Clinica Chimica Acta 439 (2015) 68–70
Contents lists available at ScienceDirect
Clinica Chimica Acta journal homepage: www.elsevier.com/locate/clinchim
Case report
Differences identified between serum and urine immunofixation electrophoresis Edward Tracy, Dorinda M. Andrews, Mary A. Constantin, Yusheng Zhu ⁎ Departments of Pathology and Laboratory Medicine, Medical University of South Carolina, Charleston, SC, United States
a r t i c l e
i n f o
Article history: Received 1 September 2014 Received in revised form 1 October 2014 Accepted 1 October 2014 Available online 13 October 2014 Keywords: Multiple myeloma Serum immunofixation electrophoresis Urine immunofixation electrophoresis Differences
a b s t r a c t Background: Different patterns between serum immunofixation electrophoresis (SIFE) and urine immunofixation electrophoresis (UIFE) happen occasionally and laboratorians should understand the mechanisms behind the differences and additional tests required for complicated cases. Methods: We investigated a complicated multiple myeloma case that showed inconsistent patterns of SIFE and UIFE. To differentiate monoclonal proteins (M-proteins), the urine sample was treated with dithiothreitol to open up the IgA molecule and urine free light chain assay was used for free light chain analysis. Results: The patient's SIFE indicated 2 IgA λ and 1 IgM κ M-proteins, while UIFE revealed monoclonal free λ light chains and a suspicious monoclonal IgA κ protein. Subsequent treatment of the urine sample with dithiothreitol and urine free light chain assay demonstrated that the suspicious monoclonal IgA κ protein was actually a monoclonal IgA λ and a free κ light chain that had similar electrophoretic mobility. Conclusions: The differences identified between SIFE and UIFE in this case are due to the limitation of immunofixation electrophoresis on different specimen types and intra-molecular disulfide bonds formation in IgA. The laboratorians must be cognizant of the strengths and limitations of the procedure being performed and the ancillary testing techniques available to solve the problem. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Multiple myeloma (MM) is a bone marrow-based plasma cell neoplasm frequently associated with a monoclonal protein (M-protein) that can be identified in the serum and/or urine. The incidence is approximately 4 cases per 100,000 in the U.S. The clinical presentation can vary widely between patients based on the extent of their disease; however anemia is the most common laboratory finding at presentation [1]. Regarding M-proteins, IgG κ or IgG λ are the most common, followed by IgA with κ or λ light chains [1,2]. Kappa light chains are more commonly involved than λ, both when associated with a heavy chain and when found alone as a free light chain [1,2]. A bi-clonal population is found occasionally, and tri-clonal proliferation is rare [3]. Serum and urine protein electrophoresis (SPEP, UPEP) and immunofixation electrophoresis (SIFE, IUFE) are commonly used in clinical laboratories for the detection and identification of M-proteins. Different patterns between SIFE and UIFE happen occasionally and Abbreviations: MM, Multiple myeloma; SPEP, Serum protein electrophoresis; SIFE, Serum immunofixation electrophoresis; UPEP, Urine protein electrophoresis; UIFE, Urine immunofixation electrophoresis; M-protein, Monoclonal protein; BME, Betamercaptoethanol; DTT, Dithiothreitol. ⁎ Corresponding author at: Department of Pathology and Laboratory Medicine, Medical University of South Carolina, 171 Ashley Avenue, MSC 908, Suite 309, Charleston, SC 29425, United States. Tel.: +1 843 792 8814; fax: +1 843 792 0424. E-mail address: [email protected] (Y. Zhu).
http://dx.doi.org/10.1016/j.cca.2014.10.002 0009-8981/© 2014 Elsevier B.V. All rights reserved.
laboratorians should understand the mechanisms behind the differences and additional tests required for complicated cases.
