Haemoglobinometry

Dr. Topesh Patle

Introduction 

  • Haemoglobinometry is the quantitative measurement of haemoglobin concentration in blood.

  • Haemoglobin is an iron-containing respiratory pigment present in red blood cells.

  • It is responsible for the transport of oxygen from lungs to tissues and carbon dioxide from tissues to lungs.

  • Haemoglobinometry is a fundamental hematological investigation.

  • It is routinely performed as part of the Complete Blood Count (CBC).

  • The test helps in the diagnosis and classification of anemia.

  • It is useful in detecting polycythemia and hemoconcentration.

  • Haemoglobinometry is essential for clinical diagnosis, treatment monitoring, and follow-up.

  • It is widely used in hospitals, diagnostic laboratories, and blood donation camps.

  • Various manual, semi-automated, and automated methods are employed for haemoglobin estimation.

Importance 

  • Haemoglobin estimation helps to assess the oxygen-carrying capacity of blood.

  • It is essential for the diagnosis of anemia and its severity.

  • Helps in the classification of anemia (mild, moderate, severe).

  • Useful in detecting polycythemia (increased hemoglobin level).

  • Important for nutritional assessment, especially iron, vitamin B12, and folate deficiency.

  • Routinely performed as a part of Complete Blood Count (CBC).

  • Essential in antenatal care to detect anemia in pregnant women.

  • Helps in pre-operative evaluation to assess surgical fitness.

  • Useful in monitoring response to treatment in anemia.

  • Important in screening programs and blood donation camps.

  • Aids in diagnosing chronic diseases such as chronic kidney disease and malignancy.

  • Helpful in assessing patients with chronic blood loss or hemorrhage.

  • Plays a role in evaluating hypoxia and respiratory disorders.

  • Important for public health assessment of anemia prevalence in the population.

 

Normal Values

Group Normal Range
Adult Male 13–17 g/dL
Adult Female 12–15 g/dL
Children 11–14 g/dL
Newborn 16–20 g/dL

 

Principle of Haemoglobinometry

Haemoglobinometry is based on the principle that:

Haemoglobin is converted into a stable colored compound, and the intensity of the color produced is directly proportional to the haemoglobin concentration in the blood.

The color intensity is measured either visually or photometrically, depending on the method used.

Common Methods of Haemoglobin Estimation

  • Sahli’s (Acid Hematin) Method

  • Cyanmethemoglobin Method (Reference method)

  • Oxyhaemoglobin Method

  • Azidemethemoglobin Method

  • Automated Hematology Analyzer

 

Sahli’s (Acid Hematin) Method

Principle

Sahli’s method is based on the principle that:

Hemoglobin reacts with 0.1 N hydrochloric acid (HCl) to form brown-colored acid hematin.
The intensity of the color produced is directly proportional to the hemoglobin concentration in the blood.

The hemoglobin concentration is estimated by visual color comparison with a standard scale.

Requirements

  • Sahli hemoglobinometer (comparator with standard brown glass)

  • Sahli graduated tube

  • Sahli pipette (20 µL)

  • 0.1 N Hydrochloric acid

  • Distilled water

  • Glass stirrer

  • Lancet

  • Blood sample (capillary or venous)

Procedure

  1. Fill the Sahli graduated tube with 0.1 N HCl up to the 2 g% mark.

  2. Using Sahli pipette, draw 20 µL of blood.

  3. Add the blood carefully into the acid in the Sahli tube.

  4. Mix thoroughly with a glass stirrer.

  5. Allow the mixture to stand for 10 minutes for complete conversion to acid hematin.

  6. Add distilled water drop by drop, mixing continuously.

  7. Continue dilution until the color matches the standard comparator.

  8. Read the hemoglobin value directly from the graduated scale in g/dL.

Advantages

  • Simple and easy to perform

  • Inexpensive method

  • Requires minimal equipment

  • Suitable for field surveys and teaching laboratories

  • Does not require electricity or sophisticated instruments

Limitations

  • Less accurate compared to photometric methods

  • Subjective error due to visual color matching

  • Not suitable for abnormal hemoglobin variants

  • Turbidity due to lipemia or high leukocyte count affects accuracy

  • Cannot measure carboxyhemoglobin and methemoglobin accurately

  • Not recommended as a reference method

 

Cyanmethemoglobin Method

Principle

The Cyanmethemoglobin method is based on the principle that:

Hemoglobin is oxidized to methemoglobin by potassium ferricyanide, which then reacts with potassium cyanide to form cyanmethemoglobin, a stable colored compound.

