Antibody Detection Relevance of cII-Specific Antibody Andrea A. Zachary, Ph.D. www.hopkinsmedicine.org/hla/

C’ cytotoxicity flow cytometry

Lymphocyte-Based Tests Cytotoxicity >35 years experience Inexpensive Require sufficient viable lymphocytes Reactivity strength affected by: Antigen expression Conditions of cells Inadequate specificity Includes reactivity from non-HLA Abs Incomplete elimination of IgM CDC: limited sensitivity

Solid Phase Immunoassays Use solubilized or recombinant HLA molecules platelets transformed cell lines Platform microtiter plates paper strips polystyrene beads Types presence/absence specificity identification

ELISA Pooled, solubilized Ags in microtiter plates Sandwich assay Colorimetric determination matrix colored product Ag

Example result: A29>A43 Negative Positive A2902 A4301

Bead Assays Flow PRA bead system tested in a standard flow cytometer

FlowPRATM

Flow PRA TM Screening Beads Pool of different 30 Class I and 30 class II microbeads Class I and II beads can be separated by their different colors. Simultaneously detect Abs to class I & II One Lambda Inc.

FlowPRA I & II Screening Test Class I 30 beads Class II One Lambda Inc. FL1

FlowPRA™ Data Acquisition Forward Scatter - Linear Side Scatter - Linear Measure fluorescence for 5,000 to 10,000 events for each sample including positive and negative serum controls Gate major population of FlowPRA™ beads on the FSC vs. SSC dot blot One Lambda Inc.

Class I & II Beads can be separated by FL2 (yellow) fluorescence FlowPRA™ Screening Test Data Analysis Class I & II Beads can be separated by FL2 (yellow) fluorescence One Lambda Inc.

FlowPRA™ Screening Test Data Analysis Neg. & Pos. Sera can be separated by the FL1(green) fluorescence Pos./neg. cut-off point should be set at the end of the peak on the FL1 negative control serum histogram One Lambda Inc.

FlowPRA™ Screening Test Data Analysis % PRA is represented by the percentage of events shifted to the right of the cut-off point One Lambda Inc.

Principle of FlowPRA Specific Test 1 3 4 5 6 7 8 2 FL1 FL2 One Lambda Inc.

FlowPRATM I Specific Tests FL2 Channel Shift FL1 Channel Shift One Lambda Inc.

Single Antigen Beads Data provided by Dr. Nancy Reinsmoen, Duke University.

Bead Assays Luminex system beads impregnated with varying proportions of two dyes to permit differentiation of up to 100 beads fluorochrome labeled antiglobulin reagent for detection of bound antibody laser excitation of fluorochrome multiplex - can run up to 100 assays in a single tube uses very small amounts of serum

Using two colors we can construct one hundred separately addressable microspheres sets. The bead sets are identified by their location on an X and Y grid of (*) infrared versus (*) red dyes. Beads that have low concentrations of both of the dyes fall into the lower regions of the bead map while beads with higher concentrations of both of the dyes fall into regions high on the map.(*)

PMT 532 nm 633 nm This is a snap shot of the picture we were just looking at, where this is the bead as it passes through the lasers.(*) On one side of the instrument we have a green 532 nm YAG laser. This is our reporter laser because it excites the fluorophores on the surface of the bead giving us our report.(*) On the other side of the instrument we have a 633 nm red laser. We call this the classification laser because it excites the two dyes inside the bead classifying the bead.(*) A PMT(photo multiplier tube) works with the green laser to interpret the light scatter and give us our report signal. Light scatter from the red laser is interpreted by a series of APDs (avalanche photo diode). The two APDs close together work to classify the bead while the third APD works with our doublet discriminator. When calibrating it is the temperature of this third APD that we monitor. Calibration is performed once a month or when the dCal temp is +/- 3 degrees.(*) APD

