Case report

Contribution of the laboratory to a diagnosis process by sequential reflective

testing: Paraprotein interference on a direct bilirubin assay
Niyazi Samet Yilmaz*1, Bayram Sen2, Ozlem Gulbahar3
1Department of Medical Biochemistry, Polatli Duatepe State Hospital, Ankara, Turkey
2Department of Medical Biochemistry, Recep Tayyip Erdogan University Research and Training Hospital, Rize, Turkey
3Department of Medical Biochemistry, Gazi University Faculty of Medicine, Ankara, Turkey

*Corresponding author: [email protected]

Abstract
Errors in laboratory medicine occur in the preanalytical, analytical, and postanalytical phases. The errors are mostly detected in the preanalytical pe-
riod. However, analytical errors are still an important source of error, despite their frequency is reduced significantly in years thanks to developments
in laboratories.
In this case, an analytical error was noticed during the verification of a patient’s results. The direct bilirubin of a 66-year-old male patient admitted
to the emergency department was higher than the total bilirubin. The patient’s symptoms were fatigue and dyspnoea. Albumin and haemoglobin
(Hb) concentrations of the patient were significantly low. After considering the patient’s demographics and laboratory results, the laboratory specia-
list suspected a paraproteinemia interference. Total protein was performed as a reflective test. The albumin/globulin ratio was reversed. Thereafter,
serum protein electrophoresis (SPEP) and immunofixation electrophoresis (IFE) were performed as another reflective tests, respectively. SPEP and
IFE results were in favour of monoclonal gammopathy. The patient was directed to a haematologist, underwent a bone marrow biopsy, and the
result was reported as Waldenstrom’s macroglobulinemia with plasma cell differentiation expressing IgM-Kappa. The patient went on a chemothe-
rapy protocol, and his condition has been improved in subsequent months.
Detection of analytical errors is of great importance, like in our case, and may be used as a tool to identify patients who have not yet been diagnosed.
The laboratory specialist must dominate the entire process of each test in the laboratory, be aware of the limitations of tests, and turn these disad-
vantages into advantages when necessary.
Keywords: direct bilirubin; interference; paraprotein; reflective testing

Submitted: November 3, 2020 Accepted: March 2, 2021

Introduction
Analytical errors have significantly been reduced Paraprotein interferences have been observed on
with factors such as automation of analysers, rea- various analytical instruments and many assays/
gent performance (the majority of reagents are methods such as turbidimetric, nephelometric,
ready to use), and participation in internal quality and spectrophotometric. Immunoassays can also
control and external quality assurance/proficiency be affected by paraproteins, though less frequent-
testing. However, each sample can present a spe- ly (3). In previous studies, spurious results caused
cific matrix that may cause irregular (individual) by paraprotein interferences have been seen on
analytical errors (1,2). Interferences may cause several analytes such as enzymes, electrolytes,
such individual analytical errors and spurious re- metabolites, proteins, hormones, cardiac markers,
sults. tumour markers, and therapeutic drug monitoring

https://doi.org/10.11613/BM.2021.020801 Biochem Med (Zagreb) 2021;31(2):020801

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Yilmaz NS, et al. From an interference to a diagnosis with reflective tests