The intensity of the color produced is directly proportional to the hemoglobin concentration and is measured photometrically at 540 nm using a colorimeter or spectrophotometer.

This method is the reference method recommended by WHO for hemoglobin estimation.

Requirements

  • Whole blood sample (capillary or venous)

  • Drabkin’s reagent
    (contains potassium ferricyanide, potassium cyanide, and buffer)

  • Cyanmethemoglobin standard

  • Test tubes

  • Pipettes / Micropipettes

  • Colorimeter or spectrophotometer

  • Cuvettes

Procedure

  1. Take 5.0 mL of Drabkin’s reagent in a test tube.

  2. Add 20 µL of well-mixed blood to the reagent.

  3. Mix gently to avoid frothing.

  4. Allow the mixture to stand for 10 minutes at room temperature for complete color development.

  5. Set the colorimeter at 540 nm using Drabkin’s reagent as blank.

  6. Measure the absorbance of the test sample.

  7. Compare the absorbance with that of the standard.

  8. Calculate hemoglobin concentration and express the result in g/dL.

Calculation

Hemoglobin (g/dL) = Absorbance of test / Absorbance of standard × Concentration of standard

Advantages

  • High accuracy and reproducibility

  • Internationally accepted reference method

  • Measures almost all forms of hemoglobin (oxyHb, deoxyHb, metHb, carboxyHb)

  • Objective and less observer-dependent

  • Suitable for routine and research laboratories

Limitations

  • Drabkin’s reagent contains cyanide (toxic)

  • Requires colorimeter or spectrophotometer

  • Sulfhemoglobin is not measured accurately

  • Proper waste disposal is essential

Precautions

  • Handle Drabkin’s reagent carefully due to cyanide content

  • Use clean cuvettes and proper wavelength (540 nm)

  • Avoid hemolyzed or clotted samples

  • Calibrate the instrument properly

 

Oxyhaemoglobin Method

Principle

The oxyhaemoglobin method is based on the principle that:

Hemoglobin is converted into oxyhaemoglobin by dilution with an alkaline solution.
The intensity of the red color formed is directly proportional to the haemoglobin concentration and is measured photometrically.

Requirements

  • Whole blood sample

  • Distilled water or dilute ammonium hydroxide

  • Test tubes

  • Pipettes

  • Colorimeter / Spectrophotometer

  • Cuvettes

Procedure

  1. Take a measured volume of distilled water in a test tube.

  2. Add a fixed volume of blood and mix well.

  3. Allow complete hemolysis and oxygenation to form oxyhaemoglobin.

  4. Measure absorbance at 540 nm or 576 nm against a reagent blank.

  5. Calculate haemoglobin concentration using a standard.

Advantages

  • Simple and rapid method

  • No toxic chemicals used

  • Suitable for routine laboratory work

Limitations

  • Measures only oxyhaemoglobin

  • Not suitable in presence of abnormal hemoglobin derivatives

  • Less accurate than cyanmethemoglobin method

 

Azidemethemoglobin Method

Principle

The azidemethemoglobin method is based on the principle that:

Hemoglobin is oxidized to methemoglobin and then reacts with sodium azide to form azidemethemoglobin, a stable colored compound.

The color intensity is measured photometrically and is directly proportional to haemoglobin concentration.

This method is widely used in modern hematology analyzers.

Requirements

  • Whole blood sample

  • Azide reagent

  • Test tubes

  • Pipettes

  • Colorimeter / Automated analyzer

Procedure

  1. Mix blood with azide-containing reagent.

  2. Allow complete reaction to form azidemethemoglobin.

  3. Measure absorbance at 570 nm.

  4. Determine haemoglobin concentration using calibration data.

Advantages

  • Does not contain cyanide (safer than Drabkin’s method)

  • High accuracy and precision

  • Stable end product

  • Suitable for automated systems

Limitations

  • Sulfhemoglobin is not measured

  • Requires analyzer or photometric equipment

 

Automated Hematology Analyzer

Principle

Automated hematology analyzers estimate haemoglobin using:

Photometric measurement after chemical conversion of hemoglobin to a stable derivative (commonly azidemethemoglobin).

Modern analyzers also simultaneously measure RBC count, WBC count, platelet count, and red cell indices.

