Hyperbilirubinemia

* Context.-Paraprotein interference in automated chemistry is uncommon. We describe 2 patients with paraproteinemia and elevated total bilirubin levels measured erroneously using the Roche total bilirubin assay.

Objectives.-To explain the mechanism of this artifactual hyperbilirubinemia and to determine its frequency in patients with monoclonal or increased immunoglobulins.

Materials and Methods.-The assay was performed manually using serum from 2 index patients and from control patients (without M proteins). Total bilirubin was also determined using another manufacturer's assay. A prospective study was then undertaken using serum from 100 consecutive patients with various monoclonal gammopathies and from 13 patients with polyclonal hypergammaglobulinemia and cryoglobulins. For all patients, serum immunoglobulin (Ig) G, IgA, IgM, total and direct bilirubin, creatinine, and a direct spectrophotometric assessment of icterus were measured.

Results.-After the addition of assay reagents, a white

precipitate formed in the reaction mixtures containing serum from the index patients, but not in other samples. This turbidity, rather than the expected color change to pink, increased the absorbance and falsely elevated the total bilirubin value. Serum from both index patients was anicteric, their direct bilirubin measurements were unaffected, and total bilirubin measured using an alternate assay was normal. Among the 113 patients studied, no additional spu rious total bilirubin values were detected.

Conclusion.-Paraprotein interference with the Roche automated total bilirubin assay is caused by precipitate formation. This interference is rare and probably idiosyncratic. Spurious hyperbilirubinemia from paraprotein interference may cause clinical confusion. If artifactual elevation of total bilirubin is suspected, the laboratory should examine the specimen for icterus (manually or by spectrophotometry) or measure total bilirubin using a different method.

(Arch Pathol Lab Med. 2003;127:55-59)

Monoclonal immunoglobulins (Igs) may interfere with a wide variety of automated nephelometric, turbidimetric, and immunologic assays.1 Their interference with total bilirubin measurements has not been reported. We identified 2 patients within a 10-month period who had paraproteins and spuriously elevated total bilirubin levels, when measured using the Roche total bilirubin assay on the Hitachi 917 automated analyzer (Roche Diagnostics, Indianapolis, Ind). The unexpected hyperbilirubinemia in these patients posed a perplexing problem, as neither patient was jaundiced. We attempted to explain the mechanism of this artifactual hyperbilirubinemia and also to determine the frequency of this phenomenon in patients with monoclonal or increased immunoglobulins.

REPORT OF CASES

Index Case 1

An 88-year-old hypertensive man with multiple myeloma was admitted to our hospital because of a right lower lobe pneumonia. He was taking metoprolol tartrate, lisinopril, and also furosemide for congestive cardiac failure. Initial laboratory tests revealed normal values for complete blood count, serum electrolytes, creatinine, and phosphate. Total bilirubin (refer to Table 2 for reference ranges) on admission, determined using the Roche assay on the Hitachi 917 analyzer, was 9.0 mg/dL (153.9 (mu)mol/ L), and the direct bilirubin was 0.1 mg/dL (1.7 (mu)mol/L). The total bilirubin was reported as 19.9 mg/dL (340.3 (mu)mol/L) 4 hours later. These serum specimens were anicteric and showed no evidence of lipemia or hemolysis. Clinically, the patient was not jaundiced, and there was no supporting evidence for hemolysis or liver disease. The IgG, IgA, and IgM levels were 6000 mg/dL (60 g/L), 12 mg/dL (0.12 g/L), and 6 mg/dL (0.06 g/ L), respectively. Serum protein electrophoresis and immunofixation electrophoresis revealed a monoclonal IgG-lambda M protein. Total bilirubin levels measured using another assay were all less than 1.5 mg/dL (25.7 (mu)mol/L). Four months later when he returned for follow-up care, the patient's total bilirubin levels using the Roche assay remained erroneously elevated, up to 20.2 mg/dL (345.4 (mu)mol/L).

