2.9. Sigma provides a stable, dry Drabkins Reagent, which is combined with a surfactant to prepare Drabkins Materials and Methods: The hemoglobin (Hb) results on 200 samples of varying Hb concentrations obtained by these two cyanide free methods were compared with a cyanmethemoglobin method on a colorimeter which is light emitting diode (LED) based. The ferrous ions (Fe 2+) of the hemoglobin molecules are oxidized by potassium ferricyanide to ferric ions (Fe 3+). Normalization of the AChE activity to the hemoglobin content, determined as cyanmethemoglobin, prevented dilution errors. 2 = ICSH (Cyanmethemoglobin method), capillary blood Bibliography HemoCue Hb 201 Operating Manuals Dacie and Potassium ferricyanide oxidizes haemoglobin to methemoglobin and then to cyanmethemoglobin. Hemoglobin consist of a prosthetic group named heam, which is combined with protein called globin (Haemoglobin = Heam + Globin). The potassium ferricyanide converts Hb to methemoglobin which is converted to cyanmethemoglobin (HiCN) by potassium cyanide. Drabkins solution on mixing with whole blood converts Hemoglobin to Cyanmethemoglobin. Due to their small size, nanoparticles have distinct properties compared with the bulk form of the same materials. Acid Hematin Method; Cyanmethemoglobin Method (CMG) a.k.a Drabkins Method syringe of the rapid reaction apparatus. Three advantages of the cyanmethemoglobin method are: i.) The etiology was investigated by hemoglobin electrophoresis in alkaline and acid pH, Hb A2 dosage and measurement Mosul Medical College. Interferences are substances such as lipids, free hemoglobin and bilirubin (also globulins and drugs) which affect the results of clinical pathologic tests, as they are being performed. In this method, the ferrous form of Hb is converted to ferric form in presence of potassium ferricyanide, converted to met-haemoglobin which is then converted to cyanmethemoglobin The erythrocytes are hemolyzed to release the hemoglobin. And then all forms of hemoglobin present in blood are completely changed to a single conjugate cyanmethemoglobin. Cyanmethemoglobin is the colored product being directly measured in the method for hemoglobin determination. This way all types of hemoglobin present in the blood are entirel Page 52 Prime CCS Instructions for Use Manual 5. Edit. Automated method on Medonic CA 530 is based on electronic impedance principle for cell counting while lysing and colorimetry for Hb estimation. Although Cyanmethemoglobin method is cheaper and often used, it takes more time. Hb estimation was done by cyanmethemoglobin method. The cyanmethemoglobin method works on the principle of conversion of hemoglobin to cyanmethemoglobin by the addition of potassium cyanide and ferricyanide, whose absorbance is measured at 540 nm in a photometer against a standard solution. In the cyanmethemoglobin method, a fixed quantity of blood is diluted with a reagent and haemoglobin concentration is determined after a fixed time interval in an accurate, well calibrated photometer. Cyanmethemoglobin (HiCN) (Drabkin s Method) Dr. Amjad H. Abid. Hemoglobin content was measured by cyanmethemoglobin method and red blood cell volume was measured by bilirubin, occult blood, ketone bodies, specific gravity by refractometry, pH by electrode reaction, and urinary electrolyte (Na+ and Ca++) by atomic absorption analysis. Whole blood was diluted with Drabkins solution, (buffered potassium ferricyanide and potassium cyanide). In order to perform this method, Drabkins solution which contains potassium ferricyanide and potassium cyanide are used and mixed with blood. Pinch together the white probe holder and remove the probe together with the S-line and air detector cable and discard. Does effective counseling play an important role in controlling 82250 R1: 1 x 50 mL: All reagents are ready for use. Blood is mixed with potassium ferricyanide and potassium cyanide to convert hemoglobin into cyanmethemoglobin which is measured spectrophotometrically at 540 nM. Some of the other methods for evaluation of Hb are Copper sulfate method, Hematocrit method by centrifuge, Lovibond type comparator method, Grey wedge photometer method, Hemocue method and cyanmethemoglobin method. constituent of erythrocytes. 