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ESTIMATIONOFHEMOGLOBINPERCENTAGE
2013(2)
ESTIMATIONOFHEMOGLOBINCONCENTRATION
March(2)
ESTIMATIONOFHEMOGLOBIN
PERCENTAGE
Hemoglobinometry
Isthemeasurementoftheconcentrationofhemoglobinintheblood
AboutMe
VenkateshSrinivasan
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METHODSofestimationofhemoglobinconcentration
Viewmycompleteprofile
Hemoglobin can be estimated by different methods it can be classified into

followingcategories:
1.Visualcolourcomparisionmethod
2.Gasometricmethod
3.Spectrophotometricmethod
4.Automatedhemoglobinometry
5.Nonautomatedhemoglobinometry
6.Othermethods
A.Visualmethods,
1.Sahilsmethod
2.Daresmethod
3.Hadensmethod
4.Wintrobesmethod
5.Haldanesmethod
6.Tallquistsmethod
B.Gasometricmethod
1.vanSlykemethod
C.Spectrophotometricmethod
1.Oxyhemoglobinmethod
2.Cyanmethemoblobinmethod
D.electronichematologyanalyszer
Automatedhemoglobinometry
E.Nonautomatedhemoglobinometry
F.Othermethods
1.Alkalinehematinmethod
2.Specificgravitymethod
3.comparatormethod.
http://drvenkie.blogspot.in/2013/03/estimationofhemoglobinpercentage.html
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3/4/2016
VISUALMETHODS
Thesemethodsaremorecommonlyusedthanphotometricmethods.InSahlismethod,
hemoglobininthebloodsampleisconvertedtoacidhematin,whichgivesbrowncolour.
Since brown is more easily matched by the human eye than red (the colour of Hb),
Sahlis method for testing hemoglobin is one of the most acceptable visual methods.
However, the error in visual methods is higher. Therefore, visual methods (especially
sahlis) are convenient and the cost of estimation is less, they are usually practiced in
hematologylaboratoriesisclinicalmedicineandforperformingpracticalsarestudentsin
physiology.
INTRODUCTION
Hemoglobin(Hb)isaconjugatedproteinpresentinredbloodcells.Itcarriesoxygenfrom
thelungstothetissues,andcarbondioxidefromthetissuestothelungs.Itismadeupof
hemeandglobin.Thehemegroupisanironcomplex,containingoneironatom.Ironis
essential for the primary function of the hemoglobin, the transport of oxygen. When
reducedhemoglobinisexposedtooxygenatincreasedpressure,oxygenistakenupat
the iron atom until each molecule of hemolecule at each iron atom. The Hb molecule
when fully saturated with oxygen, that is, four oxygen molecules combined with one
hemoglobinmolecule,iscalledoxyhemoglobin.Onegramofhemoglobincarries1.34ml
of oxygen. Hemoglobin returning with carbon dioxide from the tissues is called reduced
hemoglobin.
TYPEOFHEMOGLOBIN
Hemoglobinscanbebroadlydividedintonormalandabnormaltypes.
NormalHb:AdultHb,FetalHbandEmbryonicHb.AbnormalHb:HbS,HbC,HbD,Hb
EandUnstablehemoglobins.
NORMALHEMOGLOBINS
Adulthemoglobins
Hemoglobin A (Hb A): About 97 per cent of hemoglobin of adult red cells is Hb A. It
consistsoftwoalpha()andtwobeta()chainswiththestructuralformala22.HbA
isdetectedinsmallamountsinthefetusasearlyastheeighthweekofintrauterinelife.
DuringthefirstfewmonthsofpostnatalLife,HbAalmostcompletelyreplacesHbFand
theadultpatternisfullyestablishedinsixmonths.
HemoglobinA2(HbA2): This is the minor hemoglobin in the adult red cells. It has the
structuralformalof22.HbA2 ispresentinverysmallamountsatbirthandreaches
theadultlevelof3percentduringthefirstyearoflife.Itsconcentrationincreasesinsome
typesofanemia.
FetalHemoglobins
Fetal hemoglobin (Hb F): Hb F is the major hemoglobin in intrauterine life. It has the
structuralformalaof22.HbFaccountsfor7090percentofhemoglobinatterm.It
thenfallsrapidlyto25percentinonemonth,and5percentinsixmonths.Theadultlevel
of1percentisnotreachedinsomechildrenuntilpubertyHbFconcentrationinadults
increasesinsometypesofanemia,hemoglobinopathies,andsometimeinleukemia.
