Urbanits, MD, Ph.D.,
Department of Neurology,
Kaiser Franz Josef Hospital,
Kundratstrasse, 3 1100 Wien Austria.
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4.1 Aim of CSF Diagnostics:
What are the diagnostic questions of the
neuropathologist/neurologist to be answered by investigation of
- Inflammatory meningitis (bacterial, viral, fungal)
- Neoplastic meningitis
- Autoimmune diseases (Multiple Sclerosis, Guillain
- Degenerative diseases (Alzheimer�s Disease, Creutzfeld-Jacob
- Secondary reaction to abscesses
4.2 CSF General
- Colourless fluid, specific gravity (1.004-1.007) slightly
greater than water.
- Total volume: 80-150 ml
- normal pressure:
- Compared to blood plasma:
- less protein,
- lower pH, lower concentration of glucose, potassium, calcium,
bicarbonate and amino acids
- higher concentration of sodium, chloride, magnesium
Table 1. Comparison of CSF and serum analytes:
||(50-60% of serum) 2.5-4.2 mmol/l
|Beta 2 microglobuline
- Only few cells (12/3 cells or 4cells per �l): lymphocytes,
- CSF is formed by the choroids plexus: 500 ml per day, total
volume is replaced 2-3 times per day
diagnosis of the CSF:
How to interpret the cell picture?
We do CSF cytology on a cytospun specimen stained according May
- Inflammatory meningitis: the first common reaction invasion of
the CSF by a microorganism is granulocytic pleocytosis. It mainly
consists of neurotrophilic granulocytes and some monocytes (some of
them are activated). But in viral infections the cell picture very
quickly switches (within some hours) to a monocytic/lymphocytic
pleocytosis. Very similar changes are observed in Lyme disease with
CSF involvement. TBC and fungal meningitis reveal a mixed-cell
picture composed of granulocytes monocytes and lymphocytes with
activation. Cell count is very high in bacterial infections (>
1000-10.000/3 cells). Viral infections resemble lower cell counts
ranging from 300-1000/3 cells similar to fungal meningitis, whereas
neuroborreliosis very often shows cell counts from
- Neoplastic meningitis: In CSF with carcinomatosis and also
lymphoproliferative involvement, clusters of pleomorphic cells can
sometimes be observed. The ratio between nucleus and cytoplasm is
switched. Cytoplasm can be very dark blue. Some nuclei resemble an
increased number of nucleoli. CSF cells show a reactive change. But
not all cases are easily to diagnose as neoplastic involvement of
the CSF. Carcinoma cells may be rare and not very pleomorphic.
Lymphoproliferative CSF involvement sometimes resembles only a
small rather monomorphic lymphocytic cell population with some
reactive cells added. In such cases morphological diagnosis needs
to be completed by some more accurate diagnostic tool.
- Autoimmune diseases: Morphological changes in autoimmune
diseases may be rather unspecific. A lympho-monocytic pleocytosis
with a maximum of 100/3 cells, activation with activated
lymphocytes and plasma cells is evident, characteristic and
macrophages may vary. Oligoclonal bands are an important part of
CSF diagnoses in such conditions.
- Degenerative Diseases: The cell picture and cell count in these
diseases is not specific. The cell count may be slightly elevated
and activation of the CSF cells may be present. As CSF is
neighboured to the brain some products of cell degradation will be
found -(Tau protein, TNF-alpha, TGF-beta,14-3-3 protein) in
- Secondary reaction to abscesses: Sometimes a CSF neighboured
inflammatory process may cause a reactive granulocytic /monotypic
CSF pleocytosis with macrophages. In such cases microbiological
examinations will fail to demonstrate an underlying microorganism,
although the cell count of CSF may be up to 1000/3 cells.
investigation of CSF
In our lab we use FACS analysis for its quick and accurate
results. For the differentiation of clonal and monoclonal
lymphocytic cell populations the usual approach of immunphenotyping
by means of fluorescence labelled monoclonal antibodies is used.
Thus it is possible to distinguish between inflammatory lymphocytes
and lymphoproliferative neoplastic populations. This approach can
be used also for the follow up to demonstrate therapy success.
In cases of suspected neoplastic CSF involvement aneuploidy
measurement are helpful to evaluate malignant cell populations. For
the discrimination between mesenchymal cells and carcinoma cells
staining and gaiting with a specific antibody towards cytokeratine
that stains for carcinoma cells is helpful.
Both techniques will be presented in detail.
