Blood and Blood Products

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info
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BLOOD BLOOD AND BLOODPRODUCTS

PresentedBy:Dr.Abha Doshi (PrincipalofMETsInstituteofPharmacy,Bandra(W),Mumbai) Assistedby:AkulMehta Assisted by: Akul Mehta
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BLOOD (WholeHuman Blood)
Plasma Pl ( DriedHumanPlasma) ( HumanPlasmaProteinFraction) ( DriedHumanPlasmaProteinFraction)
RedBlood R d Bl d Corpuscles (Concentrated humanRBCs)
ClottingFactors
Serum (DriedHuman Serum)
Immunoglobulin GammaGlobulin (HumanNormalImmunoglobulin Injection)
Fibrinogen (Human Fibrinogen) Fib i )
Thrombin (HumanThrombin)
FibrinClot (HumanFibrin Foam)
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WHOLEHUMANBLOOD WHOLE HUMAN BLOOD

Definition:Itisthehumanbloodmixedwitha g suitableanticoagulant
Thatis: Th t i HumanBlood+Anticoagulant=WholeHumanBlood
ConditionsforBeingaDonor Conditions for Being a Donor

Anypersoningoodhealthisacceptedasadonorprovidedthatheor A i d h lth i t d d id d th t h she: 1) Isnotsufferingfromanydiseasethatcanbetransmittedby transfusion.Thisincludessyphilis,malaria,andserumjaundice. t f i Thi i l d hili l i d j di 2) Isnotanemic.Thehaemoglobincontentofthebloodshouldnot belessthan
12.5%forfemales 13.3%formales (checkedbyallowingadropofbloodtofallintoacoppersulphate y g p pp p solutionofspecificgravity1.053forfemales,and1.055formales.If thedropsinks,thesampleissatisfactory)
3)Hasbeentakingmedicationwhichmightprovetoxicorhave allergicreactionsinapatiente.g.antibiotics ll b
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Collectionoftheblood Collection of the blood

Bloodiscollectedasepticallyfromthemediancubitalvein,inthefrontelbow.
Thisbloodisputintoasterilecontainercontainingananticoagulantsolutionandthe bottleisgentlyshakentoensurethatbloodandanticoagulantarewellmixed,thus preventingtheformationofsmallfibrinclots.
Amaximumof420mlofbloodistakeninoneattendance.
Immediately afterwards the container is sealed and cooled to 4 6 degrees centigrade Immediatelyafterwardsthecontainerissealedandcooledto46degreescentigrade forstorage.
EquipmentUsedfortheCollection Equipment Used for the Collection

Equipmentusedfortakingthebloodismade p p fromplastics,andisdisposable Thecontainerearlierconsistedofbottles,but Plasticbagshavestartedbeingusedandare Plastic bags have started being used and are thecontainersofthefuture.
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BloodClotting Blood Clotting

Twoimportantstepsintheclottingofbloodare:
PROTHROMBIN(Soluble) InpresenceofTHROMBOPLASTIN+ In presence of THROMBOPLASTIN + IONIZEDCALCIUM
THROMBIN(Soluble)
FIBRINOGEN(Soluble)
InpresenceofTHROMBIN
FIBRIN(InsolubleClot)
Inresponsetoinjury,thetissuesandbloodplateletsfreesubstancesthatactivatetheclot p j y, p promotingenzymeTHROMBOPLASTIN. Thromboplastin,withtheassistanceofionizedcalciumandotherfactors,converts PROTHROMBINtoactiveclottingenzymeTHROMBIN. ThrombinthenactsonFIBRINOGEN,convertingitintoinsolubleFIBRIN,thematrixofthe clot. http://www.pharmaxchange.info 7
ANTICOAGULANTS
CITRATES HEPARIN
DISODIUM EDETATE
ANTICOAGULANTSUSED
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CITRATES
Thesolutionmostoftenusedasablood h l i f d bl d anticoagulantisknownasAcidcitrate dextrose(ACD),composedof:
SodiumCitrate(2.0to2.5g) Dextrose (3.0g) WaterforInjection(q.s.to120ml) j (q )
Thecitratepreventsclottingbybindingthe calciumionsasunionizedcalciumcitrate,thus calcium ions as unionized calcium citrate thus preventingavitalstepofclotting.

WhyAcidCitrateandnotNormalCitrate?? Why Acid Citrate and not Normal Citrate??

Earlierthenormal,trisodiumcitratewasusedbut ithasaveryhighalkalinepHinsolutionwhich it has a very high alkaline pH in solution which causesconsiderablecaramelisationofthe ( g) g dextrose(darkening)duringsterilizationandthe twosolutionshavetobeautoclavedseparately. p p TheAcidCitrateproducesapHofabout5and causeslittleornocaramelisation. Inaddition,itislesslikelytoinduceflakingofthe glassofthecontainer. Thehigherconcentration(2.5g/120ml)isoften preferredbecauseitmoreeffectivelyreducesthe f db ff l d h formationofsmallclots.
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WhyaddDextrose? Why add Dextrose?

Thedextrosedelayshaemolysisofthe y y p g erythrocytesinvitroandprolongstheirlife aftertransfusion. Its function is hypothesized to be connected Itsfunctionishypothesizedtobeconnected withthesynthesisofcompounds,suchasATP, thatareimportantinmakingenergyavailable h k l bl tolivingcells
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HEPARIN
Naturallyoccurringanticoagulant. ll i i l y Madebythemastcellsoftheconnective tissuesurroundingbloodvessels. Itinhibitsclottinginthecirculatorysystem. It inhibits clotting in the circulatory system Occasionally,itisusedinbloodfortransfusion whenlargevolumesmustbegiventoone when large volumes must be given to one patientandthecorrespondingamountsof citratewouldbeharmful,e.g.incardiac it t ld b h f l i di surgery.
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It quickly loses activity in blood in vitro and Itquicklylosesactivityinbloodinvitroand normalquantitiesareeffectiveforaboutaday. ACD ACDontheotherhand,prolongsthestorage h h h d l h lifetothreeweeks. Heparinisexpensiveandmaycontinueits actionevenaftertransfusion,thusneeding action even after transfusion thus needing administrationofneutralizingsubstancessuch asprotaminesulphate. t i l h t
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DISODIUMEDETATE DISODIUM EDETATE

Thi i l ThisisalsoachelatingagentlikeACD. h l ti t lik ACD Ithasastrongaffinityfordivalentmetals,and thuswillbindtocalciumfirmly. thus will bind to calcium firmly Itissometimespreferredwhenpreservationof bloodplateletsisessential,althoughthestability blood platelets is essential although the stability oftheseseemstodependmuchmoreon preventingcontactwiththeglasssurface:if preventing contact with the glass surface: if plasticbagsorsiliconetreatedglassisused,ACD isalmostaseffectiveasDisodiumEdetate. Thesurvivalofredbloodcellsindextroseedetate solutionsisasgoodasinACD.
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TESTINGOFWHOLEBLOOD TESTING OF WHOLE BLOOD

Atthetimethatbloodiscollected,twosmalladditional amountsarecollected: amounts are collected:
One,whichisoftenobtainedbydrainingthecollectingtube,is putintoasmall5mlbottleandisfirmlyattachedtothemain container.Thisisfortestingcompatibilitywiththebloodofthe t i Thi i f t ti tibilit ith th bl d f th recipient.Thisseparatespecimenavoidsthedangerous procedureofattemptingtoremoveasamplefromthemain bottlewithoutcausingbacterialcontamination. b ttl ith t i b t i l t i ti Ifaplasticbagisused,itispossibletoleavethebloodfilled collectingtubeattachedtothebagandtosealitatseveral pointswithaspecialtool;thenasectioncanbeseparatedfor i ih i l l h i b df testingwithoutcontamination Thesecond,somewhatlargersampleisusedassoonaspossible , g p p for:
Serologicaltesttoconfirmtheabsenceofsyphilisandotherdiseases To determine the ABO blood group of the cells and plasma and the Rh TodeterminetheABObloodgroupofthecellsandplasmaandtheRh groupingofthecells.
BLOODGROUPS BLOOD GROUPS

F d FundamentalAim:istopreventantigenantibody t l Ai i t t ti tib d reaction. Red cells carry an antigen that reacts with the Redcellscarryanantigenthatreactswiththe correspondingantibodyintheplasmaof individualsofcertainothergroups.Ifthecellsare individuals of certain other groups If the cells are transfusedintoanindividualwiththeequivalent antibodyinhisplasma,theyarerapidly destroyed,withseriousconsequences. Althoughsome9bloodgroupsareknown,only theABOandRhareofmajorimportanceas h d h f causesofhaemolytictransfusionreactions
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1)ABOSystem 1) ABO System

