1. Antibody Detection Relevance of cII-Specific Antibody
Andrea A. Zachary, Ph.D.

2. C’
cytotoxicity
flow cytometry

3. 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

4. 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

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

6. Example result: A29>A43
Negative
Positive
A2902
A4301

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

8. FlowPRATM

9. 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.

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

11. 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.

12. 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.

13. 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.

14. 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.

15. Principle of FlowPRA Specific Test1 3 4 5 6 7 8 2
FL1
FL2
One Lambda Inc.

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

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

18. 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

19. 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.(*)

20. 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

21. 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.(*)

22. 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.

23. Antibody Frequencies: B Locus

24. 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 -

25. 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

26. 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

27. Incidence of Antibody to Mismatches

28. 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

29. 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%)

30. Inter- and Intra-CREG Abs27 34 54 85 31
Inter-CREG AB
Intra-CREG Ab 27

31. 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

32. 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

33. 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

34. 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.