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Intrathecal synthesis of IgM and IgA in neurological diseases: comparison of two formulae with isoelectric focusing

Intrathecal synthesis of IgM and IgA in neurological diseases: comparison of two formulae with isoelectric focusing

Ciinica Chimica Acta, 216 (1993) 39-51 © 1993 Elsevier Science Publishers B.V. All rights reserved. 0009-8981/93/$06.00 ~9 CCA 05520 Intrathecal sy...

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Ciinica Chimica Acta, 216 (1993) 39-51 © 1993 Elsevier Science Publishers B.V. All rights reserved. 0009-8981/93/$06.00

~9

CCA 05520

Intrathecal synthesis of IgM and lgA in neurological diseases: comparison of two formulae with isoelectric focusing R e i n h a r d Kaiser and Carl H. Lticking Neurologische Klinik und Poliklinik der Universiti~t Freiburg (Germany)

(Received 19 August 1992; revision received 25 January 1993; accepted 3 February 1993) Key words: Isoelectric focusing; lgM; lgA; Cerebrospinal fluid; Meningoencephalitis; Neuroborreliosis

Summary The intrathecal IgM and lgA immune response was studied by detecting oligoclonal IgM and IgA bands in CSF and by determining both the lgM/IgA indices as well as the intrathecal production of IgM and IgA by the Reiber formula. A good correlation was found between the demonstration of oligocional IgM (r --- 0.890) and IgA bands (r = 0.927) and the Reiber formula. Compared with the finding of oligoclonal IgM/IgA bands calculation of an intrathecal IgM/IgA synthesis by the Reiber formula was less sensitive (86%). In two out of 22 controls and in 12 of 19 patients with polyradiculitis IgM and IgA indices were elevated while the other evaluation methods were negative. Even though oligoclonal lgM and IgA bands are considered to be a useful diagnostic tool in detecting intrathecal synthesis of IgM and IgA, i.e. in meningoencephalitis or neuroborreliosis for clinical practice, the Reiber formula provides reliable results. Detection sensitivity of assays for CSFIgM and -IgA should be at least 1.0 mg/l.

Introduction Production of immunoglobulin (lg) G within the central nervous system (CNS) is a common finding in inflammatory CNS disorders [l ]. Electrophoretic data indicate that IgG is synthesized within the CNS in at least 95% of patients with multiple Correspondence to: R. Kaiser, Neuroimmunologisches Labor der Neurologischen Klinik, D.79104 Freiburg, Hansastralk 9, Germany.

40

sclerosis (MS) [2,3]. Among patients with infectious diseases of the CNS locally produced antibodies frequently consist not only of IgG but also of IgM end IgA [4-6]. In viral encephalitis in mice durin3 the second week after infection intrathecal synthesis both of IgG and IgA shows a dramatic increase with a decline abotlt 4 to 6 weeks later [7]. Demonstration of an intrathecal synthesis of IgG is well established by different calculation methods and the finding of oligoclonal IgG bands predominantly in the cerebrospinai fluid (CSF) [8-10]. The existence of an intrathecal IgM and IgA response within the CNS is more difficult to establish; concentrations of these immunoglobulins in the CSF are often too low to be detected by methods (nephelometry) commonly used in clinical practice, ln~rathecally syn~*hesizedfractions of IgM and IgA are mostly de~ermined from IgM- and IgA-indiceS which are calculated from the formula [4]: CSF/serum ratio of IgM or IgA:CSF/serum ratio of alburr,in. Upper reference limits of these indices vary greatly, however, between different laboratories ranging from 0.06i to 1.0 for IgM [4,11,12] and from 0.34 to 0.62 for IgA [4,13,14]. The~ indices assume a linear increase of IgM and IgA with progressive impairment of the blood-CSF barrier. Taking into account the large hydrodynamic radius of IgM and, to a lesser extent, of IgA, Reiber and Felgenhauer [15] recently proposed a new formula for the calculation of !ntrathecally synthesized immunoglobulins. With this formula the increase in ratio~ between the concentrations of immunoglobulins and of albumin (indicating the progressive dysfunction of the blood-CSF barrier) is described as a hyperbolic instead of as a linear function [15]. Analogous to IgG synthesis an intrathecal synthesis of lgM and IgA may also be determined by demonstration of oligocional bands of IgM or IgA in CSF [16-18]. Experience wi'~hisoelectric focusing (IEF) of these immunoglobulins however is limited to certai'a laboratories only, so that only few data e~ist about oligoclonal IgM and IgA ba~,lds in CSF. The aim ,of this study was to investigate a correlation between the Reiber formula, the IgM a~.ld lgA indices and the finding of oligoclonal IgM and IgA bands in CSF. For IgG these studies have already been published [19]. As measurement of CSF total protein is still a part of the routine CSF examination, the practical use of measuring this parameter to decide about further determination of lgM and IgA concentrations in the CSF will be described. Materials and Methods Patients A total of 181 patients at the Department of Neurology were included in this study. Reference group. CSF and blood samples were taken from 22 patients ( 9: 10, ¢7: 12; age: median 41 years, range 17-82 years) who were examined because of headeche, spinal disc syndrome or for exclusion of an organic disease. The routine blood ~nalysis, including the erythrocy~e sedimentation rate and the concentrations of albumin, IgG, lgM and IgA in seruT~~were normal, as were the routine CSF stud-

