Quality control and QA[1].ppt [Compatibility Mode]

Quality Control Quality Assurence of Immunohematology of Reagents Speaker Prof. A.Pourazar Isfahan IRAN

Quality Assurance In Blood Bank n

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Activities that provide confidence in the systems which affect the quality of blood/ blood products are working as expected expected.. Quality can be measured if need has been defined

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Quality –need can be specified / requirement can be set Standards to be laid down by the authority e.g. FDA Quality surveillance is put into practice the authority

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Inspection should be carried out once every year to check. External Quality Assessment (EQA) program on regional and / or national level must be exercised. Internal Quality Control (IQC) by continuous monitoring all aspects of blood banking should be set. Procedural manual and Standard Operating Precedure (SOP)

Reagent Quality Control n n

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On arrival, for titer and avidity of anti anti--sera Daily basis : AntiAnti-A, Anti Anti--B and Anti Anti--AB with know A/B red cells respectively for reactivity. Anti – D with know Rh.D positive red cell for reactivity and Rh.D negative red cells specificity

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Anti-Human Globulin reagent with AntiAntiAnti-D coated red cells (Check – Cells) for reactivity and with uncoated red cells for specificity. Other reagent : like enzymes, albumin, enhancement in reactivity of IgG antianti-D And use of enzyme of reduction in reactivity of antianti-Duffy or antibodies to MNSs blood group and so on …

Standard titer and avidity of ABO reagent Antibody Anti-- A Anti Anti--A Anti Anti-- A Anti

titer with cells titer A1 A1 256 A2 A2 128 A2B A2 64

avidity second 10 20 30

Anti-B AntiAnti--B Anti

B A1B A1

256 128

10 20

Anti-AB AntiAnti--AB Anti

A11 A B

128 64

10 10

International Standards for the titer and avidity

Standard Titer and avidity of AntiAnti-A1 and AntiAnti-H lectin Antibody

titer with cells

titer

avidity scond

Anti- A1 AntiA1 16 20 Anti--H Anti O 8 * Anti – A1 extracted form Dolichus biflorus seed. n Anti Anti--H extracted from Ulex europeous seed. n Anti Anti--A1 give not agglutination reaction with O, B, A2 A2 , A2 A2B , cells

Avidity test n

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Speed and strength of agglutination reaction are tremed as avidity. The test is done by mixing on drop of antiserum with one drop of EDTA whole blood on a slide and rocking gently. with the help of a stop watch, the time for the first appearance of agglutination is recorded.

Precantion n

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Prozone is very often observed in very high titer of antibody and simultaneous presence of incomplete type of antibodies. This could be improved by suitable dilution and made it standard. Quality control is necessary for all type of antisera.

Preservation and Identification Strong bacteriostatic agent like sodium azide in final concentration of 1 : 1000 (i.e.1 (i.e.1ml of 10 10% % solution in 100ml 100 ml of antisera) is added. n The colouring agent like methylene blue for AntiAnti- A acriflavin for AntiAnti-B (zaferan in IRAN) eosin for AntiAnti-AB In concentration of 1 : 5000 are also used (i.e.2 (i.e.2ml of 1% solution in 100 100ml ml of antianti-sera)

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Quality Control for All reagent n

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Like any other typing reagent, it is important that the final processed anti sera should be sterilized Seitz filtration and sodium azide added as a preservative This is particularly important if such sera are to be distributed by post to for distances.

QC and Standardization of AntiAnti-D reagent First step Antibody

Anti – D n

titer with cells

D+(Rho)

saline titer

coombs titer

32

512 512--1024

All the negative tube should be tested by Anti – Humam – Globulin

Second step n

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Anti – Rh Rh((D) serum is accordingly tested with r′r (* (*Ccdee Ccdee), ), r′′ r′′r( r(ccdEe ccdEe)) and Ror (ccDee ccDee)) to rule out the presence of antianti-C and antianti-E Anti--D further tested with 10 Rho (D) negative Anti and 20 or so Rho(D) positive of all ABO groups It should be checked with panel of cells to exclude the presence of unwanted Ab

QC and Standardization of AHG 1- Production of the reagent n Animal immunization: n The most common animal used for the production of AHG reagent is rabbit. n Large scale production could be achieved by immunization sheep or goat n It is preferable to use young animal

Standardization of AHG Preliminary test on AHG n Preparation of sensitized O+ red cell (check cell) and non sensitized O+ red cell n Check cell: O+ red cell + Anti – D (IgG) suspended in saline n Everyday or every week we can prepare n Test : AHG + check cell give + Reation AHG + non sensitized RBC givegive- reation

Secondary test on AHG for QC n

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The standard of AntiAnti-D is used as a source of IgG antibody This AntiAnti-D should have a saline titration 1: 32 or 1:64 and coombs titration about 1:512 or 1:1024 Test should be done at 37C 37C° for 30 mins The procedure is in next table

Reaction of sensitized red cell with AHG Dilution of AHG

1

2

4

8

16

32

64

128

256

512

1024

2+ + + -

2+ + + -

+ + -

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O+ cell sensitized with AntiAnti-D

