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Cancer (Multiple Myeloma and Related Disorders) William E. Katzin Department of Laboratory Medicine, The Mt. Sinai Medical Center, Cleueland, Ohio 44106-4195 INTRODUCTION Monoclonal immunoglobulinsmay be regarded as the most important "tumormarkers" analyzedin the clinical chemistry laboratory. Also known as paraproteins or M-proteins, monoclonal immunoglobulins are the secretory products of expanded clones of B-lymphocytes. In clinical practice, the primary importanceof paraprotein detection and quantitation centers on the diagnosis and monitoring of various neoplastic disorders. From a biological perspective, the occurrence of paraproteins has provided insight into the clonal nature of neoplasia itself. The clinical significance of paraproteins is complicated by the fact that they are associated with a wide spectrum of diseases, not all of which are malignant. Even when one considers multiple myeloma alone, there are subtleties in diagnosis and management that require a comprehensiveunderstanding ofclinid aswellashiochemical parameters. The subject of paraproteins has not appeared recently in the Application Reviews of Analytical Chemistry. The following discussion therefore focuses primarily on a review of the literature published over the past five years (up to October 1992). This review includes considerations of the epidemiology and classification of plasma cell dyscrasias, the laboratory evaluation of both serum and urine paraproteins, and a general review of prognostic factors in multiple myeloma. CLINICAL SPECTRUM OF PARAPROTEINS Epidemiology. The frequency of finding paraproteins in the serum of hospitalized patients is highly dependent upon age and the sensitivity of the method used (01-03). In a review of 73 630 patients in whom serum protein electrophoresis (SPE) was performed as part of a routine screening of hospitalized patients, Vladutin (DI) reported a prevalence of 1.1% of patients with an M-protein. In that study M-proteins were defined as tall narrowspikesvisualized after Amido Black staining of agarose gel electrophoreses, apparentlyusingthecriterionofKyle(D4) who,usingdensitometry, defined a paraprotein spike as having a height a t least 4 times higher than its width at the midpoint between the base and the to . Using cellulose acetate electrophoresis, Aguzzi et al. (02) rfetected serum paraproteins in 2.9% of 35 005 hospitalized patients. In that study, the percentage of patients with paraproteins increased steeply with age and reached a plateau of around 7 % in individualsover 55 years old. Almost 80% of the monoclonal proteins were of low concentration (less than 5 /L) In a similar study, Malacrida et al. (D3) reported fining aparaprotein band by cellulose acetate SPE in 0.7% of 102 000 patients. Paraproteins of Uncertain Significance. The diagnostic approach to patients with paraproteins bas recently been reviewed by Kyle and Greipp (05, D6). On the basis of frequency of detection of paraproteins during routine screening, it is certainly apparent that monoclonal immunoglobulins are not always associated with multiple myeloma. In fact, most patients with a detectable paraprotein are best classified as having a monoclonal gammopathy of uncertain significance (MGUS). Of 856 patients with a monoclonal gammopathy seen a t the Mayo Clinic in 1990, 541 or 63% hadMGUS (D7). Thediagnosisof MGUSis basedonavariety of biochemical and clinical parameters (05,0 8 ) . The serum monoclonal protein is usually less than 30 /L anemia, renal failure, and hypercalcemia are absent; one surveys are negative; the bone marrow aspirate contains less than 10% plasma cells, without aggregates in the core biopsy; the bone marrow plasma cell labeling index is less than 1%;plasmablasts are absent; and a variety of other ancillary tests, including the &-microglobulin level are normal. Kyle and his colleagues have emphasized their belief that the term "benign monoclonal gammopathy" is generally misleading because it implies that the paraprotein will remain stable and will be associated with a benign clinical course. Kyle and Lust (D9) have reported a long-term follow-up study of 241 patients with monoclonal gammopatby who a t presen-
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Wllllam E. Kalzln earned a B.A. degree in chemistry from Oberlin College. Oberlln. OH. His graduate education included a
Ph.D. in biochemistry and an M.D.. boih from Case Western Reserve University, Cleveland. OH. After a year on the staff of The Cleveland Clinic Foundation in the department of Pathology, Dr. Katzin joined IhestaffofTheMountSinai Medicalcenter of Cleveland. OH, where he is currently a member of the Department of Laboratory Medicine and Director of the Diagnostic ImmunupaIhologyand Molecular PaIholcgy Laboratory. Or. Katzin isa member of the clinical faculty of Case Western Reserve University in the Department of Pathology.
