CEREBROSPINAL FLUID  Spinal tap

 Most commonly done; done in between L3 and L4 or L4

Transcribed by: DAPNolasco | November 2022
and L5.
Major fluid circulating the CNS and was first recognized by
COTUGNO in 1764. The volume of CSF removed is dependent on the age (adult vs. neonate) and opening
pressure of the CSF; measured when the needle enters the subarachnoid space
* ⬆ opening pressure = CSF must be withdrawn slowly
Functions:
*collect small amounts only.
 Supply nutrients to the nervous system
 Remove metabolic wastes Specimens are collected in three sterile tubes:
 Maintains intracranial pressure  Tube 1 – chemistry and serology dahil these tests are less
 Produces a mechanical barrier to cushion the brain and spinal affected by blood and bacteria introduced as a result of tap
cord procedure
 Tube 2 – microbiology
Major Constituents of CSF  Tube 3 – hematology/cell count dahil it is the least likely to
 Protein – lower than plasma due to the absence of HMW contain cells introduced by the spinal tap procedure.
molecules like fibrinogen and IgM  Tube 4 – could be for microbiology to exclude skin
 Glucose – (60-70% of plasma glucose) contamination or for additional serologic test.
 Na, Cl, Mg is higher in CSF than in plasma
 K and Ca is lower in CSF than in plasma TESTS ARE PERFORMED ON A STAT BASIS!!! And pag hindi
magagawa, specimens are maintained in the ff manner:
Formation and Physiology  Tube 1 – must be frozen
MENINGES  Tube 2 – must remain at room temp
Lines the brain and the spinal cord and has three layers:  Tube 3 – refrigerated for 4 hours
 Dura mater (hard mother) – outermost layer; lines the skull
and vertebral canal Other notes:
 Arachnoid mater (spider-web like) – middle layer; * if only one tube can be collected, it must be tested first by microbiology
filamentous inner membrane followed by hematology and chemistry/serology
 Subarachnoid space – located between arachnoid and * cells must be counted within 1 hour of collection when specimen is
pia mater; where CSF flows maintained at room temp
 Pia mater (gentle mother) – innermost layer; thin membrane * Supernatant na natira after each section has performed its test also may be
used for additional serological test.
lining the surfaces of brain and spinal cord
* excess fluid should not be discarded and should be frozen until there is
no further use for it.
CHOROID PLEXUSES
 Where CSF is produced
CSF Appearance
 A capillary network found in two lateral/lumbar ventricles
 Normal: Colorless and Crystal Clear 💎
and the third and fourth ventricles that forms CSF from
 Abnormal Variations:
plasma by mechanisms of selective filtration under
 Cloudy/Turbid/Hazy/Milky
hydrostatic pressure and active transport secretion.
-
Increased protein and lipids
THEREFORE, CSF DOES NOT RESEMBLE AN
-
Infection due to increased WBC (>200 Ul)
ULTRAFILTRATE PLASMA!
-
Centrifugation of CSF must be done in capped tubes
 Endothelial cells of choroid plexuses have very-tight fitting
-
Meningitis, production of IgG sa CNS, and
junctures that prevent the passage of many molecules called
disorder affecting blood-brain barrier
blood-brain barrier.
 Oily
o Blood brain barrier protects the brain from harmful
chemicals in the blood and also prevent the passage of - Radiographic contrast media
helpful substances including antibodies and  Xanthrochromia
medications - CSF supernatant that could either be pink, orange,
o Disruption of blood brain barrier by diseases like or yellow
meningitis allows leukocytes, proteins and additional - Most common cause: presence of RBC degradation
chemicals na makapasok sa CSF. products
- Depending on the amount of blood and the length of
*In adults, approximately 20 ml of CSF are produced per hour 😁 time it has been present, the color will vary from:
*Para mamaintain yung volume of 90-150 ml sa adults, and 10-60 ml sa o PINK - slight amount of oxyhgb
mga neonates, yung circulating CSF is reabsorbed back sa blood o ORANGE – increased hemolysis
capillaries sa arachnoid granulations. o YELLOW – oxyhgb is converted to
unconjugated bilirubin
ARACHNOID GRANULATIONS - Other causes: BCaMPR
One way valve, responding to pressure within the CNS and o Elevated serum (unconjugated) bilirubin
prevents reflux of the fluid. o Presence of carotene  increased serum levels
o Presence of melanin  meningeal
Flow of CSF melanosarcoma
Code: Come Let FM Treat Sylvia For Lunch Sa Andoks o Markedly increased serum protein
Choroid Plexus  Lateral Ventricle  Foramen of Monroe  Third (>150mg/dl)  disease affecting the b-b
Ventricle  Foramen of Sylvius  Fourth Ventricle  Foramen of barrier
Lushka  Subarachnoid space  Arachnoid villi o Other pigments: rifampin, etc.
 Bloody
Specimen Collection and Handling - RBCs ~ 6000/Ul
 Ventricular puncture (ventricle of the brain) - Tandaan: RBC begin to lyse within 1 hour and 40% of the
 Ginagawa kapag infants with open fontanels leukocytes disintegrate after two hours.
 Cisternal puncture (nape) - Hemorrhage; traumatic tap
 Dangerous; done in sub-occipital region  Clotted
 Performed if there is: - Caused by clotting factors and protein
 Blockage of spinal cord - Introduced by traumatic tap and disorders
 Vertebrae deformity affecting blood brain barrier
 Infection of the back
  Pellicle Cells are counted in the FOUR CORNER SQUARES and CENTER
- Caused by protein and clotting factors SQUARE on both sides of the hemocytometer for total cell count
- disorders affecting blood brain barrier and and WBC count.
tubercular meningitis
Differential Cell Count
Traumatic Tap vs. Intracranial Hemorrhage  Performed on a stained smear (Wright stain), not in the
Bloody CSF can be an indication of intracranial hemorrhage, but counting chamber
also may be due to the puncture of a blood vessel during the  done primarily to identify type of organism that is causing
spinal tap procedure. Therefore, it is important that we knew how meningitis
to differentiate them hehe  Specimen must be concentrated before preparing the
smear
 Concentration can be performed through:
 Sedimentation
 Filtration
 CENTRIFUGATION; most common; 5-10 minutes;
remove supernatant but do not discard
 CYTOCENTRIFUGATION  MOST
RECOMMENDED METHOD in ALL BODY FLUIDS
CELL COUNT
 NOTE: Save the supernatant for additional tests!