44 Another comparative study from Brazil showed that this method had a sensitivity of 39.5% and specificity of 93.2%. The reaction completion time for the haemoglobincyanide method was 10 minutes, whereas for the sodium lauryl sulphate method and the alkaline haematin method, it was less than 3 minutes. Objective: To evaluate accuracy of hemoglobin estimation by manual photometric method versus automated method by hematology cell counters using the same sample at the same time. whereas total conversion of HbCO requires 90-120 min. Hemoglobin was estimated by cyanmethemoglobin method by commercially available Drabkin's reagent solution (Sigma-Aldrich Co USA. However, the presence of potassium cyanide (KCN) and potassium ferricyanide (K3-Fe(CN)6) in the reagents has raised problems of laboratory and environmental pollution. The recognized reference method for total hemoglobin is the cyanmethemoglobin method, which is also known as the cyanhemoglobin method. Cyanmethemoglobin. The recognized reference method for tHb determination (tHb = total hemoglobin) is the cyanmethemoglobin method, which is also known as the cyanhemoglobin method. 1. Specific gravity. OR take 20 microliter of blood + Drabkin 5 mL = 1 : 250 dilution. * * * *Bright Intensity LED High efficiency and bright intensity LED is adopted as the light source, therefore it is sensitive and is not necessary to be changed. An interesting method of adding blood to agar media is described in Bullochs 1938 The History of Bacteriology: Human blood or the blood of animals may be used. The 700 nm reading indicates completeness of lysis. The principle of this method is that when blood is mixed with a solution containing potassium ferricyanide and potassium cyanide, the potassium ferricyanide oxidizes iron Conversion of the standard and of hemoglobin in blood samples to cyanmethemoglobin is accomplished with Drabkin's solution reconstituted from a dry powder and The produced quinoid compound with a ketoenol-system could be *Corresponding author: [emailprotected] . Direct cyanmethemoglobin method has been the gold standard for hemoglobin estimation but other methods like hemoglobin color scale, Sahli technique, Lovibond-Drabkin technique, Tallqvist technique, copper-sulfate method, HemoCue and automated haematology analyzers are also available. to count blood cells, the cyanmethemoglobin method to measure hemoglobin concentration, and latex immunoturbidimetry to measure CRP concentration. Direct cyanmethemoglobin method has been the gold standard for hemoglobin estimation but other methods like hemoglobin color scale, Sahli technique, Lovibond-Drabkin technique, Tallqvist technique, copper-sulfate method, HemoCue and automated haematology analyzers are also available. A standard hemoglobin assay utilizes the toxic cyanmethemoglobin method. 6-10. to count blood cells, the cyanmethemoglobin method to measure hemoglobin concentration, and latex immunoturbidimetry to measure CRP concentration. Stability and storage: 3 years at 18-25C. 16. The development of two kinetic methods for quantitation of hemoglobin and methemoglobin is described. Reagent for quantitative determination of hemoglobin (Hb) in whoole blood. Gravity. The conversion of carboxy hemoglobin (HbCO) to cyanmethemoglobin (MHbCN) by Drabkin's reagent has been shown to be considerably slower than is the same reaction with oxyhemoglobin(Hb02). Hemoglobin (Hgb) concentration in grams per deciliter is determined most accurately by the cyanmethemoglobin method, using a colorimeter or spectrophotometer. The method of cyanmethemoglobin - hemoglobin with the help of a special solution of Drabkin is converted into cyanmethemoglobin. the ability of superoxide dismutase to inhibit the reduction of nitro-blue-tetrazolium (NBT) Specimen: -EDT A antico a gulated blood. This oxidation results in the formation of methemoglobin. Methemoglobin combines with the cyanide ions (CN -) to form cyanmethemoglobin, a stable compound. All hemoglobin derivatives except sulfhemoglobin are converted to cyanmethemoglobin. The kinetics of the hemoglobin-methemoglobin-cyanmethemoglobin chemical system are characterized and this information is used to establish conditions for the methods. These properties are rapidly revolutionizing many areas of medicine and technology. CHYANMETHEMOGLOBIN METHOD. The blood sample is diluted 1:251 with a reagent buffering solution. Specific gravity. Direct cyanmethemoglobin method has been the gold standard for hemoglobin estimation but other methods like hemoglobin color scale, Sahli technique, Lovibond-Drabkin technique, Tallqvist technique, copper-sulfate method, HemoCue and automated haematology analyzers are also available. 12 Automated method uses a non cyanide hemoglobin method. Each hemoglobin consists of four subunits consisting of iron. The kinetics of the hemoglobin-methemoglobin-cyanmethemoglobin chemical system are characterized and this information is used to establish conditions for the methods. 