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Hemoglobin Estimation
physiology Lab.
October 2014
by: Mehdi Mafakheri



estimation method

a) Sample:
Both capillary and venous blood may be used for this test.


b. Method:
1. Cyanmethemoglobin method:
o Accurate. Because:


Acid Hematin

Coulter counter

1- depend on spectrophotometer (O.D) of Cyanmethemoglobin
2- stable for long time (about 6 month in 4Cº)
3- possibility of formation Cyanmethemoglobin from all hemoglobin type
except Sulfahemoglobin.

o Commonly used.
o Recommended by ICSH (international committee for standardization in


alkali hematin not suitable for estimation infant hemoglobin Because NaOH not
react with Hb F.


Acid hematin method: Procedure of Cyanmethemoglobin method: a) 20micl blood + 4ml diluent Principle: HiCN.interference of some compound such as plasma protein and lipid with RBCs cell membrane 2.HCl have no effected on some hemoglobin derivatives such as Sulfahemoglobin .read by naked eye Use the calculator: Absorbance of test X Conc.1 N HCL Acid Hematin. Because: 1. Cuvettes. 20 micro liter pipettes. 3. Test tube. 5-10min Blood + 0. “SAHLI’S hemoglobinometer” Measured by spectrophotometer at 540nm standard However this method is inaccurate. 5 6 Spectrophotometer c) 2.10/26/2014 a) Principle of Cyanmethemoglobin method: Blood + diluent (Drabkin’s solution) Reagent and equipment for Cyanmethemoglobin method: b) “ potassium ferricyanide + potassium cyanide” Converts: Hemoglobin (Hb) and Methemoglobin (Hi) • • • • • Cyanmethemoglobin (HiCN) Measure the absorbance of the solution by using a calorimeter at a wavelength = 540nm. mix.e. Then compare it with the standard solution of HiCN. Diluent (Drabkin’s solution) 5 ml pipette. of standard Hb (g/dl)= Absorbance of standard 7 8 . (then match the color of solution with reference solution colorimeter or colored strip) sample i.

146 9 e. Q. Glass rod.14 g/dl New born 14 .15 g/dl 6 m old child 11 . 11 10y old child 11 .  If high WBC in specimen centrifuge the specimen then use the supernatant. Graduated tube has two scales: % and g/100 ml of whole blood. • The reading in graduated tube refers to Hb level in g/dl (some tubes give reading in %. : 10% X 0.16 g/dl  In case of Hb S or C dilute the mixture in 1:1 ratio with DW then read in colorimeter. Glass dropper with rubber teat. round Hb Tube. to convert into g/dl X 0. • If the color of graduated tube is Darker add D.W -drop by drop. How can we read Hb value? Compare the color of solution in the graduated tube with that of reference strip on either side of hemoglobinometer.g.146 = (14. Cleaning brush.6 g/dl). Rubber tube with mouth piece.22 g/dl 12 . What is the unite of measurement for Hb? Whole blood Hb concentration is in g/dl 10 Reference Values Precaution  Before the sample is read the solution should be clear.10/26/2014 b) c) Procedure of Acid Hematin method : 100ul HCL + 20micl blood mix in a graduated Tube (keep for 3min) to make Reagent and equipment for Acid Hematin method: Acid Hematin A black counting chamber.18 g/dl Adult female: 12 .  In case of abnormal globins add 0. mix with glass rod until the color matches with reference strip. 20ul Pipette.with pipette.1g of potassium carbonate to the solution. Amber bottle. Adult male: 13 .

Seen in Bone morrow. Basophilic normoblast (early stage). Polychromatic normoblast (intermediate stage ). Proerythroblast The earliest precursors of erythropoiesis and do not contain hemoglobin. 2. Reticulocyte. Clinical significance: 1. finely honeycombed chromatin structure with pale blue nucleoli. Like proerythroblast in general character. Proerythroblast. 6.  Cytoplasm : darkly basophilic. 1. Orthochromatic erythroblast or Nucleated RBC (late stage ). 5. Normal erythrocytes Hb value Anemia + RBC count and indices (upcoming labs) 13 Stage formation of RBC: 14 2.10/26/2014 Stage formation of RBCs Interpretation: Reference values for Hb are variable. which disappear as the cell matures.  The nuclear-cytoplasmic ratio is shifted in favor of the cytoplasm. 2.  Nucleus: The nucleus has a dense.  Seen in BM These cells tend to be smaller than proerythroblasts. 15 16 . Basophilic Normoblast (early stage) 1.

and there is partial clumping of the nuclear chromatin.  Cytoplasm: loses more of its basophilic with a greater abundance of hemoglobin. Normal erythrocytes (Normochromic) Immature RBCs that contain cytoplasmic RNA and organelles such as mitochondria and ribosomes in various stages of maturity. Polychromatic normoblast (intermediate stage)  Nucleus : appears coarse and smudgy. 4. Reticulocyte 18 6. Orthochromatic erythroblast or Nucleated RBC (late stage ) Red blood cell with nucleus The nuclear.AIHA . central pallor which is Less than one-third of the total cell volume. 20 . The more filamentous reticula are characteristic of younger cells (brilliant cresyl blue stain) Seen in bone marrow and blood Seen in hemolytic anemia •Cells are uniform size & shape •Normal hemoglobin conc. which acquires an increasingly red tinge ultimate Seen in bone marrow and blood Seen in sickle cell disease . 19 •small.and beta-thalassaemia Seen in bone marrow 17 5.10/26/2014 3.cytoplasmic ratio is shifted in favor of the cytoplasm.

reactions to chemicals and drugs. bacteremia.g. Haemolysis due to transfusion of incompatible blood. sever burns 23 Chronic obstructive pulmonary disease (COPD) 24 . cirrhosis. Thalassemia & Sideroblastic anemia 21 22 Anaemia Hypochromic Polycythaemia High altitudes Congestive heart failure (CHF) Hemoconcentration states of blood e. carcinomatosis and systemic lupus erythematosis.g. uremia. and artificial heart valves IDA. lymphoma. hyperthyroidism. leukemia.10/26/2014 Hypochromic erythrocytes Sever Hemorrhage Systemic diseases e.

send patient’s specimen to the reference laboratory.10/26/2014 Polychromasia and Normoblasts Polychromasia • Mature RBCs with increased staining with basic stain and Hb staining. 2. 1. 27 Increased erythrocyte production & Hemolytic anemia 26 . and perform duplicate testing in your own lab. All personnel performing Hb should be checked for color blindness (Sahli’s Method). 25 Quality Control 1. If the patient’s specimen running in automated machine there are 3 levels controls should be run. Occurs in red cells have high RNA content with Hb synthesis is not yet complete. While if its running by manual method.