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 Table of Contents  
Year : 2022  |  Volume : 42  |  Issue : 2  |  Page : 98-100

Direct Bilirubin Level Greater than Total Bilirubin Level: A Bizarre Result Caused by Paraproteins in a Patient with Multiple Myeloma

1 Division of Clinical Pathology, Department of Pathology, Tri-Service General Hospital, Taipei, Taiwan
2 Division of Hematology and Oncology, Department of Internal Medicine, Tri-Service General Hospital, Taipei, Taiwan
3 Division of Clinical Pathology, Department of Pathology, Tri-Service General Hospital; Trace Element Research Center, Department of Pathology, Tri-Service General Hospital; Graduate Institute of Medical Sciences, National Defense Medical Center, Taipei, Taiwan

Date of Submission31-Dec-2020
Date of Decision08-Mar-2021
Date of Acceptance27-Mar-2021
Date of Web Publication04-Jun-2021

Correspondence Address:
Dr. Bing-Heng Yang
Department of Pathology, Division of Clinical Pathology, 3F, No. 325, Sec. 2, Chenggong Road, Neihu, Taipei City 114
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/jmedsci.jmedsci_435_20

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Paraproteins have been reported to cause interference in a sort of chemistry assays conducted by current automated chemistry analyzers. However, the nature of interference differs across different methodologies and reactions. Total bilirubin and direct bilirubin assays might yield spurious result when the sample contains excess amounts of paraproteins, which are neither reliable nor reproducible when repeated tests are applied once the interference occurs. Herein, we present the case of a 78-year-old female diagnosed with kappa light chain multiple myeloma with disease progression. On admission, she had acute hepatitis and a bizarre result with direct bilirubin level higher than total bilirubin level was noted. After further investigation, excess amounts of immunoglobulin G (IgG) were the most likely cause which led to the interference of direct bilirubin assay and caused unreliable results. This case highlights the significance of the fact that patients with excess IgG might have unreliable results of direct bilirubin assay which could mislead clinical judgment. Moreover, clinicians should be more careful and should base therapeutic decisions on the patient's clinical condition when faced with patients in a similar condition.

Keywords: Paraproteins, direct bilirubin, laboratory interference, multiple myeloma

How to cite this article:
Chen CS, Dai MS, Yang BH. Direct Bilirubin Level Greater than Total Bilirubin Level: A Bizarre Result Caused by Paraproteins in a Patient with Multiple Myeloma. J Med Sci 2022;42:98-100

How to cite this URL:
Chen CS, Dai MS, Yang BH. Direct Bilirubin Level Greater than Total Bilirubin Level: A Bizarre Result Caused by Paraproteins in a Patient with Multiple Myeloma. J Med Sci [serial online] 2022 [cited 2022 May 23];42:98-100. Available from: https://www.jmedscindmc.com/text.asp?2022/42/2/98/317820

  Introduction Top

Monoclonal immunoglobulin, also known as a paraprotein, has been reported to interfere with numerous chemistry assays, which are commonly used in automated chemistry analyzers today. The chemistry tests that have been reported to be interfered with include creatinine, total bilirubin, direct bilirubin, low-density lipoprotein cholesterol, high-density lipoprotein cholesterol, and several electrolyte assays.[1],[2] The test results can be either falsely increased or decreased.[3] Many studies have used different methods to prove that this immunoglobulin, especially immunoglobulin G (IgG), is responsible for the interference.[1],[3],[4] However, the incidence of the interference varies across different studies, but it is believed to be relatively low during the regular analysis of serum samples, which is usually lower than 2%.[1],[4] However, the interference rate was significantly elevated in cases of samples containing monoclonal protein.[3]

  Case Report Top

A 78-year-old female was diagnosed with kappa light chain multiple myeloma in 2018 and was on treatment since then. However, disease progression with elevated IgG and light chain level was noted in February 2020. She was admitted to our hospital due to general malaise and low hemoglobin level during regular follow-up at the oncology outpatient department in April 2020. After admission, she was transferred to the intensive care unit due to pneumonia complicated with acute respiratory failure.

Moreover, upon transfer to the intensive care unit, elevated liver tests were noted and the suspected diagnosis was severe sepsis associated to hepatitis or drug-related hepatitis. Although the level of aspartate transaminase and alanine aminotransferase decreased after few days of treatment, the total bilirubin level was still in a gradual increase. To determine the causes of elevated total bilirubin level, the clinician ordered tests for direct bilirubin and several biochemical profiles which were as follows [Table 1].
Table 1: Initial biochemical profile

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On the second day, the clinical doctor ordered the same assays for follow-up and a spurious test result was flagged by our laboratory information management system which found the direct bilirubin level was higher than total bilirubin level [Table 2].
Table 2: Total and direct bilirubin levels on day 2

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To find out the possible causes, we re-evaluated the patient's recent assay results which were all performed with the Beckman Coulter AU5800 auto-analyzer and found out that the sample status had shown icterus since the previous day but the level of direct bilirubin was surprisingly zero. Consequently, we checked the quality control data and the time/absorbance curves of the analysis of these two samples and found out that there were tremendous fluctuations in the optical density during the reaction which was totally different from the other normal samples which reached the plateau immediately and followed by a steady straight line. After considering the patient's underlying disease with kappa light chain multiple myeloma, we arranged for the samples of the first day to undergo repeat testing, and total protein and immunoglobulin level were also tested [Table 3]. The results showed that the total bilirubin level was quite similar to the previous test, but the direct bilirubin result was totally nonreproducible as expected. Furthermore, a high level of IgG was found in the sample which is known to cause interference in direct bilirubin assay.[1],[3],[4]
Table 3: Results of repeat testing of the samples off the first day

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Eventually, after confirming that the patient had a high level of IgG which was the most likely cause of the spurious result, we informed the clinician about the interference and advised that clinical judgment be made based on the clinical condition and not be misled by the unreliable results.