The first component is the fluidics The first component is the fluidics. The fluidics in our system are much like the fluidics of a typical flow cytometer. The beads are presented to the lasers through a quartz cuvette. When the samples are running, sheath fluid is continuously flowing in through the side of the cuvette at a rate of 90 ul/sec. The sample is injected up through the bottom of the cuvette through a sample needle at a rate of 1 ul/sec. The difference in these two flow rates causes the sheath fluid to actually create a sheath around the sample. This creates a core for the sample to travel up the center of the cuvette. The sample core is about 25 microns in diameter, and the entire opening of the cuvette is about 200 microns in diameter. As each bead passes through the lasers, the machine identifies which bead set it belongs to.(*)

The system requires some user maintenance The system requires some user maintenance. You will find a maintenance schedule in the user manual, along with detailed step by step instructions. Inside the Luminex 100 door on the instrument (*) is a 10 micron sheath fluid filter. This filter filters the sheath fluid as it comes into the system. You need to change this filter once a year. The Luminex 100 is pressure driven on the sheath side of the instrument, but syringe driven on the sample side of the instrument. Inside the horizontal door (*) is a syringe. At the top of the syringe you will see a white Teflon fitting that over time the seal will wear out. Replace the syringe fitting once every 6 months or if you begin to see a stream line of bubbles forming in the syringe. On the back of the Luminex 100 behind the metal door, there is an air intake filter. This filters the air that pressurizes the sheath fluid side of the instrument. Replace the air intake filter once every 6 months. Also on the back of the XY Platform is an air filter. This filters the air that cools the XY Platform. It will need to be replaced once every 6 months. Underneath the instrument is a ventilation filter. (*) This filters the air that cools the instrument. Because this filter is on the bottom of the instrument it is important to never put anything paper underneath the instrument. Paper would be sucked to the bottom and cause the instrument to overheat. The instrument should always sit on a solid surface. Remove this ventilation filter, rinse it with water, and replace it under the instrument once every 6 months.

Antibody Frequencies: B Locus

Solid Phase Immunoassays Increased sensitivity Increased specificity Quantifiable reactions Semi-automated: fast, high throughput CDC: 2-4 weeks plus panel preparation time ELISA 111 yes/no, 1 class, 4 hours 1 characterization - 4 hours Luminex 96 yes/no, cI and cII, 4 hours - 45% time saving 96 characterizations - 4 hours -

Solid Phase Immunoassays Conformational changes in molecules bound to solid phase Loss of gene expression Non-uniform detection of antibodies Panel constraints Less robust because of sensitivity Non-specific binding Increased sensitivity - clinical interpretation

Clinical Significance of class II-specific antibodies More than 24 cases of hyperacute or acute rejection mediated by class II-specific Abs Multiple reports demonstrating no significance to positive B cell XM Ab specificity not determined extreme end points used cII Ags are highly immunogenic and provoke crossreactive Abs

Incidence of Antibody to Mismatches

Class II CREGs DR1-10 DR3-5-6 (DRw52) DR4-7-9 (DRw53) DR1-2-9-10 DR1-4 83% of patients with anti-cII had Ab to >1 Ag

Accompanying Antibodies Antibody Groups Specificity Accompanying Antibodies DR1 DR10 (75%), DR9 (64%) DR2 DR9 (60%), DR1 (58%) DR4 DR7+9 (67%) DR9 DR7 (89%); DR1, 2 or 10 (66%)

Inter- and Intra-CREG Abs 27 34 54 85 31 Inter-CREG AB Intra-CREG Ab 27

Clinical Significance of class II-specific antibodies 16 patients in desensitization protocol (plasmapheresis + low dose CMVIg) All had Ab to donor cII but not cI 11 with no cI-specific Ab 3 with no cI mismatches

Patients with Ab to Donor cII Rej No. female regraft mean mm other risk factors flow XM + CDC XM + None 5 4 1 5.4 ACR 9 AMR 10 6 8

Patients with Ab to Donor cII Ab at or After Tx AMR Statistics None Flow XM + CDC XM + 5 1 2 = 7.4 P<0.001 7 3 9

Conclusions Current technology permits rapid, accurate, sensitive detection and characterization for HLA-specific antibodies. Antibody to donor HLA (class I or class II) are a risk factor for graft dysfunction, regardless of titer, if untreated.