Hemoglobin Barts (Hb Barts): This is the minor hemoglobin present in fetal life. It
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consists of four gamma () chains 4 . Hb Barts concentration increases in fetal life in
thalassemia.
Embryonichemoglobins
These hemoglobins are confined to the very early stage (the embryonic stage) of
development.Therearethreeembryonichemoglobins:1)HbGower1(consistingoftwo
zeta and two epsilon chains: 22 ), 2) Hb Gower 2 (consisting of two alpha and two
epsilon chains: 2 2 ) and 3) Hb Portland (consisting of two zeta and two gamma
chains:22).
ABNORMALHEMOGLOBINS
Therearefourclinicallyimportantabnormalhemoglobins:HbS,HbC,HbD,andHbE.
These are present in different hereditary hemoglobinopathies. The most commonly
encounteredhemoglobinisHbSwhichconsistsof22butinthebetachainvalineis
substitutedforglutamicacidatthesixthposition.HbSispresentinsicklecellanemia.
Unstable hemoglobins are hemoglobin variants that undergo denaturation and

precipitateintheredcellsatHeinzbodies.Unstablehemoglobinsarepresentinatypeof
congenitalnonspherocytichemolyticanemia.
HEMOGLOBINCOMPLEXES
Hb can combine with other substances besides oxygen, some normally and some
abnormally.Someofthesecommonlyencounteredcomplexesarecarbaminohemoglobin,
carboxyhemoglobin,methemoglobin,sulfhemoglobin,andcyanmethemoglobin.
Carboxyhemoglobin
Whenhemoglobinscombinewithcarbonmonoxide(CO),carboxyhemoglobinisformed.
Hemoglobin has a much greater affinity for CO than for oxygen. Therefore, it readily
combines with CO even when CO is present in low concentrations. Fortunately the
formationofcarboxyhemoglobinisreversible,so,onceCOisremovedfromtheblood,the
hemoglobin combines with oxygen. Carboxyhemoglobin is found in very low
concentrationsinnormalpersons,butinsmokersitsconcentrationrangesfrom110g/dl,
whichimpairsoxygentransportfromlungstotissues.
Methemoglobin
MethemoglobinisanabnormalHbinwhichironisoxidizedfromitsferroustoferricstate.
Therefore,itisincapableofcarryingoxygen.Normallyitispresentinlowconcentrations,
butitsformationincreasesinthepresenceofcertainchemicalsordrugs.Theformationof
methemoglobinisalsoreversible.
Sulfhemoglobin
ThisisanabnormalHbcomplexformedbytheactionofsomedrugsandchemicalssuch
assulfonamides.Onceitisformed,itisirreversibleandremainsinthecarrierRBC.Itis
incapableoftransportingoxygen.
Cyanmethemoglobin(hemoglobincyanide)
This is formed by the action of a chemical called cyanide (for example. Potassium
cyanide,KCN).Thecombinationisreversible.Hemiglobincyanideisthemethemoglobin
bonded to cyanide ions. Note: To measure accurately the total Hb in the blood, it is
essential to prepare a stable derivative that will contain all the a Hb forms (complexes)
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thatarepresentintheblood.Allformsofcirculatinghemoglobinarereadilyconvertedto
hemoglobincyanide (cyanmethemoglobin), except for sulfhemoglobin which is normally
notpresentintheblood.Therefore,thecyanmethemoglobinmethodisthemostaccurate
methodforthedeterminationofhemoglobin.
HEMOGLOBINDERIVATIES
When red blood cells are destroyed in the tissue macrophage system, hemoglobin is
degraded into heme and globin. Globin returns to the bodys metabolic pool where its
amino acids are subsequently reutilised. The porphyrin ring of heme is cleaved by the
microsomalenzyme,hemeoxidase,yieldingbiliverdin.bybiliverdinreductase.
NORMALVALUES
Adultmales:1418(162)g/dlofblood
Adultfemales:1216(142)g/dlofblood
Innewborns,hemoglobinconcentrationisnormally1622g/dl.Itdecreasesto914g/dl
byabouttwomonthsofage.Bytenyearsofage,thenormalhemoglobinconcentration
willbe1214g/dl.Theremaybeaslightdecreaseinhemoglobinlevelafter50yearsof
age.
