Lab report on Hemoglobin determination using cynmethaemoglobin method

1. KENYATTA UNIVERSITY
SCHOOL OF MEDICINE.
DEPARTMENT OF MEDICAL BIOCHEMESTRY
NAME: LANDO ELVIS OTIENO
REG NO: P29S/16344/2015.
COURSE: MBCHB
LECTURER:Dr OKUN
HANDING DATE: 18 /4/2017
SUBJECT: PRACTICAL REPPORT ON DETERMINATION OF
HEMOGLOBIN USING CYANMETHAEMOGLOBIN METHOD.
ELVIS L
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2. INTRODUCTION:
Hemoglobin,the maincomponentof the redbloodcell,functionsinthe transportationof oxygenand
CO2. Hemoglobinconsistsof 1molecule of globinand4moleculesof heme (eachcontaining1molecule
of ironinthe ferrousstate). Globinconsistsof 2 pairsof polypeptidechains. Inthe hemoglobin
molecule,eachpolypeptide chainisassociatedwith1heme group;eachheme groupcan combine with
1 molecule of oxygenorCO2.
Hemoglobincarriesoxygenfromplacesof highoxygenpressure (lungs)toplacesof low oxygenpressure
(tissues),where itreadilyreleasesthe oxygen. HemoglobinalsoreturnsCO2fromthe tissuestothe
lungs.
Hemoglobinscanbe broadlydividedintonormal andabnormal types:
Normal Hb: AdultHb,Fetal Hb and EmbryonicHb.Abnormal Hb: Hb S, Hb C, Hb D, Hb E and Unstable
hemoglobins.
NORMAL HEMOGLOBINS
Adult hemoglobins
HemoglobinA (HbA):About97 percent of hemoglobinof adultredcellsisHbA.It consistsof two alpha
(a) and twobeta (b) chainswiththe structural formala a2 b2. Hb A isdetectedinsmall amountsinthe
fetusas earlyasthe eighthweekof intrauterinelife.Duringthe firstfew monthsof postnatal Life,HbA
almostcompletelyreplacesHbF andthe adultpatternisfullyestablishedinsix months.
HemoglobinA2 (HbA2):Thisisthe minorhemoglobininthe adultredcells.Ithasthe structural formal
of a2 d2 . Hb A2 ispresentinverysmall amountsatbirthandreachesthe adultlevel of 3 percent during
the firstyear of life.Itsconcentrationincreasesinsome typesof anemia.
Fetal Hemoglobins
Fetal hemoglobin(HbF):HbF isthe major hemoglobininintrauterinelife.Ithasthe structural formala
of a2 g2 . Hb F accountsfor 70-90 percentof hemoglobinatterm.Itthenfallsrapidlyto25 percentin
one month,and 5 percentinsix months.The adultlevel of 1 percent isnot reachedinsome children
until pubertyHbF concentrationinadultsincreasesinsome typesof anemia,hemoglobinopathies,and
some time inleukemia.
HemoglobinBart’s (Hb Barts): Thisis the minorhemoglobinpresentinfetal life.Itconsistsof four
gamma (g) chains g4 . Hb Bart’s concentrationincreasesinfetallifeinthalassemia.
Embryonic hemoglobins
These hemoglobinsare confinedtothe veryearlystage (the embryonicstage) of development.There
are three embryonichemoglobins:1)HbGower1 (consistingof twozetaand twoepsilonchains: V2 e2 ),