0% average accuracy. Potassium ferricyanide converts hemoglobin to methemoglobin, which is converted to cyanmethemoglobin by the action of potassium cyanide. syringe of the rapid reaction apparatus. It measures all types of HEMOGLOBINS except Erythrocyte sedimentation rate (ESR) The ESR is a simple non-specific screening test that indirectly measures the presence of inflammation in the body. Methods: This was a prospective study that compared two groups (Anemic and Non-anemic). Accepted November 29, 1987 INTRODUCTION Numerous methods have been used for the quantitation of hemoglobin, but many have significant drawbacks. The International Committee for Standardisation in Haematology (ICSH) has recommended the cyanmethemoglobin (HiCN) method as a reference method.2v3 The advantages of HiCN over other methods are that it involves a single chemical reaction Prepare a Cyanmethemoglobin Standard Solutioncontaining 180 mg/mL of appropriate hemoglobinprepared in Drabkins Solution. Study Flashcards On HemaLab: Hemoglobin determination at Cram.com. Blood was diluted in Drabking solution and the absorbance was assessed photometrically at 540 nm on a Milton Roy Spectronic1201. While this method is straightforward and uses a single reaction solution, not all forms of hemoglobin are converted to cyanmethemoglobin at the same rate or even to completion. 15. Measures all forms of haemoglobin except sulfhemoglobin ii.) The Cyanmethemoglobin standard complies to the specifications defined by ICSH which is based on the molecular weight of Hb ( 64,458 daltons) and a millimolar extinction coefficient of 44. begins soon after conception peaks at birth, declines to <0 by three months. The absorbance of cyanmethemoglobin, directly proportional to the haemoglobin concentration, is measured at 546 nm (520-560). Glutathione peroxidase assay GP activity was determined by a modified method of Paglia and Valentine (1967) [ 1 ] Activity was determined spectrophotometrically by coupling the oxidation of glutathione and NADPH using GR. The standard clinically approved method for hemoglobin concentration measurement is called the cyanmethemoglobin method, in which a blood sample is drawn from a subject. All blood samples were collected from the jugular vein in vacuum tubes without anticoagulant. Study population and design. A study to compare its performance with more established methods found that the results did not correlate well with either the cyanmethemoglobin or CuSO 4 method. The reaction was followed at 544 (Absorption max. Serum Sample. In the first method, which is based on the reaction of cyanide ion with methemoglobin, The next method is the hemiglobincyanide or cyanmethemoglobin method is the most accurate method of measuring hemoglobin and is considered the reference standard. Conversion of the standard and of hemoglobin in blood samples to cyanmethemoglobin is accomplished with Drabkin's solution reconstituted from a dry powder and 2.1 Measurement of White Blood Cells, Red Blood Cells, and Platelets White blood cells (WBC), red blood cells (RBC), and platelets (PLT) are measured by the electrical impedance method (Fig.1). Heam carries oxygen (O2) from the lungs to the tissue cells and carbon dioxide (CO2), the gaseous waste product combines with potassium cyanide to produce hemiglobincyanide (cyanmethemoglobin). as oxyhemoglobin, carboxyhemoglobin, cyanmethemoglobin, acid and alkaline haemin, by its oxygen capacity and by iron content. This difference in reaction rate must be considered in the determination of total hemoglobin as The study is a part of the national survey of Risk Evaluation of Cancer in Chinese Diabetic Individuals, a longitudinal (REACTION) study [], which was a population-based sub-cohort study conducted amongst adults aged 40 years and older, from 2011 to 2014.The study design and methods have been described previously in detail [13,14,15]. Haematology. Can be easily standardized iii.) This method though cheaper, takes more time. certified cyanmethemoglobin solution: When using the turbidity method for detecting the presence of hemoglobin S, an incorrect interpretation may be made when there is a: a. concentration of <7 g/dL hemoglobin b. glucose concentration >150 mg/dL c. blood sugar > 2 hrs. However, Chondrex, Inc. would like to introduce a safe, non-cyanide hemoglobin assay kit (13, 14). The recognized reference method for total hemoglobin is the cyanmethemoglobin