CSF analysis and its clinical applications

Indication, sample collection, tests done,clinical applications

1. SEMINAR TOPIC: CSF ANALYSIS PRESENTER: DR VISHNUPRIYA R MODERATOR: DR ARATHI C A

2. SEMINAR PLAN INDICATIONS COLLECTION OF SAMPLE TESTS DONE CSF ANALYSIS– GROSS AND MICROSCOPY CHEMICAL AND MICROBIOLOGICAL ANALYSIS CLINICAL APPLICATIONS

3. According to American college of physicians divided into INDICATIONS OF CSF EXAMINATION Meningeal Infections (bacterial most important) Sub arachnoid hemorrhage Primary or metastatic malignancy Demyelinating disease

4. CSF is obtained by lumbar Cisternal Lateral cervical punctures Through ventricular cannulas or shunts SPECIMEN COLLECTION

5. A manometer should be attached before fluid removal to record opening pressure. CSF pressure varies with postural changes, BP, Venous return, Valsalva maneuvers and factors that alter cerebral blood flow. Normal opening adult pressure---- 90-180 mm water in lateral decubitus position with legs and neck in neutral position In sitting position------ increases upto 10 mm with respiration In obese patients----can be as high as 250 mm water OPENING PRESSURE

6. In young children and infants----10-100 mm water with adult range obtained by 6-8 yrs Opening pressure> 250 mm water ----- intracranial HTN due to meningitis, intracranial hemorrhage and tumors . If opening pressure>200 mm water in a relaxed patient, no more than 2 ml should be withdrawn Elevated pressures may be present in patients who are tense or straining and in those with congestive heart failure, meningitis, superior vena cava syndrome, thrombosis of the venous sinuses, cerebral edema , mass lesions, hypoosmolality, or conditions inhibiting CSF absorption

7. Opening pressure elevation may be the only abnormality in cryptococcal meningitis and pseudotumor cerebri Decreased CSF pressure may be present in spinal-subarachnoid block, dehydration, circulatory collapse, and CSF leakage A significant pressure drop after removal of 1 to 2 mL suggests herniation or spinal block above the puncture site, in which case no further fluid should be withdrawn. Up to 20 mL of CSF may normally be removed. Simultaneous serum glucose is to be checked. The clinician should always provide an appropriate clinical history to the laboratory .

8. CSF specimen 3 serially collected sterile tubes 1. For chemistry and immunology studies 2. Microbiological examination 3. Cell count and differential. An additional tube may be added if malignancy is suspected

9. Under certain conditions, some variations are critical Eg : If traumatic tap, it cannot be used for protein studies. Tube 1 should not be used for microbiology because it may be contaminated by skin bacteria. Glass tubes are to be avoided- --- cell adhesion to glass affects cell count and differential. Cellular degradation starts within 1 hr of collection , so is to be processed quickly. Refrigeration contraindicated for culture specimen because fastidious organisms will not survive.

10. Refrigeration also contraindicated for samples in which flow cytometry is likely to be needed for detection of leukemia and lymphoma cells: as refrigeration may affect expression and detection of certain surface antigen cells.

11. GROSS EXAMINATION Normal CSF--- crystal clear, colourless, viscosity similar to water . Abnormal CSF --- appear cloudy, frankly purulent, or pigment tinged. Turbidity or cloudiness begins to appear with leukocyte counts over 200 cells/ μL or red blood cell counts of 400/ μL Grossly bloody fluids have RBC counts greater than 6000/ μL Microorganisms (bacteria, fungi, amebas ) radiographic contrast material aspirated epidural fat protein level greater than 150 mg/dL (1.5 g/L) may also produce varying degrees of cloudiness

12. In tubercular meningitis, when csf is left to stand, a fine clot resembling a cob web may form. This is due to high level of protein in csf .

