HAEMOGLOBIN CONCENTRATION,COMPLETE BLOOD COUNT AND HAEMATOLOGY INDICES.pdf
- 1. HAEMOGLOBIN ESTIMATION,COMPLETE BLOOD COUNT AND RED BLOOD CELLS INDICES Salaam Mujeeb (PhD, MPH, MSc.) Fellowship in Chemical Pathology Department of Pathology IUIU,Kampala.Uganda 6th January,2024
- 2. Lecture Outline • Learning Objectives • Introduction • Haemoglobin Definition • Variant/Abnormal Haemoglobin • Methods of Haemoglobin Estimation • Complete Blood Count • Red Cells Indices • Classification of Anaemia based on Red Cells Indices
- 3. Learning Objectives By the end of the lecture students/Learners will be able to : • Define Haemoglobin • State normal Haemoglobins • Explain Abnormal Haemoglobin/Variants • List all methods of Haemoglobin Estimation • Explain Red Blood Cells Indices • Describe the etiology and peripheral blood film appearance of various types of Anaemia using Red Cell Indices
- 4. Introduction-Haemoglobin • Haemoglobin is the main functional constituent of the red blood cell, serving as the oxygen-carrying protein (Haemoprotein). • It consists of approximately 3.8% heme and 96.2% globin, with a molecular weight of 64,450. • Each molecule is a tetramer composed of four monomers held together by weak bonds. • It consists of two pairs of polypeptide chains, the GLOBINS, each having an attached HEME molecule composed of iron plus a PROTOPORPHYRIN molecule. • NB-The globin chain can be Alpha(α) Beta (β),Delta (σ),Epsilon (ɛ) or Gamma (γ)or Zeta (ζ)
- 5. Haemoglobin-cont’d • Oxyhemoglobin (HbO2) transports oxygen from the lungs to the tissues where the oxygen is readily released and HbO2 becomes Hb. • When Hb is exposed to certain chemicals, its normal respiratory function is blocked; for example, the oxygen in HbO2 is easily displaced by carbon monoxide, thereby resulting in the formation of fairly stable carboxyhemoglobin (HbCO) which has more affinity for Hb by 250 times • When the iron in Hb is oxidized from the ferrous(Fe 2+) to ferric state(Fe 3+), as in poisoning with nitrates and certain other chemicals, a non-respiratory compound, methemoglobin (MetHb), is formed.
- 6. Normal Haemoglobins • In humans there six kinds of normal Haemoglobins (Hb) namely : • Three embryonic Haemoglobins (Hb Gower-I, Hb Gower-II and Haemoglobin Portland.) • One foetal Haemoglobin(Hb F), and • Two adult types (Hb A, Hb A2). • There are two α globin chains containing 141 amino acid residues, and two of another kind (Beta β, Gamma γ, Delta δ, Epsilon ε, or Zeta ζ), each containing 146 amino acid residues in four of the Hb. • Hb Gower-1 has two Zeta (ζ) chains and two Epsilon(ε )chains. • The production of each kind of globin chain is controlled by a structural gene of similar Greek letter designation; normal individuals are homozygous for the normal allele at each locus.
