INTRODUCTION:

Monoclonal paraprotein in serum protein electrophoresis (SPE) establishes the diagnosis of paraproteinemia. This is identified during the investigation of patients with clinical suspicion of multiple myeloma, unrelatedsymptoms, after routine health screening in patients aged above 50 years.^1,2 This is confirmed by the presence of monoclonal(M) paraprotein by immunofixation (IF) and further quantified by SPE and/or serum-free light chain (SFLC)-assay along with other common basic biochemical parameters, imaging, and histopathological tests.³Multiple myeloma (MM) is a malignancy affecting B-cell which results in the accumulation of monoclonal plasma cells in the bone marrow.⁴

It accounts for approximately 1% of all cancers and 10% of hematologic malignancy.⁵A clinically silent premalignant stage termed monoclonal gammopathy of undetermined significance (MGUS) progresses to MM which is at the end of a spectrum of plasma cell disorders.⁶The classical complications seen in MM include hypercalcemia, renal impairment, anemia, and bony lesions which ultimately result in end-organ damages.⁷

Globally there is a higher incidence of age-standardized incidence rate (ASIR) of 2.1per one lakh persons MM in Western nations (Australia, America, and Western Europe) with a median age of 65-70 years.⁸In comparison to Western nations, data regarding the incidence of MM from India is relatively less (age-standardized incidence rate (ASIR) 0.4-0.9 per one lakh persons. The distribution of disease is attributed to the differences in environmental and lifestyle factors.^9,10The recommended tests in the diagnosis and follow-up of MM include complete blood count (CBC), peripheral smear, RFT, LFT, LDH calcium, SPE, serum-free light chain assay, Immunofixation electrophoresis. Urine BenceJones proteins.³. MM being a serious malignant disorder that is incurable, early detection can reduce the risk of disease burden for the patients.The incidence of multiple myeloma or paraproteinemia is less documented in India. Also, the clinical presentation varies with regions hence it is important to analyze all possible data obtainable with paraproteinemia.The diagnosis of multiple myeloma can be challenging, hence the current retrospective and prospective study was undertaken to find the percentage of paraproteinemia in suspected cases by serum protein electrophoresis and to correlate M –spike values with the laboratory findings.

MATERIALS AND METHODS:

This study was conducted at Kasturba Medical College Hospital,Ambedkar Circle, Mangalore. Study protocol was approved by the institutional ethical committee (IEC KMC MLR-12/2020/410). 4398 samples were received for SPE during the period 2017 January -2021 March and we included only those found to have M-band for the study. Any other variation in electrophoretogram other than M –band was excluded. Data for all other laboratory tests such as immunofixation electrophoresis (IFE), CBC, RFT, LFT, calcium, phosphorus, beta 2 microglobulins, LDH, serum-free light chain assay(SFLC) Bence Jones Proteinuria (BJP), BM biopsy, BM smear, and radiological evidence for lytic lesions were obtained from thecentral lab and MRD of hospital data management system after the consent from authorized personnel. Statistical analysis was done using SPSS version 17.0. Results were expressed as mean±SD, frequency, and percentage. Correlation of M spike with laboratory findings was done using Karl Pearson’s correlation, (P-value < 0.05 will be significant).

RESULTS:

A total of 4938 SPE data in suspected cases of paraproteinemia was screened by SPE during the period between 2017 January to 2021 March and 340 reports which showed M spike were included in the study. (Fig 1)

Fig 1: Data collection

Table 1: Descriptive characteristics of patients with M spike (n=340)

Parameters	Mean ± S.D
Age(Years)	63.01 ± 10.98
Male	63.36 ± 10.79
Female	62.54 ± 11.25
M Spike	2.19 ± 1.88
Male	2.08 ± 1.93
Female	2.34 ± 1.93
Male: Female Ratio	1 : 0.76
Albumin	3.43 ± 0.71
Globulin	4.88 ± 2.0
A/G ratio	0.79 ± 0.32

