Laboratory assessment of multiple myeloma

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Laboratory assessment of multiple myeloma Tracy Morrisona,*, Ronald A. Boothb,c, Kristin Hauffd, Philip Berardie,f, Alissa Visramg a

LifeLabs, Toronto, ON, Canada Division of Biochemistry, The Ottawa Hospital, University of Ottawa, Ottawa, ON, Canada c Department of Pathology and Laboratory Medicine, University of Ottawa, Ottawa, ON, Canada d Interior Health Corporate Office, Kelowna, BC, Canada e Ottawa Hospital Research Institute (OHRI), Ottawa, ON, Canada f Division of Anatomical Pathology, The Ottawa Hospital/University of Ottawa, Ottawa, ON, Canada g Division of Haematology, The Ottawa Hospital General Campus, Ottawa, ON, Canada *Corresponding author: e-mail address: [email protected] b

Contents 1. Introduction 2. Multiple myeloma disease classification and progression 2.1 Plasma cell and immunoglobulin biology 2.2 Myeloma disease progression 2.3 Biochemical disease features 2.4 Clinical value of the monoclonal immunoglobulin 2.5 Laboratory involvement in disease management 2.6 Molecular testing in multiple myeloma 2.7 Other disorders with monoclonal proteins 3. Laboratory testing in multiple myeloma 3.1 Serum protein electrophoresis 3.2 Serum immunofixation electrophoresis 3.3 Urine protein electrophoresis 3.4 Urine immunofixation 3.5 Serum free light chains 3.6 Additional laboratory testing in multiple myeloma patients 3.7 General interferences in laboratory testing in multiple myeloma 4. Laboratory reporting 4.1 Nomenclature 4.2 Information to be included in a protein electrophoresis report 5. Conclusion and future directions References

Advances in Clinical Chemistry ISSN 0065-2423 https://doi.org/10.1016/bs.acc.2018.12.001

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2019 Elsevier Inc. All rights reserved.

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Abstract Laboratory testing plays an essential role in the diagnosis and management of patients with multiple myeloma. A variety of chemistry and molecular assays are routinely used to monitor patient progress, response to treatment and relapse. Here, we have reviewed current literature and core guidelines on the details of laboratory testing in myeloma-related investigations. This includes the use and value of protein electrophoresis, serum free light chain and cytogenetic testing. Furthermore, we discuss other traditional chemistry assays essential to myeloma investigation, and potential interferences that may arise due to the disease nature of myeloma, that is, the presence of a monoclonal immunoglobulin. Finally, we discuss the importance of communication in protein electrophoresis results, where laboratorians are required to relate clinically relevant myeloma-relevant information to the ordering physician on the background of a complex pattern of serum or urine proteins. Laboratory testing in myeloma-related investigation relies on several traditional chemistry assays. However, we anticipate new tests and technologies to become available in the future with improved analytical sensitivity, as well as improved clinical sensitivity in identifying patients who are at high risk of progression to multiple myeloma.

1. Introduction Multiple myeloma is a plasma cell malignancy that currently affects over 250,000 people globally [1]. Neoplastic plasma cells grow in the bone marrow and displace normal cells, leading to disease onset and symptom manifestation, such as anemia and bone pain. There is currently no cure for multiple myeloma, but 50.7% of patients survive at least 5 years after diagnosis, which on average is at 62 years of age, based on the Surveillance, Epidemiology, and End Results Program from the National Cancer Institute in the United States (seer.cancer.gov). Laboratory testing has an extensive role in the diagnosis and management of multiple myeloma. Guidelines by the International Myeloma Working Group (IMWG) [2] include in their recommendations several chemistry laboratory tests for the diagnosis and management of the disease, including serum and urine protein electrophoresis, immunofixation electrophoresis and serum free light chains. These tests are performed on minimally-invasive specimens and can collectively identify the vast majority of myeloma cases. The goal of this review is to be an up-to-date resource of clinical and analytical information on multiple myeloma, with a focus on the role of the laboratory in the diagnosis and management of the disease. The

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biochemical and genetic components of disease progression are described, as well as disease categorization. Furthermore, details on analytic techniques used in myeloma investigation are discussed, as well as the importance of interpretation and reporting of electrophoretic results.

2. Multiple myeloma disease classification and progression 2.1 Plasma cell and immunoglobulin biology Multiple myeloma is a neoplastic plasma cell disorder characterized by clonal proliferation of malignant plasma cells in the bone marrow [3]. In a healthy immune system, the bone marrow produces an extensive variety of B-lymphocytes, each with the ability to facilitate neutralization of different foreign antigens. Unique B-lymphocytes undergo gene rearrangement and selection in the bone marrow, a process called V(D)J recombination, which introduces variation in the variable region of immunoglobulins. If exposed to an antigen, B-lymphocytes become activated and differentiate into plasma cells, which secrete large amounts of immunoglobulin to a presenting antigen. The system is regulated such that, eventually, plasma cells cease production. However, in myeloma, plasma B cells undergo both genetic and microenvironmental changes that contribute to cell transformation and proliferation, leading to the production of a clone of plasma cells that secrete a unique monoclonal immunoglobulin [3]. This immunoglobulin can be one of five classes (IgG, IgA, IgM, IgD and IgE) composed of both heavy and light chains attached via disulfide bonds (Fig. 1). The monoclonal immunoglobulin can also be composed of light chain only (kappa or lambda), or rarely heavy chain only.

2.2 Myeloma disease progression According to the IMWG, disease progression is defined in three stages: monoclonal gammopathy of undetermined significance (MGUS); smoldering multiple myeloma; and active multiple myeloma [4]. Progression to active multiple myeloma is thought to start with MGUS, an asymptomatic pre-malignant stage present in 3–4% of the population >50 years of age [5]. The definition of MGUS includes a serum monoclonal immunoglobulin concentration <30 g/L, clonal bone marrow plasma cells <10% and absence of symptoms (including end-organ damage attributed to hypercalcemia, renal insufficiency, anemia and bone lesions) [2]. Serum monoclonal

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Fig. 1 Immunoglobulin schematic. (A) Intact immunoglobulin is comprised of two heavy and two light chains held together by disulfide bonds. (B) Free light chain can be independently secreted from the clonal plasma cells. In its free form, part of the light chain previously bound to the heavy chain by disulfide bonds is now exposed. (C) Free heavy chain can also be secreted by the clonal plasma cell. Alternatively, it can degrade from the intact protein.

immunoglobulin concentration may be determined directly using the appropriate immunoassay or by protein electrophoresis (i.e., capillary (CE) or agarose). Identification of MGUS includes laboratory assessment of the monoclonal immunoglobulin (protein electrophoresis and immunofixation), serum calcium and creatinine and hemoglobin (complete blood count). Also, urine monoclonal immunoglobulin <500 mg/24 h is consistent with the diagnosis of free light chain MGUS. Other non-laboratory testing includes bone marrow biopsy and imaging. There is an important role for serum free light chain (sFLC) analysis in MGUS as an aid to risk prediction and probability of progression. Approximately one-third of MGUS patients have an abnormal serum free kappa: lambda light chain ratio, which is associated with a higher risk of progression [6]. An sFLC assay (The Binding Site, Birmingham, UK) has been used in the majority of diagnostic and prognostic studies and, consequently, sFLC cut-off guideline recommendations typically refer to this assay [2]. Although other assays are commercially available, they have not been as extensively characterized. MGUS patients are also monitored for progression using serum and/or urine protein electrophoresis and/or sFLC. The rate of progression to active multiple myeloma ranges between 0.5% and 2% per year, and depends on the monoclonal immunoglobulin type and concentration, sFLC ratio as well as other pathologic features [6,7]. For example, rate of progression to multiple myeloma is 1.5% for IgM MGUS [8] and 0.3% for light chain MGUS [9].

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Smoldering myeloma is an intermediate stage of disease between MGUS and active multiple myeloma. In this stage, the progression rate in the first 5 years after diagnosis is 10% [10]. The definition also includes serum monoclonal immunoglobulin <30 g/L or urine monoclonal immunoglobulin <500 mg/24 h. Similarly, end-organ damage is absent in smoldering myeloma. Although these criteria are identical to MGUS, the distinguishing feature in smoldering myeloma is that clonal bone marrow plasma cells are between 10% and 60%. According to the 2014 IMWG guidelines [2], active myeloma does not require a minimum monoclonal immunoglobulin concentration. This guideline allows for inclusion of 3% of myeloma patients with a nonsecretory myeloma, i.e., their neoplastic plasma cells do not secrete immunoglobulin. Additionally, approximately 40% of myeloma patients with end-organ damage have a monoclonal immunoglobulin <30 g/L at diagnosis [11]. Consequently, the distinction between smoldering and active multiple myeloma is the absence or presence, respectively, of myeloma-defining events (MDE). MDE include: (1) evidence of end-organ damage (attributed to hypercalcemia, renal insufficiency, anemia and bone lesions); (2) any one or more malignancy biomarkers, including (a) clonal bone marrow plasma cell percentage
60%, (b) involved:uninvolved sFLC ratio
100, and involved FLC
100 mg/L, and (c) more than one focal lesion on MRI [2]. The inclusion of MDE in the disease definition of smoldering and active myeloma is an update to previous 2003 IMWG guidelines that required evidence of end-organ damage for diagnosis of multiple myeloma. The benefit of MDE is to identify smoldering multiple myeloma patients without endorgan damage, but at higher risk for progression. For example, studies have shown that sFLC ratio
100 (involved:uninvolved free light chain) is associated with an 80% probability of progression to myeloma within 2 years [12,13]. Smoldering myeloma patients benefit from early therapy by extending overall survival [14]. According to the 2017 Canadian Cancer Statistics report, the incidence of myeloma is 9.1 for men and 5.6 per 100,000 for women with the average age of diagnosis of 62 years for men and 61 years for women (myelomacanada.ca). In the United States and United Kingdom, the incidence is similar [15,16] and globally there are approximately 154,000 cases and 101,000 deaths per year [1]. As seen in the Canadian data, the incidence can be higher in men than women. Furthermore, the incidence is two- to threefold higher in African Americans than Caucasians [1]. According to the Surveillance, Epidemiology, and End Results Program from the National Cancer Institute in the United States (seer.cancer.gov), over 50% of patients diagnosed with myeloma will survive at least 5 years.

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2.3 Biochemical disease features Multiple myeloma is suspected in patients who present with bone pain or lytic lesions, increased serum total protein, unexplained anemia or weight loss, hypercalcemia and/or acute renal failure. According to one study, 78% of patients present with normocytic, normochromic anemia, 58% with bone pain, 48% with increased creatinine, 32% with generalized weakness, 28% with hypercalcemia, and 24% with weight loss at the time of diagnosis [17]. Hypercalcemia, renal insufficiency, anemia and bone lesions are collectively referred to as CRAB features: C—hypercalcemia; R—renal insufficiency; A—anemia; and B—bone lesions. These identify end-organ damage and contribute to diagnosis of multiple myeloma. Laboratory thresholds for each CRAB parameter are outlined (Table 1). Hypercalcemia is present if serum calcium is 0.25 mmol/L above the upper normal limit or >2.75 mmol/L. Renal insufficiency is defined as a creatinine clearance <40 mL/min. Anemia is present if hemoglobin is >20 g/L below the lower normal limit or <100 g/L. Finally, bone lesions are identified by imaging. Proliferation of plasma cells in bone marrow is responsible for the top two clinical presentations. Anemia develops due to bone marrow replacement with neoplastic plasma cells. Furthermore, bone pain arises from lytic lesions identified in imaging due to plasma cell proliferation. In addition to plasma cell growth in bone marrow, another major contributor to the disease presentation is monoclonal immunoglobulin production. In the vast majority of cases, patients with myeloma produce neoplastic plasma cells that secrete monoclonal immunoglobulin. Renal disease is partially caused by free light chain deposition in the renal tubules if present at concentrations that exceed reabsorption. Other contributors to renal disease include hypercalcemia, AL amyloidosis and drug therapies. Kidney damage and dilutional effects from the presence of a monoclonal immunoglobulin further contribute to anemia.

