Method of Haemoglobin Estimation

Haemoglobin estimation method

Manual

Cyanmethaemoglobin method

Acid Haematin method

Automated

Coulter counter

Procedures

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

b. Method:

1. Cyanmethaemoglobin method:

o Accurate.o Commonly used.o Recommended by ICSH (international committee for

standardization in haematology).

Procedures

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

b) Reagent and equipment for Cyanmethaemoglobin method:

• Diluent (Drabkin’s solution)• 5 ml pipette.• Cuvettes.• Test tube.• 20 micro liter pipettes.

c) Procedure of Cyanmethaemoglobin method:

20ul blood + 4ml diluent mix, 5-10min HiCN.

Measured by spectrophotometer at 540nm

Use the calculator:Hb (g/dl)= Absorbance of test X Conc of standard Absorbance of standard

sample

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 Sahli’s haemoglobinometer.o Sahli’s pipette or Micropipette. o 0.1 N HCL.o 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).

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.

Precaution

Q. What is the unite of measurement for Hb?

Whole blood Hb concentration is in g/dl.

Adult male:13 - 18 g/dl

Adult female:12 - 16 g/dl

10y old child11 - 15 g/dl

6 m old child11 - 14 g/dl

New born14 - 22 g/dl

Reference Values

Interpretation:Reference values for Hb are variable.

Clinical significance: Hb value

+RBC count and indices (upcoming labs)

Anemia

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 RBCs

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

1. Proerythroblast

Stage formation of RBC :

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.

Decreased level of Hb

Sever Hemorrhage

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

Increased level of Hb:

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

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.

3. All personnel performing Hb should be checked for color blindness (Sahli’s Method).

Quality Control

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