Thefirstsignofthehaemolyticantigenantibody reactionisagglutinationand,therefore,redcell antigensandplasmaantibodiesarecalled gg g gg p y agglutinogensoragglutininsrespectively. Theagglutinatedcellshaemolyse,freeing haemoglobinandotherconstituentsandcausing haemoglobin and other constituents and causing jaundiceandkidneydamage:ifthelatteris extreme,thepatientmayevendie. extreme the patient may even die Fortunatelymosttransfusionreactionsaremild.
CompatibilityChart p y
Group RedCell Antigen (Agglutinogen) A B A&B Plasma Antibody (Agglutinin) AntiB AntiA None CanDonateTo: CanReceive From: AorAB BorAB AB only AorO BorO Allgroups. (Universal Recipient) Oonly A B AB
AntiAand AntiB
Allgroups. (Universal Donor
NOTE Generally,therecipientscellsarenotseriouslyharmedbythe agglutininsinthedonor splasma,becausethelatterquicklybecomesdilutedin agglutinins in the donors plasma, because the latter quickly becomes diluted in therecipientsblood(thusadonorofbloodgroupOcandonatetoalleven thoughithasagglutininsAntiAandAntiB)
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2)RhSystem 2) Rh System
Rhfactor,sonamedcauseitwasfoundinthe hf d i f di h rhesusmonkey. Theredcellsofsomeindividualscarryan g antigenthatisknownastheRhfactor. IfRh+bloodistransfusedintoanRh recipient,productionofantibodiestotheRh+ recipient production of antibodies to the Rh+ bloodmaybestimulated. If thi Ifthisoccurs,subsequenttransfusionofRh+ b tt f i f Rh+ bloodwillcauseahaemolyticreaction.
Haemolytic disease in a new born: Haemolyticdiseaseinanewborn:

IfafoetusisRh+fromitsfather,andthemotheris Rh andhastheRh+antibodyinherblood(either fromprevioustransfusionofRh+bloodorasa resultofstimulationbyantigensofthefoetus),the mothersantibodiesmaycrosstheplacentaand destroythefoetalerythrocytes. Thishaemolyticreactionmaykillthefoetusor causetheinfanttobeseverelyanaemic.
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STORAGE
Bl d ll t d Bloodcollectedmustbekeptatatemperaturebetween4 tb k t t t t b t 4 to6degreescentigrade,atalltimesexceptduringshort periodsoftransportandexamination,whichmustnot exceed30mins. Evenatthislowtemperatures,deleteriouschangesdotake place. place
Theleucocytesdisintegrateinafewhours Theplateletsdisintegrateinafewdays TheredcellsshowafallinATPandotherorganicphosphates,a reductioninoxygencarryingcapacityand,duepartlytolossof lipidfromtheirmembranes,increasedfragility.
Storageatroomtemperatureevenforaday,seriously reducesposttransfusionsurvivaloftheerythrocytes
Thefitnessofbloodfortransfusionisbasedonitsappearance.On standing,thecellssediment,leavingalayerofyellowsupernatant plasma. Ifthebloodhasbeentakenshortlyafteraheavyfattymeal,theplasma maybeturbidandshowawhitelayeroffatonitssurface.Ontopof theredcellstheremaybeacompleteorpartialgreyishlayerof leucocytes. Themostimportantfeature,however,isthelineofdemarcation betweencellsandplasma,whichmustbesharp:ifitisobscuredbya p p y diffuseredcoloration,indicatinghaemolysis,thebloodisunfitforuse. Completehaemolysis,especiallyifitoccursrapidly,isusuallyasignof bacterialinfection,butitsabsenceisnotconfirmationofsterilitysince , y certainpsychrophilicbacteria,predominatelypseudomonadsand membersofthecoliaerogenesgroup,cangrowinbloodatrefrigerator temperatureswithoutcausinghaemolysis. Manyoftheorganismsisolatedfromcontaminatedbloodhavebeen capableofusingcitrateastheirsolesourceofcarbonand,aswouldbe expected,thishasledtoclotformation,ascitratewhichisthe anticoagulantgetsassimilatedbythebacteria.
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USES
Haemorrhage,shock,burnsanduncontrollablediarrhoeaand vomiting,canallcausesignificantlossesofblood. vomiting can all cause significant losses of blood
Haemorrhageandotherdiseasesmayresultindeficiencyor absenceofvitalbloodconstituentssuchasredcells,platelets,or clottingfactors. clotting factors Thetransfusionofwholebloodcanbeofgreatvalueinallthese circumstancesbutoften,becauseoftheriskoftransfusion reactions,itisnotusedwheretheneedissolelytomakeupblood reactions it is not used where the need is solely to make up blood volumebutisrestrictedtohaemorrhageandcertaindiseases wherethereisdeficiencyofthevitaloxygencarryingerythrocytes.
N NormallywholebloodisnotadministeredunlesstheABOand ll h l bl d i t d i it d l th ABO d Rhgroupsofthedonorandrecipientareknownandasample ofthedonorsbloodhasbeentestedforcompatibilitywith thatoftherecipient. Inanemergency,groupO,Rhnegativebloodmaybegiven whiletakingnecessaryprecautions. while taking necessary precautions
CONCENTRATEDHUMANREDBLOOD CORPUSCLES
Definition:ThisisthesolutionofhumanRBCs g whichhavebeenconcentratedusing centrifugation
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Preparation
Itispreparedbyremovingmostofthecitratedplasmafromwhole bloodthatisnotmorethanafortnightoldandhasbeenallowedto blood that is not more than a fortnight old and has been allowed to standorhasbeencentrifugedtodepositthecells
Morethan40%ofthesupernatantfluidafterthesettling,issiphoned offusingsteriletubes,takingstrictasepticprecautionsthroughout. off using sterile tubes, taking strict aseptic precautions throughout. Sincethereisariskofbacterialcontaminationtheproductmustbe usedwithin12hours.
Thecellsarematchedwiththerecipientsplasmaandmaythenbe p p y mixedwithmatchedcellsfromotherbottles. Thehaemoglobincontentmustnotbelessthan15.5%
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Uses
Thisproductisusedwhenadministrationof hi d i d h d i i i f wholebloodmightovertaxthecirculation,i.e.,in treatmentofdiseases,suchaschronicanaemia t t t f di h h i i (wherebloodvolumehasnotbeenreduced), ratherthanhaemorrhage(whichwouldrequirea rather than haemorrhage (which would require a replenishmentofbloodvolumeaswellandthus wouldrequirewholeblood) would require whole blood) Anotherapplicationisinexchangetransfusionin infants:atoxicamountofcitratemightbegivenif i f t t i t f it t i ht b i if wholebloodwasused.
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DRIEDHUMANPLASMA DRIED HUMAN PLASMA

Itistheportionofthebloodwhichhasbeen p separatedfromthecellcontent,andisdried, andcanbeusedafterreconstitutionwith water. water
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DRIEDHUMANPLASMA DRIED HUMAN PLASMA