Automated Cell Count

Advantages:
 Increased precision; standardization; faster TAT
CSF count analyzers:
 ADVIA 2120i
 Sysmex XE-5000
 Iris iQ200 with body fluids module
* diseases in which damage to the b-b barrier allows increased filtration of  Beckman Coulter LH780
protein and coagulation factors also cause clot formation but usually do not  UniCel DxH800
produce bloody fluid. These conditions includes meningitis, Froin
syndrome, and blocked CSF circulation thru the subarachnoid space. Cytocentrifugation
* a classic web-like pellicle is associated with tubercular meningitis and
 0.1 mL CSF is added in a conical chamber
can be seen after overnight refrigeration of the CSF
 Cells are forced in a monolayer
 30% ALBUMIN is added to:
Calculation of CSF Cell Counts
 INCREASE CELL YIELD
 DECREASE CELLULAR DISTORTION (cytoplasmic
vacuoles, nuclear clefting, prominent nucleoli,
indistinct nuclear and cytoplasmic borders, and
cellular clumping)
 QC: Check daily for bacterial contamination using 0.2 ml
saline and two drops of 30% albumin

Cell count that is performed routinely on CSF specimens the WBC

count. Normally, RBCs are not present in CSF and is
determined only when a traumatic tap has occurred and a
correction for leukocytes or protein is desired.

