SUBCOMMITTEE ACTIVITIES (1994?1995)

A)Subcommittee Publications: Brandt JT, Triplett DA, Alving B, Scharrer I. "Criteria for the Diagnosis of Lupus Anticoagulants: An Update." Thromb Haemost (accepted May/95)

Brandt JT, Barna LK, Triplett DA. "Laboratory Identification of Lupus Anticoagulants: Results of the Second International Workshop for Identification of Lupus Anticoagulants." (submitted to SSC).

Barna LK, Triplett DA. "Third International Survey on Lupus Anticoagulants." (in process).

B) Third International Workshop on Laboratory Diagnosis of Lupus Anticoagulants. University Institute of Biomedical Sciences, Favaloro Foundation, Buenos Aires, Argentina, May 6, 1995

C) Workshop on Anticardiolipin and Anti b2 Glycoprotein I Antibodies. University Institute of Biomedical Sciences, Favaloro Foundation, Buenos Aires, Argentina, May 6, 1995

D) International Survey on Lupus Anticoagulants - 4 Participants Preliminary summary of data

METHODOLOGY
A) Anticardiolipin Assays J. Brandt

Anticardiolipin Antibodies
Where Do We Go From Here?
1. Current Status:

Multiple methods of performing the assay, including specific protein-based assays.

Multiple methods of establishing the reference (normal) range.

Multiple methods of performing the assay.

Problems with standards and reference materials.

2. Methods of performing the assay:

Is it beyond the scope of the LA/PDA Subcommittee to make specific recommendations in this fast-moving environment?

Are there recommendations that we can make in terms of validations of an assay?

Number of samples to be analyzed to demonstrate correlation between methods. Criteria for evaluating agreement between methods; linear regression analysis versus concordance between classification/titer (e.g. negative, low moderate, high)

Are there recommendations we should make regarding evaluation for background (non-specific) binding?

3. Methods for establishing reference (normal) ranges:

Minimum number of subjects to be included.

Handling of outliers (presumed positive); i.e. criteria for editing the data.

Type of subjects to include:
Gender
Age

Calculation of the reference interval
Mean ? sd (arithmetic)
Mean ? sd (log)
Percentile
Multiples of the median
a. Absorbance units
b. GPL/MPL units
Do we have adequate data to recommend a uniform approach at this time?

4. Reporting of results:

Should we recommend a uniform method? If so, which method?
Titer (e.g. negative, low/weak positive, moderate positive, high/strong positive). What criteria would be used to designate the titer?
MPL/GPL units
SD above mean or median (arithmetic or log transformed)
Multiples of the median

Do we have sufficient data to recommend one method? If not, what additional data is needed?

5. Standardization:

What form should a standard/reference material be in?
Lyophilized plasma
Lyophilized serum
Purified immunoglobulin
Pooled versus single donor

How should a value be assigned to such a material?

What would be the proposed use(s) of such a material?

Routine (day-to-day) calibration of clinical laboratory assays?
Calilbration of working standards?
Calibration of manufacturers' kits?
Research programs
B) Nature of Antigen R. Roubey
Detection of Autoantibodies to Specific Protein
Antigens for the Diagnosis of the Antiphospholipid Antibody Syndrome

1. Scientific Background
Research suggests that autoantibodies associated with the antiphospholipid syndrome and detected in lupus anticoagulant and anticardiolipin assays are directed against one or more phospholipid- binding plasma proteins or protein-phospholipid complexes. The most common antibodies are directed against epitopes expressed on b2- glycoprotein I (b2GPI) or prothrombin.

Standard anticardiolipin: ELISAs detect antibodies with at least two distinct specificities:
Antibodies to b2GPI (predominantly bovine)
Authentic anticardiolipin antibodies
? Antibodies to other calcium-independent phospholipid-binding proteins in bovine or human sera

Standard lupus anticoagulant assays detect certain antibodies to:
b2GPI
Prothrombin
? Other coagulation factors

Issue: Are the data sufficient to warrant a re-evaluation of clinical laboratory testing?

2. Implications for Clinical Testing

Potential benefits of ELISAs using purified human protein antigens:
Decreased intra- and inter-laboratory variation due to standardization and quality control of antigens.
Detection of species-specific antibodies
No detection of authentic anticardiolipin antibodies which may not be associated with the antiphospholipid syndrome (improved specificity)
Possible improved prognostic capabilities if certain autoantibodies correlate with clinical subsets

Potential disadvantages of ELISAs using purified human protein antigens
Increased expense
Represents a significant change for clinicians, clinical laboratories
Issues: Should immunoassays (ELISAs) for autoantibodies to b2GPI be developed and evaluated for clinical use?

Should such assays be used clinically at this time?

3. Issues in Immunoassay Development

Methodology
Preparation of antigen, assessment of purity
Type of microtiter plate, coating conditions
Processing of samples
Many others

Quantitation of results
End-point titration vs. single dilution/reference curve
Effect of affinity
Establishment of a normal range
Need for positive standards (workshop)
Assignment of units
Quality controls, confidence limits, coefficient of variation

Clinical correlations
Sensitivity, specificity
Predictive values

4. Nomenclature
Issue: Should terminology change and how?
NOMENCLATURE

Growing problems with confusion in the literature (e.g., Anticardiolipin Antibodies [Type A and Type B]; b2 Glycoprotein I Dependent and Independent Antibodies; Anti-b2 GPI Antibodies)

STANDARDS

Is it possible for the Subcommittee to provide monospecific or affinity-purified IgG and IgM fraction with various relevant specificities?

Plans to approach this problem (workshop)? NIH support?

College of American Pathologists Standards Committee

NIBSC - Preliminary efforts, T. Barrowcliffe

International Anti b2 GPI Workshop Letter:
D. Ware Branch
David L. McGlasson

UPDATE ON CLINICAL TRIALS

Randomized clinical trial on the management of antiphosphoslipid-antibody syndrome: a proposal

FUTURE PLANS

1996 VII International Symposium on Antiphospholipid Antibodies, New Orleans (October 9-13, 1996)

42nd Annual Meeting, Scientific and Standardization Committee of the International Society on Thrombosis and Haemostasis (June 22-24, 1996)