Lab Medicine Conference :

Cerebrospinal Fluid Analysis

Jim Holliman, M.D., F.A.C.E.P.Professor of Surgery and Emergency MedicineDirector, Center for International Emergency MedicineM. S. Hershey Medical CenterPenn State UniversityHershey, Pennsylvania, U.S.A.

Cerebrospinal Fluid (CSF)

ƒ Adults produce 450 to 500 cc per day

ƒ 150 cc in adult CNS at any one time–Neonates have 30 to 60 cc–Children have 100 cc

ƒ 80 % produced by ventricular choroid plexuses

ƒ Reabsorbed by arachnoid villiƒ Drains into dural sinuses

Suspected Diagnoses for Which CSF Exam is Indicatedƒ Meningitisƒ Encephalitisƒ Brain abscessƒ Neurosyphilisƒ Subarachnoid hemorrhageƒ Demyelinating conditions :–Multiple sclerosis–Guillian-Barre

ƒ CNS malignancies

Usual Recommended Tests to Run on Sequential Tubes of CSF from an LP

ƒ First and third tubes–Cell count & differential

ƒ Second tube–CSF total protein, glucose, +/- other chemistries

ƒ Fourth tube–Gram stain, other stains, cultures

Priority Ranking of Tests to Run If Only Small Amount of CSF Obtained

ƒ Gram stain / cultureƒ Cell count / differentialƒ Protein / glucoseƒ Chemistries

Contraindications to Lumbar Puncture

ƒ Intracranial mass lesion with impending herniation

ƒ Cutaneous infection or suspected subcutaneous abscess at LP site

ƒ Systemic coagulopathy–Could result in cord compression from para-spinal hematoma

ƒ Unrestrainable patient

ƒ Uncal or brainstem herniation–0.3 to 1.2 % mortality if papilledema present–less likely if smaller amounts of fluid removed

ƒ Arachnoiditis : can occur if needle carries in povidone-iodine

ƒ Epidermoid tumors (delayed) : from use of needle without stylet

ƒ Nerve root injury : less likely if needle bevel verticalƒ Induced meningitis ; paraspinal abscessƒ Mortality

–from hyperflexion of head & tracheal obstruction–or from vagally induced asystole

ƒ Post-procedure headache : 12 to 39 %

Potential Complications of Lumbar Puncture

CSF Exam

ƒ First step is measure the opening pressure (OP) :–normal 80 to 180 mm H2O with pt. recumbent–can be "falsely" elevated by Valsalva, head-up position, or jugular compression–should vary 5 to 10 mm H2O with respiration–Queckenstedt & Tobey Ayer tests (involving jugular compression & seeing the effect on OP) are no longer recommended

Causes of Elevated CSF Opening Pressure

ƒ Meningitisƒ Intracranial mass lesionsƒ SAHƒ CHFƒ SVC obstructionƒ Thrombosis of intracranial

venous sinusƒ Acute elevation of serum

osmolarity

Causes of Low CSF Opening Pressures

ƒ Severe dehydrationƒ Circulatory collapseƒ Chronic serum hyperosmolalityƒ Dural tears with CSF leakƒ Neurosurgical proceduresƒ Subdural hematomas in elderlyƒ Barbiturate intoxicationƒ Complete spinal subarachnoid

block

CSF Appearance

ƒ Normal is clear & consistency similar to H2O

ƒ Causes of visual turbidity :–> 200 WBC's per mm3–> 400 RBC's per mm3–Bacteria–Aspirated epidural fat–Evil aliens (this was to see if you are paying attention)

Causes of CSF Clot Formation

ƒ Traumatic tapƒ Increased protein from :

–subarachnoid block–neurosyphilis–tuberculosis

ƒ Metastatic mucinous adenocarcinoma of the meninges

Xanthochromia of the CSF

ƒ Is yellow - orange - brown coloration in supernatant of centrifuged CSF

ƒ Produced by lysis of red cellsƒ Involves 3 pigments :

–oxyhemoglobin (red) : occurs in CSF within 2 hours of a SAH–bilirubin (yellow) : converted from hemoglobin in 12 hours–methemoglobin (brown)

Causes of Xanthochromia Besides Red Cell Lysis

ƒ Direct serum bilirubin levels > 10 to 15 mg %

ƒ CSF protein levels > 150 mg %ƒ Sample contamination with

povidone iodineƒ Systemic hypercarotenemiaƒ CSF melanin from meningeal

melanosarcoma

CSF Glucose

ƒ Normally 60 to 70 % of serum levelƒ Is 100 % ratio in neonates (immature

CSF / blood barrier)ƒ In adults with serum glucose > 300 mg

%, no further increase in CSF glucose occurs

ƒ CSF level takes 2 hours to equilibrate with change in serum glucose

Causes of Hypoglycorrhachia(CSF to Serum glucose ratio < 0.6)

