MLS 419: AUBF LECTURE - These projections have small, one-way

FINALS WEEK 1 – LESSON 1 valve-like structures that allow the CSF to

Cerebrospinal Fluid enter the bloodstream of the large veins of
the head
Cerebrospinal Fluid (CSF) • Reabsorbed at the Arachnoid villae/granulations
• Discovered by Domenico Felice Cotugno • Where a volume of 90 – 150 ml is maintained
• Composition and f ormation (adults)
- CSF is the 3rd major fluid of the body • 10-60 mL in neonates
- Adult volume: 90-150 mL → CSF formation, circulation, and reabsorption into the blood
- Neonate volume: 10-60 mL make up a dynamic process that constantly turns over about
3 PROCESSES THAT GOVERN THE EXISTENCE OF CSF: 20 mL/hr if the flow path between production and
1. CSF production/f ormation reabsorption of CSF into the blood is obstructed for any
2. CSF circulation reason if there are problems in the 3 processes where CSF
3. CSF reabsorption accumulates producing hydrocephalus
→ Proper maintenance of these three processes = → Intracranial pressure – can also increase causing brain
maintains the proper volume of CSF damage, mental retardation, or death if left untreated.

1. CSF FORMATION
• CSF gates (?) the brain and spinal cord
• Produced primarily about 70% in the highly
vascularized choroid plexuses of the 4 ventricles
- The 4 ventricles: 2 lumbar ventricles, the
third ventricle, f ourth ventricle
- Choroid plexus: vascular f ringe-like f olds
present in the pia mater (directly lines the
brain and the spinal cord; the innermost
layer of the meninges)
- Ependymal cells: line the brain & spinal cord
also play a minor role in the production of CSF FUNCTIONS
CSF
• Supplies nutrients to nervous tissues
- At rate @20 ml/hr (adults)
• Removes metabolic wastes = Glutamine and
• Selective secretion/f iltration process f rom your
Ammonia
plasma under hydrostatic pressure and active
• Protects/cushions the nervous system against
transport and not as an ultraf iltration process
trauma
2. CSF CIRCULATION
BLOOD BRAIN BARRIER (Hematoencephalic barrier)
Brain and spinal cord are surrounded by 3 membranes
• Separates the brain tissues and the blood circulation
collectively termed as the meninges.
• Occurs due to tight f itting endothelial cells that
• Dura mater - the tough outermost membrane is next
prevent passage of larger molecules.
to the bone.
• Arachnoid mater - derives its name f rom its visual
resemblance to a spider web, is the middle layer.
• Pia mater - adheres to the surf ace of the neural
tissues
• CSF f lows through the subarachnoid space (space
between the arachnoid mater and pia mater), where
it gates and protects the delicate tissues of the CNS
• From its initial f ormation in the ventricles, the CSF
circulates all throughout the subarachnoid space • Parts:
towards the brain stem and spinal cord principally 1. Layer of endothelial cells – interconnected
through pressure changes caused by postural, through tight junctions and not containing
respiratory and circulatory pressures. f enestrations that is normally present in
3. CSF REABSORPTION endothelium of other tissues
• Eventually the CSF f lows f rom the subarachnoid 2. Basement membrane – consisting of the basal
space to the top outer surf ace of the brain where lamina of the astrocytes and the basal lamina of
projections of the arachnoid membrane called the endothelial cells
arachnoid granulations are present.
 3. Protrusions of astrocytes – pedicles or ➢ Routinely collected via lumbar puncture between 3 rd
vascular f eet & 4th (or lower in adults), or 4th & 5th (children) lumbar
• Functions of BBB: vertebra under sterile conditions
• Essential to protect the brain f rom pathogens ➢ Do not puncture a locally inf ected area. Avoid
• Blocks chemicals and harmf ul substances inf lamed area.
present in blood Antibodies and medications o To prevent introduction of the inf ection to the
also blocked CNS
o Larger antibodies (IgM) and medications ➢ Perf ormed aseptically af ter thorough cleansing of the
