CLINICAL SIGNIFICANCE Decreased blood levels are associated with various anaemias. Increased levels are observed In patients with polycythemia or dehydration. 1. PRINCIPLE 2. 3. Haemoglobin + KjFefCN),. ------------- > Methaemoglobin KCN --------► Cyanmethaemoglobin The brownish coloured cyanmethaemoglobin Is the product of almost all forms of haemoglobin found In blood except HbS and can be measured at 546 nM. Micro Method for discrete analysers Drabkin's Reagent.................................................... I 2.5 mL Sample ................................................................. I 0.01 mL Mix well. Incubate at RT for 5 minutes. Read O.D. at 546 nM (530 - 550 nM) against Blank. The final colour is stable for 30 minutes. Multiply results by factor or compute as given under result columns. REAGENTS SUPPLIED Cat. No. A 565 Composition 1. Drabkin's Reagent 1000 mL Potassium Ferricyanide 0.60 mMol/L Sodium Cyanide 0.77 mMol/L Phosphate Buffer 1.00 mMol/L Contains preservatives and stabilisers. 2. Cyanmethaemoglobin Standard 10 mL Haemoglobin 15.06 Gms/dL Equivalent to 15.06 Gms/dL in assay condition. (Contains buffers, stabilisers and preservatives). Preparation of Working Reagent All Reagents are ready to use. MANUAL METHOD Blank Test mL mL Pipette into 2 Test Tubes ........................... 5.00 5.00 Drabkins Reagent No.1 .............................. ----- 0.02 Sample (Fresh Blood)................................. Mix well. Incubate at RT for 5 minutes. Read at 546 nM (530-550 nM) or GREEN filter against Blank. The final colour is stable for 30 minutes. CALIBRATION GRAPH 1. Pipette into 3 Test tubes Drabkin's Reagent.................................... Cyanmethemoglobin Standard Read O.D. at 546nM (530-550nM) or GREEN filter. Corresponding Hb concentration ................. 2. Draw a line, expressing Gms/dL on the X-axis and optical densities on the Y-axis of a graph. Extrapolate the line within the limit of the dynamic range of this methodology (20 Gms/dL). NOTE: Programme the analyser using system parameters. A specific programme data sheet may be provided for each analyser upon request. STABILITY QUALITY CONTROL Drabkin's Reagent is stable at least 18 months at 20-35°C, away from light. The standard is stable at 2-8°C for 18 months. SAMPLE Fresh blood from finger prick is preferred. Anticoagulated whole blood can be used. Common anticoagulants such as Oxalate, EDTA or Heparin have no effect on this assay. (All samples should be handled as potential infective agents as no laboratory methods make conclusive findings for its safety. Therefore, adequate protective laboratory measures should be taken while handling such materials). SYSTEM PARAMETERS Reaction End-point Temperature 25°C to 35°C Wavelength 546 nM (530-550 nM) Standard Concentration (assay condition) 15.06Gms/dL Absorbance Range 0-2A0 Cuvette Path Length 1 cM Reagent Volume 2.50 mL 5.00 mL Sample Volume ______ 0.01 mL 0.02 mL Incubation 5 Minutes Linearity 20 Gms/dL Max. limit of Blank Reagent 0.070 Final Colour Stability 30 Mins. EXPECTED VALUES Since there is no acceptable Indian values established beyond arbitration, the values given here are of European references. Men 13.5 to 18.0 Gms/dL Women 12.0 to 16.0 Gms/dL Infants at birth 13.6 to 19.6 Gms/dL Children below 10 years may show slightly lower values. As with all diagnostic methods, the final diagnosis should not be made on the result of a single test as well as laboratory diagnosis must be confirmed with clinical manifestations. LIMITATIONS This reagent system is highly interfered by PAP reagent system used in GOD - POD, Cholesterol Oxidase, GPO-PAP, Uricase - PAP etc. For values higher than 20 Gms/dL dilute blood sample 1 to 2 with 0.9% Sodium Chloride an perform the assay. Multiply the result by dilution factor i.e. by 2 for 1:1 dilution. To ensure adequate quality control, each kit should be tested against a standard control material. It should be realised that the use of quality control material checks both instrument and reagent function together. Factors which might affect the performance of this test include proper Instrument function, temperature control, cleanliness of glasswares and accuracy of pippetting. It is appropriate to establish each laboratory's accuracy constant and interpret values accordingly. Similarly, laboratory findings should be established by clinical manifestations. WARNING This reagent system is for in vitro use only, This reagent system is containing preservatives and components that have not established for safety if contacted on broken skin or eye or taken orally. In case of such incidents wash off with plenty of water, or consult a physician. Bibliography 1. PRACTICAL HAEMATOLOGY J. & A Churchill (1963) 2. International committee for standardisation in Haematology, (1965) 3. British Standards Institution (1966) BIOLAB DIAGNOSTICS (I) PVT. LTD. J-245, MIDC, Tarapur, BOISAR401 501. Maharashtra, INDIA. E-mail: [email protected], [email protected] http://www.biolabdiagnostics.com (P.T.O.)