13. Clot formation May be present in patients with traumatic taps, complete spinal block ( Froin syndrome), or suppurative or tuberculous meningitis. It is not usually seen in patients with subarachnoid hemorrhage. Fine surface pellicles may be observed after refrigeration for 12 to 24 hours. Clots may interfere with cell count accuracy by entrapping inflammatory cells and/or by interfering with automated instrument counting.

14. Viscous CSF May be encountered in patients with metastatic mucin producing adenocarcinomas, cryptococcal meningitis due to capsular polysaccharide liquid nucleus pulposus resulting from needle injury to the annulus fibrosus.

15. Pink-red CSF Usually indicates the presence of blood and is grossly bloody when the RBC count exceeds 6000/ μL . It may originate from a subarachnoid hemorrhage, intracerebral hemorrhage, or cerebral infarct, or from a traumatic spinal tap.

16. XANTHOCHROMIA Xanthochromia commonly refers to a pale pink to yellow color in the supernatant of centrifuged CSF. To detect xanthochromia, the CSF should be centrifuged and the supernatant fluid compared with a tube of distilled water.

17. Although careful gross CSF inspection has good sensitivity , spectrophotometry can also help to differentiate hemoglobin derived substances from other xanthochromic pigments with different maximal absorption peaks.

18. Differential Diagnosis of Bloody CSF Distinction of a traumatic puncture from a pathologic hemorrhage is of vital importance. In a traumatic tap, the hemorrhagic fluid usually clears between the first and third collected tubes but remains relatively uniform in subarachnoid hemorrhage. Xanthochromia, microscopic evidence of erythrophagocytosis, or hemosiderin-laden macrophages indicate a subarachnoid bleed in the absence of a prior traumatic tap. RBC lysis begins as early as 1 to 2 hours after a traumatic tap. Thus, rapid evaluation is necessary to avoid false-positive results. A commercially available latex agglutination immunoassay test for cross-linked fibrin derivative d-dimer is specific for fibrin degradation and should theoretically be negative in traumatic taps .

19. MICROSCOPIC EXAMINATION Traditional manual method for cell counting in CSF samples, using undiluted CSF in a manual counting chamber, continues to be a useful approach . Improvements in the hardware and software in flow cytometers now allow reliable use of these instruments in performing automated total WBC counts and WBC differential counts and even in detecting bacteria in CSF samples. The normal leukocyte cell count in adults is 0 to 5 cells/ μL . It is higher in neonates, ranging from 0 to 30 cells/ μL , with the upper limit of normal decreasing to adult values by adolescence. No RBCs should be present in normal CSF. If numerous (except with a traumatic tap), a pathologic process is probable (e.g., trauma, malignancy, infarct, hemorrhage).

20. CELL COUNT Draw unna’s polychrome methylene blue to ‘1’ mark in RBC pipette and fill pipette to ‘101’ mark with spinal fluid. This colours white cells blue and red cells yellow. Count 9 large squares in neubauer’s counting chamber for both RBC’s and WBC’s– total multiplied by 1.1 gives number of cells per cubic mm.

21. RBC counts have limited diagnostic value, they may give a useful approximation of the true CSF WBC count or total protein in the presence of a traumatic puncture by correcting for leukocytes or protein introduced by the traumatic puncture. WBC corr = WBC obs − WBC added where WBC added =WBC BLD × RBC CSF / RBC BLD and WBC obs = CSF leukocyte count WBC added = leukocytes added to CSF by traumatic tap WBC BLD = peripheral blood leukocyte count RBC CSF = CSF erythrocyte count RBC BLD = peripheral blood erythrocyte count

22. DIFFERENTIAL CELL COUNT The sample is subjected to cytocentrifugation and cell concentrate is obtained. Manual differential cell counting on a cytocentrifuge preparation of CSF continues to be the most reliable method, even with low cell numbers

23. In adults, normal CSF contains small numbers of lymphocytes and monocytes in an approximate 70:30 ratio . A higher proportion of monocytes is present in young children, in whom up to 80% may be normal . Erythrocytes due to minor traumatic bleeding are commonly seen, especially in infants. Small numbers of neutrophils (polymorphonuclear leukocytes [PMNs]) may also be seen in “normal” CSF specimens, most likely as a result of minor hemorrhage and improved cell concentration methods.