analyses (4). Analytes such as total bilirubin, direct than total bilirubin (TBIL). Blood urea nitrogen
bilirubin, uric acid, inorganic phosphate, sodium, (BUN), creatinine, aspartate aminotransferase
creatinine, C-reactive protein (CRP), high-density li- (AST), alanine aminotransferase (ALT), alkaline
poprotein (HDL) cholesterol, vancomycin, and phosphatase (ALP), and serum electrolytes were in
many other parameters were reported to be af- the reference range. Glucose concentration was
fected by paraproteinemias (3-23). slightly high, and albumin was 29 g/L (35 - 52). His
Reflective testing is the addition of new tests and/ haemoglobin (Hb) concentration was 70 g/L (130 –
or comments to the patient’s original request by 169), whereas platelets were mildly elevated, and
the laboratory specialist after evaluating the pa- leukocyte count was normal (Table 1). The patient
tient’s demographics, clinical information in the was referred to the emergency department, and
test request, and the patient’s current results (24- DBIL was 14.2 μmol/L (0 – 3.4), while TBIL was
26). By conducting reflective tests, the laboratory measured 8.7 μmol/L (5.1 – 20.5). Serum indices
specialist can make recommendations to the pa- (haemolysis, icterus, and lipaemia) were normal
tient and clinician’s advantage, contribute to the with photometric and visual assessment. Besides,
diagnosis, and prevent unnecessary procedures there wasn’t any flag or warning on the analyser.
and interventions. In this case, after revealing an The serum was not viscous, and no gel formation
analytical interference caused by paraproteine- was present that may cause incorrect sample pi-
mia, the laboratory’s contribution of a patient’s di- petting volume.
agnosis process via sequential reflective testing The sample was analysed twice in a sample cup
has been explained. and resulted in - 4.6 and - 19.3 μmol/L for DBIL;
and 8.7 and 8.0 μmol/L for TBIL. As can be seen,
there was imprecision in the repeated direct biliru-
Case report
bin results (Table 1). The reaction monitors of the
In the postanalytical phase, during verification of patient’s DBIL and TBIL results were examined and
patient results, it was noticed that a 66-year-old then compared with the other patients’ reaction
male patient’s direct bilirubin (DBIL) was higher monitors analysed on the same day. An unusual

Table 1. Laboratory results of the patient

Parameter (unit) Result Reference interval

Haemolysis, icterus, lipaemia Normal NA
Glucose (mmol/L) 6.5 4.1 – 5.5
Blood urea nitrogen (mmol/L) 3.9 2.8 – 7.1
Creatinine (μmol/L) 69 59 – 103
Total bilirubin / Direct bilirubin (μmol/L) 8.7 / 14.2 5.1 - 20.5 / 0 - 3.4
1. Rerun 8.7 / - 4.6 /
2. Rerun 8.0 / - 19.3 /
1/3 diluted sample 10.3 / 4.6 /
Total Protein* (g/L) 91 66 – 83
Albumin (g/L) 29 35 – 52
Calcium (mmol/L) 2.19 2.20 – 2.65
Sodium (mmol/L) 137 136 – 146
Potassium (mmol/L) 4.3 3.5 – 5.1
Chloride (mmol/L) 101 101 – 109

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Yilmaz NS, et al. From an interference to a diagnosis with reflective tests

Table 1. Continued.

Parameter (unit) Result Reference interval

AST (U/L) 13 0 – 50
ALT (U/L) 11 0 – 50
ALP (U/L) 96 30 – 120
Leukocytes (x109/L) 6.3 4.5 – 13
Red blood cells (x1012/L) 3.66 4.5 – 5.9
Haemoglobin (g/L) 70 130 – 169
Haematocrit (L/L) 0.261 0.400 – 0.494
Platelets (x109/L) 530 150 – 450
MCV (fL) 71 77 – 87
MCH (pg) 19 27 – 31
MCHC (g/L) 268 320 – 360
RDW (%) 20.4 11.5 – 14.5
ESR (mm/h)† 122 0 – 15
CRP (mg/L)† 123 0–5
Beta-2 microglobulin (mg/L)* 7.37 1.42 – 3.21
Serum Protein Electrophoresis*
Albumin (%) 31.9 55.8 – 65.0
Alpha-1 (%) 5.9 2.2 – 4.6
Alpha-2 (%) 12.9 8.2 – 12.5
Beta (%) 10.3 7.2 – 14.2
Gamma (%) 39.0 11.5 – 18.6
M-protein (g/L)* 26.4 NA
Serum immunofixation electrophoresis* Monoclonal IgM-Kappa NA
Immunoglobulin G (g/L)* 11.1 7 – 16
Immunoglobulin A (g/L)* 0.3 0.8 – 4.5
Immunoglobulin M (g/L)* 52 0.5 – 3.0
FKLC (mg/L)* 142 3.3 – 19.4
FLLC (mg/L)* 18.3 5.7 – 26.3
AST - Aspartate aminotransferase. ALT - Alanine aminotransferase. ALP - Alkaline phosphatase. MCV - mean cell volume. MCH -
mean corpuscular haemoglobin. MCHC - mean corpuscular haemoglobin concentration. RDW - red blood cell distribution width.
ESR - sedimentation rate. CRP - C-reactive protein. Ig – immunoglobulin. FKLC – Free kappa light chains. FLLC – Free lambda light
chains. NA - Not available. *The tests which performed as a reflective test. †Results obtained from patient one week later.

