Download computerized analysis

SOLID PHASE IMMUNOASSAYS:
INTERPRETING PATTERNS
Julie Houp, CHS
Johns Hopkins University
School of Medicine
Division of Immunogenetics and Transplantation Immunology
Baltimore, MD
INTRODUCTION
 SPIAs are a critical part of identification of HLA antibodies
relevant to transplantation, specifically, donor-specific antibody
(DSA).
 In the pre-transplant or evaluation setting, the availibilty of donor
lymphocytes allow for crossmatch testing to be performed and
SPIA results to be correlated with the crossmatch (FCXM or CDC).
 In the assignment of unacceptable antigens, SPIAs are utilized to
virtually assess compatibility based on HLA specificity.
 In the post-transplant setting, therapeutic drugs (Rituximab,
Thymoglobulin) and the need for live donor cells make it more
difficult or impossible to perform a crossmatch. We must rely
more heavily on SPIAs and attempt to predict the strength of DSA
as it correlates to a crossmatch test.
ANALYSIS
D.4.1.4 Computer Programs
D.4.1.4.1
All computer software programs and version upgrades used for analyses must be validated for
accuracy and this validation documented, prior to release of test results.
D.4.1.4.2
The laboratory must have an ongoing process (at least annually) to ensure that all computerassisted analyses are accurate.
D.4.1.4.3
The laboratory must document historic test result review when notified by a vendor of an
update/revision/correction to analysis software or template that could yield a change, correction, or
update to the original test result and take appropriate corrective action.
COMPUTERIZED ANALYSIS
• Inclusion analysis.
The basic algorithm used by analysis.
Software.
• Statistical analysis includes chi square, correlation coefficient
(r-value), and percent inclusion.
• Certain pitfalls may exist in relying solely on computerized
analysis!
TACTICS TO IMPROVE ACCURACY OF ANALYSIS
• Consideration of the patient’s own phenotype in analysis
• Identification of sensitization (previous mismatches from
previous transplants or pregnancy)
• Analysis of sera at multiple dilutions
• Utilization of several screening methods (certain methods
allow the ability to see the strength of the reactions, etc.)
• Utilization of available crossmatch results in analysis
• Consideration of patient information (therapeutic drugs,
underlying diseases)
• Evaluation of sera over several different lots
SINGLE ANTIGEN BEAD AT 1:64 DILUTION
DP1,DP4
DP6, DP17
NO DSA PRIOR TO TRANSPLANT
REACTIVITY WITH DQ ENHANCED BEADS REFLECTS INCREASE IN DP ANTIBODY
Patient is a B35. Self reactivity?
•
Patient is B*35:03, previous transplant is B*35:01
•
B*35:01 and B*35:03 differ at pos 116
B*35:05
B*35:01
B*35:08
39 y.o. male CAPS syndrome
1st TX: LURD in 2005 No HLA specific antibody detected prior to Tx
2nd TX: LURD Repeat mismatch DR53
ANTIBODY TESTS PRIOR TO TX # 2
•
Negative?
•
Maybe DQA5/DQB2 (a previous MM)
•
SA assay #1: DQ2
•
SA assay #2: DR53 DR10
TRANSPLANT OR NOT?
• FCXM was negative
• 2 pre-tx TPE and 7 post-tx TPE
• 18 months post-transplant with no rejection
• Eculizimab
SINGLE ANTIGEN BEAD RESULTS
•
Reactivity with DR53 beads >20000MFI
•
Reactivity with DR10 moderate 12000MFI
•
Additional specificity DQA3 6000MFI
Dilution at 1:8: DR53 >10000MFI DR10 4000MFI
VIRTUAL PREDICTION: CDC POSITIVE WITH DR53; FCXM POSITIVE WITH DR10
CROSSMATCH RESULTS
• CDC One wash B cell crossmatch positive with the patient’s
spouse. DR10, DR17
• CDC One wash B cell crossmatch weaker positive with DR7,
DR14
• FCXM results confirm these findings
•
Patient had two potential donors
•
Donor #1: B7
•
Donor #2: A2
•
Donor #2 selected to donate
•
5 days later…
Patient is DR17 DQA5 DQB2
Single antigens bead assay confirms DQA2 and DQB5 reactivity
•
Patient is a DR7 DQA2 DQB2
•
Single antigen bead assay does not confirm reactivity
Donor is child: DSA DR11, DQ7
Single Antigen Bead assay correlates strength of DSA
FCXM was weakly positive, auto XM also positive
•
Single antigen bead assay confirms DQ7 reactivity >20000MFI 1:8 dilution DQ7 16000MFI
CONCLUSIONS
 Highly sensitized patient often have issues that challenge the
limitations of SPIAs
 Patient information (sensitization, medications) can
greatly assist the analysis
 HLA typing at the allele level may be needed
 Extensive expertise with the vagaries of SPIAs is needed to
correctly interpret results and provide consistent results to clinicians
ACKNOWLEDEMENTS
•
Johns Hopkins Immunogenetics Laboratory
•
Dr. Andrea A. Zachary, Dr. Mary S. Leffell, Dr. Annette Jackson and Renato Vega
CONTACT INFORMATION
Julie A. Houp, CHS
Laboratory Manager
Division of Immunogenetics and Transplantation Immunology
Johns Hopkins School of Medicine
Office 410.955.3600 x130
Mobile 410.926.7643
[email protected]