Quality ControlQuality ControlQuality AssurenceQuality Assurence

ofofImmunohematology of Immunohematology of

ReagentsReagentsSpeaker Prof. A.PourazarSpeaker Prof. A.Pourazar

Isfahan IRANIsfahan IRAN

Quality Assurance In Blood Bank Quality Assurance In Blood Bank

nn ActivitiesActivities thatthat provideprovide confidenceconfidence inin thethesystemssystems whichwhich affectaffect thethe qualityquality ofof blood/blood/bloodblood productsproducts areare workingworking asas expectedexpected..

nn QualityQuality cancan bebe measuredmeasured ifif needneed hashas beenbeendefineddefined

continuecontinue

nn Quality Quality ––need can be specified / need can be specified / requirement can be setrequirement can be set

nn Standards to be laid down by the Standards to be laid down by the authority e.g. FDAauthority e.g. FDA

nn Quality surveillance is put into practice Quality surveillance is put into practice the authoritythe authority

ContinueContinuenn Inspection should be carried out once every year to Inspection should be carried out once every year to

check.check.nn External Quality Assessment (EQA) program on External Quality Assessment (EQA) program on

regional and / or national level must be exercised.regional and / or national level must be exercised.nn Internal Quality Control (IQC) by continuous Internal Quality Control (IQC) by continuous

monitoring all aspects of blood banking should be monitoring all aspects of blood banking should be set.set.

nn Procedural manual and Standard Operating Precedure Procedural manual and Standard Operating Precedure (SOP)(SOP)

Reagent Quality ControlReagent Quality Control

nn On arrival, for titer and avidity of On arrival, for titer and avidity of antianti--seraserann Daily basis : AntiDaily basis : Anti--A, AntiA, Anti--B and AntiB and Anti--AB AB

with know A/B red cells respectively for with know A/B red cells respectively for reactivity.reactivity.

nn Anti Anti –– D with know Rh.D positive red cell D with know Rh.D positive red cell for reactivity and Rh.D negative red cells for reactivity and Rh.D negative red cells specificityspecificity

ContinueContinue

nn AntiAnti--Human Globulin reagent with AntiHuman Globulin reagent with Anti--D D coated red cells (Check coated red cells (Check –– Cells) for reactivity Cells) for reactivity and with uncoated red cells for specificity.and with uncoated red cells for specificity.

nn Other reagent : like enzymes, albumin, Other reagent : like enzymes, albumin, enhancement in reactivity of IgG antienhancement in reactivity of IgG anti--DD

nn And use of enzyme of reduction in reactivity And use of enzyme of reduction in reactivity of antiof anti--Duffy or antibodies to MNSs blood Duffy or antibodies to MNSs blood group and so on …group and so on …

Standard titer and avidity of ABO Standard titer and avidity of ABO reagentreagent

Antibody titer with cells titer avidity secondAntibody titer with cells titer avidity secondAntiAnti-- A AA A1 256 101 256 10AntiAnti--A AA A2 128 202 128 20AntiAnti-- A AA A22B B 64 3064 30

AntiAnti--B B B B 256 10256 10AntiAnti--B AB A11B B 128 20128 20

AntiAnti--AB AAB A1 128 10 1 128 10 AntiAnti--AB B AB B 64 1064 10

International Standards for the titer and avidityInternational Standards for the titer and avidity

Standard Titer and avidity of AntiStandard Titer and avidity of Anti--AA1 1 and Antiand Anti--H lectinH lectin

Antibody titer with cells titer avidity scondAntibody titer with cells titer avidity scondAntiAnti-- AA1 1 AA1 16 201 16 20AntiAnti--H O H O 8 8 --

* * Anti Anti –– AA1 1 extracted form Dolichus biflorus seed.extracted form Dolichus biflorus seed.nn AntiAnti--H extracted from Ulex europeous seed.H extracted from Ulex europeous seed.nn AntiAnti--AA1 1 give not agglutination reaction give not agglutination reaction with O, B, Awith O, B, A2 2 , A, A22B , cellsB , cells

Avidity test Avidity test

nn Speed and strength of agglutination reaction Speed and strength of agglutination reaction are tremed as avidity.are tremed as avidity.

nn The test is done by mixing on drop of The test is done by mixing on drop of antiserum with one drop of EDTA whole antiserum with one drop of EDTA whole blood on a slide and rocking gently.blood on a slide and rocking gently.

nn with the help of a stop watch, the time for the with the help of a stop watch, the time for the first appearance of agglutination is recorded.first appearance of agglutination is recorded.