Di isadvantagesof fWhole e Blood d
It h Ithaspoorkeeping k i propertiesnecessitating usewithinthreeweeks Itrequiresrefrigeration Itmustbecompatible t t e b ood o t e withthebloodofthe recipient
Advan ntageso ofDriedPlasma a
P Properlystoreditkeeps l t d it k wellforatleastfive years Ifprotectedfromlightit canbestoredatroom temperatureprovided thisisbelow20degrees centigrade Itcanbegivento It can be given to patientsofanyblood group.
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THUS,UNDERSUITABLECIRCUMSTANCES,DRIEDPLASMACANBEUSEDASA http://www.pharmaxchange.info SUBSTITUTEFORWHOLEBLOOD.
ProblemstobeOvercomeDuring Preparation
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Twomajorproblemshavetobeovercome
TransmissionofViralJaundice NeutralizationofPlasmaAgglutinins
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1]TransmissionofViralJaundice
Therearetwotypes:
Infectivehepatitis(incubationtime=5weeks,mortality rate=0.3%) t 0 3%) Homologousserumjaundice(incubationtime=20 weeks,mortalityrate 12%) weeks, mortality rate =12%)
Mostinfectionsfollowingtransfusionaremild Control is partly effected by refusing to accept Controlispartlyeffectedbyrefusingtoaccept donorswithahistoryofjaundice,butnotallcases g p arerecognizedandsinceatpresentthereisno reliabletestbywhichcarrierscanbedetected,an occasionalinfectedbottleisinevitable. Attemptshavebeenmadetokillthecausative virusesbytreatmentwithUVlight,butthemethod istechnicallydifficult. is technically difficult
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Note ifthepreparationofabloodproduct involvespoolingmaterialfromalargernumberof donors,infectioninoneortwobottleswillbe distributedthroughoutthepoolandappearin eachoftheunitsmadefromit. Nowadays,thepoolsusedformakingdriedplasma andserumarelimitedtonotmorethanten and serum are limited to not more than ten donations,andtheincidenceofjaundiceisonly slightlygreaterthanwhenwholebloodis slightly greater than when whole blood is transfused. However,inthepast,whenpoolsof300ormore bottlesweremade,theincidencewas7to12%.
2]NeutralizationofPlasmaAgglutinins
Agglutininsinthedonorsplasmausuallydonotdamagethe recipient'sredcells. Occasionally however the plasma agglutinins are very Occasionally,however,theplasmaagglutininsarevery powerfulandcancauseserioushaemolysisofthecellsofthe recipient. Thismeansthatincompatibilityproblemsarenotentirely eliminatedbyusingproductssuchasplasmaandserum,that containnocells. Theproblemhasbeenovercomesincethediscoverythatred cellagglutinogensalsooccuraswatersolubleformsinplasma, salivaandotherbodyfluids. saliva and other body fluids Consequently,bymixingplasmafromdifferentgroupsin suitableproportionsthepowerfulagglutininscanbecross neutralizedbysolubleagglutinogens,producingapreparation thatissafetotransfusetoallgroups. The most satisfactory ratio for mixing is Themostsatisfactoryratioformixingis 9ofA:9ofO:2ofBorAB. http://www.pharmaxchange.info 32
PreparationofDriedPlasma p
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Driedplasmaisusuallypreparedfromtimeexpiredcitratedblood Thebloodiscentrifugedasinthecaseofconcentratedredbloodcellsandthe p p supernatantfluidissiphonedoff. Thissiphonedfluidisthencombinedandbatchesoflessthan10bottlesare pooled,choosingthecorrectratioofbloodgroupstoneutralizethepowerful l d h i h i f bl d li h f l agglutinins
Thepoolsarekeptat4to6degreescentigradewhilesamplesaretestedfor sterilityandnopoolisusedunlessitpasses
Then400mlquantitiesaredispensedintobottlesandsubjectedtofreezedrying. q p j y g Generalaspectsoffreezedryingarefollowedwithspecialfeaturesoftheplasma process PreliminaryFreezing Pi PrimaryDrying D i Secondarydrying

PreliminaryFreezing Preliminary Freezing

Thebottlesaresealedwithbacteriologically p y g yp efficientfabricpadscoveredbyringtype closuresandthencentrifugedat18degrees centigrade. centigrade Theliquidsnapfreezesandbecomes distributedaroundtheinsideofthebottle b h f h b l
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PrimaryDrying Primary Drying

Thebottlesoffrozenmaterialaremounted h b l ff i l d horizontallyinthedryingchamberandahigh vacuumisapplied. g p Theicesublimesontoacondensingcoilkept at50degreescentigradeandasmallheater p providesthelatentheatrequiredfor q evaporation. This stage takes about 2 days after which the Thisstagetakesabout2days,afterwhichthe residualmoisturecontentisabout2%.
SecondaryDrying Secondary Drying

Thisisdoneinanotherchamberbyvacuum hi i d i h h b b desiccationoverphosphorouspentoxide. Ittakesaboutaday,andtheproductisleftwith about0.5%ofmoisture EachfabricsealisthenreplacedbyanMRCtype closureperforatedbyapluggedhypodermic needle.Thebottlesarereturnedtothesecondary dryingchamber,reevacuated,andthenthe vacuumisbrokenwithdrysterilenitrogen. Finally,theneedlesareremovedandtheclosure y isprotectedwithasterileviscosecap.
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Storage
Driedplasma,keptbelow20degreescentigradeand protectedfromlight,moisture,andoxygen,remains protected from light moisture and oxygen remains usablealmostindefinitely,althoughitiscustomaryto imposeanarbitraryexpirydateofabout5years. Itsfitnessforuseisshownbyitssolubilitywhen reconstitutedinavolumeofwaterforinjection(WFI), SodiumChlorideInjectionorasolutioncontaining2.5% Sodium Chloride Injection or a solution containing 2 5% dextroseand0.45%sodiumchloride,equivalenttothe originalvolumeofplasma. Itmustdissolvecompletelywithintenminutesatroom temperature. Gel formation or incomplete solution indicates Gelformationorincompletesolutionindicates deterioration. Afterreconstitutionitmustbeusedimmediately. After reconstitution it must be used immediately
Uses
Reconstitutedplasmaissatisfactoryalternativetowhole bloodinconditionswherethereisnolossofredcells. bl d i diti h th i l f d ll Itisofparticularvalueinthetreatmentofsevereburns andscaldswhere,becauseofextensivefluidandprotein d ld h b f t i fl id d t i loss,thereisconsiderablehaemoconcentration. It may also be given when blood is more appropriate; Itmayalsobegivenwhenbloodismoreappropriate; eitherbecausewholebloodisunavailableor,in emergency,untiltheresultsofmatchingtestsare emergency, until the results of matching tests are known. g g Becauseofitslongstoragelifeataconvenient temperature,driedplasmaismoresuitablethanblood asareservestockinasmallhospitaloraremote community.
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DRIEDHUMANSERUM DRIED HUMAN SERUM

Preparedinthesamewayasdriedplasmaexceptthat thebloodiscollectedintodrybottlesandallowedtoclot. th bl d i ll t d i t d b ttl d ll dt l t Thesupernatantserumbeingseparatedaftertheclothas retracted.
Plasmaisusuallyobtainedfrombloodthatisoutofdate,i.e.hasbeenavailable aswholebloodfor21days. y Byconvertingbloodintoserumthisperiodinreserveislostand,therefore,much lessbloodisusedforserumproduction Its,storageandusearethesameasfordriedplasma.
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LIQUIDPLASMAANDSERUM LIQUID PLASMA AND SERUM

Theonlyofficialliquidbloodproductishuman p plasmaproteinfraction. p
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Whyunmodifiedliquidplasmaand serumarenolongerrecognized??
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Pl Plasmaandserum,likeblood,areexcellentmediaforbacterial d lik bl d ll t di f b t i l growthand,therefore,theusermustbeabletodetect contamination. U f t Unfortunately,theseproductsareoftenopalescentdueto t l th d t ft l td t suspendedfat. Further,turbidityanddepositsdevelopduringstorageandasa resultofmovementduringtransport,thusmakinginfectionvery lt f td i t t th ki i f ti difficulttoidentify. Attemptstoremovefatbyfiltrationwerenotverysuccessful becauseitblockedthefilter,butcentrifugationprovedmore b i bl k d h fil b if i d satisfactory. Withserumitwasthenpossible,bypassingtheproductthrougha sterilizingpad,toobtainaclearpreparationthatwouldstore reasonablywell. Ithaspassedoutofusebecausebloodismore economicallyusedfordriedplasmaproduction
Withplasma,thereareadditionaldifficultiesbecause,unlikeserum, itcontainstheclottingfactors,whichcanbeactivatedfairlyeasily. it contains the clotting factors which can be activated fairly easily Forexample,ifplasmaisfilteredthroughfibrouspads,magnesium ionsfromtheasbestoscan,likecalciumionsinvivo,activate prothrombinandcauseclotsinthefilterand,later,thefiltrate. prothrombin and cause clots in the filter and later the filtrate Methodsweredevisedforremovingsomeoftheclottingfactorsand anunstablelipoidglobulincomplexbyadsorptionontokaolinor fractionationwithanorganicsolventatlowtemperatures. fractionation with an organic solvent at low temperatures Complexasepticmanipulationswereinvolvedand,toconfirm freedomfromcontamination,itwasnecessary,beforeissue,tostore theproductsforseveralweeksatatemperatureconduciveto the products for several weeks at a temperature conducive to bacterialgrowth(thatway,iftherewascontamination,itwould showup) Furtherprogresswasmadelessurgentbythesuccessofdried plasmabutinvestigationscontinuedandhavecontributedtothe developmentoftheproductknownashumanplasmaprotein development of the product known as human plasma protein fraction.
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THEFRACTIONATIONOFPLASMA THE FRACTIONATION OF PLASMA