- 7. Normal Haemoglobin (2) • Hb Gower-II has two Alpha(α) chains and two Epsilon(ε )chains. • Haemoglobin Portland has two zeta(ζ) chains and two gamma (γ)chains • Foetal Haemoglobin(Hb F) has two Alpha(α) chains and two gamma(γ)chains. -60% Normal Hb at birth . - Less resistant to alkali - Has > affinity for O2. - Less than 1% at 9 Months • Haemoglobin A or A1 (Hb A) has two Alpha (α) chains and two Beta(β)chains • Haemoglobin A2 (Hb A2) has two Alpha (α) chains and two Delta(β) chains –Less than 3% in Adulthood. Switch from production of delta chain to Beta Chain by 9 months Genetic Control of Haemoglobin synthesis occurs in two areas: 1.Control of Structure 2.Control of Rate and Quantity of Production
- 10. Normal Haemoglobins(3) • There are six different types of globin chains, designated by the Greek letters α, β, γ, δ, ε, and ζ. The composition of a haemoglobin is specified by a formula such as α2β2, which indicates a tetramer containing two α chains and two β chains. • The chains are coded by different genes, which are turned on and off during development in order to produce hemoglobins with the oxygen-carrying properties required at each developmental stage . • In the first three months of embryonic development, when blood cells are produced in the yolk sac, embryonic hemoglobins such as Hb Gower I (ζ2 Aε2) ,Hb Gower II (α2 Aε2) or Hb Portland (ζ2γ2) are produced. • As erythropoiesis shifts to the liver and spleen, the fetal haemoglobin Hb F (α2γ2) appears. • When erythropoiesis shifts to the bone marrow during the first year of life, the adult hemoglobins Hb A (α2β2) and Hb A2 (α2δ2) begin to be
- 11. Variants/Haemoglobinopathies Structural defects produce a group of diseases called HAEMOGLOBINOPATHIES e.g. Sickle cell disease, Haemoglobin C disease, Haemoglobin E disease, Haemoglobin Barts, HPFH etc Many abnormal haemoglobins arising from mutations have been discovered. Some have altered oxygen affinity, some are unstable, and in some the iron atom is oxidized, resulting in congenital METHAEMOGLOBINEMIA Some mutations result in a reduced rate of haemoglobin synthesis. All such conditions are known as HAEMOGLOBINOPATHIES. The most common hemoglobinopathy is SICKLE CELL DISEASE, caused by a mutation replacing the sixth amino acid in the β chain, normally glutamic acid by valine The variant Haemoglobin α2βS 2 is known as Hb S.
- 12. Thalassaemia • Defects in the rate and quantity of production result in a group of disorders called THALASSEMIAS • THALASSEMIAS may be as a result of mutations resulting in reduced synthesis of one of the chains • They can also result from deletion of the gene for a chain or from a mutation in the regulatory gene that controls the synthesis of the chain
- 13. Methods of Haemoglobin Estimation • Sahli’s Haemoglobinometry Method(Acid-Hematin) • Cyamethaemoglobin Method • Oxy haemoglobin Method • Copper Sulphate /Specific Gravity Method • Haematocrit Method • Auto analyzer Method • Haemoglobin Electrophoresis • Alkaline Haematin Method • Van Slyke Gasometric /Oxygen capacity Method • Chemical Method-Iron content measurement
- 14. Sahli’s Haemoglobinometry (Acid-Hematin /Comparator Method) • Principle: This is based on conversion of haemoglobin to Acid haematin which has a brown colour. • Anticoagulated blood is added to the 0.1 N HCl and kept for 5-7 minutes to form acid haematin. • The colour of this acid haematin is matched with the solution, present in the calibration tube. • Distilled water is added to the acid haematin until the colour matches with the brown glass standard. • The final reading is directly noted from the graduation in the calibration tube. [ • N.B :100 percent on the scale corresponds to 14.5gm % to 15gm %. • The method does not measure Carboxyhaemoglobin, Methaemoglobin and Sulfhaemoglobin • Non haemoglobin substance like proteins, lipids in plasma and cell stroma may influence the colour of the blood diluted with acid