2.4 Clinical value of the monoclonal immunoglobulin In multiple myeloma, monoclonal immunoglobulin serves as a disease biomarker that includes diagnosis, management, remission and relapse. In approximately 82% of myeloma cases, monoclonal immunoglobulin can be detected by serum protein electrophoresis (SPEP) [17]. Sensitivity increases to 93% when combined with serum immunofixation electrophoresis (SIFE) [17]. In 16% of patients with monoclonal immunoglobulin, the monoclonal immunoglobulin is composed of a free light chain only. In such

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Laboratory assessment of multiple myeloma

Table 1 Relevant laboratory values in the investigation, diagnosis and management of multiple myeloma. Clinical relevance/ interpretation Investigation Test Cut-point

Hypercalcemia

Calcium

0.25 mM greater than ULN or >2.75 mM

CRAB feature

Renal insufficiency

eGFR

eGFR <40 mL/min

CRAB feature

Anemia

Hemoglobin

20 g/L less than LLN or <100 g/L

CRAB feature

MGUS or SPEP smoldering UPEP multiple myeloma

<30 g/L

Diagnosis

Multiple myeloma

sFLC involved: uninvolved ratio


100 (plus involved FLC
100 mg/L)

Myeloma defining event

Treatment management

SPEP

>50% reduction in MIg

UPEP

>90% reduction in MIg or <200 mg/24 h

Partial response

SPEP

>90% reduction in MIg

UPEP

<100 mg/24 h

Very good partial response

SPEP


25% or
5 g/L increase in MIg

Treatment management

uPEP


200 mg/24 h

sFLC


25% or
100 mg/L difference between involved and uninvolved FLC

Relapse

<500 mg/24 h

ULN, upper limit of normal; LLN, lower limit of normal.

cases, SPEP may be insufficient to detect the free light chain due to its small size and ability to filter through the kidneys. One study suggests that serum concentrations exceeding 133 mg/L kappa and 278 mg/L lambda light chain are required to overwhelm clearance, although other parameters, such as charge of the monoclonal immunoglobulin and blood pressure, may influence this [18]. Urine protein electrophoresis (UPEP) combined with urine

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immunofixation electrophoresis (UIFE) is useful in these scenarios. Abnormal results from the sFLC assay are sufficient to diagnose myeloma in patients with clonal bone marrow cells [2]. Disease detection increases to 97% with the addition of UPEP, UIFE and sFLC [17]. The vast majority of myeloma patients have neoplastic plasma cells which secrete monoclonal immunoglobulin. Of the 97% of patients with a monoclonal immunoglobulin [17], the most common immunotype is IgG (52% of all cases). Light chain only immunotyping is present in 16% of cases, whereas IgD is present in few (2%) cases. Interestingly, 6.5% of myeloma cases have a negative SIFE [17]. Included in these are 3% of patients with non-secretory myeloma. This population consists of two categories of patients. Some have “nonmeasurable free light chain only myeloma,” i.e., negative SIFE and UIFE, whereas others have an abnormal sFLC ratio [19]. The other category has true non-secretory myeloma, i.e., negative SIFE, UIFE and a normal sFLC ratio. The latter represents about 30% of all cases [20]. Interestingly, 85% of non-secretory myeloma cases show evidence of monoclonal immunoglobulin in neoplastic plasma cells immunologically detectable by bone marrow aspirate. Interestingly, 15% of cases have no detectable neoplastic plasma cell monoclonal immunoglobulin [19].

2.5 Laboratory involvement in disease management Laboratory testing is also used to monitor treatment response, remission and relapse. Both serum and urine testing can measure different degrees of remission. The best treatment response is a stringent complete response, which includes negative SIFE, negative UIFE and normal SLFC ratio [21]. A complete response includes negative SIFE and negative UIFE only results. Partial and very good partial responses include >50% and >90% reduction in serum monoclonal immunoglobulin, respectively, and urine monoclonal immunoglobulin reduced >90% or <200mg/24 h urine, and <100 mg/ 24 h urine, respectively [21]. Furthermore, relapse is defined as a 25% increase in serum monoclonal immunoglobulin or absolute increase >5 g/L. In urine, relapse includes an increase of 25% in monoclonal immunoglobulin or absolute increase of 200 mg/24 h urine. In non-secretory myeloma, an increase of 25% or 100 mg/L in the difference between the involved and uninvolved free light chain defines relapse [21]. These thresholds are further outlined in Table 1. Importantly, it is increasingly recognized that relapse can occur due to clonal evolution wherein a new clone becomes dominant. Frequently, this may mean that a free light chain immunoglobulin becomes the major

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monoclonal immunoglobulin although previously characterized as intact. Early reports suggest that Hevylite (The Binding Site, Birmingham, UK) may provide additional information in early detection of myeloma relapse, but more studies are required to verify this approach [22].

2.6 Molecular testing in multiple myeloma Multiple myeloma development and progression is largely due to changes in plasma cell DNA. In this section, we describe the molecular abnormalities recognized as contributors to its development, as well as the role of molecular testing in diagnosis, treatment, remission and relapse. 2.6.1 Cytogenetic abnormalities and the biology of plasma cell neoplasms There are two broad classifications for cytogenetic abnormalities, primary and secondary. Primary cytogenetic abnormalities are thought to be early events that lead to the development of a clonal plasma cell, resulting in MGUS. The two main primary cytogenetic abnormalities in plasma cell neoplasms are translocations of the immunoglobulin heavy chain (IgH) locus (i.e., chromosome 14q32) and gains of chromosomes (i.e., trisomy 3, 5, 7, 9, 11, 15, 17). Translocations occur due to errors during B-cell specific DNA modification of VDJ rearrangement, somatic hypermutation and IgH switch recombination [23]. Translocation of the IgH gene often results in dys- or up-regulation of an oncogene on the partner chromosome. Common partner chromosomes include 11q13 (CCND1, cyclin D1), 4p16.3 (FGFR-3 and MMSET genes), 6p21 (CCND3, cyclin D3), 16q23 (c-MAF gene) and 20q11 (MAF-B gene) [24]. Secondary cytogenetic abnormalities are often associated with malignant transformation and disease progression. Unfortunately, the factors leading to these secondary abnormalities resulting in progression are still unknown. As such, there is no clear-cut clinical approach to predict which patients with a more indolent disease such as MGUS will ultimately progress to multiple myeloma. The main secondary cytogenetic abnormalities include del(17p), gain(1q21), t(4;14), t(14;20), MYC translocations and del(1p) [24]. 2.6.2 Laboratory detection of genetic abnormalities Metaphase karyotyping (conventional cytogenetics) is the historical method of choice for identifying cytogenetic abnormalities and determining disease risk at diagnosis. However, only 20–30% of myeloma patients have abnormal cytogenetic profiles using conventional approaches. Although

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cytogenetic karyotyping is an excellent way to identify gross cytogenetic abnormalities, it is unable to identify small or cryptic cytogenetic lesions. Furthermore, this testing method requires proliferating cells. In contrast to other hematologic malignancies, multiple myeloma often has a low burden of proliferating malignant plasma cells in the bone marrow [24]. Therefore abnormalities reported with conventional cytogenetics are likely reflective of increased plasma cell proliferation, which portends poor prognosis [25]. Metaphase karyotyping is also a useful way to identify myelodysplastic syndrome (MDS) which may develop during the course of therapy. In contrast to conventional cytogenetics, interphase fluorescence in situ hybridization (FISH) can detect cytogenetic abnormalities independent of cell proliferation. As such, the sensitivity of FISH is higher than conventional cytogenetics, but its sensitivity is limited by the percentage of plasma cells in the bone marrow. The proportion of plasma cells within bone marrow aspirates has been shown to range between 1% and 20% and there is no absolute minimum number of plasma cells to make the diagnosis of a plasma cell neoplasm [26]. Therefore, FISH performed directly on aspirate samples typically has a lower sensitivity in multiple myeloma than in other hematologic malignancies owing to the variable burden of disease at diagnosis. In order to specifically identify plasma cells and improve testing reliability, FISH testing can be performed on CD138-sorted cells or with cytoplasmic immunoglobulin enhanced FISH staining [27]. Methods for plasma cell purification include immunomagnetic bead based sorting, fluorescenceactivated cell sorting and flow cytometry, among others [26]. FISH is currently the standard method for analysis of cytogenetic abnormalities. A limitation of FISH testing is that it does not typically detect singlenucleotide variations. For example, the tumor suppressor TP53 gene is deleted in 7% of myeloma patients; however, exome sequencing has shown that this gene is mutated at a much higher frequency and subsequently would not be detected by FISH analysis [28]. Though not currently used in clinical practice, single nucleotide polymorphism mapping arrays and comparative genomic hybridization are sensitive tools to detect small numerical chromosomal aberrations or mutations [28]. Gene expression profiling (GEP) is another tool being evaluated and multiple prognostic gene expression signatures have already been reported [29]. It is conceivable that GEP-derived genetic signatures could be used to risk stratify and prognose multiple myeloma in the future [30].

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Table 2 Mayo stratification of myeloma and risk-adapted therapy (mSMART) consensus [4]. Intermediate Standard risk risk High risk

Cytogenetic abnormalities

Trisomies t(11;14) t(6;14) None

Gain(1q) t(4;14)

Del 17p t(14;16) t(14;20)

Median overall survival (years)

7–10

5

3

Percentage of newly diagnosed myeloma patients within each risk stratification (%)

75

10

15

2.6.3 Clinical relevance of cytogenetic abnormalities Multiple myeloma is a heterogeneous disease and cytogenetic testing plays an important role in risk stratification of these patients. The IMWG consensus states that translocations t(4;14), t(14;16), t(14;20) and del (17/17p) and any nonhyperdiploid karyotype are considered high risk findings in newly diagnosed patients. In general, patients with high risk disease have an overall survival less than 3 years and patients with three or more cytogenetic abnormalities have an estimated survival less than 2 years [28]. Cytogenetic abnormalities have been used to prognose the overall survival [4] (Table 2). Interestingly, certain cytogenetic abnormalities have been associated with distinct clinical presentations in myeloma. For example, 25% of patients with t(14;16) present with acute renal failure as the myeloma defining event and most patients with trisomies have bone disease at diagnosis [4,31]. Optimal therapy can also be influenced by the specific cytogenetic abnormality. In patients with t(4;14), bortezomib-based therapy ameliorates the negative prognostic impact of this translocation [28]. Overall, the landscape of myeloma treatment is rapidly evolving as novel therapeutic agents are developed and risk-adapted therapy is now an important consideration in the therapeutic approach. Consensus statements regarding how to apply risk-adapted therapy in plasma cell neoplasms are currently in development. Therefore cytogenetic testing continues to play a key role in prognosis and therapeutic decisions. 2.6.4 Future role of molecular diagnostics in multiple myeloma At present, myeloma remains an incurable disease. Deeper responses to therapy have repeatedly been shown to result in better long term outcomes.

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It has been well established that patients who achieve a minimal residual disease (MRD) negative complete remission (CR) have longer progression free survival than patients with an MRD positive CR [32]. As such, MRD testing will likely become a routine test in clinical practice for both prognosis and early prediction of disease relapse. MRD can be detected by flow cytometry or molecular methods (allele-specific oligonucleotide-quantitative polymerase chain reaction or next generation sequencing).

