Lab Medicine Conference : Cerebrospinal Fluid Analysis

Cerebrospinal Fluid (CSF) Adults produce 450 to 500 cc per day 150 cc in adult CNS at any one time Neonates have 30 to 60 cc Children have 100 cc 80 % produced by ventricular choroid plexuses Reabsorbed by arachnoid villi Drains into dural sinuses

Suspected Diagnoses for Which CSF Exam is Indicated Meningitis Encephalitis Brain abscess Neurosyphilis Subarachnoid hemorrhage Demyelinating conditions : Multiple sclerosis Guillian-Barre CNS malignancies

Usual Recommended Tests to Run on Sequential Tubes of CSF from an LP First and third tubes Cell count & differential Second tube CSF total protein, glucose, +/- other chemistries Fourth tube Gram stain, other stains, cultures

Priority Ranking of Tests to Run If Only Small Amount of CSF Obtained Gram stain / culture Cell count / differential Protein / glucose Chemistries

Contraindications to Lumbar Puncture Intracranial mass lesion with impending herniation Cutaneous infection or suspected subcutaneous abscess at LP site Systemic coagulopathy Could result in cord compression from para-spinal hematoma Unrestrainable patient

Potential Complications of Lumbar Puncture Uncal or brainstem herniation 0.3 to 1.2 % mortality if papilledema present less likely if smaller amounts of fluid removed Arachnoiditis : can occur if needle carries in povidone-iodine Epidermoid tumors (delayed) : from use of needle without stylet Nerve root injury : less likely if needle bevel vertical Induced meningitis ; paraspinal abscess Mortality from hyperflexion of head & tracheal obstruction or from vagally induced asystole Post-procedure headache : 12 to 39 %

CSF Exam 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 Meningitis Intracranial mass lesions SAH CHF SVC obstruction Thrombosis of intracranial venous sinus Acute elevation of serum osmolarity

Causes of Low CSF Opening Pressures Severe dehydration Circulatory collapse Chronic serum hyperosmolality Dural tears with CSF leak Neurosurgical procedures Subdural hematomas in elderly Barbiturate intoxication Complete spinal subarachnoid block

CSF Appearance Normal is clear & consistency similar to H2O 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 Traumatic tap Increased protein from : subarachnoid block neurosyphilis tuberculosis Metastatic mucinous adenocarcinoma of the meninges

Xanthochromia of the CSF Is yellow - orange - brown coloration in supernatant of centrifuged CSF Produced by lysis of red cells 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 Direct serum bilirubin levels > 10 to 15 mg % CSF protein levels > 150 mg % Sample contamination with povidone iodine Systemic hypercarotenemia CSF melanin from meningeal melanosarcoma

CSF Glucose Normally 60 to 70 % of serum level Is 100 % ratio in neonates (immature CSF / blood barrier) In adults with serum glucose > 300 mg %, no further increase in CSF glucose occurs CSF level takes 2 hours to equilibrate with change in serum glucose

Causes of Hypoglycorrhachia (CSF to Serum glucose ratio < 0.6) Systemic hypoglycemia Impaired glucose transport Increased CNS use of CSF 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 Normal adult range is 17 to 55 mg % Normal neonate level is up to 150 mg % Increased levels usually associated with CNS inflammatory processes, especially infections Has relation ratio with serum protein levels, so elevations of serum protein may cause elevations in CSF protein

Noninfectious Causes of Elevated CSF Protein Traumatic LP 1 mg % increase per 1000 RBC's per mm3 Interference with CSF / blood barrier Cerebral hemorrhage SAH Cerebral thrombosis Endocrine Diabetes mellitus Hyperthyroidism Hypoparathyroidism Hyperadrenalism

Other Noninfectious Causes of Elevated CSF Protein Guillian-Barre Syndrome Multiple sclerosis Collagen vascular diseases Subacute sclerosing panencephalitis Mechanical obstruction of CSF circulation tumors, abscesses, cord compression Elevated serum protein levels (multiple myeloma, etc.) Medications / toxins : Phenytoin, ethanol, heavy metals

Causes of Low CSF Protein Levels Chronic leakage from CSF otorrhea or rhinorrhea Chronic increased ICP Removal of CSF via neurosurgical procedures or repeated LP's

CSF Cell Counts Normal adult : 0 to 5 lymphs or monos Even one poly is abnormal Normal neonates have 0 to 30 cells & up to 60 % polys Increased neutrophils usually indicate infectious process

