Frequent false positive beta human chorionic gonadotropin tests in immunoglobulin A deficiency

Abstract

A patient with IgA deficiency had a series of positive serum pregnancy tests which led to medical and surgical procedures for suspected molar pregnancy. These tests were found to be falsely positive due to heterophile antibody. The aim of this study was to determine the frequency of false positive βhCG assays in sera of IgA deficient patients. Sera from a panel of IgA deficient (IgA < 7 mg/dl) patients were tested for the presence of βHCG using three different assays, and also for IgG anti-goat and anti-mouse antibodies. Patients were seen at Mount Sinai Medical Center and included 54 patients (ages 1–80 years, 32 females, 22 males) with IgA deficiency. Thirty percent of 54 IgA deficient patient sera yielded positive pregnancy tests by one or more of the three βhCG assays, however, none of the patients were pregnant. In comparison to sera of normal controls, 39% of the patient sera contained significant amounts of anti-goat antibody and 18% contained significant amounts of anti-mouse antibody. While heterophile antibodies are common in IgA deficient serum, false positive assays for βhCG in IgA deficient serum have not been previously reported. The possibility of false positive test results should be considered prior to invasive procedures in IgA deficient patients.

Introduction

Pregnancy is usually diagnosed by serum immuno assays that detect human chorionic gonadotropin (hCG), a glycoprotein hormone composed of alpha and beta subunits. While a number of immuno assays for intact hCG and its beta-subunit are in use, hormone detection relies on the incorporation in the test system of animal antibodies to hCG [1]. These assays are also essential for the successful monitoring of treatment for gestational trophoblastic disease [2,3]. If the serum to be tested contains a heterophile antibody to the animal immune globulin, false positive results can be obtained [4–7]. While uncommon, false positive hCG tests can result in unnecessary medical care and in some cases, unnecessary medications and irreversible surgical procedures [4,6–11].

The index case is a 38-year-old Caucasian woman with a history of Hashimoto's thyroiditis who had sudden onset of lower back pain, nausea, diarrhoea, lethargy and lightheadedness. Her thyroid function tests were normal and a serum βhCG test was positive. A sonogram did not show evidence of pregnancy and it was assumed she had a spontaneous abortion. Repeat βhCG measurements remained elevated (27–271 mIU/ml) and she was treated with methotrexate twice for suspected tubal pregnancy over a period of a month. Repeated sonograms again failed to show evidence of pregnancy, however, her blood continued to show fluctuating but elevated levels of βhCG. This led to laparoscopic surgery with a dilatation and curettage, again showing no indication of pregnancy. Although her menstrual periods continued on a regular schedule, subsequent βhCG levels remained elevated. A chest X-ray, CA 125 and CEA were normal. A urine pregnancy test, done for the first time after the initial positive serum test, was negative. This suggested that the elevated serum βhCG levels were false positive results, potentially due to the presence of heterophile antibodies [12–14]. It was subsequently discovered that the index case was IgA deficient (IgG = 1744 mg/dl, IgA = <7 mg/dl, IgM = 142 mg/dl).

Based on this observation, we tested a panel of stored IgA deficient sera by three immunologic assays for βhCG as well as for heterophile antibodies to goat and mouse IgG.

Materials and methods

IgA deficient subjects

Sera from 54 IgA deficient patients were analysed. All patients had IgA levels < 7 mg/dl (undetectable) by commercial nephelometry with normal levels of IgG and IgM. There were 32 females and 22 males, with an age range of 1–80 years. Patients were seen for a variety of medical issues in the Mount Sinai Immunology Clinic, including frequent infections, autoimmunity, asthma, and/or allergy; in some IgA deficiency was discovered incidentally. None were pregnant at the time of serum collection; sera was stored at−20 °C. Approval was obtained from the Mount Sinai School of Medicine IRB and patients consented to the serum collection.

Immunoassays for βHCG

Three immuno-assays for βHCG were used

• an assay for hCG dimer (intact hCG only) using mouse anti-βhCG tracer and mouse alpha subunit capture antibody (antibody 2119; gift from Unipath Inc., Bedford, UK);

• an assay using mouse monoclonal capture antibody B210 [15], which is highly specific for the urine βhCG core fragment, a component not found in the blood, with mouse tracer antibody;

• a commercial assay for whole hCG using the commercial DPC Immulite hCG (mouse monoclonal capture antibody with polyclonal goat tracer antibody) which detects all known forms of hCG and its break-down products present in serum and urine samples in pregnancy, cancer and trophoblastic disease (Diagnostic Products Corporation, Los Angles CA, USA) [16].

