Cerebrospinal Fluid

Dr. Safari wahyu Jatmiko

 CSF Formation

 CNS covered by 3 membranes

[meninges].
 Outer most membrane dura mater
 Center membrane arachnoid mater
(“spider-web like”)
 Inner membrane pia mater
 CSF between pia and arachnoid
maters
 subarachnoid space.
 CSF produced by ventricular capillary
blood vessels
 choroid plexuses
 Small volume CSF produced by
ependymal cells
 line the spaces of the brain and
meninges
 CSF Volumes and Function

 Total CSF volumes:

Adult 85 - 150 ml
Neonate 10 - 60 ml
Adult Rate of Formation 500 ml/day
Turn over = 20 mL/hour
 Action:
CNS Protection
CNS Waste Management
CNS Lubrication
CNS Nutrition
 CSF Collection Indications

1. evaluasi perdarahan serebrovaskuler

2. deteksi penyakit infeksi
3. diagnosis kelainan imunologi
4. beberapa kelainan yang melibatkan sistem saraf pusat
5. analisa LCS juga dikerjakan pada pasien unexplained seizure, fever of unknown origin,
KONTRAINDIKASI ANALISA LCS

1. kontra indikasi absolut : infeksi kulit pada tempat yang akan dilakukan LP, papiledema et causa
intrakranial mass lession,

2. kontra indikasi relatif : papiledema et causa pseudotumor serebri, bleeding diathesis, dan severe
pulmonary disease
 CSF Collection
 Lumbar puncture (LP)
 Cervical puncture
 Ventricular cannulas [Shunts]

Generally, up to 20 ml can be taken

from an adult, if pressure is normal (50-180 mm Hg);
The sample is placed into sterile tubes,
labeled 1, 2, 3 etc.
Microbiology
Chemistry Hematology

1 2 3

Small volumes – sample to micro 1st (sterility issue)

Transport to Lab STAT – store at temperature to preserve
4 analyte/constituent of interest.

Other
Graphic accessed URL http://content.jeffersonhospital.org/images/es_1650.gif, 2006
 KOMPLIKASI LP

1. herniasi otak
2. sakit kepala
3. diplopia
4. traumatic tap
5. hematom epidural
 Recommended Test Menu
Routine Causative Infectious Agents of Meningitis
 Gross exam
 Cell Counts + Diffs
 Glucose [60-70% plasma]
 Protein [15 - 40 mg/dL]
When Indicated
 Cultures
 Stains [Gram, Acid Fast]
 Cytology
 Electrophoresis
 VDRL
 CK
 Macroscopic (Gross) Examination

 Color & Clarity

 Normal CSF appearance is crystal clear
and colorless

 Pathological processes can cause fluid

to appear cloudy, turbid, bloody,
viscous, or clotted.
 PLEOCYTOSIS – increased CSF cell
numbers
 WBC > 200 cells/L
 RBC > 400 cells/L
Clinical Significance of CSF Gross Appearance
 Traumatic Tap or CNS Hemorrhage

 ~20% of LPs result in bloody

specimens.
 Pink-red CSF usually indicates the
presence of blood.
 It is extremely important to identify
the source of the blood

Graphic accessed URL http://www.thefetus.net/images/article-images/central_nervous_system/subdural_hematoma_files/image17.jpg, 2005.

Clues Useful in Differentiating Traumatic Tap from
CNS Hemorrhage

 Traumatic tap demonstrates

 maximum amount of blood in first
sample tube with progressive decrease
in subsequent sample tubes
 the sample from a CNS hemorrhage
demonstrates blood evenly mixed in all
collection tubes
 After CSF sample centrifugation,
 the supernatant from a traumatic tap is
clear
 The supernatant is “xanthochromic”
from a hemorrhage
 A very bloody tap may demonstrate blood
clots in the CSF sample, while clots are not
usually associated with CNS hemorrhage.

Graphic accessed URL http://www.webmm.ahrq.gov/media/cases/images/case69_fig2.jpg, 2005.