41

ies revealing < 5 x 10 6 leucocytes/I and albumin CSF/serum ratios < 7.5 x 10 -3 (blood-CSF barrier) [20]. Agarose-IEF of CSF and serum was normal. Study group. Seventy-five patients with clinically defined MS [21], 26 patients with acute meningoencephalitis probably due to viral infection, 24 patients with neuroborreliosis or neurosyphilis (proven by elevated agent-specific antibody indices), 19 patients with Guillain-Barr~ syndrome, and !:5 patients with acute myelitis were studied. Routine CSF examinations CSF was obtained by iumbas puncture. After immediate cell counting, the CSF was centrifuged for 5 min at 2~1,~l× g and CSF total protein was determined. Serum and CSF were assayed for If//3/IgM, IgA and albumin with the Beckmann Array Protein System [22]. With tl,~s~pparatus, detection limit for IgM is 6.9 mg/I and for IgA 11.1 mg/l. All tests wey~;/one under the manufacturer's conditions. In the case of IgM and IgA concentra/ix/~s in the CSF below the detection limit, levels in paired CSF and sera were deter/rifled by ELISA [18]. CSF total protein meas:~ment The total protein v, ?~; measured by turbidimetry using trichioroacetic acid [23]. Briefly, 50 ~l of unconco, trated CSF was mixed with 250 pl 0.2 mol/! trichloroacetic acid in a microtiter well. After standing for 10 min the optical density at 440 nm was read by a Titertek Multiscan reader (FiowLab). Appropriate dilutions of Lab-Trol Chemistry Control (Baxter) were used as a standard. Twenty samples were retested in a modified Lowry assay according to the manufacturer's conditions (Biorad Protein Assay) [24]. The correlation between the two assays was r = 0.995. ELISA Microtiter plates (Nunc Immunoplates) were coated with rabbit antiserum against human IgM or IgA antibodies (Dako, Hamburg, Germany) diluted I:l,000 in 0.5 M sodium carbonate buffer (pH 9.6). One hundred microlitres of this solution were added to each well. After incubation overnight at 4°C the antibody solution was removed from the wells and 30 mg/ml milk powder in phosphate-buffered saline (PBS; 50 mmol/I sodium phosphate (pH 7.6), 0.15 mol/I sodium chloride)containing 0.1% (v/v) Tween 20 (PBS-Tween) were added to block free protein binding sites. The plates were then incubated for I h at room temperature. Serum and CSF samples were diluted in PBS-Tween I: 10,000 and 1:100,000 (serum) and 1:10 and !: 100 (CSF). Reference sera (Behringwerke, Marburg, Germany) for each Ig class were adjusted to an IgA or lgM concentration of 200 ~g/l and diluted in two-fold steps to a final IgA or IgM concentration of 6.25 ~,g/i. The plates were washed and samples and reference samples (100 ~i per well) were added. After 2 h plates were washed again and peroxidase-conjugated rabbit anti-human IgA or IgM antibodies (1:2,000 in PBS-Tween, 100 ~l per well; Dako, Hamburg, Germany) were added for 1 h at room temperature. After repeated washing ortho.phenylenediamine (OPD) was added and the absorbance at 492 nm was measured with a Titertek Multiscan reader (FlowLab). The amounts of lgM and IgA in sampies were determined from standard

42 curves obtained from standard serum. Detection sensitivity with this a~say was 10 /~g/I for IgM and IgA.