- 15. Cyamethaemoglobin Method • Principle: Haemoglobin is oxidized to cyanmethemoglobin by the addition of cyanide, and the cyanmethemoglobin is then determined spectrophotometrically by the automated counter or manually by colorimeter. • To 5mls of a freshly prepared Drabkin’s solution (Sodium Bicarbonate, Potassium Cyanide, Potassium ferricyanide,Distilled water) is added 20μl of blood • A certified standard haemoglobin solution is diluted and a graph is obtained by plotting at he known Hb concentration against the colorimetric optical density reading of a particular unknown blood sample. • Read the O.D of sample at 540 nm(green filter) • N.B :The Drabkin’s solution is stored in a brown bottle • Cyanide solution should not be discarded in sinks or receptacle containing acids(to prevent formation of Hydrocyanic acid )
- 16. Sheard-Sandford Oxyhaemoglobin Method • Principle • This is based on oxygen capacity determination or blood iron determination • 20mls of 0.1% Sodium Carbonate is mixed with 0.1mls of blood • Optical density is read Photometrically at 540 nm within 30 mins
- 17. Copper Sulphate / Specific Gravity Method Principle: • From Specific Gravity of unknown sample its Haemoglobin is calculated • Normal Specific Gravity of Blood is 1.048-1.066( Average for Men- 1.057 ,Women 1.053.) • This is a very rapid and an uncomplicated method • Useful in screening potential blood donors for anaemia
- 18. Haematocrit /PCV • Haematocrit is the volume of Red Cells expressed as a percentage of the volume of the whole blood in the sample. • Two methods are employed : - Wintrobe tube method - Micro Haematocrit capillary method • Blood is collected into Wintrobe tube or capillary tube coated with an anticoagulant • One end of the capillary tube is sealed with sealing wax or plasticine or empty end sealed with heat. • The sealed tube is centrifuged in a special high speed centrifuge(Haematocrit centrifuge) for 3 minutes • The Haematocrit is determined by comparing the height of the packed cells to the total height of the entire specimen • Ref Range – Male 47-57% ,Female 37-47%
- 19. Autoanalyzer method • Haematology Autoanalyzer is in common use in most well equipped modern day laboratories • Both Haemoglobin concentration and PCV are measured directly by the machin • Other Blood parameters can also be measured by the autoanalyzer
- 20. Functions of Haemoglobin • Transport of Oxygen: The iron atom has a free valence and can bind one molecule of oxygen. Thus, each haemoglobin molecule can bind one molecule of oxygen. The binding of oxygen by one monomer increases the affinity for oxygen of the others in the tetramer. • To a lesser extent, Haemoglobin transports back carbon dioxide from the body to the lungs • A secondary function of haemoglobin is as part of the blood buffer system. The histidine residues in the globin chains act as weak bases to minimize the change in blood pH that occurs as oxygen is absorbed and carbon dioxide released in the lungs and as oxygen is delivered and carbon dioxide taken up from the tissues.
- 22. Introduction • Haemoglobin is the substance that carries oxygen from lungs to the cells of the body through the bloodstream • The Haematocrit describes the ratio of the volume of erythrocytes to the total blood volume (the SI unit is without dimension, e.g. 0.45). • Anaemia is the most common haematological disorder seen in general medical practice. • Risk factors include extremes of age, female gender, lactation, and pregnancy. • Anaemia can cause significant morbidity if left untreated, and is often the presenting sign of a more serious underlying condition. • The rate at which anaemia develops is often as important as the severity, as a rapid decline can overwhelm the compensatory mechanisms of the body.
- 23. Complete Blood Count • Complete Blood Count(CBC) also known as complete blood cell count(CBCC), Full Blood Count (FBC) or Full Blood Examination(FBE),is a blood panel requested by a Medical Doctor or other Medical professionals that gives information about the cells in a patients blood such as the cell count for each cell type and concentrations for various proteins and minerals. • The cells that circulates in the blood steam are generally divided into the types : -White Blood Cells(Leucocytes),-Red Blood Cells (Erythrocytes) and -Platelets (Thrombocytes) • Blood Count are among the most commonly performed blood tests as they can provide an overview of a patient’s general health status • The tests that make up complete blood count are as follows:
- 24. Complete Blood Count(2) Laboratory Tests that make up CBC are : • PCV • Hb • Haematocrit • Red Cells Count • WBC –Absolute Count • WBC-Differential Count • Platelets Count • MCV • MCH • MCHC • RDW • MPV