2.7 Other disorders with monoclonal proteins €m macroglobulinemia 2.7.1 Waldenstro Although patients with Waldenstr€ om macroglobulinemia have IgM monoclonal immunoglobulin, it is not a type of myeloma. The distinction is that the bone marrow contains lymphoplasmacytic lymphoma cells, not neoplastic plasma cells. Therefore, traditional chemistry laboratory testing alone is insufficient to distinguish it from multiple myeloma. Diagnosis includes detection of a monoclonal IgM of any size and at least 10% lymphoplasmacytic lymphoma cells (mSMART.org). Patients can experience IgM mediated symptoms including cryoglobulinemia, peripheral neuropathy and amyloidosis, as well as symptoms due to lymphoplasmacytic lymphoma such as anemia and hyperviscosity [33]. The disease has a fatality rate of 50% [33] with an incidence of approximately 4 per million annually. 2.7.2 Solitary plasmacytoma Solitary plasmacytoma is a tumor composed of plasma cells. If the tumor is exclusive to bone, it is referred to as a solitary plasmacytoma. However, if it is present outside bone in soft tissues, it is referred to as solitary extramedullary plasmacytoma. Importantly, lack of typical myeloma CRAB features is consistent with a solitary plasmacytoma diagnosis. Only bone pain presents in these patients due to the plasma tumor on bone. Based on eight studies, 24–72% of solitary plasmacytoma patients had detectable monoclonal immunoglobulin [34]. However, in general, the size of the monoclonal immunoglobulin in solitary plasmacytoma is smaller than in myeloma. Also, the incidence of non-secretory myeloma is higher in solitary plasmacytoma. Approximately one-third of these patients have no detectable monoclonal immunoglobulin. Furthermore, abnormal sFLC is associated with higher risk of progression [35].

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2.7.3 POEMS Although POEMS is a rare disorder that includes in its diagnosis the presence of a monoclonal immunoglobulin, its contribution to the disease pathogenesis is largely unknown. POEMS is an acronym for the different clinical features that present in patients: P—peripheral neuropathy, O—organomegaly, E—endocrinopathy, M—monoclonal plasma-cells proliferative disorder, S—skin changes. Not all of these clinical features are required for diagnosis. According to IMWG, there are mandatory, major and minor criteria for diagnosis [2]. The mandatory criteria include polyneuropathy and monoclonal plasma cell proliferative disorder. Organomegaly, endocrinopathy and skin changes are all considered minor. Some studies have investigated serum and urine protein electrophoresis testing in POEMS. All patients have monoclonal immunoglobulin [36] and >95% contain a lambda light chain in the intact form [37]. However, 25% of patients do not show abnormalities on SPEP and the remaining patients present with a profile consistent with a polyclonal gammopathy [38]. Therefore, SIFE is required to identify the monoclonal immunoglobulin in this subset of POEMS patients. One study has also investigated the role of sFLC in POEMS [39]. Although sFLC were increased in 90% of these patients, the ratio was abnormal in only 18% of cases. Plasma vascular endothelial growth factor (VEGF) has been suggested as marker of POEMS. Increased plasma VEGF differentiates POEMS from other plasma cell dyscrasias [40] and may be useful in prognosis and response to therapy [41,42]. 2.7.4 AL amyloidosis Immunoglobulin light chain (AL) amyloidosis accounts for 70% of all amyloidosis cases [43]. Pathogenesis is attributed to the plasma cell clone, which produces an abnormal monoclonal light chain that predisposes it to form amyloid fibrils and deposit in tissues [44]. Deposition produces irreversible organ damage. Any organ, except the central nervous system, can be affected [44]. Heart and kidney are most commonly affected [43]. The plasma cell clone in at least half of AL amyloidosis patients shows <10% infiltration in bone marrow [43]. Given that the clone can be small, some groups have recommended a combination of SIFE, UIFE and sFLC for maximum sensitivity in detecting monoclonal free light chains in these patients [45,46].

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3. Laboratory testing in multiple myeloma 3.1 Serum protein electrophoresis SPEP is the main laboratory test used for detection and management of multiple myeloma. Clinical utility is derived from monoclonal immunoglobulin quantification. This is accomplished using two predominant technologies, both of which are electrophoretic. In general, electrophoresis is a broad term that refers to the separation of charged particles in a liquid medium under the influence of an electric field [47]. Commonly used for separation of macromolecules with ionizable groups, electrophoresis is useful for, but not limited to, investigation of abnormal serum and urine proteins. Electrophoretic migration is dependent on the net size, shape and charge of the molecule, as well as the strength of the electric field, the properties of the supporting medium which provides resistance to migration and the temperature under which the system operates. In SPEP, proteins are electrophoretically resolved using a solid support (gel-) or a small diameter tube (capillary-based). Ions within the buffer flow from one electrode to the other when an electric field is applied. Positively charged ions (cations) in the sample move toward the negative electrode (cathode), while negatively charged ions (anions) move toward the positive electrode (anode). Due to their ability to exist as either positively or negatively charged molecules, proteins are considered ampholytes because they contain varying numbers of ionizable amino (-NH2) or carboxy (-COOH) groups. The overall charge on the protein will depend on its isoelectric point (pI), i.e., the pH at which the net charge is neutral. If a protein is in a buffer that is more acidic than its pI, the protein will be positively charged; conversely, if the buffer is more alkaline, the net charge will be negative [47]. The resulting migration pattern is comprised of reproducible discrete zones of proteins, according to their amino acid composition. 3.1.1 Gel electrophoresis Traditional protein electrophoretic methods are based on solid support chromatography in which the specimen is applied to an agarose or cellulose acetate gel in a low ionic strength buffer. An electric field is applied across the gel, thus facilitating migration of the charged macromolecules. Proteins are then separated into their respective zones composed of an albumin, alpha-1,

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Fig. 2 (A) Gel electrophoresis of a normal serum. Five fractions are defined. The beta-1 and -2 fractions are combined. (B) Capillary electrophoresis of a normal serum. Protein fractions are defined including separation of beta-1 and -2.

alpha-2, beta-1, beta-2 and gamma fraction. Following electrophoresis, gels are stained with a generic protein-binding dye (i.e., Coomassie Brilliant Blue), washed and then dried. Resolved proteins are then visually examined and quantified by densitometry. A normal profile containing five electrophoretic fractions is shown (Fig. 2). Coomassie brilliant blue ionically binds proteins via their positive amide groups and sulfonic acid side chains. As with any dye, not all proteins will bind equivalently. Fortunately, this dye shows sufficient sensitivity for detecting monoclonal immunoglobulins. 3.1.2 Capillary zone electrophoresis More recently, capillary zone electrophoresis has come into favor in the clinical laboratory for several reasons. For one, the platform provides an automated approach to sample processing. Also, there is minimal band broadening due to higher voltage application, i.e., improved heat dissipation [47]. Rather than a solid support, the sample migrates through a small diameter fused silica capillary (20–180 μm internal diameter) containing an appropriate buffer. Small sample volumes (1–50 nL) are injected into the anodal end by positive pressure generated by the outlet vacuum, i.e., hydrodynamic injection. Alternatively, a timed voltage may also be used to inject the sample, i.e., electrokinetic injection. An electric field is applied and the specimen migrates through the capillary separating into its traditional electrophoretic fractions, i.e., albumin, alpha-1, alpha-2, beta-1, beta-2 and gamma. In capillary zone electrophoresis, a UV-based detector is placed at the end of the capillary near the

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cathode. Peptide bonds absorb UV light (200–214 nm) resulting in an electropherogram (Fig. 2B). UV light absorption allows for comprehensive detection unlike dye-based approaches that are susceptible to differential protein binding [48]. 3.1.3 Normal protein fractions Serum zone electrophoresis generates albumin, alpha-1, alpha-2, beta-1, beta-2 and gamma fractions that contain variable amounts of different proteins. Although these fractions are generally seen by both gel and capillary electrophoresis, their relative position may vary slightly among methods. 3.1.3.1 Pre-albumin

Transthyretin, i.e., pre-albumin, migrates anodal to albumin. Due to its low concentration (0.2–0.4 g/L), this protein fraction is often not observed by gel electrophoresis but can be seen by capillary electrophoresis. In either case, it is generally uncommon for clinical laboratories to report its presence. Transthyretin is a 55 kDa protein produced by the liver with a relatively short half-life of 2 days. It acts as a main transport protein for thyroxin (T4). Although used as an indicator of protein nutritional status due to its short half-life, other markers are available [49]. 3.1.3.2 Albumin

Albumin is a 69 kDa protein produced by the liver. Normal blood concentration range is 35–55 g/L and as such is the single most abundant protein present in serum. Its main function includes maintenance of oncotic pressure and a transport protein for steroids, fatty acids, hormones, bilirubin, as well as various drugs in the circulation [50]. The migration of albumin can be affected by binding to various compounds, including penicillin and bilirubin, i.e., hyperbilirubinemia, producing a poorly resolved or “smeared” appearance. Severely decreased or complete lack of albumin results in a rare condition referred as analbuminemia. To date, 66 cases of analbuminemia have been reported (http://albumin.org). The corresponding serum protein electrophoretic pattern produced shows a compensatory increase in other protein fractions. Clinically, symptoms range from none to edema and/or lipodystrophy. Further information on analbuminemia is available at http://albumin.org. Bisalbuminemia, producing split or double albumin fractions, is most often due to an inherited electrophoretic

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Fig. 3 Bisalbuminemia. Electrophoresis performed using Sebia Hydrasys 5-band gels. Three separate electrophoretic gels shown.

abnormality produced when two albumin proteins with slightly different migration characteristics are present (Fig. 3). Clinically, there is no known pathologic state associated with bisalbuminemia. 3.1.3.3 α1 Fraction

The α1 fraction contains a number of proteins including α1-antitrypsin, α1lipoprotein, α1-fetoprotein, α1-acid glycoprotein and α1-antichymotrypsin. α1-Antitrypsin is the most abundant α1 protein and accounts for the majority of this fraction seen on electrophoresis. It is also the most clinically important in terms of SPEP reporting. α1-Antitrypsin is an acute-phase reactant and readily changes in response to inflammation. Its deficiency is a genetically inherited autosomal co-dominant disorder associated with various clinical conditions including lung disease (emphysema and bronchiectasis), liver disease (hepatitis, cirrhosis, hepatoma), skin disorders (panniculitis) and is associated with vasculitis, in particular granulomatosis with polyangiitis (GPA). For a comprehensive review of α1-antitrypsin see Ref. [51]. Decreased α1-antitrypsin is readily observed by both gel and capillary electrophoresis because it is the predominant protein in the α1-fraction. Decreased or absent α1 globulins should be noted in the interpretive report.