If peripheral blood RBC and WBC count is normal, the shortcut

formula is:
 Subtract 1 WBC for every 700 RBC seen; subtract 8 mg/dL
TP for every 10,000 RBC/uL.
 In other books, subtract 1 mg/dL TP for every 1,200 RBC QC of CSF and Other Body Fluid Cell Counts
seen.  On a BIWEEKLY basis:
 All DILUENTS must be checked for contamination
Other notes:  Examination of diluent must be done in a counting
Diluent: 3% acetic acid with methylene blue chamber under 400x magnification
Reference value:  On a MONTHLY basis:
Adult: 0-5 WBC/uL  SPEED OF CYTOCENTRIFUGE must be checked with a
Neonates: 0-30 WBC/uL TACHOMETER and the timing should be checked with a
STOPWATCH
Manual Cell Count  For nondisposable counting chambers
Uses Hemocytometer; before performing CSF cell counts,  Soak in bactericidal solution for at least 15 minutes, rinsed
CHECK THE CLARITY FIRST. with water and clean with isopropyl alcohol after each use
 If CSF sample is clear, no need for dilution
 If not: CSF Cellular Constituents
 Dilute with normal saline for total cell count  CSF Normal Cells: Lymphocytes and Monocytes
 Dilute with 3% glacial acetic acid to lyse the red cells and  NOTE: Occasional neutrophils are also observed in CSF
stain with methylene blue to differentiate neutrophils using improved concentration methods
from monocytes  In adults: Lymphocyte (70%); Monocytes (30%)
 In children: 80% are monocytes
Pleocytosis – presence of increased numbers of normal cells
which is considered abnormal.
 ⬆ CSF WBC count for Neutrophils  bacterial meningitis
 ⬆ CSF WBC count for Lymphocytes and Monocytes
(moderately elevated)  viral, tubercular, fungal, or
parasitic origin
Neutrophils
 In addition to bacterial meningitis, increased neutrophils are
also seen in the early stages (1-2 days) of viral, fungal,
tubercular, and parasitic meningitis.
 May contain cytoplasmic granules after cytocentrifugation.
 Those who are associated sa bacterial meningitis may contain
phagocytized bacteria
 May increase after hemorrhage in CNS, repeated lumbar
punctures and injection of medications or radiographic dyes
 Those with pyknotic nuclei indicate degenerating cells and
may resemble nRBCs but usually have multiple nuclei
 nRBCs are seen as a result of contamination from bone marrow
during spinal tap; nRBCs are aka metarubricytes
 capillary structures and endothelial cells may be seen after
traumatic tap
Lymphocytes and Monocytes
 common in cases of viral, tubercular, and fungal meningitis
and multiple sclerosis (demyelinating disease)
 reactive lymphocytes contains increased dark blue cytoplasm
and clumped chromatin in conjunction with normal cells
during viral infections
 increased lymphocytes is also seen in people with HIV/AIDS
Eosinophils
 seen in parasitic and fungal infections primarily Coccidiodes
immitis, and introduction of foreign material, including
medications and shunts, into the CNS
Macrophage
 appear within 2-4 hours after RBCs enter the CSF and
frequently are seen following repeated taps
 has more cytoplasm than monocytes
 indicates previous hemorrhage
 Erythropages – macrophage with ingested RBC
 Degradation of phagocytized RBC results to:
 Dark-blue or black iron-containing hemosiderin
granules
 Yellow IRON-FREE hematoidin crystal (iron-free,
consisting of hgb and unconjugated bilirubin)
Non-Pathologically Significant Cells
Seen frequently after diagnostic procedures such as
pneumoencephalography and in fluid obtained from ventricular
taps during neurosurgery. They often appear in clusters and can
be distinguished from malignant cells by their uniform
appearance.
 Choroidal cells
- From the epithelial lining of choroid plexus