- 25. Complete Blood Count(3)-Use • To monitor overall health • To confirm a diagnosis of some medical conditions • To screen for some diseases • To monitor a medical condition • To monitor changes in body caused by medical treatment
- 26. Red Cell Indices • These are blood tests that provide information about the Haemoglobin content and the size of Red Blood Cells. • Abnormal values indicate the presence of Anaemia and which type of Anaemia • Red Cell Indices include : • MCV • MCH • MCHC
- 29. Red Cells Indices(3) • Anaemia is classified based on Red Cells Indices (Cytometric scheme).Also referred to as Morphological classification • MCV-Mean Corpuscular Volume:This is the average volume of the red cells measured in femtolitres (Ref .Range 80-100 fl) .Anaemia is classified as Microcytic or macrocytic if the MCV is above or below the expected normal range. Anaemias are classified as normocytic if the MCV value is within the expected range. • MCV = Packed Cells Volume ------------------------------x 10 -¹5 fl Red Cell Count per litre N.B:1fl = 10 -¹5 • Other conditions that affect MVC include: Thalassaemia,Reticulocytosis,Alcoholism,Chemotherapy,Vit.B12 Def. and/or Folic Acid Deficiency
- 31. MCH-Mean cell Haemoglobin • MCH-Mean cell Haemoglobin .This is the average amount of Haemoglobin per red Blood Cells in the blood sample . Measured in picograms. Ref Range is 27-33 pg. N.B : 1 pg =10 ̄ ¹² This is calculated by: • MCH = Hb/RBC X 10 • MCH is diminished in Hypochromic anaemia This is the most useful classification and divides anaemia into 3 viz: i. Microcytic Hypochromic Anemia (low MCHC, low MCV). (MCV<80fl) These include: Iron deficiency anaemia ,Thalassemias and Anemia of chronic disease (rare cases) . ii. Normocytic Normochromic Anaemia- (MCV 80-100fl) further sub -classified according to the reticulocyte count as:
- 32. Red Cells Indices (4) ii.Normocytic Normochromic Anaemia- a. Hyperproliferative (reticulocyte count >2%): the proportion of circulating reticulocytes increases as part of a compensatory response to increased destruction or loss of RBCs. The cause is usually acute blood loss or haemolysis b.Hypoproliferative (reticulocyte count <2%): these are primarily disorders of decreased RBC production, and the proportion of circulating reticulocytes remains unchanged. Normochromic, normocytic anemia (normal MCHC, normal MCV). These include: Anaemias of chronic disease, Anemia of acute hemorrhage and Aplastic anaemias (those characterized by disappearance of RBC precursors from the bone marrow)
- 34. Mean Cell Haemoglobin Concentration • MCHC-Mean Cell Haemoglobin Concentration • This is calculated by: Hb / PCV X 100 • The normal mean corpuscular haemoglobin concentration , which is the average concentration of Haemoglobin within the red blood cells, is 32 to 36 g/100 ml.
- 35. Macrocytic Anaemia Showing Oval Macrocytes
- 36. Polychromasia
- 37. Red Cell Distribution Width (RDW). • Red Cell Distribution Width (RDW or RCDW).This is the variation in cellular volume of the RBC population • Ref.Range -11,5-14.5 % • It is used together with the MCV result to determine the possible cause of Anaemia • It differentiates an anaemia of mixed causes from an anaemia of single cause. • Deficiency of Vit .B12 or Folate produce a macrocytic anaemia in which RDW is elevated in about two –thirds of all cases. • However , RDW is an hallmark of Iron Deficiency Anaemia as there is an increased RDW in all cases • An elevated RDW(Red Cells of unequal sizes is known as ANISOCYTOSIS
- 38. HDN with Nucleated Red Cell
- 39. Trophozoite of Malaria Parasite
- 41. Howell Jolly Body in a Red Cell
- 45. References • Bishop M. L., Duben Engelkirk J.L., Fody E.P. Clinical Chemistry: Principles,Procedures,Correlations.2ed.J.B.Lippincott CompanyPhiladelphia • Hoffbrand V.A, Moss P.A.H .,Essential Haematology, 2012.6th Ed. Blackwell Publishers • Hoffbrand V.A,Petit J.E. ,Colour Atlas of Clinical Haematology .2000.3rdEd.Mosby Elservier,London. • Sood R,Haematology for Students and Practitioners,1996.4th ed .Jaypee Brothers Medical Publishers • www.webmd.com/a-to-z-guides/ understanding-anaemia-basic • www.Health24.com/lifestyle/yourblood/anaemia • www.haematology.org/patient/anaemia