4.5 FISH analysis on
Additionally fluorescence in situ hybridisation on a cytospun
specimen is used for detecting carcinoma cells and lymphoma cells
in CSF. Numerical chromosomal aberration can be detected on single
cell level using e.g. a probe for chromosome 1q12. Studies proved
that the cells in leptomeningeal metastases very often resemble the
same numerical content of chromosomes 1,7 and 10 as the
corresponding malignant cells in the periphery. This method is also
used for follow up examinations during specific therapeutic
FACS ANALYSIS-A NEW AND ACCURATE TOOL IN THE DIAGNOSIS OF
LYMPHOMA IN THE CEREBROSPINAL FLUID.
Urbanits S, Griesmacher A, Hopfinger G, Stockhammer G, Karimi A,
Muller MM, Pittermann E, Grisold W.
BACKGROUND: Fluorescence activated cell scanning (FACS) is a
useful tool for identifying malignant cell clones of lymphoma cells
in cerebrospinal fluid (CSF) by immunological phenotype.
METHODS: We used FACS analysis for demonstrating it to be a
quick and reliable technology that is available in most
haematological laboratories. In this study, we demonstrate the
clinical application of FACS analysis within a series of 15
lymphoma patients with suspected CSF involvement. CSF from three
patients with another diagnosis than lymphoma serves as negative
control. RESULTS AND
CONCLUSION: A malignant cell clone cannot only be identified in
CSF phenotypically, but also classified according to the
immunological surface profile. As this method improves the
diagnostic sensitivity and specificity, it should be implemented
into routine diagnosis.
METHOD: In FACS analysis, cells are analysed via a FACS scanner.
Cells are stained for several surface markers via one run. When
they are analysed and activated with light the fluorochrome
connected antibodies emit light of a given wavelength. These data
are collected and a surface profile of the analysed cells is
evaluated. According to the forward scatter also data on
granularity and cell size are available.
MEASUREMENT OF DNA-ANEUPLOIDY AND VEGF (VASCULAR ENDOTHELIAL
GROWTH FACTOR) IN THE CEREBROSPINAL FLUID OF A PATIENT WITH
MENINGEAL CARCINOMATOSIS UNDER CONTINUOUS CHEMOTHERAPY.
Urbanits S (1;2), Heinschink A (2), Stockhammer G (3), Karimi A
(2), Oberndorfer S (1), Lahrmann H (1), Grisold W (1), M�ller MM
INTRODUCTION: The evaluation of the therapeutic success in a
patient with meningeal carcinomatosis under chemotherapy allows a
more precise and better management of the patient. As the
identification of malignant cells in CSF from a cytospun specimen
has its difficulties, also the immmunocytochemical identification
of carcinoma cells with cytokeratine may be very tricky.
In the following case report we identify malignant cells in CSF
according to their aneuploidy (hyperploidy) examined by FACS.
Additionally vascular endothelial growth factor (VEGF) was measured
by ELISA technique in CSF and serum. The VEGF isoform 165 is known
to be produced by tumor cells.
These data are compared with the results of the morphological
and immmunocytochemical analysis of the cytospin specimen.
SUMMARY: The evaluation of aneuploidy and VEGF in the CSF
correlated well with the results of the morphological analysis from
cytospin specimen and with the cell count. This demonstrated
monitoring model might improve the quality of the diagnosis and the
follow up of patients undergoing chemotherapy because of meningeal
carcinomatosis. As this is only a case report many more patients
have to be evaluated according to this procedure for introducing
such a diagnostic system into the routine.
METHOD: DNA is analyzed for aneuploidy with probidium iodid with
a Becton Dickinson Scan (FACS Calibur). Cell activity is
characterized as follows: G0/G1 Phase, S-Phase and G2-M-Phase,
additionally DNA-indices were evaluated. Tumour cells were
phenotypically characterized using an anti-pan-cytokeratine
The VEGF ELISA assay (Quantikine Kit, R&D Systems,
Minneapolis, MN) detects the isoform 121 and 165.
- Single cell PCR analysis of the immunoglobulin heavy chain CDR3
using standard cerebrospinal fluid cytospins. J Neurol 2001;248: II
- Vascular endothelial growth factor in CSF: a biological marker
for carcinomatous meningitis. Neurology. 2000;54(8):1670-6.
- FACS analysis-a new and accurate tool in the diagnosis of
lymphoma in the cerebrospinal fluid. Clin Chim Acta.
- Neoplastic meningitis: a guide to diagnosis and treatment. Curr
Opin Neurol 2000;13: 641.
- Polymerase chain reaction in the diagnosis and management of
CNS infections Arch Neurol 1999;56: 1215.
- Investigating Chemokines and Chemokine Receptors in Patients
With Multiple Scierosis Opportunities and Challenges. Arch Neurol
- Labordiagnostik neurologischer Erkrankungen. Georg Thieme
Verlag Stuttgart. New York 1999.
- Meningitis. Kohlhammer Vlg 2002.
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Chemistry and Laboratory Medicine (IFCC). All rights