Antiphospholipid Antibodies
Antiphospholipid antibodies comprise a family of autoandibodies, which have a well-established association with fetal loss. They are most commonly associated with fetal loss, thrombocytopenia or thrombotic events without evidence of autoimmune disease viz. the antiphospholipid syndrome.
Primary Antiphospholipid Syndrome—must include one clinical and one serological feature.
Clinical features
1. Recurrent venous or arterial thrombosis
2. Recurrent fetal loss
3. Thrombocytopenia
Serological features
1. IgG aCL>20 GPL
2. LA
3. IgM aCL>20 MPL + LA
I MPL or GPL is equivalent to 1 ug of affinity purified aCL IgM or IgG.
Diagnostic Tests—The antiphospholipid antibodies are a diverse family of autoantibodies which share in common reactivity with negatively charged phospholipids.
There are three clinically significant members—viz. biological false positive test for syphilis.
· Lupus anticoagulant
· Anticardiolipin antibodies
I. Biological False Positive Test For Syphilis
A biological false positive test can result from :
1. Antibodies produced in response to infection by a number of non treponemal pathogens. In this situation the BFP is likely to be transient, reflecting recent activity of the pathogen. These antibodies are not associated with thromobosis or fetal loss.
2. Autoantibodies produced by patients with autoimmune discuses, particularly patients with APS and/or SLE. These autoantibodies can persist for many years and are associated with both thrombosis and fetal loss.
The BFP reaction requires
1. that an phospholipid based screening assay such as the VDRL is persistently positive for 6 months.
2. that the absence of treponemal infection is confirmed by a non-phospholipid-based assay such as the TPHA.
II. Lupus Anticoagulant
It comprises autoantibodies of either IgG or IgM class which prolong phospholipid-dependent coagulation assays by reacting with negatively charged phospholipids. Coagulation assays used to screen for LA include the activated partial thromboplastin time (APTT), Kaolin clotting time (KCT), dilute Russell Viper venom test (dRVVT) and dilule tissue thromboplastin assay (dTta).
The minimum criteria for the detection of LA proposed by Triplett & Brandt (1989) are :
1. A prolongation of a phospholipid-dependent screening test such as the APTT.
2. Demonstration that the abnormality is due to an inhibitor rather than a factor deficiency.
3. Proof that the inhibitor is directed against phospholipids.
In order to confirm the presence of an inhibitor the screening test is repeated using a mixture (1 : 1) of the patient’s plasma with normal plasma. If the abnormality is due to an inhibitor the test will remain prolonged. If the abnormality is due to a factor deficiency the normal plasma will act as a source of the factor and the test result will correct to normal.
Similarly a platelet neutralization procedure (PNP) is used to confirm the antiphospholipid nature of an inhibitor, Lysed platelets are added to the abnormal plasma, and the screening test (APTT or dRVVT) is repeated. An abnormality caused by LA will correct in a PNP whilst an abnormality due to a factor inhibitor will not.
III. Anticardiolipin Antibodies
It is detected by solid phase immune assay (enzyme-linked immuno sorbent assay (ELISA) or radio immuno assay (RIA). The aCL assay has proved to be 200-400 times more sensitive than the VDRL test and detected 90% of LA in a population.
IV. Antibodies to other phospholipids
Phospholipid antigens other than cardiolipin have been used in solid-phase assays for aPL because cardiolipin being found exclusively in the mitochondria is unlikely to be the physiological antigen for aCL as it is not exposed to circulating aPL.
Phosphatidyl serine has been used as an alternative antigen as it is located in membranes of endothelial cells and platelets. However, PS is found in the interior leaflet of non-activated cell membranes and is only exposed to circulating antibodies after cell activation causes its transfer to the exterior leaflet. Some use phosphatidyl ethanol amine which is in both the exterior and interior leaflets of cell membranes and thus is exposed to circulating aPL.