Ab t 60% f l About60%ofplasmaproteinisalbuminand, t i i lb i d therefore,itplaysamajorpartinmaintainingthe highosmoticpressurenecessarytoretainfluidin high osmotic pressure necessary to retain fluid in thebloodvessels. Averysuccessfulsolventprecipitationtechnique A very successful solvent precipitation technique wasdevelopedbywhichotherproteins,aswell asalbumin,wereseparated. Someofthese,i.e.fibrinogen,prothrombin,and gammaglobulin,provedsovaluablethatprotein fractionationofplasmaquicklybecamean f f l kl b establishedprocedure.
Conditionsfortheprocessof fractionationare:
Theprocessselectedmustnotalterthe g p p biologicalpropertiesofthefractionsnoraffect thesolubilities. It must be possible to carry it out aseptically Itmustbepossibletocarryitoutaseptically and,ideally,theconditionsshoulddiscourage bacterialgrowth. b l h Anyadditivemustbeharmlessoreasily Any additive must be harmless or easily removedafteruse
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TechniquesofProteinSeparation Techniques of Protein Separation

O Oneoftheoldestmethodsofproteinseparationis f th ld t th d f t i ti i saltingout,butthisisunsuitableforplasma g fractionationbecausehighconcentrationsofsaltare neededandthesearenotselectiveenough.Also, dialysis,atechniquethatisdifficulttoperform aseptically,isnecessarytoremovethesaltafterthe aseptically is necessary to remove the salt after the precipitation. g ( E.J.Cohnandhiscolleagues(duetotheneedfor transfusionmaterialwithalonglifeandstability,unlike wholeblood,duringtheearlypartoftheSecondWorld War),haddevelopedatechniquetoseparatealbumin War) had developed a technique to separate albumin andotherproteinsfromplasma.
Cohnstechniquewasbasedontheuseofanorganic solvent(ethylalcohol)toreducethesolubilitiesofthe solvent (ethyl alcohol) to reduce the solubilities of the proteins,andwasgivenflexibilitybyalterationsofpH, ionicstrength(i.e.,saltconcentrations)andprotein. Theuseofanorganicsolvent,insteadofsalt,asamajor precipitantconfersanumberofincidentaladvantages:
Because of its volatility it can be removed easily during the Becauseofitsvolatilityitcanberemovedeasilyduringthe freezedryingofthefinalproduct. Saltcanbeusedinlowconcentrationstoimprove resolution. Ithelpstocontrolcontaminantsbecauseofits bacteriostaticactivity. bacteriostatic activity. Beingaliquid,itiseasytoaddaseptically
Ontheotherhand,itisnecessarytokeepthe temperatureverylow(0to5degreescentigrade)to preventsolventdenaturationofproteins.

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EtherFractionationofPlasma Ether Fractionation of Plasma
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HumanPlasmaProteinFraction Human Plasma Protein Fraction

Thisisasolutionofsomeoftheproteinsfrom q p liquidplasma. Itcontainsalbuminandcertainglobulinsthat retaintheirsolubilityonheating. retain their solubility on heating Itispreparedbyfractionatingpooledcitrated plasmaandissimilartothefractionshownas crudealbumininthechartshownonthe crude albumin in the chart shown on the previousslide.
Astabilizersuchassodiumcaprylateoracetyltryptophan isadded.Thisallows thepreparationtobeheatedforseveralhoursatalowtemperaturewithout significantdenaturationofproteins. i ifi d i f i Sodiumchlorideisaddedtomakethepreparationapproximatelyisotonic. Thesolutionissterilizedbyfiltration,asepticallydistributedintobloodbottles andthenheatedat60+/ 0 5 d d h h d 60 / 0.5degreescentigradefortenhourstodestroythe i d f h d h virusesofinfectivehepatitisandhomologousserumjaundice. Althoughtheuseofabactericideorantibioticwouldhelptocontrol contamination,neitherisallowed. contamination neither is allowed
Abactericidewouldbeundesirableontoxicitygroundsinapreparationgiven intravenouslyinlargevolumes. With an antibiotic, there would be a risk of sensitization. Withanantibiotic,therewouldbeariskofsensitization.
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Thefractionationprocessinvolveconcentrationofalbuminfraction.Toensure that,asaresult,theamountofsodiumcitrateisnotraisedtoaharmfullevel, theconcentrationinthefinalpreparationislimitedto0.4%. Theproteincontentisnotlessthan4.3%w/vandtheproductexertsacolloidal osmoticpressureapproximatelyequivalenttothatofpooledliquidplasma containing5.2%w/vofprotein. Itmustbestoredbetween5to20degreescentigradeandprotectedfromlight. Sincefibrinogenhasbeenremoved,thepreparationremainsclear. Itsuseremainsthesameasdriedplasma

DriedHumanPlasmaProteinFraction Dried Human Plasma Protein Fraction

Driedhumanplasmaproteinfractionis p p preparedbyfreezedryinghumanplasma y y g p proteinfraction Its use is also the same as that of dried It suseisalsothesameasthatofdried plasma
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HumanFibrinogen Human Fibrinogen

Fibrinogenisthesolubleconstituentofplasmawhichonadditionof thrombinisconvertedtofibrin(whichisinsoluble). thrombin is converted to fibrin (which is insoluble) Afterseparationfromplasmabyfractionation,theprecipitateis collectedbycentrifugation,dissolvedincitratesaline,andfreeze dried Theairinthecontainersdisplacedbynitrogen. Thecitratepreventsspontaneousclottingwhenthematerialis reconstituted. Fibrinogendissolvesslowly.However,likemanyotherprotein solutions,itfrothsalotifshakenandthesolidstabilizedfoamisvery slowtodisperse,thusagitationshouldbelimitedtorocking. Thesolutionshouldbeusedassoonaspossibleandnotlaterthan threehoursafterpreparation. p p Thefibrinogenmustbestoredunderdryconditions,protectedfrom lightandatatemperaturebelow20degreescentigrade.Theother storageconditionsaresimilartothatofdriedserum. g
UseofHumanFibrinogen Use of Human Fibrinogen

Occasionally,fibrinogenisadministeredalone g y totreatfibrinogendeficiency. Butitismoreoftenusedinconjunctionwith thrombinaswillbeseenahead. thrombin as will be seen ahead
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HumanThrombin Human Thrombin

Th Thrombinisanenzymethatconvertsfibrinogentofibrin. bi i th t t fib i t fib i Theprothrombinobtainedfromthefractionationofplasma iswashedwithdistilledwateranddissolvedincitrate saline. ItisconvertedtothrombinbyadjustmentofpHto7and addingthromboplastinandcalciumions. adding thromboplastin and calcium ions Thesolutionisfilteredandfreezedried,andtheairinthe containersisreplacedbynitrogen. p y g Itisreconstitutedwithsalinewhenrequired. Thethrombinmustbestoredunderdryconditions, protectedfromlightandatatemperaturebelow20 t t df li ht d t t t b l 20 degreescentigrade.Theotherstorageconditionsare similartothatofdriedserum.
UsesofHumanThrombin Uses of Human Thrombin

Thefibrinclotproducedwhenthrombinismixedwith p fibrinogenisusedinsurgerytosutureseverednerves andtoassistadhesionofskingrafts. The mi t re lots at a rate that depends on the amo nt Themixtureclotsataratethatdependsontheamount ofthrombinpresentand,therefore,ifnecessary,itcan p g g j , g bekeptfluidlongenoughforadjustments,e.g.ofskin graftstobemade. Theclotalsoactsasahaemostat(whichwillbeseen aheadinHumanFibrinFoam). h di H Fib i F ) Sincethefibrinishuman,itiswelltoleratedbythe body,andnewcellspenetrateitrapidlyallowinga body and new cells penetrate it rapidly allowing a quickerandbetterhealingtooccur.
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HumanFibrinFoam Human Fibrin Foam

Thi i Thisisaspongelikemassofhumanfibrin. lik fh fib i Itispreparedbywhippingasolutionoffibrinogeninto afrothbymechanicalmeansandthenadding a froth by mechanical means and then adding thrombin. p p y Theproductispouredintotraysandfreezedried,then cutintopiecesofconvenientsizeandsterilizedbydry heatat130degreescentigradeforthreehours. The foam must be stored under dry conditions Thefoammustbestoredunderdryconditions, protectedfromlightandatatemperaturebelow20 g g g degreescentigrade.Theotherstorageconditionsare similartothatofdriedserumexceptthatfibrinfoam neednotbekeptundernitrogen.
UsesofHumanFibrinFoam Uses of Human Fibrin Foam