such as penicillin patient’s skin and application of a local anesthetic
o may sometimes pose a problem esp. in the ➢ Needle is advanced into the lumbar intersp ace and
selection of medication to treat brain you will hear a popping sound upon penetration of
diseases. Ensure that the drug chosen for the dura mater
medication is able to penetrate the BBB to ➢ Intracranial pressure (initial or opening pressure)
achieve therapeutic levels in the brain measurement taken bef ore and af ter the f luid is
• Restricts entry of large molecules withdrawn.
• Controls/restricts/f ilters blood components. So: - Measured by a manometer
- CSF composition is unlike blood’s - Opening pressure: 50-180 mm HG
- Theref ore, CSF is NOT an ultraf iltrate of o Slightly higher f rom individuals in sitting
plasma position
CSF dissolved substances o If pressure is in normal range – 20 mL of
Total protein 15-45 mg/dL CSF (saf ely)
Albumin 10-30 mg/dL o If less than or greater than normal – 1-2
IgG 1-4 mg/dL mL of CSF only
Glucose 50-80 mg/dL - Af ter removal of desired volume and bef ore needle
is withdrawn, physician must take the closing
Calcium 2.0-2.8 mEq/L
pressure
Chloride 115-130 mEq/L
- Closing pressure: 10 to 30 mm Hg less than the
Lactate 10-22 mg/dL
opening pressure.
Magnesium 2.4-3.0 mEq/L
Potassium 2.6-3.0 mEq/L
Sodium 135-150 mEq/L
Na, Cl, Mg higher in CSF
Potassium and Calcium Lower
Therefore, not ultrafiltrate of blood
4 Major categories of disease
1. Meningeal infections – bacterial or viral
2. Subarachnoid hemorrhage – or intracranial
CEREBROSPINAL FLUID TUBINGS
hemorrhage/stroke
• Assuming that the CSF volume collected is 20 mL or
3. CNS malignancy
enough
4. Demyelinating disease – e.g. multiple sclerosis;
Specimen collection and handling
destruction of myelin sheath that covers the axons
• Tube 1 – chemistries and serology
Indications for analysis
• To conf irm diagnose of meningitis – one of the most • Tube 2 – microbiology cultures
common • Tube 3 – hematology
• Evaluate f or intracranial hemorrhage • Tube 4 (if may excess) – f or microbiological or
serological studies
• Diagnose malignancies, leukemia
If the amount collected is only 1-2 mL
• Investigate central nervous system disorders – such
→ Prioritize the testing
as multiple sclerosis
→ Microbiology test (top priority)
CSF Fluid Collection
→ Hematology studies (priority #2)
• Lumbar puncture or spinal tap
→ Chemical and serology (last priority)
• Collected by trained physician
Precautions
Specimen collection
➢ Never use glass tubes because cells in CSF adheres to
➢ Lumbar puncture or spinal tap
the glass surf aces = f alsely decreased cell counts
Patient positioning
➢ Specimen potentially inf ectious
➢ Fetal position – to expose vertebra
➢ Testing considered STAT
 Clotted
• indicates increased f ibrinogen, usually due to
traumatic tap, but may indicate damage to blood-
brain barrier.
• Traumatic tap occurs if the needle inadvertently or
unintentionally has entered an epidural vein during
insertion. It gives f resh blood since introduction of
blood only happens during collection.

If immediate processing not possible:

• Tube 1 (chem-sero) f rozen
• Tube 2 (micro) room temp.
Pellicle formation
• Tube 3 (hema) ref rigerated
• Pellicle – thin skin or a cuticle membrane or f ilm, or
ANALYSIS OF THE CSF SAMPLE
a spiderweb f ormation
Physical examination
• in ref rigerated specimen associated with tubercular
Appearance - bedside evaluation (immediately af ter
meningitis.
collection of CSF)
o Pellicle f ormation –
➢ Normal - Crystal clear, colorless
picture at right (pellicle
➢ Descriptive Terms – hazy, cloudy, turbid, milky,
in L. tube, R is normal)
bloody, xanthochromic
o Requires overnight
- Of ten are qualif ied – slight, moderate, marked,
ref rigeration or 12
or grossly.
hours to f orm, at the
➢ Unclear specimens may contain increased lipids,
least
proteins, cells or bacteria. Use precautions.
Milky
• increased lipids
Oily
• contaminated with x-ray media