Anticardiolipin antibody cofactor, B2 Glycoprotein
The requirement of a cofactor to facilitate the binding of aCL to phospholipids has been described recently. The cofactor, which is present in normal serum, has been identified as B2-glycoprotein (B2-GP1 - also called apolipoprotein H).
The invitro properties of B2 - GP1 are :
1. Inhibitor of intrinsic phase of coagulation.
2. Inhibitor of platelet activation.
3. Phospholipid binding protein.
The formation of a trimolecular complex involving B2 - GP1, aCL and phospholipid prevents the normal function of B2 - GP. Also, Matsuura et al (1990)have demonstrated that, unlike aCL of autoimmune origin, aCL resulting from syphilitic or other infections bind to phospholipid in the absence of B2 - GP1. This difference in the cofactor requirements of aCL from various origins explains why only aCL of autoimmune origin are associated with thrombosis and recurrent fetal loss.
Prevalence of aPL, both LA and aCL, in the general obstetric population is 2%.
There is a definite correlation between the presence of aCL and LA. Among patients with SLE and LA, 59% will have aCL and in those with SLE and aCL, 45% will have LA. Incidence of aPL in women with three or more first trimester miscarriages varies between 14%and 42%. Parke et al (1991)found a positive frequency of aPL in women with recurrent miscarriage as 16%, women who had undergone normal pregnancy as 7% and women who had never been pregnant as 3%. Late fetal losses are also associated with aPL-incidence being 30-40%. Parazzini et al (1991)concluded that there is no apparent justification for considering aPL to be a risk factor for fetal loss among women who present with spontaneous miscarriage or fetal death and have no previous spontaneous fetal loss. Lockshin (1987)noted that fetal death occurred in 77% women with SLE with aCL alone versus 50% patients with LA alone. He concluded that aCL was more sensitive and specific for predicting fetal death than LA.
(E) PATHOGENESIS
I. Effect on Platelets
Early workers described thrombocytopenia as a frequent finding in patients with aPL. Also thrombosis associated with APS is congruent with aPL binding to and disrupting the function of platelet membrane phospholipids. However, the negatively charged phospholipids, with which aPL react, are present only in the inner leaflet. Therefore, non-activated platelets should not be antigenic for aPL. Upon activation the distribution of phospholipids in the platelet membrane is altered with the transfer of the negatively charged PS to the exterior leaflet of the membrane. This permits the platelet to participate in the processes of coagulation and may make them antigenic for aPL.
II. Effect on the Vascular Endothelium
(1) Inhibition of prostacyclin production—aPL which reacts with membrane phospholipid would inhibit the production of prostacyclin by the vascular endothelium and promote thrombosis. aPL inhibits the release of arachidonic acid from membrane phospholipid.
(2)Inactivation of the protein C/protein S/Thrombomodulin pathway—Protein C acts as an anticoagulant by inhibiting the activated coagulation factors Va, VIIIa and platelet bound Va-Xa complex. Two steps in this pathway are phospholipid dependent and may be inhibited by aPL.
a. Protein C is activated by thrombo modulin - a protein present on the surface of vascular endothelial cells. Thrombomoduln must be bound to phospholipid to yield optimum protein C activation. It has been demonstrated that aPL can inhibit the invitro activation of protein C by thrombomodulin.
b. Once activated, protein C requires a co-factor, designated protein S, which facilitates the binding of activated protein C to the platelet membrane. Once bound to the platelet membrane, the protein C/protein S complex then inhibits coagulation factors Va and Xa. aPL can interact with phospholipids and inhibit the protein S dependent anticoagulant activity of activated protein C.
(3) Inhibition of antithrombin III anticoagulant pathway—Endothelial cells express upon their surface heparin like molecules-glycosaminoglycans (GAGS)-which activate antithrombin III. The GAG heparan sulphate is believed to be the physiological activator of ATIII and is particularly important as an anticoagulant in the microcirculation. It has been proposed that aPL could cross-react with GAGS and inhibit the activation of ATIII.
Clinical Associations with APS
The hall mark of the presence of aPL is the triad of arterial and venous thrombocytopenia and fetal loss.
