session 2 - anti-d reagents selection & qualification

Anti-D Reagent Selection & Qualification

Susan T. Johnson, MSTM, MT(ASCP)SBBDirector, Clinical EducationBloodCenter of Wisconsin

Quotient Biodiagnostics Industry WorkshopOctober 24, 2011

OBJECTIVES

• List factors to consider when selecting anti-D reagents.

• Design a sampling plan for qualifying anti-D reagents.

• Discuss the importance of a process for resolution of discrepant RhD typing results.

THINGS TO CONSIDERWHEN SELECTING ANTI-D

• Who is being typed?• What reagents are available?• Where is testing being performed?• Why are you doing the Rh typing

you do?• How many samples should we test

and what type of samples?

WHO ARE YOU TYPING? PATIENTS

• Pretransfusion Samples• Surgical Patients• Oncology• OB/GYN• Pediatrics vs. Adults• Mixed Field Samples

• Prenatal Patients• Infants of Rh-negative mothers

WHO ARE YOU TYPING? DONORS

• BB/TS Standards, 27th edition, 2011

5.8.2 Determination of Rh Type for All CollectionsThe Rh type shall be determined for eachcollection with anti-D reagent. If the initial testwith anti-D is negative, the blood shall be testedusing a method designed to detect weak D.When either test is positive, the label shall read“Rh POSITIVE.”

DO YOU WANT TO DETECT WEAK/PARTIAL D?

• Blood Donors Yes• Pretransfusion Testing No• Prenatal Patients ??• Infants of Rh-Negative Mother Yes

WHAT ANTI-D REAGENTS ARE AVAILABLE & WHAT DO THEY DETECT?

• 8 “different” licensed anti-D reagents on market today

• Review manufacturer’s instructions• Review clone I.D. number

“Grey Label”

MANUFACTURER’S INSTRUCTIONS

• Determine unique aspects of different anti-D reagents

• FDA requires reactivity with partial DIV, DV & DVI RBCs be defined • All are positive at IS with DIV & DV• Most are negative at IS with DVI but positive

in IAT

MONOCLONAL IgM/IgG ANTI-D

MONOCLONAL IgM/IgG ANTI-D #1Direct Agglutination - IS

MONOCLONAL IgM/IgG ANTI-D #1Weak D Test - IAT

QUOTIENT BIODIAGNOSTICS ANTI-D REAGENTS

Method Reagent IgM IgG

Tube alpha LDM1

Tube delta*LDM1

ESD1M

Tube beta LDM3

Tube blend LDM3 ESD1

Gel ID-MTS MS201

* Detects DVI on direct agglutination

Anti-D delta ALBAclone®

RECOMMENDED FOR DONOR TESTING

• “This monoclonal IgM anti-D will directly agglutinate red blood cells from all known D categories including DVI and, therefore, is ideally suited for RhD grouping of donor samples. The reagent is not recommended for the RhD grouping of patient samples for the purpose of transfusion.”

Anti-D blend ALBAclone®

(Human/Murine Monoclonal IgM/IgG) For Slide and Tube Techniques

• “This monoclonal anti-D will directly agglutinate red blood cells from most weak D and partial RhD except DVI and, therefore, is suitable for RhD grouping of patient samples. This reagent will also detect DVI and weak D by IAT and, therefore, is also suitable for RhD grouping of donor samples.”

What Is Your Current Process & Procedure for RhD Typing?