absorbance curve containing sharp spikes were Considering the patient’s age, decreased albumin
observed in the patient’s DBIL result (Figure 1). level, anaemia, and the spurious direct bilirubin re-
It was revealed from the laboratory information sult, the laboratory specialist suspected a parapro-
system (LIS) that the patient had fatigue and dysp- teinemia, which could explain all of these findings.
noea. There was not any request for radiological For eliminating suspected paraprotein interfer-
imaging. The patient’s diagnoses made by the ence, the serum of the patient was diluted in a 1/3
doctor in the emergency department were anae- ratio. The results after dilution were TBIL = 10.3
mia and pain, unspecified. μmol/L, DBIL = 4.6 μmol/L, and the reaction kinet-

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Yilmaz NS, et al. From an interference to a diagnosis with reflective tests

Figure 1. Reaction monitors of the direct bilirubin result before and after dilution.

ics of the mentioned direct bilirubin result was Simultaneously, measurement of total protein was
normal (Figure 1), so results were verified. done, as a reflective test, to check the reversed al-
All biochemistry parameters, including bilirubin bumin/globulin ratio. Due to increased total pro-
concentrations, were analysed with Beckman tein and reversed albumin/globulin ratio, serum
Coulter reagents on AU680 automated chemistry was stored, and the next day another reflective
analyser (Beckman Coulter, Brea, USA). Direct bili- test, SPEP, was performed. The doctor in the emer-
rubin (REF: OSR6211) assay in our hospital is an gency department was informed that the patient
end-point assay. The assay was based on the for- may have a disease with monoclonal gammopa-
mation of azobilirubin at a low pH and measured thy and should be referred to haematology after
bichromatically at 570/660 nm via two cuvettes discharge because the patient had never applied
(colour and blank). The reagents for DBIL assay to our hospital’s haematology department before.
contain hydrochloric acid, sulfuric acid, and 3,5-di- Also, this recommendation was added to the pa-
chlorophenyldiazonium tetrafluoroborate as the tient’s laboratory report as a comment. A few
chromogen. The chromogen is added only in the hours later, when the patient’s file was examined,
colour cuvette (reaction cuvette). For measure- we learnt that he left the emergency department
ment of DBIL, the second cuvette is used for the voluntarily after 1U (one unit) of red blood cell
sample blank, and the blank cuvette’s absorbance transfusion.
is subtracted from the absorbance of the reaction The next day, SPEP analysis was performed in aga-
cuvette. rose gel (SAS-1 plus SAS-2, Helena Biosciences Eu-
In our case, peculiar spikes appeared after the “0” rope, UK), and a monoclonal peak in the gamma
(i.e., following the addition of the sample to the region was detected (Figure 2). M-protein concen-
cuvette, which already contains sulfuric and hy- tration was calculated as 26.4 g/L. Another reflec-
drochloric acid and mixing stage) and “10” (follow- tive test, serum IFE was performed by the Interlab
ing a stirring operation in cuvettes) photometric G26 analyser (Interlab Srl, Rome, Italy), and a path-
points (Figure 1). Neither diluted sample of the pa- ological band was seen in all lines (Figure 2). As we
tient nor other patients’ samples analysed in that saw lanes in all globulin fractions, we suspected
day did not show any spikes, and absorbance cryoglobulinemia and/or monoclonal IgM polym-
curves were parallel in these samples’ reaction erization. We could not identify the paraprotein
data. No unusual reaction curves were observed exactly with IFE because we couldn’t repeat the
for the patient’s other biochemistry assays. analysis after treating serum with 2-mercaptoetha-

