Introduction
Haemoglobin (Hb) is an iron-containing conjugated protein located inside red blood cells, and Estimation of Haemoglobin plays a crucial role in evaluating its concentration in the blood. Haemoglobin performs the essential task of transporting oxygen from the lungs to body tissues while carrying carbon dioxide back to the lungs for exhalation. When haemoglobin concentration falls below normal levels, tissues receive inadequate oxygen, which leads to fatigue, organ dysfunction, and widespread systemic complications.
Structurally, haemoglobin consists of four globin chains. Adult haemoglobin contains two alpha and two beta chains. Each globin chain binds one heme group, and each heme group carries one iron atom in the ferrous (Fe²⁺) state. This iron atom enables reversible oxygen binding, which supports efficient gas exchange throughout the body.
Clinicians and laboratory professionals rely on Estimation of Haemoglobin as one of the most frequently performed hematological investigations. The test reveals valuable information related to anemia, polycythemia, nutritional deficiency, chronic disease, blood loss, and hydration status. Because haemoglobin reflects overall oxygen-carrying capacity, even small deviations from normal values carry clinical significance.
A reduced haemoglobin level commonly indicates anemia. Iron deficiency, vitamin B12 deficiency, folate deficiency, chronic inflammation, hemolysis, or ongoing blood loss often cause this condition. In contrast, elevated haemoglobin values may point toward dehydration, polycythemia vera, smoking-related hypoxia, or chronic lung disease. Due to this broad diagnostic value, laboratories have developed several reliable methods for haemoglobin measurement.
Clinical Importance of Haemoglobin Measurement
Haemoglobin concentration directly influences tissue oxygenation and metabolic efficiency. For this reason, healthcare providers request haemoglobin testing in a wide range of clinical scenarios.
Doctors use haemoglobin values to:
- Diagnose and classify anemia
- Monitor treatment response in nutritional deficiencies
- Assess blood loss after surgery or trauma
- Screen pregnant women and children
- Evaluate chronic kidney, liver, and lung diseases
- Moreover, public health programs depend on haemoglobin screening to identify anemia prevalence in communities. Therefore, Estimation of Haemoglobin supports both individual patient care and population-level health planning.
Normal Haemoglobin Levels
Normal haemoglobin values vary based on age, sex, and physiological status. The commonly accepted reference ranges include:
- Adult Male: 13–17 g/dL
- Adult Female: 12–15 g/dL
- Children: 11–14 g/dL
- Newborn: 14–22 g/dL
- Values below the normal range indicate anemia, while values above the upper limit suggest polycythemia or dehydration. Interpretation always requires correlation with clinical findings.
Methods for Estimation
Clinical laboratories use multiple techniques for haemoglobin measurement. Each method differs in accuracy, cost, complexity, and suitability.
Common methods include:
- Sahli’s Method (Acid Hematin Method)
- Cyanmethemoglobin Method
- Tallquist Method
- Electronic Counter Method
- Direct Reading Electronic Method
- Oxyhaemoglobin Method
- Alkaline Hematin Method
- Chemical Methods
- Selection depends on laboratory infrastructure, workload, and diagnostic requirements.
A. Sahli’s Method (Acid Hematin Method)
Principle
In Sahli’s method, hydrochloric acid converts haemoglobin into acid hematin. This brown-colored compound forms a stable solution. Gradual dilution with distilled water continues until the color matches a standard comparator. The graduated tube displays haemoglobin concentration directly.
Instruments Required
Sahli’s haemoglobinometer tube
Haemoglobin pipette (20 µL)
Comparator with brown glass standards
N/10 hydrochloric acid
Distilled water
Glass stirrer
Procedure
First, fill the haemoglobinometer tube with N/10 HCl up to the 2 g% mark. Next, draw blood up to the 20 µL mark using a haemoglobin pipette. Carefully add the blood into the acid solution. After mixing, allow the mixture to stand for ten minutes so complete conversion occurs. Then, add distilled water drop by drop while stirring until the solution color matches the standard comparator. Finally, read the haemoglobin concentration from the graduated tube.
Result Interpretation
The result appears in g/dL. Visual color matching determines the final value.
Advantages
Simple technique
Low cost
Suitable for basic laboratories
Limitations
Subjective interpretation
Lower accuracy
Lighting conditions influence results
Inability to measure sulfhaemoglobin accurately
Despite these drawbacks, Sahli’s method remains popular in resource-limited settings.