 Xanthochromia
Defined as a pink, orange or yellow color of CSF supernatant.
P = oxyhemoglobin
Y = bilirubin
O = combination

2-3 days post tap

 Traumatic Tap or Not

 Traumatic
 More blood in 1st tube
 Blood concentration
decreases in subsequent
tubes
 Clear supernatant after
centrifugation
 Phagocytized RBCs by
macrophages
 Hematoidin Bodies in
macrophages

Graphics accessed http://www.medvet.umontreal.ca/clinpath/banq-im/cytology/erythophagieE.htm, http://blog.so-net.ne.jp/_images/blog/_735/case-report-by-ERP/8274703.jpg, &

http://www.ksvcp.com/bbs/zboard.php?id=ksvcp_photo&page=6&sn1=&divpage=1&sn=off&ss=on&sc=on&select_arrange=headnum&desc=asc&no=74, 2005.
 Microscopic Examinations

 Cell counts
 Total
 Leukocyte
 RBC
 Differential
 Cytology

http://www.neuropat.dote.hu/jpeg/liquor/kmcarc1.jpg
 Cell Counts
 Reference range leukocytes “normal”
adult CSF
0-5 cells/L
 mononuclears

 RBC count is of limited use, but can be

used to correct CSF leukocyte counts* Each of these small

& CSF protein values of a traumatic tap

squares is 1/25 mm square (50)

CSF.
This space is one

W* = WBCf - WBCb X RBCf

square mm. On he
hemacytometer its
depth is 1/10 of a
mm. For content

RBCb of this space, multiply

by 10 to get content
of one cubic mm

 RBC count is calculated by subtracting

the WBC count from the total cell count.
 Cell Counts
 Manual counts
 Hemocytometer
 # cells X dilution factor/ volume
 Cells/mL

 Automated Count Issues

 Linearity
 Background counts
 Correlation
 Differentials

 Performed on a stained*
smear made from CSF.
 It is recommended that
stained smears be made
even when the total cell
count is within normal
limits.  Count 100 cells in
consecutive oil-power fields.
 Report percentage of each
type of cell present.

*usually Wright’s stain

 How to Prepare CSF Smear
 Advantages of Cytoprep Making the Smear
 Ease of preparation
 Speed of preparation
 Concentrates specimen for good cell recovery
 Preparation becomes retrievable, permanent record
 Excellent preservation of cell morphology
 Disadvantages of Cytoprep - Artifacts
 Cell at interior of cell button appear smaller w/ denser
nucleus
 Increased cell destruction of abnormal/fragile cells
 Distortions of nuclei
 Segmentation
 Fragmentation
 Holes
 Prominent nucleoli
 Distortions of cytoplasm
 Stringiness
 Granule-clustering
 Peripheral vacuolization
 Perinuclear zone of lymphocytes and monocytes

Rabinovich, A and Cornbleet PJ. Body fluid microscopy in US laboratories: data from 2 CAP surveys, with practice Graphic accessed URL http://icg.cpmc.columbia.edu/cattoretti/Protocol/immunohistochemistry/CytospinPreparation.html, 2007.
Prominent Cells in CSF and Clinical Significance
Normal CSF Differential Cell Count
 Cells Observed in CSF B

CSF cytoprep, Wright-Giemsa. 1000x

A – PMNs, Lymphocytes; B – Lymphocytes; C – Monocyte.
 Graphic accessed URL http://www.geocities.com/jcprolla/481a.jpg, 2009.

Cells Observed in CSF

Image accessed URL http://bepast.org/docs/photos/anthrax/anthrax%20in%20CSF.jpg, 2009.

Anthrax in CSF. From Center for Biologic Counterterrorism and Emerging Many reactive lymphocytes and macrophages.
Disease, Med Star Health Group. Washington, D.C.