ƒ Systemic hypoglycemiaƒ Impaired glucose transportƒ Increased CNS use of CSFƒ Increased use of CSF glucose by

bacteria & leucocytes–Typical with bacterial, tuberculous, or fungal meningitis–Also sometimes with SAH, viral meningitidies, sarcoidosis, neoplasms

CSF Protein

ƒ Normal adult range is 17 to 55 mg %

ƒ Normal neonate level is up to 150 mg %

ƒ Increased levels usually associated with CNS inflammatory processes, especially infections

ƒ Has relation ratio with serum protein levels, so elevations of serum protein may cause elevations in CSF protein

Noninfectious Causes of Elevated CSF Proteinƒ Traumatic LP–1 mg % increase per 1000 RBC's per mm3

ƒ Interference with CSF / blood barrier–Cerebral hemorrhage–SAH–Cerebral thrombosis

ƒ Endocrine–Diabetes mellitus–Hyperthyroidism–Hypoparathyroidism–Hyperadrenalism

Other Noninfectious Causes of Elevated CSF Protein

ƒ Guillian-Barre Syndromeƒ Multiple sclerosisƒ Collagen vascular diseasesƒ Subacute sclerosing panencephalitisƒ Mechanical obstruction of CSF circulation–tumors, abscesses, cord compression

ƒ Elevated serum protein levels (multiple myeloma, etc.)

ƒ Medications / toxins :–Phenytoin, ethanol, heavy metals

Causes of Low CSF Protein Levels

ƒ Chronic leakage from CSF otorrhea or rhinorrhea

ƒ Chronic increased ICPƒ Removal of CSF via

neurosurgical procedures or repeated LP's

CSF Cell Counts

ƒ Normal adult : 0 to 5 lymphs or monos

ƒ Even one poly is abnormalƒ Normal neonates have 0 to 30

cells & up to 60 % polysƒ Increased neutrophils usually

indicate infectious process

Comparisons of Cell Counts in Viral Versus Bacterial Meningitis

ƒ Bacterial–Typically > 500 WBC's / mm3 & mainly polys–10 % of cases have < 50 % polys

ƒ Viral–Typically < 100 WBC's / mm3 & mainly monos–10 % of cases have > 50 % polys (especially if early)ƒ 90 % convert to mononuclear pleocytosis by 12 hours

Infectious Causes of Very Low CSF Cell Counts

ƒ Meningitis from :–Neisseria meningitidis–Hemophilus influenzae–Overwhelming Strep. pneumoniae infection

Causes of Increased Neutrophils in the CSFƒ Infectious

–Bacterial meningitis–Early tuberculous meningitis–Early viral meningitis–Early mycotic meningitis

ƒ Noninfectious–3 to 4 days post - hemorrhagic infarct–SAH or intracerebral hematoma–Injection of antibiotics or antimetabolites–Injection of contrast media–Repeated LP's

Causes of Increased Lymphocytes in the CSFƒ Infectious

–Tuberculous, fungal, or leptospiral meningitis–Partially treated bacterial meningitis–Viral or syphilitic meningoencephalitis–Subacute sclerosing panencephalitis–Measles

ƒ Noninfectious–Multiple sclerosis, Guillian-Barre Syndrome–Polyneuritis–Temporal arteritis or periarteritis–Chronic ethanol abuse–Intravenous drug abuse

Causes of Increased Eosinophils in the CSFƒ Infectious

–Bacterial, fungal, or viral meningitis–Cysticercosis

ƒ Noninfectious–Allergic reaction to foods, meds, dyes, or envenomation–Intrathecal foreign substances or contrast dye–Synthetic intrathecal shunts–Periarteritis nodosa–Allergic bronchial asthma–Acute polyneuritis–Rabies vaccination

Causes of Increased Macrophages in the CSF

ƒ Infectious–Tuberculosis

ƒ Noninfectious–Presence of erythrocytes–Acute intracranial bleeding–Mycotic meningitis–Trauma to CNS–Contrast media

Age Related Causes of Bacterial Meningitis

Intersection with line B. Join the marks on lines A & B with the ruler, and read off the probability of acute bacterial versus acute viral meningitis where the ruler intersects the central probability scale.

