Keeping a close watch means knowing how to eliminate false positive and false negative results in immunoassays.

By Amitava Dasgupta, PhD

Immunoassays, rather than high-performance liquid chromatography, are the preferred technique for drug measurement because they are automated and results are obtained rapidly, aiding in patient management. However, immunoassays are more susceptible to interference from endogenous factors, drugs with similar chemical structure and drug metabolites. Both false positive and false negative results are encountered in monitoring therapeutic drug concentrations using various immunoassays.

There are five major causes of interference in measuring therapeutic drug concentrations: 1) endogenous digoxin-like immunoreactive substances, 2) spironolactone/other steroids in digoxin immunoassays; 3) Digibind^® in total serum digoxin measurement; 4) metabolites in measurement of parent drugs; 5) interference of carbamazepine and fosphenytoin in phenytoin measurement by immunoassays.

DLIS

Several reports have indicated the presence of endogenous digoxin-like immunoreactive substances (DLIS) in human and other mammalian body fluids. Digoxin and digitoxin are cardiac glycosides that inhibit Na, K-ATPase. After discovery of endorphins, the endogenous equivalent of opiates, there is a hypothesis that endogenous equivalent of cardiac glycoside may exist.

It was further hypothesized that anti-digoxin antibody may be able to detect the presence of digoxin-like immunoreactivity in body fluid. Gruber et al. first demonstrated the presence of DLIS in the plasma of volume-expanded dogs.¹ Researchers first confirmed the presence of DLIS in human plasma and urine.² DLIS existed in plasma in three forms: tightly protein bound, weakly protein bound and free.³

The concentration of free DLIS is small and normally undetectable. Boiling of plasma releases DLIS from protein binding. DLIS has been found in various human body fluids or tissues including cord blood, placenta, amniotic fluid, bile, meconium, cerebrospinal fluid and saliva.^4,5

Different immunoassays for digoxin cross-react differently with DLIS in serum. The chemiluminescent assay for digoxin marketed by Bayer Diagnostics, Tarrytown, NY, has low cross-reactivity with DLIS. On the other hand, the fluorescence polarization immunoassay for digoxin (FPIA assay for digoxin, Abbott Laboratories, Abbott Park, IL) showed high cross-reactivity against DLIS. Usually, the concentration of DLIS in normal individuals is below the detection limit of even the FPIA assay for digoxin. However, under certain pathological conditions, such as liver disease, uremia, essential hypertension, hypertension with water volume expansion, renal and liver transplant recipients, congestive heart failure, premature baby and pregnancy can lead to a measurable concentration of DLIS in serum or plasma.^6-10

Elevated concentration of DLIS in serum is troublesome because DLIS falsely increases the digoxin concentrations. Digoxin has a narrow therapeutic range of 0.8-1.9 ng/ml. Recently, a study reported a 0.88 ng/ml digoxin equivalent DLIS concentration in a volume-expanded patient not receiving digoxin.¹¹

However, digoxin is only 25 percent bound to serum albumin while DLIS is very strongly bound to serum protein (>95 percent). Therefore, DLIS is absent in the protein free ultrafiltrate. Taking advantage of high protein binding of DLIS, the interference of DLIS in digoxin measurement can be completely eliminated by monitoring free digoxin concentration.^12,13

Spironolactone/Other Steroids

Spironolactone, a competitive aldosterone antagonist, has been used clinically in the therapy of hypertension and congestive heart failure for a long time. Spironolactone is rapidly and extensively metabolized; metabolite canrenone is also pharmacologically active. Spironolactone and canrenone are strongly bound to serum proteins (90 percent) and have structural similarity with digoxin. Because spironolactone and digoxin may be used concurrently in the management of a patient, interference of spironolactone and canrenone in therapeutic monitoring of digoxin is troublesome.

Spironolactone and its metabolite canrenone can falsely elevate serum digoxin levels if measured by FPIA, aca or Elecsys (positive interference) or falsely lower digoxin levels if measured by MEIA, IMx and Dimension (negative interference). The magnitude of interference is more significant with potassium canrenoate where concentration of its metabolite canrenone can be significantly higher.¹⁶

Our experience is that Bayer's digoxin assay is also free from such interference. Moreover, interference of spironolactone and the metabolite canrenone can be eliminated by ultrafiltration because both compounds are strongly protein bound. However, in the case of therapy with K-canrenoate (not used in the United States), complete elimination of this interference in certain digoxin assays cannot be achieved due to higher concentrations of K-canrenoate and higher concentration of its metabolite canrenone observed in plasma.¹⁷ A recent study demonstrated that low dose spironolactone as used for oral therapy does not cause clinically significant negative interference in the MEIA digoxin assay on Abbott's AxSYM analyzer.¹⁸

Digibind in Total Serum

Patients with acute digoxin overdose can be treated successfully with Digibind, manufactured by Burroughs Wellcome Company, Research Triangle Park, NC. Digibind binds free digoxin in serum and effectively removes pharmacologically active free digoxin. Therefore, the equilibrium between free and bound digoxin in serum is disturbed and digoxin bound to myocardium is released in serum and again is bound by antibody. The concentration of digoxin in myocardium is 25-35 times higher than the digoxin concentrations in serum.

