Evaluation of Antibody Frequencies and Solid Phase Assays

Andrea Zachary, John Hart

Background

This is a continuation of the workshop begun during the 15th International Histocompatibility Workshop examining the frequencies of different antibody specificities and the characterization of immunogenicity of different HLA antigens using solid phase assays.

The shortage of donor organs, the inability to use matching in transplantation of life-saving organs such as heart and lung, and the polymorphism of the HLA system have all resulted in the majority of transplants involving multiple HLA antigen mismatches. Following graft loss, patients may have antibodies to previous mismatches and antigens that share epitopes with those mismatches. Such antibody greatly reduces opportunities for subsequent transplantation and has been implicated in reduced survival of subsequent transplants. Such antibodies may also arise via other routes of exposure such as transfusion and pregnancy. We have previously presented data suggesting that different HLA antigens vary in their immunogenicity as defined by the frequencies of different antibody specificities normalized for the frequencies of antigens in the population. It is also possible that immunogenicity also varies by route of exposure. Information about immunogenicity might affect donor selection in some cases, may be useful in customizing immunosuppression, and might identify cases for which post-transplant monitoring is more critical. Current solid phase immunoassays that use soluble HLA molecules bound to beads as targets provide the opportunity for evaluating the issue of varying immunogenicity among HLA antigens. Further, the amount of data that can be accrued in an international workshop would permit meaningful studies of subgroups differentiated by race, gender, number of mismatches, and route of sensitization.

Objective

To assess the frequencies of antibodies of different HLA specificities and to assess the impact of factors that cause interference in solid phase assays.

Work Plan

There are two parts to the study. Patient information will be provided only once for all data submitted for either Part I or Part II. Laboratories performing tests on the Luminex platform are asked to submit the following on the most reactive specimen from each patient they have tested for Part I of the study.

Part I will include data only from valid tests.

Part II is for data from assays on specimens with one or more increased negative controls and/or decreased positive controls. The following criteria should be used when including specimens and their data in Part II:

• Luminex ID Assays:
  • Positive controls <10,000 MFI
  • Negative controls >300 MFI
• Single Antigen Bead Assays:
  • Positive controls <10,000 MFI
  • Negative controls >200 MFI

There is a single spread sheet for the collection of data. The spreadsheet has six tabs. Specific instructions for each tab are listed below and are also found on the spreadsheet. The six tabs include:

• Tab 1 - Laboratory Data – (entered only once)
  

Specimen Data will include:

• Tab 2 - Patient Demographic Information
• Tab 3 - Patient Typing
• Tab 4 - Sample Information – Part I (valid test data only)
• Tab 5 - Target Crossmatch (XM) Cell Phenotype – Part I
• Tab 6 - Sample Information – Part II (assays with controls out of range)

The spreadsheet can be downloaded here.

The questions to be addressed in the data analysis:

1. Do antibody frequencies match what would be predicted from random encounters with antigens?
2. Do responses to mismatches vary among the different antigens, among different degrees of mismatch, among different routes of sensitization?
3. Are the different antibody specificities identified uniformly by different laboratories?
4. Are there any technical issues with different beads that suggest stronger or weaker reactivity?

There are likely to be other questions of value to answer that will be identified as things progress.

Data File Instructions – Study Part I

1. The use of the attached spreadsheet will expedite the analysis and processing of data submitted by participants.
2. Instructions for filling out the data form are available via drop down menus, and mouse over instructions.
   
3. Laboratory Data
   In addition to contact information, this tab will summarize the methodologies used in your laboratory together with the parameters for assessing the presence or absence of HLA antibody
   • Enter the contact information for the laboratory.
   • Select the CDC crossmatch methods used in the laboratory together with details of the procedures from the drop down menus.
   • Select the Flow Cytometric Crossmatch Mean Channel Shift thresholds used in the laboratory according to your testing protocols.
   • Choose the Solid Phase Platform you use in the laboratory from the drop down menu.
   • Indicate panels used (Phenotype or Single).
   • Enter the MFI threshold for a positive reaction.
   • Enter the reaction score required to assign an unacceptable antigen for transplant.
4. Patient Information
• Patient ID entered on the Patient Typing Tab will carry over to the other spreadsheets
• Enter data as indicated by each field.
5. Patient Typings
• Enter the patient HLA type for each locus identified. Leave the second antigen at a locus blank if a second locus is not identified and homozygosity has not been determined through genotyping.
6. Sample Information – Part I
   • The Patient ID will carry over from the Patient Information tab.
   • Assign a unique identifier for each serum being analyzed. Note: If multiple assays are performed on each serum, use a separate line for each assay.
   • Select the serum treatment, if any that was performed on the serum specimen.
   • Select the appropriate assay that was performed on the serum specimen.
   • Enter the Positive control bead value for the Class I and Class II assays
   • Enter the Negative Control bead value for the Class I and Class II assays.
   • Using the most broadly reactive specimen that you have tested, enter the antibodies that were defined by your methodology, listing those that are your final interpretation from the assays you performed. Separate entries by a comma. For example: A2, B44, DR7
   • Enter the results of the crossmatch assays you performed, where appropriate. Note: Crossmatch (XM) data are not required. If a crossmatch is uninterpretable, do not enter it
     
   • Assign a unique identifier for the target cell. This will link to the target cell data on the Target Cell XM Phenotype tab.
7. Target Cell XM Phenotype (Part I)
   • Enter the Target Cell HLA type for each locus identified. Leave the second antigen at a locus blank if a second locus is not identified or homozygosity has not been determined through genotyping.
     

Data File Instructions – Study Part II

Specimens with one or more Increased Negative Control and/or decreased Positive Control

1. Sample Information – Part II
   • The Patient ID will carry over from the Patient Information tab.
   • Assign a unique identifier for each serum being analyzed.
   • Select the serum treatment, if any that was performed on the serum specimen.
   • Select the assays that were performed on the serum specimen.
   • Enter the Positive control bead value for the Class I and Class II assays.
   • Enter the Negative Control bead value for the Class I and Class II assays.
   • Enter the antibodies that were defined by your methodology, listing those that are your final interpretation from the assay you performed. Separate entries by a comma. For example: A2, B44, DR7
   • Enter the highest MFI value obtained with any antigen or phenotype.
   • Enter the highest MFI value obtained with any autologous antigen, for Single Antigen Assays only.
   • Enter the highest MFI value obtained with the donor antigen or phenotype assay containing a donor antigen, only if XM data are provided.
   • Enter the results of the crossmatch assays you performed.
   • Enter whether the crossmatch controls are within range.
   • Enter whether the patient was desensitized.

Submit Results:

Submit your completed results in the data spreadsheet to: John M. Hart jmhart [at] jhmi [dot] edu.

The spreadsheet can be downloaded here.

If you have any questions about the study or need help completing the data spreadsheets, please feel free to contact us.

Thank you for your participation.

Contacts:
Please let us know if you would like to participate in the workshop.  We will provide you with further information as the project proceeds.

Andrea A. Zachary, PhD
Director
John Hopkins University
Immunogenetics Laboratory
2041 E. Monument Street
Baltimore, Maryland 21205-2222
USA

Tel: 001 410-955-3600
Fax: 001 410-955-0431
Email: aaz [at] jhmi [dot] edu

Download a PDF of the ASHI 2010 Presentation - download PDF file.

Download a PDF of the ASHI 2011 Presentation - download PDF file.

Nov 11, 2011 Posted by Admin