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Evaluation of csf

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  • 1. EVALUATION OF CSF PRESENTER- DR S SAHA PGT MD ( Path) MODERATOR- DR P BHATTACHARYAY Assistant ProfessorDATE- 05.10.2012 DEPARTMENT OF PATHOLOGY RGKMC&H
  • 2. CONTENT
  • 3. HISTORY RELATED CSFPresence of fluid in brain known from ancient time.Hippocrates (460-375BC)commented on water surrounding brain.Discovery of CSF was attributed to Emanuel Swedenberg(1688-1722)Heinrich Irenaus Quincke first discovered lumbar puncture needle.
  • 4. INTRODUCTIONAmount adult- 60-150ml , neonate-10-60ml.Daily production -500 ml. ( @ 0.3-0.4 ml/min)Total volume replaced in every 5-7 hours.Conc. greater than plasma: Na+, Cl-, Mg++ & Glutamine.Conc. less than plasma: Glucose, K, Ca, Fe, Uric acid, Zn, Thyroxin.
  • 5. SECRETION & ABSORPTION70% of CSF secreted – choroid plexus.Remaining CSF- Ventricular ependymal lining cell & cerebral subarachnoid space.CSF pass through medial & lateral foramina, flowing over brain & spinal cord.CSF absorption occur - arachnoid villi.
  • 6. CONTD
  • 7. FUNCTION OF CSFPhysical support to brain.Protective effect against sudden changes in acute venous & arterial blood pressure.Excretory function.Maintain ionic homeostasis.Pathway for transport of certain factor in CNS.
  • 8. HYDROCEPHALUSAbnormal accumulation of cerebrospinal fluid (CSF) in the ventricles, or cavities, of the brain.Increased intracranial pressure inside the skull and progressive enlargement of the head, convulsion, tunnel vision and mental disability. 2 types –communicating & noncommunicating.
  • 9. SPECIMEN COLLECTIONCSF is collected by lumbar puncture needle.20-22 G; Pediatric 25-27 G.Flexible.12 cms in length.Parts: Needle proper & Stilette.Lumbar, cisternal, lateral cervical puncture method.L3-L4 (adult) L4-L5( children)
  • 10. CONTD…..Indications: meningeal infection. subarachnoid haemorrhage. primary or metastatic malignancy de-myelinating disease.Normal CSF pressure-  90-180 mm water in adults. 10-100 mm water in infants & children.Pressure > 250mm water- Intracranial hypertension due to ICH. Tumors. Meningitis .
  • 11. CONTD…Decreased pressure :- Spinal- subarachnoid block. Dehydration. Circulatory collapse. CSF leakage.Significant drop :- Herniation or spinal block above puncture site.Upto 20 ml CSF can be normally removed. Glass tubes should be avoided .Specimen divided serially into 3 sterile tubes :- Tube 1 : Chemical examination & immunologic study. Tube 2 : Microbiologic examination. Tube 3: Total &differential count + cytology .
  • 12. LUMBAR PUNCTURE -PROCEDURE
  • 13. PROCEDURE OF COLLECTION
  • 14. DISEASE DETECTION BY LABORATORY EXAMINATION OF CSF ↑ sensitivity; ↑specificity: Bacterial, TB & fungal meningitis. ↑sensitivity; ↔specificity: Viral meningitis, SAH, MS, CNS syphilis, Paraspinal abscess, Infectious polyneuritis. ↔sensitivity;↑specificity: Meningeal malignancy. ↔sensitivity;↔specificity: Intracranial hemorrhage, Viral encephalitis, Subdural hematoma .
  • 15. RECOMMENDED CSF LABORATORY TESTSRoutine : Opening CSF pressure. Total cell count ( WBC & RBC ). Differential cell count. Glucose ( CSF/plasma ratio ). Total protein.Useful under certain conditions : Cultures. Gram stain , acid fast stain. Fungal & bacterial antigens. Enzymes (LDH,ADA,CK-BB). Cytologic examination. Electrophoresis, PCR. Protein (crp,tau,b-amyloid). VDRL –syphilis.
