European Journal of Cardiovascular Medicine

Multiple myeloma (MM) represents malignant proliferation of plasma cells derived from a single clone. The median age at diagnosis of multiple myeloma is 70 years, and the occurrence increases with age.[1] The tumor, its products, and the host response to it result in numerous clinical presentations including anemia, hypercalcemia, clotting abnormalities, bone pain or fracture, renal failure, susceptibility to infection, neurologic symptoms and manifestations of hyper-viscosity.[2] MM is also sometimes grouped with Waldenstrom’s macroglobulinemia, primary amyloidosis and heavy chain diseases. Now a days treatment has evolved, but proper diagnosis is needed with staging.

Two cases of hyperglobulinemia, hypercalcemia, deranged renal biochemical parameters and associated anemia were detected at the Biochemistry and Pathology laboratories in a span of 6 months in a tertiary medical college (Murshidabad Medical College & Hospital) of West Bengal leading to the diagnosis of MM. Both patients had been undergoing treatment for other symptoms and routine biochemistry was advised by treating physicians for other indications.

In view of these two patients, it was considered that the threshold for suspecting Multiple Myeloma (and associated gammopathies) among non-specialized clinicians handling a large number of patients may be inadequate. On the other hand, using a diagnostic algorithm that prompts the laboratory medicine specialist to directly check for patterns consistent with MM, it is possible to identify patients of such rare diseases more efficiently, enabling faster detection, better prognosis and care. The objectives of the present study were the following:

1. To develop an algorithm-driven combination of laboratory parameters for detection of multiple myeloma without initial clinical inputs
2. To measure the diagnostic yield of such an algorithm over a period of 1 year (including the pilot phase of 6 months)
3. To list the patients who are thus detected and enumerate their clinical, hematologic, immuno-histochemistry, bone marrow and protein electrophoresis findings along with case-specific observations

2.1Subjects:

This interventional study with prospective design was conducted from 1^st January, 2022 to 30^th December, 2022 in Biochemistry and Pathology departmental laboratories for routine parameters. For advanced biochemical tests and immunohistochemistry samples were sent to authorized laboratory.All patients of age group 40 – 80 years with hyperproteinemia (>9 gm/dl) and hyperglobulinemia (>5.5 gm/dl) on routine biochemistry (performed for any indication) and whose hematological reports were available, were included in this study. Patients with chronic liver disease; any viral, bacterial and parasitic infections; auto-immune disorders (i.e., SLE, rheumatoid arthritis etc.) were excluded. Based on the nature of testing a new approach, no predetermined sample size was available.All available results during the study period were considered (inclusive sampling).The study was purely academic in nature. Permissions were obtained from the Scientific Research Committee &Institutional Ethics Committee of Murshidabad Medical College and Hospital, Berhampore, Murshidabad (ECR/1620/Inst/WB/2021dated 25.08.2022).

2.2Methods:

Routine biochemical and hematological reports in our hospital  were reviewed to identify 18 patients who presented with hyperproteinemia with increased globulin fraction, increased urea and creatinine, increased calcium and anemia.Out of these,  14 patients were contacted from hospital records and informed consent was obtained for retesting the above parameters along with serum protein electrophoresis (vide figure 1).Samples having clinico-biochemical correlations with suspected M-band in our electrophoresis were further investigated in authorized laboratory for advanced biochemical tests and bone marrow aspiration studies and Histopathology examination (HPE) along with Immunohistochemistry (IHC) for any mass lesions.

Figure 1: Patient selection process

Routine biochemical parameters including liver function test (LFT), urea, creatinine, calcium was estimated by spectrophotometric method in Erba EM 360 chemistry analyzer (ERBA Diagnostics Mannheim GmbH, Mannheim, Germany). Electrolytes were run on TransAsiaEasyLyte PLUS Automated Electrolyte Analyzer (TransAsia Bio Medicals Ltd, Mumbai, India). Next, serum protein electrophoresis was run in alkaline medium (pH 8.6) with barbitone buffer on 0.8% agarose gel by horizontal electrophoresis apparatus [3] with densitometric scanner. Immunotyping and particular free light chain identification and quantification were done by immunoturbidimetry. Further investigations include bone Marrow aspiration [4] study with Leishman-Giemsa staining, histo-pathological examination [5] with routine Hematoxylin-Eosin staining in formalin fixed, paraffin embedded section and immuno-histochemistry for antibody specific for plasma cell neoplasm (CD 138) with IHC staining kit and microwave for antigen retrieval.

Quality control is maintained with control and calibrators supplied along with the standardized kit (IQC). Tests those done from outside are from NABL/CAP accredited laboratory.