Itisusedwiththrombinasahaemostatin i d ih h bi h i surgery,whenothermethodsusedtoarrest bleedinghavebeenunsuccessful.
Apieceisdippedinthrombinsolutionandapplied tothebleedingarea. Thecombinationofthrombinandthelargerough surfaceprovidedbythespongecausestheblood toclot. Thefoamcanbeleftinsitu,whereitwillbe absorbedbecauseitisentirelyofhumanorigin.
HumanNormalImmunoglobulin Injection
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Immuno or gamma globulin is obtained from the Immuno orgammaglobulinisobtainedfromthe globulinsfractionseparatedinstage3ofthefractionation ofplasma,ashadbeenshownearlier. Theionicstrengthsarecriticalandfurtherfractionationis doneasfollows:
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Theimmunoglobulinsaredissolvedinasuitable , y , solvent,usually0.8%sodiumchloridesolution, andapreservative,e.g.0.01%thiomersal,is added. Thesolutionissterilizedbyfiltration,packedin singledosecontainersandstoredat4to6 degreescentigrade,withprotectionfromlight. d ti d ith t ti f li ht Normallypoolsofnotlessthan1500donations areusedtoensureasatisfactoryrepresentationof are used to ensure a satisfactory representation of thevarioustypesofadultantibodies. However as in the preparation of antivaccinia and However,asinthepreparationofantivacciniaand antitetanusimmunoglobulins,whichisobtained y , fromthebloodofrecentlyimmunizeddonors,the poolsofbloodcanbesmaller.
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Usedtopreventorattenuatediseasessuchas

UsesofImmunoglobulins Uses of Immunoglobulins
Measles Rubella infectioushepatitis hepatitisB hepatitis B Chickenpox Hypogammaglobulinaemia(deficiencyingammaglobulins)
Itisusedtopreparespecificimmunoglobulinssuchas:
HumanAntiVacciniaImmunoglobulin forsmallpox Human AntiTetanus Immunoglobulin HumanAnti TetanusImmunoglobulin HumanAntiDImmunoglobulin usedtosuppresssensitizationof RhvemotherstotheRh(D)antigen(Rh+veinfant) A ti HBs I AntiHB Immunoglobulin thi i till d i l b li thisisstillunderinvestigation.Itisan ti ti It i immunoglobulinforHepatitisBsurfaceantigen.
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QUALITYCONTROL OFBLOODPRODUCTS OF BLOOD PRODUCTS
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QUALITYCONTROLOF BLOODPRODUCTS BLOOD PRODUCTS
STANDARDS
LABELLING
IDENTIFICATION
WHOLEBLOOD LABELLING DRIEDPLASMA PROTEINFRACTION LABELLING
STERILITYAND STERILITY AND PYROGENS
SOLUBILITY
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Whydoqualitycontrolonblood products?
Alth Althoughallthebloodproductscansavelife,manyare h ll th bl d d t lif dangerous. If,forexample,blood,plasma,orserumbecome If for example blood plasma or serum become heavilycontaminatedwithmicroorganisms,orthe containerorsolventisnotpyrogenfree,ora bactericideisaddedtoapreparationgiveninlarge b t i id i dd d t ti i i l volumesintravenously therisktothepatientis considerable. Theofficialstandardsandlabelingaredesignedto reducethehazardstoaminimum,andqualitycontrol ofbloodproductsshouldbedoneinaccordancewith f bl d d h ld b d i d ih them.
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Identification
A ll bl d Asallbloodproductscontainproteins,thestandard d t t i t i th t d d methodusedinproteinidentificationareoftenapplicable. Precipitationtestswithspecificantiseraareusedtoshow p p thatonlyhumanserumproteinsarepresentindried serum,driedplasma,theplasmaproteinfractions, fibrinogen,thrombinandimmunoglobulin. fibrinogen thrombin and immunoglobulin Thecharacteristicmobilitiesofbloodproteinsinan electrophoreticfieldareasensitivemeansofidentifying fibrinogen,immunoglobulin,andtheplasmaprotein fib i i l b li d th l t i fraction.Forexample,intheplasmaproteinfraction there mustnotbelessthan85%oftheproteinhavingthe mobilityofalbuminandnotmorethan1%ofgamma globulin.
Proteinscanbeidentifiedbytheirsedimentationratein anultra centrifuge,whichisasuitablemethodfor an ultracentrifuge which is a suitable method for identifyingandquantifyingthedifferenttypesof g gammaglobulins. g Differencesinclottingbehaviouraresimplerbutuseful characteristics.Plasmaclotswhencalciumchlorideis added,butserumdoesnot.Fibrinogenisidentifiedby theclottingthatoccurswhenthrombinisadded,and thrombinbythesameresultwhenitismixedwith th bi b th lt h it i i d ith plasma. The determination of blood groups ABO of plasma Thedeterminationofbloodgroups ABOofplasma andcellsandRhofcells,isanidentificationtestfor wholeblood,whilethedescriptionsofthelatterandof whole blood, while the descriptions of the latter and of concentratedredbloodcorpusclesareaidstoidentify andsafeguardsagainsttheuseofanunsafeproduct.
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SterilityandPyrogens Sterility and Pyrogens

Allbloodproductsmustcomplywiththe y officialtestsforsterility,andthose preparations(i.e.immunoglobulinsandthe plasmaproteinfractions)thatareexposedto plasma protein fractions) that are exposed to specialriskofcontaminationwithpyrogens duetolengthyprocessingmustalsopassthe due to lengthy processing must also pass the testforpyrogens
65
Solubility
Completesolubilityinanappropriatevolume p oftheusualsolvent,sometimesinaspecified time,isrequiredforallsolidpreparations exceptfibrinfoam. except fibrin foam Itindicatesthattheproteinconstituentshave notdeteriorated.
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Assays
ForwholebloodandconcentratedRBCstheassayisa determinationofthehaemoglobinvalue. determination of the haemoglobin value. Fortheremainingproducts,exceptfibrinfoam(whichhas noassay)andthrombin,theproteinconstituentis determinedchemically. determined chemically Inthrombintheremustbeaminimumnumberofclotting dosespermg,aclottingdosebeingtheamountof p g, g g thrombinrequiredtoclot1mlof0.1%fibrinogeninsaline bufferedat7.2to7.3in15secondsat37degrees centigrade. DeterminationsofNaandKionsinplasmaproteinfraction ensuresthatthelevelarenothighenoughtodisturbthe electrolytebalanceoftherecipient. l t l t b l f th i i t Anassayforsodiumcitrateinthesameproductprevents toxiceffectsfromexcessofthissalt.
LabellingforWholeBlood Labelling for Whole Blood

NameofPreparation ABO Group ABOGroup Rhgroupandnatureofantiserausedfortesting TotalVolume;proportionofblood;natureandpercentageof anticoagulantandanyothermaterialintroduced DateofDonation Expiry date Expirydate StorageConditions y g Astatementthatthecontentsmustnotbeusedifthereisanysign ofdeterioration Anindicationbywhichthehistoryofthepreparationcanbetraced
LabellingofDriedPlasmaProtein Fraction
NameofPreparation
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Volumeofwaterofinjectionnecessaryforreconstitution Volume of water of injection necessary for reconstitution Totalamountofproteininreconstitutedsolution Concentrationsofpotassium,sodiumandcitrateions Namesandconcentrationsofstabilizingagentsorotheraddedsubstances N d i f bili i h dd d b ExpiryDate StorageConditions Astatementthatthecontentsmustnotbeusedif,afteraddingwater,agel formsorsolutionisincomplete An indication by which the history of the preparation can be traced Anindicationbywhichthehistoryofthepreparationcanbetraced Aninstructiontodiscardthereconstitutedsolutionifnotusedwithinthree hours
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PLASMASUBSTITUTES
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Theneedforplasmasubstitutes? The need for plasma substitutes?

