A new interference in some digoxin assays: Anti-murine heterophilic antibodies

Background: We describe a patient with cirrhotic liver disease and atria1 fibrillation who was treated with spironolactone and digoxin. He was hospitalized because of an incidental finding of a high serum digoxin level (4.2 pg/L), but he remained asymptomatic without emerging arrhythmias. Despite discontinuation of both drugs, his serum digoxin level persisted at or above 3.0 ug/L for -5 weeks, drawing into question the accuracy of the digoxin assay. Methods: Additional digoxin methods gave lower, discrepant results, providing evidence of an assay inter- ference, and several possible sources of digoxin false positivity were evaluated. This included assessment of the contribution of digoxin-like immunoreactive factor (DLIF), d i g oxin metabolites, and spironolactone. Because the routine digoxin assay used a monoclonal antibody, we also tested for another hypothetical interference: human heterophilic (“anti-mouse”) antibodies. Results: We found no contribution from DLIF, digoxin antibodies, or spironolactone to the apparent digoxin results. However, the use of protein A to complex and selectively remove immunoglobulin G molecules markedly lowered the apparent digoxin value, as did the less specific process of ultrafiltration. Conclusions: These results suggest a previously unreported cause of digoxin false positivity: heterophilic antibodies, which have been reported to bind murine monoclonal antibodies in other assays. Because newer digoxin assays now use murine monoclonal antibodies, the possible presence of heterophilic, anti-mouse antibodies should now be considered in the interpretation of a high digoxin level. (Clin Pharmacol Ther 1996;60:593-8.)

Cesar Liendo, MD, Jalal K Ghali, MD, and Steven W. Graves, PhD Shreveport, La., and Boston, Mass.

CASE REPORT A.W. is a 64-year-old white man with atria1 fibril-

lation who was treated with digoxin. His serum digoxin levels had been stable, varying between 0.6 to 0.9 p&L over the preceding 12 months. During a routine visit to the outpatient clinic of the Overton Brooks Veteran’s Administration Medical Center, he was found to have a serum digoxin level of 4.2 t.r.g/L approximately 24 hours after his last dose and was admitted to telemetry because of the potential

From the Cardiology Section, Department of Medicine, Louisi- ana State University, School of Medicine, Shreveport, and the Endocrine-Hypertension Division, Department of Medicine, Harvard Medical School, Brigham and Women’s Hospital, Boston.

Funded in part by a grant from Marion Merrell Dow, Inc., Kansas City, MO.

Received for publication March 4, 1996; accepted July 10, 1996. Reprint requests: Steven W. Graves, PhD, Endocrine-

Hypertension Division, Brigham and Women’s Hospital, 221 Longwood Ave., Boston, MA 02115.

Copyright 0 1996 by Mosby-Year Book, Inc. 0009-9236/96/$5.00 + 0 13/l/76393

toxicity. At that time, the patient had no particular medical complaint, and after a physical examination he was found to be in no acute distress. His heart rate fluctuated between 58 to 72 beats/min. No ar- rhythmia was observed with telemetry, except his underlying atria1 fibrillation with ventricular re- sponse of .%/min. Paroxysmal atria1 tachycardia with atrioventricular nodal block, bidirectional ventricu- lar tachycardia, regularization of the ventricular re- sponse to atria1 fibrillation, and other arrhythmias associated with digitalis toxicity were absent.’

Medications at the time of admission included (1) 0.25 mg./day digoxin, which was immediately dis- continued, (2) 100 mg/day spironolactone, (3) 5 mg/ day glipizide, (4) 2 mg oral terazosin nightly, (5) 150 mg ranitidine twice a day, (6) inhalers (albuterol [salbuterol] and ipratropium), and (7) 165 mg/day acetylsalicylic acid. Despite ending digoxin therapy, his serum digoxin levels remained elevated (Fig. 1): Three days after the initial level of 4.2 kg/L, the level was -3.8 t&L. Spironolactone was discontin- ued on the fifth hospital day and furosemide was

593

594 Liendo, Ghali, and Graves CLINICAL WARMA COLOGY &THERAPEUTICS

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0 0 5 10 15 20 25 30 35 61

Days after Last Digoxin Dose

Fig. 1. Apparent serum digoxin levels in the index case patient. The hospital’s routine assay (Roche) was used to monitor concentration.

substituted, but 24 days after discontinuation of spi- ronolactone and 29 days after discontinuation of digoxin, his serum digoxin level was still 3.3 l&L and his underlying tachycardia began to reemerge: His heart rate rose from 58 to 65 beats/min on the day of admission to 80 to 90 beats/min 28 days after digoxin therapy was ended (apparent digoxin level at that time was 3.1 pg/L). An echocardiogram showed good left ventricular function, left ventricu- lar hypertrophy, and a mildly enlarged left atrium (4.2 cm).