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3.1.3.4 α2 Fraction

The α2 fraction contains α2-macroglobulin, haptoglobin, ceruloplasmin and complement proteins. Similar to α1, this fraction also contains acute phase reactants. Of these, the most relevant are α2-macroglobulin and haptoglobin. α2-Macroglobin is most abundant (1.3–3.0 g/L) and functions to inhibit plasmin as well as other serum proteases including serine-, cysteine-, aspartic- and metalloproteases [52]. Increased α2-macroglobulin is seen in nephrotic syndrome due to increased synthesis and inability to pass through the glomerulus. Haptoglobin is the other relevant protein present in the α2 electrophoretic fraction. Haptoglobin functions to bind free hemoglobin released by intravascular hemolysis to protect renal damage and retain iron. The hemoglobin-haptoglobin complex is cleared from the circulation with an estimated half-life of 12 h [53]. The two major haptoglobin alleles (Hp1 and Hp2) produce three major phenotypes (Hp 1-1, Hp 1-2 and Hp 2-2) resulting in slightly different electrophoretic motility. Hp 1-1 produces a single plasma complex with the fastest mobility and migrates anodal to α2-macroglobin. Hp 1-2 and Hp 2-2 have multiple plasma complexes [54] and produce several protein bands within the α2 fraction. The hemoglobin-haptoglobin complex migrates more cathodally, i.e., between the α2- and β-fractions. Recognition of the hemoglobin-haptoglobin complex is important when interpreting electrophoretic patterns in hemolyzed specimens (Fig. 4). 3.1.3.5 β Fraction

Depending on the electrophoretic system used, the β-globulins can migrate as a single β-fraction or can be resolved into β1 and β2 fractions. The β fraction contains a number of proteins including transferrin, β1-lipoprotein, hemopexin and complement proteins C3 and C4. Transferrin is produce by the liver and is a major component (1.5–5.0 g/L) of β1 globulin. Transferrin transports non-hemoglobin bound iron in the blood. Enhanced production results in an increased β1 fraction consistent with iron deficiency. Laboratorians should be aware of the possibility of increased β1 fractions due to elevated transferrin and recognize the potential for masked β1 migrating monoclonal proteins. Decreased transferrin is seen in conditions including cirrhosis, acute inflammation and renal disease. β1 Lipoprotein (low density lipoprotein) is also present in the β fraction. It often produces an unusual appearance on gel electrophoresis with a fine darkly staining irregularly migrating band. Its mobility varies, i.e., anodal to

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Fig. 4 Hemolyzed serum. Electrophoresis performed using Sebia Hydrasys 5-band gels. Arrow heads indicate hemoglobin-haptoglobin complexes.

the β1 globulins to cathodal to C3, and is concentration dependent. β1 Lipoprotein demonstrates more consistent migration (between the α2 and β1 fractions) in capillary electrophoresis. Because IgM or IgA monoclonal protein can produce a similar migration pattern, their presence should be considered. C3 complement is second most abundant in the β fraction protein and accounts for the majority of β2 globulin when high resolution electrophoresis is performed. Increased C3 can be observed in an acute phase response, biliary obstruction and in a portion of patients with focal glomerulosclerosis. On the other hand, decreased C3 is seen in conditions that consume C3, i.e., infection, inflammatory disorders (lupus, autoimmune vasculitis) and nephritic disorders. Genetic C3 deficiency is rare [55]. The β region is a relatively common location for monoclonal protein migration. If present at low concentration, these can be difficult to identify due to the presence of normal β globulin. Although IgA is the most common β migrating monoclonal protein, monoclonal IgM and IgG can also be seen. As such, careful review is necessary to eliminate

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this possibility during visual inspection. Triggers for further investigation include hypogammaglobulinemia with or without increased or unusual β globulins (Fig. 5).

Fig. 5 β-Migrating monoclonal protein. Electrophoresis performed using (A) 5-band Sebia Hydragel and (B) 6-band Sebia Hydragel β1-β2.

3.1.3.6 γ Fraction

The γ fraction contains the immunoglobulins IgG, IgA and IgM (IgD and IgE at low concentration) along with C-reactive protein (CRP) and fibrinogen (β-γ region). Fibrinogen is a plasma protein present at 1.5–4.0 g/L and is absent in serum. A small protein band in the β-γ region suggests that a plasma or poorly clotted serum specimen was analyzed. Residual fibrinogen may also be seen in the presence of high anticoagulant therapy. Specimens suspected of containing fibrinogen can be either immunotyped or treated with thrombin. Although CRP is normally not visible by protein electrophoresis, it can substantially increase 500–1000 mg/L during inflammation. Electrophoretic migration of CRP is method dependent, but is generally found in the mid-gamma region.

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3.1.4 Monoclonal protein fractions An important clinical utility of protein electrophoresis is monoclonal protein identification and monitoring thereof in MGUS and myeloma. Identification of monoclonal protein is the sole finding in MGUS, which defines the condition. It is often the initial abnormality in myeloma. As such, accuracy is critical to identification and precision key to monitoring. An electrophoretic abnormality suspicious for monoclonal protein must be confirmed by immunotyping (IFE or immunosubtraction). Laboratorians should not consider an electrophoretic abnormality as monoclonal until confirmed as immunoglobulin. Once confirmed, however, accurate and reproducible quantitation is required. 3.1.5 Interferences Protein electrophoresis using gel- and capillary-based systems are susceptible to a number of interferences that can produce the appearance of “monoclonal” artifacts. It is imperative that laboratorians be aware of potential interferences due to their clinical consequences. Below we briefly discuss common interferences and how they affect electrophoretic patterns. 3.1.5.1 Fibrinogen

One common interferent is fibrinogen due to the use of plasma instead of serum. Residual fibrinogen may be present in patients with coagulation disorders, on anticoagulation therapy or due to an acute phase reaction. Fibrinogen migrates in the β-γ region and can be easily mistaken for a monoclonal protein (Fig. 6). As such, immunotyping confirmation should be performed prior to characterizing it as immunoglobulin. As mentioned above, specimens may be treated with thrombin and reanalyzed. Because of this issue, serum is the specimen of choice for electrophoretic analysis. 3.1.5.2 Hemolysis

Hemolyzed specimens also can produce an electrophoretic pattern suggestive of monoclonal protein. Free hemoglobin or hemoglobin-haptoglobin complexes arising from in vivo or in vitro hemolysis produce distinct electrophoretic bands that migrate in the α2-β region and can be misidentified as monoclonal protein (Fig. 6). The nature of a α2-β region migrating band as being due to hemoglobin or hemoglobin-haptoglobin can be at least partially clarified by visual inspection of the specimen. As mentioned above, confirmation should be performed by immunotyping.

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Fig. 6 Serum vs plasma. Electrophoresis performed using Sebia Hydrasys 5-band gels. Arrow indicates fibrinogen.

3.1.5.3 Cryoglobulins

Cryoglobulin also has the potential to complicate electrophoretic interpretation. Cryoglobulin is immunoglobulin that aggregates/precipitates below physiologic temperature (37 °C). Because any decrease in temperature contributes to this phenomenon, temperature maintenance is critical during collection and preanalytical processing. Failure to closely monitor all phases may lead to substantial gamma globulin loss as well as other protein constituents. Cryoglobulinemia is clinically significant. Patients are prone to hyperviscosity issues. Most are not caused by monoclonal protein, but result from infection, most commonly hepatitis C or HIV, or autoimmune disorders [56]. Cryoglobulins can be classified using the Brouet system [57]: simple cryoglobulins as Type I (single immunoglobulin component); mixed cryoglobulins (multiple immunoglobulin components) as Type II (monoclonal with polyclonal immunoglobulins); and Type III (polyclonal immunoglobulins). The presence of a cryoglobulin can have two major effects on protein electrophoresis: (1) directly, by precipitation of the cryoglobulin at the point of gel application producing the appearance of a monoclonal protein and (2) indirectly, by altering protein fraction quantitation.

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Precipitation at the point of application can be seen on both routine PEP and on IFE (Fig. 7). The atypical appearance suggests the band is not due to monoclonal protein. During immunofixation electrophoresis, cryoglobulin produces a characteristic protein band at the sample origin visible in all lanes. It is important to determine the nature of the cryoglobulin as Type I, II or III. Incorrect interpretation of type II or III cryoglobulin as a monoclonal immunoglobulin can have significant clinical consequences. Warming to 37 °C and/or treatment with β-mercaptoethanol may resolve this potential artifact. Keren [58] proposed a simple method to determine if the precipitate at the sample application point is, in fact, cryoglobulin. He suggested repeating the electrophoresis and replacing one of the antisera with buffer or saline. If the band persists, it is cryoglobulin. 3.1.5.4 IgG4 related disorders

A recently recognized group of related conditions that can produce a monoclonal-appearing pattern are the IgG4 related disorders (IgG4-RD). Clinical manifestations of IgG4-RD are broad and include a large number of organs (pancreas, thyroid, lungs, liver, kidney, skin, lymph nodes and

Fig. 7 Type I cryoglobulin IFE  β-mercaptoethanol treatment. Without treatment, a gel artifact is present in all lanes. With treatment, the band resolves into monoclonal IgM kappa. Residual staining in the IgG, IgA and lambda lanes is noted due to incomplete removal of the cryoglobulin complexes.

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blood vessels) [59]. The hallmark of IgG4-RD is increased IgG sub-class 4 (IgG4). IgG4 normally accounts for 3–6% of total IgG and alone is not visible electrophoretically. In IgG4-RD, however, increased IgG4 and its relatively restricted electrophoretic mobility (β-γ region) can appear as a low concentration monoclonal protein [60]. The κ/λ ratio can be highly skewed such that it can appear as monoclonal protein by IFE (Fig. 8). Review of clinical records can help clarify the nature of the monoclonal appearing protein. In such cases where a monoclonal appearing protein is caused by increased IgG4, clinical consultation is recommended.

3.1.5.5 Monoclonal immunoglobulin therapies

Novel monoclonal immunoglobulin-based treatments (e.g., daratumumab and elotuzumab) for multiple myeloma have improved clinical outcome. Their use, however, can complicate interpretation of protein electrophoretic profiles. Treatments are, in fact, monoclonal protein of IgG kappa isotype and are present at concentrations visualized by protein electrophoresis. As can be expected, this phenomenon can lead to clinical confusion, additional investigation, change in treatment or incorrect classification of patient response. Serum half-life of elotuzumab and daratumumab is 4.6 and 9.0 days following the first dose, respectively [61]. As such, specimens should be collected just prior to dosing. Identification of new monoclonal IgG kappa in a patient undergoing treatment for myeloma should raise concern of potential interference. Effective communication between all clinical care teams, i.e., oncology, laboratory, pharmacy, is imperative to mitigate spurious test results.

Fig. 8 IFE with anti-IgG, IgA, IgM, κ and λ sera. All three patients (A, B and C) demonstrate a focal band in the β-γ region consisting of mainly IgG antibody. Connecting light chains are strongly skewed toward κ in patient A and λ in patient C. SPE, serum protein electrophoresis. Reproduced from J.F. Jacobs, R.G. van der Molen, D.F. Keren, Relatively restricted migration of polyclonal IgG4 may mimic a monoclonal gammopathy in IgG4related disease, Am. J. Clin. Pathol. 142 (1) (2014) 76–81.

