Test Id : B2GMG

Diagnostic criteria for antiphospholipid syndrome include the presence of at least one of the following: lupus anticoagulant, anticardiolipin, and anti-beta-2 glycoprotein 1 IgG or IgM antibodies. Consider ordering CLPMG / Phospholipid (Cardiolipin) Antibodies, IgG and IgM, Serum and ALUPP / Lupus Anticoagulant Profile, Plasma concurrently with this test.

Collection Container/Tube:

Preferred: Serum gel

Acceptable: Red top

Submission Container/Tube: Plastic vial

Specimen Volume: 0.5 mL

Collection Instructions: Centrifuge and aliquot serum into a plastic vial.

If not ordering electronically, complete, print, and send 1 of the following forms with the specimen:

-General Request (T239)

-Coagulation Test Request (T753)

0.4 mL

Evaluating patients with suspected antiphospholipid syndrome by identification of beta-2 glycoprotein 1 IgM and IgG antibodies

First-line test when antiphospholipid syndrome (APS) is strongly suspected in conjunction with cardiolipin antibodies (IgG and IgM) and lupus anticoagulant testing

Estimating the risk of thrombosis and/or pregnancy-related morbidity in patients with systemic lupus erythematosus

Antiphospholipid syndrome (APS) is a systemic autoimmune disease characterized by thrombosis and/or specific pregnancy-related death. Based on the 2006 revised Sapporo consensus classification criteria, the laboratory requirements for diagnosing APS include the presence of at least one of the following: lupus anticoagulant (LAC), anticardiolipin (aCL), and anti-beta-2 glycoprotein 1 (B2GP1) IgG or IgM antibodies.(1). To avoid overdiagnosis, and to exclude patients with transient antiphospholipid (aPL) levels, the APS guidance also recommends confirmation of any positive result at least 12 weeks after the initial evaluation. Of note, aPL antibodies also occur in patients with autoimmune diseases with significant prevalence in systemic lupus erythematosus (SLE) as well as other clinical manifestations (eg, heart valve disease, livedo reticularis, thrombocytopenia, nephropathy, and neurological) often associated with APS.(1-3) Thus, in addition to the 2006 APS guidance, the 2012 derivation and validation of the Systemic Lupus International Collaborating Clinics (SLICC) classification criteria for SLE recommends testing for the criteria aPL antibody tests as well as aCL IgA and anti-B2GP1 IgA.(2)

B2GP1 is a 326-amino acid protein synthesized by hepatocytes, endothelial cells, and trophoblast cells.(4) It contains 5 repetitive structures or "sushi domains," termed domain 1 through 5, for a combined molecular weight of 54?kDa for the protein.(4-6). Autoantibodies to B2GP1 may be detected by solid-phase immunoassays (SPA) and functional coagulation assays. Unlike LAC, the SPA provides quantitative measurements and antibody isotype class determinations that are important for risk assessment. Immunoassays for B2GP1 antibodies can be performed using either a composite substrate comprised of B2GP1 plus anionic phospholipid (ie, cardiolipin-dependent B2GP1) or B2GP1 alone. Antibodies detected using B2GP1 substrate without another phospholipid (direct assays) are referred to simply as "B2GP1 antibodies." Some B2GP1 antibodies are capable of inhibiting clot formation in functional coagulation assays that contain low concentrations of phospholipid cofactors.(6) Antibodies detected by functional coagulation assays are commonly referred to as LAC. Anti-B2GP1 antibodies associated with thromboembolic events target domain 1 of the molecule and are responsible for LAC (functional, phospholipid-dependent prolongation of the clotting time) and aCL-dependent B2GP1 antibody positivity.(7)

For detection of anti-B2GP1 IgG and IgM antibodies, the APS guidance advocates for the use of values above the 99th percentile of the laboratory’s population in the establishment of reference intervals for tests. While this recommendation may be used for anti-B2GP1 IgA immunoassays, there is no consensus for their determination.(6) For aCL IgG and IgM testing, the APS classification guidance recommends antibody cutoff values greater than 40 IgG phospholipid (GPL) or IgM PL (units traceable to the Harris standards for aCL antibody assays) or more than the 99th percentile for the testing laboratory’s population for positivity.(1) The use of cutoff values greater than 40 GPL or MPL units to define positivity is not applicable to all aCL antibody immunoassays as the threshold used to distinguish moderate-to-high positive from low positive results are test dependent.(4,5,7,8) In addition, the cutoff value used at the 99th percentile of a laboratory’s testing population may not be consistent with kits from the same manufacturer or 40 GPL units in the case of aCL antibodies.(5-7).