The DPC Immulite hCG assay was preformed with added nonspecific antibodies (Scantibodies Laboratory Inc., Santee, CA, USA) a method used to reduce or eliminate heterophile antibodies. All assays are ‘sandwich assays’ which employ different mouse monoclonal antibodies against βhCG as capture antibodies bound to the well of microtitre plates, while the tracer antibody (mouse origin except for the commercial DPC which is goat) is either enzyme or chemiluminescence labelled. To test for βhCG activity, sera from IgA deficient subjects, diluted 1 : 100 in phosphate buffered saline with 0·25% Tween-20, was added in triplicate to the coated wells, incubated, and the unbound serum proteins were removed with a washing step. Next diluted detection antibody was added, followed by incubation and subsequent removal of unbound antibody by further washing. The amount of βhCG detected in each assay was normalized to a standard curve generated concurrently using pure βhCG. Human chorionic gonadotropin is normally present at levels < 0·5 mIU/ml in non pregnant women; by day 28 of pregnancy the median hCG in serum is about 100 mIU/ml (L. A. Cole, personal communication).

Detection of heterophile antibodies

The IgA deficient sera were also tested for IgG anti-goat and anti-mouse heterophile antibodies. For this, microtitre plate wells were coated with 10 µg/ml goat IgG or mouse IgG (Sigma Chemical Company, St. Louis, MO, USA) in 0·1 m sodium carbonate buffer pH 9·6 overnight. IgA deficient sera and 20 normal control sera (controls ages 10–45: 10 male and 10 female; in triplicate) were diluted 1: 100 in phosphate buffered saline containing 0·25% Tween-20 and added to the goat IgG or mouse IgG coated wells. The plates were incubated for 3–4 h at 37°C and then washed three times with the Tween buffer. Alkaline phosphatase conjugated goat anti-human IgG or mouse monoclonal anti-human IgG (Sigma) was added to the wells, incubated for 1–3 h, and then rinsed thoroughly three more times with the Tween buffer. The reaction was developed with 1 mg/ml nitrophenyl phosphate (Sigma) and stopped with 50 µl of 1 m NaOH; the wells of the plates were read at OD 405mn after 1 h. Results for the IgA deficient sera were compared to the 20 normal sera. Sera were arbitrarily considered to have significant amounts of IgG antibody to either goat or mouse IgG if the average reactivity of the triplicate samples was greater than 1·5 times the background (buffer only) since none of the normal sera tested reached this level of absorbance.

Results

βhCG assays

Of the 54 IgA deficient patients tested, 16 (30%) had positive pregnancy tests in one or more of the three assays. The features of these 16 patients are shown in Table 1 (index case is patient E). Ten (19%) samples were positive using the intact hCG dimer assay. Of these, six patients were female and four were male. Twelve (22%) IgA deficient sera, from 6 women and 6 men, tested positive for the hCG beta core fragment. In the third assay, the DPC hCG assay which incorporates blocking antibodies, there were still four positive sera (7·4%), all female. None were known to be pregnant at the time of sera collection.

Table 1.

IgA deficient patients who had positive hCG test results.

IgA deficient patients	Age (years)	Sex	IgG (mg/dl)	IgA (mg/dl)	IgM (mg/dl)	hCG 1 (mIU/ml)	hCG 2 (mIU/ml)	hCG 3 (mIU/ml)
A	2	M	665	<7	75	6·6	0	0
B	26	F	2250	<7	160	0	11·2	0
C	56	M	2904	<7	158	0	11·6	0
D	37	M	1238	<7	87	24·9	27·6	0
E	38	F	1744	<7	142	9	10	0
F	19	M	1037	<7	250	0	10	0
G	51	F	1153	<7	252	3·3	0	2·1
H	52	F	1102	<7	105	74·3	518·4	1·3
I	13	F	1380	<7	96	0	9·2	0
J	19	F	2259	<7	430	82·5	47·2	0
K	31	F	1208	<7	89	0	16	0
L	1	M	1075	<7	104	0	11·2	0
M	2	M	1172	<7	100	14·9	16·4	0
N	49	F	2290	<7	528	4·0	0	8·6
O	58	F	520	<7	40	4·7	0	1·2
P	25	M	1280	<7	237	8·0	10	0

hCG 1, Antibody 2119 (unblocked mouse-mouse hCG ELISA); hCG2, Antibody B210 (mouse-mouse urine hCG core fragment ELISA); hCG 3, Commercial DPC (mouse-goat hCG Immulite assay with added nonspecific antibody). The index case is patient E. <0·5 mIU/ml is considered normal and all samples above this level indicate pregnancy or abnormal hCG production.

