Analysis in Neuroimmunology Laboratory

1. Analysis for IgG oligoclonal bands

The validation of oligoclonal bands in patients may be used for diagnosis of MS. By separation and identification of IgG bands in cerebrospinal fluid (CSF) and plasma by use of isoelectric focusing in agarose gel, an analysis indicates if oligoclonal antibodies are present in the CSF of the central nervous system of patients.
Samples are analysed for Rigshospitalet once a week, and results are given as positive or negative for oligoclonal bands. Positive results demonstrate inflammation in the CNS with production of IgG. However, this is not specific for MS, since some other diseases also may demonstrate oligoclonal bands. In MS more than 95% of the patients generate oligoclonal bands.
The international definition of “positive for oligoclonal bands” is that at least two bands are seen in the CSF sample not present in the corresponding plasma sample. If only a single band or no bands are seen in CSF as compared to plasma then the result is negative. In the case of a single band only in the CSF and not in the plasma, we suggest sending a new sample after 3-12 months, since there may be a development to positivity over time.
It is important that the CSF and plasma samples from patients are taken within as limited time period as possible, and we accept maximum one week between the sampling.
We strive in general for the delivery of samples to the laboratory the same day. From the departments 2083, 2084, 2094, and 8501, the use of “haste-piccoline” will be used daily in the hours 8.00 until 14.30. Call tel. no. 5-1599 at Rigshospitalet to require a “haste-piccoline” for these samples. The purpose of this is to get the samples frozen within 2 hrs of tapping, which is of importance for the quality of the samples, especially for research projects. Storage of samples until delivery should be at 4°C.
The results are sent out by internal mail as soon as possible after analysis. Results are furthermore included in the MS-database at the MS Clinic. It may occur that we need to rerun the samples due to required dilution of samples or uncertainty in decision, and this can postpone the sending of results up to two weeks.

- Requisition (pdf)
On the requisition is the following information required:
- Time of sampling (spinal fluid and plasma),
- Glass-number on both sample tubes and requisition,
- patient ID-number (cpr nb),
- Name of patient, and Requiring department (section number at RH).

As tubes are polypropylene (PP) plastic used for CSF samples (Sarstedt 60.540.012, Sarstedt 62.9924.284, or Bie&Berntsen 15PPB (Barloworld Scientific)) (at least 2 mL). For plasma two 4 mL Li-Heparin-tubes (KLM130-green) are used. Remember to gently mix the heparin-tubes so that the heparin in the tube and blood are mixed properly to prevent coagulation (10 times up-down). Haemolyzed plasma is not accepted!
The paired samples are sent to Neuroimmunology Laboratory, section 6311, by use of internal “piccoline service” (call 5-1599 at RH if no agreement is made).

Price per analysis: 700 d.kr.

Responsible technician: Marie Koefoed

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2. Analysis for s-acetylcholine receptor antibodies

Autoantibodies against the acetylcholine receptor (AChR) in the neuromuscular endplate generate blockage of the neurotransmitter acetylcholine binding to the receptors on the muscular surface. This may lead to the autoimmune disease Myasthenia Gravis (MG). Demonstration of the autoantibodies has a great importance to make the diagnosis of MG, and for the follow-up of the severity of the disease.
To help for diagnostics of myasthenia gravis (MG), we perform an analysis of these autoantibodies in a serum sample from patients.
By use of a 125I-radioreceptor-assay (ACHRAB®-Assay) kit, once a week we perform the analysis in Neuroimmunology Laboratory. The samples are currently coming for analysis to the laboratory from all over the country, included the Faeroese and Greenland, and further from Iceland and Southern Sweden.
The results are sent out after analyses as a concentration (nmol/L) of antibodies to sAChR included a figure of the development of results over time for each patient. If the sample is first-time positive, it is reanalysed the following week in a series of dilutions for making a precise result. In certain cases the result may not be available before three weeks after receiving the sample due to required extra analysis.
The results should be interpreted as followed:

Negative result 0 – 0,2 nmol/l
Unclear result 0,3 – 0,4 nmol/l
Positive result >0,5 nmol/l

In case of an unclear result, it may be suggested to analyse a new sample after 2-3 months.
It should be noted that a change in the method of analysis was made in our laboratory at March 1, 2004, and results should be compared we cautiousness before and after this day in each patient. On the graphical result a vertical line may mark this day