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tation had no evidence of multiple myeloma, Waldenstrom's macroglobulinemia, amyloidosis, lymphoma, or a related disease. Twenty-four percent of those patients remained stable during a mean clinical follow-up of 19 years. In 3% of the patients the paraprotein increased to greater than 30 g/L, hut these patients did not develop evidence of a "malignant" plasma cell dyscrasia. Fifty-one percent of the patients died of causes other than a "malignant" plasma cell dyscrasia after a mean of 7.9 years. Finally, 22% of the patients developed multiple myeloma, macroglobulinemia, amyloidosis, or a related disease after a mean follow-up of 9.6 years (68% of this subset of patients developed multiple mveloma). ,~ ~. Theorcurrenreofa RenceJonesprotein (RJPJcounterpart to the typical serum MGUS is distinctly unusual. Kyle and Griepp rD10) identified seven patients with ^idiopathic" Bence Jones proteinuria, with excretion of more than 1 g of light-chain protein per 24 h, who had no evidence of overt multiple myeloma, systemic amyloidosis, or other malignant lymphoproliferative disorder. Long-term follow-up revealed progressionto some form of multiple myeloma in five patients and persistence of apparently 'benign" BJP for 7.7 and 12 years in the other two patients. No more recent or larger follow-up study of patients with idiopathic Bence Jones proteinuria was found in reviewing the literature. Using highly concentrated urine specimens analyzed by agarose gel electrophoresiaand immunofixation, Pascaliand Pezwli (Dl I , D12) were able to identify a few patients with either pure Bence Jones proteinuria of undetermined significance or transient pure Bence Jones proteinuria. In none of those patients did the excretion of BJP exceed 0.2 g/24 h. Most of the patients they identified as having pure BJP of low concentration had some type of lymphoproliferative disorder (see below). Specific Neoplastic Disorders Associated with Paraproteins. Serum monoclonal gammopathies are associated with a variety of lymphoproliferative disorders. In Kyle's of 856 patients with monoclonal ammopathies, review (07) 12% bad multiple myeloma, 9% had amyloifosis (AL type), 5% had non-Hodgkin's lymphoma, 4% had solitary plasmacytomas of bone or extramedullary plasmacytomas, 3% had chronic lymphocytic leukemia (CLL), and 2% had Waldenstrom's macroglobulinemia. In patients with paraproteins, the diagnosis of multiple myelomas is established when there are 10% or more marrow plasma cells (or aggre ate8 of plasma cells in the core biopsy) or when there are afditional otherwise unexplained clinical abnormalities such as anemia, lytic bone lesions, a hone marrow labeling index of greater than 1 %,renalinsufficiency, hypercalcemia, increased serum p2-microglobulinlevel, or light-chain isotype suppression (05). Despite these relatively specific diagnostic criteria, there are a small number of cases (perhaps 2 % of patients with monoclonal gammopathies; see ref D7) who have a plasma cell dyscrasia that is clinically intermediate between MGUS and multiple myeloma. The designation "smoldering multiple myeloma" has been proposed (D5, D13) to include patients with paraproteins greater than 30 g/L and 10% or more plasma cells in the bone marrow but with no other clinical abnormalities. The importance of this inter~
CLINICAL CHEMISTRY
mediate diagnostic category is based on the risks associated with the treatment of overt multiple myeloma. In addition to the short-term morbidity, there is a significant risk of developing acute leukemia in the longer term. Based on today’s standards of treatment, these patients should have close monitoring of serum and/or urine paraproteins. In contrast to the patients with serum paraproteins, among those patients with ure Bence Jones proteinuria there is a relatively high inciience of lymphoproliferative disorders other than multiple myeloma. In their study of 66 such patients ( D l l ) , Pascali and Pezzoli found that 32% of the atients had CLL, 27% had non-Hodgkin’s lymphoma, 35% gad hairy cell leukemia, and 15% had amyloidosis. Only 18% of the patients in that study had multiple myeloma. Nonneoplastic Disorders Associated with Paraproteins. A few specific nonneoplastic disorders may be associated with the finding of serum paraproteins. The frequency of such an association is probably quite low, and the concentration of the paraprotein is typically low. Patients with autoimmune diseases ( 0 3 )as well as both bacterial (03, 0 1 4 )and viral (03,015)infections may have either transient or persistent serum paraproteins. In addition, some patients with a peri herd neuropathy have a “benign”IgM para roteinemia (Bl6). Levinson and Keren (017)have descri ed pairs of electrophoretic restricted bands in “immunologically activated”persons that are associated with circulating immune complexes. Biochemical analysis revealed that these bands actually represent polyclonal IgG. The intensity of such bands, and the corresponding immunoglobulin concentration that they represent, are not apparent from their data.