Medical Disorders
(I) Neurological Disorders
1. Transient ischaemic attacks
2. Amaurosis fugax
3. Migraine like headaches
4. Acute ischaemic encephalopathy
5. Multi-infarct dementia
6. Degenerative myelopathy.
(II) Cardiac Disorders
1. Valvular lesions
2. Valvular or chamber thrombosis
3. Coronary artery occlusion
(III) Adrenal gland involvement
1. Addison’s disease
(IV) Skin
1. Live do reticularis
(V) Lungs
1. Pulmonary hypertension
Snedden’s Syndrome comprises the clinical triad of live do reticularis, cerebrovascular occlusion and labile hypertension in the presence of aPL. Evidence for an underlying collagen disorder can be found in 30-40% of individuals with aPL.
Drugs such as procainamide, guanidine, phenytoin, chlorpromazine, valproic acid, amoxycillin, hydrallazine and propanolol have been reported to induce aPL but drug induced aPL is generally not associated with thrombotic complications or fetal loss.
Obstetric disorders
1. First trimester miscarriage.
2. Later fetal loss with evidence of growth retardation.
3. Later fetal loss without evidence of growth retardation.
4. Placental abruption.
5. Pre-eclampsia-often severe and early onset.
6. Chorea gravidarum.
Bird Sall et al (1992)found that 33% of women who had a shill birth due to placental abrupion had aPL.
Indications for investigation of the presence of antiphospholipid
antibodies in an obstetric population
1. All autoimmune diseases
2. Thrombocytopenia
3. Previous arterial or venous thrombotic event
4. BFP VDRL
5. Recurrent (>3) first trimester miscarriages
6. All fetal losses after 20 weeks of pregnancy
7. Placental abruption (previous or current pregnancy)
8. Fetal growth retardation (previous or current pregnancy)
9. Severe early onset pre-eclampsia (previous or current pregnancy)
10. Chorea gravid arum.
Management
Specific therapy
I. Women with antiphospholipid antibodies without previous loss or significant medical disease.
—Managed with careful monitoring alone
II. Antiphospholipid syndrome with fetal loss
A. Low level antibodies (aCL<60 GPL; KCT<250s)
Most patients of this group deliver a normally grown infant at term without intervention.
These women are treated with low dose aspirin—monitoring their LA level during pregnancy and observe closely.
B. High litre antibodies (aCL>60 GPL; KCT>250s).
These patients require active treatment with close monitoring of fetus and mother.
Treatment is with low dose aspirin + (i) Heparin 10,000 IU s/c BD in patients with previous thrombotic event.
(ii) Corticosteroids to reduce KCT<200 s in patients with no thrombotic event.
Various therapies for APS
(A) Single Agent Therapy
1. Corticosteroids :
Mechanism of action—Immune suppression by inhibiting the production of Interleukin 2 by T4 cells.
Dosage—40-60 mg/day
The steroid dose is titrated against the KCT value to maintain a KCT value below 200s, usually commencing with prednisone 40 mg and measuring KCT values monthly. For maintenance low dose prednisone 10-20 mg/day is used. Administration of corticosteroids will suppress the LA but not aCL.
Side effects : Cushinoid features, acne, adrenal insufficiency, diabetes mellitus, oral candida, hypertension, osteoporosis.
Overall live birth rate—44%.
2. Heparin :
Mechanism of action—Facilitates the action of ATIII.
Dosage—Subcutaneous heparin sufficient to increase APTT to 1.5-2 times normal in those with normal APTT, or if APTT prolonged already heparin dose sufficient to achieve thrombin time of >100s.
Usually a dosage of 10,000 IU twice daily is used in women with previous thromboembolis or those with aPL in high titre.
Side effects : Bruising, thrombocytopenia, osteoporosis.
Overall live birth rate—77%.
3. Aspirin :
Mechanism of action—Inhibits cyclooxygenase in the platelet which preferentially lowers platelet thromboxane, leaving endothelial prostacyclin synthesis relatively intact.