METHODS

Interference Demonstration

Total bilirubin, direct bilirubin, IgG, IgA, IgM, and creatinine levels as well as a direct photometric measurement for icterus (I index) were determined on the Roche Hitachi 917 analyzer (Roche Diagnostics, Indianapolis, Ind). The Roche serum total bilirubin assay used is an endpoint chromogenic assay. The reaction in this assay requires the sequential addition of 2 liquid reagents (R1 and R2) to the patient's serum sample within the reaction cuvette. In this assay, after unconjugated bilirubin is solubilized by the addition of a "solubilizing agent," all of the bilirubin is coupled with a diazonium ion in a strongly acid medium (pH 1-2) at 37 deg C to form azobilirubin.2 The color (pink) intensity of the azobilirubin produced is proportional to the total bilirubin concentration. The absorbance (at 546 nm, with a bichromatic correction at 600 nm) is determined spectrophotometrically. The R1 working reagent contains 85 mmol/L of sodium acetate buffer, 110 mmol/L of sulfamic acid, surfactant, and "solubilizer." R2 contains 3 mmol/L of diazonium ion and 100 mmol/L of hydrochloric acid.

Direct photometric measurements for icterus, lipemia, and hemolysis are performed on the Hitachi 917 by adding sample to saline in a separate cuvette and taking a total of 6 absorbance readings. For the I index, the wavelengths used are 480 nm and 505 run, with corrections for the contributions from hemolysis (570 nm and 600 nm) and lipemia (660 run and 700 nm).3 The I index is calibrated such that the units correspond to the bilirubin concentration in mg/dL. Total bilirubin levels in samples from the index patients were also determined using the Dade Behring Dimension (Newark, Del) clinical chemistry system. In this assay, bilirubin (unconjugated) in the sample is solubilized by dilution in a mixture of caffeine, benzoate, acetate, and EDTA.4

Imprecision of the Roche total bilirubin assay was demonstrated by measuring the total bilirubin value several times on the Hitachi 917, using different samples received for the first index patient. Each result was then compared with the corresponding I index for that specimen. To illustrate the phenomenon causing the interference with the total bilirubin measurements, the Roche assay was performed manually for both index patients. All volumes were accordingly increased in magnitude, while still maintaining the sample-reagent ratios specified by the manufacturer. For the first patient's sample, absorbance at a wavelength of 546 run was measured after each step of the assay using a Gilford Stasar III spectrophotometer (Oberlin, Ohio). The same measurements were manually repeated on distilled water (blank) and again on serum from a control patient (with no M protein), whose total bilirubin level was 18.3 mg/dL (313 (mu)mol/L). For the second index patient's serum and for 2 control patient sampies, one with a normal total bilirubin level of 0.2 mg/dL (3A (mu)mol/L) and the other with an elevated level of 23.1 mg/dL (395 (mu)mol/L), the individual steps of the assay were recorded using digital photography. All incubations were timed as in the automated assay and performed in a 37 deg C water bath. The absorbance data for these samples from the total bilirubin assay on the Hitachi 917 were retrieved. Attempts to redissolve the precipitate from the macroscale reaction mixture of the second index patient were unsuccessful, precluding further analyses of the precipitate.

Patient Selection and Analysis

We performed a prospective study using serum samples from 100 available, consecutive patients with electrophoretically documented monoclonal gammopathy. These samples were identified during the routine interpretation of serum electrophoresis in our clinical laboratory during a 6-month period. An additional 11 individuals who presented during this period with polyclonal hypergammaglobulinemia and 2 with cryoglobulins were also studied. For all patients, serum IgG, IgA, IgM, total and direct bilirubin, creatinine, and an I index were determined on the Hitachi 917 instrument. Any discrepancies between the serum color, I index, and total bilirubin measurements were noted. Creatinine was measured because of previous reports of false measurements of this analyte due to paraprotein interference with automated methods.5-7

RESULTS

Assay Interference

Total bilirubin measurements, when repeated several times for our first patient, revealed an imprecise and positive bias (Table 1). When the assay was performed manually, a fine white flocculent precipitate, but no change in color, was noted after both R1 and R2 reagents were added to serum for both the first and second (Figure 1) patients. No such precipitate was evident in either the control or blank specimens. The precipitate remained stable during warming (37 deg C) and cooling (4 deg C) of the specimen. Only the icteric serum from control patients developed a color (clear pink) change at the end of the assay. Inspection of the absorbance readings from the total bilirubin assay on the Hitachi 917 revealed that the specimen containing the paraprotein showed increased, but delayed, absorbance when compared with the controls (Figure 2).

