Keywords
capillary zone electrophoresis - multiple myeloma - M-spike - monoclonal protein -
variable migration
Introduction
Monoclonal gammopathy (MG) comprises a wide range of conditions arising from the abnormal
clonal proliferation of plasma cells, in which monoclonal proteins can be detected
in urine and serum either as intact immunoglobulins, only light chains (kappa and
lambda) or only heavy chains (G, A, M, D, and E), or intact immunoglobulins with excess
light chains.[1]
[2] On the basis of clinical features, bone marrow findings, and quantitative measurement
of the monoclonal protein, common disorders associated with MG include malignant diseases,
such as multiple myeloma (MM), Waldenströmsmacroglobulinaemia, plasmacytoma, or benign
diseases like MG of uncertain significance (MGUS), the term being coined by the Mayo
Clinic Group Time 1971.[3]
[4]
MGUS is the common etiology of MG, with prevalence progressively increasing with age,
that is, 1, 3, and 10% in the age group of more than 50, 70, and 80 years, respectively.[2] MM contributes to nearly 10% of the hematological malignancies, with occurrence
of nearly 1 per 100,000 population worldwide.[2] Patients of MM present most commonly with complaints of back pain, spontaneous fracture,
or recurrent infections.[2] Serum protein electrophoresis (SPEP) is a routinely done investigation for suspected
cases of MG for the diagnostic, therapeutic, and prognostic evaluation of the underlying
illness. Serum proteins are separated into different regions by SPEP based on their
molecular weight and electric charge.[4] Zone electrophoresis is the most commonly used electrophoretic technique in which
serum proteins are separated into different regions and thereafter interpreted. Monoclonal
proteins usually migrate to the gamma region producing M spike; however, many times
the spike may be encountered in other regions (like α2, β1, and β2) making the diagnosis of MG challenging. So, whenever any atypical migration of M
protein is observed on SPEP, it should be further investigated with immunoelectrophoresis
(immunofixation [IF] or immunosubstraction [IS]), which would help in resolving this
diagnostic dilemma by the identification and characterization of paraproteins.
Here, we would like to present some interesting insight into electrophoretograms displaying
M spike in regions other than common location (gamma region) on capillary zone electrophoresis
(CZE).
Cases
Case 1
A 65 years old male presented to the outpatient department (OPD) with complaints of
on-and-off back pain for 6 to 7 years and intermittent fever with progressively increasing
generalized weakness since 2 years. He was hypertensive for 15 years and was diagnosed
with osteoarthritis knee 5 years back. On examination, he was overweight and pallor
was seen.
Blood investigations ([Table 1]) showed low hemoglobin level and hypercalcemia with reversal of albumin-to-globulin
ratio. No osteolytic lesion was seen on the radiological survey. Bone marrow findings
were suggestive of megaloblastic erythropoiesis with increased plasma cells in marrow
(< 10%). However, M-spike was seen in β1 fraction on SPEP which came as monoclonal IgA-lambda on immunotyping (IT) ([Fig. 1]). M protein concentration was 1.7 g/dL. The patient was provisionally diagnosed
with MGUS based on the above observations and referred for further management.
Fig. 1 (A) M-spike in the β1 region on CZE and (B) M-spike comprising of monoclonal IgA-lambda on IS.
Table 1
Blood investigations
|
Patient 1
|
Patient 2
|
Patient 3
|
Patient 4
|
Patient 5
|
|
Hemoglobin (g%)
|
7.8
|
7.6
|
7.4
|
7.8
|
6.3
|
|
TLC (per cumm)
|
7,500
|
6,400
|
4,000
|
3,200
|
5,600
|
|
Platelet (lacs per cumm)
|
3.1
|
1.8
|
1.6
|
2.3
|
1.4
|
|
Blood urea (mg/dL)
|
45
|
32
|
32
|
48
|
51
|
|
Serum creatinine (mg/dL)
|
1.6
|
1.2
|
1.2
|
2.1
|
3.1
|
|
Serum calcium (mg/dL)
|
10.2
|
7.9
|
9.1
|
9.2
|
9.6
|
|
Total serum protein(g/dL)
|
7.4
|
9.8
|
8.1
|
8.6
|
15.3
|
|
Serum albumin (g/dL)
|
2.7
|
2.4
|
2.1
|
2.3
|
3.3
|
|
A/G ratio
|
0.57
|
0.32
|
0.34
|
0.37
|
0.27
|
Abbreviations: A/G ratio, albumin-to-globulin ratio; TLC, total leukocyte count.
Note: Bold values indicate the test values not within the biological reference range
of these parameters.
Case 2
A 45 years old male presented to the OPD with complaints of progressively increasing
bony pain and generalized weakness for 6 to 7 months. He had anorexia and weight loss
for 3 to 4 months with easy bruisability and rashes since 1 month. He was a known
case of type II diabetes mellitus for 10 years, MGUS for 5 years, and chronic liver
disease for 2 years. On examination, he was of thin built and had pallor. Petechial
hemorrhages were observed on bilateral upper and lower limbs.
