Haemoglobin Estimation GCUC PDF

HAEMOGLOBIN DETERMINATION CYAN-METHAEMOGLOBIN METHOD  OBJECTIVES Know the function of Hb and appreciate it`s estimation as part of disease diagnostic process. Know different properties of Hb exploited for its quantification. Be able to prepare Drabkins solution and...

HAEMOGLOBIN DETERMINATION CYAN-METHAEMOGLOBIN METHOD  OBJECTIVES Know the function of Hb and appreciate it`s estimation as part of disease diagnostic process. Know different properties of Hb exploited for its quantification. Be able to prepare Drabkins solution and understand the function of its components. Be able to accurately determine a patient`s Hb conc using the std HiCN technique. Perform calibration charts for Hb determination & interpret correctly Hb conc result. Know application of Beer-Lambert law and its limitations in Hb conc determination.  INTRODUCTION Haemoglobin (Hb) is a protein found in the RBC`s which consists of heme (iron,10%) and globin (protein, 90%). The globin is made of two identical pairs of polypeptide chains called the alpha (α) and the non-alpha chains. 34 amino acids  Formation of Hb begins in the developing RBC at the early normoblast stage in the B.M and eventually becomes the main cytoplasmic content of the maturing RBC after the nucleus is dissolved or extruded at the late normoblast stage. The heme component of Hb is synthesized in the mitochondria and the globin component is assembled in the cytoplasm.  Genetic Control Of Human Haemoglobin Hb Gower II (α2 ɛ2) Hb Portland I (ζ 2 γ2) 3mths 3-6mths >6mths  Function of Haemoglobin Haemoglobin functions to convey oxygen from the lungs to the tissues for aerobic metabolic activities and energy generation and transport carbon dioxide from the tissues to the lungs for excretion. Whenever, the body`s level of Hb falls below normal, anaemia results. Anaemia is associated with inadequate supply of oxygen to the tissues, a condition called hypoxia. Anaemia affect the health of the individual and presents with wide range of signs and symptoms.  SIGNS & SYMPTOMS OF ANAEMIA Pallor Menstrual irregularities Fatigue Loss of libido or potency Rapid pulse Heart murmurs Shortness of breath Angenia pectoris (chest Irritability, (difficulty in pain with exertion) concentrating) Heart failure Headache Coma Dizziness Sore tongues Bleeding Brittle nails Nausea and decreased appetite  Consequences of Anaemia  Children - impairments of learning and cognitive function.  Adults - physical productivity losses  Pregnancy- still-births, low birth weight babies and risk of increased maternal mortality from excessive bleeding at parturition. Anaemia is common in developing countries than the developed world and 1.62 billion people are afflicted globally.(WHO global database on anaemia, 1993 – 2005 report).  Factors That Influence Hb Values Haemoglobin values are affected by factors such as Pregnancy- expansion in body fluid causes RBC count to fall and hence a drop in Hb conc. Altitude - less O2 at high altitudes and more oxygen at low altitudes. Diseases - a number of diseases can affect erythropoiesis & thus Hb level of a patient. Age – Hb conc varies with age. Sex- males have higher level of Hb because of their high level of androgenic hormones.  Techniques for Hb measurement Haemoglobin determination (haemoglobinometry) is the measurement of the conc of Hb in the blood. Hb conc of blood can be measured by several physical properties of the Hb molecule such as: Specific gravity Hb being a major component of blood significantly influence the s.g of blood. Hence, a change in the s.g of blood is thought to commensurate with change in Hb level. This principle is utilized at donors clinic of the blood bank to determine whether a donor has enough blood to donate through the use of CuSO4 solution. RBC with PCV > 0.38 CuSO4 solution sink to the bottom of the test tube (s.g 1.053)  Chemical composition in terms of iron content The principle is based on the fact that a molecule of Hb contains 4 atoms of iron or 0.347 g of iron per 100 g of Hb. The iron present is detached from the Hb and measured. The Hb is calculated by using the formula. Hb (g/dl) = bld iron content (mg/dl) 3.47 This is very accurate, but complex & time-consuming test. So it is not done as a routine procedure. Structure of Hb Gas analysis (measure O2 combining capacity) This relies on the facts that each iron atom of Hb binds an O2 molecule. Thus, a molecule of Hb binds 4 molecules of O2. Hence, how much O2 Hb carries can be used to determine the conc of Hb. It is estimated that 1g of Hb binds ~ 1.34 ml of oxygen. Hence, Hb conc. is given by: Hb (g/dl) = O2 binding capacity/dL of blood 1.34  Spectral property Haemoglobin is a chromo-protein thus, it is able to absorb light of a particular wavelength to an extend directly proportional to the conc of Hb. This principle is used in spectrophotometry or photo-electric colorimeters. The spectrophotometer is an instrument that separates light into its component spectrum of different wavelengths and selectively measures the intensity of a radiation after passing through a sample.  Principles of spectrophotometry When light falls on a substance it can be reflected or absorbed by the substance. Certain wavelengths can be absorbed and the remainder transmitted or reflected.  Transmittance, Absorbance & The Beer-lambert Law Transmittance is the ratio of the amount of light transmitted to the amount of light incident on the substance. Transmittance = P/Po PO P sample Incident light emergent light Absorbance = - log T = - log P/Po. Thus, absorbance is Inversely proportional to transmittance. Beer-Lambert Law Absorbance ∝ concentration of the solution and the path length of the cuvette.  Limitations of Beer-Lambert Law A major source of error in spectrophotometric analysis is applying the Beer-Lambert Law at inappropriate concentrations. The Beer-Lambert Law is strictly applicable only for DILUTE SOLUTIONS. It becomes less and less accurate as the concentration of the solution increases. The linearity of the Beer-Lambert law is limited by chemical and instrumental factors.  