Notes about blood hemoglobin estimation, lecture notes to Medical Laboratory Students at Medical Laboratory Technology, Middle Technical University, Baqubah, Iraq

1. Hemoglobin estimation
Hussein A. Abid 
Lecture: 02
Middle Technical University
Technical Institute of Baquba
Medical Laboratory Technology Department
Academic year 2019-2020 – Spring semester
Medical Laboratory Technology & Quality Control Applications
Module code: MLT113
L.No.: MLT113-20-T-02

2. Hemoglobin
• Hemoglobin (Hb) is the major constituent of the
red cell cytoplasm, accounting for approximately
90% of the dry weight of the mature cell.
• It is comprised of heme and globin.
01

3. Hemoglobin
• The hemoglobin molecule is a tetramer consisting of
two pairs of similar polypeptide chains called globin
chains, and to each of the four chains is attached
heme which is a complex of iron in ferrous form
and protoporphyrin.
02

4. 03
Methods of Hb estimation
Manual
methods
Cyanmethemoglobin
method
Acid hematin method
Automated
methods
Coulter counter
Hb meter

5. 04
Methods of Hb estimation
Measurement of iron
content
Methods based on
development of color
Sahli’s (or acid
hematin) method
Cyanmethemoglobin
method
Oxyhemoglobin
method
Alkaline hematin
method
Measurement of oxygen
combining capacity

6. Coulter counter
05
Once you review the
lecture, can you
know what the unit
of Hb here!

7. Methods:
1. Cyanmethemoglobin method:
 Accurate
 Commonly used
 Recommended by ICSH (international
committee for standardization in haematology)
06

8. Principle of cyanmethemoglobin
method:
Hb (Fe+2)
𝐊 𝟑
𝐅𝐞 𝐂𝑵 𝟔
methemoglobin (Fe+3)
𝐊𝐂𝐍
cyanmethemoglobin
(Hi) (HiCN)
07
Blood
Drabkin's
solution

9. Cyanmethemoglobin method
 Measure the absorbance of the solution by using a
spectrophotometer at a wavelength = 540nm. Then
compare it with the standard solution of HiCN.
08

10. 10
Cyanmethemoglobin method
Drabkin’s reagent
Micropipette Spectrophotometer

11. Procedure of cyanmethemoglobin
method:
10

 Measured by spectrophotometer (at 540 nm) for
 Calculate Hb (g/dL) =
Absorbance of sample
Absorbance of standard
× standard concentration (g/dL)
20 μL of blood 5 mL of blood
Mix well and left 5-10 min
HiCN
Blood sample
Standard solution (provided
with kit)

12. Methods:
2. Acid haematin method:
 Principle:
Blood + 0.1N HCL Acid haematin
 Then match the color of solution with reference solution
colorimeter or colored strip (you may dilute by D.W.)
 Other names: “SAHLI’s haemoglobinometer”
 However this method is inaccurate.
11

13. Reagent and equipment for acid haematin
method:
1. Sahli’s haemoglobinometer
2. Sahli’s pipette or Micropipette
3. 0.1N HCl
4. Dropping pipette
12 Sahli’s haemoglobinometer

14. 13
reference strips

15. Procedure of acid haematin method:
100 μL HCl + 20 μL blood
𝑚𝑖𝑥 𝑤𝑒𝑙𝑙 𝑖𝑛 𝑎 𝑔𝑟𝑎𝑑𝑢𝑎𝑡𝑒𝑑 𝑡𝑢𝑏𝑒 5 𝑚𝑖𝑛
Acid haematin
 How can we read Hb value?
Compare the color of solution in the graduated tube with
that of reference strip on either side of haemoglobinometer.
Graduated tube has two scales: % and g/100 ml of whole blood.
14

16. Procedure of acid haematin method:
• If the color of graduated tube is Darker add either 0.1N
HCl or D.W -drop by drop- with pipette; mix with glass
rod until the color matches with reference strip.
• The reading in graduated tube refers to Hb level in g/dl
(some tubes give reading in %; to convert into g/dl
multiply by 0.146)
• If read is 10  Hb = 10% × 0.146 = 14.6 g/dL
15

17. Precaution
16
• Before the sample is read the solution should be clear.
• If high WBC in specimen centrifuge the specimen then
use the supernatant.
• In case of Hb S or C dilute the mixture in 1:1 ratio with
D.W. then read in colorimeter.
• In case of abnormal globins, add 0.1g of potassium
carbonate to the solution.

18. 17
Normal ranges of Hb
1. Female: 12–16 g/dL (7.4–9.9 mmol/L SI units)
2. Males: 13–18 g/dL (8.1–11.2 mmol/L SI units)
3. Pregnancy: Decreased (dilutional)
4. Elderly: Slightly decreased
5. Newborn: Increased
Please note that:
• Normal range for females is (120 – 160 g/L) and for
males (130 – 180 g/L)

19. 18
Decreased Hb
Sever Hemorrhage
Systemic diseases (e.g. leukemia, lymphoma,
uremia, cirrhosis, hyperthyroidism,
carcinomatosis and systemic lupus
erythematosus)
Haemolysis due to transfusion of
incompatible blood, reactions to
chemicals and drugs, bacteraemia, and
artificial heart valves
Anaemia

20. 19
Increased Hb
Polycythaemia
High altitudes
Congestive Cardiac Failure
(CCF)
Haemoconcentration states of
blood e.g. sever burns
Chronic obstructive pulmonary disease (COPD)

21. Erythrocytes
20
1. Normocytic erythrocytes (normal RBCs)
 Cells are uniform size & shape
 Normal hemoglobin conc.
 Small, central pallor which is
 Less than one-third of the total cell volume.

22. Erythrocytes
21
2. Hypochromic erythrocytes (low Hb in RBCs)
 IDA
 Thalassemia
 Sideroblastic anemia

23. Erythrocytes
22
3. Hyperchromic erythrocytes (high Hb in RBCs)
 hereditaryspherocytosis

24. Quality control
23
1. If the patient’s specimen running in automated
machine there are 3 levels controls should be run.
2. While if its running by manual method; send patient’s
specimen to the reference laboratory; and perform
duplicate testing in your own lab.
3. All personnel performing Hb should be checked for
color blindness (Sahli’s Method).

25. 24
Abnormal results
1. Leaving the tourniquet in place for more than 1 minute during the
procedure will result in hemoconcentration.
2. False increases may occur with lipemic samples and when leukocytosis
is present.
3. Individuals living in high altitudes have increased hemoglobin results.
4. Smokers have increased hemoglobin levels.
5. Hemolysis of the sample can alter test results.
6. Drugs which may increase hemoglobin level: gentamicin, methyldopa.
7. Drugs which may decrease hemoglobin level: antibiotics, antineoplastic
agents, apresoline, aspirin, indomethacin, MAO inhibitors, primaquine,
rifampin, sulfonamides.