Results expressed as Mean± S.D

Table 2: Analysis of IFE data (n=39)

Characteristics	Mean ± S.D
Age (yrs)
Male	63.36±10.792
Female	62.54 ± 11.250
Ig Types	No. of Patients
IgG kappa	20
IgG lambda	4
IgA kappa	4
IgA lambda	1
IgM kappa	1
Others
IgA lambda+ Free lambda	1
IgGkappa + IgG lambda	2
IgG kappa + Free lambda + IgA kappa	1
IgG kappa + IgA lambda	2
IgG lambda + Free lambda	2
IgA kappa + IgG (with no light chain)	1

Results expressed as Mean± S.D and in numbers

Table 2 summarises the analysis of immunofixation electrophoresis reports of 39 patients. The immunoglobulin (Ig) is IgG kappa accounted for 51%. IgG lambda and IgAkappa accounted for 10% each. IgA lambda and IgMkappa accounts for 2.6% each for all the monoclonal gammopathy reported .25.6% had biclonalgammopathy.

Table 3: Correlation of M Spike with other biochemical parameters

Parameters	N	Mean ± SD	r	p
Complete Blood Count
WBC count(TC)(cells/cumm)	56	8478.57 ± 4885.51	-0.130	0.339
RDW(%))	56	16.71 ± 4.59	0.026	0.849
RBC count(10⁶/ul)	56	3.05 ± 0.94	0.041	0.765
MCV(fl)	56	90.15 ± 9.89	-0.030	0.829
MCHC( g/dl )	56	33.19 ± 4.66	0.091	0.508
MCH(pg)	56	30.14 ± 2.38	0.097	0.485
Platelet count(uL)	56	247228.07± 137182.50	-0.218	0.103
Hemoglobin(g/dl)	56	9.23 ± 2.32	-0.092	0.496
Hematocrit(%)	56	26.52 ± 7.55	-0.081	0.566
ESR(mm/hr)	74	83.04 ± 39.92	0.107	0.366
Neutrophils (%)	56	64.94 ± 13.55	-0.003	0.985
Monocyte(%)	56	7.67 ± 3.72	0.080	0.570
Lymphocytes(%)	56	25.34 ± 11.99	0.013	0.923
Eosinophils (%)	56	2.25 ± 2.46	-0.138	0.324
Basophils (%)	56	0.39 ± 0.24	-0.056	0.710
Liver Function Test (LFT)
Total Protein (g/dl)	64	8.99± 2.57	0.826	0.000*
Serum Albumin (g/dl)	62	3.31 ± 0.70	-0.473	0.000*
Serum Globulin (g/dl)	59	5.62 ± 2.74	0.798	0.000*
Alb/Glob (A/G)ratio	60	0.74± 0.41	-0.635	0.000*
Bilirubin-Total (mg/dl)	65	0.70± 0.63	-0.060	0.633
Bilirubin-Indirect (mg/dl)	58	0.46± 0.46	-0.030	0.821
Bilirubin-Direct (mg/dl)	58	0.27 ± 0.30	-0.227	0.087
ALP (U/L)	59	107.44 ± 85.30	-0.197	0.135
SGPT/ALT (U/L)	67	25.03 ± 25.73	-0.230	0.061
SGOT/AST (U/L)	59	30.29 ± 25.63	-0.181	0.169
Renal Function Test (RFT)
Serum Creatinine (mg/dl)	105	2.51 ± 2.77	-0.183	0.061
Uric Acid (mg/dl)	53	6.85 ± 2.88	0.406	0.008*
Blood Urea (mg/dl)	41	62.54 ± 51.787
LDH (U/L)	23	212.26 ± 72.90	0.279	0.197
Phosphorus (mg/dl)	48	4.31 ± 1.37
Calcium (mg/dl)	88	9.14 ± 1.24	0.054	0.618
Beta 2 microglobulin (mg/dl)	37	6.87 ± 7.17
Kappa and Lambda Lightchain Assay
Kappa Light Chain (mg/dl)	104	235.65 ± 978.85	-0.137	0.480
Kappa/Lambda Ratio	104	1042.68 ± 5562.39	-0.130	0.502
Lambda Light Chain (mg/dl)	104	103.97 ± 627.41	0.147	0.208

*Correlation is significant at the 0.01 level (2-tailed). ** Correlation is significant at the 0.05 level (2-tailed).p-value ,< 0.05 is significant.