method, which is also known as the cyanhemoglobin method.1 The blood sample is diluted 1:251 with a reagent buffering solution. DRAFT. Principle. A preweighed vial of lyophilized methemoglobin (ferrihemoglobin) provides a simple, stable, reproducible standard for colorimetric determination of total hemoglobin concentration in whole blood by the cyanmethemoglobin method. Hemoglobin (Hb) or the red pigment of RBCs is the main . The haemoglobin is estimated with the help of cyanmethemoglobin curve. The photometer is calibrated at the factory against the cyanmethemoglobin (ICSH) method, which is the international reference method for the determination of the total hemoglobin In the Drabkin's method of haemoglobin estimation haemoglobin is oxidised to methemoglobin by potassium ferricyanide, which reacts with cyanide ions of potassium cyanide to form cyanmethemoglobin. The standard clinically approved method for hemoglobin concentration measurement is called the cyanmethemoglobin method, in which a blood sample is drawn from a subject. The cyanmethemoglobin method is performed by the conversion of several different kinds of hemoglobin into stable cyanmethemoglobin, and the absorbance of cyanmethemoglobin (540 nm, =44000 l/mol per cm) is measured. It is the carrier of oxygen from the lungs to tissues and the carrier of carbon dioxide from tissues to the lungs. The cyanmethemoglobin method is the method of choice and is. Blood cells are counted using the impedance method, and hemoglobin is measured by the cyanmethemoglobin method. The latter converts Hb to methemoglobin which is converted to cyanmethemoglobin (HiCN) by potassium cyanide. In these methods blood is drawn from the patient and is used to evaluate Hb using Salt method - the blood is mixed in a test tube with a solution of hydrochloric acid. To measure CRP, the instrument uses an antigen-antibody reaction reagent to perform a latex turbidimetric immunoassay. The ratio of glycated hemoglobin can be determined by degrading a glycated hemoglobin in a whole blood sample selectively with a protease to give a glycated hemoglobin degradation product; causing a redox reaction between a glycation site The principle of this method lies in conversion of hemoglobin to cyanmethemoglobin by the addition of Potassium cyanide and ferricyanide whose absorbance is measured at 540 nm in a photoelectric calorimeter against a standard solution. The test was performed as outlined by Bhaskaram et al [ 12 ]. Beers Law, states: where A is the absorbance, e is the molar absorbitivity (L/mol cm) which is specific for each compound, b is the path length of the sample In this method, a measured sample of whole blood is added to Drabkins diluting fluid. Hemoglobin Estimation Using Cyanmethemoglobin Method. Despite the remarkable speed of development of nanoscience, relatively little is known about the interaction of nanoscale objects with living systems. Four milliliters of each blood sample were anticoagulated with 250 ~1 of EDTA, Routinely in a physiology laboratory, haemoglobin estimation is performed by Sahlis haemoglobinometer. whereas total conversion of HbCO requires 90-120 min. Hemoglobin Laboratory2 DRAFT. Which curve represents the production of delta polypeptide chains of hemog [image] Definition. Here the erythrocytes are hemolyzed and the bivalent iron in oxyhemoglobin and In this method, the ferrous form of Hb is converted to ferric form in presence of potassium ferricyanide, converted to met-haemoglobin which is then converted to cyanmethemoglobin Hemolysis can be detected by using a photometric colorimetric test (cyanmethemoglobin method) (Stadie, 1920). In the model used currently the four tangential jets to the mixing chamber were 0.5 mm bore instead of 1.0 mm bore. Total COZ content by the method of Van Slyke and Neil1 (4), pH, and usually pco3 by electrode were measured in the hemoglobin solution, in the CC, and a 1:l mixture of the two. Other. 