Large lymphoid cells with dispersed chromatin, prominent nucleoli,
and scant cytoplasm in the CSF; observed in the one case of typical
MCL (Wright-stained cytocentrifuge preparation, high magnification). Accessed URL
http://www.nature.com/modpathol/journal/v15/n10/images/3880655f2.jpg, 2009.
CSF cytology. A. Nests of anaplastic tumor cells are seen with round to oval
eccentric nuclei, coarse chromatin, prominent nucleoli and moderate homogeneous
cytoplasm (Diff-Quik, ×1000). B. Single giant malignant cells with similar morphology are also seen
(Diff-Quik, ×1000). Bilic et al. CytoJournal 2005 2:16 doi:10.1186/1742-6413-2-16
http://archive.biomedcentral.com/content/figures/1742-6413-2-16-5-l.jpg, 2009.
 Features of Malignant Cells
 Multi-layered formations
 LARGE cells
 Irregular nuclear membrane
 Multi-nucleation
 Nuclear hyperchromasia
 Unevenly distributed chromatin
 Irregularly-sized/shaped nuceloli
 Prominent nucleoli
 High N:C ratio
 Bizarre vacuolization/inclusions
 Uneven staining of cytoplasm Large cells with convoluted nuclei and moderate amounts of basophilic
cytoplasm, intermixed with some small lymphocytes (cytospin preparation of
fresh cerebrospinal fluid, stained with Diff-Quik, original magnification 3600).
(Courtesy of Dr Andrew Schriner, department of cytopathology, New York-
Presbyterian Hospital/Weill Cornell Medical Center.) URL accessed
http://theaidsreader.consultantlive.com/display/article/1145619/1362837?verify=0, 2009.
 Chemical Analysis of CSF

 Protein Glucose
 80% plasma derived Need to know plasma value
 LMW
 Transthyretin (prealbumin) Increased
 Albumin
Hyperglycemia
 Transferrin
 IgG – very small amount
Traumatic tap

 20% intrathecal synthesis Decreased

 Reference range Hypoglycemia
 15 – 45 mg/dL
Meningitis
Tumors
 Albumin and IgG
 Albumin not made in CNS
 ALB used to address blood-
brain barrier integrity
 Evaluate CSF/serum ALB
index
 Index < 9 = normal
 9 – 14 minimal impairment
 > 100 = not intact barrier
IgG sourced from inside and
outside CNS
ALB used as reference protein to
ID intrathecal source of Ig
CSF IgG index = ratio
IgGCSF/IgGserum X ALB
serum/ALBCSF
Reference range 0.3 – 0.7
> 0.7 = CNS sourced
< 0.3 = compromised BBB
 Cerebrospinal Fluid

What’s normal:
 Clear, colorless
 Cell counts less than 5 x 106/ml Adult
 Cell counts less than 30 x 106/ml Neonates
 L>M>N Adults
 L>M>N Neonates

L = lymohocytes, M = monocytes, N = Neutrophils

 CEREBROSPINAL FLUID
100 oil, Wright-Giemsa

Lymphocytes
 CEREBROSPINAL FLUID
100 oil, Wright-Giemsa

Segmented neutrophil

Lymphocyte
 CEREBROSPINAL FLUID
100 oil, Wright-Giemsa

Smudge cell
 CEREBROSPINAL FLUID
100 oil, Wright-Giemsa

Segmented neutrophils

Reactive lymphocytes
 CEREBROSPINAL FLUID
100 oil, Wright-Giemsa

Segmented neutrophil

Eosinophil
 CEREBROSPINAL FLUID
100 oil, Wright-Giemsa

Macrophages

? Cells
 CEREBROSPINAL FLUID
100 oil, Wright-Giemsa

MACROPHAGE
 References

 Fundamentals of Urine and Body Fluid Analysis by NA

Brunzel, 2004.
 Body Fluids by C. Kjeldsberg and J. Knight, ASCP Press,
1993.
 CSF Disorders - Family Practice Notebook at URL
http://www.fpnotebook.com/Neuro/CSF/index.htm
 eMedicine “ Meningitis” at
http://www.emedicine.com/EMERG/topic309.htm