Overview
Hemoglobin is the major constituent of the red cell cytoplasm, accounting for approximately 90% of the dry weight of the mature red cell. It is comprised of heme and globin, and functions as the primary medium for exchange of oxygen and carbon dioxide between the lungs and tissues.
Accurate hemoglobin estimation is one of the most frequently performed tests in any clinical laboratory. It is the cornerstone screening test for anemia, is required before every blood donation, and forms the basis for calculating red cell indices used in classifying anemia.
Learning Objectives
After this lesson you will be able toโฆ- Describe the structure of hemoglobin
- List the functions of hemoglobin
- Explain the various laboratory methods for estimation of hemoglobin
- Enumerate the advantages and disadvantages of each method
- Discuss the normal value and interpretation of abnormal results
Clinical Story
Why This MattersA 28-year-old pregnant woman comes for a routine antenatal check-up. Her physician orders a hemoglobin estimation to screen for anemia, since the WHO cut off for diagnosing anemia in pregnancy is below 11 g/dl. The lab technologist must choose a reliable, internationally accepted method โ the cyanmethemoglobin technique โ to give an accurate result that can guide whether the patient needs iron supplementation.
Core Concepts
The hemoglobin molecule is a tetramer consisting of two pairs of similar polypeptide chains called globin chains. To each of the four chains is attached heme, a complex of iron in ferrous form and protoporphyrin. The major (96%) hemoglobin in adults is HbA (ฮฑ2ฮฒ2) โ two alpha and two beta globin chains. The alpha chain gene is on chromosome 16, and the beta chain gene is on chromosome 11. Adults also have a small amount of HbA2 (less than 3.5%). During fetal life, Hemoglobin F (ฮฑ2ฮณ2) predominates (50โ90% of total Hb), falling to less than 2% by 30 weeks after birth, after which HbA becomes predominant.
| Name | Structure | Present in |
|---|---|---|
| Adult Hb (HbA) | ฮฑ2ฮฒ2 | Adults |
| Fetal Hb (HbF) | ฮฑ2ฮณ2 | Fetal life |
| Hb Portland | ฮถ2ฮณ2 | Embryonic life |
| Gower-1 | ฮถ2ฮต2 | Embryonic life |
| Gower-2 | ฮฑ2ฮต2 | Embryonic life |
Heme has the ability to bind oxygen reversibly and carry it to the tissues. It also facilitates the exchange of carbon dioxide between the lungs and tissues. Hemoglobin thus functions as the primary medium of exchange of oxygen and carbon dioxide in the body.
Methods of hemoglobin estimation fall into three broad categories: (I) Methods based on development of color โ Sahli's/acid hematin method, Cyanmethemoglobin method, Oxyhemoglobin method, and Alkaline hematin method; (II) Measurement of oxygen combining capacity; and (III) Measurement of iron content. The color-based methods, especially the cyanmethemoglobin method, are most widely used in routine practice.
Hemoglobinometers based on the cyanmethemoglobin or oxyhemoglobin method have a built-in filter and scale that gives a direct reading of Hb in g/dl. These portable instruments are widely used in field surveys and blood donation camps, but must be calibrated frequently to ensure accuracy.
Laboratory Principle
In the Cyanmethemoglobin method, blood is diluted in Drabkin's solution, which contains potassium ferricyanide and potassium cyanide. Potassium ferricyanide oxidizes hemoglobin to methemoglobin, which is then converted to cyanmethemoglobin (HiCN) by potassium cyanide. The intensity of the resulting stable colored compound is measured spectrophotometrically at 540 nm and compared against a WHO international reference standard, giving an accurate hemoglobin concentration.
Equipment Required
Reagents & Materials
| Reagent / Material | Concentration / Grade | Purpose | Storage |
|---|---|---|---|
| N/10 HCl | 0.1 N | Converts Hb to acid hematin (Sahli's method) | Room temperature |
| Drabkin's solution | pH 7.0โ7.4 (KCN 50mg, K-ferricyanide 200mg, KHโPOโ 140mg, detergent 1ml/L) | Converts Hb to cyanmethemoglobin | Brown borosilicate bottle; refrigerate if >30ยฐC; never freeze |
| WHO Reference HiCN Standard | 10ml sealed ampoule | Preparation of standard curve | Room temperature; stable for years |
Step-by-Step Procedure
Cyanmethemoglobin Method
Take 5ml of Drabkin's solution in a test tube. Confirm the solution is clear and pale yellow with zero absorbance at 540nm against water as blank.
Mix EDTA blood by gentle inversion and draw 0.02ml of blood into the Hb pipette. Wipe the outer surface of the pipette to remove excess blood.
Place the pipette into the Drabkin's solution tube and slowly expel the blood. Mix well and allow to stand undisturbed for 5 minutes.
Adjust OD to zero using Drabkin's solution as blank, then measure the absorbance of the sample at 540nm in the spectrophotometer.
Use the standard curve (prepared from serial dilutions of WHO reference HiCN standard) to read off the Hb concentration corresponding to the OD obtained.
