Hemoglobin Estimation PDF

Second stage Human Physiology Hemoglobin estimation Dr. Dalal Falah Muhsin M.Sc Mustafa Hafed B.Sc Zainab  Hemoglobin is normally present in red cells Two primary structures Globin Heme which is composed of Protoporphyrin ( ferroprotoporphyrin)...

Second stage Human Physiology Hemoglobin estimation Dr. Dalal Falah Muhsin M.Sc Mustafa Hafed B.Sc Zainab  Hemoglobin is normally present in red cells Two primary structures Globin Heme which is composed of Protoporphyrin ( ferroprotoporphyrin) Iron  IRON Iron is an essential component of hemoglobin Decreased tissue iron = cellular dysfunction Increased tissue iron = cellular destruction Regulated by absorption, not excretion Iron circulates in the plasma bound to transferrin IRON Iron Compartments/Pools GLOBIN Globin chains are composed of amino acids arranged in a specific pattern Site of synthesis is the ribosomes 4 normal chain types are produced  Alpha chain composed of 141 amino acid chains  Beta chain146 amino acid chains  Gamma  delta VARIANTS OF HAEMOGLOBIN i. Normal Haemoglobin: HbA, HbF- gama, HbA2-delta. ii. Abnormal Haemoglobin: HbS, HbC, HbD and HbE The measurement of haemoglobin concentration in the blood is called Haemoglobinometry.  Two normal Hgb forms Deoxyhemoglobin (Fe2+ without oxygen), in tissues Oxyhemoglobin (Fe2+ with oxygen), in lungs Two abnormal Hgb forms Methemoglobin (Fe3+ ,oxidized) Carboxyhemoglobin (Fe2+ with CO) Sulfhaemoglobin (SHb): (Fe2+ with s) Both are reversible 9.4. METHODS OF HEMOGLOBIN MEASUREMENT Is the measurement of concentration of Hgb in red cells (whole blood) ((((Hgb is reported in g/dL)))) There are different methods (1) Spectrophotometric a) Cyanmethemoglobin b) Hemo-Cue c) Oxyhemoglobin d) Direct Read- Out (2) Visual comparative methods a)Sahli - Hellige method b)BMS Hemoglobinometry  Determination of Hemoglobin Cyanmethemoglobin(Hemoglobin-Cyanide) method Principle: When Blood is mixed with the Drabkin’s solution, the erythrocytes are lysed by producing evenly disturbed hemoglobin solution. Potassium ferricyanide hemoglobin methemoglobin+ potassium cyanide hemiglobincyanide (cyanmethemoglobin). When the reaction is complete, absorbance of the solution is measured in a spectrophotometer at 540 nanometer. Equipment: 1-Hb pipette 2-Spectrophotometer spectrophotometer Reagents 1- Drabkin’s solution pH7.0-7.4 which Drabkin’s contains : solution b-Potassium cyanide 50 mg c-Potassium ferricyanide 200 mg d- Potassium dihydrogen phosphate 140 mg e- Nonionic detergent 1 ml f-Distilled water 1 L 2-Cyanmethemoglobin standard solution with a known hemoglobin value Sample Blood obtained from skin puncture or EDTA-anticoagulated venous blood. Procedure 1-Take 5 ml of Drabkin’s solution in a test tube and add 20 μl of blood, mix the mixture and allow to stand for at least 5 minutes. This time is adequate for the transformation of hemoglobin to cyanmethemoglobin. 2-Pour the test sample to a cuvette and read the absorbance of the test sample in a spectrophotometer at 540 nanometers. Also, read the absorbance of the standard solution. Absorbance must be read against Drabkin’s solution. 3-From the formula given below, the hemoglobin value is derived. Advantages 1-Visual error is not there as no color matching is required. 2- Cyanmethemoglobin solution is stable and it’s color does not fade with time so readings may not be taken immediately. 3- Absorbance may be measured soon after dilution. 4- A reliable and stable reference standard is available. Disadvantages 1-Diluted blood has to stand for a period of time to ensure complete conversion of Hb. 2- Potassium cyanide is a poisonous substance and that is why Drabkin’s solution must never be pipetted by mouth. 3-Abnormal plasma proteins cause turbidity when blood is diluted with Drabkin’s solution. 3- A high leucocyte count also causes turbidity on dilution of blood. Centrifuging the diluted blood can help overcome the turbidity.  Determination of Hb content = Sahli experiment = concentration of hemoglobin/100 ml blood TOOLS? Sahli apparatus, composed of : Sahli tube Sahli Comparator Sahli pipette Blood sample Hypotonic HCL??? Distilled water  SAHLI TUBES HAVE 2 GRADATIONS One for the hemoglobin content in gm/100 ml blood The other for percentage of hemoglobin content of normal (70%, 80%) PRINCIPLE? ROLE OF HYPOTONIC HCL? As it is hypotonic for the hemolysis of RBCs Convert the hemoglobin to brownish acid hematin HCl + Hb → acid hematin (brown in color) The intensity of color is proportional to hemoglobin content in blood STEPS 1. Put 0.1N HCl till the mark 10% 2. Add 0.02 ml blood (using the micro tube of the pipette) 3. Shake for 15 minutes 4. HCl + Hb → acid hematin (brown in color) 5. Add distilled water to the acid hematin drop by drop 6. Match colors at full arm length and against light. PRECAUTIONS? 