Determination of oligoclonal lgG. IgM and lg,4 bands Analysis of oligoclonal bands of IgG, IgM and IgA was performed as previously described [18]. Briefly, samples were focused on thin agarose gels (0.5 mm) containing 0.9% IEF-agarose (Pharmacia), 12% (w/v) sorbitol, !% (v/v) Nonidet P40 (NP 40, Sigma) and Pharmalyte (Pharmacia). For IEF of IgG antibodies gels were supplemented with 6.2% Pharmalyte pH 3-10, while focusing of IgM and IgA antibodies was carried out with gels containing 3.1% Pharmalyte pH 3-10 plus 3.1% Pharmalyte pH 4-6.5. Samples (10 ~1) were applied directly to the gel using a silicone application mask having 8 x 8 mm slots. Focused antibodies were blotted onto nitrocellulose strips loaded with goat antibodies to either human IgG, IgM or IgA. The nitrocellulose sheet was laid on the agarose gel carefully to avoid air bubbles, covered with one sheet of cellulose acetate, three layers of Whatman filter CHRI, a glass plate and a weight of approx. 200 g. After 60 min the nitrocellulose was removed and washed for 10 min in PBS-Tweea. Bound antibodies were detected by incubating the sheets with peroxidase-conjugated goat anti-human IgG, lgA or IgM (Dianova, Hamburg, Germany, Nos. 109-035-003, 109-035-011, 109035-043), diluted 1:1,000 in PBS-Tween. After 90 min at room temperature on a rocking platform, blots were rinsed three times with PBS-Tween and then immersed in substrate solution (3 mg/ml 4-chioronaphthoi, 3/~l/ml hydrogen peroxide in PBS) for 10 rain in the case of IgG and 20 min in the case of lgM and IgA. The blots were washed with water and air dried. Detection sensitivity for lgM and lgA bands was 0.5 mg/I (5 ng).

Calculation of indices The following methods were used to calculate an intrathecal synthesis of lgM and IgA: (i)

lg,14index [1 I ] - CSF/serumlsM:CSF/serumalbumin. Values >0.068 were con-

sidered positive. (2) IgA index [14] - - CSF/serum,gA:CSF/seruma,bumin. Values >0.34 were considered positive. (3) Reiberformula [15]: IgXlocal = [Q(lgX) - a/b ~Q2AIb~.b2+ c] . IgXserum where Q(lgX) is ratio CSF/serum concentration of IgM or lgA, QAIb is ratio CSF/serum concentration of albumin, IgXserumis serum concentration of lgM or IgA, IgXcs F is concentration of IgM/lgA in the CSF and lgXIocal is a fraction of intrathecally produced immunoglobulins. a/b: IgM = 0.65 b2: igM = 150 × 10-6 c:

IgM = 7.5 x 10 "3

IgA = 0.72 IgA= 80x 10-6 I g A = 5.1 x 10.3

43 At least 10% (IgXjoc/IgXcSF) of the igM or IgA in the CSF had to be produced intrathecally to assume an autochthonous synthesis of these immunoglobulins. (4) Detection of oligoclonal lgM and IgA bands exclusively in CSF.

Evaluation of results Sensitivity (true positive rate) was determined by: number of positive (IgM/IgAioc) by nephelometry number of positive (IgM/lgAioc) by ELISA

x 100

Demonstration of an intrathecal synthesis of IgM or IgA is only possible if levels of these immunoglobulins are measurable in the CSF. The sensitivity to determine the concentrations of these immunoglobulins in the CSF is dependent on the detection limit of the assay (ELISA/nephelometry) used. The most sensitive assay in our study was ELISA. Yield (percentage of useful results) was determined by: number of positive samples with IgMioc or lgAioc number of detectable IgM/lgA concentrations in CSF

X 100

A higher sensitivity in detecting concentrations of IgM and IgA in the CSF is not necessarily associated with a higher frequency in detecting an intrathecal synthesis of these immunoglobulins. For diagnostic purposes intrathecally produced fractions of IgM and IgA are more important than levels of these immunoglobulins in the CSF. The proportion of samples with an intrathecal synthesis of IgM/lgA in relation to the total number of samples with detectableCSF levels of these immunoglobulins by nephelometry is described as yield. Results