FUNCTION
Hemoglobinservestwoimportantfunctions.
1.Ittransportsoxygenfromthelungstothetissuesbyformingoxyhemoglobin,and
carbon dioxide from the tissues to the lungs by forming carbaminohemoglobin.
Whenfullysaturated1gofhemoglobincarries1.34mlofoxygen.
2.HemoglobinactsasabufferinmaintainingbloodpH.
SAHLISACIDHEMATINMETHOD
Principle
HemoglobinisconvertedtoacidhematinbytheactionofHCL.Theacidhematinsolution
isfurtherdiluteduntilitscolourmatchesexactlywiththatofthepermanentstandardofthe
comparatorblock.Thehemoglobinconcentrationisreaddirectlyfromthecalibrationtube.
Requirements
1.Sahlishemoglobinometer
Itcontainsacomparator,hemoglobintube,hemoglobinpipette,andstirrer.
Comparator At the middle there is a slot which accommodates the hemoglobin tube.
Nonfadingstandardbrowntintedglasspiecesareprovidedoneithersideoftheslotfor
colour matching. An opaque white glass is fitted at the back to provide uniform
illumination.
HemoglobintubeItisgraduatedononesideingrampercent(g%),from224,andon
theothersideaspercentage(%),from20140.ThistubeiscalledasSahliAdamstube.
Hemoglobin Pipette The pipette bears only one mark indicating 20 cu mm (0.2ml).
Thereisnobulbinthispipette.
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StirrerItisthinglassrodusedforstirringthesolution.
2.N/10HCl
3.Distilledwater
4.Dropper
5.Materialsforasterilefingerprick
Procedure
1.Cleanthehemoglobinometertubeandpipetteandensurethattheyaredry.
2.FillthehemoglobinometertubewithN/10HCluptoitslowestmark(10percent
or2g%)withthehelpofadropper.
3.Prick the finger with all aseptic precautions, and discard the first drop of blood
Note:Theprickshouldbedeepenoughtogivespontaneousflowofblood.Donot
squeezethefingertomakethedropofblood.
4.Allow a large drop of blood to form on the fingertip, and then dip and tip of the
hemoglobinometerpipetteintotheblooddropandsuchbloodupto20cummmark
ofthepipette,Note:Whilesuckingbloodintothepipettecareshouldbetakento
prevententryofairbubbles.Thisisdonebynotliftingthetipofpipetting.Ifanair
bubble enters, remove and discard the blood and make another drop of blood to
repipette.Ifbloodissuckedaboutthe20cummmarkofthepipette, bring down
thebloodcolumntothemarkbytappingthepipetteagainstthefinger,butnotby
usinganyabsorbentmateriallikecottonwool.
5.Wipe the tip of the pipette. Immediately transfer the 0.02ml of blood from the
pipetteintothehemoglobinometertubecontainingN/10HClbyimmersingtipof
thepipette in the acid solution and blowing out blood from the pipette. Rinse the
pipette two to three times by drawing up and blowing out the acid solution.
Withdrawthepipettefromthetube.Note: Make sure that no solution remains in
thepipette.
6.Leavethesolutioninthetubeinthehemoglobinometer,forabouttenminutes(for
maximumconversion of hemoglobin to acid hematin, which occurs in the first ten
minutes).
7.After ten minutes, dilute the acid hematin by adding distilled water drop by drop.
Mixitwiththestirrer.Matchthecolourofthesolutioninthetubewiththestandards
ofthecomparator.Note:Afteradditionofeverydropofdistilledwater,thesolution
should be mixed and the colour of the solution should be compared with the
standard. While matching, take care to hold the stirrer above the level of the
solution.But,rememberthatatnostageshouldthestirrerbytakenoutofthetube.
8.Ifthecolourofthetestsolutionisdarker,thencontinuedilutiontillitmatcheswith
thatofthestandard.
9.Note. The reading when the colour of the solution exactly matches with the
standard and express the hemoglobin content as g% Note: The reading of the
lower meniscus of the solution should be noted as the result. One more drop of
distilled water should be added and the colour should be observed to check the
result. The colour will be lighter than the standard if the previous reading was
accurate.