1. Be sure that there is no air bubbles in the blood column 2. Clean the tip of pippette from any blood to avoid false high results 3. Dilute drop by drop gradually, in case of over dilution, what to do?? 4. Compare at daylight at full arm lenghth  Normal hemoglobin content Newly born For males For females infants 15-16 gm/dl. 13-14 gm /dl 19 gm/dl. ABNORMAL HEMOGLOBIN CONTENT? Above the physiological levels = polycythemia Below the physiological levels = Anemia (all types)  BLOOD CELL COUNT RBC COUNT Purpose, of performing Total Red Blood cell count is to know whether or not patients are suffering from Erythrocytosis or Polycythemia (i.e. the increase in the no. of Red Blood Cells to more than 6.5 million/mm3) or Erythrocytopenia or Erythropenia (i.e. the Decrease in the no. of Red Blood Cells to less than 3.5 million/mm3). principle the blood specimen is diluted (usually in 1:200 ratio) with the RBCs diluting fluid (the hayem’s fluid) which preserve and fix the red blood cells. the hayem’s fluid is isotonic to the red blood cells and does not cause any damage to it. after diluting the specimen, the content is charged on hemocytometer / Neubauer’s chamber and the cells are counted in the areas specific for RBCs count. Hemocytometer / Neubauer’s Chamber The Neubauer’s Chamber has ruled the area of total 9 square mm and the depth is 0.1 mm as when the coverslip is placed on the surface of the counting chamber, the space between the bottom of the cover glass and the base of grooved area measures 0.1 mm in depth. *The central 1 square is highly ruled which is divided into 25 squares. Each square of the Central square is further subdivided into 16 small squares. so the total no. of the area to be counted for RBC Count : 16 × 5 = 80 small squares *For RBC count the cells are counted in the 5 squares of the Central square as 4 Corner squares of the Central square and 1 central square of the Larger Central Square. Materials Required 1- Blood sample (Capillary blood or EDTA anticoagulated specimen) 2- RBC diluting fluid (preferably Hayem’s fluid) 3-Gauze piece or Cotton 5-RBC pipette 6-Hemocytometer , Neubauer’s Chamber 7-Coverslip 8-Microscope Procedure 1- Fill the RBC pipette up to the 0.5 mark with the blood specimen and wipe out the pipette externally to avoid false high results. 2- Fill the same pipette with the RBC diluting fluid (Hayem’s Fluid) up to the mark 101. 3- Be cautious that there should be no air bubble in the pipette bulb. 4- Mix the Blood and Diluting fluid in the pipette by rotating the pipette (horizontally) between your palms. 5- Take out the Neubauer’s chamber / Hemocytometer from its case and clean it using a swab or gauze piece. Similarly, clean out the cover glass and place it over the grooved area of Hemocytometer. there is a special type of cover glass is used which is 0.4 mm thick with very smooth surface and even thickness so that the space between the grooved area of the chamber and cover glass is exactly 0.1 mm 6- put the RBC pipette, mix the solution present in it again and then discard 1-2 drops from the pipette before charging the chamber. 7- Gently press the rubber tube of the RBC pipette, so that the next drop of fluid is in hanging position. 8- Touch the Tip of the pipette with the hanging drop against the edge of the coverslip making an angle of 45° approximately. 9- Allow a small amount of fluid from the pipette to fill into the chamber which occurs by the Capillary action. Do not overcharge the chamber and there should be no air bubble in the Chamber. 10- After charging, wait for 3-5 min so that the cells settle down in the chamber and then focus the chamber under the microscope to calculate Red Cells. 11- focus the ruling using the 10x objective lens and then count the RBCs in 5 small squares of the central square as described above, using the 40x objective lens. 12- count the cells which are lying on the right and lower lines of the 5 small squares but not the opposite line. in case of marginal cells, count the cells on ‘l’ line that is either on right and lower lines or left and upper lines. Calculation: total RBCs count = n × 10,000 mm3  WBC count Purpose : A WBC count can detect hidden infections within human body and alert doctors to undiagnosed medical conditions, such as autoimmune diseases, immune deficiencies, and blood disorders. This test also helps to monitor the effectiveness of chemotherapy or radiation treatment in people with cancer. Principle WBC diluting fluid Turk‘s solution is used to perform the WBC count. Red cells are lysed by glacial acetic acid and Gentian violet slightly stains the leukocyte nuclei. The blood specimen is diluted 1:20 (in a White blood cell pipette with the diluting fluid, and the cells are counted under the low power of the microscope using a counting chamber.The number of cells in undiluted blood is reported per cumm( μl) of the whole blood. Materials Required 1- Blood sample (Capillary blood or EDTA anticoagulated specimen) 2- WBC diluting fluid (Turk’s fluid) 3-Gauze piece or Cotton 5-WBC pipette 6-Hemocytometer, Neubauer’s Chamber 7-Coverslip 8-Microscope Composition of WBC Diluting Fluid Turk‘s solution is used. It is a 3% solution of acetic acid (for destruction of red cells) with trace of Gentian Violet (for staining the white cells). Procedure: 1-Draw the EDTA anticoagulated blood to 0.5 mark on the capillary end of the WBC pipette. 2- wipe excess blood outside the pipette using cotton. 3-Draw the diluting fluid up to 11 marks. 4-Mix the contents in a pipette and after 5 minutes, discard a few drops, fill the counting chamber and allow the cells to settle for 2- 3 minutes. 5-Focus on 1 of the ” W” marked areas( each with 16 small squares) by turning the objective to 10X low power Count cells marked in all 4 ” W” corner squares. Calculation: total RBCs count = n × 50 mm3