- seen singularly and in clumps
- nucleoli is absent
- nuclei have uniform appearance
 Ependymal cells
- Lines the ventricles and neural canal
- Less defined cell membranes
- Frequently seen in clusters
- Nucleoli are often PRESENT
 Spindle-shaped cells
- Lines the arachnoid
- Usually seen in clusters
- May be seen with systemic malignancies
Malignant Cells of Hematologic Origin
 Lymphoblasts, Myeloblast and Monoblast  acute
leukemias
 Lymphoma cells  indicates dissemination from the
lymphoid tissue; resembles as large and small lymphocytes
usually appearing in clusters of large, small, or mixed cells
based on the classification of lymphoma. Nucleoli may
appear cleaved, and prominent nucleoli are present.
Malignant Cells of Nonhematologic Origin
 Metastatic carcinoma cells – primarily from lung, breast, renal
and gastrointestinal malignancies
 Cells from primary CNS tumors – astrocytomas,
retinoblastomas, medulloblastomas; appears in clusters and
must be distinguished between the normal clusters of
ependymal, choroid plexus, lymphoma, and leukemia cells
CHEMICAL TESTS
CSF Protein
 Pinaka common na performed na chemical test
 Determines whether increased IgG is due to synthesis within
CSF or due to damage in the blood-brain barrier, serum and
CSF levels of albumin are compared
 CSF/serum albumin index: evaluates the integrity of the
blood-brain barrier
 CSF IgG index: measures IgG synthesis within the CNS
 Reference value: 15-45 mg/dl
 Note: reference value is higher among infants and
people over age 40.
 Increased protein are seen in cases of:
 Multiple sclerosis
 Meningitis
 Intracranial hemorrhage
 Normal CSF protein constituents:
 Albumin – major protein; most abundant
 Prealbumin – aka transthyretin; second most
prevalent fraction
 Alpha globulins include primarily ceruplasmin (a
copper transporting protein) and haptoglobin (a
carrier of hemoglobin)
 Gamma-globulins: majors globulin is IgG with some
IgA
 Beta globulin includes transferrin (a circulating iron)
- Separate carbohydrates-deficient transferrin
fraction, referred to as tau is seen in CSF and not
in serum
 CHON not found in CSF are IgM, beta-lipoprotein and
fibrinogen
Qualitative Test for CSF Total Protein
 Ross Jones
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uses 3% ammonium sulfate
 Nonne - Apelt (+) results: white ring appearance
 Pandy’s Test
 Reagent: Phenol
 (+) Result: Faint blue cloud
* positive results equals increase in CSF protein
Quantitative Test for CSF Total Protein
Turbidimetric method
 Tricholoroacetic Acid (TCA): PREFFERED PRECIPITATING
AGENT because it precipitates both albumin and globulin
 Sulfosalicylic (SSA): PRECIPIATATES ALBUMIN ONLY.
Remedy: Add sodium/ammonium sulfate to precipitate globulin
Ma’am Delcie: Bakit quantitative is turbidimetric method? Yun ay dahil
gingradean yung turbidity na napoproduce; pagtindi ng turbidity, pagdami
rin ng total protein na present.
 CSF Glucose
Dye Binding Method  Glucose enters the CSF via selective transport
 Uses Coomasie Brilliant Blue Dye  Reference value: 60-70% of blood glucose (approx: 65 mg/dl)
 Original color: Red Dye +Protein  blue color  Blood glucose test must be run for comparison
 Pag intense ng color blue, pagincrease ng protein present  Blood glucose should be drawn about 2hrs before spinal tap to
 In spectrophotometer, the blue color is red allow time for equilibration between the blood and fluid.
 INCREASED CSF glucose IS NOT SIGNIFICANT
 Decreased CSF glucose determines cause of meningitis
Type of Markedly
Markedly decreased
meningitis increased
Bacterial CSF Glucose Neutrophils
Tubercular CSF Glucose Lymphocytes
Viral Normal CSF glucose Lymphocytes