2. Case report A 65-y-old male with a past medical history of myocardial infarction, hypertension, and arthritis presented to his primary care physician for a routine annual physical exam. Routine laboratory studies revealed an anemia and, during the course of the workup of the anemia, the patient was discovered to have multiple myeloma (MM). He was referred to hematology–oncology for further work-up and treatment. Serum protein electrophoresis (SPEP) was performed to detect the presence of a monoclonal protein (M-protein) and to quantify it and serum immunofixation electrophoresis (SIFE) was conducted to further characterize the patient's monoclonal gammopathy. SPEP and SIFE revealed 2 monoclonal IgA λ proteins at 24.3 g/l and 2.6 g/l with a total of 26.9 g/l in the beta zone and beta-gamma interzone, respectively, and a monoclonal IgM κ protein at a level too low to quantitate in the gamma zone (Figs. 1A and 2A). The patient also demonstrated hypogammaglobulinemia. Subsequent urine protein electrophoresis (UPEP) and urine immunofixation electrophoresis (UIFE) revealed monoclonal free λ light chains and a suspicious monoclonal IgA κ protein, but the κ light chain migrated slightly cathodic to the IgA heavy chain (Fig. 2B). Since the monoclonal IgA protein identified in
E. Tracy et al. / Clinica Chimica Acta 439 (2015) 68–70
69
Free λ IgA λ (1)
IgA λ (2) ? Albumin
α2
α1
β
γ
Albumin
A
α1
α2
γ
β
B
Fig. 1. Serum and urine protein electrophoresis. A. SPEP showing 2 monoclonal IgA λ proteins in the beta zone [IgA λ [1]] and beta-gamma interzone [IgA λ [2]], respectively, with hypogammaglobulinemia. B. UPEP showing a suspicious monoclonal IgA protein labeled with a question mark (?) and a monoclonal free λ light chain protein.
the urine did not match the 2 monoclonal IgA λ proteins in the serum, further testing of the specimen was performed. 3. Discussion This case presents challenges to laboratorians and clinicians who are unfamiliar with interpreting serum and urine protein electrophoresis. On SIFE, there were 2 monoclonal IgA λ protein bands (likely monomers and dimmers from the same plasma cell clone) and 1 monoclonal IgM κ protein, but on UIFE, only a suspicious monoclonal IgA κ protein and a monoclonal free λ light chain protein were present (2 free λ light chain bands were seen due to antigen excess) (Figs. 1 and 2). The presence of monoclonal IgM κ and monoclonal IgA λ in the serum and the lack of these M-proteins in the urine are easily understandable. This is a product of the kidney's filtration ability based on the size of the glomerular pores and the charge of the glomerular membrane. IgM usually exists as a pentamer in the serum with a molecular weight of 900 kDa and IgA has a molecular weight of 150 kDa. A normally functioning kidney will only allow molecules up to 60– 70 kDa to pass through the glomerular membrane and into the tubules [4]. In the instance of damage to the glomerular membrane, high molecular weight molecules, such as globulins, can appear in the urine. Also identified on UIFE were free λ light chains that were not seen on SIFE. The explanation for this lies in the processing of different types of specimens. For serum, the specimen is diluted at predetermined ratios for each immunoglobulin lane (e.g. IgG 1:12, IgA 1:6, IgM 1:2, κ and λ light chains 1:6) to reduce the background. Regarding urine specimens, free light chains can pass through the glomerular membrane due to small molecular sizes and weights (22.5 kDa, free κ monomer and 45 kDa, free λ dimer) and are normally reabsorbed in the tubules. In patients with MM, the amount of free light chains filtered through the glomerular membrane exceeds the absorption capacity of the tubules, and therefore, they can be detected in the urine. Furthermore,
compared to serum, the total protein concentration in the urine is much lower; thus urine specimens are generally concentrated prior to electrophoresis. This concentrating effect can allow for detection of very low level free light chains in the urine which are unseen on SIFE. It is worth noting that there are 2 free λ light chain bands in the λ lane, which is due to the prozone or hook effect. One band was seen when the specimen was diluted (data not shown). Although we can explain the difference in monoclonal IgM κ, IgA λ and free λ light chains between SIFE and UIFE, we still need to determine why a suspicious monoclonal IgA κ was present on the UIFE, but not on SIFE. When the UIFE gel was reviewed by an experienced reviewer, it was noticed that the migration of the κ light chain was slightly cathodic to the IgA heavy chain. Therefore, it was postulated that the κ light chain band might represent a free κ light chain and the IgA heavy chain band might be a monoclonal IgA λ in which the associated λ light chain was not detected for unknown reasons, because the migration of the IgA heavy chain band on the UIFE was the same as that of 1 of the IgA λ on the SIFE. To determine if the κ light chain band on UIFE was a free κ light chain, a urine free light chain assay was performed. The urine free light chain assay showed that both elevated free κ (78.9 mg/l, reference interval: 1.4–24.2 mg/l) and free λ (2340 mg/l, reference interval: 0.2– 6.7 mg/l) light chains. In the context of polyclonal gamma globulin suppression on the SIFE gel, the urine free κ light chain elevation was not due to renal failure and buildup of polyclonal free κ light chain, which indicated that the κ light chain band on UIFE was a monoclonal free κ light chain. We next determined if the IgA heavy chain band represented a monoclonal IgA λ. It has been reported that some monoclonal immunoglobulins, especially IgA, can have quaternary structure changes that result in indiscernible associated light chains on UIFE. The usual structure of a monomeric immunoglobulin molecule is a “Y” configuration, with 2 heavy chains forming the backbone of the structure, and the
γ β α2 α1 Albumin
A
B
C
Fig. 2. Serum and urine immunofixation electrophoresis. A. SIFE demonstrating 2 monoclonal IgA λ proteins in the beta zone and beta-gamma interzone, and a monoclonal IgM κ protein in the gamma zone. B. UIFE demonstrating a suspicious monoclonal IgA κ protein and a monoclonal free λ light chain protein (2 free λ light chain bands were seen due to antigen excess). C. UIFE following DTT treatment demonstrating an IgA λ protein, which corresponds to the IgA protein on initial UIFE and the IgA λ protein on SIFE. A monoclonal free κ light chain is also seen.