Dosage—75-80 mg per day.
Overall live birth rate—83%.
(B) Combination therapy
1. Coricosteroid + low dose aspirin
Over all live birth rate—68%
2. Heparin plus low-dose aspirin
Overall live birth rate—88%
3. Azathioprine plus corticosteroid
(75-100 mg)
4. Immunoglobulin prednisone plus low dose aspirin
Overall live birth rate—71%.
General Care
Woman with APS is at significant risk of hypertension, venous thrombosis, pulmonary embolism or thrombocytopenia. Besides she may develop activation of her autoimmune disease.
(C) Asymptomatic Antinuclear Antibodies
Significantly greater prevalence of low-titer antinuclear antibodies (ANA) has been reported in patients with unexplained fetal losses before viability than in normal control subjects. High frequency of positive ANA titres also occurs in patients with fetal losses caused by nonimmunologic factors such as uterine anatomic malformations and luteal phase defect. ANA titre is between 1 : 20 and 1 : 160 and the fluorescent pattern is usually speckled or homogenous. Occasionally there will be a positive history of nasal or oral ulcerations, skin sensitivity, or unexplained recurrent musculo-skeletal pain. A positive ANA tite in a patient with prior early pregnancy losses indicates the need for further investigations of autoimmune factors viz. LAC; ACA and BFP-ST. If any of these tests is positive, autoimmunity is most probably responsible for pregnancy losses.
(F) CLINICAL FEATURES
The clinical types of abortions which obstetrician usually encounters regarding early pregnancy losses are
1. The patient with first trimester vaginal bleeding sub grouped into
a. Threatened abortion
b. Inevitable abortion :
Complete
Incomplete
2. The patient with fetal death or second trimester abortion—Missed Abortion
3. The patient that has a history of multiple early pregnancy losses—Recurrent Abortion.
Threatened Abortion
The most common symptom of a patient with impending abortion is vaginal bleeding. In most patients there is an interval of several days between the onset of symptoms and the actual miscarriage. However, in some the symptoms progress rapidly so these patients should be examined within a reasonable time.
At the time the ovum is becoming embedded in the uterus, that is, at the time the first menstrual period is missed or soon after, a slight implantation hemorrhage is not uncommon. This diagnosis can only be made in retrospect and the safe rule is to regard any bleeding, no matter how little, which is not explained by a lesion on the vagina or cervix as evidence of threatened abortion. The bleeding is indicative of some degree of separation of the chorion from the decidua and it varies in amount, duration and type. At first the discharge is bright red. When it changes to dark brown it means that active bleeding has ceased and that old blood in the uterus is undergoing dissolution.
Sometimes the patient also complains of backache and mild lower abdominal discomfort due to uterine contractions. However the pain is usually mild in these cases.
Inevitable Abortion
Abortion becomes inevitable if, in addition to the clinical features of threatened abortion, there are painful uterine contractions, dilatation of the cervix, or extrusion of some part of the conceptus through the os. Ballooning of upper vagina, tenderness of the uterus and pyrexia are other suggestive signs.
Many abortions occur with a minimum of warning and upset, others are characterized by recurrent haemorrhage and this may produce serious shock. Retention of the products in the canal can itself produce shock.
Incomplete Abortion—The bleeding does not get progressively less but varies from day to day, becoming heavy from time to time. It continues intermittently for weeks and months and may be accompanied by periodic uterine colic. A normal menstrual rhythm is not re-established.
In long standing cases, the attachment of the chorion to the uterus becomes organized into fibrous tissue and becomes a placental polyp.
On examination the uterus is slightly enlarged and often softened. Internal os is patulous —a very important sign indicating that there is something within the uterus.
Complete Abortion
The bloody discharge decreases progressively and usually ceases within 7-10 days. The first menstrual period occurs 4-6 weeks later.
