Excerpt: Characterization of Antiphospholipid Antibodies in Women with Recurrent Spontaneous Abortions


Dr. William Matzner, Simi Valley, California

Characterization of Antiphospholipid Antibodies in Women with Recurrent Spontaneous Abortions

This  is an excerpt of an article originally published in The Journal of Reproductive Medicine and was co-authored by Dr. William Matzner.  The full article is available here. 

Introduction

It is estimated that approximately 40% of women with systemic lupus eythematosus (SLE) will have antibodies to negatively charged phospholipid, with reported ranges of 30% to 70% having suffered thrombotic events. In 1986 a group of women without known autoimmune diseases who had recurrent spontaneous abortions (RSA) or vascular thrombosis was described. These women had antibodies to a negatively charged phospholipid, cardiolipin, with high titers primarily of the IgG isotype. The mechanisms of action defined were platelet membrane damage, endothelial wall injury, inhibition of prostacyclin and inability to activate protein C; thus, RSA would occur via placental vascular insufficiency. In 1985 Harris showed that anticardiolipin antibody (aCL) bound equally to all negatively charged phospholipids. Subsequent studies that addressed issues of antiphospholipid antibodies (aPLs) therefore assumed nondifferential binding or concentrated only on aCL.

In 1985 Lockshin et al demonstrated that the level of aCL was useful as an early predictor of fetal distress or death in patients with SLE. Recent work has implicated antiphosphoserine antibody, predominantly of the IgM isotype, as an inhibitor of placental formation and a cause
of RSA.

In the study described below, aPLs were characterized in a group of women who have suffered recurrent fetal loss.

Materials and Methods

Patients

Three hundred fifty-two patients with a history of two or more consecutive spontaneous pregnancy losses were evaluated. They were premenopausal (aged 2145; mean, 39), and all were recruited between March 1991 and May 1992. Women with collagen vascular disease were excluded. Phospholipid antibodies of the IgM and IgG isotypes to cardiolipin, phosphoethanolamine, phosphoinositol, phosphoserine, phosphatidic acid and phosphoglycerol were measured.

Controls

The control group consisted of 43 people without known immunologic or rheumatologic diseases. None of the women in the control group had suffered RSA or any other thromboembolic phenomena.

aPL Assay

The assay used has been described previously. Briefly, six purified phospholipids were coated separately on Immulon 2 96-well enzyme-linked immunosorbent assay plates overnight. The plates were blocked the next day with phosphate-buffered saline (PBS) and 10% newborn calf serum for two hours and then washed (Biotech BT500) in PBS.

Fifty microliters of patient serum was added to the appropriate wells and incubated for one hour followed by a second wash with PBS. Alkaline phosphatase-conjugated goat antihuman IgG and IgM was incubated for an hour, then washed in PBS. Sigma Substrate 104 in diethanolamine buffer was added to the wells and incubated for 30 minutes at 37°C, and the reaction was stopped with NaOH. The trays were read in a BioTech BT2000 microtiter reader at 405 nm. Delta optical densities were calculated by subtracting out the background (wells without the phospholipid antigens).

The results were compared to the mean of the delta optical densities for the control group. Positivity was defined as 3 SD above the mean of the controls.

Results

The prevalence of one or more antibodies to any of the six phospholipids was 59.1% (208/352) in the study population. In the control group, only 4.6% (2/43) had a positive aPL.

Two hundred eight patients had 439 antibodies of the IgG or IgM isotype to the phospholipids. The most frequently identified aPLs in order of decreasing frequency were to phosphoserine, 20.5% (90/439); phosphoethanolamine, 19.1% (84/439); phosphatidic acid, 18.7% (82/439); cardiolipin, 16.4% (72/439); phosphoglycerol, 16.2% (71/439); and phosphoinositol, 9.1% (40/439). In the two positive controls there were 2 anitibodies, both of the IgG isotype, 1 each to cardiolipin and phosphatidic acid. In the RSA patients, 75.2% (330/439) of the antibodies were of the IgM isotype, and 24.8% (109/439) were of the IgG isotype.

Of all of the patients studied, 18.2% (64/352) had aCL versus 40.9% (144/352) with any other combination excluding aCL. Of all the patients with any aPL, 30.8% (64/ 208) were to cardiolipin. Eighty-one patients had antibodies to only one epitope: 37.0% (30/81) were to phosphoethanolamine, 14.8% (12/81) to phosphatidic acid and 16.0% (13/81) to cardiolipin. Of this group, 83.9% (68) were IgM and 16.1% (13) were IgG isotypes.

The remainder of the patients (132) had antibodies to multiple epitopes. They had 358 antibodies of the IgG or IgM isotype. In this group, in order of decreasing frequency, were antibodies to phosphoserine, 22.9% (82/ 358); phosphatidic acid, 19.6% (70/358); phosphoglycerol, 18.2% (65/358); cardiolipin, 16.5% (59/358); phosphoethanolamine, 15.1% (54/ 358); and phosphoinositol, 7.8% (28/358). In these patients, 73.2% (262/358) of the antibodies were of the IgM and 26.8% (96/358) of the IgG isotype.

About William L. Matzner, M.D., PhD, FACP

Dr. William Matzner works in the area of healthcare economics consulting at Healthcare Analytics, LLC, in California. He graduated Phi Beta Kappa from Stanford University. He received his M.D. with Honors from Baylor College of Medicine. In 1988, he was the Solomon Scholar for Resident Research at Cedar Sinai Medical Center. Dr. Matzner subsequently was awarded a PhD in Neuro Economics from Claremont Graduate University. He is board certified in Internal Medicine and Palliative Medicine. He has researched and published extensively on the issue of reproduction and immunology in medical literature. He has been in private practice since 1989, specializing in Reproductive Immunology and Internal medicine.

Consulting Website: https://healthcareanalytics.biz

 

William Matzner, MD (Simi Valley, California), has been practicing medicine since 1989, Internal Medicine and Reproductive Immunology. M.D. with Honors from Baylor College of Medicine.

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