Traumatic collection vs Cerebral/Intracranial

hemorrhage
When blood is found in the CSF sample, there is need to
Xanthochromia – Yellow discoloration of supernatant (may
determine whether the blood present is due to an actual
be pinkish, or orange).
bleeding in the nervous system (cerebral hemorrhage) or due
• “xantho” = yellow; “chromia” = color to trauma during collection (traumatic tap)
• Due to unconjugated bilirubin Cerebral hemorrhage
• May also be pinkish = slight amount of → old blood in the CSF
oxyhemoglobin; or orange = heavy hemolysis 1. Even distribution of blood in the numbered tubes
• Unique to CSF samples 2. Clot f ormation improbable
• Most commonly due to presence of ‘old’ blood = long ▪ No clots in CSF
standing blood (at least 2 hrs) ▪ Fibrinogen, which is responsible for
o Lysis of blood transpires only af ter 2 hrs and clotting, is not normally f ound in
xanthochromia may only be manif ested in CSF
the CSF if hemolysis already occurred 3. Xanthochromic supernatant
• Other causes include increased serum bilirubin, - RBCs must have been in CSF @ 2+ hours
carotene, increased protein, and melanoma pigment ➢ D-dimer negative, f ibrin degradation product f rom
hemorrhage site
➢ Microscopic presence of erythrophages,
siderophages, or hemosiderin granules
o Take time to f orm
Traumatic tap
→ Fresh blood
→ During puncture
 → Puncture of adjacent blood vessels, will introduce Correction of WBC count for traumatic tap
enough f ibrinogen that will enable CSF to f orm clots contamination
→ Less than 2 hrs contact time of blood and CSF = will • Uses ratio of WBCs to RBCs in blood and compares
not allow hemolysis to happen = no hemoglobin it to same ratio (WBC/RBC) in CSF
degradation = no bilirubin = no yellow • If patient’s peripheral cell counts are normal, can
collor/xanthochromia subtract 1 WBC f or each 700 RBCs counted in CSF.
→ Positive f or D-dimer test • Great chance f or considerable error, makes this of
little value
Expected results
• Normally 0 RBCs/uL regardless of age
• WBCs – depends upon patient’s age
Adult up to 5 mononuclear WBCs/uL
Newborn up to 30 mononuclear WBCs/uL
Hemorrhage Traumatic tap Children (1-4) up to 20 mononuclear /uL
Children (5+) up to 10 mononuclear / uL
Increased numbers Pleocytosis (can be neutrophilic,
lymphocytic, or mixed cell)