• Direct Agglutination (IS) only

• D IAT or Weak D Testing on all negatives at IS

• Other

Typing for D Antigen - Traditional

• Anti-D reagents

Pt’s cells Anti-D

Spin & Read

D+

D-

Incubate @ 37C, Wash, Add

Anti-IgG

Spin & Read

If still (-) Report D-

If now + Report D+

If (-) or weak +

Direct AgglutinationDirect Agglutination IAT or Weak D TestingIAT or Weak D Testing+

Typing for D – Another Approach

• Anti-D Reagent

Direct Direct AgglutinationAgglutination

Pt’s cells Anti-D

Spin & Read

3-4+ D+

D-

1-2+ D – or D+

0 D-

Typing for D – Another Approach

• Anti-D Reagent

Direct AgglutinationDirect Agglutination IAT or Weak D IAT or Weak D TestingTesting+/-

Pt’s cells Anti-D

Spin & Read

3-4+ D+

D-

Incubate @ 37C, Wash, Add

Anti-IgG

Spin & Read

If still (-) or < 2+

Report D-

If now 2+ or more Report D+

If 1-2+

VALIDATION

• The laboratory must verify for each method the performance specifications for accuracy, precision, clinical sensitivity, clinical specificity, and any other applicable performance characteristic appropriate for measuring test performance.

QUALIFICATION

• The reagent must be tested to ensure that it is performing to each laboratories specification/requirements.

VALIDATION QUALIFICATION

REAGENT MANUFACTURER LABORATORY

SOURCES OF REQUIREMENTS

• Customer• Source reference materials are a key

source • They define the minimum requirements of

what needs to be proven or challenged.

SOURCE REFERENCE DOCUMENTS

• Package Inserts & Operators Manuals• SOPs• Regulations and Standards• Industry Standards• Scientific/Medical Literature• Customer contracts / agreements• Product and/or Service Acceptance Criteria

SELECTING REQUIREMENTS

• Requirements are the “must haves” for the process or test. • Choose requirements that you would actually fail if

they aren’t met. • Requirements should be measureable and can be

expressed in a clear qualitative or quantitative result.

• For Antisera - test & compare to current reagent in use.

REQUIREMENTS

ACCEPTANCE CRITERIA

PREDICTABLE RESULTS

VALIDATION/QUALIFICATION

How Many Samples Should I Test?

SCIENCE

SAMPLE PLAN PLANNING

Population

Sample

Account for Sources of Variability

SAMPLE PLAN– WHERE TO START?

Factors to Consider:• Sources of Variability – How many conditions need to

be tested?• What level of confidence is required in the result of the

validation/qualification?• How many boundary conditions do I need to evaluate?• Availability and quantity of samples available for

testing?• What level (number) failures can be accepted?

I want to be ___% certain that ___% of times the process/test is performed that I get the expected result.

In choosing this sampling plan, I am willing to accept __ (#) of failures in my validation.

Sample Plan Planning Quantitative Approach

Page 35

Putting the Sampling Plan TogetherAcademic Medical Center

Population

Sample10

Spread the sampling plan across the Sources of Variability

Rh Neg patients

Weak D/Partial D

Rh pos patientsMixed Field

Samples – 48 hours old

10

10

2010

Putting the Sampling Plan TogetherCommunity Hospital

Population

20

Sample 410

Spread the sampling plan across the Sources of Variability

Weak D/Partial D

Rh Pos Patients Rh Neg patients

Rh Neg Mothers

Cord Bloods

6

4

Putting the Sampling Plan TogetherImmunohematology Reference Lab

Population

Sample10

Spread the sampling plan across the Sources of Variability

Weak D/Partial D

Rh Neg patients

Mixed Field

R1r

6

20

10

R2r

10

Ro

20

Develop a Process for Handling Discrepant RhD Typing

• Historical mismatch• What testing will be performed to interpret

results• D IAT?• Different anti-D clones• Partial D Kit• Molecular characterization

• How results will be reported

Anti-D (std method)Negative

No

Inconclusive Rh NegativeRh Positive

Yes

No

Report Rh pos Test with Different Anti-D Reagents

Send out for confirmation

No

Anti-D (std method)>2+ agglutination score

No

Yes YesMatches historical

?

Matches historical

?

Yes

Partial D Kit or Molecular Typing

Rh Discrepancy Algorithm – 1 Example

Report Rh neg

OBJECTIVES

• List factors to consider when selecting anti-D reagents.

• Design a sampling plan for qualifying anti-D reagents.

• Discuss the importance of a process for resolution of discrepant RhD typing results.