24. No general consensus regarding an upper limit of normal for PMNs has been established. Many laboratories accept up to 7% neutrophils with a normal WBC count . Over 60% neutrophils has been reported in high-risk neonates without meningitis. The number of PMNs may be decreased by as much as 68% within the first 2 hours after lumbar puncture owing to cell lysis.

25. Traumatic puncture may result in the presence of bone marrow cells, cartilage cells, squamous cells, ganglion cells, and soft-tissue elements. In addition, ependymal and choroid plexus cells may rarely be seen . Moreover, blastlike primitive cell clusters, most likely of germinal matrix origin, are sometimes found in premature infants with intraventricular hemorrhage

26. Cluster of blast like cells in premature new born

27. In early bacterial meningitis, the proportion of PMNs usually exceeds 60%. However, in about one-quarter of cases of early viral meningitis, the proportion of PMNs also exceeds 60%. Viral-induced neutrophilia usually changes to a lymphocytic pleocytosis within 2 to 3 days Neutrophilic meningitis (over 1 week) may be noninfectious or due to less common pathogens such as Nocardia, Actinomyces, Aspergillus, and the zygomycetes INCREASED CSF NEUTROPHILS

28. Lymphocytosis (>50%) may occur in early acute bacterial meningitis when the CSF leukocyte count is under 1000/ μL . Reactive lymphoplasmacytoid and immunoblastic variants may be present, particularly with viral meningoencephalitis. Blast like lymphocytes may be seen admixed with small and large lymphocytes in the CSF of neonates. Increased CSF lymphocytes

29. Plasma cells, not normally present in CSF, may appear in a variety of inflammatory and infectious conditions , along with large and small lymphocytes, and in association with malignant brain tumors . Multiple myeloma may also rarely involve the meninges PLASMA CELLS

30. Eosinophils are rarely present in normal CSF may be increased in a variety of CNS conditions. For example, eosinophilia is frequently mild (1%–4%) in a general inflammatory response, but in children with malfunctioning ventricular shunts, it may be marked A suggested criterion for eosinophilic meningitis is 10% eosinophils Parasitic invasion of the CNS is the most common cause worldwide . EOSINOPHILS

31. Macrophages with phagocytosed erythrocytes ( erythrophages ) appear from 12 to 48 hours following a subarachnoid hemorrhage or traumatic tap . Hemosiderin-laden macrophages ( siderophages ) appear after about 48 hours and may persist for weeks Brownish yellow or red hematoidin crystals may form after a few days MACROPHAGES

32. TUMOR CELLS Morphologic CSF examination for tumor cells has moderate sensitivity and high specificity (97%–98%) Sensitivity depends on the type of neoplasm. CSF examination of leukemic patients has the highest sensitivity (about 70%), followed by metastatic carcinoma (20%–60%) and primary CNS malignancies (30%). Sensitivity may be optimized by using filtration methods with larger fluid volumes or by performing serial punctures in patients in whom a neoplasm is strongly suspected. Processing of CSF samples using liquid-based thin-layer methods also increases sensitivity in the detection of neoplastic cells and enhances preservation of these cells for potential immunocytochemical analysis. These liquid-based methods are now commonly used for cytopathologic examination of CSF and other body cavity fluid specimens.

33. Leukemic involvement of the meninges is more frequent in patients with acute lymphoblastic leukemia than in those with acute myeloid leukemia . Both are significantly more common than CNS involvement in the chronic leukemias. A leukocyte count over 5 cells/ μL with unequivocal lymphoblasts in cytocentrifuged preparations is commonly accepted as evidence of CSF involvement. The incidence of CNS relapse in children with lymphoblasts but cell counts lower than 6 cells/ μL appears to be low and is not significantly different from cases in which no blasts are identified.