Clinical assessment of patients with
first trimester vaginal bleeding
Firstly the gestational age of the pregnancy should be estimated by clinical dating and by assessment of uterine size. A large for date uterus may indicate a Hydatidiform-mole whereas a small uterus suggests a blighted ovum. Presence of a tender adnexal mass suggests an ectopic pregnancy. Also, the pelvic examination may show cervical changes if the process is advanced.
For pregnancy of less than 6 weeks GA, serum hCG is done to decide whether an ultrasound should be done and how to interpret its results. It is possible to see a gestational sac inside the uterus using TVS only when S.hCG is 1000 MIU/ml or more.
Patients with first trimester bleeding and serum hCG levels below this critical value should have a repeated quantitative hCG evaluation 3 days later.
· If the hCG value doubles—pregnancy is likely to be intrauterine and there is a high probability of a normal outcome.
· If the hCG value does not double and initial progesterone and estradiol concentrations are low (progesterone < 15 ng/ml estradiol < 200 mg/ml), then the pregnancy is abnormal, either a blighted ovum or an ectopic.
The predictive accuracy of low values for hCG, progesterone, and estradiol is 90-95% and most patients exhibiting this combination have spontaneous abortions or tubal pregnancies. The prediction of a normal outcome based on favorable hormone concentrations early in gestation is accurate in 80% of the cases. Also sonographic evidence of normal fetal cardiac activity in these patients indicates a low risk for abortion. Simpson et aldemonstrated that the fetal loss rate after documenting the presence of fetal heart motion by USG at 8 weeks is only 3.2%. This finding has been confirmed by other investigators.
Patients with first trimester vaginal bleeding as well as reassuring ultrasound and hormonal findings should be told that the possibility of spontaneous abortion is small (2.5% to 3.2%). Bed rest + progesterone supplementation in cases with serum value < 15 ng/ml is recommended in these cases.
Patients with blighted ova need karyotyping of the products of conception. If the karyotype of the blighted ovum reveals an autosomic trisomy, the patient will be at higher risk for a subsequent trisomy offspring and genetic amniocentesis should be recommended in subsequent pregnancies. If the karyotype shows structural rearrangement of the chromosomes, parents should go for karyotyping to rule out the possibility that one is a carrier of a translocation or an inversion.
Cases with inevitable abortion require evaluation of the uterus under general anesthesia. However patients in shock should be resuscitated first.
Missed Abortion
Sometimes the fetus dies in utero but the uterus fails to respond normally by expelling it. The fetus then becomes macerated or mummified, the liquor amnii is absorbed and the placenta becomes pale and thin. Carneous (Blood) Mole is one variant caused by multiple hemorrhages in the choriodecidual space. A mass of partly organized blood clot and chorion is formed eventually after absorption of the dead fetus.
Patient usually has complained of slight uterine bleeding following symptoms and signs of a normal pregnancy. The discharge clears up temporarily and this pregnancy is apparently progressing normally until it becomes clear after some weeks of observation that the uterus is not growing, indeed it becomes smaller and harder in consistency. Sooner or later hemorrhage recurs or there may be an intermittent brown discharge. Breast signs retrogress and symptoms such as nausea disappear. Diagnosis is usually made by ultrasound.
Complications include disseminated intravascular coagulation caused by products of placental degeneration but is unlikely to occur for at least 3 weeks after the death of the pregnancy. Stimulation of the uterus can precipitate the trouble presumably by raising the intrauterine pressure to drive thromboplastin into the circulation. So blood tests for fibrinogen content and for clot stability before and during the induction of abortion are must. Normal level of fibrinogen is 350-450 mg%. The danger level is < 100 mg%.
Treatment of afibrinogenaemia is only evacuation of uterus, which causes correction in 12-14 hours.
Histologic and microbiologic examination of the placenta is a fundamental part of the evaluation of these patients as the most common reasons for early fetal demise include chromosome abnormalities, antiphospholipid syndrome, ascending infection, subchorionic hematomas and abnormal placentation. Placenta will show extensive acute inflammatory changes in patients with ascending infection and typical lesions in patients with chronic villitis caused by cytomegalic virus infection. Thrombosis of fetal and maternal vessels will be seen in patients with protein C deficiency—possibility of fetal chromosome abnormalities can also be diagnosed.