CSF Lactate
Valuable aid in dx and managing meningitis cases
 reference value is 10-24mg/dl
 increased CSF lactate is primarily due to oxygen deprivation
o meningitis
o head injuries
o hydrocephalus
o intracranial hemorrhage (RBC contains high amounts of
Immunofixation electrophoresis (IFE) and Isoelectric lactate)
focusing (IEF)  Inversely proportional to CSF glucose
We measures the rate of migration based on electrical charges;  Increased CSF lactate are seen in cases of fungal, tubercular
we uses electrophoretic field and is highest in bacterial meningitis (>35 mg/dl)
 Uses silver stain to visualize the bands  Anong meningitis ang normal csf lactate and normal ang glucose?
 Method of choice when determining whether a fluid is VIRAL MENINGITIS
actually a CSF  Pano natin dinidifferentiate ang type of meningitis?
o Dinidetermine natin ang specific type of wbc na predominant
CSF Electrophoresis o Determine csf glucose
 Primary purpose: detection of oligoclonal bands and pag o Determine csf lactate
may nadetect na ganito ibig sabihin there is an increase
concentration in IgG MENINGITIS
 Must be done simultaneously together with serum Bacterial Meningitis
electrophoresis Causative agent (encapsulated):
 Needed in dx of neurologic disorders associated with  From birth to 1 month  Group B streptococcus
abnormal CSF particularly multiple sclerosis (S.agalactiae, neonatal meningitis)
 Nagkakaroon ng oligoclonal bands kapag may multiple  1 month to 5 years old  H.influenzae
sclerosis  5 years old to 29 years old  N.meningitidis
 NOTE: pag may multiple sclerosis ang isang tao, pwedeng  >29 years old  S.pneumoniae
may oligoclonal bands siya sa CSF pero wala sa serum  Newborns, elderly, immunosuppressed px  Listeria
monocytogenes
Lab Tests
 Increased CSF WBC, neutrophils, increased LD4 and LD5,
increased protein, decreased glucose and increased lactate
>35mg/dl)
 Limulus Lysate Test – POSITIVE
 Test for bacterial endotoxin (for gram negative
bacteria only)
 Reagent: Blood of Horseshoe crab (Limulus
Polyphemus) (blue color because of copper)
Possible complication/interpretation Oligoclonal Bands  Amoebocytes in crab release CHON (lysate) in the
SERUM CSF presence of endotoxin
Leukemia + +  + Result: CLUMPING
Lymphoma
Viral infections (HIV infection)
Multiple sclerosis - + Viral Meningitis
Encephalitis  Causative Agent: Enterovirus (Echovirus, Coxsackie, and
Neurosyphilis Polio virus, Arbovirus)
Guillain-Barre Syndrome  Increased WBC count predominantly lymphocytes
Neoplastic disorders (increased LD2 and LD3)
 Increased protein, normal glucose and lactate
Note: In multiple sclerosis, oligoclonal
banding remains upon remission but
disappears in other condition Fungal Meningitis
 Causative agent: Cryptococcus neoformans (capsulated)
Myelin Basic Protein  Increased WBC count predominantly lymphocytes and
 Myelin – protects our axons (carries information away monocytes
from the neurons) and neurons (demyelination)  Increased protein, decreased glucose, increased lactate
 Presence in the CSF indicates recent destruction in the (>25 mg/dl)
myelin sheath  Lab Test: India Ink (an indirect/negative stain – capsule is
 Also provides a valuable measure of the effectiveness of unstained and the background is black)
current and future treatments
Tubercular Meningitis
 Causative agent: Mycobacterium tuberculosis
 Increased WBC count predominantly lymphocytes and
monocytes
  Increased protein, decreased glucose, increased lactate  Note: both culture and staining methods are performed
(>25 mg/dl) on CSF precipitate
 Hallmark: pellicle formation (web-like clot)
 Lab test: AFB (+) Red staining bacilli Gram Staining
 Upon refrigeration of CSF there would be pellicle formation  Routinely performed on CSF from all suspected cases of
after 12-24 hours meningitis
 Performed on concentrated specimens
Pellicle Formation in CSF  CSF is centrifuge at 1500 g for 15 minutes
 Enhanced by refrigeration of the sample  Organisms most frequently encountered include:
 Macroscopically, it appears as small fine clots seen after o Streptococcus pneumonia
refrigeration of CSF for a period of 12-24 hours o Haemophilus influenzaae
 Microscopically it is consists of white blood cells against a o Escherichia coli
fibrinous background and must be examined for bacteria thru o Neisseria meningitides
gram stain and culture o Streptococcus agalactiae and Listeria
 Normal: no clots due to absence to the absence of fibrinogen monocytogenes (encountered in newborns)
 Variations:
o Small clots – paresis (incomplete paralysis) Acid-fast Staining
o Large clots – associated with purulent meningitis Not routinely performed unless tubercular meningitis is
o Web-like clots – TB meningitis suspected. Tuberculosis is not stationary in the lungs. It can also
o Clotting en masse – blockage in CSF circulation disseminate in the meninges.

