Demonstration in children of oligoclonal IgG bands in unconcentrated CSF using agarose isoelectric focusing and immunolabeling

Demonstration in children of oligoclonal IgG bands in unconcentrated CSF using agarose isoelectric focusing and immunolabeling

Demonstration in C dren of Oligoclonal IgG Bands in Unconcentrated CSF Using lsoelectric and olabeling Vasilios Kostulas*, Orvar Eeg-Olofssont, Agam...

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Demonstration in C dren of Oligoclonal IgG Bands in Unconcentrated CSF Using lsoelectric and olabeling Vasilios Kostulas*,

Orvar Eeg-Olofssont,

Agamse isoelecttic focusing, followed by protein transfer to cellulose nitrate m e m b r a n e and doubleantibody avidin-biodn peroxidase staining (avidinbiotin agarose isoelecttic focusing), was used to demonstrate oligodonal I g G bands in unconcentrated cest6mspinal fluid (CSF) and serum; 161 consecutive pediatric patients, ages 6 m o n t h s to 16 years with a variety o f mainly neumlogic disorders, were studied. The procedure was standardized for agarose isoelectric focusing (AIF) using 5 01 specimens containing 125 ng o f IgG. Oligoclonal bands were found in the CSF of 12% o f the patients; bands were f o u n d simultaneously in the CSF and serum o f 10% o f the patients, mostly those with nervous system infectious, b u t also those with central nervous system tumors, seizures, or migraine. In about 5 0 % o f positive cases, oligodonal bands constituted the only CSF abnormality, reflecting an a b n o r m a l h u m o r a l i m m u n e response within the CSF-central nervous system c o m p a r t m e n t . Avidinbiotin AIF can be r e c o m m e n d e d as an integrated part o f routine CSF examinations in children. Kostulas V, Eeg-Olofsson O, Olsson T, Link H. Demonstration in children of oligoclonal I g G bands in unconcentrated CSF using agarose isoelectric focusing and immunolabeling. Pediatr Neurol 1986;2:286-9.

Introduction Separation of cerebrospinal fluid (CSF) by electrophoresis or isoelectric focusing for the demonstration o f oligoclonal bands reflecting an i m m u n e response within the central nervous system (CNS)-CSF comp a r t m e n t , is important in the diagnosis o f inf l a m m a t o r y nervous system diseases [1]. The patient's serum must be obtained simultaneously and examined in parallel with the CSF. C o m m o n l y used techniques require 5-10 ml o f concentrated CSF to conduct examinations, a limiting factor in pediatric practice.

From the *Department of Neurology: Karolinska Institute Medical School, Huddinge University Hospital; Huddinge, Sweden and the "I'Departmentof Pediatrics, University Hospital; Linkoping, Sweden.

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Tomas Olsson*, and Hans Link*

This study, however, presents results from examinations of unconcentrated CSF and serum from 161 consecutive pediatric patients using agarose isoelectric focusing (AIF) followed by protein transfer to cellulose nitrate membrane and double-antibody avidin-biotin peroxidase staining, as described previously [2]. Methods One hundred and sixty-one consecutive CSF and corresponding serum specimens received from 63 girls and 98 boys ages 6 months to 16 yearswere included. The diagnoses are listed in Table 1. CSF (1-2 ml) and serum (0.5 ml) specimens were obtained and processed simultaneously. Phase-contrast microscopy was used for cell-counting and cell-differentiarion into polymorphonuclear and mononuclear cells and etythrocyres. After cell counting, the CSF was centrifuged at 200 x g for 10 min. IgG and albumin in unconcentrated CSF and serum were measured by automated immunoprecipitation nephelometry. For each specimen we calculated a CSF IgG index, equal to (CSF/serum IgG): (CSF/serum albumin) [3]. This index takes into account fluctuations in concentrations of serum IgG as well as damage to the blood-brain barrier; an abovenormal value (> 0.7) indicates synthesis oflgG within the CNS. The blood-brain barrier was evaluated by calculation of the CSF/serum albumin ratio [3]. Age-dependent upper reference values described previously were used [4]. For avidin-biotin AIF performed as previously described [2], 5/A of unconcentrated CSF and diluted serum were used. Briefly, 5 V.Iof CSF containing 20-30 mg of IgG/L was diluted wkh O.15 tool/L NaCI; 5 v.l of the patient's serum was diluted to the same IgG concentration; then both were applied side by side to 0.5 x 6.0 mm troughs in agarose gel and subjected to AIF. Five Vaof pooled blood donor serum (25 mg of IgG/L) and 5 V.Iof carboxyhemoglobin solution (40 gin/L) were run as references on each plate. After immunoblotting and staining, plates were inspected for IgG patterns. The occurrence of two or more homogeneous bands in CSF within the pH range 5,5-9.5 in comparison with the protein pattern of pooled blood donor serum was considered to represent oligoclonal IgG. Results Twenty of the 161 children (12%) displayed oligoclonal I g G bands in CSF. Most positive findings were encountered a m o n g the children with encephalitis or aseptic meningitis (Table 1). Thus oligoclonal IgG bands in CSF, but not in the corresponding serum, were demonstrated in seven patients with encephalitis,