B. Cyanmethemoglobin Method (HiCN Method)
Principle
This internationally recommended method uses Drabkin’s reagent, which contains potassium ferricyanide and potassium cyanide. Ferricyanide oxidizes haemoglobin to methemoglobin. Cyanide then converts methemoglobin into cyanmethemoglobin, a stable colored compound. A spectrophotometer measures absorbance at 540 nm.
Instruments Required
Spectrophotometer (540 nm)
Test tubes
Micropipette
Drabkin’s reagent
Standard haemoglobin solution
Technique
Add 5 mL of Drabkin’s reagent into a test tube. Introduce 20 µL of blood sample and mix gently. Allow the mixture to stand for five to ten minutes. Measure absorbance at 540 nm against a reagent blank.
Calculation
Hb (g/dL) =
(Absorbance of Test ÷ Absorbance of Standard) × Standard Concentration
Advantages
High accuracy
Excellent reproducibility
International standard
Suitable for quality control
Disadvantages
Toxic cyanide reagent
Requires laboratory equipment
Unsuitable for field screening
C. Tallquist Method
Principle
This method involves placing a drop of blood on filter paper and visually comparing it with a standardized color chart.
Advantages
Very simple
No equipment required
Useful for quick field surveys
Disadvantages
Very low accuracy
High subjectivity
Unsuitable for clinical diagnosis
Most laboratories now consider this method obsolete.
D. Electronic Counter Method
Automated hematology analyzers commonly perform haemoglobin measurement in modern laboratories.
Principle
The analyzer lyses red cells and measures haemoglobin photometrically. Many systems use non-cyanide reagents for safety.
Advantages
High precision
Rapid results
Simultaneous CBC analysis
Reduced human error
Disadvantages
High cost
Regular calibration required
E. Direct Reading Electronic Method
Portable haemoglobinometers provide immediate haemoglobin values.
Principle
Capillary blood enters a cuvette or strip. The device measures haemoglobin using photometric analysis.
Advantages
Portable
Quick results
Suitable for outreach programs
Disadvantages
Consumable strip cost
Slight variation from analyzer results
F. Oxyhaemoglobin Method
Principle
Oxygen exposure converts haemoglobin into oxyhaemoglobin. A spectrophotometer measures absorbance.
Limitation
Low stability limits routine use
G. Alkaline Hematin Method
Principle
An alkaline solution converts haemoglobin into alkaline hematin. Color intensity reflects concentration.
Limitation
Limited standardization restricts widespread adoption.
H. Chemical Methods
Chemical reactions produce measurable color changes after haemoglobin interacts with reagents. Automation has largely replaced these methods.
Factors Affecting Accuracy
Several factors influence test reliability:
Sample collection technique
Anticoagulant selection
Proper mixing
Timely analysis
Instrument calibration
Strict adherence to laboratory protocols improves result accuracy.
Conclusion
Estimation of Haemoglobin remains a cornerstone of hematological investigation. Various laboratory methods serve different diagnostic settings, from basic rural laboratories to advanced automated facilities. Sahli’s method offers simplicity, while the cyanmethemoglobin method provides reference-level accuracy. Automated analyzers dominate modern practice due to efficiency and precision. Proper technique, method selection, and interpretation ensure meaningful clinical outcomes.
Disclaimer
This article serves educational purposes only. Laboratory professionals must follow institutional protocols, standard operating procedures, and professional guidelines while performing haemoglobin estimation and interpreting results.
Frequently Asked Questions
Q1. Why is haemoglobin estimation important?
Haemoglobin estimation helps diagnose anemia, monitor treatment, and assess oxygen-carrying capacity.
Q2. Which method offers the highest accuracy?
The cyanmethemoglobin method provides the highest accuracy and international standardization.
Q3. Why do laboratories prefer automated methods today?
Automated analyzers deliver rapid, precise results with minimal human error.
Written by Jambir Sk Certified Medical Laboratory Technologist
Disclaimer: This content is for educational purposes only and should not be consideredas medical advice. Always consult a qualified doctor.We do not provide professional medical advice, diagnosis, or treatment.All health-related content is based on research, knowledge, and general awareness.Always consult a licensed healthcare provider for any medical concerns.HealthSeba.com will not be responsible for any loss, harm, or damage caused by the use of information available on this site.
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