3. 2) Hb Gower 2 (consistingof twoalphaandtwo epsilonchains: a2 e2 ) and 3) Hb Portland(consistingof
twozeta and twogamma chains:V2 g2).
Abnormal hemoglobins
There are fourclinicallyimportantabnormal hemoglobins:HbS,Hb C,Hb D, andHb E. These are present
indifferenthereditaryhemoglobinopathies.The mostcommonlyencounteredhemoglobinisHbS which
consistsof a2 b2 butin the betachain valine issubstitutedforglutamicacidatthe sixthposition.HbSis
presentinsickle cell anemia.
Unstable hemoglobins are hemoglobinvariantsthatundergodenaturationandprecipitate inthe red
cellsat Heinzbodies.Unstablehemoglobinsare presentinatype of congenital nonspherocytic
hemolyticanemia.
Hemoglobincomplexes
Hb can combine withothersubstancesbesidesoxygen,some normallyandsome abnormally.Some of
these commonlyencounteredcomplexesare carbaminohemoglobin,carboxyhemoglobin,
methemoglobin,sulfhemoglobin,andcyanmethemoglobin.
Carboxyhemoglobin
Whenhemoglobinscombinewithcarbonmonoxide (CO),carboxyhemoglobinisformed.Hemoglobin
has a much greateraffinityforCOthanfor oxygen.Therefore,itreadilycombineswithCOevenwhen
CO ispresentinlowconcentrations.Fortunatelythe formationof carboxyhemoglobinisreversible,so,
once CO is removedfromthe blood,the hemoglobincombineswithoxygen.Carboxyhemoglobinis
foundinverylowconcentrationsinnormal persons,butinsmokersitsconcentrationrangesfrom1-
10g/dl, whichimpairsoxygentransportfromlungstotissues.
Methemoglobin
Methemoglobinisanabnormal Hb inwhichironis oxidizedfromitsferroustoferricstate.Therefore,it
isincapable of carryingoxygen.Normallyitispresentinlow concentrations,butitsformationincreases
inthe presence of certainchemicalsordrugs.The formationof methemoglobinisalsoreversible.
Sulfhemoglobin
Thisis an abnormal Hb complex formedbythe actionof some drugsand chemicalssuchas
sulfonamides.Once itisformed,itisirreversible andremainsinthe carrierRBC.It isincapable of
transportingoxygen.
Cyanmethemoglobin(hemoglobin-cyanide)
Thisis formedbythe actionof a chemical calledcyanide (forexample.Potassiumcyanide,KCN).The
combinationisreversible.Hemiglobincyanide isthe methemoglobinbondedtocyanide ions. Note:To
measure accuratelythe total Hb inthe blood,itis essentialtoprepare astable derivativethatwill

4. containall the a Hb forms(complexes) thatare presentinthe blood.All formsof circulatinghemoglobin
are readilyconvertedtohemoglobin-cyanide (cyanmethemoglobin),exceptforsulfhemoglobinwhichis
normallynotpresentinthe blood.Therefore,the cyanmethemoglobin method isthemostaccurate
method forthe determinationof hemoglobin.
Hemoglobinderivaties
Whenred bloodcellsare destroyedinthe tissuemacrophage system,hemoglobinisdegradedinto
heme andglobin.Globinreturnstothe body’smetabolicpool where itsaminoacidsare subsequently
reutilised.The porphyrinringof heme iscleavedbythe microsomal enzyme,heme oxidase,
yieldingbiliverdin. bybiliverdinreductase
Methodsfor hemoglobinometrycanbe groupedinto4 mainclassesdependingonthe basictechnique
employedwithvariantswithineachclass:
1. ColorimetricMethods
2. GasometricMethods
3. SpecificGravityMethods
4. Chemical Methods
The methodof choice for hemoglobindeterminationisthe cyanmethemoglobinmethod(Thisisatype
of colorimetricmethod).
Three advantagesof the cyanmethemoglobinmethodare:
1. measuresall formsof hemoglobinexceptsulfhemoglobin
2. can be easilystandardized
3. cyanmethemoglobin reagent(alsocalledDrabkin'ssolution)isverystable
Normals: women 12 - 16 g/100 ml blood(g/dl) (g%)
: men 14 - 18 "
:newborn 14 - 20 "
PRINCIPLES:
The principle of thismethodisthatwhenbloodismixedwithasolutioncontainingpotassium
ferricyanide andpotassiumcyanide,the potassiumferricyanideoxidizesirontoformmethemoglobin.
The potassiumcyanide thencombineswithmethemoglobintoformcyanmethemoglobin,whichisa
stable colorpigmentreadphotometricallyata wave lengthof 540nm.
REAGENTS:

5. 1. Potassiumferricyanide =200 mg
2. Potassiumcyanide =50 mg
3. Potassiumdihydrogenphosphate =140 mg
4. Non-ionicdetergent=1 ml
5. Distal water= Make up to 1000 ml (1 L)
MATERIALS
12 x 75 tubes
20 l capillarypipettes
aspirator
Hgb standard
cyanmethemoglobinreagent(Drabkin'ssolution)
testtube rack
spectrophotometer
PROCEDURE
1. Take 20 microlit.of blood+Drabkin4 mL = 1 : 200 dilution.
2. OR take 20 microliterof blood+ Drabkin5 mL = 1 : 250 dilution.
3. Nowmix well.
4. Readwithin6 hoursof mixingongreenfilter510 550nm.
5. Readagainstblankof drabkinsolution(Drabkinsolutioncanbe usedasblank).
6. Alsoreadthe standard solution(12G/dL) withthe same dilutionlike testsample.
7. Calculationmethod=( OD of test/OD of std) Xconc.of stand.= Hb of testsample.
OBSERVATIONS AND RESULTS
BLANK(ml) STD(ml) SAMPLE(ml)
Hb workingreagent - 5ml 5ml
Distilledwater 5ml - -
Standard(Sd) - 0.02 -
SAMPLE (test) - - 0.02
ABSORBANCE 0.00 0.139 0.271