  • 16. DEPARTMENT OF PATHOLOGY
  • 17. GROSS EXAMINATIONNormally- crystal clear & colourless ; viscosity like water. May be turbid , cloudy frankly purulent or pigmented.Turbidity – WBC > 200 cells/µl,RBC >4OO cells/µl micro organisms, radiographic contrast, aspirated epidural fat, protein > 150 mg/dl.Clot formation – traumatic taps, complete spinal blocks, suppurative & T.B meningitis.Viscosity increased – metastatic mucin producing adenocarcinomas, cryptococcal meningitis.Pink- red CSF – traumatic tap, sub arachnoid haemorrhage, ICH, cerebral infarct.
  • 18. XANTHOCHROMIAPale-pink to yellow colour to the supernatant of the centrifuged CSF.After centrifugation, compare with a tube of distilled water.Due to RBC lysis & Hb breakdown.Orange: OxyHb.Yellow: Bilirubin.Yellow green: Biliverdin.Brown: Meningeal metastatic melanoma.
  • 19. D/ D OF BLOODY CSFIn traumatic tap, hemorrhagic fluid clears between1st & 3rd tubes; remains uniform in SAH.Xanthochromia, Erythrophagocytosis & Hemosiderin-laden macrophages indicate a sub- arachnoid bleed in absence of prior trauma.Commercial latex agglutination immunoassay: For fibrin derivative D-Dimer, is specific for fibrin degradation, is negative in traumatic tap
  • 20. MICROSCOPIC EXAMINATIONCell counts are performed on undiluted CSF in a manual counting chamber.Automated counting of WBC & RBC ( Talstad-1984) – poor precision.Recently- automated flow cytometry of CSF using UF-100 flow cytometer –yields rapid, reliable count; Van Acker,2001.The normal leucocyte count in adults- 0-5 cells/µl. in neonates- 0-30 cells/µl.No RBC should be present in the CSF.
  • 21. DIFFERENTIAL CELL COUNTDC performed in a counting chamber has poor precision.Direct smear of centrifuged specimen- cellular distortion.Filtration & sedimentation are other methods.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, blast-like primitive cell clusters, most likely of germinal matrix origin, are sometimes found in premature infants with intraventricular hemorrhage .
  • 22. CHOROID PLEXUS CELLS IN CSF.CLUSTER OF BLAST LIKE CELLS IN CSF OF PREMATURE NEWBORN.
  • 23. CONTDAmong acellular component –Corpora amylacea- spherical proteinaceous structure, seen commonly brain in elderly, occasionally found in CSF.Powder crystal- By starch granules from powder use in gloves, crystal may be mistaken with spore of cryptoccous.
  • 24. MANUAL COUNTER
  • 25. CSF REFERENCE VALUES FOR DIFFERENTIAL CYTOCENTRIFUGE COUNTSCell type Adult (%) Neonate (%)Lymphocyte 62 34 20 18Monocyte 36 20 72 22Neutrophil 2 5 3 5Histiocyte Rare 5 4Ependymal cell Rare RareEosinophil Rare Rare
  • 26. CAUSE OF CSF NEUTROPHILIAMeningitis Bacterial meningitis Early viral meningoencephalitis Early tuberculous meningitis Early mycotic meningitis Amebic encephalomyelitisOther infections Cerebral abscess Subdural empyema AIDS-related CMV radiculopathyFollowing seizuresFollowing CNS hemorrhage Subarachnoid IntracerebralFollowing CNS infarctReaction to repeated lumbar puncturesInjection of foreign material in subarachnoid space . metastatic tumor in contact with CSF
  • 27. CSF NEUTROPHIL Shortcut to csf neutrophil.lnk
  • 28. CAUSE OF CSF LYMPHOCYTOSISMeningitis: Viral, Tb, Fungal, Syphilis, Leptospira, Early bacterial.Degenerative disorders: MS, GBS, Drug abuse encephalopathy.Handl Syndrome: Headache+ Nero deficit+ Lymphocytosis.Sarcoidosis.Polyneuritis.CND periarteritis.
  • 29. CSF CYTOLOGY (LYMPHOCYTE TO MONOCYTE DISTRIBUTION RATIO 70:30).
  • 30. PLASMA CELL IN CSFPlasma cells, not normally present in CSF, may appear in a variety of inflammatory conditions along with large and small lymphocytes and in association with malignant brain tumors.Multiple myeloma may also rarely involve the meninges.Causes of CSF plasmacytosis :-  Acute viral infections Guillain–Barré syndrome Multiple sclerosis Parasitic CNS infestations Sarcoidosis Subacute sclerosing panencephalitis Syphilitic meningoencephalitis Tuberculous meningitis
  • 31. CONTD….