CSF Gram Stain

ƒ Should be done on uncentrifuged CSF if CSF cloudy

ƒ Should be done on centrifuged CSF if CSF clear

ƒ Identifies 80 % of bacterial CSF infections

ƒ False positive only if LP tray or stain itself is contaminated

CSF gram stain showing E. coli

CSF gram stain showing Listeria monocytogenes

CSF gram stain showing Neisseria meningitidis

CSF gram stain showing Streptococcus pneumoniae

CSF gram stain showing Staphylococcus aureus

CSF gram stain of Pneumococcal meningitis

Use of Acrinidine Orange Stain (AOS) for CSF

ƒ Is fluorochrome stain for bacterial nucleic acids

ƒ Bacteria stain bright orange–Background of cellular debris stains yellow - pale green

ƒ Takes 2.5 minutes to prepare (versus 3.5 minutes for gram stain)

ƒ Useful if bacteria not seen on gram stain (increases pickup rate > 75 %)

Other CSF Tests for Meningitis

ƒ Lactic acid–Levels > 35 mg % in 90 % of bacterial meningitis–Numerous false positives (neoplasm, injury, etc.)

ƒ LDH–Elevated (especially LDH-5) with bacterial meningitis, but is nonspecific

ƒ C-reactive protein–If elevated has high sensitivity & specificity for bacterial meningitis, but is a technically difficult assay

ƒ Quelling Reaction–Antisera cause swelling in pneumococci & Hemophilus influenzae

Other CSF Tests for Meningitis (cont.)ƒ Limulus amebocyte lysate assay

–Requires 60 minutes–Not 100 % sensitive

ƒ CSF amino acids–Elevated with bacterial meningitis–May be useful for dx if partial treatment

ƒ Countercurrentimmunoelectrophoresis CIE)–Takes 30 to 60 minutes–Precipitant line forms between bacterial antigens & serum with known antibodies–Can be useful in partially treated meningitis–False positives & cross-reactions occur

Causes of False Negative CIE

ƒ Amount of antigen too small (if < 10,000 bacteria per ml.)

ƒ If infection early, not enough time for antigen to dissolve off the bacteria

ƒ Poor antibody quality for some strians (as for group B meningococcus & pneumococci types 7 & 14)

Sensitivity of CIE in Meningitis

ƒ Meningococcal : 50 to 90 %ƒ Strep. pneumoniae : 50 to 100

%ƒ Hemophilus influenzae : 80 %ƒ Group B strep : 60 to 90 %

Latex Agglutination Antigen Tests for Meningitis

ƒ More sensitive than CIE for pneumococci & meningococci

ƒ Only takes 15 minutes to perform

ƒ Not affected by antigen excessƒ Less false negatives than CIE

Other Tests to Consider for Suspected Non-Bacterial, Non-Viral Meningitis

ƒ Acid fast stainƒ Mycobacterial cultureƒ India ink prep (for

Cryptococcus)ƒ Cryptococcal antigenƒ Fungal culture

Charges at H.M.C. for CSF Cultures & Microbial Stains

ƒ Gram stain & culture : $ 48ƒ Sensitivity (antibiotic) : $ 45 to $

105–Agar diffusion vs. dual vs. add anerobic

ƒ Fungal smear : $ 21ƒ Fungal culture : $ 48ƒ AFB smear & culture : $ 50ƒ CIE : $ 37

Charges at H.M.C. for Other Standard Studies on CSF

ƒ Cell count & diff. : $ 67 (stat)ƒ Glucose (stat) : $ 35ƒ Protein (stat) : $ 35ƒ Cryptococcal antigen : $ 35ƒ Lactate : $ 26

Charges at H.M.C. for Miscellaneous Studies on CSF

ƒ Darkfield exam : $ 54ƒ VDRL : $ 16ƒ India ink prep : $ 22ƒ IgG : $ 20ƒ Immunochemistry eval. : $ 126ƒ ph by electrode : $ 26ƒ Sperm count (rule out sperm

embolus) : $16

Total Charges at H.M.C. for Different Patterns of CSF Test Ordering

ƒ CBC/diff., gm. stain / culture, glucose, protein : $ 185

ƒ All standard, & culture / sensitivity studies : $ 322

ƒ All standard, & culture / sensitivity, & misc. studies : $ 462

Summary of Lab Studies on CSF for Meningitis

ƒ Measure opening pressureƒ Send four tubesƒ Check gram stainƒ If gram stain negative :

–Consider AOS–Consider CIE +/- LA

ƒ If clinical suspicion for meningitis, start broad spectrum antibiotics prior to initial lab results