Flow Diagram
Quality Control
Check the pH of Drabkin's solution every month. Discard the solution if it becomes turbid, if pH falls outside range, or if its absorbance is not zero at 540nm. A standard curve should be re-verified periodically against the WHO HiCN reference standard to ensure accuracy across the testing range.
A reliable and stable reference standard is available from the World Health Organisation for direct comparison, making the cyanmethemoglobin method ideal for participation in external proficiency testing schemes across laboratories.
Reference Values
Normal Rangesโ ๏ธ Reference ranges may vary between laboratories. Always apply your laboratory's established reference intervals.
Clinical Interpretation
| Finding | Possible Significance | Action / Follow-up |
|---|---|---|
| Hb within normal range | No anemia detected | Routine follow-up as per clinical context |
| Hb below WHO cut-off | Anemia present โ must investigate underlying cause | Order red cell indices, peripheral smear, iron studies as indicated |
| Hb > 17 g/dl (males) / >15 g/dl (females) | Possible polycythemia or dehydration | Correlate with hematocrit and clinical findings |
Common Errors & How to Avoid Them
Cause: Color of acid hematin fades with time, and dilution must be done exactly at 10 minutes when color development is maximum.
Prevention: Time the procedure precisely; if the matching point is passed, repeat the entire test.
Cause: Abnormal plasma proteins or a high leucocyte count can cause turbidity when blood is diluted with Drabkin's solution.
Prevention: Centrifuge the diluted blood sample to clear turbidity before measuring absorbance.
Cause: The rate of conversion of blood containing carboxyhemoglobin (e.g., smokers) is slowed considerably.
Prevention: Prolong the reaction time to 30 minutes to ensure complete conversion.
Laboratory Tips from the Bench
Never pipette Drabkin's solution by mouth โ potassium cyanide is a poisonous substance. Always use mechanical pipetting aids.
Once OD readings are taken, prepare a table of OD-to-Hb-value correlations from your standard curve โ this is far more convenient than reading off a graph for every sample in a busy laboratory.
Remember "HiCN" โ the cyanmethemoglobin method converts Hemoglobin to cyanide-bound HiCN, the most stable and internationally standardized form for measurement.
Important Notes
All forms of hemoglobin except sulphemoglobin are converted to HiCN in the cyanmethemoglobin method, making it nearly universal but not perfectly comprehensive.
Not all hemoglobins (oxyhemoglobin, sulphemoglobin) are converted to acid hematin in Sahli's method, so the value obtained is typically lower than the actual hemoglobin concentration.
Interactive Quiz
Test Your KnowledgeFlashcards
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Clinical Case Study
Apply Your KnowledgeMrs. Rao presents for her routine antenatal visit complaining of fatigue and mild breathlessness on exertion over the past month. She has no significant past medical history and is not on any iron supplementation.
The hemoglobin of 9.2 g/dl is well below the WHO cut-off of 11 g/dl for pregnant women, confirming anemia. The cyanmethemoglobin method, being internationally standardized, gives a reliable basis for this diagnosis and for monitoring response to treatment.
- โThe WHO anemia cut-off for pregnant women (11 g/dl) is stricter than for non-pregnant women (12 g/dl)
- โCyanmethemoglobin method should be used for accurate, comparable results across antenatal visits
- โHb alone confirms anemia but further tests (red cell indices, iron studies) are needed to establish the underlying cause
Frequently Asked Questions
The cyanmethemoglobin method converts nearly all forms of hemoglobin (except sulphemoglobin) to a single stable compound, has no visual matching error since it is read on a spectrophotometer, and has a globally available WHO reference standard for comparison. Sahli's method is less accurate, subject to individual visual variation, and underestimates true hemoglobin values.
Drabkin's solution contains potassium cyanide, a highly poisonous substance. Accidental ingestion can be fatal, so mechanical pipetting devices must always be used.
No. Hemoglobin estimation is a screening test for the presence of anemia, but it cannot determine the underlying cause. Red cell indices, peripheral smear examination, and further tests like iron studies or vitamin B12/folate levels are needed for a complete diagnosis.
Quick Revision
10-Minute ReviewKey Takeaways
- The hemoglobin molecule is a tetramer of globin chains each bound to heme (iron + protoporphyrin)
- HbA (ฮฑ2ฮฒ2) is the major adult hemoglobin; hemoglobin's primary function is Oโ/COโ exchange
- Color-development methods, especially cyanmethemoglobin, are the mainstay of Hb estimation
- Sahli's method is quick and easy but less accurate than cyanmethemoglobin
- The cyanmethemoglobin method uses Drabkin's solution and is read spectrophotometrically at 540nm
- Hb estimation is the principal screening test for detection of anemia
Competency Checklist
Track Your MasteryReferences
- Dacie JV, Lewis SM. Practical Haematology. 11th ed.
- World Health Organisation. Haemoglobin concentrations for the diagnosis of anaemia and assessment of severity.
- NIOS Vocational Course โ Hematology and Blood Bank Technique, Lesson 3.