PrecantionPrecantion

nn Prozone is very often observed in very high Prozone is very often observed in very high titer of antibody and simultaneous presence of titer of antibody and simultaneous presence of incomplete type of antibodies.incomplete type of antibodies.

nn This could be improved by suitable dilution This could be improved by suitable dilution and made it standard.and made it standard.

nn Quality control is necessary for all type of Quality control is necessary for all type of antisera.antisera.

Preservation and IdentificationPreservation and Identification

nn Strong bacteriostatic agent like sodium azide in final Strong bacteriostatic agent like sodium azide in final concentration of concentration of 1 1 : : 1000 1000 (i.e.(i.e.11ml of ml of 1010% % solution in solution in 100100ml of antisera) is added.ml of antisera) is added.

nn The colouring agent like The colouring agent like methylene blue for Antimethylene blue for Anti-- A A acriflavin for Antiacriflavin for Anti--B (zaferan in IRAN)B (zaferan in IRAN)eosin for Antieosin for Anti--ABAB

In concentration of In concentration of 1 1 : : 5000 5000 are also used (i.e.are also used (i.e.22ml of ml of 11% % solution in solution in 100100ml of antiml of anti--sera)sera)

Quality Control for All reagent Quality Control for All reagent

nn Like any other typing reagent, it is important Like any other typing reagent, it is important that the final processed anti sera should be that the final processed anti sera should be sterilizedsterilized

nn Seitz filtration and sodium Seitz filtration and sodium azideazide added as a added as a preservative preservative

nn This is particularly important if such sera are This is particularly important if such sera are to be distributed by post to for distances.to be distributed by post to for distances.

QC and Standardization of AntiQC and Standardization of Anti--D D reagentreagent

First stepFirst step

Antibody titer with cells saline titer coombs titerAntibody titer with cells saline titer coombs titer

Anti Anti –– D D+(Rho) D D+(Rho) 32 51232 512--10241024

nn All the negative tube should be tested by Anti All the negative tube should be tested by Anti –– Humam Humam ––GlobulinGlobulin

Second stepSecond step

nn Anti Anti –– RhRh((D) serum is accordingly tested with D) serum is accordingly tested with rr′′rr (*(*CcdeeCcdee), r), r′′′′r(r(ccdEeccdEe) and ) and RorRor ((ccDeeccDee) ) to to rule out the presence of antirule out the presence of anti--C and antiC and anti--EE

nn AntiAnti--D further tested with D further tested with 10 10 Rho (D) negative Rho (D) negative and and 20 20 or so Rho(D) positive of all ABO or so Rho(D) positive of all ABO groupsgroups

nn It should be checked with panel of cells to It should be checked with panel of cells to exclude the presence of unwanted exclude the presence of unwanted AbAb

QC and Standardization of AHGQC and Standardization of AHG

11-- Production of the reagentProduction of the reagentnn Animal immunization: Animal immunization: nn The most common animal used for the The most common animal used for the

production of AHG reagent is rabbit.production of AHG reagent is rabbit.nn Large scale production could be achieved by Large scale production could be achieved by

immunization sheep or goat immunization sheep or goat nn It is preferable to use young animal It is preferable to use young animal

Standardization of AHG Standardization of AHG

Preliminary test on AHGPreliminary test on AHGnn Preparation of sensitized O+ red cell (check Preparation of sensitized O+ red cell (check

cell) and non sensitized O+ red cell cell) and non sensitized O+ red cell nn Check cell:Check cell: O+ red cell + Anti O+ red cell + Anti –– D (IgG) D (IgG)

suspended in saline suspended in saline nn Everyday or every week we can prepareEveryday or every week we can preparenn Test :Test : AHG + check cell give + Reation AHG + check cell give + Reation AHG +AHG + non sensitized RBC givenon sensitized RBC give-- reation reation

Secondary test on AHG for QCSecondary test on AHG for QC

nn The standard of AntiThe standard of Anti--D is used as a source of D is used as a source of IgG antibodyIgG antibody

nn This AntiThis Anti--D should have a saline titration D should have a saline titration 11: : 32 32 or or 11::64 64 and coombs titration about and coombs titration about 11::512 512 or or 11::10241024

nn Test should be done at Test should be done at 3737CC°° for for 30 30 mins mins nn The procedure is in next tableThe procedure is in next table

Reaction of sensitized red cell with Reaction of sensitized red cell with AHGAHG

Dilution of AHG Dilution of AHG 1 2 4 8 16 32 64 128 256 512 10241 2 4 8 16 32 64 128 256 512 1024

