Module 13 Haemoglobin Synthesis Function and Measurement Acknowledgments Ministry of Health Guyana Centers for Disease Control and Prevention CDC Global AIDS Program GAP Guyana Centers for Disease Control and Prevention CDC Atlanta American Society for Clinical Pathology ASCP Objectives Discuss the following aspects of haemoglobin biochemistry Molecular structure synthesis and breakdown Iron metabolism and compartments pools Normal types of adult haemoglobin A A 2 F including approximate percentages Oxygen carrying function of haemoglobin Oxyhaemoglobin Deoxyhaemoglobin Haemoglobin forms unable to transport oxygen Methaemoglobin Carboxyhaemoglobin Objectives Describe the principle of the cyanmethaemoglobin method or modifications of haemoglobin determination including specimen requirements Describe sources of specimen collection error preanalytic that can cause inaccurate results Discuss potential sources of error when measuring haemoglobin photometrically i e substances that interfere with photometric measurements Discuss quality control and checks utilized to establish test validity and prevent erroneous results Objectives Discuss reference ranges and the significance of haemoglobin values on the basis of age and sex Explain the correlation between red blood cell count haemoglobin and haematocrit results Haemoglobin Synthesis Structure Haemoglobin is normally present in red cells only Carries oxygen and removes CO2 Three building blocks Globin Protoporphyrin and Iron Haeme Iron must be in 2 state Iron Compartments Pools Iron is an essential component of haemoglobin Decreased tissue iron cellular dysfunction Increased tissue iron cellular destruction Regulated by absorption not excretion Iron circulates in the plasma bound to transferrin Iron essential trace metal if absent detrimental to health because component of heme it is the iron that binds the oxygen 4 molecules of O significant amount has to come from diet After 120 days spherocytes rigid lose membrane flexibility are then destroyed spleen recycles and contents are reabsorbed certain amount of Iron that was bound to O is reabsorbed is recycled back through digestion Fe 2 is stored and bound by transferrin Fe 3 is bound at this state Protoporphyrin this plus heme makes hemoglobin Protoporphyrin III 9 Site of synthesis is the mitochondria in RBC cytoplasm HAEME Vitamins are essential for certain physiological functions come from outside source body does not produce 4 hemes for every hemoglobin molecule 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 beta gamma delta Haemoglobin Molecule Consists of 4 globin chains 4 haeme groups haeme groups are identical Different globin chains determine the haemoglobin type 3 normal haemoglobin types by 6 months of age Hgb A alpha2beta2 Hgb A2 alpha2 delta2 Hgb F alpha2gamma2 Hgb A is predominant Hgb F fetal hemoglobin during embryogenesis A stands for adult Based on differences in molecular size hemoglobin electrophoresis So majority is Hgb A Oxygen Transport Ability of Hgb Oxygen binds to central iron atom in haeme Two normal Hgb forms Iron must be Fe 2 ferrous state to transport oxygen Each haemoglobin molecule can carry up to 4 oxygen molecules Deoxyhaemoglobin Fe 2 without oxygen tissues Oxyhaemoglobin Fe 2 with oxygen lungs Two abnormal Hgb forms Cyanosis hypoxia coma death Methaemoglobin Fe 3 oxidized Carboxyhaemoglobin Fe 2 with CO Both reversible HGB RBC Breakdown Aged 1 lost daily or defective red cells are mainly removed by splenic macrophages RES Albumin circulating protein If problem with liver or any tissues or organs so that bilirubin cannot be conjugated remains conjugated it stays in the tissues jaundice Liver plays significant role in this process Haemoglobin Measurement Haemoglobin measurement of concentration of Hgb in red cells whole blood reported in g dL Cyanmethaemoglobin method reference method EDTA whole blood or capillary samples Photometric semi or fully automated instruments Drabkin s reagent causes red cell lysis release of haemoglobin and conversion to cyanmethaemoglobin Pigment is measured photometrically 540 nm Proportional to Hgb concentration All haemoglobin forms measured EDTA HemoCue photometer whole blood Haemoglobin HemoCue HemoCue photometer Dry reagent system cuvettes Determines concentration of azide methaemoglobin photometrically Electronic check and whole blood control samples must be run to monitor instrument function and reagent Procedure overview Turn on HemoCue instrument Run electronic calibration check red control cuvette Fill specimen cuvette with EDTA or capillary blood Place cuvette in instrument insert to measure position Haemoglobin result will be displayed in g dL Haemoglobin HemoCue Specimen cuvette Electronic calibration red cuvette Sources of Error Pre analytical errors are a common cause of inaccurate results Testing process begins with sample collection Clotted sample haemoconcentration haemodilution wrong patient identification improper capillary collection Haemolysis does not cause haemoglobin error Technical errors Control samples with known assayed values are used to check result reliability Controls detect invalid results caused by errors in testing technique reagents or instrument malfunction Do controls detect specimen collection errors Quality Control Control samples monitor the correct functioning of equipment stability of reagents and testing technique Controls do not detect specimen collection errors Haemoglobin Interference Interference with photometric Hgb measurement can cause falsely high results Lipids or bilirubin cause cloudiness Extremely high WBC count causes cloudiness Detect by checking data correlation between Hgb and Hct values Haemolyzed Lipemic plasma plasma Normal plasma haemolysis lipids Icteric plasma bilirubin Fluid called plasma because anticoagulated Not anticoagulated serum Toward bottom of tube reddish brownish line erythrocytes Greyish band is white cells platelets called buffy coat Upper part is called plasma In second one erythrocytes have been lysed destroyed Can tell because does not appear normal Can be result of blood drawing technique or disease In third one have too much Hardee s The top part is fat cloudy and whitish pinkish 2 types of cholesterol cell membrane phospholipid bilayer good hdl bad ldl Triglycerides Liver cannot metabolize all those fats and stays in circulation