Cerebrospinal Fluid Proteins and their study

Transcription

1 PRESENTS

2 Cerebrospinal Fluid Proteins and their study

3 Functions of the CSF Volume = 140 ± 30 ml (adult) Protein concentration: 0.30 ± 0.15 g/l Protection of the brain mechanical immunological physico-chemical environment maintenance Elimination of the brain metabolism products

4 Origins of CSF proteins Filtrated from serum filtration through the blood/brain barrier selectivity Proteins specific of CSF or more concentrated in CSF than in serum transthyretin (prealbumin), desialylated transferrin (tau fraction), cystatin From lymphoplasmocytic origin immunoglobulins

5 CSF proteins Serum proteins transthyretin (prealbumin) desialated transferrin (tau fraction) cystatin albumin alpha 1 alpha 2 beta gamma globulins orosomucoid α1-antitrypsin α1-antichymotrypsin α lipoprotein Gc globulin, α2 macroglobulin, haptoglobin, ceruloplasmin β lipoprotein transferrin, hemopexin C3 HYDRAGEL 15 HR

6 Mechanisms of pathological CSF protein variation Transudation - Increase of blood-csf barrier permeability Relative decrease of proteins specifically found in CSF (transthyretin, tau fraction) by dilution with serum proteins Local synthesis relative increase of proteins specifically found in CSF (transthyretin, tau fraction, cystatin) local synthesis of immunoglobulins Combination of both

7 Blood-brain barrier damage Transudative pattern in non inflammatory neurological diseases Associated pathologies : Compressions Inflammatory syndromes Central nervous system tumors Ischemic strokes

8 Local immune reactions Specific CSF oligoclonal pattern Associated pathologies : Frequences Multiple sclerosis >95 % Chronic meningitis >95 % Subacute sclerosing panencephalitis 100 % Untreated neuro - syphilis >95 % HIV infection 80 % Lyme neuro - borreliosis 100 % Cerebral cysticercosis 100 % Neuro systemic lupus erythematosus 50 % Neuro - sarcoidosis 40 % Neuro - Behcet 50 % Paraneoplastic syndroms 60 % CNS tumors: germinoma < 5%

9 Criteria for an effective method to study CSF protein discriminate between transudation and local synthesis detect qualitative variations of protein composition work on small puncture volume

10 Total protein quantification in CSF Methods Turbidimetric methods Colorimetric methods (Lowry, Coomassie blue/sds, pyrogallol red/sds) Quantifying proteins does not discriminate between origins of CSF proteins Quantifying proteins allows only detection of quantitative abnormalities. Qualitative abnormalities are not in evidence Reference values : 0.3 ± 0.15 g/l. (age dependant)

11 Specific protein quantifications Immuno-nephelemetric or immuno-turbidimetric methods Quantified proteins: albumin IgG (local synthesis marker): CSF IgG = mg/l albumin (transudation marker): CSF albumin = mg/l Protein must be quantified in serum and CSF in serum and CSF Calculations from these values : albumin ratio, IgG indexes,

12 Albumin ratio Normal value < 0.65 Alb (CSF) albumin ratio = x 100 Alb (serum) Albumin ratio is increased in case of damage of the blood-brain brain barrier Albumin is a transudative marker as its synthesis is the liver origin. The ratio is age dependant

13 Q Albumin according the age Age Q albumine New born 0,8 2,80 1 month 0,5 1,50 2 months 0,3 1 3 months 0,2 0,5 4-6 months 0,05 0,35 6 months 15 years < 0, years < 0, years < 0,8O > 60 years 0,80 0,90

14 IgG index (Tibbling( and Link) IgG index = IgG (CSF) / IgG (serum) Alb (CSF) / Alb (serum) Normal value < 0.65 IgG index increase in case of local synthesis of IgG Some pathological changes in local synthesis of IgG (oligoclonal, ) do not modify the IgG index

15 IgG CSF IgG ser 0,1 0,5 0,2 0, Reiber s diagram Normal Transudation Transudation and intrathecal synthesis of IgG 0,005 0, ,002 0,005 0,01 0,2 0,5 0,1 4 Alb CSF Alb ser Intrathecal synthesis of IgG

16 Tourtellotte Formula ( Formula (in mg/24 h) h (IgG (LCR) -IgG (sérum ) ) - (Alb (LCR) -Alb (sérum) ) (IgG (sérum ) ) Alb (sérum) (0,43).5 The local synthesis is showed when this value is > 3.3 mg/day

17 Other immunoglobulins normal values IgA CSF IgM CSF 1 5 mg/l 0,1 1 mg/l IgA index 0,10 0,45 IgM index 0,01 0,2 The IgA or IgM local synthesis shows a local immune response after an infection with a micro-organism organism or after a reaction with an auto antigen