Blood investigations ([Table 1]) revealed low hemoglobin level and high total serum protein level with reversal
of albumin-to-globulin ratio. Radiological investigations showed osteopenia with multiple
punched-out lytic lesions in pelvic bones and proximal shafts of bilateral femur.
As the patient was a diagnosed case of MGUS, its progression to MM was suspected and
subsequently M-spike was characterized as monoclonal IgG-lambda type on IT ([Fig. 2]). M protein concentration was 4.8 g/dL. These findings were further supported by
bone marrow result which was suggestive of plasma cell myeloma (> 15% plasma cells)
with CD138+ and CD19 − . Therefore, the patient was diagnosed with MM and referred
for further management and treatment.
Fig. 2 (A) M-spike in the fused β-γ region on CZE and (B) monoclonal IgG-lambda corresponding to M-spike on SPEP.
Case 3
A 53 years old male presented to the emergency with complaints of black tarry stool
and generalized weakness for 4 days. He had a loss of appetite for 20 to 30 days,
night sweats for 1 to 2 months, and persistent backache for 4 to 5 months. He had
a fracture of D11 and L2 vertebrae a year back. He was a known case of type II diabetes
mellitus for 17 years and been an alcoholic for 20 years. On examination, he was of
normal built and had pallor.
Blood investigations ([Table 1]) revealed low hemoglobin level and marginal decrease in leukocytes and platelet
count. He also had hypercalcemia and high total serum protein level with reversal
of albumin-to-globulin ratio. On X-ray skull rain drop appearance was seen and X-ray
spine showed old D11 and L2 vertebral fracture. This raised the suspicion of plasma
cell gammopathy and SPEP was requested on which M-spike was seen in β fraction (merged
β1 and β2 fractions) which on IT revealed the presence of monoclonal IgA-lambda and on quantification
its value was 3.2 g/dL ([Fig. 3]). On the basis of the above findings, bone marrow aspiration was recommended for
a definitive diagnosis of MM and subsequent treatment. The patient did not show for
follow-up, so bone marrow aspiration could not be performed.
Fig. 3 (A) M-spike in the merged β1 and β2 region on CZE and (B) monoclonal IgA-lambda corresponding to M-spike on SPEP.
Case 4
A 58 years old female presented to OPD with complaints of pain in left forearm since
3 months. She also had generalized weakness and persistent backache for 5 years. She
had menopause 4 years back. There was no history of trauma or any chronic illness.
Pallor was observed on general examination.
Blood investigations ([Table 1]) revealed low hemoglobin level and low leukocyte counts. She also had hypercalcemia
and high total serum protein level with reversal of albumin-to-globulin ratio. On
X-ray, forearm ulnar fracture was seen. SPEP was advised. On SPEP, M-spike in β2 fraction was detected followed by IT, which revealed the presence of monoclonal IgA-lambda
in the region corresponding to M spike on SPEP ([Fig. 4]). M protein concentration was 0.4 g/dL. Bone marrow findings showed increased plasma
cells in marrow (< 10%). On the basis above findings, a provisional diagnosis of MGUS
was made and the patient was referred for further management.
Fig. 4 (A) M-spike in the β2 region on CZE and (B) monoclonal IgA-lambda forming M-spike on SPEP.
Case 5
A 60 years old female presented to OPD with complaints of progressively increasing
weakness for 7 months and on-and-off backache since 3 years. She was on treatment
for post-menopausal bleeding. On general examination, pallor was seen.
Blood investigation reports ([Table 1]) showed low hemoglobin and low platelet counts. Raised total serum calcium and serum
protein were also reported. Considering low hemoglobin levels with others suggestive
of MG, SPEP was requested. M-spike appeared in gamma fraction on SPEP, which was followed
by IT where the monoclonal protein IgA kappa was seen corresponding to the position
of M spike on SPEP ([Fig. 5]). The M protein concentration was 4.2 g/dL. There was no significant finding on
the radiological survey. She was provisionally diagnosed with MGUS and referred for
further management and treatment.
Fig. 5 (A) M-spike in the gamma region on CZE and (B) monoclonal IgA kappa corresponding to M-spike on SPEP.
Discussion
Nearly 100 serum samples were processed on the two capillaries automated capillary
electrophoresis system (Minicap flex piercing, Sebia, France) installed in our setup
during a period of 8 months (August 2020–March 2021). Among them, we observed M-spike
in 14 CZE electrophoretograms. The M-spike was present in the gamma region in 10 samples,
while in four samples the M-spike was detected in regions other than gamma. Quantitative
estimation of monoclonal proteins can provide valid interpretation in diagnosis, evaluation,
and monitoring of MGs; therefore, from capillary electrophoresis, the absolute concentration
of monoclonal protein was calculated from the value of serum total protein obtained
on a dry chemistry analyzer (Vitros5600 Ortho clinical diagnostics).
