Title page
Type of manuscript: Case report
Title of the study: Elevated IgG causing spurious elevation in serum Total bilirubin assay
Total number of authors: Four
Authors:
1. Kriti Singh, MD postgraduate student, Department of Biochemistry, Kasturba
Medical College, Manipal University, Manipal- 576104, Karnataka
Phone: 9886815540,
Email: drskriti@gmail.com
2. Pragna Rao, Professor and Head, Department of Biochemistry, Kasturba Medical
College, Manipal University, Manipal- 576104, Karnataka
Phone: 0820-2922326,
Email: drpragnarao@gmail.com
3. Priyanka Datta, MD postgraduate student, Department of Biochemistry, Kasturba
Medical College, Manipal University, Manipal- 576104, Karnataka
Phone: 9742151074,
Email: dattapriyanka12@gmail.com
4. Vijetha Shenoy Belle, MD postgraduate student, Department of Biochemistry,
Kasturba Medical College, Manipal University, Manipal- 576104, Karnataka
Phone: 9844667820,
Email: vijethashy@gmail.com
Declaration
To,
The Editor
Asia Pacific Journal of Research,
I hereby declare that the Case report titled ”Elevated IgG causing spurious elevation in serum
Total bilirubin assay” has not been submitted or published elsewhere.
Dr. Kriti Singh
MD postgraduate student
Department of Biochemistry
Kasturba Medical College
Manipal University
Manipal – 576104
Karnataka
India.
Phone: 9886815540
Email: drskriti@gmail.com
Elevated IgG causing spurious elevation in serum Total bilirubin assay
Abstract
Introduction: Monoclonal gammopathy is characterized by the presence of an M-protein that has
homogeneous structural and functional properties. At very high concentrations it may cause
significant interference in clinical chemistry assays. Incidence of interference in total bilirubin
measurement is described in this study.
Methodology: A 43 years old diabetic female, known case of multiple myeloma of 6 years, on
chemotherapy, presented to OPD. Routine tests were inconspicuous except for elevated total
bilirubin and globulins. Liver enzymes were normal. Total bilirubin estimation was done by
diazonium ion method.
Result: Repeat testing after dilution of sample revealed normal total bilirubin.
Discussion: Paraprotein interference with the Roche automated total bilirubin assay is caused by
precipitate formation which may cause clinical confusion. If false elevation of total bilirubin is
suspected the laboratory should measure total bilirubin using a different method.
Conclusion: The increase in serum total bilirubin of the patient was due to interference in the
assay caused by high IgG levels of the patient. Every laboratory should be able to identify such
incidences and have a standardized method to overcome the interferences. Sample dilution or
precipitation of interfering substances can overcome the interference.
Keywords: Clinical biochemistry, Interferences, Gammopathy, Hemolytic index, Multiple
Myeloma, Total Bilirubin
Introduction:
Paraproteinemia can cause false elevations in total and direct bilirubin levels (Smogorzewska A
et al 2004, Chung et al 2005). These occurrences are not just limited to bilirubin. Interference in
the measurement of serum total bilirubin by Roche Cobas 6000 auto analyzer, in a patient of
multiple myeloma is described in the subsequent sections.
Case Description:
A 43 years old diabetic female, known case of multiple myeloma of 6 years, on chemotherapy,
presented to oncology OPD, with complaints of headaches and cough for a week. At the time of
admission the patient was on progression phase of Bortezomib, Cyclophosphamide and
Dexamethasone (VCD) protocol. She was not receiving any treatment for diabetes except dietary
restrictions. The patient received a cycle of VCD chemotherapy on day 2 of admission and an
episode of plasmapheresis on day 5 of admission.
Laboratory investigations done on the day of admission are represented in Table 1.
