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2% per year and it was related to the amount of monoclonal protein found at the initial diagnosis of MGUS (e.g., at the initial 0.5 g/dL and 3 g/dL, the risk was about 0.6% and 3% per year, respectively). Interestingly, the amount of monoclonal protein at diagnosis of multiple myeloma is not a predictive factor of the outcome of the disease. Patients with monoclonal IgA and IgM had an increased risk of progression compared to monoclonal IgG. More recent findings indicate that biclonal MGUS have a higher risk of progression than previously thought. In only 2% of the patients, the initial small monoclonal protein (with a measurable spike) disappeared. Opposite conclusions were drawn whether or not detectable Bence Jones proteinuria is a risk factor for the progression. Age, sex, the presence of connective tissue disorder, viral hepatitis, solid tumors, iatrogenic immunosuppression, serum albumin and 2-microglobulin did not appear to be risk factors. However, normal range concentrations of the latter two proteins as well as of CRP 1-antitrypsin and thymidine kinase are believed to be , prerequisite for MGUS classification. The reduction of polyclonal immunoglobulin background (hypogammaglobulinemia), although a controversial risk factor for progression, must always be interpreted with caution. It might be the first laboratory indication of a lymphoproliferative disorder associated with limited or absent secretion of a monoclonal protein into serum, e.g., in light chain disease (LCD), amyloidosis, lymphoma, etc. The level of bone marrow plasma cells (>10%) and in some studies hypo-gammaglobulinemia were identified as independent risk factors requiring stricter monitoring of the MGUS patients. From the patients who progressed into a malignant disorder, most developed multiple myeloma. Other notable disorders were IgM lymphoma, primary amyloidosis, Waldenstrms macroglobulinemia and non-Hodgkins lymphoma. The undisputed conclusion: since the benign nature of MGUS is generally impossible to ascertain, the patients diagnosed with MGUS should be monitored for the rest of their lives. Usually the first follow-up testing is suggested six months after the initial diagnosis. If the monoclonal protein and other biochemical markers remain stable in the asymptomatic patient, next follow-up in one year seems to be sufficient. There is still much to be learned about MGUS. The molecular and cellular changes in the progression of clonal cells to a malignant condition have not been clearly delineated. Structural chromosomal abnormalities and chromosomal translocations seem to occur early in the clonal development; however, none of these changes are specific for all MGUS. It is also unclear whether or not smoldering and indolent myeloma are the necessary or only occasional transitory stages in the progression of MGUS. Currently, there is still no way to tell whether the progression will happen and when or whether the clonal proliferation will stay arrested in the benign form. If you are interested in receiving information concerning Sebias Hydragel Protein (155) or Hydragel Immunofixation (156) assays, please circle the appropriate number on the Reader Response Card or visit our website (http://www.sebia-usa.com/products/reagents.html).

issue 3 vol. 4 2003

Separations
Oligoclonal IgG Banding Detection: The Most Sensitive Method
By: Bonny Champagne, Marketing Product Specialist

The most sensitive method for the detection of oligoclonal immunoglobulin bands is isoelectric focusing.I Investigators analyzed large numbers of CSF by IEF and showed that 95% to 100% of clinically definite MS patients had oligoclonal IgG bands in their CSF.2 Electrophoresis of cerebrospinal fluid (CSF), in order to detect oligoclonal banding, is one of the most useful laboratory tests in the evaluation of a patient suspected of having multiple sclerosis (MS). As quoted above, a great percentage of MS patients exhibit oligoclonal banding during the course of the disease; banding can appear very early in the disease and generally persists even through remission and intensified cycles. The detection of oligoclonal banding can be carried out by many methods including: agarose gel electrophoresis (AGE) and isoelectric focusing (IEF). For many years, AGE was the most commonly used method in the clinical laboratory. However, many studies have been released stating that IEF is much more sensitive than AGE in detecting oligoclonal bands. IEF is, in very basic terms, the separation of IgG on the basis of different charges or isoelectric points. In order to visualize IgG bands, IEF is then generally followed by either an immunofixation or immunoblotting step. There are many compelling advantages of immunofixation over immunoblotting including procedural simplicity and result quality. The immunofixation procedure requires fewer reagents and does not require the continued attention of a technologist, i.e. is more walkaway. In a study, oligoclonal IgG band patterns seen after immunoblotting were compared with those of conventional immunofixation and the resolution and intensity of oligoclonal IgG bands were... better after immunofixation. Since immunofixation is simpler than immunoblotting,

