Session 2 - Anti-d Reagents Selection & Qualification
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Transcript of 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.