10. what is the principle of cyanmethemoglobin method? The cyanmethemoglobin procedure is the most widely used and preferred method for hemoglobin assay (2). Sodium deoxycholate haemolyses erythrocytes and hemoglobin is released. The cyanide then reacts with the methemoglobin to form the stable. This method is simple, rapid and reliable. Cram.com makes it easy to get the grade you want! Measures all forms of haemoglobin except sulfhemoglobin ii.) Color intensity is proportional to total hemoglobin concentration. When Blood is mixed with a solution of potassium cyanide, potassium ferricyanide, and Drabkins solution, the erythrocytes are lysed by producing evenly disturbed hemoglobin solution. There are specific changes in the acrosome region which are collectively called as acrosomal reaction. Sodium nitrite converts hemoglobin to methemoglobin which, together with sodium azide, gives azidemethemoglobin. a. CuSO4 method on donor deferral and concluded that the CuSO4 method does not give quantitative results, has a subjective end point and is inaccurate [15]. cyanmethemoglobin method (Drabkins reagent, Sigma Chemical, St. Louis, MO). Detection of SARS-CoV-2 virus using an alternative molecular method and evaluation of biochemical, hematological, inflammatory, and oxidative stress in healthcare professionals. Blood is mixed in a solution containing potassium cyanide and potassium ferricyanide. ), 580 (shoulder), 500 (valley), and 700 (turbidity check) nm (top to bottom traces). The absorbance of blood + Drabkin 4 mL = 1 : 200 dilution. of Biochemistry. Drabkin reagent: ( drab'kin ), a solution used in the cyanmethemoglobin method of measuring hemoglobin. A two-stage random sampling method was used to select 30 clusters on the basis of probability proportional to size. The cyanmethemoglobin method works on the principle of conversion of hemoglobin to cyanmethemoglobin by Product Information. From the Home screen, press the Tool Box icon. Total COZ content by the method of Van Slyke and Neil1 (4), pH, and usually pco3 by electrode were measured in the hemoglobin solution, in the CC, and a 1:l mixture of the two. The conversion of carboxy hemoglobin (HbCO) to cyanmethemoglobin (MHbCN) by Drabkin's reagent has been shown to be considerably slower than is the same reaction with oxyhemoglobin(Hb02). recommended by the Technical Subcommittee on Hemoglobinometry of the. cyanmethemoglobin. Potassium ferricyanide transforms hemoglobin into methemoglobin, and methemoglobin combines with potassium cyanide to produce hemiglobincyanide (cyanmethemoglobin). Accordingly, what is the principle of estimation of Hb by Sahli's method? Cyanmethemoglobin method. Edit. 44 Another comparative study from Brazil showed that this method had a sensitivity of 39.5% and specificity of 93.2%. cyanmethemoglobin by addition of potassium cyanide (KCN). Three advantages of the cyanmethemoglobin method are: i.) Hemoglobin concentration was measured in solution as cyanmethemoglobin. In particular, Fe 3+-methemoglobin or Fe 3+-cyanmethemoglobin was produced by exposing Hb to air for 24 h or by its reaction with the Drabkins reagent (Sigma-Aldrich, St. Louis, MO, USA), whereas Fe 2+-carboxyhemoglobin by exposing Hb to CO as described in the previous section. Th e SFHb and polySFHb samples were diluted at a ratio of 1:200 with PPB (50 mM, pH 7.0) to a total volume of 5 ml; while the SFHb mixed with Conversion of Hb02 to MHbCN is complete in 10 min. Detection of SARS-CoV-2 virus using an alternative molecular method and evaluation of biochemical, hematological, inflammatory, and oxidative stress in healthcare professionals. Initially screening for anemia was performed by manually measuring hemoglobin (Bioclin Kit); the results were subsequently compared with an automated screening method (Coulter T-890). Cyanmethemoglobin method (Calorimetry) Alkaline hematin method; Oxyhemoglobin method; CuSO4 specific gravity method; Spectrophotometry; Hemoglobin estimation by sahlis method. This reaction take place after the attachment of the sperm to the egg surface. The azide-MetHb reaction has been adapted for POCT hemoglobinometers. REAGENT COMPOSITION Vial R1 REAGENT (50 x concentrated) Working reagent Phosphate Buffer 50 mmol/L 1 mmol/L Potassium cyanide 37.5 mmol/L 0,75 mmol/L Save. Quickly memorize the terms, phrases and much more. recommended by the Technical Subcommittee on Hemoglobinometry of the. Cyanmethemoglobin Standard is used for direct comparison with blood. an hour ago. This process is experimental and the keywords may be updated as the learning algorithm improves. The broad absorption peak of cyanmethemoglobin permits its measurement using both wide and narrow bandwidth instruments (530550 nm). This method used separated alkaline ferricyanide and cyanide reagents. Our proposed method of hemoglobin concentration measurement using the modified BeerLambert law is not intended to replace the cyanmethemoglobin method, rather, it is intended to be used as a screening test for In this method, ferricyanide and potassium cyanide convert hemoglobin to a more stable cyanmethemoglobin form that is measured photometrically. METHODS Measurement of the Velocity Constant of the Reaction CO + Hb - COHb A double beam stopped-flow apparatus was used as described by Sirs and Roughton (25) with modifications as previously described (14, 16). In this method, ferricyanide and potassium cyanide convert hemoglobin to a more stable cyanmethemoglobin form that is measured photometrically. Nowadays in many laboratories, the Hemoglobin estimation is done by using Automatic Hematology Analyzers but still in many other labs the following method is Commonly used to determine the Hemoglobin concentration in patients blood. 