NEUBAUER HEMOCYTOMETER/COUNTI NG CHAMBER

- Same as taught in Hematology; apply the same
concepts
Cerebrospinal fluid (CSF) – Procedures - General f ormula f or cell counts:
• All specimens should be examined microscopically – 𝒂𝒗𝒆 . # 𝒄𝒆𝒍𝒍𝒔 𝒄𝒐𝒖𝒏𝒕𝒆𝒅 × 𝒅𝒊𝒍𝒖𝒕𝒊𝒐𝒏
hematology # 𝒔𝒒𝒖𝒂𝒓𝒆𝒔 𝒄𝒐𝒖𝒏𝒕𝒆𝒅 × 𝒗𝒐𝒍𝒖𝒎𝒆 𝒐𝒇 𝒆𝒂𝒄𝒉 𝒔𝒒𝒖𝒂𝒓𝒆
• Stat priority
- RBCs and WBCs begin to lyse in 1 hour, 40%
WBC disintegrates in 2 hrs.
• Electronic counters generally unusable so…
o Manual cell count is needed because the
number of cells present in the CSF are too
low to be counted accurately using
automated cell counters
1. Total Cell Count
• Clear specimens - No dilution usually required
(use saline if needed) Quality Control for CSF processes
• TCC = WBC count + RBC count • CSF controls
2. RBC Count o Should be used regularly to verif y the
accuracy of the techniques
• not routinely perf ormed
o Biweekly – diluents by examination in a
• Perf ormed only during traumatic tap
counting chamber under 400X magnif ication
• RBC Count = TCC - WBC Count
o Monthly – speed of cytocentrif uge using
➢ Standard Neubauer hemocytometer counting
tachometer; timing should be checked with a
chamber – same lang sa Hema
stopwatch
3. WBC counts
• Check techniques
➢ 3% acetic acid can be used to lyse RBC
• Decontaminate all counting chambers in bleach
o Clear – undiluted
water f or 15 minutes.
o Slightly Hazy – 1:10
o Hazy - 1:20 • Rinse thoroughly with water
o Slightly Cloudy - 1:100 • Cleaned again with isopropyl alcohol.
▪ Example: 30 ul sample + 2,970 ul
diluent CSF Differential
o Slightly Bloody – 1:200 CSF Slide Dif f erential
o Bloody – 1:10,000 • Wrights stained smear of concentrated sediment.
➢ Methylene blue staining will improve o Because cells in the CSF are usually at low
visibility – stains the nuclear details of WBC levels
Concentration Techniques:
• Centrif ugation – 5-10 minutes
 • Sedimentation – preserves cellular morphology
• Filtration - preserves cellular morphology
• Cytocentrif uge (cytospin) – f luid is added to a conical
chamber
- Cells are f orced into a monolayer within a
6mm diameter circle on the slide
- Increases number of cells to evaluate,
however, risk of cell distortion f rom the
centrif ugation process. L – lymphocytes & macrophages
- Use of 30% albumin to: reduces cell
distortion and increase yield
mono/macro, segs and lymph

• Count and dif f erentiate 100 nucleated cells.

• Any cell f ound in peripheral blood may be seen in Eosinophils
CSF, other nucleated cells and malignant cells can • Of ten associated with parasitic / f ungal inf ections
also be f ound. • Introduction of f oreign materials into the CSF,
• Cells f rom the tissues f orming the membrane may including medications and shunts
also be seen – ex. Ependymal cells – normally f ound
in the CSF
• Entire smear should be evaluated f or:
- abnormal cells, inclusions within cells,
clusters, presence of intracellular organisms
• Normal dif ferential values
o Adults: 70% lymphocytes, 30% monocytes.
o Children/newborns: reverse is true
Choroidal cells
• Types of cells
• Epithelial lining of the choroid plexus
o Lymphocyte - Normal
• Singular or in clumps
o Neutrophils – Bacterial
o Eosinophil • Nucleoli absent
o Monocyte – Viral Tubercular Fungal • Nuclei have a unif orm appearance
o Plasma Cells - MS
o Macrophages – increase f ollowing CVA
o Ependymal cells, and normal lining cells can
also be seen.
→ Read more about these cells daw in Strasinger
ana si sir ☺
So again...
• Entire smear should be evaluated f or
o abnormal cells Ependymal cells
o inclusions within cells • Normal cells but not of ten f ound, unique to CSF
o clusters • Line the ventricles and neural canal, produce CSF
o presence of f luid
intracellular organisms • Large cell with distinct round/oval nucleus,
sometimes f ound in sheets
o Lymphocytes &
monocyte/macrophages
 • Suspicious/unclassif ied or malignant cells are • Hematoidin crystals
reported as “other” or “unclassif ied” o When the digestion process continued along
o send unstained slide to cytology/pathology f urther in the siderophage, all that will remain
or histopathology for accurate identif ication is the indigestible hematoidin crystal
• Blasts (appearance similar to peripheral blood, o Primarily composed of unconjugated
always consult with hematology bilirubin
specialist/pathologist)