LABORATORY EVALUATION OF PARAPROTEINS The detection and quantitation of serum or urine paraproteins can be accomplished using several different laboratory methods. Electrophoretic methods including agarose gel and cellulose acetate membrane electrophoresis, immunoelectrophoresis, and immunofixation have been considered the standard methods against which other approaches have been compared. In the interest of cost savings and improved turnaround time automated methods based on turbidimetric or nephelometric measurements have been evaluated by a number of different groups. When differences in the sensitivity and precision of various methods are being considered, it is important to consider the impact of laboratory data on clinical management. In this regard it is of interest to note that, in one hospital, when serum protein electrophoresis was used as a routing screening test, the detection of a paraprotein was probably ignored in 59% of the positive cases (01).The laboratory investigation of paraproteinemia,includin general recommendations,has recent1 been reviewed by Wiicher et al. (018) and by Riches and hobbs (019). Detection of Serum Paraproteins. Serum protein electrophoresis is the standard method used to screen for paraproteins. Plasma is not recommended because fibrinogen forms a band in the fast y region. Hemolysis can also be problematic since the hemoglobin-haptoglobin complex forms a band between the a-2and 0 regions and free hemoglobin migrates as a band in the 0 region (019). Good quality electrophoresis is critical for several reasons. As a screening test, the method must be sensitive. Furthermore, densitometric evaluation of SPE patterns is often used to quantitate paraproteins; this quantitative analysis is an important parameter for guiding patient management. Routine electrophoresis can be performed using either cellulose acetate membranes or agarose el. Both media are relatively inert and have large pore sizes. nder the conditions of SPE, the net charge on the protein is therefore the major property influencing migration. Agarose el electrophoresis ma achieve better resolution, particular y in the 0 region, andlis therefore considered by some as the superior method (020). However, Whicher et al. (018)have suggested a minor modification in cellulose acetate membrane electrophoresis (namely, the inclusion of 0.3-0).5%(w/v) Tween 20 in the buffer used to hydrate the membranes prior to electrophoresis) to achieve high reaolution using that medium. With either method, the sensitivity for detecting paraproteins is below 5 g/L ( 0 2 , 020).
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Failure to detect paraproteins b SPE may arise from several causes (018). First, the ban& of para roteins of low concentration (less than 5 g/L) may be hid en by normal 8-region bands. Paraproteins composed of monoclonal IgD or free heavy chains may undergo degradation in vivo or in vitro to form diffuse bands of low concentration. Some paraproteins may com lex with other proteins, their migration is thus altered, and tteir resence may be masked. Cryoinsoluble paraproteins mayie lost during processing. Finally, some araproteins, particularly those of the IgM class, have a tengncy to form polymers that become insoluble and fail to migrate from the origin of the electrophoretic separation. Dense stainin at the application point should suggest this possibility. T b s problem can often be overcome by sulfydryl reduction (1 mg/mL dithiothreitol) of serum prior to electrophoresis. In an attempt to overcome some of the limitations of SPE as a screening method, several authors have advocated using the quantitative analysis of immunoglobulin light chains as an adjunct to SPE (021-024). In each of these studies rate nephelometry was used to measure the serum concentrations of K- and A-containing immunoglobulins. In the study by Keren et al. (021),336 sera were referred to the laboratory for evaluation of a possible monoclonal gammopathy. Amon those cases, 81 patients were found to have monoclon proteins. In 10 of those patients, the SPE was either normal or had only “minor alterations” but the K/Aratio (KLR) was abnormal and the presence of a paraprotein was confirmed by immunofixation. Among those 10 patients, there were two cases of chronic lymphocytic leukemia and two cases of multiple myeloma. In a similar stud of 4173 patients, Jones et al. (023)detected 34 atients w i d a n abnormal KLR and with a paraprotein conkmed by immunofixation in whom the SPE pattern was normal. By itself the KLR is a relatively insensitive test that also suffers from a significant lack of specificity (021-026). Based on the data from several different laboratories, it is also clear that each center using the KLR as an adjunct to SPE for screening must establish its own normal range. For example, in the multicenter study of Jones et al. (023), the lower limit of the normal range among four laboratories varied from 0.42to 1.06and the upper limit varied from 2.50 to 3.03. Part of the variation among different laboratories may result from differences in the antisera used (027). Although it seems fairly predictable that the simple measurementof serum albuminand total serum protein would be of little use in screening for paraproteins, the “globulin measurement” has in fact been examined as a potential lowcost screening test in which the required data are often already . this study of 539 patients, the finding of available (028)In a high globulin concentration (total serum rotein minus albumin) had a sensitivity of only 30% for Ltecting paraproteins compared to SPE. Furthermore, there were 17false positive results. Identification/Typing of Serum Paraproteins. When a paraprotein is detected by a dependable screenin method, its presence must be confiied and its immunoglob&nheavychain type and light-chain class should be defined. There