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3.1.5.6 Radiocontrast dyes

Radiocontrast dyes specifically interfere with capillary electrophoresis due to their ability to absorb UV light. Contaminating dyes are typically detected as monoclonal appearing peaks in the α2 or β regions (Table 3). Migration patterns and washout periods are dye dependent, i.e., most have elimination half-lives of 60–120 min [62]. Although dye may be removed by desalting, it is preferable to collect the specimens prior to administration or after sufficient washout period (>5 half-lives). Electrophoretograms showing the various radiocontrast dyes are presented (Fig. 9). Many antibiotics also absorb light at 214 nm, but only a few have had reported interferences with capillary electrophoresis. In contrast, there is minimal clinical risk with gel-based IFE because the band will not be visualized. In immunosubtraction, the band will not correct with specific antisera. 3.1.6 Challenges in serum protein electrophoresis testing 3.1.6.1 Quantitation of bands

Accurate quantitation of monoclonal proteins by electrophoresis can be challenging, particularly if the monoclonal protein has a relatively low

Table 3 Common interferences in UV detection of proteins. Absorption Drug Migration maxima (nm)

Elimination T(1/2)

Meglumine iotroxate

Pre-albumin

237–244

Meglumine amidotrizoate

Anodal α2 globulin

237–244

60–120 min

Ioxitalamic acid; Iobitridol

Middle α2 globulin

237–244

120 min

Iopamidol; Iohexol; Iopromide

Cathodal α2 globulin

237–244

121 min

Meglumine ioxaglate

Anodal β globulin

237–244

Ioversol; Iomeprol

Middle β globulin

237–244

Piperacillin–Tazobactam (IV)

Anodal β1 globulin

214

0.7–1.2 h

Sulfamethoxazole– Trimetoprime

Anodal albumin

214

9h

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Fig. 9 Common contrast dyes that interfere with CE. Red arrow indicates the location of the contrast dye peak. Modified from C.R. McCudden, et al., Recognition and management of common, rare, and novel serum protein electrophoresis and immunofixation interferences. Clin. Biochem. 51 (2018) 72–79.

concentration relative to the polyclonal background. Quantitation is achieved by densitometric scan of the protein-stained gel and assignment of protein fraction quantitation based on the total protein and percent area of each fraction. In contrast, CE assigns fraction quantitation based on direct peptide bond UV detection. In either case, fraction identification is imperative for accurate monoclonal protein quantitation. Various techniques for monoclonal quantitation have been proposed, including perpendicular baseline quantitation, compensated perpendicular baseline quantitation and tangent skimming quantitation (Fig. 10). Perpendicular baseline quantitation involves placing the fraction delimiters at the intersection point of the monoclonal fraction and the polyclonal immunoglobulins. This technique tends to over-estimate the monoclonal protein fraction, particularly when the monoclonal protein concentration is low (<10 g/L) and the polyclonal background is not suppressed [63]. Large monoclonal proteins with minimal polyclonal immunoglobulin interference

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Fig. 10 Monoclonal peak quantitation. Serum protein CE electropheretograms (Sebia Capillarys) was performed. Quantitation by: (A) standard perpendicular drop method (shaded) where the demarcation begins where the monoclonal protein meets the polyclonal region below it (arrows) and then proceeds straight down to the baseline including all the area above and below. This measures 14.7 g/L (1.47 g/dL) and is the most frequently used technique currently; (B) corrected perpendicular drop where an attempt is made to narrow the area measured to compensate for the inclusion of the polyclonal area below. The same monoclonal protein here measures 11.5 g/L (1.15 g/dL); and (C) tangent skimming method described by Schild et al. [63]. As shown by the arrows, this method attempts to cut off only the area above the peak. The monoclonal protein now measures 9.5 g/L (0.95 g/dL). Reproduced from D.F. Keren, L. Schroeder, Challenges of measuring monoclonal proteins in serum. Clin. Chem. Lab. Med. 54(6) (2016) 947–61.

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can be accurately quantitated in this manner. This method is easily implemented for most electrophoretic systems. Compensated perpendicular baseline quantitation involves compensating for the polyclonal immunoglobulins by tightening the delimiters around the monoclonal peak. In this approach, underlying polyclonal immunoglobulin which contributes to the quantitation is compensated for. This method, if implemented consistently, can generate relatively accurate monoclonal protein quantitation. Unfortunately, there are no strict guidelines for selecting the amount of compensation. As such, this approach can generate significant operator induced variation in monoclonal quantitation. Tangent skimming quantitation, as proposed by Schild [63], is likely the most accurate method for quantitation. This practice, however, can be difficult to operationalize and is software dependent. The tangent skimming method is performed by identifying the two points where the monoclonal peak intersects the polyclonal immunoglobulins and measuring the area under the monoclonal peak, above this tangent line. Keren and Schroeder provide an in-depth discussion and comparison of the three methods [64]. 3.1.6.2 Reference change value

Monitoring of the monoclonal protein concentration is essential in the follow-up of MGUS, smoldering myeloma and multiple myeloma. The IMWG define specific response criteria which include changes in the monoclonal protein concentration (Table 1) [21,65]. These criteria are used to determine response category, but may be insensitive to detect early progression or response. A potentially more sensitive and objective criteria for assessing clinically relevant changes in monoclonal protein concentration is use of a reference change value [66]. A reference change value is the critical difference between serial biomarker values to determine if the difference is due to a true change in the clinical status of the patient or is due to analytical and normal biological variation. It is calculated knowing the analytical variation and normal biological variation (Eq. 1). qffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi pffiffiffi (1) RCV ¼ 2  Z  CV 2A + CV 2I where CVA is the analytical variation, CVI is the biological variation, and Z is the number of standard deviations appropriate to the desired probability, 1.96 for P < 0.05. Katzmann et al. and Salamatmanesh et al. determined the biologic variation of monoclonal protein in stable MGUS and generated change

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criteria, i.e., 27.5% change at a 95% CI and RCV for low (5.6 g/L) and high (32.2 g/L) concentration monoclonal protein of 36.7% (low) and 39.6% (high), respectively [67,68]. RCV determination should be performed using in-house analytic variation. Ideally, incorporation of the change criteria in the PEP report would be useful to interpret monoclonal protein change. 3.1.6.3 Co-migrating monoclonal proteins

Monoclonal components that migrate with other protein can be particularly difficult to quantitate. Options include: (1) quantitation of the monoclonal band along with the co-migrating normal β or α globulin; (2) choosing to not report the monoclonal fraction and recommending quantitation by more direct immunonephelometric method; and (3) semi-independent monoclonal quantitation if there is only partial overlap of the monoclonal protein with normal β or α globulin. Schroeder et al. proposed an interesting approach to deal with quantitation of β or α globulin migrating monoclonal proteins [69]. They suggest use of CE immunosubtraction to differentially quantitate the fraction including both normal globulins and monoclonal protein with and without the presence of the monoclonal protein, the difference being the amount of monoclonal protein present. This method is limited to those using CE and immunosubtraction, and, at this point, must be performed manually. However, those laboratories with suitable equipment could consider this approach. Regardless of the technique chosen for monoclonal quantitation, laboratories should clearly communicate the method used and when necessary identify the potential for overestimation of monoclonal protein quantitation. Effective communication becomes more problematic in the event of acute phase reactions wherein globulins are changing. As such, a median and range of normal globulin concentration should be provided so that the relative background contribution can be taken into account.

3.2 Serum immunofixation electrophoresis SIFE is required for confirmation of abnormalities detected by SPEP and to improve detection of monoclonal immunoglobulin not detected by SPEP alone. The sample is applied to a total of six lanes on the gel. Following electrophoretic separation, antisera specific to immunoglobulin heavy and light chains are applied. For control purposes, one lane, typically the first, is stained for total protein stain. The next five lanes are overlaid with antisera to human IgG, IgA, IgM, kappa and lambda.

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Periodically, the pattern seen on IFE will be missing a corresponding heavy chain (Fig. 11A). In this case, testing using anti-sera for a specific free light chain, IgD or IgE can be used to determine the identity of the monoclonal protein (Fig. 11B). In extremely rare conditions, a monoclonal heavy chain may be identified with no corresponding light chain, i.e., heavy chain disease [70]. In some circumstances, there will be multiple bands on the gel. On occasion, this will represent a bi- or even tri-clonal protein, but often, particularly if there are multiple bands in the same lanes, or bands in multiple lanes at the same migration, these multiple bands are aggregates of a single clone. To rectify, β-mercaptoethanol and/or fluidil may be added to the specimen to reduce disulfide bonds. The specimen may then be electrophoresed and interpreted. Disappearance of multiple bands with β-mercaptoethanol treatment supports immunoglobulin aggregation typically seen with dimeric IgA and pentameric IgM (Fig. 12A and B). Lack of band resolution following treatment supports the presence of a biclonal immunoglobulin (Fig. 12C and D). 3.2.1 Immunosubtraction An alternative to IFE is immunosubtraction CE. Five aliquots of the sample are pre-incubated with antisera to human IgG, IgA, IgM, kappa or lambda light chains. Following aggregation, CE is performed and the electropherotogram compared to the original. Each immunoglobulin will be subtracted as their larger size causes them to migrate differently. The combination of heavy and light chain antisera that causes the suspected monoclonal peak to disappear confirms the immunotyping. Although easily interpreted with large monoclonal proteins, it becomes more difficult with

Fig. 11 SIFE positive for serum free lambda light chain. (A) The lambda lane shows positive detection, but no corresponding heavy chain is detected. (B) Same sample as in (A) is analyzed for IgD, IgE and free lambda. Results show positive for free lambda light chain only.

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Fig. 12 β-Mercaptoethanol treatment effect on SIFE. (A) Sample with multiple IgMkappa bands. (B) Same patient sample treated with β-mercaptoethanol. Note: multiple bands observed in (A) have resolved. (C) and (D) Sample without and with β-mercaptoethanol treatment, respectively. No difference in band resolution was observed following treatment. Biclonal IgG lambda shown.

co-migrating clones and small clones on a polyclonal background. One of the main detractions from common use of this technique is the difficulty in interpretation, particularly for small underlying bands, i.e., light chain disease. As this technique uses CE, aggregated proteins are less often a source of interference.

3.3 Urine protein electrophoresis UPEP is commonly performed during the workup of patients with suspected myeloma and in monitoring disease activity. Unlike serum, urine does not normally contain large amounts of protein, i.e., healthy individuals secrete <150 mg/24 h. The glomerulus serves as a barrier to proteins where it effectively retains larger proteins >100 kDa, freely allows proteins <10–15 kDa and selectively retains mid-sized proteins. Normally, proteins that pass through the glomerulus are resorbed. Various pathologic states modify the amount and profile of urine proteins generating unique electrophoretic profiles. These include glomerular, tubular, non-selective (mixed) and overflow proteinuria.

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The principle of electrophoretic separation for urine is the same as serum. Electrophoresis of urine from a healthy individual generally shows no protein or very small amount of albumin. Low urine protein content may be mitigated by concentrating the urine specimen prior to electrophoretic analysis. Gel electrophoresis can be performed using standard 5- or 6-band serum agarose gels or high-resolution gels that produce up to 15 protein bands. Alternatively, urine specific gels can be used without the need to concentrate the urine. Whether concentrated urine or neat, both methods show similar sensitivity for detection of monoclonal protein [71]. Concentrated healthy urine (50–100  by volume) shows very low amount albumin and globulin following electrophoresis. Glomerular filtrate contains various medium and small molecular weight proteins, the most common being α1-acid glycoprotein (orosomucoid), α1-microglobulin, β2-microprotein, retinol binding protein and γ-trace protein. Disruption of glomerular function alters the composition of the filtrate and ultimately, urinary protein. The glomerular proteinuria pattern can be identified by presence of albumin and substantially lower amounts of β-globulin alone (selective glomerular) or α1, α2 and β globulin (non-selective glomerular) (Fig. 13). The albumin fraction is dominant (50 to >70% total protein).

Fig. 13 Glomerular proteinuria. Electrophoresis performed on unconcentrated urine by Sebia Hydrasys Hydragel HR. Gels show various patterns.

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Fig. 14 Tubular proteinuria. Electrophoresis performed on unconcentrated urine by Sebia Hydrasys Hydragel HR. Gels show various patterns.

Identification of monoclonal protein present in the β fraction can be complicated by β globulin protein. Comparison of the increased β fraction relative to albumin can aid identification and prompt further characterization by IFE. Tubular proteinuria is identified by the presence of two protein bands in the α2 region by high-resolution electrophoresis (Fig. 14). Low-resolution electrophoresis does not routinely show the α2 doublet. Although albumin does not predominate, it is visible due to its relative abundance in the circulation and does not represent glomerular dysfunction. Identification of low concentration monoclonal protein in tubular proteinuria can be difficult. Visual examination of the protein patterns can facilitate identification of suspected monoclonal protein, i.e., tubular proteinuria bands are usually of similar concentration. Suspected bands may be further characterized by IFE. Non-selective proteinuria has an electrophoretic pattern similar to that of serum with all fractions represented. The presence of polyclonal

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immunoglobulin indicates non-selective proteinuria due to their high molecular weight (>150 kDa). Overflow proteinuria occurs when plasma protein overwhelms tubule resorption. Significant inflammation and the presence of a monoclonal gammopathy result in this phenomenon. In inflammation, increased low molecular weight acute-phase proteins are circulating spill into the urine producing multiple bands on electrophoresis. In myeloma, overproduction of monoclonal protein, i.e., intact immunoglobulin or light chain, simply overwhelms renal capacity. UPEP shows monoclonal protein which present as a single band or multiple bands depending on the clonal expansion (Fig. 15).