1991 CAP 30 CSF

hematoidin crystal/bilirubin
crystal

CHEMISTRY
• Blood-brain barrier causes selective f iltration
• Abnormal values of CSF analytes (electrolytes,
proteins, glucose, etc.)
• Bottom are malignant cells
o f rom altered permeability
• Top are leukemic cells f ound in CSF
o Increased production
• Remember – we classif y them as ‘other’ or
o Increased metabolism
‘unclassif ied’ and take the slide to the Cerebrospinal fluid (CSF) - Protein
cytologist/pathologist
• Very low compared to serum/plasma
• Normal 15 – 45 mg/dL
o Inf ants – 150 mg/dL
o Immature – 500 mg/dL
• Decreased levels – not signif icant but may signify
CSF leakage
o Otorrhea – if CSF leaks into ear
o Rhinorrhea – if CSF leaks into nose
Cellular inclusions
• Increased levels
• Erythrophage
o Damaged BBB (as in meningitis or
o Remember that whenever there is bleeding
hemorrhage) - allow inf lux of protein towards
in the cerebral spaces, red cells will have to
CSF f rom plasma
be scavenged by macrophages
o Production of immunoglobulins within CNS
o Macrophage that has engulf ed an
(multiple sclerosis)
erythrocyte
o Decreased protein clearance
o Neural tissue degeneration
Protein Fractions
ASCP 21 CSF
• Major CSF Protein – Albumin
erythrophage, with few
iron granules forming • 2nd most prevalent – Transthyretin/Prealbumin
• Alpha globulins – Haptoglobin (f ree Hb),
Ceruloplasmin (copper)
• Beta-globulin – B2 Transf errin or TAU
• Siderophage o Carbohydrate-def icient
o An erythrophage eventually becomes a o Seen in CSF, not in serum
siderophage as the digestion of RBCs takes • Gamma – IgG and IgA
place because we will now have the • Not f ound in normal CSF – IgM (too large),
hemosiderin granules f rom the degradation Fibrinogen, beta lipoprotein
of the RBCs inside

