Session 2 - Anti-d Reagents Selection & Qualification
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  • ACOG guidelines based on this testing.

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  • 1. Anti-D Reagent Selection & Qualification
    • Susan T. Johnson, MSTM, MT(ASCP)SBB
    • Director, Clinical Education
    • BloodCenter of Wisconsin
    Quotient Biodiagnostics Industry Workshop October 24, 2011
  • 2. 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.
  • 3. THINGS TO CONSIDER WHEN 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?
  • 4. 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
  • 5. WHO ARE YOU TYPING? DONORS
    • BB/TS Standards , 27 th edition, 2011
    5.8.2 Determination of Rh Type for All Collections The Rh type shall be determined for each collection with anti-D reagent. If the initial test with anti-D is negative, the blood shall be tested using a method designed to detect weak D. When either test is positive, the label shall read “ Rh POSITIVE.”
  • 6. DO YOU WANT TO DETECT WEAK/PARTIAL D?
    • Blood Donors Yes
    • Pretransfusion Testing No
    • Prenatal Patients ??
    • Infants of Rh-Negative Mother Yes
  • 7. 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”
  • 8. 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
  • 9. MONOCLONAL IgM/IgG ANTI-D
  • 10. MONOCLONAL IgM/IgG ANTI-D #1 Direct Agglutination - IS
  • 11. MONOCLONAL IgM/IgG ANTI-D #1 Weak D Test - IAT
  • 12. QUOTIENT BIODIAGNOSTICS ANTI-D REAGENTS * Detects DVI on direct agglutination Method Reagent IgM IgG Tube alpha LDM1 Tube delta* LDM1 ESD1M Tube beta LDM3 Tube blend LDM3 ESD1 Gel ID-MTS MS201
  • 13. 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. ”
  • 14. 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.”
  • 15. 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
  • 16. 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 Agglutination IAT or Weak D Testing
  • 17. Typing for D – Another Approach
    • Anti-D Reagent
    Direct Agglutination 0  D- Pt’s cells Anti-D Spin & Read 3-4+  D+ D- 1-2+  D – or D+
  • 18. Typing for D – Another Approach
    • Anti-D Reagent
    Direct Agglutination IAT or Weak D Testing +/- 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+
  • 19. 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.
  • 20. QUALIFICATION
    • The reagent must be tested to ensure that it is performing to each laboratories specification/requirements.
  • 21. VALIDATION QUALIFICATION REAGENT MANUFACTURER LABORATORY
  • 22. SOURCES OF REQUIREMENTS
    • Customer
    • Source reference materials are a key source
      • They define the minimum requirements of what needs to be proven or challenged.
  • 23. 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
  • 24. 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.
  • 25. REQUIREMENTS ACCEPTANCE CRITERIA PREDICTABLE RESULTS
  • 26. VALIDATION/QUALIFICATION How Many Samples Should I Test? SCIENCE
  • 27. SAMPLE PLAN PLANNING Population Sample Account for Sources of Variability
  • 28. 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?
  • 29.
    • 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.
  • 30. Sample Plan Planning Quantitative Approach Page 35
  • 31. Putting the Sampling Plan Together Academic Medical Center Population Sample 10 Spread the sampling plan across the Sources of Variability Rh Neg patients Weak D/Partial D Rh pos patients Mixed Field Samples – 48 hours old 10 10 20 10
  • 32. Putting the Sampling Plan Together Community Hospital Population 20 Sample 4 10 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
  • 33. Putting the Sampling Plan Together Immunohematology Reference Lab Population Sample 10 Spread the sampling plan across the Sources of Variability Weak D/Partial D Rh Neg patients Mixed Field R 1 r 6 20 10 R 2 r 10 R o 20
  • 34. 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
  • 35. Anti-D (std method) Negative No Inconclusive Rh Negative Rh 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 Yes Matches historical? Matches historical? Y es Partial D Kit or Molecular Typing Rh Discrepancy Algorithm – 1 Example Report Rh neg
  • 36. 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.