CSF levels of lgM and lgA The range of IgM in the CSF of the reference group of patients was between 0.01 and 0.8 mg/I with a median of 0.29 mg/l. CSF-IgA ranged from 0.2 to 5.9 mg/I with a median of 1.7 mg/i. The median and range of the different subgroups of the study group are shown in Table I. CSF total protein: predictive value on detection of IgM and IgA in CSF The predictive value of CSF total protein for the determination of IgM and IgA in the CSF was investigated for IgM >_. 6.9 mg/I and IgA > l l.l mg/I (Beckmann Array Protein System) and for IgM and IgA >_ 1.0 mg/i (i.e. Behring nephelometer). The lowest CSF total protein concentration for detectable CSF IgM and IgA concentrations with the Beckmann nephelometer was 600 mg/! for IgM and 550 mg/~ for IgA. Only two of l l4 patients (1.75%) with a CSF total protein < 600 mg/I had measurable levels of IgA in the CSF. One patient was suffering from chronic neurosyphilis (550 rag/l), the other from ~.cute encephalitis (580 rag/l). Results are shown in Table II.

44 TABLE !

Protein variables (median and range) in CSF of 181 patients N

" '.SF-lgM (rag/l)

lgM-index

CSF-lgA (mg/l)

igA-index

Control

22

0.29 0.01-0.80

0.034 0.006-0.160

! .7 0.2-5.9

0.2 0.05-0.46

MS

75

0.75 0.0 i -6.0 30.4 l.O-IO0.O 4.5 0.2-22.0 2.4 0.02-13.5 5.9 0.3-26. "~

O.!08 0.003-0.610 i. 180 0.070-4.100 0.254 0.003-0.790 0.1 ! 0.02-0.34 O. 16 O.Ol-O.S6

2.3 O. I - 1 8 . 0 28.2 I.O-100.0 12.7 0.3-41.2 6.2

0.2 0.01-0.56 0.54 0.04-1.6 0.65 0.02-2.3 0.3 0.03-0.7 0.36 0.05-0.16

NBS

24

ME

26

Myelitis

15

GBS

19

O.1-25.5 27.9 2.5-100.0

MS, multiple sclerosis; NBS, neuroborreliosis/neurosyphilis; ME, meningoenccphalitis; GBS, GuillainBarr~ syndrome.

In patients with IgM or lgA levels in the CSF _> 1.0 mg/! the CSF total protein was generally ~ 200 mg/I with the lowest CSF total protein measured being 270 mg/! for IgM and 200 mg/I for IgA. In 77 of 181 individuals (42.5%) IgM concentrations in the CSF were ~ 1.0 mg/l; IgA levels in the CSF > 1.0 mg/! were detected in 150 individuals (82.8%).

Intrathecai synthesis of igM and IgA: dependency of positive results flora detectable immunoglobulin levels in CSF Employment by ELISA provided measurable levels of lgM and IgA in the CSF of all samples. Intrathecal synthesis of these immunoglobulins was assessed from the finding of oligoclonal IgM and lgA bands in the CSF which was demonstrated in 45 samples for lgM and in 25 samples for IgA. By nephelometry (Beckmann Array, cut-off: lgM :~ 6.9 rag/I; IgA: ~ I !. i rag/I) levels of lgM and lgA in the CSF could TABLE !!

Detectable levels of lgM and lgA in CSF in dependence on total CSF protein CSF protein < 600 mg/I

CSF protein ~ 600 mg/I

N= 114 114 0

N=67

N-- 181

IgM < 6.9 mg/l IgM m 6,9 mg/l

33 34

147 34

IgA < II.I mgll IgA :- II.I mgll

I12 2

20 47

132 49

45 Detection

Sample

Total

Serum

IgM

CSF

IEF-Pattern

1141

~

:l/m

pH-gradient m

IEF-Pattern Total

[ Serum

IgA

[

CSF

pH-gradient

4

6

Fig. I. (a) Oligoclonal lgM bands predominantly in CSF indicating autochthonous synthesis of lgM. (b) Oligoclonal IgA bands predominantly in CSF indicating autochthonous synthesis of igA.

be determined only in 34 of 181 and in 49 of 181 samples, respectively. In 23 out of 34 (yield: 67.6%) and in 19 out of 49 samples (yieid: 38.7%) IEF disclosed oligoclonal bands of IgM and IgA in the CSF. Regarding the findings from nephelometry, sensitivity in detecting oligoclonal bands of IgM was 51.1% (23/45) and of IgA 76% (19/25). If concentrations of IgM and IgA in CSF were examined by a more sensitive nephelometer (i.e. Behring nephelometer, cut-off: IgM and