we recommend that clinical laboratories use immunofixation after IEF to detect oligoclonal IgG bands in CSF.3 Sebia now offers an FDA-cleared IEF assay, in which paired CSF and serum samples (diluted such that the IgG levels are equivalent) are run in parallel on the semi-automated HYDRASYS instrument. IEF is then followed by immunofixation with enzyme-labeled anti-IgG for visualization purposes; no immunoblotting is necessary. As a result of the anti-IgG utilized, banding is specifically IgG in nature. This is an advantage over silver staining methods since multiple non IgG-specific bands are a nuisance to the interpreter. Oligoclonal bands present in CSF, but absent in serum, indicate local synthesis of IgG. Sebias new CSF IEF assay is completed in less than half the time of other IEF assays! Up to 18 tests or nine paired specimens can be completed on the Hydragel CSF IEF gel in just over two hours. If you are interested in receiving information concerning Sebias Hydragel CSF IEF assay, please circle number 154 on the Reader Response Card or visit http://www.sebiausa.com/products/csfIsoelectric.html
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Oligoclonal IgG Banding Detection: The Most Sensitive Method

(continued) References
1

Andersson M, Alvarez-Cerrneno J, et al. Cerebrospinal fluid in the diagnosis of multiple sclerosis: a consensus report. Journal of Neurology, Neurosurgery, and Psychiatry 1994 Aug; 57(8): 897-902. 2 Mehta, PD. Diagnostic Usefulness of Cerebrospinal Fluid in Multiple Sclerosis. Crit Rev Clin Lab Sci. 1991; 28(3): 233-51. 3 Mehta, PD, Patrick BA, Black J. Comparison of oligoclonal immunoglobulin G bands in multiple sclerosis cerebrospinal fluid by immunoblotting and immunofixation. Electrophoresis. 1988 Mar; 9(3): 126-8.

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consensus: no bone marrow biopsy and no treatment. In serum protein or immunofixation electrophoregrams, MGUS can usually be seen as a small restriction having the appearance of a weak monoclonal band. In one study spanning 25 years, the median of the serum monoclonal protein at diagnosis was 1.2 g/dL (<0.5 g/dL, 24%; 0.5<1.0 g/dL, 19%; 1.0<2.0 g/dL, 51% and maximum 3 g/dL; <1%). The BJP in urine was (g/24 hours) <0.1, 74%; 0.1<0.3, 15%; 0.3<1, 10%. At least in the initial stage, MGUS is caused by an apparently benign proliferation of plasma cells. The reports on the frequency and distribution widely differ. According to some, it affects about 0.15% of the general population. It is rare before the age of 40. MGUS is expected in ~2% of the apparently normal individuals age >50 years and in ~3% (as high as 10% was reported) of the elderly population >75 years. Others found MGUS in 5% of healthy adults of all ages, in 11% of hospitalized patients for no-malignant disorders >45 years of age and in about 50% of patients having a monoclonal protein in serum. MGUS is often found in conditions associated with chronic antigenic stimulation or immunosuppression, such as seen with restricted heterogeneity patterns, in connective tissue disorders and chronic liver disease. Experience suggests that these so-called secondary MGUS are benign and the associated condition does not represent a risk factor. Although the genesis of MGUS is not understood, the increased incidence with age, the often observed reduction of uninvolved immunoglobulins (in approximately 40% cases) and association with immune stimulation or suppression, point to possible involvement of immunoregulatory defects. Several studies reported analysis of patients data accumulated over long periods of time (up to several decades). The average risk of progression of MGUS to malignant plasmaproliferative or lymphoproliferative disorders was 1-