70 ml of hydrochloric acid 25 % GR for analysis overnight. Determination of CAT activity Th e established method has been described in the study paper by Zhu and Chang [21]. Can be easily standardized iii.) SENSITIVITY: This procedure has a sensitivity of 0.04g/dL per 0.001 absorbance unit. Colorimetric method of DRABKIN (Cyanmethemoglobin). This procedure describes a method to measure hemoglobin adducts of acrylamide and its primary metabolite glycidamide in human whole blood or erythrocytes. Now mix well. Source From the System Menu select Replace Probe and wait for pump to stop. The reaction in the microcuvette is a modified azide-methemoglobin reaction. Preparation of a zinc chloride solution 0.1 mol/l volumetric standard: Approx. peeweealmondiagili_11781. Store tightlycapped and refrigerated (28 C) in the dark.2. Dilution of blood samples (1:100) stops secondary reactions in the presence of inhibitor (organophosphate) and reactivator (oxime). Cyanmethemoglobin Method The principle of this method lies in conversion of hemoglobin to cyanmethemoglobin by the addition of Potassium cyanide and ferricyanide whose absorbance is measured at 540 nm in a photoelectric calorimeter against a standard solution. Then, the absorbance of cyanmethemoglobin is measured in a spectrophotometer at 540 nm. The development of two kinetic methods for quantitation of hemoglobin and methemoglobin is described. Term. Click card to see definition . Linearity: up to 25 g/dL (15.5 mmol/L). cyanmethemoglobin absorbs green light (540 nm) well and thus the Hgb concentration can be determined from the degree of light absorption. (cyanmethemoglobin). Conversion of Hb02 to MHbCN is complete in 10 min. ctHb results by this method are comparable to results obtained by reference HiCN method; this is an acceptable alternative manual method. The KleihauerBetke ("KB") test, KleihauerBetke ("KB") stain, Kleihauer test or acid elution test, is a blood test used to measure the amount of fetal hemoglobin transferred from a fetus to a mother's bloodstream. method have been used [9]. This oxidation results in the formation of methemoglobin. In 1958 the National Research Council (NRC) recommended adoption of the cyanmethemoglobin procedure based on field trials conducted by the Army Medical Department.6,7 In 1966 the International 6.54 g of zinc (Volumetric Standard) are analy tically exactly weighed, transferred to an exactly weighed 1-l volumetric flask and dissolved in approx. RESULTS The cyanmethemoglobin method works on the principle of conversion of hemoglobin to cyanmethemoglobin by The haemoglobin is estimated with the help of cyanmethemoglobin curve. Tap again to see term . Anemia was diagnosed by estimating the hemoglobin concentration in the blood with the use of the indirect cyanmethemoglobin method. Here the erythrocytes are hemolyzed and the bivalent iron in oxyhemoglobin and des-oxyhemoglobin are oxidized by the reagent potassium hexa- Tap card to see definition . Interferences. Automated method on Mindray cell counter is based on principle of electrical impedance for cell counting and colorimetry for HB estimation. Drabkins Solution reacts with all forms of hemoglobin except sulfhemoglobin, a pigment that normally occurs in only minute concentrations in blood. a. University. The photometer follows the reaction and presents the result only when the reaction has stopped. The cyanmethemoglobin (HiCN) method has been adopted as the international standard procedure for hemoglobin (Hb) determinations due to the accuracy and stability of result. When the cyanmethemoglobin complex is formed, a single large absorption band is generated with a maximum at 540 nm and a shoulder at 560 nm. It consists of sodium bicarbonate, potassium cyanide, and potassium ferricyanide. Common methods used are sahlis acid hematin method, photometric cyanmethemoglobin method with hemoglobin estimation by colorimeter, automated method by cell counter.

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