Thelimitedsuppliesofplasma,thecostof p producingthedriedformandtheriskof g transmittingserumhepatitisstimulated attemptstofindsubstitutesofnon human attempts to find substitutes of nonhuman originthatcouldbeusedtorestoretheblood volumetemporarilywhiletherecipient volume temporarily while the recipient replacedthelostprotein
71
PropertiesofanIdealPlasma Substitute
Thesamecolloidalosmoticpressureaswholeblood. Th ll id l ti h l bl d Aviscositysimilartothatofplasma. Amolecularweightsuchthatthemoleculesdonoteasilydiffuse throughthecapillarywalls. 4. Afairlylowrateofexcretionordestructionbythebody. 5. Eventual and complete elimination from the body. Eventualandcompleteeliminationfromthebody. 6. Freedomfromtoxicity,e.g.noimpairmentofrenalfunction. 7. Freedomfromantigenicity,pyrogenicity,andconfusingeffectson importanttestssuchasbloodgroupingandtheerythrocyte important tests such as blood grouping and the erythrocyte sedimentationrate. 8. Isotonicity,insolution,equaltothatofbloodplasma. 9. Highstabilityinliquidformatnormalandsterilizingtemperatures f andduringtransportandstorage. 10. Easeofpreparation,readyavailabilityandlowcost.
1. 1 2. 3.
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GumSaline
Plasma Substitutes
Dextran
Polyvinyl P l i l pyrrolidone
73
GumSaline Gum Saline

ThisisasynonymforInjectionofSodiumChloride hi i f j i f S di Chl id andAcacia,whichwasofficialinthe1932British Pharmacopoeia. Ph i IntheFirstWorldWarBaylissexperimentedwith solublestarch,dextrin,andgelatinasplasma substitutesandfinallyused6%acaciain0.9% SodiumChloridesolution. S di Chl id l i Itwastransfusedextensivelyuntilsignsofliver dysfunctiondisclosedthatthegumwasnot metabolizedbutstoredinvariousorgans.
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Polyvinylpyrrolidone
IntheSecondWorldWar,theGermans y introducedasyntheticcolloid, polyvinylpyrolidone,forthetreatmentof shock. shock Itwasmarketedinthe1950sbutwaslater withdrawnbecauseofsuspected h b f carcinogenicity.
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Dextran
Todatethisisthemostsatisfactoryplasma substitute. Itisapolysaccharideproducedwhenthe bacteriumLeuconostocmesenteroidesis bacterium Leuconostoc mesenteroides is growninasucrosecontainingmedium. Inthesugarindustryitoccursasaslimethat clogspipesandfiltersandinterfereswith clogs pipes and filters and interferes with crystallization.
Theorganismsecretesanenzymethatconverts sucrosetodextranaccordingtothefollowing d d h f ll equation
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Differentstrainsproducedextransoftwomain groups groups

Long,practicallyunbranchedchainsofglucoseunits joinedby16glucosidiclinkages. joined by 16 glucosidic linkages Highlybranchedpolymersconsistingofshortchains of16unitsjoinedby14and13linkagesto of 16 units joined by 14 and 13 linkages to branches.
Branched chains are more likely to give rise to Branchedchainsaremorelikelytogiveriseto allergicreactionswheninjected,andin dextransusedforplasmasubstitutesthe dextrans used for plasma substitutes the linkagesshouldbealmostentirelyofthe16 type.Thisisachievedbychoosingasuitable hi i hi d b h i i bl speciallydevelopedstrainoftheorganism thatproducesdextraninwhichabout95%of thelinkagesare1 6. the linkages are 16.
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Production
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Productioninvolveslaboratoryculturefollowedbygrowthin seedtanksinthefactoryandthenin4500cubicdm seed tanks in the factory and then in 4500 cubic dm fermenters.(similarprocesstoantibioticproduction). Becausesynthesisoftheenzymeanditsactiononthe sucrosearerapid,thehighdegreeofasepsismaintainedin f antibioticfermentationisnotnecessaryhere. Also as the process is inhibited by aeration there is no need Also,astheprocessisinhibitedbyaeration,thereisnoneed foracostlysupplyofsterileair. Anotherspecialfeatureistheneedtopreventthehydrolysis ofsucrosetoglucoseandfructoseduringsterilizationofthe f t l df t d i t ili ti f th culturemedia.Ifthisoccurs,dextranwillnotbeproduced becauseinnaturetheconversiondoesnotinvolveinversion butisastraighttransglycosidation.Preventivemeasures includeadjustmentofthemediatoneutralpHbefore sterilization,andtheavoidanceofoverheating. , g
Whenmaximumconversiontodextranhasbeenobtained itisprecipitatedbyaddingasuitableorganicsolvent. it is precipitated by adding a suitable organic solvent Naturaldextranconsistsofchainsofapproximately 200,000glucoseunitswithmolecularweightsuptoabout 200,000 glucose units with molecular weights up to about 50million. e y a ge o ecu es e t ose t a o ecu a e g t Verylargemoleculesi.e.thosewithamolecularweight aboveabout250,000haveseriousdrawbacks:
Theyyieldveryviscoussolutionsthataredifficultto administer. Theymaycauserenaldamageandallergicreactions. Theyinterferewithbloodmatchingandsedimentationtests h f h bl d h d d bycausingrouleauxformation.Rouleauxareaggregatesofred cellsthatresemblepilesofplates. cells that resemble piles of plates. Theyproducecolloidalosmoticpressuresthatarelowerthan thoseofsmallmolecules.
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Thereforetoproduceamaterialsuitablefor medicaluseitisnecessarytoreducethesizeof p thenaturalmolecules.Thiscanbeaccomplished inseveralways: A id H d l i (th AcidHydrolysis(themethodmostwidelyused) th d t id l d) ThermalDegradation g UltrasonicDisintegration S di SeedingtheFermenter h F
81
AcidHydrolysis The dextran is adjusted to a pH of 2 a is heated at 90 degrees ThedextranisadjustedtoapHof2aisheatedat90degrees centigrade. Ashydrolysisproceeds,thepreparationbecomeslessviscous andthereactionisstoppedattherequiredviscosity. Acidhydrolysisisthemethodmostoftenused.Thehydrolyzed productcontainsmoleculesrangingfrom10,000to1millionin product contains molecules ranging from 10 000 to 1 million in molecularweight. ThermalDegradation Asolutionofdextranisheatedunderpressureat160degrees centigradeinthepresenceofsodiumsulphite,toprevent centigrade in the presence of sodium sulphite to prevent oxidativedeterioration,andcalciumcarbonate,toneutralize acidity. Themethodisslowerthanacidhydrolysisbuttheyieldofthe preferredmoleculesisbetterandfewerreducinggroupsare p produced.
http://www.pharmaxchange.info Page 42 UltrasonicDisintegration Bombardmentwithultrasonicwavessplitsthemolecules Bombardment with ultrasonic waves splits the molecules intofragmentsofapproximatelythesamesizeandthe p productisclinicallyacceptable,unlikethematerialfromthe y p , previouslymentionedmethods,whichrequires considerablefractionation. Unfortunately,thistechniqueismuchmoreexpensiveto use.
SeedingtheFermenter Ifalowmolecularweightdextranisaddedintheculture mediumbeforefermentation,theorganismwilluseitasa templateonwhichtobuildmoreglucosemolecules. t l t hi h t b ild l l l Theaveragemolecularweightoftheproductismuchlower thanifnotemplatedextranisprovided. than if no template dextran is provided
Theverysmallmolecules,i.e.thoseofbelowa molecularweightofabout60,000alsohave disadvantages

Theyarerapidlyexcretedintheurine. They pass into the tissue fluids causing an adverse Theypassintothetissuefluidscausinganadverse osmoticpressure
Th f Therefore,theproductshouldcontainthe th d t h ld t i th minimumofmoleculesofmolecularweightless than60,000(i.e.thenumberofmoleculeswitha g , molecularweightoflessthan60,000shouldbe kepttoaminimum)

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Tosummarize To summarize
Thissuggeststhattheidealfractionshouldhavea j y g majorityofmoleculeswithintherangeof 100,000to250,000withabiastowardsthelower end.

