Practical Hematology Lecture 3 PDF

Practical Hematology Laboratory Technique Department Third stage Lecture Three Hemoglobin Determination Introduction and Principle: Hemoglobin is the pigment inside the red blood corpuscles which bind w...

Practical Hematology Laboratory Technique Department Third stage Lecture Three Hemoglobin Determination Introduction and Principle: Hemoglobin is the pigment inside the red blood corpuscles which bind with oxygen and carries the oxygen from the lungs to the tissues. The amount of hemoglobin can be estimated by different methods, The cyanmethaemoglobin (haemoglobincyanide) method for haemoglobin determination is the reference method. Objectives: 1- At the end of this session the students will be able to estimate hemoglobin concentration in a provided blood sample. 2- Identify on the normal value of hemoglobin according to age. Materials and Methods:  Colorimetric method. Spectrophoto-meter. 1  Figure (1) spectrophotometer (colorimetric method) Drabkin's solution. Principle: cyanmethemoglobin reagent (drabkin's solution) containing potassium cyanide and potassium ferricyanide is added to the provided blood sample the ferricyanide will convert hemoglobin iron from the ferrous (Fe2+) to the ferric (Fe3+) state to form methemoglobin. Then combines with potassium cyanide to give the stable pigment cyanmethemoglobin. The absorbance of this pigment is measured with a spectrophoto- meter at a wavelength of 540 nm.  The diluent should be clear and pale yellow in colour.  When measured against water as a blank in a spectrometer at a wavelength of 540 nm, absorbance must be zero.  If stored at room temperature in a brown borosilicate glass bottle, the solution keeps for several months.  If the ambient temperature is higher than 30°C, the solution should be stored in the refrigerator but brought to room temperature before use. 2   It must not be allowed to freeze.  The reagent must be discarded if it becomes turbid, if the pH is found to be outside the 7.0–7.4 range or if it has an absorbance other than zero at 540 nm against water blank. Method 1. Make a 1 in 201 dilution of blood by adding 20 µl of blood to 4 ml of diluent. 2. Stopper the tube containing the solution and invert it several times. 3. Let the test sample stand at room temperature for at least 5 min (to ensure the complete conversion of haemoglobin to haemiglobincyanide). 4. Then pour it into a cuvette and read the absorbance in a spectrometer at 540 nm or in a photoelectric colorimeter with a suitable filter against a reagent blank. 5. The absorbance of the test sample must be measured within 6 h of its initial dilution. The absorbance of a commercially available HiCN standard (brought to room temperature if previously stored in a refrigerator) should also be compared to a reagent blank in the same spectrometer or photoelectric colorimeter as the patient sample. 6. The standard should be kept in the dark and, to ensure that contamination is avoided, any unused solution should be discarded at the end of the day on which the ampoule is opened. Calculation of Haemoglobin Concentration Sources of Error and Comments: 1. Cyanmethaemoglobin reagent is sensitive to light. It should be stored in a brown bottle or in a dark place. 3  2. A high WBC count (greater than 20 X 109/L) or a high platelet count (greater than 700 X 109/L) can cause turbidity and a falsely high result. In this case, the reagent-sample solution can be centrifuged and the supernatant measured. 3. Lipaemia also can cause turbidity and a falsely high result. It can be corrected by adding 0.01 mL of the patient’s plasma to 5 mL of the cyanmethaemoglobin reagent and using this solution as the reagent blank 4. Cells containing Hb S and Hb C may be resistant to haemolysis, causing turbidity; this can be corrected by making a 1:2 dilution with distilled water (1 part diluted sample plus 1 part water) and multiplying the results from the standard curve by 2. 5. Carboxyhaemoglobin takes 1 hour to convert to cyanmethaemoglobin and theoretically could cause erroneous results in samples from heavy smokers. The degree of error is probably not clinically significant, however. 6. Because the haemoglobin reagent contains cyanide, it is highly toxic and must be used cautiously. Consult the safety data sheet supplied by the manufacturer. Acidification of cyanide in the reagent releases highly toxic hydrogen cyanide gas. A licensed waste disposal service should be contracted to discard the reagent; reagent-sample solutions should not be discarded into sinks. Result: Age Adult Adult 6-12years 2-6 years 1year 3-6 Man Women Months Hemoglobin 150±20 135±15 135±20 125±15 126±15 126±15 Concentration g/l Age 2Months 1Months 14 day 7 day 3day Birth Hemoglobin 112±18 140±25 165±4 175±4 180±30 180±40 Concentration g/l 4 Questions and Discussion: 1. Is this test enough to diagnose anemia from hemoglobin estimation? 2. What the difference between hemoglobin and hematocrit? Good Luck Assist. Lect. Iman. H. Jirjees 5