Bacterial Viral Tubercular Fungal India Ink

Elevated WBC Elevated WBC Elevated WBC Elevated WBC Detects the presence of thickly encapsulated Cryptococcus
count count count count neoformans
Neutrophils Lymphocytes Lymphocytes Lymphocytes  Gram stain reaction of Cryptococcus neoformans = classic
and monocytes and monocytes
starburst patten
Increased Increased Increased Increased
protein protein protein protein
Decreased Normal Decrease Normal to Latex Agglutination Test
glucose level glucose level glucose level decrease  Detects the presence of C.neoformans antigen in serum and
glucose level CSF; MORE SENSITIVE METHOD than INDIA INK PREP
Lactate level > Normal lactate Lactate level Lactate level  Presence of rheumatoid factor causes false-positive
35 mg/dl level >25mg/dl; >25mg/dl reactions
pellicle
formation
Lateral Flow Assay (LFA)
Positive gram Positive india
stain and ink with  Rapid method for detecting C.neoformans (yeast fungus
bacterial Cryptococcus with capsule)
antigen tests neoformans  Utilizes a reagent strip coated with monoclonal antibodies
that react with the cryptococcal polysaccharide capsule
Positive
immunologic ELISA
test for
Mas rapid and sensitive kesa kay culture
C.neoformans
An immunoassay; Detects the interaction with antibodies
between antigens. It also detects the ff organisms and antigens:
CSFGlutamine
 S.streptococcus, group B
 Normal value: 8-18 mg/dl
 H.influenzae, type B
 by product of ammonia and a-ketoglutarate
 S. pneumonia
 removes toxic ammonia
 concentration is directly proportional to serum ammonia  N.meningitidis A, B, C, Y, W135
 Mycobacterium tuberculosis
 preferred over direct measurement of ammonia since it is
 E.coli K1 antigens
more stable than ammonia
 testing for CSF glutamine is done for px with coma of
Miscellaneous
unknown origin (depletion of a-ketoglutarate)
Naegleria fowleri
 increased in cases of Reye’s syndrome – a childhood
 Opportunistic parasite found in ponds, small lakes, and even
complication following aspirin intake by px with viral
chlorinated swimming pools
infections
 Enters the nasal passages and migrates along the olfactory
nerve to invade the brain
CSF Lactate Dehydrogenase
 Motile trophozoites can be observed microscopically by
 In serum (normal): LD2>1>3>4>5
examining a wet prep of CSF
o In myocardial infarction: LD1>LD2
 Nonmotile trophozoites may be seen on cytocentrifuged
 In CSF (normal): LD1>2>3>4>5
stained smears accompanied by increased WBCs and no
o If there is a problem in the CNS: LD2>LD1
bacteria
Microbiological Tests for CSF
Serological Testing
Identifies the causative agent in meningitis
 Used to dx neurosyphilis
 Primary Tests:
 CDC recommended for dx of neurosyphilis: Venereal Disease
 Gram Staining: differentiate gram negative from
Research Laboratories (VDRL)
gram positive or if its bacilli or coci
 Other tests:
 Acid-fast staining: para malaman kung ang
 Fluorescent treponemal antibody-absorption (FTA-
meningitis ng px ay tubercular; if ang nagcacause ay
ABS) = avoid serum contamination! (serum remains
mycobacterium
positive even after treatment)
 India-ink preparation: for fungal meningitis;
 Rapid plasma reagin (RPR) test = not recommended
Cryptococcus neoformans; the organism is clear, while
because it is less sensitive than the VDRL
the background is dark brown (indirect staining)
 mas maganda ang vdrl kesa kay RPR dahil mas sensitive siya
 Latex agglutination tests
 Confirmatory test: CSF culture