3.4 Urine immunofixation UIFE is required to confirm UPEP findings such as a suspected monoclonal component. Although hemolysis may also produce a monoclonal-like abnormality, this finding can be easily evaluated by UIFE. The combined use of SPEP, UPEP, SIFE and UIFE significantly increases probability of detecting myeloma.

Fig. 15 Urine monoclonal protein. Electrophoresis performed on unconcentrated urine by Sebia Hydrasys Hydragel HR. Arrowheads indicate monoclonal immunoglobulin.

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For maximum sensitivity, a 24-h urine collection is required for UIFE [11]. Although IMWG guidelines indicated that UIFE can be performed on a first-morning or random specimen, sensitivity is reduced. To enhance sensitivity, urine is typically concentrated prior to electrophoretic analysis. Some, however, have shown that unconcentrated urine may be used for UIFE with minimal false negative detection rate [71]. Unique to UIFE is the antisera applied to the gel. Because free light chains are filtered through the renal glomerulus, they can be detected by UIFE if present in sufficient quantity. The goal of UIFE is detection of monoclonal free light chain, i.e., Bence-Jones protein. To maximize sensitivity, various antisera combinations may be used including: antisera to individual heavy chains IgG, IgA and IgM; antisera to combined IgG/A/M; antisera to bound and free kappa or lambda; and antisera to free kappa or lambda only. Common findings in UIFE include detection of a monoclonal free kappa or lambda light chain protein (Fig. 16A) and detection of intact heavy and light chain monoclonal protein (Fig. 16B). The former is consistent with myeloma and may be the only evidence supporting a diagnosis. The latter is also consistent with myeloma and supports the presence of renal damage due to the presence of monoclonal heavy chain in urine. A direct quantitative nephelometric assay for urine free light chains is commercially available. Although comparable results may be obtained using direct and electrophoretic methods [72,73], sensitivity of the former may be higher.

3.5 Serum free light chains Intact immunoglobulin is composed of two heavy chains (IgG, IgA, IgM, IgD or IgE) and two light chains (kappa or lambda) held together by disulfide

Fig. 16 Urine monoclonal protein. UIFE electrophoresis was performed. (A) Positive for free lambda light chains (arrow). (B) Positive for intact monoclonal immunoglobulin. Unable to distinguish heavy chain due to combined G/A/M antisera (lane 2). Light chain is kappa. No free light chain detected.

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bonds (Fig. 1). The synthesis of immunoglobulin protein results from random recombination events leading to functional variability. Although this intrinsic variability is excellent for immune function, this phenomenon results in a very heterogeneous product. Such heterogeneity complicates laboratory analysis, i.e., all immunoglobulin is not equal. Degradation of immunoglobulin to their independent peptides can also occur with kappa light chain cleared at approximately twice that of lambda light chain. Although normal kidneys can process 10–30 g/24 h free light chain [74], accumulation of sFLC is clinically significant as they may bind TammHorsfall protein, i.e., uromodulin, and accumulate in distal tubules. These waxy casts disrupt the basement membrane and interstitium causing renal dysfunction [74]. 3.5.1 Methods of serum free light chain measurement Patients presenting with severe bone pain/fractures having urine proteins that precipitate at 60 °C and dissolve at 75 °C were first described by Dr. Bence Jones in 1847. Unfortunately, it was not until 2001 that an assay was developed to differentiate free from total light chain protein (The Binding Site, Birmingham UK). Using nephelometric or turbidimetric approaches, this assay employed polyclonal antibodies to kappa and lambda light chains specific for epitopes only exposed when free from heavy chains. Because of the variable nature of polyclonal antibodies, this manufacturer limited their reagents to an in-house designed turbidimetric instrument [75]. To overcome heterogeneity, alternative methods have been commercially developed. These include a nephelometric method using monoclonal antibodies (Siemens, Berlin, Germany) [76] and a polyclonal ELISA-based method (Sebia, Norcross, GA, USA) [77]. 3.5.2 Interpretation of serum free light chains As might be expected, sFLC assay variation contributes to differences in results making inter-laboratory comparison technically and clinically difficult. These differences in absolute measure further contribute to differences in kappa to lambda ratio especially for polyclonal increases, i.e., inflammation. Although both kappa and lambda will be increased under these circumstances, their relative levels will not change. A monoclonal increase will, however, result in a change in the relative level of light chain protein. Although oligoclonal patterns may occur in infection or post autologous transplant, changes to the ratio will likely be transient.

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Given its historical use, the original assay forms the basis for current diagnostic and therapeutic guidelines. These include reference intervals for kappa FLC (3.3–19.4 mg/L), lambda FLC (5.7–26.3 mg/L) and the kappa/lambda ratio (0.26–1.65). The latter represents 100% of the healthy population, allowing assay specificity to reach 100%, while reducing sensitivity to 97% [35]. Decreased renal function will increase kappa FLC, thus increasing the ratio (0.37–3.1) [74]. Interestingly, the nephelometric method cited above measures a higher level of lambda FLC in normal specimens thus impacting the ratio in patients with renal decline [76].

3.6 Additional laboratory testing in multiple myeloma patients Although it is outside of the scope of this chapter to describe all methods in detail, the most relevant analytes will be briefly discussed. For a more thorough review of analytes, the reader is directed to traditional references, i.e., Tietz and Kaplan. 3.6.1 Immunoglobulins Different methods are required for each immunoglobulin class due to their range of concentration, i.e., a million-fold difference for IgG vs IgE [78]. IgD or IgE plasma cell dyscrasias are rarely seen and represent 10% and 1% of all neoplasms, respectively [79]. Because each method measures all immunoglobulins within a class, tumor burden would be overestimated in a polyclonal increase. Conversely, quantitation mirrors tumor burden when the immunoglobulin production is suppressed. As such, quantitation methods alone are of insufficient sensitivity to diagnose or monitor myeloma [11]. Antigen-antibody binding, i.e., immunometric methods, has been widely used to measure numerous physiologically relevant analytes including immunoglobulin. These include turbidimetric, nephelometric as well as enzymatic methods. Nephelometric methods are, however, limited by the amount of incident light and background scatter from dust, lipids and other proteins in the specimen. This issue may be overcome by using kinetic-based measurements [80]. More recently, turbidimetry has shown enhanced sensitivity due to the incorporation of latex particles to form larger immunocomplexes. Nephelometric and turbidimetric methods are frequently used to quantitate IgG, IgA and IgM. Although highly reliable, these approaches are susceptible to prozone or hook effect in the presence of high immunoglobulin concentration as in myeloma. Under these circumstances, specimen dilution may be necessary to obtain an accurate result [81]. Despite their rarity, IgD

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and IgE may be measured by nephelometry. Due to its very low concentration, the latter may be detected via a sandwich biotin-streptavidin immunoassay. 3.6.2 Total protein In myeloma, total protein is performed for two reasons: (1) immunoglobulins are included in measurement of total protein; (2) total protein determination is essential for densitometric quantitation of SPEP fractions. Proteins are a diverse group of compounds made up of carbon, hydrogen, oxygen and an average of 16% by weight nitrogen. This is held together in a variety of configurations by peptide bonds and resulting in complex diversity, i.e., enzymes, immunoglobulins, transport proteins, based on their amino acid composition. Nearly all UV absorption in serum is attributable to protein. Absorption in the lower region (200–225 nm) is due to peptide bonds while upper region (270–290 nm) absorption is due to aromatic amino acids, i.e., tryptophan, tyrosine and phenylalanine. The preferred reference method for determination of total protein is the Biuret method, which relies on the reaction of copper (Cu2+) ions with peptide bonds in an alkaline solution. The formation of the violet-colored product can be read as a kinetic or end-point reaction at 540 nm forming the basis for most clinical laboratory methods [82]. As mentioned above, urine total protein concentration is significantly lower than serum, requiring a more sensitive method of measurement. One approach is to denature and precipitate urine protein with trichloroacetic acid (TCA), benzethonium chloride or ammonium chloride. Protein can be measured as a fine suspension using turbidimetry [83]. 3.6.3 Calcium The IMWG defines hypercalcemia as an increase of 0.25 mmol/L above the upper limit of normal as a defining feature of myeloma [2]. Hypercalcemia is one of the least common features at time of diagnosis (28%), but predominates in late stages [70]. The most common form of calcium measurement uses a dye, i.e., o-cresolphthalein or arsenazo III, to form a colored complex. Reaction of calcium with the former under alkaline conditions (pH 10–12) results in the production of a red colored complex that can be quantitated (570–575 nm) [84]. Dry slide technology favors arsenazo III dye binding under acidic conditions to produce a blue-violet colored complex at 680 nm.

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3.6.4 Creatinine Serum creatinine, a widely used measure of renal function, is typically measured by the chemical method of Jaffe utilizing the reaction kinetics with picric acid, or by an enzymatic method. Urine creatinine is routinely used to assess urine concentration. The IMWG recommendation for renal damage constituting a myeloma defining event is an estimated glomerular filtration rate (eGFR) <40 mL/min, as calculated by MDRD or the CKD-EPI equations [2]. Increased creatinine occurs in 48% of newly diagnosed patients [70]. Methods in use today have been standardized to the isotope dilution mass-spectrometry method to allow universal calculation of the eGFR [85]. The use of a kinetic colorimetric assay may compensate for, but not eliminate the non-specific reaction of picrate with non-creatinine chromogens. Fortunately, many of these non-specific chromogens have different rates of reaction to picrate. If monitored over time, formation of the orange-red Janovski complex is far more specific for creatinine. Due to mass enzyme production, methods utilizing creatinine-degrading enzymes have become a viable alternative. The conversion of creatinine to creatine is catalyzed by creatininase (EC 3.5.2.10; creatinine amidohydrolase). Consumption of reactant or production of end-product is typically measured by absorbance. These can vary based on the approach used. For example, creatininase and creatinase method (EC 3.5.3.3; creatine amidinohydrolase) yields sarcosine and urea as products. Sarcosine is oxidized to form glycine, formaldehyde and hydrogen peroxide via sarcosine oxidase (EC 1.5.3.1). Hydrogen peroxide formation can be measured in a variety of ways. Formaldehyde can be monitored by reduction of NAD by formaldehyde dehydrogenase (EC 1.2.1.46) at 340 nm [85]. Endogenous interferences can be removed by a pre-incubation step. 3.6.5 Albumin/creatinine ratio The urine albumin/creatinine ratio provides a relative measure of protein loss to assess renal function. Increased urine albumin occurs due to increased glomerular leak or decreased tubular uptake. In contrast to serum, the concentration of albumin in urine is significantly lower (<30 mg/L) requiring a more robust approach including immunometric analysis with anti-albumin antibodies, i.e., microalbumin. Accurate measurement is complicated by heterogeneity of urine albumin due to its susceptibility to undergo fragmentation and other chemical modifications. This variability likely contributes to inter-assay differences.

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It should be noted that these fragmentation products can undergo further breakdown during storage. The albumin/creatinine ratio minimizes the effect of hydration on albumin concentration. The first morning void remains the preferred specimen. Increased muscle mass creatinine may also contribute to inaccuracy by underestimating excretion. Fortunately, urine creatinine assays are less impacted by non-creatinine chromogen interference and can, thus, be measured by most methods. 3.6.6 Hematology Anemia occurs in myeloma. Common causes include anemia of chronic disease, relative erythropoietin deficiency from renal impairment and myelosuppressive effects of chemotherapy. Anemia is typically normochromic and normocytic due to decreased red blood cell production. The IMWG myeloma defining level of hemoglobin, reduction of >20 g/L below the lower limit of normal or <100 g/L, occurs in most patients (73%) at diagnosis [2,70]. Anemia often resolves with complete remission [86]. 3.6.7 Bone pain/fractures Clonal plasma cell disorders frequently result in bone density change resulting in bone pain in many (58%) newly diagnosed patients [70]. IMWG lists osteoporosis with compression fractures as a myeloma defining event. This is, however, not an uncommon finding in the aged making assignment more complicated. Evidence of one or more osteolytic bone lesions >5 mm in size (PET-CT, low-dose whole body CT, 18F-fuorodeoxyglucose-PT or MRI) with more than one skeletal focal lesion defines the presence of multiple myeloma.