  • 32. CAUSES OF EOSINOPHIL IN CSFGenerally rare. Most commonly found in parasitic infections.Most common causes of CSF eosinophilia are :-  Acute polyneuritis CNS reaction to foreign material (drugs, shunts) Fungal infections Idiopathic eosinophilic meningitis Idiopathic hypereosinophilic syndrome Parasitic infectionsInfrequently associated with :-  Bacterial meningitis Leukemia/lymphoma Myeloproliferative disorders Neurosarcoidosis Primary brain tumors.
  • 33. CONTD….
  • 34. MONOCYTE & MACROPHAGE IN CSF Increased CSF monocytes lack diagnostic specificity, usually part of a mixed cell reaction, seen in tuberculous and fungal meningitis. Macrophages with phagocytosed erythrocytes (erythrophages) appear from 12-48 hours following a subarachnoid hemorrhage or traumatic tap.
  • 35. OTHER CELLS IN CSFCerebrospinal fluid examination for tumor cells has moderate sensitivity and high specificity (97-98%).Sensitivity depends on the type of tumor.Leukemic patients has the highest sensitivity (about 70%), followed by metastatic carcinoma (20-60%) and primary CNS malignancies (30%).Leukemic involvement of the meninges is more frequent in ALL than in AML.A leukocyte count over 5 cells/μL with unequivocal lymphoblasts in cytocentrifuged preparations is commonly accepted as evidence of CSF involvement.High grade NHL like large cell immunoblastic,lymphoblastic & burkitt’s lymphoma may involve meninges.
  • 36. BLAST CELL IN CSF
  • 37. LYMPHOMA CELL IN CSF
  • 38. DEPARTMENT OF BIO- CHEMISTRY
  • 39. PROTEIN MEASUREMENT Protein Plasma: CSF80% Of CSF protein derived from blood, in Prealbumin 14 concentration of less Albumin 236 than 1% of plasma Transferrin 142 level. Ceruloplasmin 366 IgG 802Normal value-15-45 IgA 1346 mg/dl. (adult)90mg/dl (term infant) Fibrinogen 4940115 mg/dl(preterm infant)
  • 40. CONDITION ASSOCIATED WITH INCREASED CSF PROTEINTraumatic spinal punctureIncreased blood–CSF permeability Arachnoiditis (e.g., following methotrexate therapy) Meningitis (bacterial, viral, fungal, tuberculous) Hemorrhage (subarachnoid, intracerebral)Endocrine/metabolic disorders Milk–alkali syndrome with hypercalcemia Diabetic neuropathy Hereditary neuropathies and myelopathies Decreased endocrine function (thyroid, parathyroid) Other disorders (uremia, dehydration)Drug toxicity Ethanol, phenothiazines, phenytoinCSF circulation defects Mechanical obstruction (tumor, abscess, herniated disk) Loculated CSF effusionIncreased IgG synthesis Neurosyphilis, multiple sclerosis Subacute sclerosing panencephalitisIncreased IgG synthesis and blood–CSF permeability Guillain–Barré syndrome Collagen vascular diseases (e.g., lupus, periarteritis) Chronic inflammatory demyelinating polyradiculopathy
  • 41. METHODOLOGYTurbidimetric method – based on TCA or sulphosalicylic acid & sodium sulphate for protein precipitation. This method is simple, rapid, require no special instrumentation.Colorimetric methods – Lowry method, dye binding method using CBB, & modified biuret method.CBB method is rapid, highly sensitive, use in small sample size.Immunologic method measure specific protein, require 25-30µl of CSF, simple to perform.
  • 42. CSF SERUM ALBUMIN RATIOAssess permeability of BBB.Normal ratio- 1:230.CSF/ serum albumin index- CSF albumin/ serum albumin. <9= Intact barrier. 9-14= Slight impairment of barrier. 14-30= Moderate impairment of barrier. >30= Severe impairment of barrier.