is still some controversy regarding the best laboratory approach to this problem. In a recent review of multiple myeloma, Kyle (07)recommended using immunoelectrophoresis(IEP) or immunofixation (IF) or both. In two other reviews (018, Dl 9) the authors recommended usin immunofixation. IEP is certainly the time-honored methofof defining monoclonal proteins (D29),and in man laboratories there is wealth of experience in interpreting I&P patterns. Studies comparing these two methods f i s t appeared around 1980 (030-033) and indicated a significant advantage in the sensitivity of IF. Nevertheless, Merlini et al. (033)emphasized that IF is more susceptible to “technical artifacts” and recommended usin IEP for difficult cases. However, there are several technic3 problems with IEP. In an era when short turnaround time is a premium, the long incubation time required for diffusion (16-24h) is a disadvantage. In addition, electrophoretic loss of resolution occurs as a consequence of this long diffusion time. Perha s the most significant drawback of IEP is the so-called umErella effect (034)that occurs when a si nificant amount of polyclonal immunoglobulin (usually 108) masks identification of relatively small monoclonal proteins. As more laboratories switch to IF as the routine method for typing
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monoclonal proteins, it is also likely that more authors will claim difficulty in interpretation of IEP patterns as a significant drawback. Three recent studies (024,025,035) have directly compared IEP and IF. Gerard et al. (035)prospectively studied 32 samples including 28 serum specimens and 4 urine s ecimens in which routine electrophoresis revealed an a normal band. Thirty-one of the 32 specimens were definitively characterized by IF whereas only 18 (60%) were fully characterized by IEP. In one specimen a monoclonal immunoglobulin was not identified b either method. The most common situation that precludd identification of a paraprotein b IEP was a high background of polyclonal immunoglobdns. Another problem with IEP involved identification of IgM monoclonal roteins; two out of three of those paraproteins required reluction with mercaptoethanol prior to identification by IEP. A similar reduction step was not required for successful IF. Finally, these authors found that intersupplier variability of anti-X antibody markedly influenced the interpretability of IEP patterns, but did not have such an influence on IF. The authors did concede that antibody rea ent cost was somewhat greater for IF compared to IEP, {ut stated that this difference was mostly offset by fewer repeat IF testa and could be minimized by reducing the size of the antibody-soaked cellulose acetate strips. Duc et al. (025) evaluated 153serum samplesfrom patients SUB ded of having amonoclonalimmunoglobulin,using both IEpand IF. Both methods were negative in 70 samples, and both were positive in 67 samples. In the remaining samples, only IF revealed a monoclonal protein in 15 cases and only IEP revealed a monoclonal protein in one case. The one case with a positive result only by IEP was an example of an essential mixed (type 11) cryoglobulin, which was evaluated at 37 OC by IEP but at room temperature by IF. In 7 of the 15 cases “missed“by IEP, the concentrationof the paraprotein was less than 10 g/L. In those cases the discrepancy was therefore attributed to the greater sensitivity of IF. In the remaining cases, the discrepancy was attributed to the “umbrella”effect. These authors emphasized the importance of ade uately diluting sera that have high concentrations of monoclonal protein in order to avoid antigen excess. In their method, anti-immunoglobulinantisera were directly applied to the electrophoretic gels as opposed to application through soaked cellulose acetate strips. They stated that the concentration margin of antigen detectable by IF is greater with this modification. They concluded,on the basis of increased sensitivity, simplicity, speed, ease of interpretation, and absence of the “umbrella effect”, that IF is superior to IEP. Guinan et al. (024) compared IEP and IF in several different types of specimens. In a series of 127 consecutive samples referred to their laboratory for routine paraprotein analysis, IF was found to be a more sensitive test, in terms of both the number of samples found to contain paraproteins and the total number of paraprotein bands detected. In a separate group of 132 serum sam les in which difficulty had been experienced in typin using PEP, these authors reported increased success with,!I particularly in cases where the paraprotein was present in low concentration (less than 7 g/L), when the paraprotein was I M type, and when there were multiple paraprotein bands. bespite these advanta es the authors recommended the combination of SPE and fE$ for the routine analysis of paraproteins. They claimed that the greater sensitivity of IF was offset by the “need for continuous o erator involvement” and by ita “technical complexity”. %hey recommended using IF only for difficult cases and for evaluation of patients in whom the concentration of the serum paraproteins could be expected to be low. Just as immunoglobulin heavy- and light-chain measurementa have been proposed as a screening tool for detecting paraproteins, some authors have suggested using these quantitative analyses as a means of t in monoclonal immunoglobulins (036,037). Whicher anycofeagues (036) used either an end-point turbidimetric aasay or rate nephelometry to quantitate serum IgG, IgA, IgM, K , and A. The lightchain measurements in both assaye were calibrated with Kallestad reference material, which yields values for K and X light-chain concentrations that reflect the “immunoglobulin e uivalent molar weight”. Therefore, in the ideal situation wxere only IgG, IgA, and IgM are present and are normally