Since the advent of capillary protein electrophoresis (CE), detection and quantification
of monoclonal immunoglobulins in serum and urine have been improved to a larger extent
because of less chances of variation otherwise seen due to different dye-binding capacities
of proteins in agarose gel electrophoresis.[5] CE also provides better differentiation of the β region into β1- and β2-globulin fractions, thus improving further their separation.[5]
[6] Better resolution has been seen in CE when compared with agarose gel electrophoresis
as it is based on the principle of endosmosis and also employs a high voltage (R)
and which, in turns, facilitates separating the proteins into β1 and β2 fractions constituted majorly by globulin proteins transferrin, low-density lipoprotein,
and complement proteins (C3,C4). The α1 fraction is majorly constituted by α1 antitrypsin
and α1 acid glycoprotein (orosomucoid). Being acute-phase reactants, an increase in
α1 fraction may be seen in clinical conditions associated with an acute inflammatory
response like trauma, inflammatory joint disease exacerbation, burns, etc.[5] Similarly, haptoglobin and ceruloplasmin in the α2 fraction are also acute-phase
reactants, but this fraction is also contributed to by α2 macroglobulin. An increase
in α2 macroglobulin is characteristically seen in nephrotic syndrome because it is
a large-sized protein and, hence, hardly passes through the glomerular membrane. Another
reason for the rise seen in serum α2 macroglobulin is due to its increased synthesis
to maintain the normal plasma oncotic pressure as a compensatory mechanism for significantly
decreased serum albumin levels in nephrotic syndrome.[7] Monoclonal proteins and physiologically present serum proteins in the β1 and β2 fractions may co-migrate and, thus, contribute to the unusual elevation seen in these
fractions. In patients with iron deficiency anemia, pesudomonoclonal elevation in
beta fraction may be seen due to increased serum transferrin levels, and this pattern
may also be seen in hemolyzed samples (a preanalytical variable) because of the comigration
of free hemoglobin as released from breakdown of RBCs. For β2 fractions, in the electrophoretic pattern seen in blood samples of patients undergoing
dialysis or on anticoagulant therapy (e.g., heparin), such an unusual elevation may
be appreciated because of falsely high fibrinogen levels. The gamma fraction is the
most common region for monoclonal protein migration; however, extremely high serum
CRP levels, uremia, rheumatoid factor, or old degraded sample may also contribute
to pseudo-monoclonal increase seen in this fraction. Polyclonal IgA may result in
the confluence of β2 and gamma fractions depicted as β-γ bridging in the electrophoretogram.[5] CE in combination with IS helps in the identification and characterization of even
weak monoclonal immunoglobulin components in SPE and in differentiating between pseudo-monoclonal
proteins and paraproteins. Biochemical assays of these normally present serum proteins
as well as free light chain assay and serum Ig levels may further help in confirmation
of monoclonality. While both CE and agarose gel electrophoresis are 99% specific,
CE has a sensitivity of 95% compared with gel electrophoresis which has 91%.[2]
SPEP gives an idea about the concentration of important serum protein fractions in
addition to paraproteins if present, most commonly observed in the gamma fraction.
However small monoclonal components do not always follow this and may appear in fractions
other than the gamma region and which may be seen as curve irregularity on electrophoretograms.
The most common immunoglobulin presenting irregularity due to their variable migration
is IgA, and the possible explanation for this is their property to polymerize.[2] Sometimes, it is difficult to tell whether the irregularity is because of raised
levels of proteins normally found in that fraction or because of monoclonal protein
migration.[5] This dilemma can be resolved by using the technique immunoelectrophoresis (IF or
IS).
IS is performed using specific antisera against both heavy and light chains, and the
IS occurred during the process of immunoelectrophoresis is identified and interpreted.[6] The specific antisera binds to its respective immunoglobulin if present in the sample,
and the complex formed is precipitated and thus does not migrate, causing reduction
or disappearance of peak as seen in CE. This reduction or disappearance of peak, characterizing
a particular type, is appreciated when original capillary SPEP trace is overlayed
on trace obtained by the addition of one of each five specific antisera to the patient's
serum.[6] By this method, it is possible to recognize as well as quantify monoclonal proteins.
Thus, capillary electrophoresis along with IS aids in the diagnosis and management
of patients with MG.
Conclusion
The M spike is seen most commonly in the gamma region. However, it is reported that
at times monoclonal protein can migrate anywhere from α2 to gamma region, especially IgA which often appears in the β region. Here, we have
enumerated few cases where M protein has been seen in fractions other than the gamma
region. Thus, one needs to be cautious about the variable appearance of M-spike during
the interpretation of SPEP as some physiological proteins if elevated can also give
rise to similar spike sometimes referred as the pseudo-monoclonal pattern.