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Yilmaz NS, et al. From an interference to a diagnosis with reflective tests

Figure 2. Serum protein electrophoresis and immunofixation electrophoresis results. A. The arrow assigns a monoclonal peak in the
gamma region. B. A pathological band which seen in all lines.

nol (a reducing agent that breaks down disulfide department. We also advised him to go on all his
bands in protein precipitates) to dissolve parapro- planned requests and procedures in case of any
tein precipitation (13,27). Meanwhile, nephelomet- other malignancies. A few weeks later, the patient
ric quantification revealed IgM-Kappa increase wanted to share his results with us. Informed con-
(Beckman Coulter Immage 800, Brea, USA). Al- sent was obtained from the patient to report these
though the clinician had been informed and the findings for scientific purposes. According to the
possible paraproteinemia mentioned as a com- patient’s results, the faecal occult blood test was
ment in the patient’s laboratory report in previous negative, urinalysis wasn’t significant, either. Ab-
days, for patient safety, we decided to contact the dominal ultrasonography and colonoscopy were
patient in the light of new results obtained. normal; endoscopic biopsy resulted in atrophic
The patient was contacted by phone and asked gastritis. Cryoglobulinemia test result was nega-
about his previously diagnosed diseases and med- tive in another laboratory. After seen by a haema-
ications. We learnt that the patient hadn’t had any tologist, the patient underwent a bone marrow bi-
haematological diagnosis yet. He’d been taking opsy. The bone marrow biopsy result was report-
valsartan+thiazide, metoprolol, salicylate, clopi- ed as Waldenstrom’s Macroglobulinemia (lym-
dogrel, and metformin for hypertension, coronary phoplasmacytic lymphoma) with plasma cell dif-
artery disease, and type 2 diabetes mellitus. One ferentiation expressing IgM-Kappa. The patient
week ago, the patient applied to his family doctor went on a chemotherapy protocol, and his condi-
with fatigue, dyspnoea, and weight loss (8 kg in tion has been improved in subsequent months.
last year). After the family doctor noticed the pa-
tient’s Hb concentration was 78 g/L in the com- Discussion
plete blood count, she planned urinalysis, faecal
occult blood test, abdomen ultrasonography, en- In this case report, the laboratory’s contribution to
doscopy, and colonoscopy for suspected malig- the diagnosis process of a patient is presented. A
nancy. The patient was kindly invited to our labo- spurious DBIL result arose from a paraprotein in-
ratory to give him information about his reflective terference. Thanks to the awareness of our labora-
test results and directed him to the haematology tory about M protein interference, sequential re-

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Yilmaz NS, et al. From an interference to a diagnosis with reflective tests