M spike positively correlated with total protein(r=0.826;p=0.000), serum globulin (r=0.798;p=0.000 and serum uric acid (r=0.406;p= 0.000). M spike negatively correlated with serum albumin (r = -0.473;p=0.000) and A/G ratio(r=-0.635;p=0.000).

Fig. 2: Peripheral smear reports of patients with M spike (n=53)

Results expressed in%

Fig. 3: Bone marrow smear examination reports of patients with M spike (n=22)

Results expressed in%

Fig. 4: Bone marrow biopsy (n=13)

Results expressed in%

Fig. 5: Radiological reports (n=23)

Results expressed in%

DISCUSSION:

This study was taken up to estimate the finding of paraproteinemia, analyze the types and correlate the spike value with biochemical and hematological parameters to understand the most influential determinants. In this laboratory-based retrospective time-bound study we analyzed 4398 serum protein electrophoresis (SPE) reports in suspected cases of paraproteinemia. 7.7 % of SPE reports were found to have a M spike. Studies describing the disease burden of paraproteinemia in India are very few. A study by Bora et al⁷observed that the occurrence of MM varied in different parts of India due to the differences in their lifestyle and environmental factors which have led to a finding that the frequency of MM was higher in developed areas in the northern and southern parts of the country compared to the northeastern region. On the other hand, a hospital-based study of 300 patients who had at least one criteria to categorize the cases as MM reported the incidence of MM as 23.3%.⁹The worldwide distribution of MM cases was reported by a study done by Cowan et al⁶they observed that MM cases were more pronounced in the western population but during the time period between 1990-2016 they saw a shift in this trend and more MM cases were reported from all regions of the world and this was attributed to disease affecting the older age group people. The mean age reported in several studies was between 60-75 years with a predominance in males.^9,5 In our present study, we found the mean age for males as 64years and for females as 63years which is more akin to a study done during 2009-2010 by the American Cancer Society.⁹ Our study reported male: female ratio to be 1: 0.76 (4:3) which is in contrast to the other studies by Mikhael et al ⁵and Ricomiet al¹⁰which show a male predominance.

CRAB indices, staging system likeDurieSalmon, International Staging System (ISS), Revised –ISS based on biochemical and hematological parameters such as SPE, IFE CBC, RFT, LFT, calcium, phosphorus, beta2-microglobulin, LDH, Bence Jones Proteinuria, bone marrow biopsy, bone marrow smear and radiological evidence for lytic lesions are used to categorize the predominant features of MM.^3,13We collected the available data for these parameters in our laboratory-based study. In paraproteinemia, myeloma cells secrete M protein which isa gamma globulin.¹⁴Accordingly, we found a significant association of total protein, serum globulin with M spike in SPE. This study also reported a negative correlation with serum albumin and A/G ratio with M spike. The decrease in albumin seen in myeloma is due to a chemical messenger, IL6 which blocks the synthesis of albumin, a finding similar to a study byPoudelet al.¹³Hence, estimation of serum globulins and its association with M spike could be used for monitoring patients with paraproteinemia.

Bhandari et al¹⁴reported an interesting case where they found a presenting feature of jaundice and further workup detected myeloma. Myeloma presenting with acute liver disease is uncommon. In the current study, liver enzymes were found to be elevated in the MM cases, however, there was no significant correlation with the M spike value. Another study by Torreloetal¹⁵reported increased levels of conjugated bilirubin in patients which was an incidental finding in MM patients without clinical signs so concluded that it could be used in the early diagnosis of MM in suspected cases of paraproteinemia.

