The study used 70 consecutive patients with definite MS according to the criteria of Poser  and a primary relapsing course. All patients had at least two spinal taps, mostly during disease exacerbations. Three lumbar punctures (LP) were performed in 26 patients, four in 12 patients and five in 7 patients. Patients were characterized clinically by age, sex, disease duration, course of the disease and the expanded disability status scale (EDSS), documented at the time of the first LP. Furthermore, the progression index was calculated as the ratio of the EDSS and the disease duration for each patient. Patients with corticosteroid treatment in the last four weeks or with immunomodulatory or immunosuppressive therapy in the last 3 months prior to the first LP were excluded. Cerebrospinal fluid and serum sample pairs were analyzed for cell count in the CSF, oligoclonal bands in serum and CSF, local IgG synthesis, IgG index and antibody index for the following antigens: measles, rubella and varicella zoster virus. We focused only on these specific antigens because of the frequent detection of these antigens within CSF in the case of MS described in prior studies .
The CSF cell count was determined immediately after LP. For this purpose, 90 μl of CSF were stained with 10 μl dye containing 20% crystal violet solution, 20 % glacial acetic acid and 60% H2O. Cells were enumerated in a Fuchs-Rosenthal counting chamber.
The intrathecal IgG production was quantitated by the IgG index. For this purpose, albumin and IgG were measured in matched serum and CSF pairs by nephelometry according to the manufacturer's instructions using commercially available kits (antiserum against human albumin or IgG, respectively) and the BN 100 nephelometer (both Dade Behring GmbH, Marburg, Germany). The IgG index was calculated according to the following formula: (CSF IgG × serum albumin) / (serum IgG × CSF albumin). An IgG index above 0.7 was indicative of an intrathecal IgG synthesis .
Local IgG synthesis
We quantitated the local IgG synthesis within the CSF with the help of a method described by Reiber and colleagues . Therefore, we used the following formula: Igloc = [QIg - Qlim (Ig)] × Ig(serum) (Q = quotient CSF: serum). Details of the calculation have been described previously .
Oligoclonal bands (OCB)
All samples were analyzed directly after performing the LP without freezing, to minimize post-sampling changes. The presence of oligoclonal bands in the CSF indicated an intrathecal IgG production. For detection of OCB, isoelectric focusing was performed on matched serum and CSF sample pairs. The serum and CSF samples were diluted to the same IgG concentration and run on polyacrylamide gel precoated with ampholytes (pH between 4.5 and 10.0) at increasing voltage according to the manufacturer's instructions (Servalyt Precotes, Serva Electrophoresis GmbH, Heidelberg, Germany). Subsequently, a silver staining with commercially available reagents was done as indicated by the manufacturer (Serva Electrophoresis GmbH). The patterns were interpreted qualitatively by comparing the presence or absence of OCB in the serum and CSF.
Antibody specific index (AI)
During the whole study period, antibody production against measles, rubella and varicella zoster virus was analysed in matched serum and CSF pairs by commercially available, specific enzyme immune assays (Enzygnost by Dade Behring) on an ELISA II processor using the alpha-method (Dade Behring). The AI was calculated according to the following formula: AI = Q spec. / Q lim. with Q spec. = CSF antibodies / serum antibodies and Q lim. being calculated from the CSF / serum albumin ratio according to the Reiber formula . AI larger than 1.4 indicate an intrathecal antibody synthesis against the given antigen.
This study was carried out as retrospective case-control study. The mean values of cell count, IgG index, local IgG synthesis and AI were compared for the whole group at the different time points (LP 1 to 5) by non-parametric tests for dependent samples (Friedman test). The percentage of patients positive for OCB or with a positive AI against measles at the 1st and 2nd LP, respectively, was compared with the Fisher's Exact test.
Different clinical subgroups, i.e. those with a high or low EDSS, were analyzed for their mean measles AI with the help of a median split. The mean measles AI in the subgroups was compared with a non-parametric test for independent samples (Mann Whitney U test).