ASCP 6 macrophage,
lymphocyte,
siderophage
CSF Protein Determination ▪ Values greater than 0.70
Total Protein indicate IgG production within
• Turbidimetric – makes use of acids to precipitate the CNS
proteins out the CSF ▪ Rules out possibility of a
o Sulf osalicylic acid (SSA) – Precipitates damaged BBB-associated
albumin more than globulin: add sodium increase of CSF IgG
sulf ate (to enhance precipitation of Electrophoresis
globulins) ➢ Another way to help establish diagnosis of MS is
o Trichloroacetic acid (TCA) – reagent of by subjecting both serum and CSF to
choice; both precipitates albumin and electrophoresis
globulin equally (no need to add extraneous ➢ CSF Electrophoresis f or MS
sodium sulf ate) • Done in conjunction with serum
• Dye-binding electrophoresis
o Used to measure CSF protein, where the • For the detection of oligoclonal bands in the
protein will couple with the dye or other gamma region
molecule that can be easily measured by • Oligoclonal bands – atleast 2 bands seen in
using spectrophotometer CSF with no corresponding bands in the
o Coomassie Brilliant Blue – CHON binds to serum
dye: Red to blue • 2 or more Oligoclonal Bands in CSF but not
▪ The blue color is proportional to the in serum may support diagnosis of MS
amount of protein
▪ f ollows Beer’s Law
o The alkaline biuret procedure has been used
but the Coomassie brilliant blue is pref erred
CSF – MS Panel
Multiple Sclerosis – increased proportion of CNS IgG
→ autoimmune disease
→ symptoms vary among patients because location
and severity can be really dif f erent
→ episodes can last f or days, weeks, or months; in
• Same pattern in: Encephalitis,
between attack, patient may have f ew or no
Neurosyphilis, Guillain-Barre Syndrome,
symptoms at all
and some other Neoplastic Disorders
→ Dif f icult diagnosis, of ten diagnosed af ter everything
• To accurately diagnose MS: correlate other
else has already been ruled out or eliminated
test results (IgG index), clinical symptoms
• In lab testing, increased CSF IgG should be determined if
and manif estations
it was a result of actual production of IgG in CNS (in MS)
or if it was just because serum blood IgG reaching the
Myelin Basic Protein
CSF which can result from a damage BBB
• Abnormal protein that indicates demyelination of
• To distinguish between these 2 probabilities, we compute neuron axons
for the CSF/serum albumin index and the IgG index
• 70% lipid, 30% protein
• CSF/Serum Albumin index - measure
• In MS, there is demyelination or destruction of the
intactness of BBB
myelin sheath releasing the MBP into the CSF
𝑪𝑺𝑭 𝒂𝒍𝒃𝒖𝒎𝒊𝒏 (𝒎𝒈⁄𝒅𝑳 )
= • Not specif ic f or MS; also produced in other
𝑺𝒆𝒓𝒖𝒎 𝒂𝒍𝒃𝒖𝒎𝒊𝒏 (𝒈⁄𝒅𝑳)
demyelinating diseases
o Index less than 9: Intact BBB
• Present only in acute exacerbation of MS
▪ Increase in CSF IgG could have
• Measurement used to monitor course of disease and
been a result of increased
ef f ectiveness of treatment against MS
production in the CNS mismo
(just as is the case of MM)
o Index of 100 indicates: total destruction
of BBB
• IgG levels (both serum and CSF)
o IgG synthesis rate
𝑪𝑺𝑭 𝑰𝒈𝑮 (𝒎𝒈⁄𝒅𝑳 )/ ⁄𝒔𝒆𝒓𝒖𝒎 𝑰𝒈𝑮 (𝒈⁄𝒅𝑳)
=
𝑪𝑺𝑭 ⁄𝑨𝒍𝒃 𝑰𝒏𝒅𝒆𝒙
Cerebrospinal Fluid (CSF) – Glucose o
Has 3 isoenzymes: CK-1.-2, -3
• Selectively transported across blood-brain barrier o
Isoenzyme CK1/ CK-BB f rom brain tissue
• Normal values: 60-70% of blood glucose Following cardiac arrest, patients’ CSF with
o
• STAT procedure, glycolysis reduces level quickly. CK-BB levels <17 mg/dL have f avorable
• Procedure perf ormed as f or and in conjunction with outcome.
blood specimen ▪ If levels are low, patient is doing well
o Measure both CSF and blood glucose DIFFERENTIAL DIAGNOSIS OF MENINGITIS
o Blood glucose must be drawn about 2 hours BY LABORATORY RESULTS
bef ore the lumbar tap to allow time for Bacterial Viral Tubercular Fungal
Increased Increased Increased Increased
equilibration between the blood and CSF
WBC count WBC count WBC count WBC count
• Decreased levels seen in dif f erent types of
Neutrophils Lymphs Lymphs & Lymphs &
meningitis (bacterial, tubercular, f ungal) Monos Monos
o Hypoglycemia Marked ↑ Mod. ↑ Mod-marked Mod-marked ↑
o Brain tumors protein protein ↑ protein protein
o Leukemias Marked ↓ Normal ↓ glucose Normal to ↓
o Damage to CNS glucose glucose glucose
• Increased levels – result of plasma elevation Lactate > 35 Lactate Lactate > 25 Lactate > 25
mg/dL normal mg/dL mg/dL
CSF Lactate + gram stains Pellicle + India
formation ink/nigrosin
inversely proportional to glucose
with
• Normal values = 11-22 mg/dL
Cryptococcus
• Increase as result: neoformans
o Bacterial Meningitis - >35 mg/dL + bacterial +
o Tubercular Meningitis - >25 mg/dL antigen tests immunological
o Fungal >25 mg/dL test for C. neo
o Normal in viral
o For monitoring of treatment f or meningitis *all have increased WBC count but WBC types differ in each
▪ Serum lactate remain elevated ✓ bacterial – neutrophils
during treatment of meningitis but it ✓ viral – lymphocytes
will f all rapidly if treatment is ✓ tubercular and fungal – combination of increased
successf ul (sensitive method to lymphocytes & monocytes
evaluate ef f ectiveness of antibiotic) *remember the inverse relationship of glucose and lactate
o Increased in Hypoxia *C. neoformans is encapsulated, use India ink/nigrosin to
o Xanthochromic and hemolyzed CSF - f alsely demonstrate capsule
elevates lactate CSF - MICROBIOLOGY
CSF Glutamine • to determine causative agent
→ product of ammonia and a-ketoglutarate by the brain cells • Gram stain - routinely perf ormed on all CSF
– a process by the body which removes the toxic metabolic suspected of meningitis
waste (ammonia) from CSF, since ammonia induces coma o Extremely important f or early diagnosis of
• Normal: 8-18 mg/dL bacterial meningitis
• Increased levels associated with liver disorders o CSF should be concentrated: 1500g f or 15
• As the concentration of ammonia in the CSF minutes
increases, the supply of a-ketoglutarate becomes ▪ To increase chance of being able to
depleted; glutamine can no longer be produced to extract the organism present in CSF
remove the toxic ammonia, and coma ensues. ▪ Sediment used f or GS and culture
• Reye’s Syndrome - swelling of the brain and • Even when using concentrated samples, 10% f alse
degeneration of the liver negatives occur thus…
CSF Enzymes • Blood culture should be taken
• Lactate dehydrogenase (LDH or LD)
o Isoenzymes 1 2 3 4 5;
▪ LD1 & LD2 are in brain tissue
▪ LD 2 and 3 = Lymphocytes
▪ LD 4 and 5 = Neutrophil
o Application: ex: in bacterial meningitis, LD4
and LD5 will be most abundant
• Creatine kinase (CK)
Organisms
Birth-1 month S. agalactiae

