Determination of Hemoglobin concentration
Hemoglobin is a conjugated protein present in red blood cells. It carries oxygen from lungs to tissue cells, and carbon dioxide- the gaseous waste from cells to lungs.
Hemoglobin consists of two components; “haem” (iron+protoporphyrin) and “globin” (amino acid chains).
After the normal life- span of red blood cells is over (120 days), the red blood cells are destroyed by reticuloendothelial cells (specially the spleen) and the components of hemoglobin undergo metabolic degradation.
The iron component of haem is recycled and used up again in hemoglobin synthesis. The globin breaks down into its constituent amino acids which are also recycled.
The only component which cannot be recycled is protoporphyrin and it leads to the formation of bilirubin. Defects in hemoglobin, called hemoglobinopathies, are congenital diseases (e.g. sickle cell disease). Such defects originate from the abnormalities in the chemical structure of globin and its amino acid sequence. Several methods are available for determination of hemoglobin of which the Cyanmethemoglobin Method is most reliable.
Clinical Significance of Hemoglobin
A decrease in hemoglobin concentration in blood below normal values is a sign of anemia.
The hemoglobin concentration is lower in adult women as compared to adult males. Hemoglobin values further drop during pregnancydue to hemodilution (a proportionately greater increase in plasma volume as compared to the increase in red cell mass). Children also have values lower than those seen in adult males.
An increase in hemoglobin concentration can occur due to hemoconcentration (loss of body fluid e.g. in severe diarrhea, vomiting), reduced oxygen supply (e.g. congenital heart disease, emphysema) and in a rare condition polycythemia. A decrease or increase in hemoglobin concentration must be reported as it is a sign of disease requiring further investigations.
Normal Values of Hemoglobin Concentration
Men: 14 g/dL to 17 g/dL Women: 13 g/dL to 15 g/dL
Sample should be an EDTA anticoagulated venous blood but the use of other anticoagulants, e.g. heparin, double oxalate will also yield the same result. Capillary blood can be used directly.
Methods of Hemoglobin Hetermination
Acid Hematin Method
The acid hematin method is a common laboratory test used to determine the concentration of hemoglobin in a blood sample.
Hemoglobin is a protein found in red blood cells that carries oxygen throughout the body.
The acid hematin method involves the conversion of hemoglobin to acid hematin, which is then measured calibration tube.
This is also known as Sahli’s method. It is recommended for places where a colorimeter is not available.
It is not a recommended method because all forms of hemoglobin are not converted to acid hematin and colour does not vary strikingly due to change in hemoglobin concentration.
Acid Hematin Method Principle
Hemoglobin is converted to acid hematin by the action of hydrochloric acid. The acid converts the hemoglobin molecule to acid hematin by removing the iron from the heme group, resulting in the formation of a stable compound. The acid hematin solution is further diluted with the acid until its colour matches exactly that of the permanent standard of the comparator block. The hemoglobin concentration is read directly from the calibration tube. The amount of acid hematin produced is proportional to the amount of hemoglobin in the sample.
Sahli’s method requirements
- 0.1 N hydrochloric acid
- Sahli’s hemoglobinometer
- Sahli’s pipette (20 μL)
- Calibrated tubes of hemoglobinometer
Acid Hematin Method Procedure
- The acid hematin method involves the following steps:
- Collect a blood sample from the patient using a venipuncture.
- Transfer a measured volume of the blood sample to a test tube.
- Fill the specially marked calibrated tube of hemoglobinometer up to the 10 mark with 0.1 N HCI.
- Draw the blood specimen into Sahli’s pipette (20ul).
- Make sure the anticoagulated blood is mixed so that no air bubble has entered into the pipette while taking the blood sample.
- Wipe the outside of sahli’s pipette with a cloth or cotton.
- Check the level of the blood in the pipette
- Blow the blood into the acid solution inside the graduated tube.
- Wash out the contents of the pipette by repeated drawing in and blowing out of the diluting fluid.
- Allow the mixture to stand at room temperature for 10 minutes and then place the graduated tube in the hemoglobinometer and face a window for better lighting from the back.
- Compare the color of the diluted blood in the tube with the reference tube.
- If the colour is the same, then the hemoglobin concentration is recorded.
- If the colour is darker, then the contents of the tube is diluted with 0.1 N HCI or distilled water drop by drop and stirred using glass rod till the colour of the contents match with the comparator.
- Hemoglobin concentration is then recorded.
Acid Hematin Method Results
If the colour is the same, then the hemoglobin concentration is recorded.
If the colour is darker, then the contents of the tube is diluted with 0.1 N HCI or distilled water drop by drop and stirred using glass rod till the colour of the contents match with the comparator.
Hemoglobin concentration is then recorded.
