SOLID PHASE IMMUNOASSAYS: INTERPRETING PATTERNS Julie Houp, CHS

SOLID PHASE IMMUNOASSAYS: INTERPRETING PATTERNS

Julie Houp, CHSJulie Houp, CHS

Johns Hopkins UniversityJohns Hopkins UniversitySchool of MedicineSchool of Medicine

Division of Immunogenetics and Transplantation ImmunologyDivision of Immunogenetics and Transplantation ImmunologyBaltimore, MDBaltimore, 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.

ANALYSISD.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 computer-assisted 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*35:01

B*35:05

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 DSAFCXM 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

• Johns Hopkins Immunogenetics Laboratory

• Dr. Andrea A. Zachary, Dr. Mary S. Leffell, Dr. Annette Jackson and Renato Vega

ACKNOWLEDEMENTS

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]

CONTACT INFORMATION