Operational definition:

The new definition of active Multiple Myeloma is [6]:

Clonal bone marrow plasma cells >10% or biopsy-proven bony or extramedullary plasmacytoma and any one or more of the following CRAB features and myeloma-defining events:

1. Evidence of end organ damage that can be attributed to the underlying plasma cell proliferative disorder, specifically:

1. Hypercalcemia: serum calcium >0.25 mmol/L (>1mg/dl) higher than the upper limit of normal or >2.75 mmol/L (>11mg/dl)
2. Renal insufficiency: creatinine clearance <40 ml per minute or serum creatinine >177mol/L (>2mg/dl)
3. Anemia: hemoglobin value of>20g/L below the lowest limit of normal, or a hemoglobin value <100g/L (<10 g%)
4. Bone lesions: one or more osteolytic lesion on skeletal radiography, CT, or PET/CT. If bone marrow has <10% clonal plasma cells, more than one bone lesion is required to distinguish from solitary plasmacytoma with minimal marrow involvement

2. Any one or more of the following biomarkers of malignancy (MDEs):

1. 60% or greater clonal plasma cells on bone marrow examination
2. Serum involved / uninvolved free light chain ratio of 100 or greater, provided the absolute level of the involved light chain is at least 100mg/L (a patient's involved free light chain either kappa or lambda is the one that is above the normal reference range; the uninvolved free light chain is the one that is typically in, or below, the normal range)
3. More than one focal lesion on MRI that is at least 5mm or greater in size.

2.3Statistical analysis:

There would be calculation of the diagnostic yield as a percentage of total samples which are positive.

All samples from out-patient departments were scrutinized for 1 year.  Considering  a total of 5283 samples with adequate data, 18 patients were initially suspected based on the criteria mentioned above. Out of 18 these, 14 could provide consent and 6 were positively diagnosed as MM with a diagnostic yield of 42.85%. The age of the patients ranged from 45-72 years with mean age 61 years. The common presenting symptoms were fatigability, lower limb weakness, bone pain and chronic backache, decreased appetite etc. without any atypical findings (i.e. skin changes, endocrinopathies, retinopathy etc.).

Six patients were confirmed on clinical, biochemical, pathological basis and each patient is described in brief along with the baseline test characteristics shown in table 1 and serum protein electrophoresis shown in table 2.

Table 1: Baseline parameters of study population

Table 2: Serum Protein Electrophoresis Quantification by Densitometric Scanning/ Capillary Electrophoresis

Case 1: A 63-year-old male patient presented with generalized weakness, headache for 1 month and rapidly developing shortness of breath for 3 days. Bence Jones protein were detected in urine by The Heat test and Osgood and Haskin’s test. Chest X-ray showed massive left pleural effusion with underlying collapse and consolidation. M spike is noted on serum electrophoresis on gamma globulin region. Bone marrow aspiration revealed 20% plasma cells.

                         Electrophoresis & bone marrow aspiration of case 1

Case 2: A 70 years old male presented with generalized weakness for months and sudden onset shortness of breath for 5 days. In his routine blood examination, anemia, increase total protein with hypoalbuminemia were detected. His urea, creatinine and calcium were also increased. On serum protein electrophoresis, M spike is noted in gamma globulin region (Figure 4). His bone marrow aspiration study revealed 24% plasma cells, which is conclusive of diagnosing multiple myeloma along with other findings.

                               Figure 4: Electrophoresis pattern of Case 2

Case 3: A 45-year-old female patient presented with rapidly progressing low back pain for 2 months with lower limb weakness. Routine blood examination showed  anemia, raised ESR, high globulin, leading to a quick CECT of lumbosacral spine (Figure 5) which revealed expansile lytic lesion containing soft tissue in right ala of sacrum (5X5.5 cm). CT-guided Tru-cut biopsy from right sacral mass showed predominantly monomorphic cells with scattered large cells having atypical hyperchromatic nuclei diffusely involving the core and cells having granular nuclear chromatin and fried-egg appearance at places, suggestive of non-epithelial primary neoplastic lesion of plasma cells. Cell block was prepared from cytology aspirate, revealed kappa light chain positive plasma cell neoplasm. Serum protein electrophoresis showed M spike in gamma globulin region. All these findings confirmed diagnosis of multiple myeloma.

                        Figure 5: CECT of lumbo-sacral spine of Case 3

Case 4: A 72-year-old male patient presented with back pain for 2 months along with weakness, anorexia, weight loss. His routine blood examination revealed severe anemia along with increased total protein with altered A:G ratio and hypercalcemia. Urgent  CECT of dorso-lumbar spine and thorax showed right paravertebral intrathoracic mass at level of T2 vertebral body with adjacent bony erosion with well-defined tiny focus of calcification (33X22X26 mm). CT-guided FNAC from right paraspinal mass showed dyscohesive dispersed cells with abundant bluish cytoplasm with eccentric nuclei suggestive of non-epithelial neoplasm. CT-guided Tru-cut biopsy from upper right paravertebral mass showed sheets of monomorphic plasmacytoid cell with eccentric nuclei and granular chromatin. A diagnosis of plasma cell neoplasm/multiple myeloma was given. Serum protein electrophoresis with immunofixation revealed M spike at gamma globulin region which is of kappa band of IgG in nature.