3.7 General interferences in laboratory testing in multiple myeloma Potential analytical interferences can occur in the presence of increased monoclonal protein, even those that are small and clinically irrelevant. Interferences include those that affect the assay by precipitation, increased viscosity or chemical interference, and those that impact the analyte itself. 3.7.1 Turbidity Because of the wide use of nephelometric and turbidimetric assays, interference via monoclonal immunoglobulin turbidity and/or precipitation is not unexpected. Interference may be either positive or negative depending on

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assay kinetics and turbidity. Heterogeneity of clonal protein makes it impossible to predict inter-individual variability. IgM clones are, however, the most often cause of turbidity due to their pentameric structure [87]. One of the most well-known interferences from turbidity is falsely increased phosphorus due to monoclonal protein precipitation during the late phase of the molybdenum blue reaction [88]. Precipitation of IgG monoclonal protein has been reported to falsely increase creatinine when enzymatic methods are used [89]. Interestingly, this phenomenon may occur at monoclonal protein <5 g/L. Jaffe-based methods may also be susceptible due to clonal protein interference with picric acid [87]. However, it would be exceedingly rare to find monoclonal protein showing interference in both methods of analysis. A number of strategies have been commercially employed to mitigate creatinine interference including use of buffer blanks, bichromatic analysis, kinetic measurements and incorporation of detergents. Despite these efforts, heterogeneity of clonal proteins makes it virtually impossible to prevent interference in all cases. 3.7.2 Pseudohyponatremia Pseudohyponatremia may be triggered by increased serum protein, i.e., monoclonal components, when sodium is measured by indirect ionselective electrodes. When non-aqueous components are >7%, the calculation used to determine total plasma volume results in an underestimation of sodium concentration. 3.7.3 Hyperviscosity Increased specimen viscosity, due to the presence of cryoglobulin and in Waldenstr€ om macroglobulinemia, will physically interfere with analysis. Some, but not all, will benefit from pre-dilution or warming prior to analysis. In other cases, plasma cell dyscrasia may result in differential serum separation in gel separator tubes (unpublished findings). The use of non-gel tubes and rapid separation of serum may overcome this limitation. 3.7.4 Heterophilic antibodies and hook effect Two well-known interferences in immunoassay methods are heterophile antibodies and hook effect. Heterophile antibodies present in the specimen bind to antibodies in the assay creating pseudo-complex formation in the absence of analyte, i.e., false positivity. Alternatively, heterophile antibodies

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may block interaction causing falsely negative results. These are nearly impossible to detect, but are assay specific [87]. In methods that directly measure monoclonal protein, i.e., sFLC, the concentration of the monoclonal component may exceed assay capacity. In non-competitive assays, the concentration of the capture and detection antibodies must exceed that of the analyte. The absence of adequate reactants leads to a falsely decreased results. Commercial suppliers have recognized this limitation and have developed assays to mitigate or at least alert the user to the potential prozone effect. In the past, the sFLC assay suffered from this problem across manufacturers. 3.7.5 Other Pseudohyperphosphatemia can also result from the ability of monoclonal protein to bind substrate including ions such as phosphate. Although a similar effect has been noted in pseudohypercalcemia, this phenomenon can be overcome by measurement of ionized calcium [87]. Macroenzymes are another well-known phenomenon mediated by monoclonal protein, particularly IgG. Although polymerization reduces renal clearance, it may not interfere with enzyme activity. The clinical significance of these complexes is, however, unclear. A similar phenomenon may occur with hormones as well as lipids. The former may result in significant clinical consequences including hypoglycemia due to insulin-antibody complex formation [87]. Interpretation of anion-gap can become confusing with increased clonal protein. At physiologic pH, monoclonal IgG behaves as cations causing compensatory retention of anions, chloride and bicarbonate. In contrast, IgA clonal protein behaves as anions [87].

4. Laboratory reporting Clinical practice guidelines have been developed by and for clinical users of PEP, the most widely being that from the IMWG [2]. It should be noted that these guidelines were primarily developed for clinicians and not for laboratorians. In 1999, Keren et al. [90] generated a set of nine guidelines for laboratory evaluation of monoclonal gammopathy. The Australia and New Zealand Working Party on Standardized Reporting of Protein Electrophoresis [91] provided some guidance on reporting and more recently the Canadian Society of Clinical Chemists Monoclonal

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Gammopathy Working Group [92] generated a comprehensive set of candidate recommendations that require formal ratification. This section will discuss the various elements of the PEP report including suggestions on how to structure the report and what elements are necessary and useful clinically.

4.1 Nomenclature Medical communication requires clear, accurate, concise and precise language. To facilitate transfer of information, laboratorians should use standardized nomenclature. For example, Logical Observation Identifiers Names and Codes (LOINC) is one nomenclature standard adopted by organizations worldwide for quantitative and qualitative test results. Unfortunately, this system does not provide guidance on interpretive comments which is of consequence for PEP. The language used in PEP interpretations is a mixture of accurate formal scientific nomenclature and informal colloquial terms, which could be regional, national or international. For example, an abnormal PEP finding may be referred to as a monoclonal band and/or component, an abnormal band, an M-spike, a monoclonal spike, a paraprotein, etc. Various groups have recommended use of common terminology including the Australia and New Zealand Working Party [91] and the Canadian Society of Clinical Chemists [92]. Ideally, laboratorians should strive to use formal harmonized nomenclature when reporting interpretations. Nomenclature recommended by these groups includes: • Monoclonal protein or paraprotein—to describe an unknown monoclonal immunoglobulin prior to immunotyping or in non-specific reference to a monoclonal immunoglobulin. The term encompasses monoclonal immunoglobulins, monoclonal free light chains and monoclonal free heavy chains when present in either serum or urine. A commonly used abbreviation is M-protein. • Monoclonal immunoglobulin—for whole molecule monoclonal immunoglobulin, i.e., monoclonal IgG kappa, monoclonal IgG lambda, monoclonal IgA kappa, etc. • Monoclonal free light chain—when referring to monoclonal immunoglobulin light chain not associated with an immunoglobulin heavy chain, i.e., monoclonal free kappa or free lambda protein. This can be used for serum or urine monoclonal protein. Although Bence-Jones protein is commonly used to describe monoclonal free light chain in urine, it is

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not the preferred term because it refers to an antiquated technique used to identify the presence of a monoclonal free light chain rather than the protein itself. • Monoclonal free heavy chain—when referring to monoclonal immunoglobulin heavy chain not associated with an immunoglobulin light chain, i.e., monoclonal IgG heavy chain, IgA heavy chain, and IgM heavy chain. At a minimum, nomenclature should be harmonized within an institution and across both physical (electronic medical record, printed reports) and verbal communication mechanisms. Standardization allows the clinician to correctly interpret the information provided by the laboratory. Harmonized reporting would be practiced within the health system.

4.2 Information to be included in a protein electrophoresis report Although a prime clinical utility of PEP is workup and monitoring of monoclonal gammopathy, it is often used as a screening tool in patients with anemia, back pain, hypercalcemia, rouleaux cell formation, renal insufficiency, unexplained pathologic fractures and peripheral neuropathy [93]. Due to its broad utility and coupled with the often unknown underlying pathology, the PEP report must be interpretable by specialists, i.e., oncologists, as well as general practitioners. Interpretation of electrophoretic patterns and clinical reporting thereof are inherently tied together. Intrinsic to this endeavor is having appropriate knowledge, technical understanding and clinical skill. In this section we will cover reporting of results, strategies for reporting monoclonal protein as well as suggested report content and format. 4.2.1 Electrophoresis report The PEP report should contain all relevant information needed to guide clinical decision making with respect to diagnosis and/or treatment. The report should be easily interpretable and include an appropriate comment on the electrophoretic pattern including quantitation of monoclonal protein, if present. The report should include quantitative values for normal protein fractions. The Canadian Society of Clinical Chemists Monoclonal Gammopathy Working Group recommendations for PEP reporting [92] include the following elements: • Information reported in a consistent format and location on the report. • The same quantity of information in the same format. • Reports should be consistent between interpreters.

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Should include the presence of a monoclonal immunoglobulin, isotype (if previously known) and concentration. PEP and results from isotyping (when required) should be reported concurrently. It should be stated that an abnormality is present and that additional testing is recommended when confirmation testing is not immediately available.

4.2.2 Protein fraction reporting Inclusion of quantitative values for normal serum protein fractions in the electrophoresis report provides the clinician with additional information not provided in the interpretive comment. Inclusion of these values has been helpful in the assessment of hepatic dysfunction, inflammation, renal disease, α1-antitryrpsin deficiency, etc. The availability of more highly specific diagnostic tests does, however, question the need to provide these values going forward. For laboratorians, comparison to normal values is extremely important for accurate SPEP interpretation. For example, identification of increased α- or β-fraction can indicate the presence of an underlying monoclonal protein or cryoglobulin. At a minimum, protein fractions should be compared to validated reference intervals during interpretation. When SPEP or UPEP is performed to monitor disease, the monoclonal protein must be quantitated and consistently reported to minimize variability. Individual quantitative values should be reported when more than one monoclonal protein is present. The monoclonal fraction should be labeled so the clinician can unambiguously identify the monoclonal protein(s) (Fig. 17). 4.2.3 Non-monoclonal patterns Although the electrophoretic profile can reveal other conditions, the sensitivity and specificity of this approach is far lower than targeted diagnostic testing. For example, Chan et al. [94] made practice recommendations for reporting of non-monoclonal SPEP patterns (Table 4). Here we discuss those patterns that may be clinically significant for reporting. α1-Antitrypsin deficiency is an autosomal co-dominant genetic disorder with a prevalence of 1:2000 to 1:5000 depending on ethnicity [51]. Deficiency is associated with a broad array of clinical conditions including emphysema and bronchiectasis, chronic hepatitis, cirrhosis and hepatoma, skin disease and ANCA-associated vasculitis. Due to its heterogeneous presentation, diagnosis can be significantly delayed. Therefore, early identification is of critical importance. Electrophoretic patterns containing a

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Fig. 17 Strategies for reporting SPEP. (A) Possible reporting nomenclature options. (B) Monoclonal protein report using generic nomenclature. (C) Report using isotope specific nomenclature for identification of monoclonal proteins. (D) Report using isotope specific nomenclature for beta region co-migrating monoclonal IgA lambda.