Following Digibind treatment, the total digoxin concentrations in serum increase significantly because the protein precipitation step in the digoxin assay releases digoxin bound both to albumin and FAB fragment. If TDx is used for measuring total digoxin concentration on Abbott's FPIA assay, the observed total digoxin concentrations should be elevated significantly because in the sample pretreatment phase, digoxin bound to both albumin and digibind can be released due to protein precipitation. However, monitoring free digoxin concentration is useful to monitor the therapy with Digibind.

In one case study, a young patient took digoxin tablets for suicide. On admission to a hospital, her total digoxin concentration was 28.7 ng/ml, while the free digoxin concentration was 18.9 ng/ml (FPIA assay). Digibind was administered immediately. Half an hour after initiation of treatment, her free digoxin concentration was significantly reduced to 0.4 ng/ml, while her total digoxin concentration was increased dramatically to 90 ng/ml. At 31 hours post treatment, the patient was discharged with a free digoxin concentration of 2.2 ng/ml.

Digoxin assays that do not require sample pretreatment do not truly reflect total digoxin in patients treated with Digibind. In contrast, the values may be closer to free digoxin concentrations, although some overestimation of free digoxin levels may occur. Therefore, these assays can be used for monitoring patients treated with Digibind without the necessity of sample ultrafiltration, but some caution should be exercised.^14,15

Antibodies used in immunoassays for measuring digoxin have much higher affinity for digoxin than albumin. Assay incubation times are sufficient for albumin-bound digoxin to dissociate from albumin to bind with the antibody. Because these assays measure total digoxin, total digoxin range should be used in monitoring patients being treated with Digibind. Moreover, if incubation time is prolonged, a small amount of digoxin bound to Digibind may also dissociate and may bind with the assay antibody.

Metabolites: Carbamazepine

Carbamazepine 10, 11-epoxide is the metabolite of carbamazepine, which is present in 15-20 percent of total carbamazepine concentration at the steady state. The concentration of metabolite may be significantly higher in carbamazepine overdose or in patients with renal failure. The cross-reactivity of carbamazepine 10, 11-epoxide with different immunoassays for carbamazepine may vary between 0 percent (Vitros analyzer) to 94 percent (Dade Dimension^® from Dade Behring, Deerfield, IL).

Drug metabolites are candidates for monitoring if the metabolites are active. Carbamazepine 10-11-epoxide is an active metabolite, but it is not routinely monitored, partly because no immunoassay for the epoxide is available in the market and an HPLC method is necessary. Dade Dimension assay equally measures carbamazepine and the epoxide, as there is no clinically significant difference between values obtained by the immunoassay and carbamazepine + epoxide value obtained by HPLC. Nevertheless, cross-reactivity of inactive metabolite carbamazepine diol is another issue.¹⁹

In patients with renal failure, measurement of both carbamazepine and the epoxide with an immunoassay is probably the best approach. Carbamazepine immunoassays can still be used if the steady state concentration of epoxide is within normal limit, as is the case with many patients.

Carbamazepine, Fosphenytoin

Carbamazepine–False positive serum tricyclic antidepressant (TCA)–screen due to the presence of carbamazepine has been reported.²⁰ Researchers reported false positive TCA levels of 80 ng/ml and 130 ng/ml in two patients who never received any tricyclics.²¹

These values are significant because TCA has a narrow therapeutic range (90-130 ng/ml for nortriptyline). Although the manufacturer of FPIA for TCA–Abbott Laboratories–only recommends its use in the diagnosis of an overdose, the assay is sometimes used in therapeutic drug monitoring.²² Carbamazepine 10, 11-epoxide interferes much less than carbamazepine in the TCA FPIA assay. HPLC is the best method for therapeutic drug monitoring of TCA.

Fosphenytoin–This is a phosphate ester prodrug of phenytoin that provides improved efficacy and safety when given intravenously or intramuscularly. After systemic administration, the phosphate moiety is rapidly cleaved to form an unstable intermediate that breaks down to yield phenytoin and formaldehyde. A study reported falsely elevated phenytoin values when measured by immunoassays compared to HPLC. More interestingly, no fosphenytoin was detected by HPLC in those specimens. For example, in one with renal failure, the phenytoin concentration measured by HPLC was 5.3 mg/ml.

The corresponding phenytoin concentrations as measured by immunoassays were 22.0 (ACS: 180), 12.7 (AxSYM) and 28.0 mg/ml (TdxII). The authors studied seven hospitalized renal failure patients receiving IV fosphenytoin. The authors observed falsely increased total and free phenytoin values in all patients using various immunoassays. HPLC measurement of phenytoin showed much lower values. The authors proposed the presence of a novel metabolite of fosphenytoin, which has a very high cross-reactivity with antibodies, used in immunoassays for phenytoin.²³

Conclusion

Monitoring free digoxin can eliminate interference of DLIS, Digibind and spironolactone in serum digoxin measurement. Carbamazepine immunoassays with high cross-reactivity with carbamazepine 10, 11-epoxide complements immunoassay with low cross-reactivity because a difference between carbamazepine values obtained by these immunoassays can be used to roughly estimate epoxide concentration. Results obtained by FPIA assay for TCA should be interpreted with the knowledge of patient's drug history. HPLC is recommended for therapeutic drug monitoring of TCA.

References

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Dr. Dasgupta is professor of Pathology and Laboratory Medicine at University of Texas-Houston Medical School.