Requirements

  • EDTA anticoagulated blood

  • Automated hematology analyzer

  • Reagents specific to the analyzer

Procedure

  1. Collect blood in an EDTA tube.

  2. Load the sample into the analyzer.

  3. The analyzer aspirates the sample automatically.

  4. Hemoglobin is chemically converted and measured photometrically.

  5. Results are displayed digitally and printed.

Advantages

  • Highly accurate and reproducible

  • Rapid processing of large number of samples

  • Minimal human error

  • Provides complete CBC parameters

  • Suitable for high-throughput laboratories

Limitations

  • Expensive equipment and maintenance

  • Requires trained personnel

  • Instrument calibration and quality control essential

 

MCQs

1. Haemoglobin is primarily responsible for:

A. Immunity
B. Blood clotting
C. Oxygen transport
D. Hormone synthesis

Answer: C

2. Haemoglobin is present in:

A. Plasma
B. White blood cells
C. Platelets
D. Red blood cells

Answer: D

3. Haemoglobinometry refers to:

A. Measurement of RBC count
B. Measurement of hemoglobin concentration
C. Measurement of hematocrit
D. Measurement of plasma proteins

Answer: B

4. Normal hemoglobin level in adult males is:

A. 10–12 g/dL
B. 11–14 g/dL
C. 13–17 g/dL
D. 18–22 g/dL

Answer: C

5. Normal hemoglobin level in adult females is:

A. 9–11 g/dL
B. 10–12 g/dL
C. 12–15 g/dL
D. 15–18 g/dL

Answer: C

6. Low hemoglobin level indicates:

A. Polycythemia
B. Leukocytosis
C. Anemia
D. Thrombocytosis

Answer: C

7. Increased hemoglobin level is seen in:

A. Iron deficiency anemia
B. Polycythemia vera
C. Hemolytic anemia
D. Megaloblastic anemia

Answer: B

8. Haemoglobin estimation is part of:

A. Liver function test
B. Renal function test
C. Complete blood count
D. Lipid profile

Answer: C

9. Sahli’s method is also known as:

A. Cyanmethemoglobin method
B. Oxyhaemoglobin method
C. Acid hematin method
D. Azide method

Answer: C

10. In Sahli’s method, hemoglobin is converted into:

A. Cyanmethemoglobin
B. Methemoglobin
C. Oxyhaemoglobin
D. Acid hematin

Answer: D

11. Reagent used in Sahli’s method is:

A. Drabkin’s reagent
B. Sodium azide
C. 0.1 N Hydrochloric acid
D. Ammonium hydroxide

Answer: C

12. Main disadvantage of Sahli’s method is:

A. Expensive
B. Toxic reagent
C. Subjective color matching
D. Time consuming

Answer: C

13. Reference method recommended by WHO for hemoglobin estimation is:

A. Sahli’s method
B. Oxyhaemoglobin method
C. Cyanmethemoglobin method
D. Azidemethemoglobin method

Answer: C

14. Cyanmethemoglobin method uses which reagent?

A. Sahli reagent
B. Drabkin’s reagent
C. Sodium citrate
D. EDTA

Answer: B

15. Cyanmethemoglobin absorbance is measured at:

A. 400 nm
B. 480 nm
C. 540 nm
D. 620 nm

Answer: C

16. Drabkin’s reagent contains:

A. Potassium oxalate
B. Sodium azide only
C. Potassium ferricyanide and potassium cyanide
D. Hydrochloric acid

Answer: C

17. Major drawback of cyanmethemoglobin method is:

A. Low accuracy
B. Subjective error
C. Presence of toxic cyanide
D. Cannot measure hemoglobin

Answer: C

18. Oxyhaemoglobin method measures hemoglobin as:

A. Acid hematin
B. Cyanmethemoglobin
C. Oxyhaemoglobin
D. Methemoglobin

Answer: C

19. Oxyhaemoglobin method is less accurate because:

A. Uses cyanide
B. Measures only oxyhaemoglobin
C. Requires automation
D. Is time consuming

Answer: B

20. Safer alternative to cyanmethemoglobin method is:

A. Sahli’s method
B. Oxyhaemoglobin method
C. Azidemethemoglobin method
D. Haldane method

Answer: C

21. Azidemethemoglobin method forms:

A. Cyanmethemoglobin
B. Acid hematin
C. Azidemethemoglobin
D. Oxyhaemoglobin

Answer: C

22. Azidemethemoglobin method is commonly used in:

A. Sahli apparatus
B. Blood banks