Thrombosis and obstetric complications are common clinical events in the general population and are not unique to APS; therefore, the presence of aPL antibodies is an absolute requirement for the diagnosis of definite APS.(1,5,7) Furthermore, aPL antibodies are heterogeneous with overlapping tendencies; the lack of aPL test harmonization or standardization requires the use of all 3 tests for optimal APS diagnosis.(1,5-7) The APL antibodies were traditionally determined using the classic enzyme-linked immunosorbent assay, with more diverse methods recently developed and adapted for clinical testing. Recognizing the analytical and diagnostic challenges associated with aPL antibody testing, initiatives to support assay harmonization and utilization, including the development of calibrators, test development, and validation efforts, as well as preanalytical, analytical, and postanalytical measures, have been published.(5-7) Based on these and other published studies, the interpretation and relevance of APL antibody tests are dependent on factors such as the type of aPL (LAC, aCL or anti-B2GP1), the source of cardiolipin and/or B2GP1, aPL antibody class (IgG, IgM or IgA) and level, as well as whether antibody positivity is single, double or triple.(1,5-8)

In conclusion, although the APS classification criteria were not established for routine clinical use, in the absence of formal diagnostic guidelines, these have widely been adopted to diagnose or assess risk for APS and the need for treatment or prophylaxis. Therefore, in clinical practice, if suspicion for disease is high but criteria aPL antibody tests are inconclusive or negative, deviation from the APS diagnostic criteria may be justified. This may include testing for non-criteria aPL antibody tests such as the aCL IgA and anti-B2GP1 IgA recommended in 2012 SLICC guidance for SLE and/or evaluation of anti-phosphatidylserine/prothrombin IgG and IgM autoantibodies, amongst others.(2,6,8-10)

BETA 2 GLYCOPROTEIN 1 (GP1) ANTIBODIES IgG:

<15.0 SGU (negative)

15.0-39.9 SGU (weakly positive)

40.0-79.9 SGU (positive)

> or = 80.0 SGU (strongly positive)

Results are reported in standard IgG anti-beta 2 GP1 units (SGU).

Reference values apply to all ages.

BETA 2 GLYCOPROTEIN 1 ANTIBODIES IgM:

<15.0 SMU (negative)

15.0-39.9 SMU (weakly positive)

40.0-79.9 SMU (positive)

> or = 80.0 SMU (strongly positive)

Results are reported in standard IgM anti-beta 2 GP1 units (SMU).

Reference values apply to all ages.

Positive results for beta-2 glycoprotein 1 (B2GP1) IgG and IgM antibodies, in association with specific clinical manifestations, may be diagnostic for antiphospholipid syndrome (APS).

Low levels of B2GP1 IgG or IgM antibodies, especially in the absence of other criterial phospholipid antibodies should be interpreted with a high degree of suspicion. Compared to B2GP1 IgG, low and isolated levels of B2GP1 IgM antibodies have been reported to demonstrate a low risk for APS.

Documentation of persistent anti-B2GP1 antibodies is a requirement for the diagnosis of definite APS. Antibodies must be detected on 2 or more occasions at least 12 weeks apart to fulfill the laboratory diagnostic criteria for APS.

Detection of B2GP1 antibodies using the enzyme-linked immunoassay method or other solid-phase immunoassays is not affected by anticoagulant treatment.

Immunoassays for the detection of antiphospholipid (aPL) antibodies including beta-2 glycoprotein 1 may not completely distinguish between autoantibodies specific for antiphospholipid syndrome (APS) and those antibodies produced in response to infectious agents with or without thrombosis. Since these antibodies may be transiently produced, documentation of persistence as outlined for aPL IgG and IgM antibodies in the 2006 revised Sapporo guidance for definite APS is required (see Clinical Information).