O+ cell sensitizedO+ cell sensitizedwith Antiwith Anti--DD

11::1 1 + + + + + + 22+ + 33+ + 44+ + 44+ + 33+ + 22+ + 22+ ++ +11::2 2 + + + + 22+ + 44+ + 44+ + 44+ + 33+ + 22+ + + + + + + + 11::4 4 -- 22+ + 33+ + 44+ + 44+ + 44+ + 22+ + 22+ + + + + + --11::8 8 -- + + 22+ + 33+ + 44+ + 33+ + + + + + -- -- --11::16 16 -- -- + + 22+ + 33+ + 22+ + + + -- -- -- --11::32 32 -- -- + + + + 2 2 + + -- -- -- -- --

Non sensitized Non sensitized -- -- -- -- -- -- -- -- -- -- --O+,O+, A+, B+ cellsA+, B+ cells

Standardization of AHG by dilution of Standardization of AHG by dilution of AntiAnti--D (STD)D (STD)

Difution of Difution of 11::2 12 1::4 14 1::8 18 1::16 116 1::32 132 1::64 164 1::128 1128 1::256 1256 1::512512AntiAnti--DDNo ofNo of 1 2 3 4 5 6 7 8 9 1 2 3 4 5 6 7 8 9

tubetubeAdd O+Add O+ 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 dropdrop55% % RBC RBC

Incabate for Incabate for 30 30 min at min at 3737CC°° serofuge for serofuge for 30 30 second at second at 1000 1000 rpmrpmResult +Result +4 4 ++4 4 ++3 3 ++2 2 ++1 1 -- -- -- --

All the negative tube (All the negative tube (66--1010) washed ) washed 3 3 time with saline Add two drop AHG and time with saline Add two drop AHG and 30 30 second second serofuge at serofuge at 1000 1000 rpm rpm

Coombs Coombs Result +Result +4 4 ++3 3 ++2 2 ++11

Final result : saline titration Final result : saline titration 11::32 32 and coombs titration and coombs titration 11::512512

Use of AHGUse of AHG

nn In routine blood group immunohematology In routine blood group immunohematology reagent is used to detect reagent is used to detect

**The coating of antibody molecules on the red The coating of antibody molecules on the red cell incell in--vivo as in HDN and vivo as in HDN and

**autoimmune hemolytic anemia AIHA or autoimmune hemolytic anemia AIHA or **transfusion reaction due to blood group transfusion reaction due to blood group

incompatility incompatility Direct and Indirect coombs test or AHGDirect and Indirect coombs test or AHG-- test test

The reagent is used for detection of incomptete The reagent is used for detection of incomptete antibody in blood circulationantibody in blood circulation

**Immunization of pregnant lady Immunization of pregnant lady **Autoimmune DiseaseAutoimmune Disease**Transfusion reaction Transfusion reaction **CrossCross--match before transfusionmatch before transfusionIndirect coombs test ICTIndirect coombs test ICT--test test

Besides the conventional method for Besides the conventional method for direct and indirect coombsdirect and indirect coombs

**Used for very weak antibodies Used for very weak antibodies **antibodies which are complement fixingantibodies which are complement fixing**for detecting antibodies to leucocytes and for detecting antibodies to leucocytes and

platelets platelets **and research oriented problem in and research oriented problem in

ImmunohematologyImmunohematology

Some Procedure Controls:Some Procedure Controls:

nn AntiAnti--human Globulin test (AHG): Check cells human Globulin test (AHG): Check cells on negative results.on negative results.

nn Elution: Last wash testing in eluate Elution: Last wash testing in eluate preparation.preparation.

nn SalivaSaliva--inhibition test:inhibition test: addition of saline as the addition of saline as the dilution control in saliva inhibition test ( for dilution control in saliva inhibition test ( for secretor status of an individual).secretor status of an individual).

Equipment QCEquipment QC

nn Specification as per requirement: what has been Specification as per requirement: what has been received is what have we asked while placing the received is what have we asked while placing the order?order?

nn Preventive maintenance should be planned.Preventive maintenance should be planned.nn Functional a assessment immediately after Functional a assessment immediately after

installation/ repairs.installation/ repairs.nn DayDay--toto--day check in routine useday check in routine usenn Periodicity of servicing: depends on observed Periodicity of servicing: depends on observed

frequency of deviation, differ from one equipment to frequency of deviation, differ from one equipment to other:other:

Personnel Surveillance:Personnel Surveillance:

Staff training:Staff training:nn To all the staff with basic objective being same To all the staff with basic objective being same

but the knowledge required varies with the but the knowledge required varies with the staff category.staff category.

nn First training while joining and then on regular First training while joining and then on regular interval as CMEinterval as CME

nn Competency/ proficiency test helps to find Competency/ proficiency test helps to find reliable workers, allocate specific duties and reliable workers, allocate specific duties and predict training need.predict training need.