  • 43. CSF SERUM IMMUNOGLOBULIN RATIOCSF/ serum IgG ratio = CSF IgG (mg/dl) serum IgG (g/dl)Normal ratio is 1:390.CSF IgG index = CSF IgG ( mg/dl) x serum albumin (g/dl) serum IgG ( g/dl) x CSF albumin (mg/dl)It can increase in intrathhecal IgG synthesis or increased IgG crossover. Normal level- 3.0-8.7.% of CSF IgG increases in multiple sclerosis.Increased CSF IgM & kappa light chains- marker of MS.
  • 44. OTHER CSF PROTEIN Proteins Major diseases Alpha-2-macroglobulin Subdural hemorrhage, bacterial meningitis. Beta-amyloid and tau proteins Alzheimers disease. Beta-2-microglobulin Leukemia/lymphoma & Bechet’s syndrome. C-reactive protein Bacterial and viral Meningitis. Fibronectin Lymphoblastic leukemia,AIDS Methhemoglobin Mild subarachnoid/subdural haemorrhage. Myelin basic protein Multiple sclerosis, other tumors. Protein 14-3-3 Creutzfeldt–Jakob disease Transferrin CSF leakage (otorrhea)
  • 45. CSF GLUCOSEDerived from blood glucose.Fasting CSF glucose- 50-80 mg/dl .Normal CSF/ plasma glucose level – 0.3-0.9.CSF values below 40 mg/dl or ratios below 0.3 are considered to be abnormal.Hypoglycorrhachia is a characteristic finding of bacterial, tuberculous, and fungal meningitis.Decreased CSF glucose – due to increased anaerobic glycolysis in brain tissue by leukocytes and impaired transport into the CSF.
  • 46. CONTDCSF glucose levels normalize before protein levels and cell counts during recovery from meningitis , 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.
  • 47. CSF LACTATECSF & blood lactate level are largely independent.Normal level Newborn-10-60 mg/dl. Older child & adult-9-26 mg/dl.Lactate measurement - helps in differentiating viral meningitis from bacterial, mycoplasma, fungal, and tuberculous meningitisIn viral meningitis - lactate levels are usually below 25 mg/dL and almost always less than 35 mg/dL, whereas bacterial meningitis has levels above 35 mg/dl.Persistently elevated ventricular CSF lactate levels are associated with a poor prognosis in patients with severe head injury.
  • 48. CSF F-2 ISOPROSTANESF-2 isoprostane level are inceased in patients with alzheimer disease.
  • 49. CSF ENZYMESAdenosine deaminase (ADA) Since ADA is particularly abundant in T lymphocytes, which are increased in tuberculosis – useful in the diagnosis of pleural, peritoneal, and meningeal tuberculosis. More recently, ADA levels greater than 15 U/L were found to be a strong indication of tuberculous meningitis since nontuberculous meningitis consistently had levels less than 15 U/L.Creatine kinase (CK) Increased CSF CK activity has been reported in numerous CNS disorders including hydrocephalus, cerebral infarction, various primary brain tumors, and subarachnoid hemorrhage. Since the CK-BB isoenzyme comprises about 90% of brain CK activit, so CK isoenzyme measurements are more specific for CNS disorders than total CK.
  • 50. CONTD…Lactate dehydrogenase- LDH-1, LDH-2 isoenzyme is very high in brain. CSF –LDH level is high – CNS leukaemia, lymhoma, metastatic carcinoma, bacterial meningitis, SAH.Lysozyme – Since the enzyme is particularly rich in neutrophil and macrophage,its activity is very low in normal CSF. However, CSF lysozyme activity is significantly increased in patients with both bacterial and tuberculous meningitis.Ammonia, Amines, and Amino Acids – CSF ammonia levels vary from 30-50% of the blood values. Elevated levels are generally proportional to the degree of existing hepatic encephalopathy.
  • 51. CSF ELECTROLYTEOsmolality 280–300 mOsm/LSodium 135–150 mEq/LPotassium 2.6–3.0 mEq/LChloride 115–130 mEq/LCarbon dioxide 20–25 mEq/LCalcium 2.0–2.8 mEq/LMagnesium 2.4–3.0 mEq/LLactate 10–22 mg/dLpH Lumbar fluid 7.28–7.32
  • 52. CSF TUMOR MARKERSCarcinoembryonic antigen (CEA)- Metastatic carcinoma of leptomeninges.Human chorionic gonadotropin (HCG)- Choriocarcinoma and malignant germ cell tumors with a trophoblastic component.Alpha-fetoprotein- Increased in germ cell tumors with yolk sac elements.Elevation of CSF ferritin is a sensitive indicator of CNS malignancy but has very low specificity since it is also increased in patients with inflammatory disorders.