70
E. Tracy et al. / Clinica Chimica Acta 439 (2015) 68–70
light chains binding to the shorter arms of the heavy chain backbone. If an intra-molecular disulfide bond occurs, the quaternary structure of the molecule changes, such that the shorter arms are bent “down” at an acute angle to the backbone forming a “↑” configuration, hiding the light chain epitopes behind the heavy chains. The antiserum is not able to penetrate and bind with the light chain epitope; hence, no band can be seen [5]. Treatment of specimens with beta-mercaptoethanol (BME) or dithiothreitol (DTT) can be used to reduce intra-molecular disulfide bonds in this instance, or repeating IFE with antisera from a different manufacturer may also visualize the light chain. We mixed 100 μL of concentrated urine with 10 μL of 1M DTT and incubated the mixture at 45 °C for 20 min. Similarly, we mixed 100 μL of concentrated urine with 10 μL of H2O and incubated the mixture at 45 °C for 20 min as a control. On UIFE after the DTT treatment, it was revealed that the immunoglobulin in question was actually an IgA λ, which is consistent with the patient's SIFE (Fig. 2C). The final issue is that 2 monoclonal IgA λ bands were identified on SIFE, while only one was detected on UIFE with DTT. This is an issue of relative concentration. The IgA λ band identified on UIFE with DTT corresponds to the beta-zone IgA λ on SIFE, which was present at much higher concentrations than the other IgA λ band in the betagamma interzone. It is worthy to note, that the densities of the heavy chains of the 2 IgA λ bands on SIFE were so different, but the staining of the 2 corresponding λ light chains looked similar. This phenomenon
was also most likely related to the cryptic/covered λ light chain of IgA λ in serum due to the changes in molecular configuration (Fig. 2A). In conclusion, the differences identified between SIFE and UIFE in this case are due to the limitation of immunofixation electrophoresis on different specimen types and intra-molecular disulfide bonds formation in IgA. This case presents many interesting issues that the laboratorians may encounter when performing immunofixation electrophoresis. The interpreter must be cognizant of the strengths and limitations of the procedure being performed and the ancillary testing techniques available to solve the problem. An intimate knowledge of the physiology of kidney function and the molecular structure of immunoglobulins is required to ensure that the most accurate information is provided to our clinical colleagues and thus to our patients. References [1] Kyle RA, Gertz MA, Witzig TE, Lust JA, Lacy MQ, Dispenzieri A, et al. Review of 1027 patients with newly diagnosed multiple myeloma. Mayo Clin Proc 2003;78:21–33. [2] Keren DF. Monoclonal gammopathies. In: McClatchey, editor. Clinical laboratory medicine. 2nd ed. Philadelphia: Lippincott Williams & Wilkins; 2002. p. 1448–57. [3] Attaelmannan M, Levinson SS. Understanding and identifying monoclonal gammopathies. Clin Chem 2000;46:1230–8. [4] Zhang A, Huang S. Progress in pathogenesis of proteinuria. Int J Nephrol 2012;1–14. [5] Janik B. Guide to electrophoresis and immunofixation in clinical diagnosis: the proteins of serum, urine and cerebrospinal fluid. Sebia electrophoresis; 2006.