  Discussion Top

As mentioned above, paraproteins can lead to interference in the estimation of several biochemical assays.[1],[2] However, the causes of the interference vary depending the reaction and methodology type. The possible cause of the interference in direct bilirubin assay has been reported as formation of fine white precipitate when sample with paraproteins adding to the reagent especially under strongly acidic condition.[1],[5]

Although the methodology of the total and direct bilirubin assay of Beckman Coulter is quite similar, the biggest difference is that the total bilirubin assay reagent contains solubilizing agent and surfactant to detect the level of indirect bilirubin amount. On the other hand, the direct bilirubin assay does not only have the substances as above but also uses strongly acidic medium to eliminate conjugated isomers of bilirubin. Nevertheless, the direct bilirubin assay does contain protein stabilizing agents to avoid precipitation; it might still happen when it comes to excess protein levels in the sample.[1],[4]

The paraprotein interference in bilirubin assay has mostly been report as falsely positive results in samples which are not icterus.[5],[6] However, the interference might also cause negative values or unreliable results which seem to be normal. Moreover, in our case, the patient had underlying acute hepatitis which might make the judgment even harder.

To solve this problem, many studies have proposed several solutions to prevent this interference. However, some of the solutions focus on how to identify interference during the reaction or prevent spurious reports. Currently, none of these methods is cost-effective, provides an easy means of eliminating the interference, or yields the true value of the assay. Using other methodologies such as dry chemistry methods[1],[3] can prevent paraprotein interference and obtain the true value; however, it is costly and not practical in the current medical systems. Using precipitation techniques to eliminate paraproteins is probably an easy and cheap solution; however, the reagents are not always available or routinely used during daily analysis in the clinical setting.[7],[8] Dilution is another common method to eliminate interference used in chemical assays, which can also be done by the auto-analyzer itself. However, how to determine the result is reliable without interference or unaffected by the dilution remains a big question.[8]

  Conclusion Top

Further study is still needed to come up with a practical solution and protocol to get the true value of the assay. Especially when the patient is severe illness, it is important to know the correct result to monitor the patient's infection status or hepatitis condition. This case gives us a hint that when evaluating data from patients with progressed multiple myeloma, there might be interference which could mislead clinical decisions. Moreover, the importance of communication between the laboratory and clinic cannot be overemphasized.

Ethical considerations

This study proposal was approved by the Institutional Review Board of Tri-Service General Hospital. TSGHIRB No.: A202005182.

Declaration of patient consent

The authors certify that they have obtained all appropriate patient consent forms. In the form the patient has given his consent for his images and other clinical information to be reported in the journal. The patient understands that his name and initials will not be published and due efforts will be made to conceal his identity, but anonymity cannot be guaranteed.

Financial support and sponsorship

The study was funded by the Medical Affairs Bureau of Taiwan's Ministry of National Defense (MAB106-036, MAB107-023).

Conflicts of interest

There are no conflicts of interest.

  References Top

Song L, Kelly KA, Butch AW. Monoclonal and polyclonal immunoglobulin interference in a conjugated bilirubin assay. Arch Pathol Lab Med 2014;138:950-4.  Back to cited text no. 1
Pantanowitz L, Horowitz GL, Upalakalin JN, Beckwith BA. Artifactual hyperbilirubinemia due to paraprotein interference. Arch Pathol Lab Med 2003;127:55-9.  Back to cited text no. 2
Yang Y, Howanitz PJ, Howanitz JH, Gorfajn H, Wong K. Paraproteins are a common cause of interferences with automated chemistry methods. Arch Pathol Lab Med 2008;132:217-23.  Back to cited text no. 3
Nauti A, Barassi A, Merlini G, d'Eril GV. Paraprotein interference in an assay of conjugated bilirubin. Clin Chem 2005;51:1076-7.  Back to cited text no. 4
Sheppard CA, Allen RC, Austin GE, Young AN, Ribeiro MA, Fantz CR. Paraprotein interference in automated chemistry analyzers. Clin Chem 2005;51:1077-8.  Back to cited text no. 5
Dutta AK. A curious case of hyperbilirubinemia. Indian J Clin Biochem 2012;27:200-1.  Back to cited text no. 6
Bakker AJ, Mücke M. Gammopathy interference in clinical chemistry assays: Mechanisms, detection and prevention. Clin Chem Lab Med 2007;45:1240-3.  Back to cited text no. 7
Berth M, Delanghe J. Protein precipitation as a possible important pitfall in the clinical chemistry analysis of blood samples containing monoclonal immunoglobulins: 2 Case reports and a review of the literature. Acta Clin Belg 2004;59:263-73.  Back to cited text no. 8


  [Table 1], [Table 2], [Table 3]


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