Population Analysis

The total and direct bilirubin, monoclonal immunoglobulin, and serum creatinine measurements for all patients are shown in Table 2. No spurious total bilirubin or creatinine measurements using Roche assays on the Hitachi 917 analyzer were detected in any of our patients with monoclonal gammopathy, polyclonal hypergammaglobulinemia, or cryoglobulins.

COMMENT

We identified 2 patients whose serum paraproteinemia resulted in a spuriously elevated total bilirubin measurement using a Roche assay on the Hitachi 917 automated analyzer. Our first patient with myeloma had a monoclonal IgG-lambda, and our second patient with Waldenstrom macroglobulinemia had a monoclonal IgM-kappa. It seems clear from the clinical histories, serum color, direct bilirubin levels, and I index measurements on the Hitachi 917, as well as the total bilirubin values determined using the Dade Behring Dimension instrument, that the Roche total bilirubin levels were falsely elevated and that the most likely cause of the interference was the paraprotein.

The absence of a solubilizing agent in the direct bilirubin assay strongly suggests that it is this agent that caused the interfering precipitate. The turbidity of this suspended precipitate, and not a color change, resulted in increased light absorption when measured spectrophotometrically, which artifactually raised the total bilirubin measurements. It is interesting that even with the bichromatic correction used in this assay on the Hitachi 917 the untoward effect of the turbidity is not eliminated. Along these lines, one can ascribe the marked imprecision of the Roche total bilirubin values on these samples to the fact that this precipitate likely formed at different times, in varying amounts, and with different particle sizes on each separate occasion. It is ironic that the human eye, but not the Hitachi 917, can easily distinguish the pink color of the genuine diazo reaction from the white turbidity of the precipitate. In contrast, the Hitachi 917 can detect the icterus (or lack thereof) of a serum sample more precisely than the human eye. Unfortunately, it cannot use the discrepancy between the I index and the measured total bilirubin to alert the operator to the problem under discussion.

Monoclonal immunoglobulins have been shown to interfere with various automated measurements. False measurements due to paraprotein interference with automated methods have been reported for creatinine,5-7 urea,8 phosphate,9-13 calcium,14 acetaminophen,15 serum iron,16 hemoglobin,17-18 C-reactive protein, and certain microbiological serological tests.1 In these reports, the interfering paraproteins were either monoclonal IgG or IgM, but never IgA proteins. The method of interference documented in these assays, as was demonstrated in this study, was attributed to the precipitation of the paraprotein, usually in an acid medium. The likelihood of this interference in most of these reports was also unrelated to either the paraprotein type or concentration. Only a single report in the Japanese literature noted a human IgG-lambda-type M protein that interfered with the automated determination of direct bilirubin.19 However, paraprotein interference has never before been reported in the literature for total bilirubin measurements. Abnormal bilirubin binding to paraproteins can occur in myeloma patients,20 but this reaction has not resulted in falsely elevated total bilirubin levels. Interference with the measurement of total bilirubin due to propranolol with certain automated diazo techniques has been documented.21-23 Our 2 index patients had not been on propranolol therapy, and none of the drugs being taken by these patients, including rituximab, have been reported to erroneously affect bilirubin values.

A customer bulletin received from Roche Diagnostics informed our clinical laboratory that a positive bias may indeed occur with their liquid total bilirubin assay in myeloma patient samples,24 but to date they have provided no explanation or information regarding the frequency of this phenomenon. Based on our series of patients with monoclonal gammopathies, the incidence of paraproteins interfering with the Roche total bilirubin assay is very low. This interference is probably idiosyncratic and is not dependent on either the paraprotein type or concentration. The interference in our 2 index patients occurred with both an IgG-lambda and IgM-kappa paraprotein, and failed to occur in the other 100 patients studied, among whom a wide variety of monoclonal immunoglobulins was exhibited. Furthermore, concentrations as high as 8784.4 mg/dL (87.8 g/L) for monoclonal and 3751 mg/dL (37.5 g/L) for polyclonal immunoglobulins did not demonstrate this phenomenon.