Contents lists available at ScienceDirect
Clinica Chimica Acta journal homepage: www.elsevier.com/locate/clinchim
Case report
Differences identified between serum and urine immunofixation electrophoresis Edward Tracy, Dorinda M. Andrews, Mary A. Constantin, Yusheng Zhu ⁎ Departments of Pathology and Laboratory Medicine, Medical University of South Carolina, Charleston, SC, United States
a r t i c l e
i n f o
Article history: Received 1 September 2014 Received in revised form 1 October 2014 Accepted 1 October 2014 Available online 13 October 2014 Keywords: Multiple myeloma Serum immunofixation electrophoresis Urine immunofixation electrophoresis Differences
a b s t r a c t Background: Different patterns between serum immunofixation electrophoresis (SIFE) and urine immunofixation electrophoresis (UIFE) happen occasionally and laboratorians should understand the mechanisms behind the differences and additional tests required for complicated cases. Methods: We investigated a complicated multiple myeloma case that showed inconsistent patterns of SIFE and UIFE. To differentiate monoclonal proteins (M-proteins), the urine sample was treated with dithiothreitol to open up the IgA molecule and urine free light chain assay was used for free light chain analysis. Results: The patient's SIFE indicated 2 IgA λ and 1 IgM κ M-proteins, while UIFE revealed monoclonal free λ light chains and a suspicious monoclonal IgA κ protein. Subsequent treatment of the urine sample with dithiothreitol and urine free light chain assay demonstrated that the suspicious monoclonal IgA κ protein was actually a monoclonal IgA λ and a free κ light chain that had similar electrophoretic mobility. Conclusions: The differences identified between SIFE and UIFE in this case are due to the limitation of immunofixation electrophoresis on different specimen types and intra-molecular disulfide bonds formation in IgA. The laboratorians must be cognizant of the strengths and limitations of the procedure being performed and the ancillary testing techniques available to solve the problem. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Multiple myeloma (MM) is a bone marrow-based plasma cell neoplasm frequently associated with a monoclonal protein (M-protein) that can be identified in the serum and/or urine. The incidence is approximately 4 cases per 100,000 in the U.S. The clinical presentation can vary widely between patients based on the extent of their disease; however anemia is the most common laboratory finding at presentation [1]. Regarding M-proteins, IgG κ or IgG λ are the most common, followed by IgA with κ or λ light chains [1,2]. Kappa light chains are more commonly involved than λ, both when associated with a heavy chain and when found alone as a free light chain [1,2]. A bi-clonal population is found occasionally, and tri-clonal proliferation is rare [3]. Serum and urine protein electrophoresis (SPEP, UPEP) and immunofixation electrophoresis (SIFE, IUFE) are commonly used in clinical laboratories for the detection and identification of M-proteins. Different patterns between SIFE and UIFE happen occasionally and Abbreviations: MM, Multiple myeloma; SPEP, Serum protein electrophoresis; SIFE, Serum immunofixation electrophoresis; UPEP, Urine protein electrophoresis; UIFE, Urine immunofixation electrophoresis; M-protein, Monoclonal protein; BME, Betamercaptoethanol; DTT, Dithiothreitol. ⁎ Corresponding author at: Department of Pathology and Laboratory Medicine, Medical University of South Carolina, 171 Ashley Avenue, MSC 908, Suite 309, Charleston, SC 29425, United States. Tel.: +1 843 792 8814; fax: +1 843 792 0424. E-mail address: [email protected] (Y. Zhu).
http://dx.doi.org/10.1016/j.cca.2014.10.002 0009-8981/© 2014 Elsevier B.V. All rights reserved.
laboratorians should understand the mechanisms behind the differences and additional tests required for complicated cases.