The results of the acid hematin method are reported as the hemoglobin concentration in grams per deciliter (g/dL) of blood. Normal values for hemoglobin concentration vary depending on age, sex, and other factors. In adult males, the normal range is 13.5-17.5 g/dL, while in adult females, the normal range is 12.0-15.5 g/dL.
Clinical Manifestations of Acid Hematin Method
The acid hematin method is used to measure the concentration of hemoglobin in the blood, which is important for the diagnosis and management of various medical conditions such as anemia, bleeding disorders, and polycythemia. The test is also used to monitor the response to treatment in patients with these conditions.
Uses
- The acid hematin method is a commonly used laboratory test to measure hemoglobin concentration in the blood.
- It is used for the diagnosis and management of various medical conditions such as anemia, bleeding disorders, and polycythemia.
- The test is also used to monitor the response to treatment in patients with these conditions.
Limitations
- The acid hematin method has some limitations. It requires careful handling of the blood sample to ensure accurate results.
- The test may be affected by hemolysis, lipemia, or icterus, which can interfere with the accuracy of the results.
- In addition, the acid hematin method may not detect certain types of hemoglobin variants, which can result in false-negative results.
- Finally, the test may be affected by other factors such as altitude, smoking, and pregnancy, which can affect the hemoglobin concentration in the blood.
Cyanmethemoglobin Method
The cyanmethemoglobin method is another laboratory test used to determine the concentration of hemoglobin in a blood sample. It involves the conversion of hemoglobin to cyanmethemoglobin, which is then measured spectrophotometrically.
Cyanmethemoglobin Method Principle
Cyanmethemoglobin Method is a colorimetric procedure for determining hemoglobin concentration. An aliquot of well mixed whole blood is taken and reacted with a solution of potassium cyanide and potassium ferricyanide (Drabkin’s solution).
The chemical reaction yields a product of stable colour called cyanamethemoglobin. The intensity of the colour is proportional to the hemoglobin concentration and obeys Beer’s law. The hemoglobin in the blood sample is converted to cyanmethemoglobin by the addition of a solution containing potassium ferricyanide and potassium cyanide.
The potassium ferricyanide oxidizes the heme group of hemoglobin to form methemoglobin, while the potassium cyanide reacts with the methemoglobin to form cyanmethemoglobin, which is stable and can be measured using a spectrophotometer. The amount of cyanmethemoglobin produced is proportional to the amount of hemoglobin in the sample.
Requirements for Cyanmethemoglobin Method
- Colorimetre adjusted at 540 nm
- Sahli’s pipette (20 μL capacity)
- Test tubes
- Drabkin’s solution
| Composition | Amount |
|---|---|
| Potassium Cyanide | 50 mg |
| Potassium Ferricyanide | 200 mg |
| Distilled Water | 1000 ml |
Cyanmethemoglobin Method Procedure
- The cyanmethemoglobin method involves the following steps:
- Collect a blood sample from the patient using a venipuncture.
- Transfer a measured volume of the blood sample to a test tube.
- Label the test tubes as test (T) and blank (B)
- Pipette out 5 ml of Drabkin’s solution into both the tubes with the help of automated dispenser or rubber bulb (avoid pipetting by mouth)
- Mix the blood gently
- Draw 20ul blood in the tube labeled as (T) test.
- Mix the contents and wait for 10 minutes
- Take the absorbance of test at 540 nm against blank.
- Take the absorbance of standard directly by taking the standard solution in the cuvette.
- Read the concentration of standard on the vial.
Hemoglobin (g/dl) = Absorbance of test x Concentration of standard
Cyanmethemoglobin Method Results
The results of the cyanmethemoglobin method are reported as the hemoglobin concentration in grams per deciliter (g/dL) of blood. Normal values for hemoglobin concentration vary depending on age, sex, and other factors. In adult males, the normal range is 13.5-17.5 g/dL, while in adult females, the normal range is 12.0-15.5 g/dL.
Uses
The cyanmethemoglobin method is a commonly used laboratory test to measure hemoglobin concentration in the blood. It is used for the diagnosis and management of various medical conditions such as anemia, bleeding disorders, and polycythemia. The test is also used to monitor the response to treatment in patients with these conditions.
Limitations
The cyanmethemoglobin method has some limitations. It requires careful handling of the blood sample to ensure accurate results.
The test may be affected by hemolysis, lipemia, or icterus, which can interfere with the accuracy of the results. In addition, the cyanmethemoglobin method may not detect certain types of hemoglobin variants, which can result in false-negative results.
Finally, the test may be affected by other factors such as altitude, smoking, and pregnancy, which can affect the hemoglobin concentration in the blood.
Clinical Manifestations of Cyanmethemoglobin Method
The cyanmethemoglobin method is used to measure the concentration of hemoglobin in the blood, which is important for the diagnosis and management of various medical conditions such as anemia, bleeding disorders, and polycythemia.
The test is also used to monitor the response to treatment in patients with these conditions.