Communications should be addressed to: Dr Kostulas: Department of Neurology; Karolinska Institute Medical School, Huddinge University Hospital: S-141 86; Huddinge, Sweden. Received May 2, 1986; accepted May 29, 1986.

Table 1.

Diagnoses and CSF findings in 161 pediatric patients

Diagnoses

No. of Children

OLigodonal IgG Bands in

a: CSF

N u m b e r of Children with IgG Index

CSF / Serum Albumin

> 0.70

ratio > 5

Pleocytosis > 5/VA

b: CSF + serum

a

b

a

b

a

b

15

7

4

3

1(4)

3

0(3)

4

0(5)

Aseptic meningitis

9

4

0

1

0(2)

3

0(7)

3

1(9)

Bacterial meningitis

5

0

2

0

0

0

1(4)

1

1(5)

Demyelinating diseases

4

2

1

0

0

1

0(2)

0

1(2)

CNS tumors

6

2

0

0

0

1

1(2)

1

0(1)

Febrile seizures

7

0

0

0

0

0

0(2)

0

0(1)

Epilepsy

12

1

1

0

0

o

o0)

o

o

Mental retardation, cerebral palsy and seizures

36

1

2

0

1(1)

0

1(2)

0

0

Acute lymphatic leukemia

6

0

3

0

0

0

0

0

0

Spina bifida occulta

3

0

1

0

0

0

0

0

0

Neurofibromatosis

11

0

0

0

0(1)

0

0(1)

0

0

3

0

0

0

0

0

0

0

0

Migrainous disorders

18

3

0

0

0

0

0(3)

0

0

Miscellaneous*

26

0

2

0

0

0

0(4)

o

o

161

20

16

4

2(8)

8

3(31)

9

3(23)

Encephalitis

Stroke

Total

Numbers within parentheses refer to total number of children with abnormal findings in each diagnostic group. *Peripheral neuropathy, myopathy, degenerative encephalopathy, parkinsonism, ischemic lumbago, syncope, vertigo, deafness, diabetes mellitus, anisocoria, skull trauma, hysteria, upper respiratory tract infections, hematologic diseases (excluding leukemia).

four with aseptic meningitis, and nine with a variety of other neurologic diseases. The following diagnoses were encountered in the last group: two patients with demyelinating diseases (a lO-year-old boy with adrenoleukodystrophy, and an l 1-year-old girl with probable multiple sclerosis); two girls with CNS tumors (a 1 5 - y e a r - o l d with dysgerminoma, and an 11-year-old with tuberculoma); a 4-year-old boy with epileptic seizures at night and with bilateral synchronous paroxysmal episodes on EEG; a 5-year-old girl with mental retardation, cerebral paresis, and partial seizures with secondary generalization; three patients with migrainous disorders (a boy, 4 years of age, with benign paroxysmal vertigo and a family history of migraine; a girl, 10 years of age, with migraine and a family history of migraine; a boy, 11 years of age, with headache and migraine).