TABLE !!1 Findings from various evaluation methods

IgM Index • ~V >0.068 Control MS NBS ME Myelitis GBS

22 75 24 26 15 19

2 29 23 20 7 12

l~

l~

Synthesis a OCB b Index > 10% + >0.34

Synthesisa OCB b Index > 10% + >0.7

Synthesis a > 10%

OC Bb

0 10 19 9 ! 0

0 3 7 iI ! 0

0 65 20 8 7 2

0 75 24 II ? 0

0 15 19 9 2 0

2 12 18 14 4 12

0 3 8 12 2 0

0 65 20 8 6 !

+

MS' multiple sclerosis; NBS, neuroborreliosis/neurosyphilis; ME, meningoencephalitis; GBS, GuillainBarr~ syndrome; aCalculation by the Reiber formula. bOligoclonal bands of lgM, IgA or lgG.

46 igA ~ 1.0 mg/l) measurable levels of IgM and IgA would have been determined in 77 out of 181 and 150 out of 18 i samples, respectively. The yield in demonstrating an intrathecal synthesis of immunoglobulins fell to 49.3% (38/77) for IgM and to 16% (25/150) for IgA. Sensitivity in detecting an intrathecal immune response however increased to 84.4% (38/45) for IgM and to 100% (25/25) for lgA. Of the seven patients (7/45) with an intrathecal synthesis of oligoclonal IgM and CSF-IgM levels < 1.0 mg/I (0.5-0.9 mg/l) five suffered from MS and two from myelitis. Intrathecal synthesis of oligoclonal IgG was a common feature in these patients.

Relationship between different evaluation methods The intrathecal synthesis of IgM and IgA was determined by the IgM and IgA indices, the Reiber formula, and the demonstration of oligoclonal lgM or IgA bands in the CSF (Fig. !). Results for different subgroups of the study group are shown in Table llI. Based on elevated IgM- and IgA-indices only (IgM: 93/181, IgA: 62/181) a considerably higher proportion of patients were assessed positive for intrathecal synthesis of these immunoglobulins than by calculation from the Reiber formula (IgM: 39/181, IgA: 22/181) or by oligoclonal IgM/lgA bands in CSF (lgM: 45/181, IgA: 25/181). However two controls with a spinal disk syndrome and no other symptoms for CNS inflammation (Table Ill) also had elevated indices. The highest index for controls was 0.160 for IgM and 0.46 for lgA (Table !). In patients with oligoclonal IgM bands in CSF and a positive finding assessed from the Reiber formula the IgM index generally was > 0.230. in a small number of patients (n = 6) with oligoclonal IgM bands in CSF and negative findings from the Reiber formula, the IgM index extended from 0.080 to 0.220. in patients with oligoclonal lgA bands in CSF and an intrathecal synthesis of lgA calculated from the Reiber formula the lgA index in general was > 0.5. In three patients with oligocional IgA bands and negative results from the Reiber formula the lgA index ranged from 0.12 to 0.48. Both for IgM (r = 0.890, P < 0.001) and lgA (r = 0.927, P < 0.001) the best correlation was found between results from the Reiber formula and findings in IEF. Correlations between results from the Reiber formula and the lgM or lgA index (IgM: r = 0.512, IgA: r = 0.666, P < 0.001) and between the lgM or IgA index and the finding of oligoclonal lgM or IgA bands (IgM: r = 0.527, IgA: r = 0.680, P < 0.001) were less intense.

lntrathecal synthesis of lgM and IgA in inflammatory diseases of the CNS The predictive value of an intrathecal synthesis of IgM and/or lgA for a suspected diagnosis was investigated. Intrathecal immune response was established from oligoclonal lgM and igA bands in CSF. Nineteen of 45 patients with a local production of IgM suffered from neuroborreliosis (42.2%), 15 from MS (33.3%), nine from meningoencephalitis (20%), and two from myelitis (4.4%). Intrathecal synthesis of IgA was determined in 25 patients: 12 patients suffered from meningoencephalitis (48%), eight from neuroborreliosis (32%), three from MS (12%), and two from myelitis (8%). The frequency of an intrathecal production of IgM and lgA was compared with that of IgG in different diseases. The criterion of an intrathecal synthesis was