OTHERMETHODS
Gasometricmethod
GasometricmethodofestimationofhemoglobinbyusingvanSlykeapparatusisthemost
accurate method. But it is not used routinely in clinical laboratories because it is time
consumingandtheprocessofestimationiscomplex.Itisusedasareferencemethodto
obtain the hemoglobin concentration in blood samples used for standardization of
hemoglobinestimationprocedures.Thisisthepreferredmethodforresearch.
Spectrophotometricmethod
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Thesemethodsarerapidandgiveaccurateresults.
a.Oxyhemoglobinmethod
Ammonium hydroxide (0.04ml / dl) is used to hemolyse the red cells and
convert the hemoglobin to oxyhemoglobin for measurement in the
spectrophotometer. This conversion is complete and immediate and the
resultingcolourisstable.
b.Cyanmethemoglobinmethod
ModifiedDrabkinsreagentisusedinthismethod.Drabkinsreagentcontains
sodium bicarbonate, potassium ferricyanide, and potassium cyanide. This
reagent takes at least ten minutes for complete conversion of hemoglobin to
cyanmethemoglobin. It also produces turbid solutions caused by protein
precipitation or incomplete hemolysis. In modified Drabkins reagent,
potassium phosphate is used for sodium bicarbonate, which shortens the
conversion time to three minutes, and minimizes turbidity and enhances red
celllysis.
Variousautomatedtechniqueshavebeenemployedtomeasurehemoglobin.Automatic
pipettors and dilutors are used for pipetting and diluting blood in many procedures.
Hemoglobin estimation done by an automated instrument applies the same principle as
thatdescribedforthemanualmethods.
Nonautomatedhemoglobinometry
Disposable, selffilling, selfmeasuring diluting micropipettes are commercially available
for the determination of hemoglobin. One such system is the Unopette. These systems
are easy to use and are available with a series of different diluting fluids for different
purposes.
The Unopette system for hemoglobin determination consists of a selffilling, self

measuring pipette attached to a plastic holder. The pipette is filled with the blood
automatically by capillary action. A plastic container called a reservoir is filled with
modified Drabkins regent. The pipette containing blood is inserted into the regent
reservoir, emptied and rinsed according to the manufacturers instruction. The blood is
mixedwellwiththereagentandisthenreadytobereadinthespectrophotometer.
Alkalinehematinmethod
Thealkalinehematinmethodisausefulancillarymethodunderspecialcircumstancesas
itgivesatrueestimateoftotalhemoglobinincludingmethemoglobinandsulfhemoglobin.
Atruesolutionisobtained,andplasmaproteinsandlipidshavelittleeffectonthecolour.
Theprincipleistoconverthemoglobinintoalkalinehematin,whichisinthetruesolution.
Therearetwomethodsthestandardmethod,andtheacidalkalinemethod.
Specificgravitymethod
Thismethodusestheprinciplethatwhenadropofwholebloodisdroppedintoasolution
ofcoppersulfate,whichhasagivenspecificgravity,thedropwillmaintainitsowndensity
for approximately 15 seconds. The density of the drop is directly proportional to the
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amountofhemoglobininthatdrop.Ifthatdropisdenserthanthespecificgravityofthe
solution, the drop will sink to the bottom if not, it will float on the surface. It is not a
quantitative test. However, it is a quick, easy, and a reasonably accurate technique to
screenblooddonorsforpossibleanemia.Itisalsousedtodetecthematocrit.
Comparatormethod
Thisisavisualmethodsimilartothatoftheacidhematinmethod,exceptthatthediluent
used is an alkali solution (ammonia solution 0.04 percent). After mixing with dilute
ammoniasolution,theintensityofthecolourofthehemolyzedsolutionofredbloodcells
is compared against a standard colour disc in the comparator. This method has all the
disadvantageofSahlisacidhematinmethod.
Tallquistmethod
This method involves direct disual matching of the red colour of a drop of whole fresh
blood on a filter paper with colour standards on a paper. This technique is totally
unsatisfactorywithahighdegreeoferror,throughitisoneofthequickestmethods.
Haldanemethod
In this method, hemolysis of red cells is produced by mixing blood with a hypotonic
solutionlikedistilledwater.Carbonmonoxideisaddedtothemixture.Thecolourofthe
solutioniscomparedwiththestandardone.