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distributed, and where no free light chains are resent, the sum of IgG, IgA, and IgM is equal to the sum O ~ and K A. In order to type a para rotein, the KIAratio is first calculated (their normal range Eased on 183 sera that did not contain a paraprotein was 1.29-2.61). When the KLR indicated an excess of K the minimum concentration of the K-containing monoclonal immunoglobulin is represented by [ K ] - ([AI X 2.61). Similarly, when the KLR indicated an excess of A, the minimum concentration of the A-containing monoclonal immuno lobulinis equal to [XI - ([~1/1.29).The heavy-chain class of %e monoclonal immunoglobulin was assigned when a single heavy-chain class of the monoclonal immunoglobulin had a concentration equal to or greater than that of the monoclonal light chain. Usin this ap roach, this “immunochemical evaluation” or IC# methdcorrectly t the paraprotein in 89% of the cases. The failure rate was ‘ghest for paraproteins of low concentration (lessthan 5 g/L), where the success rate dropped to 68%. Using the ICE method no paraprotein was incorrectly typed. Using essentially the same method,Jonesetal. (037)evaluated1lOseraknowntocontain a monoclonal protein. The method correctly classified 67% of the cases and misclassified 8%. Half of the misclassified cases were IgA myelomas. In addition to the KLR, several studies have addressed the utility of the heavy-chain to lightchain ratio in typing paraproteins. The sensitivit and one specificity of this approach are both unsatisfactory. the “Ig excess ratio” had a false negative rate of study (OB), 58% and a false positive rate of 25%. In theory the ratio of heavy chains to light chainsusing quantitative measurementa of I G, IgA, IgM, K , and X could be of help in identifyin cases wit[ free serum light chains and BJP or IgD m efomas. However, the preliminary results of a recent stu y (038) indicated very poor sensitivity and specificity of the ICE approach even for that subset of paraproteinemias. While it is possible that the automated ICE method could replace many of the labor-intensive IF analyses with a consequent reduction in cost, Jones et al. (037) emphasized that these savin s may largely be offset by the need to perform IF on sampfes with no band detected by SPE, but with a KLR outaide the normal range. In summary, on the basis of recent literature, either IF or IEP should be used to confirm the presence of a serum monoclonal paraprotein and to t e the heavy- and lightchain components of the immunogl%din protein. In contrast to immunoelectrophoresis,IF lacks the inherent mechanism whereby diffusion effective1 titrates antigen and antibody. Therefore, properly dilutedr antigen (paraprotein) and attention to the variation of antibody affinity, avidit , and specificity are critical (039). Kahn has recommendedTusin standard dilutions of serum for IF as follows: 1:lO for 1 0 4 1:5 for IgA, I M, and K light chain; and 1:2 for X light chain (039,040). sing this approach they found IF to provide a very sensitive meana of detectin IgM araproteins in serum (sensitivity of at least 0.25 g/L) (B40). fn using IF one should also be aware of the minor cross-reactivities of commercial antisera against components such as C3, C4, transferrin, and fibrinogen (041,042). The effective use of IEP also requires careful attention to several issues. As in IF, the quality of the commercial antiserum is critical. This ap ears to be . ietection of especially true for anti4 antibodies (035)The I M ara roteins is a particular problem with IEP; however, tfis frawgack can usually be overcome by reduction of serum using 0-mercaptoethanol(D43). IEP is also not the optimal method when multiple paraprotein bands are present. Finally, one must be aware of the limited sensitivity of IEP compared to IF and the “umbrella effect” caused by high concentrations of polyclonal immuno4lobulins. In most situations, the limited sensitivity of IEP i s not a problem; the detection of monoclonal proteins at very low concentrations is generally of uncertain clinical significance. However, in specific clinical situations including patients sua ected of having amyloidosis, a paraprotein-associated perip eral neuropathy, or lymphoproliierativedisorders other than multiple myeloma, the added sensitivity of IF may be important. Detection and Typing of Urine Paraproteine. The detection and typin of monoclonal immunoglobulin light chains in urine or 8ence Jones proteins is an important parameter in the diagnosis and man ement of patients with multiple m eloma as well as other r2ated disorders. This is highlightelby the fact that in approximately 20% of all
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patients with multiple myeloma BJP may be the only . aspects of the labodetectable para rotein (044)Several rato approac to BJP have recently been evaluated. Brigxn and colleagues (045)studied the effect of the manner of urine collection on the detection of BJP. They compared 24-h, early-morning, and random urine collections. All specimenswere concentrated100-foldprior to electrophoresis. In their study of 20 patients with known BJP, they found the early-morning specimen was positive in 19 atienta. The 24-h specimen was positive in 17 patients, anzall of the random specimens were positive in only 14 patients. In one patient both the early-mornin and 24-h specimens were negative. On the basis of these %ata,the concluded that the earlymorning urine s ecimen is re&rred to a random sample. ) emphasized Apart from the gtection of EJP, Kyle (07has the need for collection of a 24-h urine specimen for quantitation of the monoclonal protein. This measurement is an important parameter for patient monitoring and is an excellent indication of the effect of chemotherapy (07). The degree of concentration of urine prior to electrophoresis