Figure 6: HPE slide of Case 4

Case 5: A 56-years-old male was presented with LBP with lower limb weakness for >6 months. On his routine blood examination anemia, increased total protein and globulin were detected. Therefore, in this case instead of searching for central causes of the weakness, urgent MRI scan of LS spine revealed altered signal intensity of vertebral bodies with involvement of posterior elements along with disc protrusion at L4-L5 & L5-S1 and disc bulge at L1-L2, L2-L3, L3-L4 levels compressing the exiting nerve roots on both sides. HPE from D12-S1 lesion revealed plasma cell neoplasia suggestive of multiple myeloma. On IHC tumor cells are positive for CD138 and kappa LC that conclude the diagnosis of multiple myeloma. His renal function tests and calcium were within normal limit and no M spike noted in serum protein electrophoresis.

Case 6: A 60-years female patient presented with rapidly developing back pain. Her routine blood tests showed anemia, hyperglobulinemia and altered renal function, thereby prompting the MRI scan of DL spine which revealed multifocal areas of punctate T1 & T2 hypointense marrow areas involving whole of dorso-lumbar, sacrum vertebrae, bilateral iliac blades and multi-level compression fracture of dorso-lumbar vertebrae along with circumferential disc bulge at L5-S1 with narrowing of right lateral recess & neural foramen compressing right S1 traversing & L5 exiting nerve roots. Serum protein electrophoresis with immunofixation revealed M spike at gamma globulin region which is kappa light chain of IgG in nature.

Multiple myeloma is a malignant neoplasm of the terminally differentiated plasma cells, typically occurs in elderly patients. Normally, the plasma cells constitute 1% of the cells in the bone marrow, but as the disease advances, the tumor load in the bone marrow increases up to 80%, depending upon the disease severity. These malignant plasma cells synthesize monoclonal antibodies which are released into the circulation. Therefore, the monoclonal protein (antibody) level in the serumincreases.

In our study 42.85% cases were found to have monoclonal gammopathy or para-proteinemia, whereas Col. Chopra et al., found 24.4% samples to be positive for the M protein by SPEP.[7] In our study 5 patients had positive M band. Among the 5 M band positive cases that we studied, there was an M spike in the gamma region in 4 cases (80%) and 1 case (20%)had an M band in the beta-2 region (vide Table 2). Sunita Tripathy et al reported that, 87.5% of the cases had an M band in the gamma (γ) region and that 12.5% cases had an M spike in the beta(β) globin region.[8] The most common type of M protein found in MM is IgG followed by IgA and light chain only. Our study showed that IgG increased in all cases more than 3 – 4 times of upper reference limit (Figure 2). Hussain et al reported that IgG accounted for 40.8% of cases and IgA accounted for 18.5% of patients. In our study 4 cases had free kappa light chain whereas rest 2 had free lambda light chain (Figure 3). As per Col. Chopra et al., IgG with κ chain was seen in 40%, IgG with λ chain was seen in 50%.

Figure 2: Immunoglobin quantification

Figure 3: Free light chain quantification

In this present study all patients presented with anemia (Hb% <10 g%), 2 patients had hypercalcemia where total calcium raised upto 14 mg/dl and 2 patients had serum creatinine >2 mg/dl (Table 1). Hussain et al reported that initially anemia (Hb <10 gm/dl) was found in 50.5% cases and was mostly normocyticnormochromic; hypercalcemia (>11 mg/dl) was seen in 11.3% and serum creatinine level > 2.0 mg/dL was seen in 27.2% of cases.[9]

It has been reported that hyponatremia can be observed in plasma with extremely high protein values; however, it is usually classified as pseudohyponatremia. Pseudohyponatremia is defined as spuriously low plasma sodium concentration. The measured sodium concentration is low, but the actual sodium concentration in the plasma is normal.[10] This measurement artifact is evident in cases of extremely high levels of lipids (hyperlipidemia) or proteins (hyperproteinemia) in the blood.