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ESTIMATIONOFHEMOGLOBINCONCENTRATION
Hemoglobinometry
Isthemeasurementoftheconcentrationofhemoglobinintheblood
METHODSofestimationofhemoglobinconcentration
Hemoglobin can be estimated by different methods it can be classified into

followingcategories:
1.Visualcolourcomparisionmethod
2.Gasometricmethod
3.Spectrophotometricmethod
4.Automatedhemoglobinometry
5.Nonautomatedhemoglobinometry
6.Othermethods
A.Visualmethods,
1.Sahilsmethod
2.Daresmethod
3.Hadensmethod
4.Wintrobesmethod
5.Haldanesmethod
6.Tallquistsmethod
B.Gasometricmethod
1.vanSlykemethod
C.Spectrophotometricmethod
1.Oxyhemoglobinmethod
2.Cyanmethemoblobinmethod
D.electronichematologyanalyszer
E.Nonautomatedhemoglobinometry
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3/4/2016
F.Othermethods
1.Alkalinehematinmethod
2.Specificgravitymethod
3.comparatormethod.
VISUALMETHODS
Thesemethodsaremorecommonlyusedthanphotometricmethods.InSahlismethod,
hemoglobininthebloodsampleisconvertedtoacidhematin,whichgivesbrowncolour.
Since brown is more easily matched by the human eye than red (the colour of Hb),
Sahlis method for testing hemoglobin is one of the most acceptable visual methods.
However, the error in visual methods is higher. Therefore, visual methods (especially
sahlis) are convenient and the cost of estimation is less, they are usually practiced in
hematologylaboratoriesisclinicalmedicineandforperformingpracticalsarestudentsin
physiology.
INTRODUCTION
Hemoglobin(Hb)isaconjugatedproteinpresentinredbloodcells.Itcarriesoxygenfrom
thelungstothetissues,andcarbondioxidefromthetissuestothelungs.Itismadeupof
hemeandglobin.Thehemegroupisanironcomplex,containingoneironatom.Ironis
essential for the primary function of the hemoglobin, the transport of oxygen. When
reducedhemoglobinisexposedtooxygenatincreasedpressure,oxygenistakenupat
the iron atom until each molecule of hemolecule at each iron atom. The Hb molecule
when fully saturated with oxygen, that is, four oxygen molecules combined with one
hemoglobinmolecule,iscalledoxyhemoglobin.Onegramofhemoglobincarries1.34ml
of oxygen. Hemoglobin returning with carbon dioxide from the tissues is called reduced
hemoglobin.
TYPEOFHEMOGLOBIN
Hemoglobinscanbebroadlydividedintonormalandabnormaltypes.
NormalHb:AdultHb,FetalHbandEmbryonicHb.AbnormalHb:HbS,HbC,HbD,Hb
EandUnstablehemoglobins.
NORMALHEMOGLOBINS
Adulthemoglobins
Hemoglobin A (Hb A): About 97 per cent of hemoglobin of adult red cells is Hb A. It
consistsoftwoalpha()andtwobeta()chainswiththestructuralformala22.HbA
isdetectedinsmallamountsinthefetusasearlyastheeighthweekofintrauterinelife.
DuringthefirstfewmonthsofpostnatalLife,HbAalmostcompletelyreplacesHbFand
theadultpatternisfullyestablishedinsixmonths.
HemoglobinA2(HbA2): This is the minor hemoglobin in the adult red cells. It has the
structuralformalof22.HbA2 ispresentinverysmallamountsatbirthandreaches
theadultlevelof3percentduringthefirstyearoflife.Itsconcentrationincreasesinsome
typesofanemia.
FetalHemoglobins
Fetal hemoglobin (Hb F): Hb F is the major hemoglobin in intrauterine life. It has the
structuralformalaof22.HbFaccountsfor7090percentofhemoglobinatterm.It
thenfallsrapidlyto25percentinonemonth,and5percentinsixmonths.Theadultlevel
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of1percentisnotreachedinsomechildrenuntilpubertyHbFconcentrationinadults
increasesinsometypesofanemia,hemoglobinopathies,andsometimeinleukemia.
Hemoglobin Barts (Hb Barts): This is the minor hemoglobin present in fetal life. It
consists of four gamma () chains 4 . Hb Barts concentration increases in fetal life in
thalassemia.
Embryonichemoglobins
These hemoglobins are confined to the very early stage (the embryonic stage) of
development.Therearethreeembryonichemoglobins:1)HbGower1(consistingoftwo
zeta and two epsilon chains: 22 ), 2) Hb Gower 2 (consisting of two alpha and two
epsilon chains: 2 2 ) and 3) Hb Portland (consisting of two zeta and two gamma
chains:22).