- Requisition (pdf)
Information of necessity for the analysis: date of sampling, Sample ID-number (on both tubes and requisition), name and ID (cpr-number) of patient, the addresses whereto the result of the analysis and invoice is sent, and finally, in Denmark, the EAN-number is important.
The blood sampling is made in a tube for serum, and at least 4 mL blood is required. After leaving the blood for coagulation at room temperature for at least 30 min., the sample is centrifugated and serum transferred to another tube. Haemolyzed serum is not accepted! At least 2 mL is required for the analysis. The sample tube together with the requisition is sent to us by ordinary postal service and at room temperature, if sent from day to day. However, do not send samples the day before weekends and holidays. In these cases the best is to store the serum sample at 4°C until after the weekend or holidays and then send it. Otherwise they may be sent on ice.

Price per analysis: 850 d.kr.

Responsible technician: Michael K. Jensen

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3. Quantification of in vivo MxA mRNA induction in mononuclear blood cells

Treatment of Relapsing-Remitting Multiple Sclerosis (RR MS) with interferon-ß (IFN-ß) reduces the average attack frequency by 30-40% and reduces the progression of disease. However a subgroup of the MS-patients respond badly on the interferon treatment, which may be explained by circulating neutralizing antibodies. IFN-ß binds to its specific receptor IFNAR on leucocytes and thereby a specific upregulation of MxA mRNA occur in the cells.
As a biological response to treatment of MS-patients with IFN-ß, the MxA mRNA-expression is measured. In a limited time period (4-12 hrs after injection of IFN-ß a blood-sample is taken in two PAXgene Blood RNA Tubes (obtained from Neuroimmunology Laboratory), and from this RNA is isolated, and then converted to cDNA. The integrity of RNA is checked by electrophoresis. The amount of cDNA for each patient is measured by RT-PCR to evaluate if there has been a blood cellular response to the treatment. The MxA protein is a “second messenger”, which is induced by type I interferons only, it functions as a marker for a cellular interferon response.

Blood to be taken in the PAXgene tubes are done following the use of a glass for waste at first. The tubes with blood is turned up and down 10 times immediately and is left at room temperature for 2 to 12 hrs before freezing and sending. Please send on dry ice if the sample is not received in our laboratory within 12 hrs. If the samples are not sent the same day, they are stored at minus 20°C in a freezer until sending, after they have incubated for 2 hrs. Please, do NOT send on Fridays or before holidays.
The samples are sent to “Neuroimmunologisk Laboratorium, afsnit 6311, Rigshospitalet, Blegdamsvej 9, 2100 København”. Please mark the envelope with “MxA”.
The result of the analysis is in general send within 3 weeks after receiving the sample.

To cover our expenses for the analysis: 1.500 d.kr per sample.

- Requisition for Rigshospitalet (pdf)
- Requisition for other hospitals than Rigshospitalet (pdf)

Responsible technician: Joy Mendel-Hartvig

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4. Analysis for P-Interferon beta antibodies (RH6311)

Interferon beta is a naturally occurring protein in the human circulation. However, it has been demonstrated to have beneficially effects on the treatment of a group of MS-patients. It is given to the patients for treatment, but the generation of antibodies to interferon beta may influence its effects. Therefore it is important to analyze the presence of interferon beta binding antibodies in the blood of MS-patients.
The samples are tested for the presence of interferon beta binding antibodies by an ELISA-assay.
The glass tube to be used for the blood sample is a KLM-104 (red 6 mL dry glass).
At least 3 mL serum is needed for the analysis. Time and day for blood taking is noted together with the patient data. The tubes are marked with identification number as referred to on the requisition.
The tube with the blood sample coagulates for at least 30 minutes at room temperature and is centrifugated within 1 hour after sampling. The centrifugation procedure is normally at 3000xg for 10 minutes at room temperature. Haemolyzed serum is not accepted!
Please send on dry ice if the sample is not received in our laboratory within 12 hrs. If the samples are not sent the same day, they are stored at minus 20°C in a freezer until sending. Please, do NOT send on Fridays or before holidays.
The samples are sent to “Neuroimmunologisk Laboratorium, section 6311, Rigshospitalet, Blegdamsvej 9, 2100 Copenhagen”. Please mark the envelope with “Bab”.

The results are given as negative and positive.