34. Non-Hodgkin lymphomas involving the leptomeninges are usually high-grade tumors (lymphoblastic, large-cell immunoblastic, and Burkitt lymphomas) Low-grade lymphomas and Hodgkin lymphoma are significantly less common T cells predominate in normal and inflammatory conditions, whereas most lymphomas, especially those occurring in immunocompromised hosts, are of B-cell lineage. Lymphoblastic lymphoma, the most common T-cell lymphoma to involve the CSF, can be detected by terminal deoxynucleotidyl transferase stain.

35. Multiparameter flow cytometric immunophenotypic studies, deoxyribonucleic acid (DNA) analysis by polymerase chain reaction (PCR), and, more recently, DNA sequence analysis have been shown to significantly improve diagnostic sensitivity and specificity in CSF samples involved by leukemic or lymphoma cells. Amebas , fungi (especially Cryptococcus neoformans), and Toxoplasma gondii organisms may be present on cytocentrifuge specimens but may be difficult to recognize without confirmatory stains.

36. CHEMICAL ANALYSIS Normal CSF protein levels -- 15 to 60 mg/dL Elevated CSF protein levels may be caused by Increased permeability of the BBB, Decreased resorption at the arachnoid villi, Mechanical obstruction of CSF flow due to spinal block above the puncture site, An increase in intrathecal immunoglobulin (Ig) synthesis.

37. Low lumbar CSF total protein levels may ---- Normally occur in some young children between 6 months and 2 years of age In patients with conditions associated with increased CSF turnover. (1) removal of large CSF volumes; (2) CSF leaks induced by trauma or lumbar puncture; (3) increased intracranial pressure, probably due to an increased rate of protein resorption by the arachnoid villi (4) hyperthyroidism

38. Glucose Derived from blood glucose, fasting CSF glucose levels are normally 50 to 80 mg/dL (2.8–4.4 mmol/L)—about 60% of plasma values. The normal CSF/plasma glucose ratio varies from 0.3 to 0.9, with fluctuations in blood levels caused by the lag in CSF glucose equilibration time. A good rule of thumb is that CSF glucose is about two-thirds of the level of serum glucose. This enables the detection of low CSF glucose in patients with abnormal glucose levels.

39. CSF values below 40 mg/dL (2.2 mmol/L) or ratios below 0.3 are considered to be abnormal Hypoglycorrhachia is a characteristic finding of bacterial, tuberculous, and fungal meningitis Other causes of low CSF glucose: Meningeal involvement by a malignant tumor , Sarcoidosis Cysticercosis Trichinosis Ameba (Naegleria) Acute syphilitic meningitis Intrathecal administration of radioiodinated serum albumin Subarachnoid hemorrhage Symptomatic hypoglycemia Rheumatoid meningitis

40. Decreased CSF glucose results from increased anaerobic glycolysis in brain tissue ,leukocytes and from impaired transport into the CSF. Bacteria are usually present in insufficient quantities to be a major contributor to the use of glucose in CSF CSF glucose levels normalize before protein levels and cell counts during recovery from meningitis, making it a useful parameter in assessing response to treatment. Increased CSF glucose is of no clinical significance, reflecting increased blood glucose levels within 2 hours of lumbar puncture. A traumatic tap may also cause a spurious increase in CSF glucose.