6. CALCULATIONS
Hemoglogin=(ABSTest/ABSStandard) x conc:std
Conc of std= 15g/dL
(0.271/0.139) x 15g/dl
=29.24gL
NORMAL VALUES:
women:12-16 g/100 ml blood (g/dl) (g%)
men :14-18 g/100 ml blood(g/dl) (g%)
newborn:14-25g/100 ml blood(g/dl) (g%)
small children:11–14 g/100 ml blood(g/dl) (g%)
Olderchildren:12- 16 g/100 ml blood(g/dl) (g%)
INTERPRETATION:
The hemoglobinvalueis decreasedinanemiaandincreasedinpolycythemiaanddehydration. The
sample testhemoglobinisincreased.therefore ispolycythimic.
DISCUSIONSAND CONCLUSION
Accurate determinationof hemoglobinconcentrationisacommonelementinassessingthe extentof
anemiaandmakinga decisionwhethertransfusionisnecessaryornot.Thisdecisionshouldbe made
basedon reliableandrapidlyassessedlaboratorytests.In settingswhereacentral laboratoryisusedfor
the purposesof testingandtransfusionmonitoring,the timelossforbloodsample transportationcreate
delayswhichmayleadtothe lossof lives
Cyanmethemoglobin (Drabkin's) methodof haemoglobin estimationhaemoglobinisoxidisedto
methemoglobinbypotassiumferricyanide,whichreactswithcyanide ionsof potassiumcyanide toform
cyanmethemoglobin.The haemoglobinisestimatedwiththe helpof cyanmethemoglobincurve.The
advantagesof thismethodare i) error due to subjective visual matchingisavoidedas
spectrophotometerisusedandhence readingisprecise andreliable,ii)measuresall formsof
haemoglobinexceptsulphaemoglobin.iii)singlestepprocedureusingsingle reagent.iv)
cyanmethemoglobinformedproducesbroadabsorbentbandat530 rim v) good stable haemoglobin
standardsare available.
Physiological variationof Hb:

7. 1. Strenuousphysical exercise.
2. There isdiurnal variationwithhighestlevel inthe morningandlow inthe morning.
3. Highaltitude increase the Hbconcentration.
False causes of raisedHb:
1. Hemoconcentrationdue todehydration,andburns.
2. Immediatelyafterhemorrhage.
3. If takenduringthe I/V infusionif itcontainsiron.
Sources ofError
A. Inadequate mixingof bloodsample
B. Incorrectlycalibratedpipettes
C. Incorrectlycalibratedspectrophotometer
D. Incomplete conversionof Hgbtocyanmethemoglobin
E. Lipemicspecimen
F. Highconcentrationof WBC's or platelets
G. Doesnot measure sulfhemoglobin.
Clinical Significance
Many anemiasare detectedbyroutine laboratoryscreeningperformedbefore the patientis
symptomatic.Whenthe patientdoeshave symptomsfromanabnormalityinthe hemoglobinlevel,the
symptomsare oftena nonspecificweaknessorfatigue.The onlyfindingonphysical examinationmaybe
pallor;additional changesinthe nail beds(suchasspooning),glossitis(redtongue),or
hepatosplenomegaly(enlargedliverorspleen) maygive aclue tothe etiologyof the anemia.Symptoms
are usuallyrelatedtothe levelof hemoglobin,itsabruptnessof onsetanditsduration.A patientwith
perniciousanemiamayfeel well atthe same level of hemoglobinthatwouldcause severeweaknessina
patientwithacute gastrointestinal hemorrhage.Thisisdue tovolume compensationbyplasmaand
shiftsinthe oxygendissociationcurve whichoccurovertime.
Whenfirstconfrontedwithanabnormal hemoglobinorhematocritlevel,the nextstepistoassessthe
redcell indices,peripheralsmear,andthe reticulocytecountinlightof the patient'shistoryandphysical
examination.

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