INTRODUCTION The recent confirmation of digoxin’s safety and

efficacy in the management of heart failure,2’3 along with the increasing prevalence of this syndrome,4 make likely a steady increase in the prescription of digoxin. However, as this case illustrates, there can be problems in the assay of digoxin. Interferences include a substance(s) that crossreacts with digoxin antisera, thereby producing falsely elevated digoxin levels.‘-’ These digoxin-like immunoreactive factors (DLIF) are frequently increased in patients with kidney and liver disease and may be elevated in other medical conditions5-’ A second cause of falsely elevated serum digoxin levels, one at least theoretically possible for some digoxin assays, is an increase in digoxin metabolites.8 In general, less than 5% of digoxin is metabolized; however, some patients appear to metabolize substantially more.’ These metabolites interact with some digoxin anti- bodies more avidly than digoxin itself, producing apparent concentrations 200% to 300% greater than those actually present.8 Falsely elevated digoxin lev- els have also been described in patients taking spi-

ronolactone, although the evidence suggests that spironolactone and its principal metabolite can- renone are not directly responsible.” These three explanations were considered here and each in turn tested, but none explained the apparent digoxin lev- els seen in this patient. Because the digoxin assay used to monitor this patient employed a murine anti-digoxin monoclonal antibody, we considered an additional possibility. The presence of heterophilic (or human “anti-mouse”) antibodies has been re- ported to interfere with other monoclonal immuno- assays,lr although such an interference has not been previously described for digoxin assays. This expla- nation was tested.

METHODS Assessment of specimen procurement and digoxin

assay reliability. The specimen was drawn -24 hours after the patient’s last digoxin dose and the result was reproducible on reassay in the initial hospital assay (FARA 2 digoxin assay, Roche, Branchberg, N.J.; a monoclonal digoxin enzyme immunoassay (EIA), used according to the manufacturer’s proto- col), confirming as well that the correct patient’s specimen had been assayed. Serum specimens from this time were reassayed by other digoxin immuno- assays (TDX, Abbott Laboratories, Chicago, 111.; which includes a pretreatment precipitation step; ACA, DuPont, Wilmington, Del.; and a digoxin ra- dioimmunoassay [RIA] from New England Nuclear, Billerica, Mass.)r2 and compared to the hospital’s assay (Roche). Dilution linearity (seen with true digoxin) was tested as an assessment of interfering factors. A specimen from the first day of hospital- ization was diluted by one-half and one-fourth with

Liendo, Ghali, and Graves 595

assay zero standard and reassayed (Roche digoxin assay). On the basis of the results of these initial experiments, other experiments, assessing known sources of false positivity, were also carried out.

Assessment of the presence of DLIF. Previous stud- ies have found that the DLIF is highly protein boundI and hence that ultrafiltration (Centrifree micropartition system, Amicon, Beverly, Mass., 30,000 MW cutoff) at 4” C results in quantitative DLIF reten- tion by the filter with DLIF removal from the filtrate.i4 In contrast, true digoxin is not retained by the filter and >90% is recovered in the filtrate.i4”’ An aliquot of patient serum was ultrafiltered as previously de- scribed and compared to unfiltered specimen.i3%i4 Two methods were then used to assay the untreated and the ultrafiltered specimen: the Roche method, which has a monoclonal antibody predicted to have low DLIF crossreactivity, and the New England Nuclear digoxin method, known to have relatively high DLIF crossre- activit.y.‘6,17

Determination of the contribution by digoxin metab- olites and spironohzctone. Another serum specimen during this period (apparent digoxin of 3.6 &L) was extracted with chloroform and methanol as de- scribed previouslyi” to remove digoxin and its me- tabolites. The chloroform was collected, the organic phase was reduced to ~1.0 ml, 10 parts water were added, and this solution was loaded onto a C,, reversed-phase HPLC column (VYDAC [The Sep- arations Group], Hesperia, Calif.), followed by gra- dient elution at 1.0 mlimin as described elsewhere.15 Sequential fractions were collected (1.0 minute), divided, dried thoroughly, and assayed for activity by the New England Nuclear and the Roche digoxin assay. The New England Nuclear digoxin RIA has been previously shown to have the following cross reactivities with digoxin metabolites: for a gravimet- rically prepared concentration of 2.0 l&L, digoxi- genin: 219%; digoxigenin-mono-digitoxoside: 140%; and digoxigenin-bis digitoxoside: 180%.’ The loca- tion of digoxin, digoxigenin, and its mono- and bis- digitoxoside were determined with true standards of each. Spironolactone would be extracted by this same technique and would elute during the HPLC elution region collected for assay.