unanswered, whether or not MGUS will stay arrested in the benign state or will progress to an aggressive, malignant state (either directly or via smoldering myeloma), and what are the molecular and cellular changes causing and accompanying such progression. Although such a state of uncertainty is not favored by those who prefer black and white interpretation of laboratory results, this is exactly what MGUS is - a monoclonal gammopathy of undetermined significance. The criteria for classification of MGUS proposed in the past, namely the cutoff concentrations of monoclonal proteins in serum and urine, differed somewhat from study-to-study. The International Myeloma Working Group has reviewed the criteria for diagnosis and classification with the aim of producing easy to use definitions based on routinely used investigations. The criteria were presented at the 9th International Workshop on Multiple Myeloma, May 2003, in Salamanca, Spain. According to the consensus, a patient with MGUS is characterized as follows: low levels of monoclonal protein in serum < 3g/dL (<1.5-3 g/dL IgG and <1-2 g/dL IgA) <10% plasma cells in bone marrow no evidence of multiple myeloma, other B-cell proliferative disorders, or amyloidosis no evidence of ROTI (Related Organ or Tissue Impairment) ROTI is typically manifested by CRAB (increased Calcium, Renal insufficiency, Anemia or Bone lesions) attributed to the plasma cell proliferative process Other criteria that have also been considered include: 01 g/dL BJP in urine labeling index in bone marrow <0.2% stability of biochemical and clinical features absence of hypo-albuminemia Concerning diagnostic and therapeutic considerations, there is a clear

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LIFE IN THE LABORATORY

By: Josie C. Roberts, MT (ASCP), NCA

Please circle the reader service numbers of those items on which you would like more information.

article 154

article 155

Life to save, life to lose, new life to bring Into the world of the laboratory For to adequately fit into its space Education to fulfill obtained In the hospital, clinic, doctors office Night and day, all hours, all seasons Tests on injured, sick, diseased Help maintain, evaluate, educate Ever current procedure, protection

article 156
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Monoclonal Gammopathy of Undetermined Significance (MGUS)

MGUS always has been a very popular topic when it comes to interpretation of weak and unusual immunofixation patterns when their diagnostic significance is far from obvious. Sometimes I feel the MGUS category is abused as it serves as a garbage disposal for hazy results. Such a situation is not due to insufficient interest in resolving the mystery of MGUS. The classification and diagnostic significance of the monoclonal gammopathies that are grouped under MGUS is a frequent subject of discussions by the experts and matter of confusion to others. Not enough is known about the cause of MGUS, their origin and further development. MGUS, also referred to as essential monoclonal gammopathy, is typically associated with low levels of a monoclonal protein produced by a single clone of plasma cells that appear to be in a benign or pre-neoplastic state. Some argue that considering MGUS a benign condition might be misrepresentation. The early trend to amplify the M component might be slow but clonal evolution to overt MM or kindred B cell malignancies does occur in 1-2% cases per year. Eventually, given enough time some believe, all cases of MGUS would convert. The most intriguing question thus remains

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Light or heavy chain, alpha, gamma, or beta Areas for culture, coag, hematology, chemistry Bilirubin, blood counts, B-cell or T-cell Online results relayed to chart Results confirmed, rule out, or monitor As protime, fibrinogen, urinalysis Josie C. Roberts, MT (ASCP), NCA Total protein, cardiac enzyme Saint Francis Medical Center, Monroe, LA On blood, urine, body fluids Ready to rush to the task Yearly professionals nationwide assemble to examine, evaluate, educate

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LIFE IN THE LABORATORY was submitted to Sebia by Ms. Josie C. Roberts at this years AACC show in Philadelphia, PA. Josie has been a laboratory scientist for 36 years and finds the field just as enjoyable today as it was in the very beginning. Josie is employed at Saint Francis Medical Center in Monroe, LA. During her 21 years at Saint Francis Medical Center, Josie has worked in the microbiology lab and, most recently, the hematology lab. On behalf of all the Sebia staff, thank you for your time and creativity in writing and submitting this truly wonderful laboratory poem!

In the meantime, keep sending in those questions. You may send them to me by mail at Sebia, 400 - 1705 Corporate Drive, Norcross, GA 30093, attn. Ask Borek, by fax at 770-446-8511 attention Ask Borek, or by e-mail at borek.janik@sebia-usa.com. Whichever method you choose, include your name, laboratory name and phone number should I have questions for you.