and/or immunofixationwill certainly have a direct im act on the sensitivity of detecting BJP. Pascali and PezzoE (011, 012,044-047) have advocated using relatively high degrees of urine concentration combined with immunofiition in order to achieve maximum sensitivity. In their procedure, the concentrationof urine is graded according to protein content; sam lee having less than 1 g/L protein are concentrated as mu& as 300-600-fold. The sensitivity of their method is in the range of 1m /L of the original urine. Usin this approach, they found BJ% in 96% of patients with muftiple myeloma (047), in 65% of patients with CLL (0481, and in 61% of patients with non-Hodgkin’s lymphoma (049). Pascali has also pointed out that electrophoresis of “adequately” concentrated urine also has the advantage of providing information useful for classification of proteinuria into glomerular and/or tubular types. On the basis of the resent management of multiple myeloma and related rymphoproliferative disorders, there appears to be little clinical utility in detecting very low levels of BJP. O’Connor and his colleagues (050) point out that agarose gel electrophoresis is capable of detecting protein bands at a concentration, as applied, of 1 /L. A 20-fold concentration of urine would therefore allow ietection of BJP at 0.05 g/L. Even this level of sensitivity is sufficient to detect BJP in at least 80% of patients with systemic amyloidosis (011). Kyle (07) recommends a 15Cb200-fold concentration. O’Connor (050) also raises the im ortant question of what uantity of monoclonal free ligit chain in the urine s h o d be called “Bence Jones protein”. They suggest the phrase ”trace amount of monoclonal free light chain” for cases where the paraprotein concentration is less than 0.5 124 h (or per gram of creatinine or er liter). NO matter w L t terminology is used, it is clear tfat it is not only the presence of detectable BJP that is clinically si ificant, but the concentration of monoclonal protein in’ t urine is also a critical piece of information. Studies aimed at establishing the significance of BJP should quantitatively define what is considered a “positive” test. Immunoelectrophoresis and immunofixation are the standard methods for typing BJP. As is true for serum paraproteins, immunofiiation is more sensitive and is especially useful when there is a background of polyclonal light chains as occurs in the nephrotic syndrome (07). When interpretin urine IF patterns, one must be aware of the restrictea electrophoretic heterogeneity of polyclonal immunoglobulin light chains. This feature has been variously referred to as the “ladder light chain” or “pseudo-oligo clonal” pattern. Its greatest importance rests on the fact that it may be misinterpreted as evidence of Bence Jones proteinuria (see refs D51 and D52). Harrison (053) and MacNamara et al.(054) both performed elegant studies to determine the chemical nature of these re arly spaced, low-concentration bands detected b IF. ey concluded that the bands represent polyclonal ight chains that are invariably resent in the urine of patients with tubular proteinuria. &e relative1 faint banding pattern is seen about 6 times more frequent7 with K than mth X light chains (053). Three to eight band5 may be seen with anti-r antisera, and two to five may be seen mth anti-A. In distinguishi these polyclonal proteins from BJP, MacNamara (054)emBasizes the even spacing of the bands
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which are of maximum staining intensity at the cathodal side of the center of the pattern and diminish anodally and cathodally. In the back ound of the “ladder light chain” atterns, the presence monoclonal protein is suggested Ey finding a band of distinctly increased density or a band that disrupts the regular pattern. As an alternative to immunoelectrophoretic methods, Boege et al. (055) has pro osed using a rapid, automated nephelometric method for t t e identification and quantitation of BJP. As a first step, cyl-microglobulin, albumin, transferrin, and IgG are measured by nephelometry using s ecific antisera. In addition, total protein is measured by nepgelometry using trichloroacetic acid rotein recipitation. Bence Jones proteinuria is indicateaby a dikerence between the sum of the four markers and the total protein concentration of greater than 31% (Le, an excess of total protein). In those cases, the KIAratio is calculated based on nephelometricmeasurements using monospecific antibodies to bound as well as free light chains. A K / X ratio less than 1or greater than 5.2 indicated a monoclonal li ht chain. In analyses of urine sam lea from 84 patients wit[ monoclonal gammopathies, the Jagnostic sensitivity of the assa was 100% and the s ecificity was 97 % when IF was useJas the standard. Base: on the small sample size, which included BJP of relatively low concentration, this method appears to be a reasonable alternative to IF, especially when one considers the ease of ita ap lication. The reliability of this approachwill require larger s& studies. Furthermore,consider’ the variable reactiwty between BJP and commercially a v a i s l e antibodies to K and X light chains, it is questionablewhether or not the quantitative data derived from this type of analysis are reliable. Levinson (056) has also provided a limited amount of data suggestin that the KLR method may be useful for identifyin BJb. Other methods for detecting BJP, including immuno%lottin (057), isoelectric focusing with immunoblotting (0581, SDbypolyacrylamide gel electrophoresis (D59), and immunoisotachophoresis (0601, have also been proposed; however, their advantages over IF are not clear. It is possible that methods using isoelectric focusing will have some clinical utility, since the nephrotoxicity of Bence Jones proteins is thought to be due, at least in part, to their PI(061).