Renal impairment is a documented fact in patients with MM due to infiltration by plasma cells and light chain accumulation.¹⁸This is evident by elevated levels of creatinine, urea, and uric acid which are markers of renal involvement. Several studies have documented a positive correlation of M spike with serum creatinine.^{14, 11} Although in the present study serum creatinine level was increased more than the normal level in the patients, however, we did not find an association with M spike, but we found an association of uric acid with M spike and this finding is similar to the study done by Torreloet al.¹⁵ Hence, estimation of uric acid may also indicate an abnormal renal function in patients with MM.

Malignant plasma cells produce an excess of either intact immunoglobulin or free light chains which contributed to a significant increase in serum-free light chains (SFLC).²¹, Increased levels of lambda light chain, kappa light chain, kappa/lambda ratio were observed in the present study. The serum-free light chain has been implicated as a cause for renal damage. Talbot et al¹⁹concluded that SPEtogether with FLC analysis more effective in diagnosing plasma cell disorders. Hence serum free light chain assay could be used for assessing the progression of asymptomatic patients to MM. But in the current study, only 30% of SPE reports had FLC assay data which indicates that FLC assay has been underutilized in assessing the prognosis of MM patients. Unlike some of the studies, data on Bence Jones proteinuria was very meager and hence was not included in the study.

The Immunofixation electrophoresis (IFE) is an ideal method used to confirm both the presence of M protein and to distinguish heavy chain and light chain.²¹ The study by Mangano et al²¹reported that IgG kappa, IgAkappa, and IgAlambda in the asymptomatic patients could progress to MM and the most common type is IgG kappa. Our study also confirms that the common type of monoclonal gammopathy is IgG kappa despite being a small number of IFE reports.

Hemostatic abnormalities due to passive interference by the paraprotein in the coagulation process or on platelet function are seen in MM patients.²¹ Anemia is a common clinical presentation in MM patients. In the current study, we reported 63% of patients presented with normocytic normochromic anemiawhich may result due to increased plasma volume even in the absence of hematopoietic dysfunction or a decrease in red cell mass. We also observed plasmacytosis in bone marrow smear which is in consensus with several studies where the proliferation of plasma cells produces an abnormal amount of paraprotein.^3,7,20Hence plasmacytosis in conjunction with other hematological and biochemical investigations and clinical features is useful in the diagnosis of MM. The radiological data in patients with M spike showed the presence of osteolytic lesion which is one of the CRAB indices frequently seen in MM patients.^7,6,2 In the present study, only 16% of SPE reports had one or more investigations so a complete workup is essential for diagnosis and assessing of risk in patients with MM.

A laboratory audit of 4398, suspected cases of paraprotein showed the presence of M spike in 7.7% of cases.M spike showed a significant positive association with total protein, globulin, and uric acid and negative association with albumin. Associated biochemical, hematological, and radiological correlates of the positive cases could be documented in only less than 20% of the positive cases. A total workup is required for implementing appropriate therapy. One of the most important reasons may be that ours is a tertiary care hospital and the associated lab is also a reference lab where samples from in and around the city are tested. After the preliminary SPE, it could be possible that the patients were further evaluated and continued treatment at some other facility.

CONCLUSION:

For a more conclusive opinion, reports of all 340 were not available. It could not be ascertained if the SPE was for the first time or if it was a follow-up SPE as case records were not seen. However, it was made sure that there are no duplicates of the same patient. Whenever there was more than one report of the patients, the earliest one was considered for the study. As it was a lab-based study, the clinical profile of the patients like presenting symptoms, therapy, and the outcome could not be documented.

CONFLICT OF INTERESTS:

The authors declare that they have no conflict of interest

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Received on 11.01.2022 Modified on 13.07.2022

Research J. Pharm. and Tech 2023; 16(8):3583-3587.

DOI: 10.52711/0974-360X.2023.00591