The analysis of mirror pattern bands synthesized in patients with neurological disorder during Oligoclonal immunoglobulin isoelectric focusing electrophoresis CURRENT

Background The analysis of oligoclonal band (OCB) of cerebrospinal fluid is of vital importance for the clinical diagnosis and differential diagnosis of neurological disease. However, there still exists limited knowledge about the interpretation for the OCB patterns especially the mirror patterns for the neurological disorder during the clinal course. In this study, we thoroughly analyzed the 12 patients of mirror pattern results with laboratory and clinical information. Methods specimens were obtained from patients who had CSF and serum analyzed by isoelectric focusing (IEF) in the Special Examination Laboratory of XuanWu Hospital over a 4-year period. 12 patients with mirror patterns were screened out by isoelectric focusing electrophoresis from 5892 patients and then immunofixation electrophoresis were performed on the 12 patients. All three types of mirror connected M protein from peripheral serum. However,not all patients with the patterns present positive M out of 12 patients (58.3%) were blood system-related None the were Multiple in CSF: cerebrospinal fluid, TP: total protein, OCB: oligoclonal bands, QALB: CSF/serum albumin quotient, IgG-Syn: intrathecal IgG synthesis, C-IgG: CSF IgG, S-IgG: Serum IgG, MS: multiple sclerosis, SIE: serum immunofixation electrophoresis, P3: pattern 3, P4: pattern 4, P5: pattern 5, ND: not detected; MGUS: monoclonal gammopathy of undermined significance; MM: myeloma, POEMS: POEMS syndrome, MGAPU: monoclonal gammopathy-associated neuropathy; ALS: Amyotrophic lateral sclerosis DLB: Dementia with Lewy body, IMD:

serves as not only an especially valuable tool for differential diagnosis but also for prognosis [6] ADDIN EN.CITE , and even for predicting the conversion of optic neuritis and clinically isolated syndromes (CIS) to MS [7]. For such a qualitative and clinically significant technique, the correct recognition and interpretation of CSF oligoclonal band patterns is utmost crucial. A general consensus is that there are five typical types of band patterns [8] ADDIN EN.CITE , of which the presence of oligoclonal bands in CSF but not in serum means local synthesis of immunoglobins in the central nervous system, which is most likely observed in patients with MS [8,9] ADDIN EN.CITE . Relatively little attention has been paid, however, to the mirror patterns, which are quite uncommon and the clinical significance is still rarely clarified. There are three types of abnormal mirror patterns, the first type is termed "type 3", which performs both identical bands in CSF and serum and additional CSF restricted oligoclonal bands. The second is so-called "type 4", which presents identical oligoclonal bands (irregularly spaced bands) in CSF and serum, and indicates a systemic instead of intrathecal immune reaction [4]. The third type is "type 5", which symmetrically displays monoclonal bands both in the CSF and serum sample [10]. The monoclonal bands have the characteristic of space in symmetric steps among bands and tend to be evenly distributed [11,12] ADDIN EN.CITE . The monoclonal proteins form clusters and are more prominent, with higher concentrations and stronger immunoreaction, which actually makes this band patterns more easier to recognize [13]. This identical multiple bands in both CSF and serum (mirror pattern) may be relative to the following disorders, such as the systemic inflammation, probable MS, systemic lupus erythematosus, paraneoplastic syndrome, vascular disease, peripheral neuropathies and so on [2,11,14] ADDIN EN.CITE . However, in clinical practice, there are still many discrepancies among different research institution in interpreting the above three CSF/serum mirror patterns, which is to a large extent attribute to the very rare occurrence [15] ADDIN EN.CITE . Quite a few neurologists usually unreasonably downplayed the clinical significance of mirror patterns when the paired CSF and serum samples were analyzed for OCB, instead played more emphasis on the classical pattern 2 which to a large extent indicates MS.
CSF analysis is undoubtedly an important tool for diagnosis for those with neurological disorders [16,17] ADDIN EN.CITE . Analysis of paired CSF/serum for OCB has been a conventional laboratory method in our laboratory for the patients with neurological disfunction but without clear pathogeny. In the past four years, among 5892 patients who received OCB detection in our hospital, just only 12 cases presented the mirror patterns. In this study, we focus on the 12 patients with comprehensive clinical and laboratory information in order to further evaluate the potential value of OCB mirror patterns and to what extent OCB mirror patterns results influence the final diagnosis.   [12,22] ADDIN EN.CITE . Therefore, IgG isoelectric focusing followed by immunofixation electrophoresis is increasingly employed to be a routine laboratory technique for those patients with neuropathy to perform oligoclonal band analysis in order mainly to aid early differential diagnosis of multiple sclerosis. The mirror patterns are usually an incidental finding during CSF OCB analysis [23].
Due to the rarity, the laboratory interpretation to the mirror patterns is still often ambiguous [13], not to mention most neurologists involuntarily weaken the clinical significance of this patterns [4]. In this retrospective study, we spent more than four years focusing on the value of OCB analysis in the  and two cases (No.1 and 12) were multiple myeloma (MM). The 58.3% positive rate of diagnosis of blood system-related disease was established on the premise that we didn't rule out the possibility that patients with mirror patterns who didn't diagnosed with blood system disorder in the short time were more likely to be diagnosed later. The presence of mirror patterns is though almost excluded the possibility of MS, provide an additional and powerful evidence to the diagnosis of blood system diseases. Amounts of researches show that MGUS is present in more than 3% to 4% of population older than age 50 years [24] and is regarded as a premalignant precursor of MM and often categorized as low-tumor-burden diseases as well [25]  Lumbar puncture and all specimens (CSF and serum) of these 12 patients were obtained for routine determination of IgG and albumin quantitation both in CSF and serum, evidence of intrathecal immunoglobulin synthesis (IgG-Syn) was based on the calculation according to the method of Reiber-Felgenhauer [18]. IgG-Syn demonstrates an immunologic reaction in the CNS, the upper limit of reference>9 is thought to have immune response [19]. CSF/serum albumin quotients (QALB=CSF albumin/serum albumin) is to assess the integrity of blood-CSF barrier. The upper reference limit of QALB×103>8 for the patients over 40 years predicts blood-CSF barrier disfunction [20]. In addition to laboratory parameters, we also analyzed the 12 patients in great detail with clinical characteristics including first clinical manifestations and suspected or confirmed diagnosis.

Oligoclonal band determination
IgG and albumin in CSF and serum were quantified respectively by kinetic nephelometry (Beckman Coulter IMMAGE800). Paired serum/CSF samples were used to analyze the IgG-specific OCB performed with IgG-isoelectric focusing on agarose gel followed by immunofixation (HYDRASYS FOCUSING, Sebia, France) [21]. Before IEF, serum samples were diluted in deionized water to reach the same IgG concentration as that of parallel CSF samples. Serum samples are run in parallel to CSF samples. The patterns were interpreted qualitatively by comparing the presence or absence of OCB in CSF and serum. The present of two or more bands in the electrophoresis lanes are considered to be positive reaction [12].

Consent for publication
All the authors have read and approved the paper for publication.
Peichang Wang made the critical revision of the manuscript. All authors have given final approval of the version to the published.