2. Case report A 65-y-old male with a past medical history of myocardial infarction, hypertension, and arthritis presented to his primary care physician for a routine annual physical exam. Routine laboratory studies revealed an anemia and, during the course of the workup of the anemia, the patient was discovered to have multiple myeloma (MM). He was referred to hematology–oncology for further work-up and treatment. Serum protein electrophoresis (SPEP) was performed to detect the presence of a monoclonal protein (M-protein) and to quantify it and serum immunofixation electrophoresis (SIFE) was conducted to further characterize the patient's monoclonal gammopathy. SPEP and SIFE revealed 2 monoclonal IgA λ proteins at 24.3 g/l and 2.6 g/l with a total of 26.9 g/l in the beta zone and beta-gamma interzone, respectively, and a monoclonal IgM κ protein at a level too low to quantitate in the gamma zone (Figs. 1A and 2A). The patient also demonstrated hypogammaglobulinemia. Subsequent urine protein electrophoresis (UPEP) and urine immunofixation electrophoresis (UIFE) revealed monoclonal free λ light chains and a suspicious monoclonal IgA κ protein, but the κ light chain migrated slightly cathodic to the IgA heavy chain (Fig. 2B). Since the monoclonal IgA protein identified in
E. Tracy et al. / Clinica Chimica Acta 439 (2015) 68–70
69
Free λ IgA λ (1)
IgA λ (2) ? Albumin
α2
α1
β
γ
Albumin
A
α1
α2
γ
β
B
Fig. 1. Serum and urine protein electrophoresis. A. SPEP showing 2 monoclonal IgA λ proteins in the beta zone [IgA λ [1]] and beta-gamma interzone [IgA λ [2]], respectively, with hypogammaglobulinemia. B. UPEP showing a suspicious monoclonal IgA protein labeled with a question mark (?) and a monoclonal free λ light chain protein.
the urine did not match the 2 monoclonal IgA λ proteins in the serum, further testing of the specimen was performed. 3. Discussion This case presents challenges to laboratorians and clinicians who are unfamiliar with interpreting serum and urine protein electrophoresis. On SIFE, there were 2 monoclonal IgA λ protein bands (likely monomers and dimmers from the same plasma cell clone) and 1 monoclonal IgM κ protein, but on UIFE, only a suspicious monoclonal IgA κ protein and a monoclonal free λ light chain protein were present (2 free λ light chain bands were seen due to antigen excess) (Figs. 1 and 2). The presence of monoclonal IgM κ and monoclonal IgA λ in the serum and the lack of these M-proteins in the urine are easily understandable. This is a product of the kidney's filtration ability based on the size of the glomerular pores and the charge of the glomerular membrane. IgM usually exists as a pentamer in the serum with a molecular weight of 900 kDa and IgA has a molecular weight of 150 kDa. A normally functioning kidney will only allow molecules up to 60– 70 kDa to pass through the glomerular membrane and into the tubules [4]. In the instance of damage to the glomerular membrane, high molecular weight molecules, such as globulins, can appear in the urine. Also identified on UIFE were free λ light chains that were not seen on SIFE. The explanation for this lies in the processing of different types of specimens. For serum, the specimen is diluted at predetermined ratios for each immunoglobulin lane (e.g. IgG 1:12, IgA 1:6, IgM 1:2, κ and λ light chains 1:6) to reduce the background. Regarding urine specimens, free light chains can pass through the glomerular membrane due to small molecular sizes and weights (22.5 kDa, free κ monomer and 45 kDa, free λ dimer) and are normally reabsorbed in the tubules. In patients with MM, the amount of free light chains filtered through the glomerular membrane exceeds the absorption capacity of the tubules, and therefore, they can be detected in the urine. Furthermore,
compared to serum, the total protein concentration in the urine is much lower; thus urine specimens are generally concentrated prior to electrophoresis. This concentrating effect can allow for detection of very low level free light chains in the urine which are unseen on SIFE. It is worth noting that there are 2 free λ light chain bands in the λ lane, which is due to the prozone or hook effect. One band was seen when the specimen was diluted (data not shown). Although we can explain the difference in monoclonal IgM κ, IgA λ and free λ light chains between SIFE and UIFE, we still need to determine why a suspicious monoclonal IgA κ was present on the UIFE, but not on SIFE. When the UIFE gel was reviewed by an experienced reviewer, it was noticed that the migration of the κ light chain was slightly cathodic to the IgA heavy chain. Therefore, it was postulated that the κ light chain band might represent a free κ light chain and the IgA heavy chain band might be a monoclonal IgA λ in which the associated λ light chain was not detected for unknown reasons, because the migration of the IgA heavy chain band on the UIFE was the same as that of 1 of the IgA λ on the SIFE. To determine if the κ light chain band on UIFE was a free κ light chain, a urine free light chain assay was performed. The urine free light chain assay showed that both elevated free κ (78.9 mg/l, reference interval: 1.4–24.2 mg/l) and free λ (2340 mg/l, reference interval: 0.2– 6.7 mg/l) light chains. In the context of polyclonal gamma globulin suppression on the SIFE gel, the urine free κ light chain elevation was not due to renal failure and buildup of polyclonal free κ light chain, which indicated that the κ light chain band on UIFE was a monoclonal free κ light chain. We next determined if the IgA heavy chain band represented a monoclonal IgA λ. It has been reported that some monoclonal immunoglobulins, especially IgA, can have quaternary structure changes that result in indiscernible associated light chains on UIFE. The usual structure of a monomeric immunoglobulin molecule is a “Y” configuration, with 2 heavy chains forming the backbone of the structure, and the
γ β α2 α1 Albumin
A
B
C
Fig. 2. Serum and urine immunofixation electrophoresis. A. SIFE demonstrating 2 monoclonal IgA λ proteins in the beta zone and beta-gamma interzone, and a monoclonal IgM κ protein in the gamma zone. B. UIFE demonstrating a suspicious monoclonal IgA κ protein and a monoclonal free λ light chain protein (2 free λ light chain bands were seen due to antigen excess). C. UIFE following DTT treatment demonstrating an IgA λ protein, which corresponds to the IgA protein on initial UIFE and the IgA λ protein on SIFE. A monoclonal free κ light chain is also seen.