Whenever high total bilirubin levels are found in patients with plasma cell dyscrasias, Waldenstrom macro-- globulinemia, and possibly lymphomas associated with abnormal immunoglobulin synthesis, in the absence of signs and symptoms of jaundice, the possibility of a spurious hyperbilirubinemia due to an interfering paraprotein should be entertained. Awareness of this phenomenon may help prevent unnecessary concern and expensive invasive investigations. If artifactual elevation of total bilirubin is suspected, we recommend the following:

1. check the serum color by eye to see if it is truly icteric;

2. obtain the spectrophotometric measurement using an I index, if available on the autoanalyzer (such as the Hitachi 917);

3. rerun the assay to demonstrate any imprecision beyond what is typically observed;

4. measure direct bilirubin on the same specimen, as this assay does not require any solubilizing agent;

5. measure the total bilirubin using a different method; and

6. correlate the total bilirubin result with the clinical information, if available.

References

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2. Total bilirubin [package insert]. Indianapolis, Ind: Roche Diagnostics; 2001. 3. Reference Guide: Boehringer-Mannheim/Hitachi 917. Indianapolis, Ind: Boehringer-Mannheim; 1998:245-256.

4. Total bilirubin Flex reagent cartridge [package insert]. Newark, Mass: Dade Behring Inc; 1999.

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istics of monoclonal immunoglobulins that interfere with serum inorganic phosphate measurement. Ann Clin Biochem. 1994;31:249-254.

13. Cohen AM, Magazanik A, van-der Lijn E, Shaked P, Levinsky H. Pseudohyperphosphataemia incidence in an automatic analyzer. Eur] Clin Chem Clin Biochem. 1994;32:559-561.

14. John R, Oleesky D, Issa B, et al. Psuedohypercalcaemia in two patients with IgM paraproteinaemia. Ann Clin Biochem. 1997;34:694-696.

15. Hullin DA. An IgM paraprotein causing a falsely low result in an enzymatic assay for acetaminophen. Clin Chem. 1999;45:155-156.

16. Bakker AJ. Influence of monoclonal immunoglobulins in direct determinations of iron in serum. Clin Chem. 1991;37:690-694.

17. Goodrick MJ, Boon RJ, Bishop RJD, Copplestone JA, Prentice AG. Inaccurate haemoglobin estimation in Waldenstrom's macroglobulinemia: unusual reaction with monomeric IgM paraprotein. J Clin Pathol. 1993;46:1138-1139.

18. Roberts WL, Fontenot JD, Lehman CM. Overestimation of hemoglobin in

a patient with an IgA-K monoclonal gammopathy. Arch Pathol Lab Med. 2000; 124:616-618.

19. Aoki YJ, Kameko M, Fujita K. A case of IgG4-lambda type monoclonal immunoglobulin that interfered with determinations for albumin, direct bilirubin and iron in serum [in Japanese]. Rinsho Byori. 2001;49:686-689.

20. Gulian JM, Dalmasso C, Pesquie M, Harle JR, Charrel M. Abnormal bilirubin binding to human serum albumin in a patient with unusual myeloma immunoglobulin G. EurJ Clin Chem Clin Biochem. 1993;31:115-119.

21. Stone WJ, McKinney TD, Warnock LG. Spurious hyperbilirubinemia in uremic patients on propranolol therapy. Clin Chem. 1979;25:1761-1765.

22. AI-Damluji S, Meek JH. Interference of a propranolol metabolite with serum bilirubin estimation in chronic renal failure. Br Med]. 1980;280:1414.

23. Belsey R, Mueggler P, Swanson JR. Propranolol and spurious hyperbilirubinemia. JAMA. 1984;251:38.

24. Multiple Myeloma Interference in Total Bilirubin. Customer bulletin 00117. Indianapolis, Ind: Roche Diagnostics; June 30, 2000.

Liron Pantanowitz, MD; Gary L. Horowitz, MD; Jan N. Upalakalin, MD; Bruce A. Beckwith, MD

Accepted for publication July 17, 2002.

From the Department of Pathology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Mass.

Reprints: Department of Pathology, Beth Israel Deaconess Medical Center, Harvard Medical School, 330 Brookline Ave, Boston, MA, 02215 (e-mail: lpantano@caregroup.harvard.edu).

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