Hb – Hemoglobin Estimation – Methods, Merits, and Demerits
Introduction
- Hemoglobin is a conjugated protein present in red blood cells (RBCs).
- It consists of a pigment part called heme which contains iron and a protein part called globin and
- Hemoglobin plays a vital role in transporting oxygen from the lungs to tissues and returning carbon dioxide from tissues to the lungs.
- After the normal life span of RBC (over 120 days), the red cells are destroyed by the reticuloendothelial cells (specially in the spleen) and the components of the hemoglobin undergo metabolic degeneration.
Normal Hemoglobin Values
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Men: 13.5 – 17.5 g/dL
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Women: 12.0 – 15.5 g/dL
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Children: 11.0 – 13.0 g/dL
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Newborns: 14.0 – 24.0 g/dL
Clinical Importance of Hemoglobin Estimation
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Diagnosis of anemia and polycythemia.
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Monitoring response to treatment in anemic patients.
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Evaluation of blood loss.
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Pre-operative assessment.
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Nutritional assessment in populations.
Methods of Hemoglobin Estimation
Several methods are available for the estimation of hemoglobin. The most commonly used are:
1. Sahli’s (Acid Hematin) Method
2. Cyanmethemoglobin Method (Drabkin’s Method)
3. Hemoglobin Color Scale (WHO Scale)
4. Automated Hematology Analyzer
5. Tallqvist Method (Historical/Obsolete)
1. Sahli’s (Acid Hematin) Method
Principle:
- Hemoglobin is converted into brown-colored acid hematin by reacting with hydrochloric acid (N/10 HCl).
- The color is matched against a standard comparator.
Procedure:
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Add 0.1N HCl into the Sahli’s hemoglobinometer tube up to the mark.
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Add blood using a pipette (usually 20 µL).
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Mix and let it stand for 10 minutes.
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Dilute with distilled water till the color matches the standard.
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Read the value on the graduated tube.
Merits:
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Simple and inexpensive.
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Suitable for fieldwork and small labs.
Demerits:
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Subjective color matching.
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Less accurate.
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Interference by abnormal hemoglobins or lipemia.
2. Cyanmethemoglobin Method (Drabkin’s Method)
Principle:
Blood is mixed with Drabkin’s reagent which contains potassium ferricyanide and potassium cyanide. Hemoglobin is converted into stable cyanmethemoglobin which is measured spectrophotometrically at 540 nm.
Procedure:
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Add 20 µL blood to 5 mL Drabkin’s reagent.
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Mix and wait for 5–10 minutes.
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Measure absorbance at 540 nm.
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Compare with standard or calibration curve.
Merits:
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Highly accurate and reproducible.
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International reference method.
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Measures all types of hemoglobin except sulfhemoglobin.
Demerits:
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Toxic reagents (cyanide).
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Requires spectrophotometer.
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Not ideal for field settings.
3. Hemoglobin Color Scale (WHO Scale)
Principle:
Capillary blood is compared with a printed color scale to estimate hemoglobin.
Merits:
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Inexpensive.
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Easy to use in remote areas.
Demerits:
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Highly subjective.
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Less accurate.
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Not suitable for clinical diagnosis.
4. Automated Hematology Analyzer
Principle:
Measures hemoglobin through photometric or electrical impedance method. Usually uses cyanmethemoglobin or sodium lauryl sulfate (SLS) method.
Merits:
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Accurate and fast.
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Provides complete blood count (CBC) with hemoglobin.
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Ideal for large laboratories.
Demerits:
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Expensive instruments.
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Requires maintenance and electricity.
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Not suitable for small clinics or field use.
5. Tallqvist Method (Historical/Obsolete)
Principle:
Blood on filter paper is compared with a color scale.
Merits:
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Very simple and portable.
Demerits:
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Crude and inaccurate.
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Not used in modern clinical practice.
Conclusion
Hemoglobin estimation is a vital diagnostic procedure. The method chosen depends on the available resources, setting (lab vs. field), and purpose (screening vs. diagnosis). For clinical accuracy, the cyanmethemoglobin method is the most reliable, while Sahli’s method remains a practical tool in limited-resource settings.