Testing for anti-mouse and anti-goat antibodies

Seventeen (39%) of the 44 IgA deficient sera available for further testing (10 samples had inadequate amounts for further assays), including serum from our index patient, contained significant amounts of IgG anti-goat IgG antibody, an amount greater than 1·5 times the buffer only control wells, a level not reached by any of the 20 control sera tested concurrently. Similarly sera of 8 (18%) IgA deficient patients, not including our index patient, demonstrated significant amounts of IgG anti-mouse IgG antibody. Seven of the sera with anti-goat or anti-mouse antibody tested positive in one or more of the hCG tests; 12 sera tested positive for mouse and/or goat heterophile antibody but did not test positive to one of the hCG tests.

Discussion

IgA deficiency is the most common of the primary immunodeficiencies with a prevalence from 1/223 to 1/1000 in European populations [17–20]. The highest frequency appears to be in Finland with the lowest in Asian countries such as Japan [21,22]. Most IgA deficient patients are healthy [18], but those who have illnesses may experience recurrent sinopulmonary infections, allergies, autoimmune diseases, gastrointestinal disorders, or very rarely, transfusion reactions [20,23,24].

Heterophile antibodies have specificity for serum immunoglobulins, or other proteins, of another species such as horse, goat or mouse [25]. Such antibodies can appear in serum due to polyclonal activation; for example during primary EBV infection, the Paul Bunnell Davidsohn heterophile antibody is generated by activated B cells [26]. This heterophile antibody agglutinates sheep or horse erythrocytes [27,28]. Heterophile antibodies can also develop after parenteral administration of animal immunoglobulins for treatment or diagnostic purposes, immunotherapy, dietary exposure, animal handling, or administration of vaccines containing animal tissue [29]. While detectable antibodies to immunoglobulin from other species can be found in almost all normal serum, the titre is generally low [30]. It has been known for some time that sera of patients with IgA deficiency often contain heterophile antibodies [31,32]; the reasons are not clear, although increased mucosal antigen exposure to the systemic circulation is likely [33,34].

False positive serum βhCG tests have been described in numerous reports; in many cases these have resulted in unnecessary, and even irreversible, medical procedures such as bilateral salpingo-oophorectomy and hysterectomy [4,6–11]. Here we show that up to 30% of the IgA deficient sera analysed lead to a false positive pregnancy test. Our index patient was subjected to numerous unnecessary procedures based on her false positive pregnancy test. Since the majority of IgA deficient individuals are undiagnosed, false positive or negative results due to heterophile antibody production may go unrecognized.

Since the majority of IgA deficient patients remain undiagnosed, the increasing use of sandwich assays in medicine may to lead to incorrect results in many areas, including obstetrics/gynaecology, cardiology, infectious diseases, oncology, endocrinology and rheumatology [13,29,35–44]. Caution is required for interpretation of immunoassay results in IgA deficient patients.

The interference of heterophile antibody can be decreased by incorporating sufficient species appropriate nonspecific immune globulin into the immunoassay [12,26,43,45,46]. Utilizing these nonspecific antibodies increases the cost of the test and thus may be impractical for use in all assays, but should be considered when a test result does not fit with the clinical scenario or prior to invasive procedures based primarily on immunoassay test results [47]. However, even with the incorporation of nonspecific antibodies, 4 of 54 of our IgA deficient sera still tested positive for hCG, illustrating the imperfect nature of this additional step. Discussion with the clinical laboratory should be done in cases where the laboratory testing is not consistent with the clinical presentation to assist in the selection of further tests or repeating the test with a different assay. Other methods, such as nonantibody based affinity protein incorporation into an ELISA assay [48], need further investigation as possible solutions. Alternatives to serum immunoassay based testing should be considered if the patient's clinical history is not consistent with laboratory findings, especially for patients with IgA deficiency.