Causes of non-linearity include: Deviations in absorptivity co-efficients at high concentrations (>0.01M) due to electrostatic interactions between molecules in close proximity Scattering of light due to particulates in the sample Fluorescence or phosphorescence of the sample Changes in refractive index at high analyte conc. Shifts in chemical equilibria as a function of conc. Non-monochromatic radiation, deviations can be minimized by using a relatively flat part of the absorption spectrum such as the maximum of an absorption band Stray light.  Techniques for Hb determination using spectral properties Numerous techniques exist for spectrophotometric determination of Hb, however, not all are routinely used due to challenges encountered with their usage. Oxyhaemoglobin technique This involves measuring the light absorption of a diluted blood in the oxyhaemoglobin form. The challenge is oxyhaemoglobin fades with time and this has made preparing acceptable standard oxyhaemoglobin solution impossible. Alkaline haematin technique This involves conversion of Hb to alkaline haematin through denaturation with an alkaline. HbF and Hb Barts` are resistant to alkaline denaturation, unless the soln is heated for about 4 mins before accurate measurement can be made. Carboxyhaemoglobin technique Hb measurement in the form of carboxyhaemoglobin is more stable and can be used to overcome instability challenges encountered in other techniques, however, standard carboxyhaemoglobin solutions are not wholly satisfactory. Acid haematin technique This involves conversion of Hb to acid haematin using dil. HCL. The principle was used in Haden-Hausse and Sahli- Hellige methods where the brownish-coloured acid haematin formed is visually compared with a std to determine the Hb conc. Cyanmethaemoglobin (Hemiglobincyanide) technique The technique permits the conversion of all haemoglobin variants with the exception of sulphaemoglobin to the most stable cyanmethaemoglobin (HiCN). The reagents needed to form the stable HiCN can be prepared together in a solution form called Drabkin`s solution.  Principles of HiCN technique This relies on the conversion of haemoglobin and it`s derivatives with exception of sulphaemoglobin into a final stable solution of HiCN whose absorbance is spectrophotometrically determined at an optimum wavelength of 540nm and compared to that of the standard HiCN solution.  Method Blood is mixed with Drabkin's solution, which contains ferricyanide and cyanide. The ferricyanide oxidizes the iron in the Hb, thereby changing Hb to methaemoglobin. Methaemoglobin then combines with cyanide to form cyanmethaemoglobin (hemiglobincyanide or (HiCN). HiCN develops a colour which is measured in a colorimeter, or spectrophotometer. The intensity of the colour relates directly to the conc of Hb in the blood.  How to calculate the Hb conc of a patient`s blood 1. The Hb conc of a patient`s blood can be determined by using the formula Test [Hb] = Std [Hb] × Abs test Abs Std 2. By using a prepared haemoglobin calibration chart of absorbance against conc, to determine the concentration of patient`s blood sample.  Plotting the calibration graph This consists of a plot of absorbance versus concentration for a series of standard solutions whose concentrations are accurately known. Calibration curves are used in reading off the unknown concentrations, thus their accuracy is of absolute importance. A serial dilution of a std HiCN is made and the absorbance of each dilution is measured to generate a table as shown below.  CALIBRATION CHART Vol. HiCN 5 4 3 2 1 0 Vol. Drabkins 0 1 2 3 4 5 Conc. Of Serial dilution Abs. of serial dilution The of std [HiCN] solution is always indicated on it`s vial. Drabkins` solution is used for the serial dilution. The conc of each serial dilution is obtained by the formula Volume of std HiCN X std [HiCN] Total vol. of solutions  GRAPH OF CALIBRATION PLOTS The Beer-Lambert Law (A α l c) implies that when concentration is equal to zero (c = 0), absorbance must also be zero (A = 0). In other words, the calibration line must pass through the origin. The conc of the patient`s blood can be determined using the measured absorbance to trace the corresponding conc.  Drabkin`s solution-Components & function 1.Potassium dihydrogen phosphate or sodium bicarbonate This is the buffer component of the solution which maintains the alkaline pH of the medium at 7.0 – 7.4 needed by the reaction. The alkaline medium created also reduces the tendency of plasma proteins precipitating to cause turbidity which could affect photometric measurement of haemoglobin. 2.Potassium ferricyanide This compound oxidizes the iron in haemoglobin to form methaemoglobin. 3.Potassium cyanide The cyanide component of this compound combines with methaemoglobin to form cyanmethaemoglobin (hemiglobincyanide) which is very stable, permitting photometric measurement of haemoglobin. 4.Non – ionic detergent This catalysis the rate of the chemical reaction, thus hastening the conversion and reducing the duration for the reaction to reach completion from minimum of 10 minutes to 3-5 minutes. Eg of non-ionic detergent is Nonidet P40.  Preparation of Drabkin`s solution (volume = 1 liter) Potassium Dihydrogen Phosphate ……140mg or Sodium Bicarbonate…………………….2g Potassium Ferricyanide………………….200mg Potassium Cyanide……………………….50mg Non – Ionic detergent…………………….. 1mL Distilled H2O ……………….. 1liter (1000mL)  Care of Drabkin`s solution Drabkins` solution is photodegradable hence should be stored in brown borosilicate bottles at room temperature or dark cupboard, away from direct sunlight where the reagent could last for 2months. Plastic absorbs cyanide from Drabkins` and should not be used to store the reagent. Freezing destroys the potassium ferricyanide but this can be prevented by adding ethanol, methanol or glycerol.