To cover our expenses for the analysis: 300 d.kr per sample.

- Requisition for Rigshospitalet (pdf)

Responsible technician: Marie Koefoed

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5. Analysis of the presence of Tysabri antibodies

Tysabri (Natalizumab) is a humanized, recombinant monoclonal antibody that binds to the alpha4-subunits of alpha4beta1 and alpha4beta7 integrin on mononuclear blood cells, and block their binding to the adhesion molecule VCAM-1 among others on endothelial cells. The important effect of this in MS-patients is that mononuclear cells, which may cause inflammation, in less degree enter the central nervous system.
Antibodies against the Tysabri in the the blood may generate blockage of its binding to the integrin. Demonstration of the antibodies has a great importance for the validation of the effects of Tysabri on MS-patients.
To help for the validation of the beneficial effects of Tysabri on the MS-patient, we perform an analysis of a serum sample from patients by an ELISA developed by Biogen Idec Inc.
The results are given for the presence of antibodies to Tysabri or not.

The glass tubes to be used for the blood sample are two KLM-104 tubes (red 4 ml glass) or one KLM-101 tube (red 10 ml glass).
The samples are to be taken at least 1 week after treatment of patients and at Tysabri-treatment after 3, 6, and 12 months.
At least 3 mL serum is needed for the analysis. Time and day for blood taking is noted together with the patientdata. The tubes are marked with identification number as referred to on the requisition. Haemolyzed plasma is not accepted!
The tube with the blood sample coagulates for at least 30 minutes at room temperature and is centrifugated within 1 hour after sampling. The centrifugation procedure is normally at 3000xg for 10 minutes at room temperature.

Please send on dry ice if the sample is not expected received in our laboratory within 12 hrs. If the samples are not sent the same day, they are stored at minus 20°C in a freezer until sending. Please, do NOT send on Fridays or before holidays.
The samples are sent to “Neuroimmunologisk Laboratorium, section 6311, Rigshospitalet, Blegdamsvej 9, 2100 København”. Please mark the envelope with “TAb”.
The result of the analysis is send within 3 weeks after receiving the sample.

To cover our expenses for the analysis: 550 d.kr per sample.

Requisition for Rigshospitalet (pdf)
Requisition for other hospitals than Rigshospitalet (pdf)

Responsible technician: Marie Koefoed

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6. Analysis for neutralizing IFN-b antibody (RH6311)

Interferon beta (IFN-b) treatment is beneficial in relapsing-remitting and some secondary progressive MS patients. In some patients treatment results in the development of antibodies to IFN-b that may neutralize the biological effects of treatment. Therefore it is important to analyse for the presence of IFN-b neutralizing antibodies in the blood of MS-patients.

The blood samples are tested by a luciferase reporter gene assay using the human cell line HT-1080 (HL-116 clone, Dr. G. Uzé, France) transfected with the firefly luciferase gene linked to an interferon-stimulated response element. When IFN-b binds to the cell surface receptor, the reporter system is activated, leading to the expression of luciferase protein in the cell. The amount of luciferase is quantified by the chemiluminescence signal generated from a specific substrate. The chemiluminescence measured when diluted serum and a known amount of IFN-b is added to the cells reflects the concentration of neutralizing anti-IFN-b antibodies in the serum sample. The concentration of neutralizing antibody in samples testing positive for neutralizing antibodies is expressed as the tenfold reduction unit (TRU), also known as the Kawade titre.
Blood samples are taken for analysis from the MS-patient at 6, 12, 18 and 24 months after start on IFN-b treatment.
The glass tube to be used for the blood sample is a yellow gel tube (SSTII) with coagulation activator (KLM-105) (for 3,5 ml serum). At least 3 ml serum is needed for the analysis. The tube with the blood sample coagulates for at least 30 minutes at room temperature and is centrifuged within 1 hour after sampling. The centrifugation procedure is normally at 3000xg for 10 minutes at room temperature. Haemolysed serum is not acceptable!
Time and day for blood taking is noted together with the patient data. The tubes are marked with identification number as referred to on the requisition (see below).
Please see details concerning sample sending on the requisition.
 
The samples are sent to “Neuroimmunologisk Laboratorium, section 6311, Rigshospitalet, Blegdamsvej 9, 2100 Copenhagen”. Please mark the envelope with “NAb”.

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