TheAmericantypeofdextranhasanaverage molecularweightofabout75,000. Theaimsaretorestorecolloidalosmotic The aims are to restore colloidal osmotic pressurequicklyandtoensurefairlyrapid eliminationoftheforeigncolloidfromthebody. elimination of the foreign colloid from the body Thelatteradvantage,combinedwitha reductioninrouleauxformationisconsidered g g g tooutweighthedisadvantageofthehigh dosagenecessarytocompensateforexcretion losses. losses
86
TheBritishdextranofthe1963BP(Dextran150 injection)hasanaveragemolecularweightof injection) has an average molecular weight of about150,000andproducedamoreprolonged effectduetothepreponderanceoflarger effect due to the preponderance of larger moleculesthatarenotlostfromthebloodvessels. Thispreparationcausedrouleauxformation,a This preparation caused rouleaux formation a conditionknownassludgingbecauseitslowsthe flowofbloodinthecapillariesandpost capillary flow of blood in the capillaries and postcapillary veins. To overcome the problem which is less evident Toovercometheproblem,whichislessevident withlowermolecularweightfractions,the1968BP hasreplacedDextran150withDextran110,which has replaced Dextran 150 with Dextran 110 which hasanaveragemolecularweightof110,000.
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Toobtaintheclinicalrangesofmolecularweight, theneutralizedhydrolysateissubjectedtoalong the neutralized hydrolysate is subjected to a long processoffractionalprecipitation. Awater miscibleorganicsolvent,inwhichthe A watermiscible organic solvent, in which the polysaccharideisinsoluble(e.g.acetoneor alcohol)isaddedunderverycarefullycontrolled conditionsandtherequiredfractionisgradually separatedbyrepeatedretreatmentofeitherthe precipitateorsupernatantfluid. precipitate or supernatant fluid Costdecidesthenarrownessofthefractionfinally accepted. accepted Inallsamples,becauseofentrainmentofone fractionwithanother,therearesmallproportions fraction with another there are small proportions ofverylargeandverysmallmolecules.
Theselectedfractionstillrequiresconsiderablepurificationto remove remove ReducingSugars byfurthersolventprecipitation.Themain contaminantisfructose,thebyproductoffermentation. Fractionationsolvents byevaporationunderreduced pressure. Inorganic salts by demineralization in a mixed bed ion Inorganicsalts bydemineralizationinamixedbedion exchanger.Itisparticularlyimportanttoremovephosphates becausetheycauseprecipitationduringsterilizationand storage. storage Colour byadsorptionontoactivatedcharcoal. Pyrogens by adsorption on to asbestos, or cellulose Pyrogens byadsorptionontoasbestos,orcellulose derivatives. Microorganisms byfiltration.Betweeneachtreatmentthe preparationispassedthroughafibrouspadandjustbefore ti i d th h fib d dj tb f bottlingamembranefilterisused.
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89
Thesolutionisdilutedtoaconcentrationof5% ineither5%DextroseInjectionorSodium ChlorideInjection,packedinsulphurtreated sodalimebottles,andclosedwithlacquered rubberplugs. rubber plugs. Finally,itissterilized,usuallybyheatinginan autoclave. l
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ControlforDextran Control for Dextran

Thefollowingtestsfromtheofficialspecification h f ll i f h ffi i l ifi i forDextran110Injectionillustratethe precautionstakentoconfirmthattheproductis ti t k t fi th t th d ti suitableasaplasmasubstitute. Chemicaltechniqueslimittheamountoflead, acetoneandalcohol,reducingsugars,nitrogen (fromculturemedium)andacidandalkali. (f l di ) d id d lk li Biologicalmethodsshowthatthepreparationis notpyrogenic,issterile,andisfreefromproteins thatcouldcauseanaphylaxis.
Thedextrancontentisdeterminedbypolarimetry andtherearelimittestsforsmallandlarge and there are limit tests for small and large molecules.
The former involves the injection into rabbits; the urine Theformerinvolvestheinjectionintorabbits;theurine collectedthroughoutthesucceeding48hoursmustnot contain more than 30% of the injected dose. (as small containmorethan30%oftheinjecteddose.(assmall moleculesareexcretedintheurine). Thelatternecessitatesprecipitationofthetop10%ofthe fractionwithalcoholanddeterminingitsintrinsic viscosity;thismustnotbegreaterthan0.4%whichis equivalenttoanaveragemolecularweightofabout equivalent to an average molecular weight of about 240,000. The intrinsic viscosity of the fraction as a whole is also Theintrinsicviscosityofthefractionasawholeisalso foundandmustindicateanaveragemolecularweightof about110,000.
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Dextran40Injection Dextran 40 Injection

Anumberofconditions,includingsevereburns,crush injuriesandacuteperitonitis,areaccompaniedbyasevere injuries and acute peritonitis are accompanied by a severe degreeofsludgingintheblood. ThiscanbereducedbytheadministrationofDextran40 injectionwhich,becauseitcontainspolymersoflow molecularweight,lowersplasmaviscosityandimproves capillaryflow. capillary flow Bothchangesreducecellaggregationandthisinturn, p furtherimprovestheflow. Acrudedextranoflowmolecularweightismanufactured byincludingverysmalltemplatemoleculesinthe fermentationmedium. fermentation medium Thenfractionationisusedtoproducetheclinicalmaterial whichhasanaveragemolecularweightof40,000. which has an average molecular weight of 40 000
AbsorbableHaemostats bso bab e ae ostats
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AbsorbableHaemostats Absorbable Haemostats

Thesematerialsareusedtocontrolbleeding y whenitcannotbecheckedbymore conventionalmeans. They are gradually absorbed by the tissue and Theyaregraduallyabsorbedbythetissueand, therefore,ifusedduringsurgerycanbeleftin thebodywhentheincisionisclosed,andif h b h h l f appliedtoasurfacewoundneednotbe removedwhenthedressingischanged.
Therearefourimportanttypes: p yp
HumanFibrinFoam(whichhasbeencovered earlier) GelatinSponge O idi d ll l Oxidizedcellulose CalciumAlginate
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AbsorbableGelatinSponge Absorbable Gelatin Sponge

Thisispreparedbyaddingasmallpercentageof formaldehydetoawarmsolutionofgoodqualitygelatin.
Whi h i th Whichisthenwhiskedintoafoamandfreezedried. hi k d i t f df di d
Theporousproductiscutintopiecesofsuitablesizeand sterilizedbydryheatat140degreescentigrade. sterilized by dry heat at 140 degrees centigrade
97
Itismarketedaswhileornearwhite,rectangular,very p porouspiecesthatareextremelylightandhaveapapery p y g p p y feel. Itabsorbsmanytimesitsownweightofbloodandthe officialstandardforabsorbencyrequiresabsorptionof ffi i l d df b b i b i f notlessthanthirtytimesitsweightofwater. When pressed tightly on to a bleeding area blood is Whenpressedtightlyontoableedingarea,bloodis takenupandclottingisencouragedbythelargerough surfacewhichcausesplateletdisintegration. Thespongealsoactsasaplugbystickingtothe underlyingtissuesandmechanicallysupportingtheclot overtheoozingvessels. over the oozing vessels Sometimesitispreviouslysoakedinsaline,antibioticor , p thrombinsolution,whenitmustbepressedtoremove airandexcessliquidbeforeapplication.
98
Sincesomeorganismsliquefygelatinitshould notbeusedinsepticwounds;norisitsuitable not be used in septic wounds nor is it suitable forarrestinghaemorrhagefromlargevessels. Absorptionoccursinfourtosixweeks. p g g Thespongeisnonantigenicandtissuereactions havebeenmild. The standards include a test for digestibility in Thestandardsincludeatestfordigestibilityin acidpepsinsolution. U l Unlessaseptichandlinghasbeenperfectitis ti h dli h b f t it i advisabletodiscardanyunusedpartofthe contentsofacontainer.Resterilizationshould f i R ili i h ld notbeattempted.
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OxidizedCellulose Oxidized Cellulose

C ll l Cellulosecanbeconvertedintopolyanhydroglucuronic b t di t l h d l i acid,anabsorbablehaemostaticmaterial,byoxidationwith nitrogendioxide. Earlyproductsweremadefromcottonintheformofwool, lint,andparticularly,gauze. More recently a knitted material in which cotton is Morerecentlyaknittedmaterial,inwhichcottonis replacedbyregeneratedcellulose(viscoserayon)hasbeen introduced.Fibresofthelatterareofuniformdiameterand thesizedistributionofthepolymersislesswideandmore th i di t ib ti f th l i l id d constantthanincotton.Asaresulttheoxidationcanbe carriedoutinamorereproduciblemannerandtheproduct isclaimedtoshowlessvariationintheabsorptiontime, haemostaticefficiency,andtissuereactivity.
Oxidationiscarriedoutonthefabricated dressinginvolvesconversionofabout20%of theprimaryalcoholgroupstocarboxyls. Gaseoussterilization,oftenbyformaldehyde,is usedbecauseheatcausesseriousdeterioration. used because heat causes serious deterioration Thematerialhastheappearanceoftheoriginal dressingexceptthatitmaybelesswhitein colour. Ithasafaintodourandacidtaste. Incontactwithblooditturnsdarkbrownand swellstoagelatinouscoagulum.
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101
Itshaemostaticactivitymaybepartlyduetochemical reactionbetweenthepolyuronicacidandhaemoglobinor reaction between the polyuronic acid and haemoglobin or otherbloodproteins. g Butthefabricalsoactsasascaffoldingforclotformation andaplugatcutendsofthevessels. Itismoreeffectifuseddry. Smallpiecesareabsorbedin2to7daysbutverylarge amountsmaytakeseveralweeks. It inactivates thrombin unless previously neutralized with Itinactivatesthrombin,unlesspreviouslyneutralizedwith sodiumbicarbonateinjection,andisincompatiblewith penicillin. Itshouldnotbeusedinbonesurgerybecausecallus formationisinhibited Unusedpiecesfromanopenedcontainershouldbe d f d h ld b discarded.
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CalciumAlginate Calcium Alginate