47 TABLE IV lntrathecal synthesis of igM and lgA established from oligoclonal bands (OCB) of lgM or lgA in CSF, from IgM/lgA index values and Reiber's formula (Rei) in different disease categories

lgM-index > 0.068 IgMoce lgMR©i > 10% lgA-index > 0.34 lgAoc B lgARei > 10%

Early MS N = 30

Relapsing/chronic progressive MS N = 45

13 (43%) 8 (26%) 5 (16%)

20 (44%) 8 (I 7%) 6 (13%)

4 (13.3%) 0 0

8 (17.7%) 4 (8.8%) 4 (8.8%)

established from the finding of oligoclonal IgG, IgM or IgA bands in IEF. Individual data of various study groups are listed in Table Ill. Simultaneous synthesis of oligoclonai IgG and IgM was demonstrated in 19 of 24 patients with neuroborreliosis (79°/0), in seven of 26 patients with meningoencephalitis (26.9%), in 15 of 75 patients with MS (20%) and only in two of 15 (13%) with myelitis. Simultaneous intrathecal synthesis of IgG and IgA was detected in eight patients with neuroborreliosis (33.3%), in seven patients with meningoencephalitis (28%), in three individuals with MS (4%) and in no patients with myelitis. Frequency of simultaneous intrathecal synthesis of IgM and IgA was similiar in patients with meningoencephalitis (26.9%) and in neuroborreliosis (25%). lntrathecal synthesis of lgM or IgA (derived from oligoclonal igM or IgA in CSF and positive results according to the Reiber formula) was a notable finding in five patients with meningoencephalitis and no intrathecal synthesis of oligoclonal IgG.

Predictive value of intrathecal lgM synthesis in MS The MS patients were classified as those with early probable or latent MS (n = 30) and relapsing and/or progressive MS (n = 45). Findings from the different methods for the determination of an intrathec~tl synthesis of lgM and igA are listed in Table IV. No significant differences could be established (x2-test) for autochthonous lgM synthesis in early or relapsing MS by any of the methods used. Values for local production of IgA were too low to calculate significant differences by x2-test. Discussion

One aspect of this study was to investigate the diagnostic and possible predictive value of the intrathecal synthesis of lgM and IgA in different inflammatory diseases of the CNS. Our results not only agree with previous reports of Felgenhauer et al. [5,2?] referring to the predominance of an autochthonous IgM response in neuroborreliosis but also confirm experimental studies of viral encephalitis in mice indicating the significance of an autochthonous synthesis of lgA in viral infections of the CNS [7]. In five patients with meningoencephalitis the intrathecal immune re-

48

sponse was restricted to IgA or IgM and not to IgG, which is investigated routinely. Even though simultaneous synthesis of IgG and IgM most frequently was detected in neuroborreliosis (79%), a considerable proportion of patients with meningoencephalitis (26.9%) and MS (20%) revealed the same constellation. Predictive value of these parameters alone is limited. Taking into account further criterions such as the CSF/serum albumin ratio and the leucocyte count in the CSF, laboratory differentiation between MS and infectious diseases of the CNS is greatly facilitated. Unlike Sharief et al. we could not demonstrate a correlation between the presence of oligoclonal IgM bands in CSF and the duration of disease in patients with MS [27]. Due to the detection limit in IEF (lgM: 5 ng) we were not able to demonstrate oligoclonal IgM bands in CSF containing less than 0.5 rag/! of IgM. Amounts of locally produced IgM in patients with MS in general were much lower than in patients with infectious diseases of the CNS (neuroborreliosis, meningoencephalitis). We agree with Giles and Wroe [28] that in patients with MS significance of oligoclonal IgM, has to be determined by further studies especially in cases with CSF-IgM concentrations below 1.0 mg/I. Another purpose of this study was to investigate a relationship between three different methods of demonstrating an intrathecal synthesis of IgM and IgA. In a former report [18] a highly sensitive immunoblotting technique has been presented which has meanwhile been improved to detect oligoclonal IgM and IgA bands even at CSF-IgM/IgA levels of 0.5 l.ig~. The finding of oligoclonal lgG b~nds in IEF has been proposed as a 'gold standard' to demonstrate an intrathecal synthesis of IgG [3,19]. Analogous to lgG studies, oligoclonal bands of lgM and lgA might therefore serve as a reference for the intrathecai synthesis of these immunoglobulins. With this assumption sensitivity of the Reiber fo,"mula in this study was 86% for lgM and 88% for lgA. In a considerable number of patients calculation o~"the indices provided positive results (lgM: n = 52, lgA: n = 38) without confirmation by the Reiber formula or the demonstration of oligoclonal lgM or igA band,~ in CSF. Elevated IgM and lgA indices were found, for example, in two controls as well as in 12 out of 19 patients with polyradiculitis (GBS syndrome). These findings are partially in agreement with former studies of Sharief et al. [24]: of the 105 patier~ts with no oligoclonal bands 38 and 40 patients had elevated index and Reiber formula values, respectively; on the other hand, of the 54 patients of their study with oligoclonal IgM bands, 26 had normal index values, and 20 patients showed no abnormal lgM synthesis by Reiber's formula (sensitivity: 62.9%). While Sharief et al. found a good correlation between values from the Reiber formula and the IgM-index, in our study results from the Reiber formula were better correlated with the finding of oligoclonal IgM or igA bands in CSF. Comparison of both studies however is hampered by two facts. First, two different techniques were used to investigate oligoclonal IgM bands in the CSF [18,25]. Second, using the Reiber formula, in Sharief's study an intrathecai synthesis of IgM was considered positive if at least 0.9 x IO"3 rag/! of igM was produced intrathecally while in our study more than 10% of the igM in the CSF had to be produced intrathecally. Additionally, the constants (a/b, b 2 and c) of the Reiber formula were modified in Sharief's study. The proposal of oligoclonal IgM bands to be superior to quantitative formulae in demonstration of an intrathecal igM response holds