None of the five children with bacterial meningitis had bands only in CSF; two of them had bands of identical numbers and migration properties in both CSF and serum. Fourteen additional children displayed oligoclonal IgG bands of identical numbers and mobilities in both CSF and serum. The number of bands varied between 2-8. Of these children, 6 had infectious CNS diseases. Results from cell counting and determinations of CSF/serum albumin ratio and CSF IgG index also are presented in Table 1. Of the 20 children with oligoclonal bands only in CSF, 9 had pleocytosis and 4 had elevated CSF IgG index. No fewer than 10 of the children had oligoclonal bands with normal CSF cell count and IgG index values. Among the 16 children with identical numbers of oligoclonal bands in CSF and serum, only 3 had elevated cell counts and 2 had

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Figure 1. Patterns from avidin-biotin amplified agarose isoelectric focusing of diluted serum and unconcentrated CSF. Column 1 corresponds to pooled normal serum; columns 2 and 3 depict results from sera of a patient with tension headache; columns 4 and5 depict results from sera of patients with dysgerminoma; columns 6 and 7 depict results from sera o f patients with aseptic meningitis; columns 8 and 9 depict results from sera of patients with encephalitis.

elevated CSF IgG indexes. Thus, the majority of children had otherwise normal CSF findings. Discussion

Avidin-biotin AIF is a useful method for demonstrating CSF oligoclonal bands in pediatric patients. The method can be adopted easily for use with 5/A of unconcentrated CSF. As has been demonstrated in adult neurologic patients, the sensitivity of avidinbiotin AIF is similar to that of AIF performed on concentrated CSF with Coomassie Blue staining of separated proteins [2]. Furthermore, avidin-biotin AIF has unsurpassed specificity because the visible bands consist only of IgG. This study demonstrated that oligoclonal IgG bands may be present in a substantial number of children with a variety of neurologic diseases. The dominating diagnoses were CNS infections as has been reported previously [5,6]. However, we observed oligoclonal bands in diseases in which inflammatory reactions were not expected, such as CNS tumors, seizures, migraine, and vertigo. Studies for infectious etiologies and superimposed infections should be performed in such patients. Two of the children had demyelinating diseases (i.e., adrenoleukodystrophy, probable multiple sclerosis). Both of these conditions are known to be accompanied by oligoclonal bands in CSF [7,8]. Tuberculous meningitis also is accompanied by oligodonal bands in CSF [9] which have been demonstrated to contain specific antibodies [10]. The epileptic seizures en-

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countered in the two patients with oligoclonal bands may be postencephalitic. Even after ordinary mumps meningitis, oligoclonal bands may persist in CSF for more than two years [11]. Oligoclonal bands have been reported previously in patients with CNS tumors [12,13], but CSF studies rarely are performed in cerebral tumor patients. Our observations of oligoclonal bands in CSF from three patients with migrainous disorders are of special interest. In all 9 patients, the CSF IgG index was normal which indicates that the intra-blood-brain barrier IgG production was low, presenting as oligoclonal bands but no elevation of the IgG index. The present study also demonstrated the necessity of concomittant serum examination. No fewer than 16 of our patients (10%) had oligoclonal IgG bands with identical numbers and migration properties in CSF and serum. Although the occurrence of oligoclonal IgG bands with identical numbers and mobilities in both CSF and serum has been observed previously in bacterial meningitis, selective intra-blood-brain barrier production of oligoclonal IgG in this disease seems to be rare [14]. It is not clear whether the oligoclonal tgG in these cases was synthesized outside the CNS and present in CSF secondary to transudation into the CSF compartment, or synthesized within the CNS, or produced in both these compartments. The first alternative seems most likely because the IgG index increased in only two of these patients; a more pronounced intrathecal IgG synthesis, which is manifested primarily by oligoclonal IgG bands in CSF and secondarily in the serum, should be reflected by high CSF IgG indices. The frequency of oligoclonal bands demonstrable in CSF is dependent on the procedure used for protein separation. Isoelectric focusing has been demonstrated to yield a somewhat higher incidence of positive results in comparison with electrophoresis [15,16]. Fur. thermore, standardization of the amount of IgG applied to the gel is essential, irrespective of the separation procedure used, because application of too small an amount of CSF IgG may make oligoclonal bands undetectable [17]. Separation of CSF proteins after concentration of the fluid in a standardized manner in relation to total protein, as performed by others [5,6], leads to the application of varying amounts of CSF IgG, frequently too small. This practice may explain why oligoclonal bands were only rarely found in neurologic disorders other than infectious CNS diseases. Oligoclonal IgG bands in CSF may be the only demonstrable CSF abnormality. This observation stresses the value of routine examination of CSF by electrophoresis or by isoelectric focusing for detection of oligoclonal IgG bands. Avidin-biotin AIF enabled the detection of CSF oligoclonal bands in only 12 % of 161 children with a

