Single HLA Antigen Bead DataInterpretation: Normalized Ratios

Peter Stastny

Transplantation Immunology DivisionDepartments of Internal Medicine and Pathology

UT Southwestern Medical CenterDallas, Texas, USA

Determine a Cut-off for each of the Single

Antigen Beads

NORMALIZED RATIOS

• Normalized ratios are a method of expressing the results of SA assays developed at UT Southwestern.

• Normalized ratios : [(Raw MFI x ag density corr. factor) - NC)]/NHS Mean+3SD

• Each bead has a different cutoff value.

• NR of >2x are considered positive. Above 10x strong positive.

Antigen Density on the Beads

ANTIGEN DENSITY

Monoclonal antibodies usedfor antigen density correction

• HLA Class I: w6/32• HLA Class II

– HLA-DR: L243– HLA-DQ: SPVL3 (Beckman)– HLA-DP: SPM421 (Abcam)

The Reactions of Normal Human Sera

HLA Class I Beads

HLA Class II Beads

Mean plus 3 SD

99.6 %

NHS Mean plus 3SD

Variation in Ranges of Binding of IgG from Normal Human Sera to SA Class I Beads

2X (Mean Plus 3SD)

MFI

SA Beads HLA Class I

Cut-off at 1000 MFI

Cut-off at 500 MFI

Variation in Ranges of Binding of IgG from Normal Human Sera to SA Class II Beads

NHS Mean plus 3SD

2X (Mean Plus 3SD)M

FI

SA Beads HLA Class II

NORMALIZED MFI

• No correction for antigen density.• No individual cutoff based on NHS panel. • Fix 1000 NMFI cutoff.• Normalized MFI are obtained by

subtracting negative ctrl from raw MFI.

METHOD

• Analyze two consecutive sera from 30 patients using normalized ratios and fixed 1000 NMFI cutoff and compared the results.

NR vs NMFI: Number of positive beads

875

588

1039+

18.7%

+ 15.6%

Num

ber o

f pos

itive

be

ads

Patients with additional specificities identified by normalized ratios

21/30 patient with additional specificities

14/30 patient with additional specificities

Analysis of consecutive sera for the 21 discrepant patients for class I antibodies

Analysis of consecutive sera for the 14 discrepant patients for class II antibodies

64.3%

35.7%

57.1%

42.8%

Antibodies detected as positive with normalized ratio but negative with normalized MFI can cause

a positive crossmatch

T CELL FLOW CYTOMETRY XM

DSANormalized

RatioNormalized

MFIA0203 >2x 478A0201 >2x 413

Cw0304 3x 450

MESF Delta MESFNegative ctrl 2062 -

Patient 5738 3676Positive ctrl 35455 33393

Positive Cutoff: Delta MESF of 2250

Negative ctrlPatient

Positive ctrl

Antibodies detected as positive with normalized ratio but negative with normalized MFI can cause

a positive crossmatch

B CELL FLOW CYTOMETRY XM

DSANormalized

RatioNormalized

MFICw07 3X 720.5

MESF Delta MESFNegative ctrl 1022 -

Patient 5430 4408Positive ctrl 70013 68991

Positive Cutoff: Delta MESF of 2169

Negative ctrlPatient

Positive ctrl

Cw07 is a regraft antigen.

CONCLUSIONS

• Normalized ratios generally identified more weak positive specificities than normalized MFI.

• Normalized ratios yielded more reproducible results when comparing sequential serum samples than normalized MFI.

• Normalized ratios has a different cutoff for each bead.

• Specificities that were identified with normalized ratios but not with normalized MFI could yield a positive crossmatch.

• Methods used to analyze SA tests can impact the antibody specificities identified.

An early batch from Source A

Class I Beads sorted by MFI

Antigen Density with W6/32 Source B

Class I Beads sorted by MFI

Trim

med

Mea

n Fl

uore

scen

ce

SA Beads for HLA Class I

Antigen Density with W6/32 A more recent batch from Source A

Normalized Ratios

• MFI/(Mean +3SD) • Weak positive: 2X to 10X• Strong positive: greater than

10X

Analysis of Results obtained with Single Antigen Beads

• Antigen density correctionMonoclonal antibodies for HLA class Iand class II antigens

• Ig binding from normal seraMean + 3SD

• Normalized ratiosMFI/(Mean +3SD for each bead)

IgG Antibodies against Donor HLA Antigens

Primary Kidney Allografts

Effect of DSA detected with SA beads when T-cell flow-cytometry crossmatch

was negative.

Heart Transplants

Association of anti-donor HLA antibodies with transplant-related coronary artery disease.

Donor-Specific Antibodies

Effect of antibodies against HLA antigens not present in the graft

From: Stastny et al, Antibodies against donor HLA and the outcome of cardiac allograftsin adults and children, Transplantation 84: 738, 2007.

Antibodies against HLA antigens not expressed in the graft did not harm the heart transplant in 5 ½ years