Thisisderivedfromalginicacid,acolloidal hi i d i d f l i i id ll id l substanceobtainedfromseaweedsLaminaria digitataandLaminariacloustoniwhichgrowoff di it t d L i i l t i hi h ff thescottishandIrishcoasts. Alginicacidisapolyuronidebuiltupfromd mannuronicacidunits. Itscarboxylgroupsreactwiththemetallicionsto formalginatesand,sincetheparentacidis unstable,thewatersolublesodiumsaltisusedas thesourceofotheralginates.
Ifionizedcalciumsaltisaddedtosodium alginatesolutioninstantaneousprecipitationof calciumalginateoccurs,asensitivereaction thatcanbeusedforpreparingfoams,fabrics andotherphysicalforms. and other physical forms. Thesecanbesterilizedbyautoclavingordry heat.
104
Calciumalginatedressingshaveamarked haemostaticeffectthatisprobablyduemainly tomechanicalpressure. Aslongastwelveweeksmaybenecessaryfor completeabsorptionand,althoughitispossible complete absorption and although it is possible tomakeproductsthatareabsorbedinabout10 days,byincludingasmallproportionofsodium d b i l di ll i f di alginate,thetendencyistorestricttheuseof alginatestothearrestofexternalbleeding,e.g. o su g ca c s o s, toot soc ets, a d s tes fromsurgicalincisions,toothsockets,andsites fromwhichgraftshavebeentaken.
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Atransparentprotectivefilmcanbemadein situoveraburn,woundorincisionbyapplyinga situ over a burn wound or incision by applying a solutionofsodiumalginateandthenspraying withcalciumchloridesolution.Thisfilmis ith l i hl id l ti Thi fil i impervioustowaterbutpermeabletowater vapour. Alginatedressingscanberemoved,ifnecessary, bywashingwithasolutionofsodiumsalt,e.g. 5%sodiumcitrate,whichreversesthereaction shownintheequationabove. Theyarecompatiblewithpenicillinandcanbe They are compatible with penicillin and can be resterilizedifnecessary.
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REFERENCES
Mainreference CooperandGunn s TutorialPharmacy Cooper and Gunns Tutorial Pharmacy Pleaseaddpointsfrom Bentley sTextBookofPharmaceuticsbyE.A. Bentleys Text Book of Pharmaceutics by E A Rawlin
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http://www.pharmaxchange.

BLOOD BLOOD AND BLOODPRODUCTS

BLOOD (WholeHuman Blood)

Plasma Pl ( DriedHumanPlasma) ( HumanPlasmaProteinFraction) ( DriedHumanPlasmaProteinFraction)

RedBlood R d Bl d Corpuscles (Concentrated humanRBCs)

Serum (DriedHuman Serum)

Immunoglobulin GammaGlobulin (HumanNormalImmunoglobulin Injection)

Fibrinogen (Human Fibrinogen) Fib i )

FibrinClot (HumanFibrin Foam)

WHOLEHUMANBLOOD WHOLE HUMAN BLOOD

ConditionsforBeingaDonor Conditions for Being a Donor

Collectionoftheblood Collection of the blood

Thisbloodisputintoasterilecontainercontainingananticoagulantsolutionandthe bottleisgentlyshakentoensurethatbloodandanticoagulantarewellmixed,thus preventingtheformationofsmallfibrinclots.

EquipmentUsedfortheCollection Equipment Used for the Collection

BloodClotting Blood Clotting

Thecitratepreventsclottingbybindingthe calciumionsasunionizedcalciumcitrate,thus calcium ions as unionized calcium citrate thus preventingavitalstepofclotting.

WhyAcidCitrateandnotNormalCitrate?? Why Acid Citrate and not Normal Citrate??

WhyaddDextrose? Why add Dextrose?

DISODIUMEDETATE DISODIUM EDETATE

TESTINGOFWHOLEBLOOD TESTING OF WHOLE BLOOD

BLOODGROUPS BLOOD GROUPS

1)ABOSystem 1) ABO System

Allgroups. (Universal Donor

Haemolytic disease in a new born: Haemolyticdiseaseinanewborn:

Thecellsarematchedwiththerecipientsplasmaandmaythenbe p p y mixedwithmatchedcellsfromotherbottles. Thehaemoglobincontentmustnotbelessthan15.5%

DRIEDHUMANPLASMA DRIED HUMAN PLASMA

DRIEDHUMANPLASMA DRIED HUMAN PLASMA

Advan ntageso ofDriedPlasma a

THUS,UNDERSUITABLECIRCUMSTANCES,DRIEDPLASMACANBEUSEDASA http://www.pharmaxchange.info SUBSTITUTEFORWHOLEBLOOD.

Then400mlquantitiesaredispensedintobottlesandsubjectedtofreezedrying. q p j y g Generalaspectsoffreezedryingarefollowedwithspecialfeaturesoftheplasma process PreliminaryFreezing Pi PrimaryDrying D i Secondarydrying

PreliminaryFreezing Preliminary Freezing

PrimaryDrying Primary Drying

SecondaryDrying Secondary Drying

DRIEDHUMANSERUM DRIED HUMAN SERUM

Plasmaisusuallyobtainedfrombloodthatisoutofdate,i.e.hasbeenavailable aswholebloodfor21days. y Byconvertingbloodintoserumthisperiodinreserveislostand,therefore,much lessbloodisusedforserumproduction Its,storageandusearethesameasfordriedplasma.

LIQUIDPLASMAANDSERUM LIQUID PLASMA AND SERUM

THEFRACTIONATIONOFPLASMA THE FRACTIONATION OF PLASMA

TechniquesofProteinSeparation Techniques of Protein Separation

Ontheotherhand,itisnecessarytokeepthe temperatureverylow(0to5degreescentigrade)to preventsolventdenaturationofproteins.

EtherFractionationofPlasma Ether Fractionation of Plasma

HumanPlasmaProteinFraction Human Plasma Protein Fraction

DriedHumanPlasmaProteinFraction Dried Human Plasma Protein Fraction

HumanFibrinogen Human Fibrinogen

UseofHumanFibrinogen Use of Human Fibrinogen

HumanThrombin Human Thrombin

UsesofHumanThrombin Uses of Human Thrombin

HumanFibrinFoam Human Fibrin Foam

UsesofHumanFibrinFoam Uses of Human Fibrin Foam

UsesofImmunoglobulins Uses of Immunoglobulins

Measles Rubella infectioushepatitis hepatitisB hepatitis B Chickenpox Hypogammaglobulinaemia(deficiencyingammaglobulins)

QUALITYCONTROL OFBLOODPRODUCTS OF BLOOD PRODUCTS

QUALITYCONTROLOF BLOODPRODUCTS BLOOD PRODUCTS

WHOLEBLOOD LABELLING DRIEDPLASMA PROTEINFRACTION LABELLING

STERILITYAND STERILITY AND PYROGENS

SterilityandPyrogens Sterility and Pyrogens

LabellingforWholeBlood Labelling for Whole Blood

Theneedforplasmasubstitutes? The need for plasma substitutes?

GumSaline Gum Saline

Theorganismsecretesanenzymethatconverts sucrosetodextranaccordingtothefollowing d d h f ll equation

Differentstrainsproducedextransoftwomain groups groups

Thereforetoproduceamaterialsuitablefor medicaluseitisnecessarytoreducethesizeof p thenaturalmolecules.Thiscanbeaccomplished inseveralways: A id H d l i (th AcidHydrolysis(themethodmostwidelyused) th d t id l d) ThermalDegradation g UltrasonicDisintegration S di SeedingtheFermenter h F

Theverysmallmolecules,i.e.thoseofbelowa molecularweightofabout60,000alsohave disadvantages

Th f Therefore,theproductshouldcontainthe th d t h ld t i th minimumofmoleculesofmolecularweightless than60,000(i.e.thenumberofmoleculeswitha g , molecularweightoflessthan60,000shouldbe kepttoaminimum)