Fetal autopsy to rule out genetic syndrome, fetal blood for karytyping as well as for bactrial cultures for mycoplasma, ureaplasma, chlamydia and listeria may be done.
ANA titer, anticardiolipin antibody, lupus anticoagulant and a TORCH titer may also help searching for the cause. HSG is recommended in unexplained cases a few weeks after their miscarriage to rule out a uterine anomaly. Induction with laminaria tents for cervical dilation followed by prostaglandin suppositories or high dose oxytocin are preferable over uterine evacuation and curettage—so as to get an intact fetus and placenta.
Recurrent Abortions—Incidence is 1%. Three consecutive pregnancies ending in spontaneous abortion constitute the definition of recurrent abortion.
Clinical Assessment and Diagnostic Work-up
Majority of patients will have recurrent anembryonic abortions or blighted ova in this group. Following history and findings are suggestive of recurrent blighted ova :
i. Finding of molar degeneration in the histological examination of the products of conception of a prior abortion is suggestive of triploidy.
ii. Previous abortions showing empty sacs on ultrasound examination is suggestive of trisomy 16.
iii. History of a malformed infant in the family suggests balanced translocation in one of the parents.
iv. Interval infertility is again suggestive of balanced translocation in the parents.
v. History of repetitive abortions occurring before 12 weeks.
(a) The probabily of a successful pregnancy in such cases is 62%without any treatment.
(b) Recurrent abortions with early fetal demise—Need evaluation of following factors.
i. Anatomic abnormalities of uterus and cervix best assessed by HSG (Hysterosalpingography) surgery, cervical cerclage can correct the defect.
ii. Corpus luteum deficiency :
Results in progesterone deficiency leading to pregnancy loss in first trimester especially between 8 and 12 weeks when the production of progesterone switches from the corpus luteum to the developing placenta. Typically, these patients have uterine contractions for several days preceding the onset of bleeding and the abortion. Diagnosis is made by endometrial biopsy or serum progesterone estimation during the secretory phase of the cycle.
iii. PCOD & Hyperandrogenism diagnosed by serum luteinizing hormone/follicle-stimulating hormone (LH/FSH) ratio and total testosterone concentrations. Other endocrinal problems like thyroid gland dysfunction should be treated. GnRH agonists for pituitary suppression followed by induction of ovulation with hCG is helpful in PCOD. For hyperandrogenism prednisone is used.
iv. Growth retarded but chromosomally normal fetuses. A biopsy of placental implantation area (obtained by curetting at the time of delivery) shows the lack of physiologic changes in the spiral arteries.
v. Autoimmune disorders diagnosed by ANA titer, lypus anticoagulant, VDRL, anticardiolipin and SS-A antibodies. Testing for HLA sharing or antipaternal antibodies has no role in establishing a prognosis. Low dose aspirin + prednisone 40-60 mg/day or low dose aspirin + subcutaneous heparin 5000 U 12 hrly is recommended.
vi. Repeated ascending infections due to group B streptococci, mycoplasma, ureaplasma and chlamydia can cause repetitive early pregnancy losses in patients who are carriers of the organism causing the infection and in patients with some degree of cervical in competence.
Cerclage operation + antibiotic treatment is recommended in these cases.
(G) CONCLUSION
Differentiation of all abortion cases into embryonic and anembryonic pregnancies before abortion occurs with the advent of ultrasonography has simplified the counselling of patients with threatened abortions and of patients who had one or more spontaneous abortions. Couples with blighted ova do not require extensive workup, whereas patients who have aborted cytogenetically normal fetuses need as extensive search for nongenetic factors responsible for the pregnancy loss.
Eventually it can be concluded that the obstetrician should investigate each pregnancy loss until the underlying cause is discovered. Otherwise, the diagnosis and management of patients with repetitive early pregnancy losses will be affected by inadequate information
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