18 Some pathological changes in local synthesis of IgG (oligoclonal, )) do not modify IgG indexes. The abnormality is qualitative and it is detected only by electrophoresis

19 Standard electrophoretic methods Procedure steps preparation of samples application and electrophoresis running fixation and staining of proteins Advantages industry-scale preparation of gels (standardization) easy handling Drawback CSF must be concentrated The staining process does not differentiate Igs from proteins migrate in the gamma zone

20 Protein concentration required according to the procedure electrophoresis on cellulose acetate : red Ponceau staining : g/l (normal CSF must be concentrated 100 times, it requires high volume of CSF) electrophoresis on agarose gel : amido black staining : g/l (normal CSF must be concentrated 50 times) acid violet staining : 6-10 g/l (normal CSF must be concentrated 15 to 30 times) silver staining : no concentration, but poorly reproducible with agarose due to high background

21 Three RULES CSF and serum must be collected the same day CSF and serum must be run same gel run side by side side on the Protein concentrations of the applied samples must be the same for the couple CSF / serum : adjust protein concentrations

22 High-resolution agarose gel electrophoresis (HYDRAGEL( HR) Principle Better resolution lengthened migration multifractionation Higher sensitivity acid violet (sensitivity threshold : 100 to 250 mg/l per band)

23 High-resolution agarose gel electrophoresis (HYDRAGEL( HR) Six CSF-serum couples were analyzed on HYDRAGEL 15 HR with HYDRASYS

24 Interpretation The local synthesis of Igs is suspected if: - Bands are observed in CSF without any corresponding fractions in serum - Extra bands are found in CSF compare to the bands observed in serum This local Ig synthesis must be confirmed with an immunological procedure (Proteins other than Igs can migrate in gamma zone)

25 Immunofixation: HYDRAGEL IF IgG local synthesis

26 Drawback of CSF testing by HR and IF gels: CSF must be concentrated On HR gel: between 6-10 g/l of total proteins On IF gel: between g/l of Ig

27 Agarose gel electrophoresis and immunofixation (HYDRAGEL( 3/6 CSF) Principle CSF protein are run on high resolution agarose gel, without prior concentration (serum must be diluted at the same concentration as CSF : between 5 and 10 mg/l IgG) Igs are fixed by immunoreaction with specific antibodies, labelled with peroxidase (G,A,M,kappa and lambda) Peroxidase activity is revealed : high-sensitive method (a protein down to µg/l can be detected)

28 Hydragel 6 CSF Six CSF-serum couples were analyzed on HYDRAGEL 6 CSF on HYDRASYS, and revealed with a labelled-anti-igg

29 Hydragel 3/6 CSF : result interpretation CSF and serum must be analyzed at the same time Only differences between CSF and serum pattern can be interpreted HYDRAGEL 6 CSF is part of the diagnosis of neuropathies ; in any way diagnosis can be made with the sole electrophoresis

30 Clinical cases Diagnosis and Link Index 1 : Amyotrophic lateral sclerosis (0.52) 2 : Dementia (0.37) 3 : Paraneoplasic Stiff-Man syndrome (0.57) 4 : Multiple Sclerosis (1.10) 5 : Multiple Sclerosis (2.08) 6 : Viral Meningoencephalitis (0.84)

31 Clinical cases Diagnosis and Link Index 1 : Myelitis (0.91) 2 : Multiple Sclerosis (2.69) 3 : Paraneoplasic Encephalitis (0.70) 4 : Multiple Sclerosis (0.63) 5 : Multiple Sclerosis (3.60) 6 : Multiple Sclerosis (0.74)

32 Clinical cases Hydragel 6 CSF 1 : Multiple sclerosis 2 : Sciatic 3 : Optic neuritis 4 : Optic neuritis 5 : Unorganic pathology 6 : Multiple sclerosis Hydragel 15 HR Link Index 3,09 0,52 0,56 0,53 0,46 0,83

33 Clinical cases 1 : Syphilis 2 : Multiple sclerosis 3 : Multiple sclerosis 4 : Undetermined pathology 5 : Multiple sclerosis 6 : Peripheral neuropathy Hydragel 6 CSF Hydragel 15 HR Link index 0,64 0,90 1,19 0,44 0,52 0,46

34 Clinical cases 1 : Multiple sclerosis 2 : Multiple sclerosis 3 : Optic neuritis 4 : Multiple sclerosis 5 : Undetermined pathology 6 : Multiple sclerosis Hydragel 6 CSF Hydragel 15 HR Link index 0,63 0,51 0,49 0,65 2,63 1,44

35 Clinical cases 1 : Undetermined pathology 2 : Ophtalmoplegia 3 : Peripheral neuropathy 4 : Dementia 5 : Ischemic vascular injury 6 : Multiple sclerosis Hydragel 6 CSF Hydragel 15 HR Link index 0,45 0,42 0,35 0,37 0,42 1,33