Features of a modern Blood ServiceFeatures of a modern Blood Service

nn Centralized managementCentralized managementnn Organized on a regional/community basisOrganized on a regional/community basisnn Total coverage of target region/communityTotal coverage of target region/communitynn Based wholly on voluntary blood donorsBased wholly on voluntary blood donorsnn Every citizen assured of blood productsEvery citizen assured of blood products

Case: Iran Blood Transfusion Case: Iran Blood Transfusion ServiceService

nn Blood Collection CentersBlood Collection Centersnn Blood Processing CentersBlood Processing Centersnn Organized on a Pre future basisOrganized on a Pre future basisnn Blood Processing Centers run on the lines of Blood Processing Centers run on the lines of

Manufacturing facilities following full GMP Manufacturing facilities following full GMP

What is GMP?What is GMP?

nn GMP is that part of Quality Assurance which ensures GMP is that part of Quality Assurance which ensures that products are consistently produced and controlled that products are consistently produced and controlled to the Quality Standards appropriate to their intended to the Quality Standards appropriate to their intended useuse

nn GMP is primarily intended at diminishing the risks GMP is primarily intended at diminishing the risks inherent in any pharmaceutical production which may inherent in any pharmaceutical production which may broadly be categorized into two groups: cross broadly be categorized into two groups: cross contamination/mixcontamination/mix--ups and false labellingups and false labelling

Why GMP in Blood Banks?Why GMP in Blood Banks?

nn Blood Banks use blood from donors as raw Blood Banks use blood from donors as raw material to material to produce products for therapeutic produce products for therapeutic useuse

nn Blood products should conform to stringent Blood products should conform to stringent standards of safety and qualitystandards of safety and quality

nn This can be accomplished only by strict This can be accomplished only by strict process control and control over the quality of process control and control over the quality of inputs and equipment inputs and equipment

ISHIKAWA DIAGRAMISHIKAWA DIAGRAM

MAN

METHOD MATERIALS

MACHINE

QUALITY OUTCOME

Determinants of Quality outcomes Determinants of Quality outcomes

nn All elementsAll elements of this fish bone structure must of this fish bone structure must work well in order to get quality outcomeswork well in order to get quality outcomes

nn The The Quality SystemQuality System of the Blood bank must be of the Blood bank must be designed to ensure that this happensdesigned to ensure that this happens

nn This is why GMP is importantThis is why GMP is important

MANMAN

nn Trained personnel are crucial to quality Trained personnel are crucial to quality outcomesoutcomes

nn Studies have shown that Studies have shown that 9090% % of Transfusion of Transfusion related complications are due to human errorrelated complications are due to human error

MachineMachine

nn Blood processing uses sophisticated equipment Blood processing uses sophisticated equipment designed to deliver standard products designed to deliver standard products

nn The right equipment needs to be procured The right equipment needs to be procured capable of accomplishing the outcomes capable of accomplishing the outcomes intendedintended

MethodMethod

nn Every stage in the processing of blood has to Every stage in the processing of blood has to conform to strict controlled processesconform to strict controlled processes

nn These processes have to be validated using the These processes have to be validated using the equipment and disposables generally used by equipment and disposables generally used by the blood centerthe blood center

nn Robust QA systems should be put in place to Robust QA systems should be put in place to ensure that SOPs are followed and all ensure that SOPs are followed and all abnormalities are recorded and analyzedabnormalities are recorded and analyzed

MaterielMateriel

nn All consumables and material used must All consumables and material used must conform to strict specificationsconform to strict specifications

nn The most important raw material used in a The most important raw material used in a Blood Bank is the blood collected from a Blood Bank is the blood collected from a donor donor

Quality of BloodQuality of Blood

nn Studies have clearly shown that blood drawn Studies have clearly shown that blood drawn from a from a voluntary repeat donorvoluntary repeat donor has a much has a much higher assurance of safetyhigher assurance of safety

nn We cannot simply rely on screening tests to We cannot simply rely on screening tests to assure safetyassure safety

nn Blood Banks therefore need to take steps to Blood Banks therefore need to take steps to create a donor pool of voluntary donors willing create a donor pool of voluntary donors willing to donate regularly to donate regularly

SummarySummary

nn The The quality of blood collectedquality of blood collected is the single is the single most important determinant of quality and most important determinant of quality and safety of blood productssafety of blood products

nn GMP in all Blood Processing operations alone GMP in all Blood Processing operations alone can ensure quality outcomescan ensure quality outcomes

nn GMP is a GMP is a multimulti--dimensional conceptdimensional concept covering covering MAN, METHOD, MACHINE, and MAN, METHOD, MACHINE, and MATERIEL MATERIEL