HEMOGLOBIN ESTIMATION

Hemoglobin refrence values:

HGB Men 13.0-18.0 g/dL Women 11.5-16.5 g/dL Infants (full-term, cord blood) 13.5-19.5

g/dL Infants, 3 months 9.5-13.5 g/dL Infants, 1 yr. 10.5-13.5 g/dL Children, 3-6 yr. 12.0-14.0 g/dL Children, 10-12 yr. 11.5-14.5 g/dL

Hemoglobin estimation

•Physical – Based on Specific gravity •Chemical –Based on Iron content of Hb •Gasometric-Based on O2 combining

capacity of Hb •Calorimetric-Based on colour •Photoelectric – Cynmethhemoglobin

method •Automated methods

Sahli’s Haemoglobinometer

principle Blood added to N/10 HCl which converts

Hb into acid hematin. Brown colour of acid hematin , matched

against comparimeter

Procedure

•Take N/10 HCL in the tube up to lowest mark. •Fill the Hb pipette with 0.02ml of blood & wipe off

the excess blood at nozzle and transfer it to the tube.

•Mix the acid and blood by shaking the tube •Allow it to stand for 10 minutes •dilute the solution with distilled water and match

the colour with the comparator. •The upper meniscus is noted and reading on the

tube is read as gm/dl

Source of error If blood not mixed If Hb pipette is not caliberated correctly If instrument is not caliberated correctly Fading of comparator

Disadvantages Error due to visual matching Lower values if read after 10 min due to

fading Acid does not convert all Hb into hematin

like carboxy, met, sulfhaemoglobin

SPECTROPHOTOMETER OR PHOTOELECTRIC COLORIMETER

haemiglobincyanide (HiCN; cyanmethaemoglobin) method

oxyhaemoglobin (HbO2) method

HAEMIGLOBINCYANIDE (CYANMETHAEMOGLOBIN) METHOD

Principle:

Hb oxidised by potassium ferricyanide to MetHb,stabalised by potassium cynide to cynmethemoglobin

Reagent

Drabkin's reagent Potassium ferricyanide (0.607 mmol/l) 200

mg Potassium cyanide (0.768 mmol/l) 50 mg Potassium dihydrogen phosphate (1.029

mmol/l) 140mg

Nonionic detergent 1 ml Distilled or deionized water To 1 litre

Method 5ml of drabkin’s solution in test tube Add 0.02 ml of blood by pipette Mix and wait for 10 minutes Switch on colorimeter for 5 min Adjust OD at 0 by test solution Put test sol. into cuvette and take OD

Calculation of Haemoglobin Concentration

Hb = OD of test sample × conc of std.×250

OD of std.× 1000

advantages No visual error All Hb are converted to

cyanmethemoglobin Colour does not fade with time allows direct comparison with the

reference standard and that the readings need not be made immediately after dilution so batching of samples is possible

disadvantages KCN is potent poison Turbidity in Drabikin’s solution alter OD In India no reliable Hb standards available

OXYHAEMOGLOBIN METHOD

Principle RBCs lysed by 0.04% liquid ammonia

and OD of oxyhemoglobin obtained is measured at 540 nm in colorimeter

Advantage --- quick and simple ---not affected by

S.bilirubin Disadvantage --- no stable HbO2

standard --- not reliable in

carboxy,met,SHb

alkaline-haematin method N/10 NaOH is used and colour of alkaline

hematin is compared Not affected by carboxyhaemoglobin

(HbCO), Hi, or SHb plasma proteins and lipids have little

effect cumbersome and less reliable method

Standard method – heating method Acid alkali method – acid method

DIRECT SPECTROMETRY Hb of a diluted blood sample can be

determined by without a standard, if spectrometer has been correctly calibrated

Hb = A540 HiCN×16114×dilution factor 11×d×1000

Haldane’s method RBC are lysed and CO is added to

convert Hb to carboxyhemoglobin

DIRECT READING PORTABLE HAEMOGLOBINOMETERS

Colour Comparators compare the colour of blood against a

range of colours representing haemoglobin concentrations.

Used for anaemia screening

Portable Haemoglobinometers

based on the HbO2 method. a built-in filter and a scale calibrated for

direct reading of haemoglobin The HemoCue system The DHT Haemoglobinometer

Noninvasive Screening Tests

Methods developed for using near infrared spectroscopy at body sites, mainly a finger, to identify the spectral pattern of haemoglobin in an underlying blood vessel.

There is an approximate correlation with blood haemoglobinometry

Estimation of fetal Hb By Alkali denaturation method --- Brinkman and Jonix method --- Singer & Shernoff method

Hematology autoanalyser

Hematology autoanalyser Red cells are lysed by lysate provided

and colour is matched against the in built standard colour

A cynide free bio-degradable reagent is used

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