Faculty of allied medical sciences

1. Faculty of allied medical sciences Clinical chemistry (MLCC-203)

2. CEREBROSPINAL FLUID EXAMINATION Presented by : Dr.Eman El-Attar MLC-203

3. ILOs • Define CSF and its formation . • Recognize physiological functions. • Define lumbar puncture why and how? • Examine CSF: physical, chemical, microscopic. • Interpret tests results.

4. CSF formation: • Cerebrospinal fluid (CSF) is mostly derived the choroid plexuses by ultrafiltration and active secretion. • CSF flows through the subarachnoid space between the arachnoid and pia mater

6. Total volume of CSF It is about 90-150 ml Medicine School of Shandong University

7. To supply nutrients to the nervous system • To remove metabolic wastes • To produce a mechanical barrier to cushion the brain and spinal cord against trauma. . Functions

9. Specimen Collection and handling CSF is collected by lumbar puncture between third, fourth, fifth lumbar vertebrae. It requires certain precautions and careful technique to prevent the introduction of infection or the damaging of neural tissue.

10. CSF is collected in three sterile tubes • Tube 1 – used for chemical and serologic test: centrifuge and use supernatant for chemistry analysis (glucose, protein, chloride) • Tube 2 – used for microbiology lab • Tube 3 – used for hematology (cell count) Cell counts done as soon as possible after the fluid is collected as cellular degradation occurs rapidly. If postponed, refrigerate for up to one hour.

13. Who to LP? Indications: • Meningitis, demeylinating disease , meningeal involvement in malignant disease Contraindications: • INR > 1.5 • Platelets < 50,000 • intracranial mass • partial / complete spinal block • acute spinal trauma

14. Risks of LP • Post-lumbar puncture Headache. • Bleeding; spinal hematoma. • Infection (poor sterile technique)

15. Normal CSF values • Aspect: Crystal Clear color and viscosity comparable to water • < 5 RBCs / mm3 • < 5 WBC’s / mm3 • Protein : 15-45mg/dl • Glucose : 60% of serum level (75-100)

16. Abnormal appearance • Cloudy or turbid: • increased protein or lipids • presence of WBC • Hemolyzed or bloody: • Traumatic tap • Tumor or hemorrhage • Xanthochromic: • Slight hemolysis • Jaundice • Marked increase in proteins • Clot formation: • traumatic tap • meningitis

17. xanthochromia Should be examined 1 Hr or less after collection to differ between cell lysis before or after collection. Medicine School of Shandong University

18. Microscopic examination

19. Microscopic examination

20. Microscopic examination RBCs: Always send tube #1 and #3 for cell count and compare RBCs Traumatic tap:  RBC in tube 1, nil in tube 3 • RBC : WBC ratio should be the same as in blood (approx 1000 RBC : 1 WBC) • In the CBC (RBCs are in millions and WBCs are in thousands) Subarachnoid Hemorrhage : RBC in tube 1 AND tube 3 • “Crenated RBCs” and xanthochromia (yellow supernatant after centrifuge)

21. WBC count • Performed promptly as 40% of leukocytes may lyse after 2 hrs at room temp • Normal adult 0 – 5 WBCs/µL • Children 30 mononuclear cells/µL ( 200WBC/ 400RBCs) - DIFFERENTIAL COUNT ON A CSF SPECIMEN • It should be performed on a stained smear and not from the cells in the counting chamber.

22. Typical Bacterial Meningitis • CSF WBC > 1000, PMN predominance • CSF protein > 500mg/dl • CSF glucose < 45 mg/dl

23. Typical Viral Meningitis • CSF WBC elevated, but < 250 (PMNs in early disease, then lymphocytes) • CSF protein elevated, but < 150 • Glucose > 50% of serum concentration

24. Chemical examination • Glucose: • Blood glucose should be drawn simultaneously. • The normal glucose is about 60% compared to serum level. • Normal 50~80mg/dl • Elevated Glucose: within 2 hrs preceeding lumbar puncture. (Diabetes mellitus) • Decreased Glucose: Systemic hypoglycemia, Bacterial Meningitis, TB meningitis, amoebic meningitis Medicine School of Shandong University

25. Total Protein • Over 80% of CSF protein content is derived from the plasma by ultrafiltration. • Normal level 15~45mg/dl. • In premature and full term neonates it reach up to 130mg/dl and 120 mg/dL respectively. • Determination of protein to assess permeability of BBB or intrathecal synthesis of protein. Medicine School of Shandong University

26. Elevated CSF protein • Increased permeability of the blood-brain barrier dt brain tumour, intracranial hemorrhage, traumatic injury. • Increased intrathecal synthesis of IgG as in Multiple schlerosis • Mechanical obstruction of CSF flow above the puncture site. Medicine School of Shandong University

27. Elevated CSF protein • Arachnoiditis • Meningitis • Hemorrhage • Endocrine/Metabolic disorders Medicine School of Shandong University

28. Decreased CSF proteins: • Less than 10mg/dl Dt- CSF leakage from dural tear. -Hyperthyroidism Medicine School of Shandong University

29. Laboratory consideration: • Determination of total protein by: -Turbidimetric method.(need big volume) -Dye binding method by CBB underestimate globulins. - Immunochemical methods. Determination of specific proteins by: -Electrophoresis. - Immunoturbidimetry - Nephelometry. - RID - RIA - Electroimmunodiffusion Pandys test for globulin determination Medicine School of Shandong University

30. Assessment of increased permeability • CSF albumin (mg/dl)/ Serum albumin(g/dl) Index. • Index less than 9= intact barrier Medicine School of Shandong University

31. CSF LACTATE In neonates (10-40mg/dl) . In adult or older children (10-22 mg/dl) • It refers to anaerobic metabolism. It increase in bacterial, tubercular and fungal meningitis. Not in viral meningitis. • Brain abscess-Intracranial hge- hypoxia-hydrocephalus-traumatic brain injury.

32. CSF GLUTAMINE • Glutamine is produced in the CNS by the brain cells from ammonia and alpha-ketoglutarate. This process serves to remove the toxic metabolic waste product ammonia from the CNS. • Elevated levels associated with liver failure,septic encephalopathy , respiratory failure. Medicine School of Shandong University

33. CSF ENZYMES • LDH – LD1, LD2, LD3, LD4, LD5%: -Increase LD5 in metastatic brain tumor. -Increase all fractions in 1ry brain tumor. -Increase LD4,LD5 in bacterial meningitis. • CK – BB: -Increase in: epileptic patient Brain tumor cerebral infarction Medicine School of Shandong University

34. MICROBIOLOGY TEST GRAM STAIN Is routinely performed on CSF from all suspected cases of meningitis although its value lies on the detection of bacterial and fungal organisms. • Organisms most frequently encountered: • S. pneumoniae (gram positive cocci) • H. influenzae ( pleomorphic gram negative rods) • E. coli (gram negative rods)

35. Study Questions • - How many tubes of CSF should be collected for CSF examination? • - What are the chemical analytes done to a CSF sample • - What abnormal findings suggest a traumatic tap ? Medicine School of Shandong University

36. Thank you for your attention Medicine School of Shandong University