Basic methods in haematology

Basic methods in haematology

1. BASIC METHODS IN HAEMATOLOGY ESTIMATION OF HAEMOGLOBIN (Hb) CONCENTRATION presented by MISS TASMIA ZEB

2. ESTIMATION OF HAEMOGLOBIN (Hb) CONCENTRATION Whole blood haemoglobin concentration can be estimated by a number of methods. Most commonly used methods are: • Cyanmethaemoglobin method • Alkaline haematin method • Acid haematin method Each of these methods has its advantages and disadvantages. Most commonly used method is cyanmethaemoglobin method. Major advantage of this method is the availability of a stable and reliable standard preparation. This method, however, does not measure sulphhaemoglobin (SHb). Acid haematin method has the advantage of being useful without a colorimeter (Sahli's haemoglobinometer) but is the least accurate of all. Alkaline haematin method has the advantage that it can measure carboxyhaemoglobin, methaemoglobin and sulphhaemoglobin but it does not measure foetal haemoglobins (HbF and Hb Barts').

3. CYANMETHAEMOGLOBIN METHOD PRINCIPLE The principle of this method is that blood sample is diluted in a solution containing potassium cyanide and potassium ferricyanide (Drabkin's solution). It converts haemoglobin (Hb) and methaemoglobin (Hi) to cyanmethaemoglobin (HiCN), which is a stable compound. The absorbance of the solution is measured in a photoelectric colorimeter with a yellow green filter or in a spectrophotometer at a wavelength of 540 nm and is compared with a standard solution of HiCN. OR The hemoglobin is converted into cyanmet haemoglobin by the addition of Drabkin's solution. the intensity of colour produced is directly propotional to the amount of haemoglobin present and is measured at 540 nm wavelength.

4. Requirements 1. Diluent (Drabkin's solution) Potassium ferricyanide 200 mg Potassium cyanide 50 mg Potassium dihydrogen phosphate 140 mg Nonidet P40 (Sigma) 1 ml Distilled water up to 1000 ml

5. The pH should be between 7.0-7.4 and the solution should be clear and pale yellow in colour. It should give zero absorbance against water at 540 nm. The reagent is stored at room temperature in a brown borosillicate glass bottle. If Nonidet is not available then reaction time is to be increased, as haemolysis may be slow.Reagent can be obtained in prepared concentrate form. If stored properly, the reagent is fit for use for several months. The reagent is discarded if it becomes turbid or the absorbance changes.

6. Cyanmethaemoglobin reference solution: The cyanmethaemoglobin reference preparation is used for direct comparison with blood, which is also converted to HiCN. Solutions of different concentrations are commercially available and if unopened are stable for years. But once opened, it is only stable for few hours. It is therefore recommended that a calibration curve should be prepared with the help of these solutions and future readings should be taken from it. But it is necessary that with each batch of tests or at least few times a day the calibration is checked by a fresh cyanmethaemoglobin reference solution or an internal reference prepared against it. The manufacturer’s inset with the pack of standards gives the Hb g/L equivalent of HiCN concentration of the standard.

7. Cyanmethaemoglobin reference solution: The cyanmethaemoglobin reference preparation is used for direct comparison with blood, which is also converted to HiCN. Solutions of different concentrations are commercially available and if unopened are stable for years. But once opened, it is only stable for few hours. It is therefore recommended that a calibration curve should be prepared with the help of these solutions and future readings should be taken from it. But it is necessary that with each batch of tests or at least few times a day the calibration is checked by a fresh cyanmethaemoglobin reference solution or an internal reference prepared against it. The manufacturer’s inset with the pack of standards gives the Hb g/L equivalent of HiCN concentration of the standard.

8. PROCEDURE Venous blood collected in EDTA or free flowing capillary blood can be used. Measurement can be carried out on blood that has been stored at 4°C for several days, provided it is free from infection and contamination. 20 µl of blood is added to 4 ml of diluent and well mixed by inverting the tube several times. It is allowed to stand at room temperature for 3-5 min so that all Hb is converted to HiCN. The absorbance is then measured in the spectrophotometer at 540 nm. Hb level can be directly read from previously prepared calibration curve or chart. Alternatively, absorbance of known standard is also read in the spectrophotometer with each batch of tests And Hb is calculated by the formula:

9. PREPRATION OF CALIBRATION CURVE/CHART The hemoglobin level can also be calculated from standard curve or calibration curve.the stndard curve is used to measure concentration of test suibstance,the absorbance of which is reffered to it. the time is saved which is spent on calculation by using the formula it also helps to check the linearity of the instrument.

10. Precautions • Performance of equipment and calibration curve should be quality controlled by testing simultaneously a commercial or in-house reference preparation with each batch of tests and maintaining quality control chart. For details see the chapter on quality control. • If Nonidet has not been added to the diluent, then 10-15 min should be given for reaction to complete and reading should be taken immediately. • Abnormal plasma proteins and high white cell count may result in turbid reaction mixture. This should be centrifuged and clear supernatant should be used for taking the reading.

11. SAHLI'S ACID HAEMATIN METHOD PRINCIPLE The method is based on the principle that haemoglobin is converted into acid haematin by addition of 0.I N Hydrochloric acid. The resultant solution is then compared with a reference solution in a colorimeter or coloured strip. OR THE HEMOGLOBIN IS CONVERTED TO ACID HAENATIN BY THE ADDITION OF 0.1N HCL. THE RESULTANT SOLUTION IS THEN COMPARED WITH REFENCE SOLUTION IN A COLOURED STRIP. Details of procedure, if a photoelectric calorimeter is used, are the same as for cyanomethaemoglobin method. Details of procedure, when Sahli's haemoglobinometer is used are given below:

12. Requirements • Sahli's haemoglobinometer • Sahli's pipette • 0.1N HCl • Dropping pipette • Blood sample

13. Procedure 1. Fill the tube of Sahli's haemoglobinometer up to mark with 0.1N hydrochloric acid. 2. Venous or capillary blood may be used. The Sahli's pipette is filled up to the 20 mark by gentle suction. Wipe outer side of pipette clean. There should be no air bubbles in blood column. 3. Blow the blood into the graduated tube of the Sahli's haemoglobinometer and suck the solution in and out of pipette 2-3 times. 4. Allow to stand for 5 min, so that haemoglobin gets converted into acid haematin. 5. Compare the colour of the solution in the graduated tube with that of the reference strips on either side of the haemoglobinometer. 6. If the colour of the graduated tube is darker, add drop by drop either 0.1N HCl or distilled water by the dropping pipette and mix with glass rod, until the colour matches with the reference strips. 7. Note the reading on the graduated tube. This is the haemoglobin level in g/dl. Some tubes also give level in percentage. To convert percentage into g/dl multiply the percent figure by 0.146.

14. NORMAL VALUES MEN 13-18 g/dl WOMEN 11.5-16 g/dl at birth 14-19 g/dl