1:1 1:2 1:4 1:8 1:16 1:32

+ + -

+ + 2+ + -

+ 2+ 3+ 2+ + +

2+ 4+ 4+ 3+ 2+ +

3+ 4+ 4+ 4+ 3+ 2

4+ 4+ 4+ 3+ 2+ +

4+ 3+ 2+ + + -

3+ 2+ 2+ + -

Non sensitized O+, A+, B+ cells

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-

-

-

-

-

-

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Standardization of AHG by dilution of Anti--D (STD) Anti Difution of 1:2

1:4 1:

1:8 1:

1:16 1:

1:32 1:

1:64 1:

1:128 1:

1:256 1:

6

7

8

1:512 1:

Anti--D Anti

No of

1

2

3

tube Add O+ 5% RBC

1

1

1

4

5

1

1

1

1

9

1

Incabate for 30 min at 37 37C C° serofuge for 30 second at 1000 rpm Result +4 +4 +4 +3 +2 +1 -

1 drop

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All the negative tube (6 (6-10 10)) washed 3 time with saline Add two drop AHG and 30 second serofuge at 1000 rpm Coombs Result +4 +4 +3 +2 +1

Final result : saline titration 1:32 and coombs titration 1:512

Use of AHG In routine blood group immunohematology reagent is used to detect *The coating of antibody molecules on the red cell inin-vivo as in HDN and *autoimmune hemolytic anemia AIHA or *transfusion reaction due to blood group incompatility Direct and Indirect coombs test or AHGAHG- test n

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

*Immunization of pregnant lady *Autoimmune Disease *Transfusion reaction *Cross Cross--match before transfusion Indirect coombs test ICTICT-test

Besides the conventional method for direct and indirect coombs *Used for very weak antibodies *antibodies which are complement fixing *for detecting antibodies to leucocytes and platelets *and research oriented problem in Immunohematology

Some Procedure Controls: n

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Anti-human Globulin test (AHG): Check cells Antion negative results. Elution: Last wash testing in eluate preparation. Saliva--inhibition test: addition of saline as the Saliva dilution control in saliva inhibition test ( for secretor status of an individual).

Equipment QC n

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Specification as per requirement: what has been received is what have we asked while placing the order? Preventive maintenance should be planned. Functional a assessment immediately after installation/ repairs. Day--to Day to--day check in routine use Periodicity of servicing: depends on observed frequency of deviation, differ from one equipment to other:

Personnel Surveillance: Staff training: n To all the staff with basic objective being same but the knowledge required varies with the staff category. n First training while joining and then on regular interval as CME n Competency/ proficiency test helps to find reliable workers, allocate specific duties and predict training need.

Features of a modern Blood Service n n n n n

Centralized management Organized on a regional/community basis Total coverage of target region/community Based wholly on voluntary blood donors Every citizen assured of blood products

Case: Iran Blood Transfusion Service n n n n

Blood Collection Centers Blood Processing Centers Organized on a Pre future basis Blood Processing Centers run on the lines of Manufacturing facilities following full GMP

What is GMP? n

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GMP is that part of Quality Assurance which ensures that products are consistently produced and controlled to the Quality Standards appropriate to their intended use GMP is primarily intended at diminishing the risks inherent in any pharmaceutical production which may broadly be categorized into two groups: cross contamination/mix--ups and false labelling contamination/mix

Why GMP in Blood Banks? n

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Blood Banks use blood from donors as raw material to produce products for therapeutic use Blood products should conform to stringent standards of safety and quality This can be accomplished only by strict process control and control over the quality of inputs and equipment

ISHIKAWA DIAGRAM

MAN

MACHINE

QUALITY OUTCOME

METHOD

MATERIALS

Determinants of Quality outcomes n

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All elements of this fish bone structure must work well in order to get quality outcomes The Quality System of the Blood bank must be designed to ensure that this happens This is why GMP is important

MAN n

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Trained personnel are crucial to quality outcomes Studies have shown that 90% 90% of Transfusion related complications are due to human error

Machine n

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Blood processing uses sophisticated equipment designed to deliver standard products The right equipment needs to be procured capable of accomplishing the outcomes intended

Method n

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Every stage in the processing of blood has to conform to strict controlled processes These processes have to be validated using the equipment and disposables generally used by the blood center Robust QA systems should be put in place to ensure that SOPs are followed and all abnormalities are recorded and analyzed

Materiel n

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All consumables and material used must conform to strict specifications The most important raw material used in a Blood Bank is the blood collected from a donor

Quality of Blood n

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Studies have clearly shown that blood drawn from a voluntary repeat donor has a much higher assurance of safety We cannot simply rely on screening tests to assure safety Blood Banks therefore need to take steps to create a donor pool of voluntary donors willing to donate regularly

Summary n

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The quality of blood collected is the single most important determinant of quality and safety of blood products GMP in all Blood Processing operations alone can ensure quality outcomes GMP is a multi multi--dimensional concept covering MAN, METHOD, MACHINE, and MATERIEL