ABNORMALHEMOGLOBINS
Therearefourclinicallyimportantabnormalhemoglobins:HbS,HbC,HbD,andHbE.
These are present in different hereditary hemoglobinopathies. The most commonly
encounteredhemoglobinisHbSwhichconsistsof22butinthebetachainvalineis
substitutedforglutamicacidatthesixthposition.HbSispresentinsicklecellanemia.
Unstable hemoglobins are hemoglobin variants that undergo denaturation and

precipitateintheredcellsatHeinzbodies.Unstablehemoglobinsarepresentinatypeof
congenitalnonspherocytichemolyticanemia.
HEMOGLOBINCOMPLEXES
Hb can combine with other substances besides oxygen, some normally and some
abnormally.Someofthesecommonlyencounteredcomplexesarecarbaminohemoglobin,
carboxyhemoglobin,methemoglobin,sulfhemoglobin,andcyanmethemoglobin.
Carboxyhemoglobin
Whenhemoglobinscombinewithcarbonmonoxide(CO),carboxyhemoglobinisformed.
Hemoglobin has a much greater affinity for CO than for oxygen. Therefore, it readily
combines with CO even when CO is present in low concentrations. Fortunately the
formationofcarboxyhemoglobinisreversible,so,onceCOisremovedfromtheblood,the
hemoglobin combines with oxygen. Carboxyhemoglobin is found in very low
concentrationsinnormalpersons,butinsmokersitsconcentrationrangesfrom110g/dl,
whichimpairsoxygentransportfromlungstotissues.
Methemoglobin
MethemoglobinisanabnormalHbinwhichironisoxidizedfromitsferroustoferricstate.
Therefore,itisincapableofcarryingoxygen.Normallyitispresentinlowconcentrations,
butitsformationincreasesinthepresenceofcertainchemicalsordrugs.Theformationof
methemoglobinisalsoreversible.
Sulfhemoglobin
ThisisanabnormalHbcomplexformedbytheactionofsomedrugsandchemicalssuch
assulfonamides.Onceitisformed,itisirreversibleandremainsinthecarrierRBC.Itis
incapableoftransportingoxygen.
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3/4/2016
Cyanmethemoglobin(hemoglobincyanide)
This is formed by the action of a chemical called cyanide (for example. Potassium
cyanide,KCN).Thecombinationisreversible.Hemiglobincyanideisthemethemoglobin
bonded to cyanide ions. Note: To measure accurately the total Hb in the blood, it is
essential to prepare a stable derivative that will contain all the a Hb forms (complexes)
thatarepresentintheblood.Allformsofcirculatinghemoglobinarereadilyconvertedto
hemoglobincyanide (cyanmethemoglobin), except for sulfhemoglobin which is normally
notpresentintheblood.Therefore,thecyanmethemoglobinmethodisthemostaccurate
methodforthedeterminationofhemoglobin.
HEMOGLOBINDERIVATIES
When red blood cells are destroyed in the tissue macrophage system, hemoglobin is
degraded into heme and globin. Globin returns to the bodys metabolic pool where its
amino acids are subsequently reutilised. The porphyrin ring of heme is cleaved by the
microsomalenzyme,hemeoxidase,yieldingbiliverdin.bybiliverdinreductase.
NORMALVALUES
Adultmales:1418(162)g/dlofblood
Adultfemales:1216(142)g/dlofblood
Innewborns,hemoglobinconcentrationisnormally1622g/dl.Itdecreasesto914g/dl
byabouttwomonthsofage.Bytenyearsofage,thenormalhemoglobinconcentration
willbe1214g/dl.Theremaybeaslightdecreaseinhemoglobinlevelafter50yearsof
age.
FUNCTION
Hemoglobinservestwoimportantfunctions.
1.Ittransportsoxygenfromthelungstothetissuesbyformingoxyhemoglobin,and
carbon dioxide from the tissues to the lungs by forming carbaminohemoglobin.
Whenfullysaturated1gofhemoglobincarries1.34mlofoxygen.
2.HemoglobinactsasabufferinmaintainingbloodpH.