36 Clinical Case IgG IgA IgM Bacterial meningoencephalitis (Staphylococus aureus) Patient with CSF-protein = 8.5 g/l ; CSF-IgG = 1.15 g/l

37 Sensitivity Samples from 22 patients with multiple sclerosis were analyzed : 14 (63,6 %) have an increased Link index 20 (90,9 %) were detected with Hydragel HR 21 (95,5 %) were detected with Hydragel 6 CSF Hydragel 6 CSF Hydragel 15 HR Link index 1,10 2,08 2,69 0,63 0,47 0,51

38 Conclusion The diagnostic sensitivity is better by electrophoresis than by the quantitative measurements and calculations of the different ratios The Hydragel CSF procedure is itself better than the standard electrophoresis with a total protein staining. It present a very good practicability and follows all the criteria given by the European Concensus except that is not an isoelectrofocusing technique

39 1994: Criteria from the European Consensus - CSF must be analyzed without concentration - CSF and serum must be analyzed in parallel at the same IgG concentration - Immunological characterization of oligoclonal IgG must be done - Proteins must be separated by Isoelectrofocusing

40 Isoelectric focusing (IEF) principle IEF is a high resolution technique for separating proteins on the basis of their isoelectric points Migration occurs in a linear ph gradient generated by ampholytes. When proteins are applied, they migrate toward the electrode with the opposite charge. When a given protein reaches the ph zone equal to its own isoelectric point, the resulting charge is 0 and protein precipitates into the gel

41 Zone electrophoresis and isoelectrofocusing comparison Zone electrophoresis ph gradient 3-10 Isoelectrofocusing procedure is a more resolutive technique

42 IgG immunodetection after immunoblotting of CSF and serum proteins Isoelectrofocusing on agarose gel Immunoblotting on PVDF membrane immunodetection with labelled IgG antibodies «Home made» procedure

43 HYDRAGEL 3/9 CSF Isofocusing Sebia It is the consensus reference method but without transfer on nitrocellulose which is still a manual procedure with a lot of long incubations and a lot of washing steps (which can lead to some errors and artifacts). It is a difficult technique to perform in a routine laboratory and it is technician dependant Sebia succeeded to set up a much easier procedure using a direct immunodetection on the gel similar to the Hydragel CSF technique. The analysis is performed on unconcentrated CSF because of the high sensitivity obtained with the anti IgG labeled with peroxydase (CSF and serum are analyzed at 20 mg/l, usually 20 μl of CSF are sufficient).

44 Hydragel CSF Isofocusing

45 HYDRAGEL 3/9 CSF Isofocusing procedure Dry chamber: 20 min Application: 5 min Migration: 45 min Antiserum incubation: 10 min Blotting: 3 min Washing: 5 min Blotting: 3 min Washing: 5 min Substrate incubation: 15 min Blotting: 3 min Drying: 3 min Washing: 10 min Drying: 8 min Total time: 2h 15 mn

46 Interpretation of 3/9 CSF Isofocusing The intrathecal synthesis, within the central nervous system (CNS), is indicated by the presence of IgG bands in the immunofixation pattern of CSF that are not in the serum pattern from the same patient. In theory, a single IgG band in CSF that is absent in serum is doubtful of intrathecal synthesis but in practice, two or more bands are taken as dependable indication for the supportive evidence of local synthesis. It should be noted that the number of bands in the oligoclonal patterns does not correlate with the diagnostic of the disease nor with its severity and prognosis.

47 Interpretation 3/9 CSF isoelectrofocusing Usually 5 types are describe Normal IgG local synthesis IgG local synthesis No local synthesis No local synthesis Monoclonal band splitted in several bands by IEF

48 Hydragel CSF Hydragel CSF Isofocusing

49 Hydragel CSF Hydragel CSF Isofocusing

50 Hydragel CSF Hydragel CSF Isofocusing

51 Hydragel CSF Hydragel CSF Isofocusing

52 Hydragel CSF Hydragel CSF Isofocusing

53 Hydragel CSF Hydragel CSF Isofocusing

54 Hydragel CSF Hydragel CSF Isofocusing

55 IEF advantages No special required equipment. IEF is performed with regular Hydrasys modified with a high voltage power supply unit (very easy to adapt) or with an Hydrasys Focusing All the other steps are similar to the 3/6 CSF procedure : immunodetection, washing, blotting and detection steps

56 Conclusion The Sebia IEF procedure follows all the recommendations given by the concensus report of European countries This technique is very easy to handle, with a total manipulation time of only 2 h 15 min, very fast compared to the reference manual procedure. The sensitivity and reproducibility are very good, similar to the Hydragel CSF ones and concensus reference method. The Sebia Hydragel CSF procedure allows to study the IgA and IgM local synthesis which are not studied with the isoelectrofocusing technique.

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