Assessment of the presence of heterophilic antibodies. Heterophilic antibodies represent human immuno- globulin G (IgG) molecules having a high affinity for mouse antibodies.18,‘y Hence, in addition to the ul- trafiltration experiments, a patient serum sample (apparent digoxin of 2.9 u&L) was incubated with protein A, a polypeptide isolated from Staphylococ-

cus aureus that tightly binds the Fc region of human IgG molecules.*’ The preparation of protein A used here was attached to a solid support to allow for its easy removal. Specifically, 0.1 and 0.2 ml of protein A coupled to a 250 km acrylic bead (Sigma Chem- ical Co., St. Louis, MO.; capacity: -10 mg human IgGiml) were washed with a phosphate buffered saline (PBS) solution (pH, 7.4). Just before use the excess PBS solution was drained off and 50 ~JJ of the patient specimen was added to displace the remain- ing PBS solution. Then 300 pJ of patient serum was added to the resin and the resin and serum were rocked overnight at 4” C in a small column with a closed fritted end. The treated serum was drained from the column (the particles with protein A being retained), and the eluate assayed in conjunction with an untreated aliquot of the same specimen with use of the Roche digoxin assay.

RESULTS Assessment of assay reliability. The initial specimen

was drawn appropriately and the initial value of 4.2 kg/L was reported by the laboratory to be reproduc- ible in its assay (Roche). Dilutions of serum (initial apparent digoxin level of 3.6 l.~.g/L) showed marked deviation from linearity (predicted concentrations: 3.6, 1.8, and 0.9 F~/L; measured concentrations: 3.6, 2.3, and 1.9 (~.giL [Roche]). In addition, single spec- imen comparison between the Roche and ACA, Roche and TDX, or Roche and New England Nu- clear methods gave markedly different results (3.6 kg/L for Roche versus 1.8 l.~g/L for ACA; 3.6 l~.g/L for Roche versus 0.2 u&L for TDX; 3.6 pg/L for Roche versus 0.29 pg/L for New England Nuclear).

Assessment of DLIF contribution. With use of an antiserum known’“,17 to have relatively high affinity for the DLIF (New England Nuclear), sera from this patient showed dramatically lower apparent concen- trations of digoxin compared with the routine method (3.6 l~,g/L for Roche versus 0.29 l~,g/L for New England Nuclear), opposite of what would have been anticipated had DLIF been present. In addition, two specimens were ultrafiltered to rc- move DLIF. This treatment produced no reduction in the digoxin RIA (New England Nuclear) values (serum No. 1,0.29 l~,g/L for unprocessed versus 0.35 kg/L for ultrafiltrate; serum No. 2, 0.25 kg/L for unprocessed versus 0.27 kg/L for ultrafiltrate).

HPLC assessment of digoxin metabolites. Extraction of digoxin and its common metabolites from serum with methanol and chloroform has been described previously. ” This same technique was applied to a

596 Liendo, Ghali, and Graves CLINICAL PHARMACOLOGY & THERAPEUTICS

NOVEMBER 1996

specimen from this patient (apparent digoxin, 3.6 u&L [Roche]). The extract, which contained any digoxin and its metabolites, was subsequently frac- tionated on an analytical C,, HPLC column and the sequential fractions assayed by two different digoxin methods (New England Nuclear and Roche). None of the fractions, including those that would contain digoxin and its metabolites, digoxigenin, and its mono- and bis-digitoxoside, showed any activity in the Roche assay. When measured by the New En- gland Nuclear RIA, only a small amount of activity (digoxin -0.2 kg/L) was found in the fraction pre- dicted to contain digoxin. Other fractions that would contain spironolactone and other steroidal com- pounds produced undetectable concentrations of apparent digoxin immunoactivity.

Assessment of the presence of heterophilic antibodies. Filtration of the patient’s serum through a 30,000 dalton exclusion membrane caused a precipitous drop in apparent digoxin levels measured in the filtrate by the Roche assay (3.1 &L for unfiltered serum versus 0.0 t&L for filtrate). Likewise, a tri- chloroacetic acid pretreatment step (TDX, which results in near total precipitation of proteins) caused a dramatic decrease in measured digoxin compared with the original value (apparent digoxin, 3.0 pg/L for Roche versus 0.5 &L for TDX). Treatment of sera from this patient with protein A to reduce IgG concentration showed a reduction in apparent digoxin concentration that depended on the amount of protein A used: with 100 PL resin, there was a small drop in apparent digoxin levels (4.2 pg/L for untreated versus 3.8 pg/L for treated). When -200 ~1 resin was used with a second specimen, a greater reduction was observed (2.9 ug/L for untreated ver- sus 1.9 &L for treated). Protein A treatment of a control specimen produced no effect on the assay.