variety of neurologic diseases; simultaneous detection of oligoclonal bands in CSF and serum specimens occurred in an additional 10%. Although occurring most commonly in nervous system infections, this abnormality also was observed in disorders such as seizures, cerebral tumors, and migrainous disorders, implying an inflammatory component. Since avidinbiotin AIF has high sensitivity and specificity, and requires only 5 IA of unconcentrated CSF and of diluted serum, and yields important information about the presence of humoral immune response within the CNS compartment, it can be recommended as an integral part of most routine CSF examinations performed in children. This study was supported by the Swedish Medical Research Council (Grant No. 3381) References [1] Link H. Characteristics of the immune response within the CNS in neurological disorders. Acta Neurol Scand 1978;57(Suppl 67):177-90. [2] Olsson T, Kostulas V, Link H. Improved detection of oligoclonal IgG in cerebrospinal fluid by agarose isoelectric focusing, double-antibody peroxidase and avidin-biotin amplification. Clin Chem 1984;30:1246-9. [3] Tibbling G, Link H, Ohman S. Principles of albumin and IgG analysis in neurological disorders. Part I. Establishment of reference values. Scand J Clin Lab Invest 1977; 37: 385-90. [4] Eeg-Olofsson O, Link H, Wigertz A. Concentrations of CSF proteins as a measure of blood brain barrier function and synthesis of IgG within the CNS in "normal" subjects from the age of 6 months to 30 years. Acta Paediarr Scand 1981 ;70:167-70. [5] Siemes H, Siegert M, Hanefeld F. Occurrence of oligoclonal gammaglobulin in the CSF of children with prolonged and chronic CNS infections. Acta Paediatr Scand 1981;70:91-9.

[6] Gerbaut L, Ponsot G. Semeiology of the gammaglobulinic patterns of cerebrospinal fluid in children. In: Peeters H, ed. Protides of the biological fluids. Oxford: Plenum Press, 1984; 195-8. [7] Bdtton DE, Houff SA, Eiben RM, Madden DL, Sever JL. Studies of viral antibodies, oligoclonal IgG, in situ central nervous system IgG production, and lymphocyte rosetting in sex-linked recessive adrenoleukodystrophy. Neurology 1977;27: 396. [8] Link H. Immunoglobulin G and low molecular weight proteins in human cerebrospinal fluid. Chemical and immunological characterization with special reference to multiple sclerosis. Acta Neurol Scand 1967 ;43(Suppl 28): 1-136. [9] Laterre EC. Les proteines du liquide cephlo-rachidien a l'etat normal et pathologique. Brussels: Edition Arscia, 1964. [10] Kinnman J, Fryden A, Eriksson S, Moiler E, Link H. Tuberculous meningitis: Immune reactions within the central nervous system. Scand J Immunol 1981; 13: 289-96. [11] Fryden A, Link H, Norrby E. Cerebrospinal fluid and serum immunoglobulins and antibody titers in mumps meningitis and aseptic meningitis of other etiology. Infect Immun 1978;21:852-61. [12] Laterre EC, Callewaert A, Heremans JF, Sfaello Z. Electrophoretic morphology of gamma globulins in cerebrospinal fluid of multiple sclerosis and other diseases of the nervous system. Neurology 1979;20:982-90. [13] Link H, Muller R. Immunoglobulins in multiple sclerosis and infections of the nervous system. Arch Neurol 1971 ;25:326-44. [14] Forsherg P, Fryden A, Link H. Immunoglobulin abnormalities in cerebrospinal fluid during bacterial meningitis. J Neuroimmunol. In Press. [15] Laurenzi MA, Link H. Comparison between agarose gel electrophoresis and isoelectric focusing of CSF for demonstration of oligoclonal immunoglohulin bands in neurological disorders. Acta Neurol Scand 1978;58:148-56. [16] Hershey LA, Trotter JL. The use and abuse of the cerebrospinal fluid IgG profile in the adult: A practical evaluation. Ann Neurol 1980;8:426-34. [17] Link H. Comparison of electrophoresis on agar gel and agarose gel in the evaluation of gamma-globulin abnormalities in cerebrospinal fluid and serum in multiple sclerosis. Clin Chim Acta 1973;46:383-9.

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