Hb estimation from dried blood spot by cyanmethaemoglobin method

Anaemia is the most common nutritional deficiency in IndiaAnaemia cannot be diagnosed clinically as the symptoms andsigns are not sensitive or specificDiagnosis of anaemia is made on the basis of Hb estimationCyan methaemoglobin method is the gold standard methodproviding accurate estimation of Hb at affordable cost in primaryhealth care settingsIn laboratory or hospital settings 20 µl blood collected from fingerprick is directly deposited into 5 ml of Drabkin’s solution and thereading taken in colorimeter / spectrophotometer ( direct method )In community settings 20 µl blood collected from finger prick isdeposited on a filter paper, dried, put into plastic envelope andtransported to the lab for estimation of Hb within the next 30 days(indirect method)In the following slides collection of blood in community settingsand estimation of Hb from the dried blood spot are described

Collecting blood for Hb estimation

Items required for bloodcollection on filterpaper for Hb estimation

If the blood sample isto be collected on thefilter paper, ethershould be used forwiping the finger.

Alcohol/spirit,should not be usedfor wiping the finger.

Alcohol denaturesproteins. If alcohol isused for wiping fingerand then blood iscollected on filterpaper, the blood spotwill not elute fully.

Dampen the cotton with solvent ether solution

Wipe the left middle finger tip with ether soaked cotton

Gently squeeze the finger tip Prick the finger tip with the lancet

Wipe the drop of bloodA drop of blood wells up

Squeeze the finger tipso that one drop ofblood wells up

Pipette out 20 microlitresof blood from the drop ofblood

20 microlitres ofblood deposited onthe filter paperlabeled as number1 in pencil.

Arrows indicateareas where bloodfrom the tip hasbeen deposited.

Pipette containing20 microlitres ofblood

Insert filter paper containingdried blood spot into the plasticbag and seal it.

Filter papercontainingdried bloodspot has beeninserted intothe plasticziplockenvelope andsealed

The plastic ziplockenvelope comeswith a stickerwith spaces toenter details ofidentification ofthe person whosesample is put inthe envelope

Blood has beendrawn beyond20 microlitremark

Removing excessblood from thepipette usingtissue paper

Only 20microlitre isleft in thepipette

Errors in blood collection –air bubbles in the blood column in the pipette

Blood outside the pipette has not been wiped outbefore depositing the blood on the filter paper.

Blood in the pipette not fully blownout on to the filter paper

Errors in blood deposition on filter paper

As soon as blood is deposited on the filter paper, rinse thepipette twice with Drabkin’s solution and blow out into tissuepaper /cotton.Then rinse the pipette twice in distilled water and blow outthe water onto tissue paper/cotton.Dry the pipette by pipetting ether twice and blowing it out.Once it is dry, the pipette is ready for use.

Cleaning and drying the pipette

Hb estimation from dried blood spot by cyanmethaemoglobin method

Hb estimation from dried blood spot by

cyanmethaemoglobin method

Test tubes in test tube rack

Drabkin’s solution

5ml dispenser

Numbered dried blood spots containing 20micolitres of blood areremoved from their plastic covers, placed on a white sheet.Identification data checked with the sticker on the envelope andSchedule II (the lab data sheet).

Test tubes containing filterpapers from which bloodspot has been fully eluted.

Test tubes containingDrabkin’s solution in whichblood from filter paper hasbeen fully eluted.

Drabkin’s solution in which blood spot has been eluted has beentransferred to a cuvette.Test tube now contain clear filter papers from which blood spot hasbeen completely eluted.

Colorimeter

Cuvette with Drabkin’s solution

(Blank )

Calorimeter

Hbstandard

Cuvette with Drabkins

solution in which blood spot

has been eluted

Calorimeter

reading when

cuvette containing

plain Drabkin’s

solution is

inserted

Calorimeter reading for

the cuvette containing

Drabkin’s solution with

eluted blood from

blood spot is inserted