1 month-5 years Haemophilus influenzae

5 years-29 years
29 years
Inf ants, elderly,
immunocompromised
Immunocompromised -
Fungal
Neisseria meningitidis
S. pneumoniae
L. monocytogenes
Cryptococcus neoformans
Candida albicans,
Coccidioides, or any
opportunistic organism

Limulus Lysate Test

• Diagnosis of meningitis caused by gram-negative
bacteria
• Detection of endotoxin (remember that endotoxin is
Serology
not produced by GP bacteria)
VDRL (Venereal Disease Research Laboratory)
• Reagent: Amebocytes f rom Limulus polyphemus
• CDC recommended f or preliminary detection of
(horseshoe crab)
neurosyphilis (when T. pallidum already reached the
➢ Amebocytes release lysate in the presence of
nervous system)
GNE at 37°C 1 hour
• On CSF test it has low sensitivity, but great
➢ Result is clotting (positive result)
specif icity – it may not detect all cases of
➢ If there is clotting it means that lysate was
neurosyphilis, but a positive result is a true positive
released from the amebocytes = endotoxin is
result
present = causative agent: GN organism such as
FTA-ABS
Neisseria meningitidis
➢ What if L. monocytogenes? Answer = negative, • Fluorescent treponemal antibody absorption test
because it is a GP organism • Used to detect antibodies f ormed against T. pallidum
• also used on CSF
• more sensitive than VDRL, but its use in the
diagnosis of neurosyphilis is still controversial as it is
extremely sensitive to the ef f ects of blood
India ink contamination.
• Negative stain to view the encapsulated • Must prevent blood contamination.
Cryptococcus neoformans Rapid Plasma Reagin
- of ten AIDS/immunocompromised complication • not recommended
• remember that C. neof ormans is encapsulated, • Not that sensitive compared to VDRL
when the specimen containing the organism is mixed
w/ the india ink and when viewed under the
microscope = capsule pushes away stain
• Instead of stain, can also use dark f ield microscopy
f or same ef f ect
• Gram stain - starburst pattern
• Latex agglutination tests to detect the f ungal antigen
in serum and CSF
- More sensitive
- Should be conf irmed by culture and…India ink
- Rheumatoid f actor interf erence - False Positive
results
• Naegleria fowleri
- Observe trophozoite in CSF sample
- Motile in wet preparation
- Nonmotile in cytocentrif uged stained smears
with increased WBC