SAHLISACIDHEMATINMETHOD
Principle
HemoglobinisconvertedtoacidhematinbytheactionofHCL.Theacidhematinsolution
isfurtherdiluteduntilitscolourmatchesexactlywiththatofthepermanentstandardofthe
comparatorblock.Thehemoglobinconcentrationisreaddirectlyfromthecalibrationtube.
Requirements
1.Sahlishemoglobinometer
Itcontainsacomparator,hemoglobintube,hemoglobinpipette,andstirrer.
Comparator At the middle there is a slot which accommodates the hemoglobin tube.
Nonfadingstandardbrowntintedglasspiecesareprovidedoneithersideoftheslotfor
colour matching. An opaque white glass is fitted at the back to provide uniform
illumination.
HemoglobintubeItisgraduatedononesideingrampercent(g%),from224,andon
theothersideaspercentage(%),from20140.ThistubeiscalledasSahliAdamstube.
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3/4/2016
Hemoglobin Pipette The pipette bears only one mark indicating 20 cu mm (0.2ml).
Thereisnobulbinthispipette.
StirrerItisthinglassrodusedforstirringthesolution.
2.N/10HCl
3.Distilledwater
4.Dropper
5.Materialsforasterilefingerprick
Procedure
1.Cleanthehemoglobinometertubeandpipetteandensurethattheyaredry.
2.FillthehemoglobinometertubewithN/10HCluptoitslowestmark(10percent
or2g%)withthehelpofadropper.
3.Prick the finger with all aseptic precautions, and discard the first drop of blood
Note:Theprickshouldbedeepenoughtogivespontaneousflowofblood.Donot
squeezethefingertomakethedropofblood.
4.Allow a large drop of blood to form on the fingertip, and then dip and tip of the
hemoglobinometerpipetteintotheblooddropandsuchbloodupto20cummmark
ofthepipette,Note:Whilesuckingbloodintothepipettecareshouldbetakento
prevententryofairbubbles.Thisisdonebynotliftingthetipofpipetting.Ifanair
bubble enters, remove and discard the blood and make another drop of blood to
repipette.Ifbloodissuckedaboutthe20cummmarkofthepipette, bring down
thebloodcolumntothemarkbytappingthepipetteagainstthefinger,butnotby
usinganyabsorbentmateriallikecottonwool.
5.Wipe the tip of the pipette. Immediately transfer the 0.02ml of blood from the
pipetteintothehemoglobinometertubecontainingN/10HClbyimmersingtipof
thepipette in the acid solution and blowing out blood from the pipette. Rinse the
pipette two to three times by drawing up and blowing out the acid solution.
Withdrawthepipettefromthetube.Note: Make sure that no solution remains in
thepipette.
6.Leavethesolutioninthetubeinthehemoglobinometer,forabouttenminutes(for
maximumconversion of hemoglobin to acid hematin, which occurs in the first ten
minutes).
7.After ten minutes, dilute the acid hematin by adding distilled water drop by drop.
Mixitwiththestirrer.Matchthecolourofthesolutioninthetubewiththestandards
ofthecomparator.Note:Afteradditionofeverydropofdistilledwater,thesolution
should be mixed and the colour of the solution should be compared with the
standard. While matching, take care to hold the stirrer above the level of the
solution.But,rememberthatatnostageshouldthestirrerbytakenoutofthetube.
8.Ifthecolourofthetestsolutionisdarker,thencontinuedilutiontillitmatcheswith
thatofthestandard.
9.Note. The reading when the colour of the solution exactly matches with the
standard and express the hemoglobin content as g% Note: The reading of the
lower meniscus of the solution should be noted as the result. One more drop of
distilled water should be added and the colour should be observed to check the
result. The colour will be lighter than the standard if the previous reading was
accurate.
OTHERMETHODS
Gasometricmethod
GasometricmethodofestimationofhemoglobinbyusingvanSlykeapparatusisthemost
accurate method. But it is not used routinely in clinical laboratories because it is time
consumingandtheprocessofestimationiscomplex.Itisusedasareferencemethodto
12/16
3/4/2016
obtain the hemoglobin concentration in blood samples used for standardization of
hemoglobinestimationprocedures.Thisisthepreferredmethodforresearch.