C. Automated hematology analyzers
D. Field surveys

Answer: C

23. Advantage of azidemethemoglobin method is:

A. Very cheap
B. Visual matching
C. Cyanide-free and safe
D. No instrument needed

Answer: C

24. Sulfhemoglobin is not measured accurately by:

A. Sahli’s method
B. Cyanmethemoglobin method
C. Azidemethemoglobin method
D. Automated analyzers

Answer: C

25. Sample used for hemoglobin estimation is:

A. Serum
B. Plasma
C. Whole blood
D. Urine

Answer: C

26. Anticoagulant used for automated Hb estimation is:

A. Sodium citrate
B. Heparin
C. EDTA
D. Oxalate

Answer: C

27. Automated hematology analyzers estimate hemoglobin by:

A. Visual color comparison
B. Gravimetric method
C. Photometric method
D. Titration

Answer: C

28. Major advantage of automated analyzers is:

A. Low cost
B. Manual calculation
C. High accuracy and speed
D. No reagents required

Answer: C

29. Automated analyzers provide:

A. Only hemoglobin value
B. Only RBC count
C. Complete blood count
D. Only WBC count

Answer: C

30. Hemoglobin estimation is especially important in:

A. Hypertension
B. Diabetes mellitus
C. Anemia
D. Hyperlipidemia

Answer: C

31. Hemoglobin estimation is routinely done in:

A. Blood sugar test
B. Urine analysis
C. Antenatal screening
D. ECG

Answer: C

32. Normal hemoglobin level in newborn is:

A. 8–10 g/dL
B. 10–12 g/dL
C. 12–14 g/dL
D. 16–20 g/dL

Answer: D

33. Hemoglobin estimation helps to assess:

A. Renal function
B. Oxygen-carrying capacity
C. Liver enzymes
D. Immune status

Answer: B

34. Color matching error is mainly seen in:

A. Cyanmethemoglobin method
B. Automated analyzers
C. Sahli’s method
D. Azide method

Answer: C

35. Hemoglobin is an example of:

A. Enzyme
B. Hormone
C. Glycoprotein
D. Metalloprotein

Answer: D

36. Iron in hemoglobin is present in which form?

A. Ferric (Fe³⁺)
B. Ferrous (Fe²⁺)
C. Ferritin
D. Hemosiderin

Answer: B

37. Hemoglobin estimation is useful for monitoring:

A. Antibiotic therapy
B. Response to anemia treatment
C. Blood pressure
D. Blood sugar

Answer: B

38. Which method is least accurate?

A. Automated analyzer
B. Cyanmethemoglobin
C. Azidemethemoglobin
D. Sahli’s method

Answer: D

39. Hemoglobin estimation should be avoided in which sample?

A. Fresh blood
B. EDTA blood
C. Clotted blood
D. Capillary blood

Answer: C

40. High hemoglobin value may be seen in:

A. Chronic blood loss
B. Iron deficiency
C. Dehydration
D. Pregnancy

Answer: C

41. Hemoglobin estimation in blood donation is done to:

A. Check blood group
B. Detect infection
C. Prevent anemia in donor
D. Measure platelet count

Answer: C

42. Hemoglobinometry mainly reflects:

A. RBC size
B. RBC shape
C. RBC oxygen-carrying function
D. RBC lifespan

Answer: C

43. Hemoglobin estimation is a:

A. Biochemical test
B. Hematological test
C. Immunological test
D. Microbiological test

Answer: B

44. Which method uses visual comparison?

A. Automated analyzer
B. Cyanmethemoglobin
C. Sahli’s method
D. Azidemethemoglobin

Answer: C

45. Azidemethemoglobin absorbance is measured around:

A. 450 nm
B. 500 nm
C. 570 nm
D. 620 nm

Answer: C

46. Most commonly used method in modern labs is:

A. Sahli’s method
B. Oxyhaemoglobin method
C. Automated analyzer
D. Haldane method

Answer: C

47. Hemoglobin estimation helps in diagnosing:

A. Leukemia
B. Anemia
C. Thrombocytopenia
D. Hemophilia

Answer: B

48. Hemoglobin is synthesized in:

A. Liver
B. Bone marrow
C. Spleen
D. Kidney

Answer: B

49. Which method is suitable for field surveys?

A. Automated analyzer
B. Cyanmethemoglobin
C. Sahli’s method
D. Azidemethemoglobin

Answer: C

50. Haemoglobinometry is best described as:

A. Qualitative test
B. Semi-quantitative test
C. Quantitative test
D. Screening test only

Answer: C