OR
QUANTITATION OF SERUM AND URINE PARAPROTEINS The concentration of monoclonal immunoglobulins in patients with multiple m eloma represents an important measure of the “tumor burlen” and as such is used to monitor patients and thereby guide clinical management. Serum paraproteins are routinely measured by electrophoresis followed by scanning densitometry or by more automated methods such as immunoturbidimetry or nephelometry.The precision and reliability of paraprotein determinations by agarose gel electrophoresis and densitometry has been evaluated by Stermerman et al. (018). The interassay variability of serum paraprotein concentrations was determined b repetitively assaying 27 sera, including 21 cases of Igd paraprotein, 5 cases of IgA paraprotein, and 1 case of IgM paraprotein. For all of the samples, regression analysis of the paraprotein concentration versus the standard deviation of the paraprotein concentrationrevealed a linear relationship between these variables. For IgG paraproteins the increase in standard deviation with increasing paraprotein concentration was statistically significant (P= O.OOO1). Based on their data, the authors constructed a very useful table indicating the minimum differences in paraprotein measurements that indicated “true” differences between sera. Their data apply specifically to IgG araproteins, and although the table can serve as a rough guile for monitoring patients with IgA and IgM paraproteins, the authors indicate that the error may be somewhat lar er for these non-IgG monoclonal immunoglobulins. In t%e same study, Stemerman and his colleagues evaluated the accuracy of electrophoresis and scanning densitometryby comparing the values obtained with those determined by a quantitative ELISA method. The correlation coefficient for this com arison for IgG was 99 % . The paraprotein values determinefelectrophoreticallywere slightly lower than those determined by the ELISA method, presumably due to pol clonal immunoglobulins detected by the latter method. ‘?he accuracy of the electrophoretic ANALYTICAL CHEMISTRY, VOL. 65, NO. 12, JUNE 15, 1993
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method was maintained up to a concentrationof 60 g/L. Above that level the authors recommended diluting the serum 1:l with phos hate-buffered saline. Liu et al. (062) also emphasized t1e potential problems that may be encountered in using the electrophoretic method to quantify monoclonal proteins of high concentration. They recommended careful visual inspection of the stained electrophoretic pattern and described several different types of abnormalitiesin the shape of the monoclonal protein band. These abnormalities result in underestimation of the paraprotein concentration. They also recommended dilution of serum to achieve a more accurate determination. To assess the adequacy of the dilution and accuracy of the determination, they compared the albumin concentration determined by densitometry with that determined by a standard automated method. Since serum albumin is routinely measured in hospitalized patients, this simple comparison could provide a rough measure of the reliability of electrophoretic/densitometric quantitations. Ne helometry and immunoturbidimetry are potentially usefur automated approaches to quantitation of monoclonal immunoglobulins. Vuorinen et al. (063) evaluated the Behring nephelometer for this purpose. They examined a total of 24 patient samples including eight cases each of IgG, IgA, and I M paraprotein. In seven cases, almost one-third of the totaf the difference in the measurements by nephelometry and electrophoresis followed by densitometrywas greater than 20%. These relatively lar e differences occurred for each class of immunoglobulin. fn some cases the ne helometric determination was higher and in others the gnsitometric determination was higher. The within-series coefficients of variation (CV) for the ne helometricmethod were similar for each class of immunoglo∈ the CV ranged from 2.37 % to 5.85 7%. In the case of IgM, the authors emphasized that special care must be taken to adjust the sample dilution so that the nephelometer functions in the most accurate part of the calibration curve. Others have also found consistent overestimationof IgM paraproteins by nephelometry (064). Sinclair et al. (065com ) ared immunoturbidimetry(IT)with electrophoresis followefby densitometry for quantitation of araproteins. They evaluated 84 sera including 34 cases of gG paraprotein, 23 cases of IgA paraprotein, and 25 cases of IgM paraprotein. For all three classes of paraprotein, IT gave higher mean results compared to electrophoresis/ densitometry. In the case of IgM monoclonal proteins, they concluded that the determinations made by IT overestimated the paraprotein concentration. The within-batch re licate determinations was 4.5% for IgG paraproteins and)2.8% for IgA paraproteins. On the basis of the available data, it appears that either nephelometry or IT can be used to monitor serum IgG and IgA paraprotein concentrations. Free immuno lobulin light chains ma also be measured by IT (066). I g h paraproteins are progably best quantitated by electrophoresis/densitometry.There are some problems with these automated methods. First, there are variations in the immunoreactivity among different para roteins and between paraproteins and the calibration stanlards. Second, the presence of polyclonal immunoglobulins limits their sensitivity. Similar problems also arise with electrophoresis/densitometry. There is some variation in dye binding by different paraproteins. Furthermore, the “ ating” of the paraprotein spike is somewhat subjective and tEe densitometric measurement may be affected by background polyclonal immunoglobulins or, when migration of the para rotein is outside the y re ’on,by other protein bands as well. %hatever method is use8 it should be kept in mind that there are unpredictable differences in the results of quantitation of paraproteins by different methods. Therefore, when the course of a given patient’s disease or response to therapy is being followed, the same method should be used each time (07).