Spectrophotometricmethod
Thesemethodsarerapidandgiveaccurateresults.
a.Oxyhemoglobinmethod
Ammonium hydroxide (0.04ml / dl) is used to hemolyse the red cells and
convert the hemoglobin to oxyhemoglobin for measurement in the
spectrophotometer. This conversion is complete and immediate and the
resultingcolourisstable.
b.Cyanmethemoglobinmethod
ModifiedDrabkinsreagentisusedinthismethod.Drabkinsreagentcontains
sodium bicarbonate, potassium ferricyanide, and potassium cyanide. This
reagent takes at least ten minutes for complete conversion of hemoglobin to
cyanmethemoglobin. It also produces turbid solutions caused by protein
precipitation or incomplete hemolysis. In modified Drabkins reagent,
potassium phosphate is used for sodium bicarbonate, which shortens the
conversion time to three minutes, and minimizes turbidity and enhances red
celllysis.
Variousautomatedtechniqueshavebeenemployedtomeasurehemoglobin.Automatic
pipettors and dilutors are used for pipetting and diluting blood in many procedures.
Hemoglobin estimation done by an automated instrument applies the same principle as
thatdescribedforthemanualmethods.
Nonautomatedhemoglobinometry
Disposable, selffilling, selfmeasuring diluting micropipettes are commercially available
for the determination of hemoglobin. One such system is the Unopette. These systems
are easy to use and are available with a series of different diluting fluids for different
purposes.
The Unopette system for hemoglobin determination consists of a selffilling, self

measuring pipette attached to a plastic holder. The pipette is filled with the blood
automatically by capillary action. A plastic container called a reservoir is filled with
modified Drabkins regent. The pipette containing blood is inserted into the regent
reservoir, emptied and rinsed according to the manufacturers instruction. The blood is
mixedwellwiththereagentandisthenreadytobereadinthespectrophotometer.
Alkalinehematinmethod
Thealkalinehematinmethodisausefulancillarymethodunderspecialcircumstancesas
itgivesatrueestimateoftotalhemoglobinincludingmethemoglobinandsulfhemoglobin.
Atruesolutionisobtained,andplasmaproteinsandlipidshavelittleeffectonthecolour.
Theprincipleistoconverthemoglobinintoalkalinehematin,whichisinthetruesolution.
Therearetwomethodsthestandardmethod,andtheacidalkalinemethod.
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Specificgravitymethod
Thismethodusestheprinciplethatwhenadropofwholebloodisdroppedintoasolution
ofcoppersulfate,whichhasagivenspecificgravity,thedropwillmaintainitsowndensity
for approximately 15 seconds. The density of the drop is directly proportional to the
amountofhemoglobininthatdrop.Ifthatdropisdenserthanthespecificgravityofthe
solution, the drop will sink to the bottom if not, it will float on the surface. It is not a
quantitative test. However, it is a quick, easy, and a reasonably accurate technique to
screenblooddonorsforpossibleanemia.Itisalsousedtodetecthematocrit.
Comparatormethod
Thisisavisualmethodsimilartothatoftheacidhematinmethod,exceptthatthediluent
used is an alkali solution (ammonia solution 0.04 percent). After mixing with dilute
ammoniasolution,theintensityofthecolourofthehemolyzedsolutionofredbloodcells
is compared against a standard colour disc in the comparator. This method has all the
disadvantageofSahlisacidhematinmethod.
Tallquistmethod
This method involves direct disual matching of the red colour of a drop of whole fresh
blood on a filter paper with colour standards on a paper. This technique is totally
unsatisfactorywithahighdegreeoferror,throughitisoneofthequickestmethods.
Haldanemethod
In this method, hemolysis of red cells is produced by mixing blood with a hypotonic
solutionlikedistilledwater.Carbonmonoxideisaddedtothemixture.Thecolourofthe
solutioniscomparedwiththestandardone.
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PostedbyVenkateshSrinivasanat10:02PM
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2comments:
MehulPatel January2,2015at4:03AM
ThankyouforgiveInformation,Itisveryuseful.
Reply
northernlight August1,2015at6:30AM
Canyoupleaseelaborateonoxyhemoglobinmethod?Irequestforitsprocedure.Thanks
Reply
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Hemoglobin can be estimated by different methods it can be classified into

Unstable hemoglobins are hemoglobin variants that undergo denaturation and

The Unopette system for hemoglobin determination consists of a selffilling, self

Hemoglobin can be estimated by different methods it can be classified into

Unstable hemoglobins are hemoglobin variants that undergo denaturation and

The Unopette system for hemoglobin determination consists of a selffilling, self

+2 Recommend this on Google

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