DISCUSSION The narrow therapeutic range and significant tox-

icity of digoxin requires its routine monitoring.“21 The use of immunoassay, although almost universal for this purpose, has been shown to produce incor- rect and potentially misleading results in some pa- tients. Factors known to interfere with digoxin im- munoassays include DLIF,5-7 digoxin metabolites,‘T9 and some drugs. lo This case, with highly suspicious digoxin levels, had all of these potential sources of interference and illustrates how complicated inter- preting a potentially toxic digoxin level can be. Close clinical observation may be necessitated early, but relatively simple procedures provided evidence of

falsely elevated digoxin levels and supported less intensive surveillance or treatment.14’15

Probably the most common interference in digoxin immunoassays is the crossreactivity of a DLIF.7-9 Certain patient groups (e.g., those with kidney or liver disease) have measurable “apparent” digoxin levels, even in the absence of digoxin ther- apy, and falsely elevated digoxin levels when receiv- ing digoxin therapy.16 DLIF typically produces a nonlinear dilution pattern, but the degree of nonlin- earity is antibody dependent. In this patient the dilution pattern even over a small range was mark- edly nonlinear compatible with, but not limited to, a DLIF interference.

DLIF crossreactivity varies greatly from digoxin antibody to antibody. The New England Nuclear antibody, with relatively high crossreactivity for DLIF, measured very little apparent digoxin, whereas the Roche monoclonal antibody, which should be less affected by DLIF, measured substan- tial apparent digoxin, suggesting a limited contribu- tion from DLIF. Finally, DLIF is highly protein bound and is removed by ultrafiltration, whereas digoxin and its metabolites are not (at 4” C). No apparent digoxin concentration (by the New En- gland Nuclear DLIF-sensitive RIA) was lost with ultrafiltration, ruling out a measurable contribution of DLIF to the apparent digoxin level of this patient. Although other measures have been used to elimi- nate the influence of DLIF,14Y22 they were unneces- sary here.

The contribution of digoxin metabolites was also considered. Newer monoclonal antisera have less digoxin metabolite crossreactivity than conventional RIAs.~~,~~ Hence, the New England Nuclear assay should have had higher apparent digoxin levels than the Roche assay if metabolites were the source of apparent digoxin. That was not the case. Moreover, when we extracted and fractionated the serum of this patient on HPLC, assessing digoxin and its com- mon metabolites, neither the New England Nuclear nor the Roche assay detected any metabolite and mea- sured only small amounts of digoxin (co.25 pg/L).

Patients receiving spironolactone can have appar- ent digoxin-like activity. lo The extraction and HPLC fractionation of the serum of this patient would have included spironolactone and canrenone in the HPLC eluate. No digoxin activity was found. More- over, the biological half-lives for both spironolac- tone and canrenone are less than 24 hours.25 After discontinuation of spironolactone, its influence should have fallen to undetectable levels within a

Liendo, Ghali, and Graves 597

few days; however, the apparent digoxin levels for this patient persisted, unaffected by discontinuation of spironolactone.

Although not previously reported for the mea- surement of digoxin, we were aware of another po- tential assay interference. Immunoassays that use murine monoclonal antibodies for a variety of other analytes (e.g., thyrotropin”) have been noted to show falsely increased apparent levels in some pa- tients.i8’i9 These patients have endogenous antibod- ies that bind tightly to the mouse monoclonal anti- bodies, producing the assay interference. These human antibodies have been referred to as anti- mouse antibodies and appear to occur often in the general population (estimated as high as -9% in some reports’“), suggesting that they may be part of the body’s normal ensemble of antibodies and are not necessarily a consequence of animal bites or other direct antigen exposure, such as murine anti- bodies given as therapeutic agents.2”,2’ Our data were consistent with the presence of such hetero- philic antibodies. Marked reductions in apparent digoxin concentrations (Roche) after ultrafiltration or acid protein precipitation, although not specifi- cally demonstrating their presence, are consistent with their being present and responsible for most of the apparent digoxin activity. The reduction of ap- parent digoxin concentration after protein A treat- ment suggests strongly the presence and contribu- tion of heterophilic human IgG or anti-mouse antibodies to the factitiously elevated serum digoxin level in this patient.

In conclusion, when faced with toxic serum digoxin levels, particularly in the absence of signs of toxicity, the potential presence of mouse antibodies should be considered among other factors that falsely increase apparent digoxin levels.

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