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Table 4 Interpretive commenting for SPE findings without an identified monoclonal protein. SPE pattern Pattern description Practice suggestion

No unusual findings

Absence of “areas of Avoid using the word restricted mobility,” all “normal.” No special fraction values lie within comment recommendeda reference intervals, and no unusual findings

Hypoalbuminemia

Decreased Alb fraction

No special comment recommendeda

Bisalbuminemia

Split Alb band

No special comment recommendeda

Haptoglobin variant

Split A2G fraction

No special comment recommendeda

Nephrotic pattern

Decreased Alb fraction with increased A2G fraction with/without increased BG fraction

No special comment recommendeda

Alpha1 antitrypsin (A1AT) deficiency

Decreased A1G fraction

Reflexive testing and/or special comment recommending A1AT measurement to rule out congenital deficiency

Increased beta

Increased total BG fraction Reflexive testing and/or or individual B1G and/or special comment B2G fractions recommending specified follow-up testing (e.g., IFE) because of possible co-migrating M-protein and BG

Hypogammaglobulinemia Decreased GG fraction

Reflexive testing and/or special comment recommending specified follow-up testing based on local protocols

Acute inflammatory pattern

No special comment recommended

Increased A1G and/or A2G fractions with/ without decreased albumin

Continued

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Table 4 Interpretive commenting for SPE findings without an identified monoclonal protein.—cont’d SPE pattern Pattern description Practice suggestion

Beta-gamma bridge

Increased protein in the No special comment beta to gamma recommended transitioning zone resulting in a merged appearance of BG and GG fractions

Polyclonal gammopathy

Increased broad diffuse protein pattern in GG fraction

Reflexive testing and/or special comment indicating absence of any obvious M-protein detected. Diffuse increases in gamma fraction consistent with polyclonal immunoglobulin increases. Further investigative workup may be indicated if clinical suspicion for MG is high

a A comment is not essential but if given may indicate the absence of any obvious M-protein and direct to further serum and/or urine testing if clinical suspicion for monoclonal gammopathy is high. Alb, Albumin; A1G, Alpha1 globulin; A2G, Alpha2 globulin; BG, Beta globulin; B1G, Beta1 globulin; B2G, Beta2 globulin; GG, Gamma globulin. Reproduced from P.-C. Chan, Y. Chen, E.W. Randell, Monoclonal Gammopathy Interest Group (MGIG) Canadian Society of Clinical Chemists, On the path to evidence-based reporting of serum protein electrophoresis patterns in the absence of a discernible monoclonal protein—a critical review of literature and practice suggestions, Clin. Biochem. 51 (2018) 29–37.

significantly reduced or absent α1 fraction should be immediately reported. Under these circumstances, the interpretative comment should read “Reduced α 1 globulin level. Suggest α1-antitrypsin measurement to exclude congenital deficiency” [94]. Other electrophoretic patterns of questionable clinical utility include acutephase, nephrotic, beta-gamma bridging, polyclonal hypergammaglobulinemia and hypogammaglobulinemia. 4.2.4 Pattern interpretation and reporting McCudden et al. [95] have proposed synoptic style reporting to improve standardization of PEP reporting. A synoptic report is defined as a clinical documentation method that uses a structured format to produce complete,

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consistent medical reports. A synoptic-style report provides the clinician with a structured report that includes only the clinically relevant pieces of information. It also allows the user to quickly review and understand the interpretation without having to search for relevant information buried in a paragraph. When adopted by all laboratories within a health region it enables harmonization of resulting that ultimately can enable clinicians to make better and more consistent clinical decisions regarding diagnosis and treatment. Although synoptic reporting has been adopted by Anatomic and Heme Pathology [96–98], it has not been routinely used in Clinical Pathology. The elements suggested by McCudden et al. [95] include identification of an abnormal band or monoclonal protein, a description of the band, inclusion of previous history if available, an interpretation and recommendations as well as identification of the interpreter. Shown are a brief description and templates for SPEP/UPEP (Table 5) and immunofixation/immunotyping (Table 6) as well as examples of each (Fig. 18). For an in-depth discussion of PEP synoptic reporting see McCudden et al. [95]. In lieu of synoptic reporting, it is beneficial to harmonize interpretation and reporting. Commonly encountered pattern interpretations are shown (Table 7). Table 5 Synoptic reporting template for SPEP and UPEP. Field Contents

Abnormal bad

Present/absent/equivocal

Band description

Number and position (if relevant) of abnormal band(s). Limitation of band quantitation as relevant to interpretation, e.g., co-migrating bands

Previous history

If available, history of previous analyses (SPE and IFE). Source of orders from other hospitals would be provided where relevant

Interpretation

Concise summary of collective pattern and if changes are noted as relevant

Recommendations Description of whether repeat testing or alternative testing is recommended (e.g., UPE, sFLC); frequency of repeat testing. Use available literature and guidelines where applicable Interpreter

Who interpreted the results, contact info

Modified from C.R. McCudden, et al., Synoptic reporting for protein electrophoresis and immunofixation. Clin. Biochem. 51 (2018) 21–28.

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Table 6 Synoptic reporting template for immunofixation/immunotyping. Field Contents

Monoclonal protein Present/absent/equivocal Isotype

Isotype of intact immunoglobulin, free light chain, or heavy chain

Abnormal band description

Number and position (if relevant) of abnormal band(s). Align description with quantitation as relevant

Previous history

History of previous analyses (IFE). Source of orders from other hospitals would be provided where relevant

Immunosuppression Present/absent Interpretation

Concise summary of collective pattern and if changes are noted as relevant

Recommendations

Description of whether follow up and/or repeat testing; frequency of repeat testing. Use available literature and guidelines where applicable

Interpreter

Who interpreted the results, contact info

Modified from C.R. McCudden, et al., Synoptic reporting for protein electrophoresis and immunofixation. Clin. Biochem. 51 (2018) 21–28.

5. Conclusion and future directions Current laboratory work-up in the diagnosis and management of multiple myeloma includes a variety of biochemical testing, electrophoretic analysis and monitoring of end-organ damage parameters, i.e., creatinine and eGFR. As evident in most recent IMWG guidelines [2], selection of tests for disease diagnosis continues to expand and supports the use of sFLC in active disease. In the future, test options beyond the traditional will likely broaden to include flow cytometry and mass spectrometry. As described above, both flow cytometry and molecular diagnostic are clearly relevant tools in disease remission and relapse. Furthermore, studies are demonstrating the ability of mass spectrometry to supplant traditional approaches such as PEP and IFE [99–101]. The introduction of novel assays such as Hevylite to specifically quantitate monoclonal heavy chain has shown potential in risk stratifying MGUS and smoldering myeloma [102] as well as predicting relapse [22]. Despite these advancements, it is likely that traditional PEP techniques will continue to play an important role in the diagnosis and management of

A

B

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Fig. 18 Synoptic reporting. (A) Serum with a single unidentified abnormal band. (B) Serum with a single previously identified monoclonal IgM κ. (C) Urine with a glomerular proteinuria pattern. (D) Urine with a single previously identified monoclonal free kappa light chain. (E) SIFE of a monoclonal IgG kappa and monoclonal free kappa light chain. Modified from C.R. McCudden, et al., Synoptic reporting for protein electrophoresis and immunofixation. Clin. Biochem. 51 (2018) 21–28.

C

Fig. 18—Cont’d

E

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Table 7 Example serum and urine electrophoresis pattern interpretations. Pattern Interpretation

Serum Normal

No abnormal bands seen

Repeat specimen

Pattern unchanged. Monoclonal (insert isotype; e.g., IgG kappa) in the gamma region

Hypergammaglobulinemia

Polyclonal hypergammaglobulinemia; no abnormal bands seen

Hypogammaglobulinemia

Hypogammaglobulinemia; no abnormal bands seen. Suggest urine for protein electrophoresis if clinically indicated

New possible monoclonal protein

Abnormal protein in gamma region. Immunofixation follows

Beta-region co-migrating monoclonal protein

Pattern unchanged. Monoclonal (insert isotype; e.g., IgA kappa) that overlaps with beta globulins and cannot be quantitated separately. Abnormal band includes normal beta globulins

Inflammation

Pattern suggestive of inflammatory/reactive changes. No abnormal bands seen

Beta-gamma bridging

Diffuse increase in gamma globulins in the betagamma region. Pattern suggestive of intestinal/ respiratory tract, skin diseases or liver disease. No abnormal bands seen

Nephrotic pattern

Pattern suggestive of protein loss (nephritic syndrome/gastroenteropathy). No abnormal bands seen

Urine Albumin present. No bands suggestive of a monoclonal protein seen Albumin and globulins present. No bands suggestive of a monoclonal protein seen Glomerular proteinuria pattern. No bands suggestive of a monoclonal protein seen Tubular proteinuria pattern. No bands suggestive of a monoclonal protein seen Non-selective proteinuria; pattern similar to serum. No bands suggestive of a monoclonal immunoglobulin seen Pattern unchanged. Albumin and globulins with monoclonal (insert isotype; e.g., free kappa light chains) in the gamma region

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myeloma. Furthermore, it is imperative that standardization of test methods and harmonization of reporting format be adopted in order to provide the most accurate and consistent test results.

References [1] C. Fitzmaurice, et al., Global, regional, and national cancer incidence, mortality, years of life lost, years lived with disability, and disability-adjusted life-years for 32 cancer groups, 1990 to 2015: a systematic analysis for the global burden of disease study, JAMA Oncol. 3 (4) (2017) 524–548. [2] S.V. Rajkumar, et al., International Myeloma Working Group updated criteria for the diagnosis of multiple myeloma, Lancet Oncol. 15 (12) (2014) e538–e548. [3] A. Palumbo, K. Anderson, Multiple myeloma, N. Engl. J. Med. 364 (11) (2011) 1046–1060. [4] S.V. Rajkumar, Multiple myeloma: 2016 update on diagnosis, risk-stratification, and management, Am. J. Hematol. 91 (7) (2016) 719–734. [5] O. Landgren, et al., Racial disparities in the prevalence of monoclonal gammopathies: a population-based study of 12,482 persons from the National Health and Nutritional Examination Survey, Leukemia 28 (7) (2014) 1537–1542. [6] S.V. Rajkumar, et al., Serum free light chain ratio is an independent risk factor for progression in monoclonal gammopathy of undetermined significance, Blood 106 (3) (2005) 812–817. [7] C. Cesana, et al., Prognostic factors for malignant transformation in monoclonal gammopathy of undetermined significance and smoldering multiple myeloma, J. Clin. Oncol. 20 (6) (2002) 1625–1634. [8] R.A. Kyle, S.V. Rajkumar, Criteria for diagnosis, staging, risk stratification and response assessment of multiple myeloma, Leukemia 23 (1) (2009) 3–9. [9] R.A. Kyle, et al., Clinical course of light-chain smouldering multiple myeloma (idiopathic Bence Jones proteinuria): a retrospective cohort study, Lancet Haematol 1 (1) (2014) e28–e36. [10] R.A. Kyle, et al., Clinical course and prognosis of smoldering (asymptomatic) multiple myeloma, N. Engl. J. Med. 356 (25) (2007) 2582–2590. [11] The International Myeloma Working Group, Criteria for the classification of monoclonal gammopathies, multiple myeloma and related disorders: a report of the International Myeloma Working Group, Br. J. Haematol. 121 (5) (2003) 749–757. [12] E. Kastritis, et al., Extensive bone marrow infiltration and abnormal free light chain ratio identifies patients with asymptomatic myeloma at high risk for progression to symptomatic disease, Leukemia 27 (4) (2013) 947–953. [13] J.T. Larsen, et al., Serum free light chain ratio as a biomarker for high-risk smoldering multiple myeloma, Leukemia 27 (4) (2013) 941–946. [14] M.V. Mateos, et al., Lenalidomide plus dexamethasone for high-risk smoldering multiple myeloma, N. Engl. J. Med. 369 (5) (2013) 438–447. [15] R.A. Kyle, et al., Incidence of multiple myeloma in Olmsted County, Minnesota: trend over 6 decades, Cancer 101 (11) (2004) 2667–2674. [16] K.J. Phekoo, et al., A population study to define the incidence and survival of multiple myeloma in a National Health Service Region in UK, Br. J. Haematol. 127 (3) (2004) 299–304. [17] R.A. Kyle, et al., Review of 1027 patients with newly diagnosed multiple myeloma, Mayo Clin. Proc. 78 (1) (2003) 21–33. [18] T. Dejoie, et al., Comparison of serum free light chain and urine electrophoresis for the detection of the light chain component of monoclonal immunoglobulins in light chain

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