Table 1: Laboratory reports from day one of admission
Samples
Patient sample
Reference ranges
Day of admission
-
25.7
0.2-1
0.3
0- 0.2
AST (IU/L)
35
15- 40
ALT (IU/L)
24
15- 40
ALP (U/L)
40
Total Proteins (g/dL)
12.6
6- 8
Albumin (g/dL)
3.6
3.5- 5.5
Globulin (g/dL)
9.0
2.5- 3.5
Na+ (mEq/L)
138
135- 145
K+(mEq/L)
4.0
3.5- 5.5
Urea (mg/dL)
32
20- 40
Creatinine (mg/dL)
1.0
0.6- 1.2
SPE
M band in γ region
-
Serum Immunofixation
IgG and λ bands
-
Serum
IgA (mg/dL)
<14
70-400
Immunoglobulins
IgG (mg/dL)
6610
700-1600
IgM (mg/dL)
22
40-230
Complete blood
Hb (g/dL)
12.2
12-15
counts
RBC
4.9 x 106
4.2 – 5.4 x 106
WBC
12,800
11,000- 14,000
Tests
Time of collection
Total bilirubin
(mg/dL)
Direct bilirubin
LFTs
Electrolytes
RFTs
(mg/dL)
Legend: AST- Aspartate Aminotransferase, ALT- Alanine Aminotransferase, ALP- Alkaline
Phosphatase, LFTs- Liver function tests, RFTs- Renal function tests, SPE- Serum protein
electrophoresis, IgA- Immunoglobulin A, IgG- Immunoglobulin G, IgM- Immunoglobulin
M, Hb- Hemoglobin, RBC- Red blood cells, WBC- White blood cells
Clinically, the patient was not jaundiced, and there was no supporting evidence for liver
disease. From Table 1, it is evident that apart from serum total bilirubin (25.7 mg/dL) and
serum globulins (9 g/dL) no other abnormality is seen in the patient sample.
In vitro hemolysis as a potential interfering factor was ruled out by estimating the H index
which was within acceptable range. The only interfering factor in this case was elevated
serum IgG levels of 6610 mg/dL (Reference range: 700-1600).
Repeat total bilirubin estimation was done after diluting the sample in a ratio of 1:50. The
total bilirubin level was then found to be 0.8 mg/dL
Another sample sent on day six of admission after an episode of plasmapheresis showed
following findings (Table 2)
Table 2: Laboratory reports from day six of admission
Samples
Patient sample
Reference ranges
Day 6 of admission
-
0.5
0.2-1
0.3
0- 0.2
AST (IU/L)
18
15- 40
ALT (IU/L)
25
15- 40
ALP (U/L)
88
Total Proteins (g/dL)
9.3
6- 8
Albumin (g/dL)
2.9
3.5- 5.5
Globulin (g/dL)
6.4
2.5- 3.5
Na+ (mEq/L)
133
135- 145
K+(mEq/L)
3.9
3.5- 5.5
Urea (mg/dL)
28
20- 40
Creatinine (mg/dL)
0.9
0.6- 1.2
SPE
-
-
Serum Immunofixation
-
-
Serum
IgA (mg/dL)
-
70-400
Immunoglobulins
IgG (mg/dL)
2875
700-1600
IgM (mg/dL)
-
40-230
Complete blood
Hb (g/dL)
12.4
12-15
counts
RBC
5.0 x 106
4.2 – 5.4 x 106
WBC
12,400
11,000- 14,000/ μL
Tests
Time of collection
Total bilirubin
(mg/dL)
Direct bilirubin
LFTs
Electrolytes
RFTs
(mg/dL)
Legend: AST- Aspartate Aminotransferase, ALT- Alanine Aminotransferase, ALP- Alkaline
Phosphatase, LFTs- Liver function tests, RFTs- Renal function tests, SPE- Serum protein
electrophoresis, IgA- Immunoglobulin A, IgG- Immunoglobulin G, IgM- Immunoglobulin
M, Hb- Hemoglobin, RBC- Red blood cells, WBC- White blood cells.
From Table 2, after plasmapheresis, serum IgG level reduced to 2875 mg/dL along with a
reduction in serum total bilirubin to 0.5 mg/dL.