P
Ffl;
PROGNOSTIC FACTORS I N MULTIPLE MYELOMA In evaluating prognostic factors in plasma cell dyscrasias, it is important to keep several clinical facts in mind. No therapy is available for patients with monoclonal gammopathies of undetermined significance that will prevent the development of overt multiple m eloma. This occurs in a significant percentage of patients. n! frank multiple myeloma 980R
ANALYTICAL CHEMISTRY, VOL. 65, NO. 12, JUNE 15, 1993
there are no indications for s ecific forms of therapy, since none is curative (067, 068). kevertheless, there is a subset of patients with aggressive multiple myeloma in whom aggressive chemotherapy or even bone marrow transplantation is considered appropriate (069,070) and it is in these patients that the prognostic factors can play an important role. Of the biochemical markers used to redict prognosis in multi le myeloma, serum 8-2 microglob& is clearly the most usefuf(D67,071,079). The 8-2 micro lobulin concentration corrected for serum creatinine (see refb8O) is afairly reliable indicator of total body tumor mass at diaposis in multiple myeloma (076). The uncorrected 8-2 microglobulin level, which in addition to the tumor mass also reflects the degree of renal dam e, is consistently identified as one of the most important inxpendently si ificant variables in predicting survival (072,073,078). #e serum concentration used as a cutoff to divide patients into groups with good and poor prognoses varies from 4 (074) to 8 mg/L (071). Obviously, a higher cutoff will produce prognostic information with a greater degree of statistical celtcunty but will identify fewer patients in the poor prognostic category. On the basis of the prognostic im ortance of 19-2 microglobulin, a number of investigators Kave proposed “staging” systems based on combinations of this serum marker with other factors includingC-reactive protein (0791,serum albumin and patient age (0781, percent bone marrow “myelomatous” cells ( 0 7 9 , and bone marrow plasma cell labeling index (073). From the analytical point of view, 8-2 microglobulin can be measured using a variety of different methods including an enzymelinked immunosorbentassay (073,radioimmunoassay (074, 076,078, 079), and radial immunodiffusion (075). A number of other biochemical markers have also received some attention as possible factors for predicting prognosis and for measuring tumor cell mass in patients with multiple myeloma. These recently described serum markers include IL-6 (081-083), thymidine kinase (084), lactate dehydrogenase (0851, neopterin (0861, and IL-2 (087). Of these, IL-6 has generated the most interest. Both ELISA (088) and radioimmunoassays (0811 have recent1 been described for measuring serum IL-6. This polypeptiL cytokine has a number of biological activities including effectson the immune system, hematopoiesis, and acute phase reactions (089).As a growth factor, IL-6 may play a central role in the malignant transformationof the plasma cells in multiple myelomas (079, 090-093). It is also possible that a thorough understanding of the role of IL-6 in multiple myeloma may provide a basis for designing new therapeutic strategies for this as yet incurable malignancy (094). LITERATURE CITED (Dl)VladuUu, A. 0.Ann. Clh. Lab. Scl. 1987, 77, 157-181. (D2)Aguul. F.; Bergaml, M. R.: Qasperro, C.; Bellom, V.; M i n l , 0. fv.J. &-&I. 1992, 48, 192-195. (D3)Malacriba, V.; De Francesco, D.; Banfi, G.; Porta, F. A,; Rlches, P. 0. J. C h . Bthd. 1987, 40, 793-797. (D4)Kyle, R. A.; Bayrd, E. D.; McKenrle. 8. F.: Heck, F. J. J. Am. M,Assoc. 1960, 774, 245-251. (D5) Grelpp, P. R. E&odRev. 1989, 3, 222-238. (D8) Kyle, R. A.; Qreipp, P. R. W. Rev. Oncd. Hemtd. 1988, 8, 93-152. (D7)Kyle, R. A. Hemtol. O n d . Clln. North Am. 1992, 6. 347-358. (D8) Kyle. R. A. Am. J. M.1978, 64, 814-828. (DS) Kyle, R. A.; Lust, J. A. Sem/n. I.lemetd. 1989, 26, 176200. (D10)Kyle, R. A.; Grelpp, P. R. N. €w.J. M.1982, 306, 584-587. (Dll)Pascall. E.; Peudl, A. Cancer 1988, 62, 2408-2415. (D12)Peuoll. A.; Pascall. E. Am. J. CUn. Pathd. 1989, 91. 473-475. (D13)Kyle, R. A.; Grdpp, P. R. N. E@. J. M.1980, 302, 1347-1349. (D14)Robeti, C.S.:Roland, J. M.: Slmpm, K. R.; Fakham, S. A. Postgrad. Med. J. 1991. 67, 295-298. (D15)Clapper, R. M.: Deam, D. R.; MacKay, 1. R. Am. J. Clln. pethd. 1987, 88, 348-351. (D18)Rank, R.: Horsfall, K.; Caron, J. CUn. Chem. 1986, 32, 1141. (D17)Levlnm, S. S.: Keren, D. F. CNn. CMm. Acta 1988, 182, 21-30. (D18)Whlcher, J. T.; Calvin, J.; Rlches, P.; Warren, C. Ann. G#n. Bkchem. 1987, 24, 119-132. (Dl9)Riches, P. Q.; Hobbs, J. R. J. CUn. PeW. 1988, 47, 778-785. (D20)Stemerman, D.; Papedea, C.; MartimSattzman, D.; OConnell, A. C., hmallne, 8.: Austln, G. E. Am. J. CUn. Patho/. 1989, 91, 435-440. (D21)Keren, D. F.; Warren, J. S., Lowe. J. B. CUn. chcwn. 1988, 34, 21982201. (D22)FIRsU, R.; Keller, I. Ann. Clln. & & e m . 1990, 27, 327-334. (D23)Jones. R. Q.: Aguul, F.; Bienvenu. J.: Blenchl, P.;@spano, C.: Bergami, M. R.: Perinet, A.; BenxJn. H.; Penn, G. M.; Keller, I.; Whlcher. J. T. Clln. 1991. 37, 1917-1921. (D24)Qulnan, J. E.; Kenny, D. F.; Gatenby. P. A. P a M . 1989, 27, 35-41.
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