Comparisons of Cell Counts in Viral Versus Bacterial Meningitis Typically > 500 WBC's / mm3 & mainly polys 10 % of cases have < 50 % polys Viral Typically < 100 WBC's / mm3 & mainly monos 10 % of cases have > 50 % polys (especially if early) 90 % convert to mononuclear pleocytosis by 12 hours

Infectious Causes of Very Low CSF Cell Counts Meningitis from : Neisseria meningitidis Hemophilus influenzae Overwhelming Strep. pneumoniae infection

Causes of Increased Neutrophils in the CSF Infectious Bacterial meningitis Early tuberculous meningitis Early viral meningitis Early mycotic meningitis 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 CSF Infectious Tuberculous, fungal, or leptospiral meningitis Partially treated bacterial meningitis Viral or syphilitic meningoencephalitis Subacute sclerosing panencephalitis Measles Noninfectious Multiple sclerosis, Guillian-Barre Syndrome Polyneuritis Temporal arteritis or periarteritis Chronic ethanol abuse Intravenous drug abuse

Causes of Increased Eosinophils in the CSF Infectious Bacterial, fungal, or viral meningitis Cysticercosis 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 Infectious Tuberculosis Noninfectious Presence of erythrocytes Acute intracranial bleeding Mycotic meningitis Trauma to CNS Contrast media

Age Related Causes of Bacterial Meningitis

Intersection with line B 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 Should be done on uncentrifuged CSF if CSF cloudy Should be done on centrifuged CSF if CSF clear Identifies 80 % of bacterial CSF infections 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 Is fluorochrome stain for bacterial nucleic acids Bacteria stain bright orange Background of cellular debris stains yellow - pale green Takes 2.5 minutes to prepare (versus 3.5 minutes for gram stain) Useful if bacteria not seen on gram stain (increases pickup rate > 75 %)

Other CSF Tests for Meningitis Lactic acid Levels > 35 mg % in 90 % of bacterial meningitis Numerous false positives (neoplasm, injury, etc.) LDH Elevated (especially LDH-5) with bacterial meningitis, but is nonspecific C-reactive protein If elevated has high sensitivity & specificity for bacterial meningitis, but is a technically difficult assay Quelling Reaction Antisera cause swelling in pneumococci & Hemophilus influenzae

Other CSF Tests for Meningitis (cont.) Limulus amebocyte lysate assay Requires 60 minutes Not 100 % sensitive CSF amino acids Elevated with bacterial meningitis May be useful for dx if partial treatment 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 Amount of antigen too small (if < 10,000 bacteria per ml.) If infection early, not enough time for antigen to dissolve off the bacteria Poor antibody quality for some strians (as for group B meningococcus & pneumococci types 7 & 14)

Sensitivity of CIE in Meningitis Meningococcal : 50 to 90 % Strep. pneumoniae : 50 to 100 % Hemophilus influenzae : 80 % Group B strep : 60 to 90 %

Latex Agglutination Antigen Tests for Meningitis More sensitive than CIE for pneumococci & meningococci Only takes 15 minutes to perform Not affected by antigen excess Less false negatives than CIE

Other Tests to Consider for Suspected Non-Bacterial, Non-Viral Meningitis Acid fast stain Mycobacterial culture India ink prep (for Cryptococcus) Cryptococcal antigen Fungal culture

Charges at H.M.C. for CSF Cultures & Microbial Stains Gram stain & culture : $ 48 Sensitivity (antibiotic) : $ 45 to $ 105 Agar diffusion vs. dual vs. add anerobic Fungal smear : $ 21 Fungal culture : $ 48 AFB smear & culture : $ 50 CIE : $ 37

Charges at H.M.C. for Other Standard Studies on CSF Cell count & diff. : $ 67 (stat) Glucose (stat) : $ 35 Protein (stat) : $ 35 Cryptococcal antigen : $ 35 Lactate : $ 26

Charges at H.M.C. for Miscellaneous Studies on CSF Darkfield exam : $ 54 VDRL : $ 16 India ink prep : $ 22 IgG : $ 20 Immunochemistry eval. : $ 126 ph by electrode : $ 26 Sperm count (rule out sperm embolus) : $16

Total Charges at H.M.C. for Different Patterns of CSF Test Ordering CBC/diff., gm. stain / culture, glucose, protein : $ 185 All standard, & culture / sensitivity studies : $ 322 All standard, & culture / sensitivity, & misc. studies : $ 462

Summary of Lab Studies on CSF for Meningitis Measure opening pressure Send four tubes Check gram stain If gram stain negative : Consider AOS Consider CIE +/- LA If clinical suspicion for meningitis, start broad spectrum antibiotics prior to initial lab results