All chemistry parameters were estimated using standard traceable assays in Roche Cobas
6000 auto analyzer. Sample used was plain serum collected in a red capped evacuation tube.
Total bilirubin estimation was done by diazonium ion method (Jendrassik L et al. 1938)
Immunofixation was done using Helena Biosciences SAS MX Immunofix apparatus which
includes gel electrophoresis followed by immunoprecipitation and staining.
Serum protein electrophoresis was done using Helena biosciences SAS MX serum protein
technique setup using Tris- barbital buffer system (pH- 8.6).
Hemolytic index of both the patient samples was measured in Roche COBAS c501 subunit
(cut off <60 mg/dL of free Hb i.e. visible hemolysis) (Salvagno GL et al, 2012). It was found
to be within acceptable range in both the patient samples.
Furthermore, the normal haemoglobin levels in both the patient samples (12.2 g/dL and 12.4
g/dL) along with normal RBC counts excluded in vivo hemolysis. Another point to be noted
here is that the patient’s serum globulin levels were 9g/dL initially on day 1. After
plasmapheresis, serum globulin levels reduced to 6.4 g/dL.
Discussion:
Interference is defined as "the effect of a substance present in the sample that alters the
correct value of the result for an analyte" (Pantanowitz L et al, 2003). The paraproteinemia
consistent with multiple myeloma (Saibaba KSS et al, 1998) is known to interfere with
estimation of several parameters especially where the mode of measurement employs
photometric or turbidimetric assays (Smogorzewska A et al. 2004, Puppalwar P. V 2012,
Alexander D. D 2007). This interference is due to precipitation of these paraproteins in the
reaction mixture causing turbidity and thereby increasing absorbance at every wavelength
due to scattering of light. The Roche total bilirubin assay is a 1-cuvette, 2-point end point
assay. The addition of R2 to the reaction mixture changes the assay environment to an acidic
pH of 1–2, which precipitates paraproteins causing the interferences (Dimeski G 2008).
Smogorzewska et al (2004) hypothesized that the Roche solubilising agent is in fact the cause
of this artefact. The direct bilirubin assay shows no such interference. In our patient a fall in
serum IgG levels coincided with fall in total bilirubin levels. This indicates that the cut-off
levels of IgG that interfere with total bilirubin measurements may fall somewhere in the
range of 2000-3000 mg/dL, but this is our hypothesis and warrants further analysis.
A possible factor leading to an actual increase in total bilirubin could have been the long term
chemotherapy, as literature states that lenalidomide and bortezomib may have adverse effects
on liver functions (Coehn M.A 1970, Drug safety update 2013, Jollift CR et al 1982, Smith
AF et al 2006). But the patient had no such clinical features.
Hydration status of the patient is another cause for fluctuations in bilirubin levels (Cornelis T
et al 2012). However, an elevation in serum total bilirubin due to dehydration would coincide
with an increase in other parameters such as urea, creatinine and red cell indices.
Routine clinical chemistry assays are designed keeping in mind that proteins, normal or
otherwise, should not interfere with their values. However, when the monoclonal antibodies
proliferate to a significant degree the “precipitation becomes relevant and influences the
assay” (Canadian Paediatric Society Statement 1999). Proteins are least soluble at their
isoelectric point. At this pH their net charge is zero, causing low electrostatic repulsion.
Precipitation can be significantly reduced by adding surfactants to impair hydrophobic
interactions, and by modifying the pH to increase electrostatic repulsion between individual
M-protein molecules (Bakker AJ 2007). Because dispensing correct reports is of utmost
priority for any laboratory it is imperative that every laboratory has a standardized protocol to
combat interferences. While we applied dilution as a corrective technique, precipitation of
interfering factors can also be performed (Canadian Paediatric Society Statement 1999,
Bakker AJ 2007). It is up to the concerned laboratory to come up with suitable measures for
correction.
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