Comparative studies and interlaboratory proficiency surveys indicate that results of phospholipid antibody tests can be highly variable, and results obtained with different commercial immunoassays may yield different results.(1,5,7,8)

1. Miyakis S, Lockshin MD, Atsumi T, et al: International consensus statement on an update of the classification criteria for definite antiphospholipid syndrome (APS). J Thromb Haemost. 2006 Feb;4(2):295-306

2. Petri M, Orbai AM, Alarcon GS, et al: Derivation and validation of the Systemic Lupus International Collaborating Clinics classification criteria for systemic lupus erythematosus. Arthritis Rheum. 2012 Aug;64(8):2677-2686

3. Sciascia S, Amigo MC, Roccatello D, Khamashta M: Diagnosing antiphospholipid syndrome: 'extra-criteria' manifestations and technical advances. Nat Rev Rheumatol. 2017 Sep;13(9):548-560

4. Lozier J, Takahashi N, Putnam FW: Complete amino acid sequence of human plasma beta 2-glycoprotein 1. Proc Natl Acad Sci USA. 1984 Jun;81(12):3640-3644. doi: 10.1073/pnas.81.12.3640

5. Lakos G, Favaloro EJ, Harris EN, el al: International consensus guidelines on anticardiolipin and anti-beta2-glycoprotein I testing: report from the 13th International Congress on Antiphospholipid Antibodies. Arthritis Rheum. 2012 Jan;64(1):1-10

6. Audrain MAP, El-Kouri D, Hamidou MA, el al: Value of autoantibodies to beta(2)-glycoprotein 1 in the diagnosis of antiphospholipid syndrome. Rheumatology (Oxford). 2002 May;41(5):550-553

7. Pengo V, Bison E, Denas G, Jose SP, Zoppellaro G, Banzato A: Laboratory diagnostics of antiphospholipid syndrome. Semin Thromb Hemost. 2018 Jul;44(5):439-444

8. Devreese KMJ: Solid phase assays for antiphospholipid antibodies. Semin Thromb Hemost. 2022 Sep;48(6):661-671. doi: 10.1055/s-0042-1744364

9. Abisror N, Nguyen Y, Marozio L, et al: Obstetrical outcome and treatments in seronegative primary APS: data from European retrospective study. RMD Open. 2020 Aug;6(2):0. doi: 10.1136/rmdopen-2020-001340

10. Nakamura H, Oku K, Amengual O, et al: First-line, non-criterial antiphospholipid antibody testing for the diagnosis of antiphospholipid syndrome in clinical practice: a combination of anti-beta 2 -glycoprotein I domain I and anti-phosphatidylserine/prothrombin complex antibodies tests. Arthritis Care Res (Hoboken). 2018 Apr;70(4):627-634

Purified beta-2 glycoprotein (B2GP1) antigen is bound to the wells of a polystyrene microwell plate under conditions that will preserve the antigen in its native state. Prediluted controls and diluted patient sera are added to separate wells, allowing any B2GP1 IgM or IgG antibodies present to bind to the immobilized antigen. Unbound sample is washed away, and an enzyme-labeled antihuman IgM or IgG conjugate is added to each well. A second incubation allows the enzyme-labeled antihuman IgM or IgG to bind to any patient antibodies that have attached to the microwells. After washing away any unbound enzyme-labeled antihuman IgM or IgG, the remaining enzyme activity is measured by adding a chromogenic substrate and measuring the intensity of the color that develops. The assay can be evaluated spectrophotometrically by measuring and comparing the color intensity that develops in the patient wells with that of a 5-point calibration curve. Semiquantitative results are reported in standard IgM and IgG anti-B2GPI units (SMU and SGU, respectively).(Package inserts: QUANTA Lite beta 2 GP1 IgM ELISA. Inova Diagnostics; Revision 16, 09/2018; QUANTA Lite beta 2 GP1 IgG ELISA. Inova Diagnostics; Revision 19, 07/2020)

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This test has been cleared, approved, or is exempt by the US Food and Drug Administration and is used per manufacturer's instructions. Performance characteristics were verified by Mayo Clinic in a manner consistent with CLIA requirements.

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