49 good

for laboratories that are experienced with IEF of large molecules. In our opin-

ion of an intrathecal synthesis of IgM and IgA by the Reiber formula provides good results for practical use. Some practical aspects were considered in this study. In most clinical laboratories concentrations of immunoglobulins in CSF and serum are measured by nephelometry even though measurement by ELISA is much more sensitive. btection limit of the current nephelometers range from 1 to 11 mg/l for IgM and IgA depending on the instrument used. As measurement of immunoglobulins is costly, especially if values are below the detection limit, we examined whether the CSF total protein provides information about the probability of detectable CSF IgM and IgA concentrations. In the case of the Beckmann Array Protein System a CSF total protein of 600 mg/l was found to be a good indicator for further determination of CSFIgM and -1gA concentrations, as below this cut-off only 1.75% of patients had detectable levels of these immunoglobulins in CSF. Reference levels of CSF-IgM and -1gA in our study were within the range previously reported by other authors [ 11,13,14]. These concentrations are hardiy detectable by usual nephelometry. The need for a more sensitive detection system in clinical practice however arises from the finding that a considerable proportion of patients with an autochthonous synthesis of IgM and IgA had levels of these immunoglobulins in the CSF between 1.0 and 10 mg/l. The desirable detection sensitivity of at least 1.0 mg/l for IgM and IgA has been provided recently by an improvement of the Behring nephelometer. With this detection sensitivity prescreening for total CSF protein seems unnecessary. Even though we could demonstrate an autochtonous synthesis of IgM in seven patients with CSF-IgM levels below 1.0 mg/l, the clinical relevance of this finding remains to be determined. Routine examination of the CSF already had demonstrated an intrathecal synthesis of IgG in all

these patients. In view of clinical practice determination of an intrathecal synthesis of IgM and IgA provides a useful supplementation to routine examination of CSF. Detection sensitivity for CSF-IgM and -1gA should be about 1.0 mg/l.

Acknowledgements We thank Gisela Fleig for her expert and skillfirl technical assistance. Stephen Batsford is gratefully acknowledged for help in editing the manuscript. This study was supported by grant no OlKI9001/0 from the German Federal Ministry for Research and Technology (Bundesministerium fur Forschung und Technologie). References Frick E, Scheid-Seydei L. Untersuchungen mit J ‘31-markikrtemGammagiobufin zur Frage der Abstammung der LiquoreiweiDkGrper.Klin Wochenschr 1958;36:857-863. Link I-I,Miiiler R. Immunoglobuiins in multiple sciero;is and infections of the nervous system. Arch Neuroi 1971;25:326-344. McLean BN, Luxton RW. Thompson EJ. A study of immunogiobuiin G in the ccrebrospinai fluid of 1,007 ;Datientswith suspected neurological disease using isoelectric focusing and the log IgGindex. Brain 199O;i13:1269-1289.

50 4 5 6

7 8 9

10

Ii

12 13

14 15

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