In this study all patients had hyponatremia during presentation (Table 3). Usually, it is mild, self-limiting, and not associated with symptoms. Potassium and chloride are also influenced by the electrolyte exclusion effect in the same manner as sodium because they are electrolytes contained in the water portion of plasma and are measured using the same methodology. Though we had used direct ISE method for estimating these electrolytes but still we had such results on freshly collected sample in fasting which were tested in duplicates. The anion gap evaluates the difference between measured and unmeasured anions in the plasma and is usually used to assess acid-base disorders or to provide quality control for measuring all electrolytes. In all patients, anion gap is significantly decreased. Low values are an infrequent finding and most commonly indicate laboratory error or hypoalbuminemia (low albumin in the blood). Laboratory errors occur when there is an underestimated sodium concentration due to elevated proteins in the plasma. Hypoalbuminemia is the second most common cause of a low anion gap. The negative charges of albumin make up most of the unmeasured anions, so changes in the plasma albumin would be expected to alter the plasma anion gap. Studies have found that for every 1 g/dL decrease in albumin, there is a 2.3- to 2.5-mmol/L decrease in the anion gap.[11]

The majority of patients presented with pain as a symptom. Imaging was a major contributor in diagnosis once these patients were identified on basis of hyperglobulinemia, otherwise many of them would have gone through standardized care for back pain involving rest, analgesics, physiotherapy etc. Imaging also was quicker in narrowing down the diagnostic possibilities and should be considered more liberally in settings with adequate resources. The patients did not always show M band indicating that every tertiary care institution should have more specialized diagnostic capability including CD-138 and similar tests.

The study could detect only six cases in one year. The issue of obtaining informed consent meant that once routine tests showed abnormality, the laboratory medicine expert had to either track down the patient from hospital database or contact the clinician responsible for the patient to obtain additional tests and samples. It meant additional time on behalf of the laboratory medicine specialist had to be devoted for this project. This study is not a multicentric study so the diagnostic approach or algorithm utilized needs to be re-validated by other groups before it can be widely adopted.

The study aimed to develop a method or algorithm for detection of MM with limited resource and available skill. Based on the results, it may be concluded that implementing the system requires additional time and resource on the part of the laboratory medicine specialist. Also, availability of CD-138 or similar tests will definitely help in improving procedures. Whether significant diagnostic yield is obtained from such an approach remains to be verified by other research groups.The issue of considering the additional presence of hyponatremia and decreased anion gap for further improving the diagnostic algorithm may be examined by other studies in future.

6.CONFLICT OF INTEREST:

One of the authors (No.4) is a member of Institutional Ethics Committee, so he  excused himself from the meeting in which the study was discussed.

7.PROTECTING PATIENT PRIVACY & AUTONOMY:

The study was not utilized the full name or address of the patients nor the patient be denied of any service should they choose not to participate in the study. The test results were always shared with the patient or the caregiver as the case may be.As soon as the diagnosis of MM or relevant other disease was established the authors arranged for oncology referral so the treatment of the patient is not delayed.

8. FINANCIAL BURDEN:

Cost for further investigations were borne by the authors on equal share basis and no additional expense were incurred by the participants for the tests. In case the patient needs to travel for such testing, the actual cost incurred was borne by the authors on a case – to case basis.

1. Dash NR, Mohanty B. Multiple myeloma: a case of atypical presentation on protein electrophoresis. Indian J Clin Biochem [Internet]. 2012 Jan 1 [cited 2022 Aug 9];27(1):100–2. Available from: https://europepmc.org/articles/PMC3286584
2. Loscalzo J, Fauci AS, Kasper DL, Hauser SL, Longo DL, Jameson JL, editors. Harrison’s principles of internal medicine. 21st edition. New York: McGraw Hill; 2022. 1 p.
3. International Myeloma Working Group (IMWG) criteria for the diagnosis of multiple myeloma [Internet]. International Myeloma Foundation. [cited 2022 Aug 9]. Available from: https://www.myeloma.org/international-myeloma-working-group-imwg-criteria-diagnosis-multiple-myeloma
4. Chopra G, Gupta P, Mishra D. Evaluation of Suspected Monoclonal Gammopathies: Experience in a Tertiary Care Hospital. Med J Armed Forces India [Internet]. 2006 Apr [cited 2023 Jan 16];62(2):134–7. Available from: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4921976/
5. Tripathy S. The Role of Serum Protein Electrophoresis in the Detection of Multiple Myeloma: An Experience of a Corporate Hospital. J Clin Diagn Res [Internet]. 2012 Nov [cited 2022 Aug 9];6(9):1458–61. Available from: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3527769/
6. Hussain A, Almenfi HF, Almehdewi AM, Hamza MS, Bhat MS, Vijayashankar NP. Laboratory Features of Newly Diagnosed Multiple Myeloma Patients. Cureus [Internet]. [cited 2022 Aug 9];11(5):e4716. Available from: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6650180/
7. Pseudohyponatremia [Internet]. [cited 2023 Jan 16]. Available from: https://acutecaretesting.org/en/articles/pseudohyponatremia