Hemoglobin Concentration Determination

Hemoglobin Concentration Determination  Provides an estimate of the oxygen carrying capacity of blood  A standard curve must be set-up before testing patient specimens  Cyanmethemoglobin standard is only commercially available standard used in routine hematology; all other products for QC maintainance are known as controls  The CURVE is set up by diluting cyanmethemoglobin certified standard with cyanmethemoglobin reagent and measuring the absorbance of each diluents 540nm  In the cyanmethemoglobin method, whole blood is mixed with K ferricyanide and KCN to convert Hb to cyanmethemoglobin which is very stable. The absorbance of the solutions is directly proportional to the amount of hemoglobin present. All forms of Hb are measured completely except sulfhemoglobin STANDARD CURVE PREPARATION Procedure 1. For instruments that require cuvette volumes of 5.0ml or less make dilutions to achieve certain concentration. The amount of dilution will depend on the concentration of the standard and the total volume required by the cuvette. 2. Transfer these solutions to matched cuvettes and read absorbance of each dilution against the blank on a spectrophotometer at 540nm. Use same instrument and wavelength for standards and unknowns. 3. On linear graph paper plot absorbance of each standard against its concentration. Alternatively, on semi logarithmic paper, plot %T against concentration. The curve may be used to read Hb concentration of controls and unknowns.  Cyanmethemoglobin standard curve is developed on the basis of the following percent transmission (T) readings obtained after preparion the hemoglobin concentration as illustrated in the Table: 0 g/dL = 100%, 5 g/dL = 75%T, 10 g/dL = 56%, 15 g/dL = 42.5%, 20 g/dL = 32%T  This curve is presented for illustrative purposes only. For e.g. Using this graph, if a patient’s sample gave a T reading of 52%, the Hb concentration (using these reagents and equipment) would be approximately 11g/dL  Cyanmethemoglobin standard curve developed on the basis of the following percent transmission (T) readings obtained after preparing the Hb concentration as illustrated in the Table : 0g/dL = 100%, 5 g/dL = 75%T, 10 g/dL = 56%, 15 g/dL = 42.5%, 20 g/dL = 32%T BLOOD INDICES 1. 2. 3. 4. 5. MCV – Mean Corpuscular Volume MCH – Mean Corpuscular Hemoglobin MCHC – Mean Corpuscular Hemoglobin Concentraition CI – Color Index VI – Volume Index N.V. 80-100cuu 27-32uug 30-36% 0.9-1.1 0.9-1.1 6. MCT – Mean Corpuscular Thickness 7. MCD – Mean Corpuscular Diameter 8. SI – Saturation Index 1.7-2.5u 6.7-7.7u 0.8-1.2 1. MCV – average volume of an erythrocyte in terms of cuu. N.V. 80-100cuu S.I. 80-100fL MCV = Hct x 10 Note: 1fL=10-15/L RBC in millions (femtoliter) e.g. Hct or PCV = 45% RBC = 5.0M = 45 x 10 5.0 Normocytic 80-100cuu Microcytic <80cuu Macrocytic >100cuu = 90 cuu (normocytic) 4. CI – based on the comparison of the average Hb concentration of an RBC with that of the normal RBC. N.V. 9 – 1.1 e.g. Hb = 15 gm% Hb = 14.5 gm% RBC = 5.5 M RBC = 5.0 M Normal CI = % of Normal Hb % of Normal RBC = gm% Hb 14.5___ RBC in M 5.0 = 103 110 = 0.93 2. MCH – measures the weight of hemoglobin in the average red corpuscle and expresses the result in micromicrograms. N.V. S.I. 27-32uug 27-32pg MCH = Hb x 10 RBC in M e.g. Hb 14.8 gm% 14.8 x 10 = 29.6uug (normocytic) 5.0 Normocytic 27-32uug Hypochromic <27 uug Hyperchromic >32 uug Note: 1pg = 10-12 = 1 micromicrogram (uug) (picogram) 3. MCHC – it denotes the average hemoglobin concentration per unit volume of packed RBC. N.V. 30-36% MCHC = Hb x 100 S.I. 30-36gm/dL Hct e.g. Hb = 14.8gm% (148gm/L) = 14.8 x 100 Hct = 45% (.45) 45 = 32.88 *microcytic hypochromic *macrocytic hypochromic *normocytic normochromic 15 x 100 14.5 __ 5.5 x 100 5.0 5. Volume Index – based on the comparison of the volume occupied by an average RBC with that of the normal RBC. (Hct = 43%) e.g. 26% = Hct (RBC = 5.0M) 4.0M = RBC VI = 26 x 100 43 ____ 40 x 100 5.0 = 60.4 80 = 0.76 6. Saturation index – average Hb concentration per unit volume of the RBC as compared to the normal. N.V. 0.8 – 1.2 SI = Color index e.g. = 0.93 = 1.2 Volume index 0.76 7. MCAT – average thickness of an erythrocyte N.V. 1.7 – 2.5u MCAT = __MCV__ MCD 2 2 *Valuable in detecting spherocytes and target cells 8. MCD N.V. 6.7 – 7.7 u Average = 7.2 u The measurement of cell diameters can be accomplished by: 1. Price Jones Method - Measurement of cell diameters as seen in the microscope, by placing a micrometer disk on the eyepiece (complete 500 cells or more). The diameter of the RBCs maybe recorded graphically to show the number of cells of various sizes – PRICE JONES CURVE Purpose of Determining Blood Indices 1. Correlate fundamental results obtained in CBC 2. Classify the type of anemia 3. RBC variations may be specifically studied 4. Obtain an accurate picture of RBC morphology *RDW SAMPLE EXERCISES HEMOGLOBINOMETRY I. Colorimetric Method 1. Direct Matching Method 2. Acid Hematin 3. Alkali Hematin 4. Cyanmethemoglobin 5. Oxyhemoglobin Method 6. Cyanhematin 7. Pyridine Hemochromogen Method II. Chemical Method 1. Iron Content Determination III. Gasometric Method II. Chemical Method Iron Content Method (Wong Test) - Most accurate/used chiefly for calibration purposes 2. Halometer or Diffraction Method 1. Iron is liberated from Hb by the action of Sulfuric and K persulfate 2. Proteins are precipitated by Tungstic acid and the Iron in the PFF is allowed to form ferricthiocyanate (salmon pink) 3. The density of the resultant color is measured photometrically and compared with the color of the standard solution of known iron content Based on the constant iron content of Hb; according to the ratio 1.0 gram of Hb = 3.47mg of Fe e.g. 34.7 mg of Fe ++ how many grams of Hb? Mg of Fe/100ml 34.7 1:3.47 = X: 34.7 3.47X = 34.7 = Hb (gm/100ml) X = 10gm% III Gasometric Method - Uses Van Slyke apparatus - Hb is allowed to be saturated with O 2 or CO. When the bound O2 or CO is released and measured, the amount of Hb to which it was bound can be calculated: O 2 Gasometric method/CO Application: makes use of the Hufner’s factor 1 gm of Hb = (binds) 1.34ml of O 2 Volume of 02/100ml 1.34 = grams Hb/100ml e.g. 14.7ml of 02 was bound to Hb in 100ml of blood 1: 1.34 = X: 14.7 1.34X = 14.7 X = 10.07 gm%