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Hb estimation

This document describes methods for estimating haemoglobin levels, including automated and manual methods. The cyanmethaemoglobin and acid haematin methods are described in detail. The cyanmethaemoglobin method is accurate and commonly used, involving converting haemoglobin to cyanmethaemoglobin which is then measured spectrophotometrically. The acid haematin method involves mixing blood with acid and comparing the color to a reference strip on a haemoglobinometer. Reference ranges for haemoglobin are provided for adults and children. Clinical significance and causes of abnormal haemoglobin levels are also summarized.

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Method of Haemoglobin Estimation
Haemoglobin estimation method Automated Manual Cyanmethaemoglobin method Acid Haematin method Coulter counter
Procedures a) Sample: Both capillary and venous blood may be used for this test.
Procedures b. Method: 1. Cyanmethaemoglobin method: o Accurate. o Commonly used. o Recommended by ICSH (international committee for standardization in haematology).
a) Principle of cyanmethaemoglobin method: Blood + diluent (Drabkin’s solution) “ potassium ferricyanide + potassium cynaide” Converts: Haemoglobin (Hb) and Methaemoglobin (Hi) Cyanmethaemoglobin (HiCN)
Measure the absorbance of the solution by using a calorimeter at a wavelength = 540nm. Then compare it with the standard solution of HiCN. Spectrophotometer
Reagent and equipment for Cyanmethaemoglobin method: b) • • • • • Diluent (Drabkin’s solution) 5 ml pipette. Cuvettes. Test tube. 20 micro liter pipettes.
c) Procedure of Cyanmethaemoglobin method: 20ul blood + 4ml diluent HiCN. mix, 5-10min sample Measured by spectrophotometer at 540nm standard Use the calculator: Hb (g/dl)= Absorbance of test X Conc of standard Absorbance of standard
2. Acid haematin method: a) Principle: Blood + 0.1 N HCL Acid Haematin. (then match the color of solution with reference solution colorimeter or colored strip) i.e. “SAHLI’S haemoglobinometer” However this method is inaccurate.
b) Reagent and equipments for Acid Haematin method: o o o o Sahli’s haemoglobinometer. Sahli’s pipette or Micropipette. 0.1 N HCL. Dropping pipette.
• The standard set of Haemometer consists of: A black counting chamber, round Hb Tube, 20ul Pipette, Rubber tube with mouth piece, Cleaning brush, Glass dropper with rubber teat, Glass rod, Amber bottle.
c) Procedure of Acid Haematin method : 100ul HCL + 20ul blood mix in a graduated Tube (keep for 5min) Acid Haematin How can we read Hb value? Compare the color of solution in the graduated tube with that of reference strip on either side of haemoglobinometer. Graduated tube has two scales: % and g/100 ml of whole blood.
• If the color of graduated tube is Darker add either 0.1N Hcl or D.W 7 -drop by drop- with pipette; mix with glass rod until the color matches with reference strip. • The reading in graduated tube refers to Hb level in g/dl (some tubes give reading in %; to convert into g/dl X 0.146 So, e.g. 10% X 0.146 = (14.6 g/dl).
Precautio n  Before the sample is read the solution should be clear.  If high WBC in specimen centrifuge the specimen then use the supernatant.  In case of Hb S or C dilute the mixture in 1:1 ratio with DW then read in colorimeter.  In case of abnormal globins add 0.1g of potassium carbonate to the solution.
Q. What is the unite of measurement for Hb? Whole blood Hb concentration is in g/dl.
Reference Values Adult male: 13 - 18 g/dl 10y old child 11 - 15 g/dl Adult female: 12 - 16 g/dl 6 m old child 11 - 14 g/dl New born 14 - 22 g/dl
Interpretation: Reference values for Hb are variable. Clinical significance: Hb value Anemia + RBC count and indices (upcoming labs)
Stage formation of RBCs 1. Proerythroblast. 2. Basophilic normoblast (early stage). 3. Polychromatic normoblast (intermatiedate stage ). 4. Orthochromatic erythroblast or Nucleated RBC (late stage ). 5. Reticulocyte. 6. Normal erythrocytes
Stage formation of RBC: 1. Proerythroblast The earliest precursors of erythropoiesis and do not contain hemoglobin. Nucleus: The nucleus has a dense, finely honeycombed chromatin structure with pale blue nucleoli, which disappear as the cell matures. Cytoplasm : darkly basophilic. Seen in BM
2. Basophilic Normoblast (early stage) These cells tend to be smaller than proerythroblasts. Like proerythroblast in general character.  The nuclear-cytoplasmic ratio is shifted in favor of the cytoplasm. Seen in BM.
3. Polychromatic normoblast (intermatiedate stage ) Nucleus appears coarse and smudgy, and there is partial clumping of the nuclear chromatin.  Cytoplasm loses more of its basophilic with a greater abundance of hemoglobin . Seen in bone marrow
4. Orthochromatic erythroblast or Nucleated RBC (late stage ) Red blood cell with nucleus The nuclear- cytoplasmic ratio is shifted in favor of the cytoplasm, which acquires an increasingly red tinge ultimate Seen in bone marrow and blood Seen in sickle cell disease ,AIHA ,and beta-thalassaemia
5. Reticulocyte Immature RBCs that contain cytoplasmic RNA and organelles such as mitochondria and ribosomes in various stages of maturity. The more filamentous reticula are characteristic of younger cells (brilliant cresyl blue stain) Seen in bone marrow and blood Seen in haemolytic anaemia
6. Normal erythrocytes (Normochromic) •Cells are uniform size & shape •Normal hemoglobin conc. •small, central pallor which is Less than one-third of the total cell volume.
Sever Hemorrhage Systemic diseases e.g. leukemia, lymphoma, uremia, cirrhosis, hyperthyroidism, carcinomatosis and systemic lupus erythematosis. Haemolysis due to transfusion of incompatible blood, reactions to chemicals and drugs, bacteraemia, and artificial heart valves Anaemia
Hypochromic erythrocytes IDA, Thalassemia & Sideroblastic anemia
Hypochromic
Polycythaemia High altitudes Congestive cardiac failure (CCF) Haemoconcentration states of blood e.g. sever burns Chronic obstructive pulmonary disease (COPD)
Polychromasia • Mature RBCs with increased staining with basic stain and Hb staining. Occurs in red cells have high RNA content with Hb synthesis is not yet complete.
Polychromasia and Normoblasts Increased erythrocyte production & Hemolytic anemia
Quality Control 1. If the patient’s specimen running in automated machine there are 3 levels controls should be run. 2. While if its running by manual method; send patient’s specimen to the reference laboratory; and perform duplicate testing in your own lab. 1. All personnel performing Hb should be checked for color blindness (Sahli’s Method).
Hb estimation