41. Although changes in opening pressure, total cell and differential counts, total protein, and glucose suggest an infectious origin : Gram stain, culture, and other relevant studies are critical for a definitive diagnosis Bacterial Meningitis The most common agents of bacterial meningitis are 1)group B streptococcus (neonates) 2) Neisseria meningitidis (3 months and older) 3)Streptococcus pneumoniae (3 months and older), 4)Escherichia coli and other gram-negative bacilli ( newborn to 1 month), 5) Haemophilus influenzae (3 months to 18 years) 6) Listeria monocytogenes (neonates, older adults, alcoholics, and immunosuppressed MICROBIOLOGICAL EXAMINATION

42. H. influenzae, once the most common bacterial cause of meningitis in young children, has decreased dramatically from widespread use of H. influenzae type b vaccine. CSF shunts, head trauma, and neurosurgery place patients at risk for CNS infections from Staphylococcus species, gram-negative bacilli, and Propionibacterium species. The Gram stain remains an accurate, rapid method by which to diagnose CNS infection. All specimens should be concentrated by centrifugation before Gram stain and culture. Viral Meningitis Enteroviruses (echoviruses, coxsackieviruses, polioviruses) and arboviruses are responsible for the majority of meningitis cases.

43. Most patients present with a CSF pleocytosis; although neutrophils may be observed early in the infection, patients soon develop a predominance of lymphocytes . In recent years, PCR-based assays have evolved as the gold standard for the increasingly rapid diagnosis of viral meningitis secondary to enterovirus, HSV, cytomegalovirus, varicella zoster, and JC virus. Human Immunodeficiency Virus A wide variety of CSF abnormalities may be found in HIV-positive patients with or without neurologic disease, including lymphocytic pleocytosis, elevated IgG indexes, and OCBs Serious fungal infections may exist in the presence of few or no CSF parameter abnormalities

44. Fungal Meningitis Cryptococcus neoformans is the most frequently isolated fungal pathogen from CSF. India ink or nigrosin stains for cryptococcus capsular halos have a sensitivity of about 25%, increasing to 53% with multiple lumbar punctures, and to greater than 90% in untreated HIV-infected patients. If clinical suspicion for dimorphic or filamentous fungi is high, large volumes of CSF (approximately 15–20 mL) are optimal for culture to improve the recovery of fungal organisms

45. INDIA INK PREPARATION A drop of india ink is added to the drop of sediment on a glass slide, coverslip is placed, and examined under x40 objective Cryptococcus appear yeast forms, 2-10microm in diameter surrounded by a large unstained capsule.

46. Tuberculous Meningitis Abnormal CSF with elevated protein and lymphocytic predominance are the hallmark features of tuberculous meningitis. The sensitivity of CSF acid-fast stains for the diagnosis of tuberculous meningitis is highly variable, ranging from 10% to 12% to greater than 50% . Large volumes of CSF, often obtained from multiple lumbar punctures with the use of concentration techniques, are recommended to improve the sensitivity of both acid-fast stain and culture PCR nucleic acid amplification for detecting Mycobacterium tuberculosis DNA-specific sequences have shown great promise in the rapid and accurate diagnosis of tuberculous meningitis

47. Primary Amebic Meningoencephalitis This rare disease is caused by the free-living ameba Naegleria fowleri , Acanthamoeba species, and Balamuthia mandrillaris . Naegleria and Balamuthia are more likely to cause an acute inflammatory response with a neutrophilic pleocytosis, decreased glucose level, elevated protein concentration, and the presence of erythrocytes. Gram stain is always negative Motile Naegleria trophozoites may be visualized by light or phase-contrast microscopy in direct wet mounts, allowing rapid diagnosis. Intact and degenerating organisms may be identified on Wright- or Giemsa-stained cytospins but must be distinguished from macrophages Acridine orange stain is useful in differentiating amebas (brick red) from leukocytes (bright green)

48. Immunocytochemical staining of CSF specimens using an antibody to Naegleria fowleri has been shown to improve microscopic detection of this organism Multiplex PCR based methods are an even more sensitive modality for detecting multiple amebic organisms in CSF

49. REFERENCES HENRY’S CLINICAL DIAGNOSIS AND MANAGEMENT BY LABORATORY METHODS—24 TH EDITION HARRISON’S PRINCIPLES OF INTERNAL MEDICINE—19 TH EDITION TEXTBOOK OF MEDICAL LABORATORY TECHNOLOGY--- RAMNIK SOOD

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