitology, Panikar
4. Swayam Prabha lectures
5. Text book of P. Godkar
6. Images drawn by student, kindly refer book for
correction.

13. 1. Volume of blood taken in the Hb pipette for
Haemoglobin estimation.
 10ul 5 ul 50 ul 20 ul
2. Sahli method is based on principle –
 Acid Haematin Alkaline Haematin
Neutral Haematin None
3. Colorimetry method is based on principle –
 Acid Haematin Alkaline Haematin
cyanamethamoglobin None
4. Haemoglobin is decreased in----
 Anaemia Polycythemia
 Heart disease None
5. Haemoglobin is increased in----
 Anaemia Polycythemia
 Pregnancy None

14. DR. RAJASHREE
MUTHA
MBBS,DPB
(Path&Bact)