70
E. Tracy et al. / Clinica Chimica Acta 439 (2015) 68–70
light chains binding to the shorter arms of the heavy chain backbone. If an intra-molecular disulfide bond occurs, the quaternary structure of the molecule changes, such that the shorter arms are bent “down” at an acute angle to the backbone forming a “↑” configuration, hiding the light chain epitopes behind the heavy chains. The antiserum is not able to penetrate and bind with the light chain epitope; hence, no band can be seen [5]. Treatment of specimens with beta-mercaptoethanol (BME) or dithiothreitol (DTT) can be used to reduce intra-molecular disulfide bonds in this instance, or repeating IFE with antisera from a different manufacturer may also visualize the light chain. We mixed 100 μL of concentrated urine with 10 μL of 1M DTT and incubated the mixture at 45 °C for 20 min. Similarly, we mixed 100 μL of concentrated urine with 10 μL of H2O and incubated the mixture at 45 °C for 20 min as a control. On UIFE after the DTT treatment, it was revealed that the immunoglobulin in question was actually an IgA λ, which is consistent with the patient's SIFE (Fig. 2C). The final issue is that 2 monoclonal IgA λ bands were identified on SIFE, while only one was detected on UIFE with DTT. This is an issue of relative concentration. The IgA λ band identified on UIFE with DTT corresponds to the beta-zone IgA λ on SIFE, which was present at much higher concentrations than the other IgA λ band in the betagamma interzone. It is worthy to note, that the densities of the heavy chains of the 2 IgA λ bands on SIFE were so different, but the staining of the 2 corresponding λ light chains looked similar. This phenomenon
was also most likely related to the cryptic/covered λ light chain of IgA λ in serum due to the changes in molecular configuration (Fig. 2A). In conclusion, the differences identified between SIFE and UIFE in this case are due to the limitation of immunofixation electrophoresis on different specimen types and intra-molecular disulfide bonds formation in IgA. This case presents many interesting issues that the laboratorians may encounter when performing immunofixation electrophoresis. The interpreter must be cognizant of the strengths and limitations of the procedure being performed and the ancillary testing techniques available to solve the problem. An intimate knowledge of the physiology of kidney function and the molecular structure of immunoglobulins is required to ensure that the most accurate information is provided to our clinical colleagues and thus to our patients. References [1] Kyle RA, Gertz MA, Witzig TE, Lust JA, Lacy MQ, Dispenzieri A, et al. Review of 1027 patients with newly diagnosed multiple myeloma. Mayo Clin Proc 2003;78:21–33. [2] Keren DF. Monoclonal gammopathies. In: McClatchey, editor. Clinical laboratory medicine. 2nd ed. Philadelphia: Lippincott Williams & Wilkins; 2002. p. 1448–57. [3] Attaelmannan M, Levinson SS. Understanding and identifying monoclonal gammopathies. Clin Chem 2000;46:1230–8. [4] Zhang A, Huang S. Progress in pathogenesis of proteinuria. Int J Nephrol 2012;1–14. [5] Janik B. Guide to electrophoresis and immunofixation in clinical diagnosis: the proteins of serum, urine and cerebrospinal fluid. Sebia electrophoresis; 2006.