flective tests were performed. The patient under- when protein concentrations are much higher
went the biopsy process quickly and was diag- than usual concentrations of serum proteins. The
nosed with Waldenstrom’s Macroglobulinemia. probable mechanism of interference in our case
It has been shown that Beckman Coulter conjugat- was the precipitation of excessive M-protein in a
ed bilirubin assay may be sensitive to parapro- strong acid medium and resulting turbidity (5,8-
teinemia interference. The interference rate of the 12,18,23,28,29).
samples with monoclonal protein was found be- As seen in our case, paraprotein interference may
tween 1.5 - 44% (10-16). The prevalence of mono- cause irreproducible results in direct bilirubin as-
clonal gammopathies in laboratories, different M- say (10,12,13,15). There may be a fluctuation pat-
protein concentrations, and the criteria used to tern; the DBIL results may be negative, or higher
define the DBIL interference may be the causes of than the TBIL results (10,12). Aggregation/precipi-
variable interference rates between studies (12). tation suspended in a solution due to parapro-
Precipitation of paraproteins, sample turbidity, teinemia can scatter light and interfere with the
binding of the M-protein to the analyte or a com- absorbance measurements (30).
ponent in the measuring system, volume displace- For demonstrating the paraprotein interference,
ment effect, prozone effect, hook effect, hypervis- the assay may be performed in a test tube via
cosity and cryoglobulinemia are the mentioned manually adding reagents (5,6,8-12,29). The test
mechanisms of paraproteinemia interference may be analysed in a different manufacturer’s as-
(3,19,21,23,28). However, the most common inter- say or another method with the same manufactur-
ference mechanisms are paraprotein precipitation er (20). The parameter can also be measured with
and increased sample turbidity (23,28). a slide-based dry technology assay (6,7,10-12). In
Similar to our case report, Bora and Chutia ob- our case, we could not perform these methods.
served peculiar spikes in a patient with monoclo- Removal of paraproteins by ultrafiltration or de-
nal gammopathy in Beckman Coulter TBIL assay proteinization, treatment of the sample with poly-
(9). They mentioned that transient turbidity oc- ethylene glycol, and dilution of the sample are the
curred in the cuvettes due to paraprotein precipi- methods recommended for eliminating parapro-
tation when the serum and TBIL reagent were tein interference (3,6,11,13,17,18,20,30). Since we
mixed, which led to the first peculiar spike (after eliminated the interference with the dilution, we
the 0 point). Then, transient turbidity reappeared did not perform any additional procedure.
when the autoanalyser performed a stirring oper- Each paraprotein is unique and may cause individ-
ation between “10” and “11” points, which was re- ual analytical errors due to interferences. Some
sponsible for the second spike. We also saw pecu- approaches or protocols may be useful to detect
liar spikes right after the addition of the sample in these individual analytical errors systemically. Pre-
the cuvette, then following the second mixing venting the verification of negative test results or
stage. providing flags and warnings via LIS, interferences
Direct bilirubin assay carries out in a strongly acid- on several assays may be detected (14). Also, im-
ic medium (13). Precipitation of paraproteins in a plementations like delta checks or consistency
strongly acidic pH or mixing the constituents in checks on patient results may be applied with LIS
the cuvettes could cause these peculiar spikes. or middlewares (15). Implementing warning flags
Physicochemical properties of the M-protein, pH, from instruments and reviewing the photometric
ionic strength, and assay additives affect precipita- reaction data can also be useful for detecting
tion (4). In clinical chemistry assays, reagents in- these individual analytical errors systematically
clude protein stabilizing agents for avoiding pre- (7,16,29). Our case is an example that such an ana-
cipitation (10,11,13). The solubilisation capacity of lytical interference can be used to identify non-di-
protein stabilizing agents is probably not sufficient agnosed patients with monoclonal gammopathy.

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Yilmaz NS, et al. From an interference to a diagnosis with reflective tests

Conclusion
In this case, the laboratory detected a parapro- Detection of analytical errors is of great impor-
teinemia interference and contributed to the pa- tance, such in our case, and may be used to identi-
tient’s diagnosis. Communication of the laborato- fy patients who have not yet been diagnosed. The
ry specialist with the clinician and the patient has laboratory specialist should be aware of the limita-
also facilitated this process. In the postanalytical tions of tests and turn these disadvantages into
phase, in the light of the laboratory’s knowledge, advantages when necessary.
laboratory data were transformed for the benefit
of the clinician and the patient, and future action Potential conflict of interest
plans for patient care were proposed. None declared.

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