  • 53. DEPARTMENT OF MICROBIOLOGY
  • 54. BACTERIAL MENINGITISA thorough and prompt microbiologic examination of CSF – useful for a definitive diagnosis.Bacterial Meningitis- The most common agents of bacterial meningitis are-group B streptococcus (neonates)Escherichia coli (newborn to 1 month)Neisseria meningitidis (3 months and older)Streptococcus pneumoniae (3 months and older)Haemophilus influenzae (3 months to 18 years)Listeria monocytogenes (neonates, elderly, alcoholics, and immunosuppressed) Cerebrospinal fluid shunts, head trauma, and neurosurgery place patients at risk for CNS infections from Staphylococcus species, aerobic Gram-negative bacilli, and Propionibacterium species.
  • 55. CSF GRAM STAIN SHOW GRAM NEGATIVE DIPLOCOCCI
  • 56. CSF GRAM STAIN SHOW GRAM POSITIVE ROD
  • 57. CONTD….The Gram stain remains an accurate, rapid method to diagnose CNS infections.All specimens should be concentrated by centrifugation before Gram stain and culture.Recent tools used -  Binax NOW® Streptococcus pneumoniae antigen test-an immunochromatographic membrane assay. Latex agglutination bacterial antigen tests (BAT) -detect H. influenzae, N. meningitidis, S. pneumoniae, and beta- hemolytic group-B streptococcus. The limulus lysate assay - very sensitive test for the presence of endotoxin, a product of most Gram-negative bacteria. It is particularly useful as a rapid test in the newborn where early diagnosis and treatment are critical. Polymerase chain reaction .
  • 58. SPIROCHETAL MENINGITIS.The diagnosis of CNS infection in patients with syphilis relies primarily on CSF parameters and serologic testing.Abnormalities in CSF protein and cell counts are common in syphilitic meningitis, although they are nonspecific.The standard nontreponemal test performed on CSF is the VDRL . If there are few erythrocytes contaminating the CSF, the VDRL specificity is high but sensitivity is low.Treponemal tests, such as the treponemal antibody absorption (FTA-ABS), are both sensitive and specific for syphilis.
  • 59. VIRAL MENINGITISEnteroviruses (echoviruses, Coxsackieviruses, polioviruses) are responsible for up to 80% of meningitis cases, with a seasonal peak in late summer.Most patients present with a CSF pleocytosis,neutrophils may be observed early in the infection, patients soon develop a predominance of lymphocytes.Reverse transcriptase polymerase chain reaction (RT-PCR) is significantly more sensitive than cell culture – evolving as the ‘gold standard’ for the diagnosis of viral meningitis.PCR amplification of HSV-2 DNA in CSF may be useful in the early diagnosis of HSV encephalitis.False negatives might occur in very early infections and bloody taps.Serum and CSF serologies for HSV antibody may be useful, when PCR becomes negative ( after 2 weeks.)
  • 60. HIV MENINGITISA wide variety of CSF abnormalities are lymphocytic pleocytosis, elevated IgG indexes, and oligoclonal bands.Identifying opportunistic infections is the most important indication for examining the CSF.Serious fungal infections may exist in the presence of little or no CSF parameter abnormalities
  • 61. FUNGAL MENINGITISCryptococcus is the most frequently isolated fungal pathogen from CSF.India ink or nigrosin stains show cryptococcus capsular halos.Detection of cryptococcal antigen from sera or CSF using latex agglutination has high sensitivity, ranging from 60-95%False negatives due to a prozone effect, low concentration of polysaccharide, Early disease, intraparenchymal infection, infection with nonencapsulated Cryptococcus neoformans variants may occur.Conversely, sera or CSF from patients with rheumatoid factor or Trichosporon beigelii infections may be falsely positive.If clinical suspicion for dimorphic or filamentous fungi is high, large volumes of CSF (approximately 15-20 mL) are optimal for culture to improve recovery of fungal organisms.
  • 62. TUBERCULAR 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.PCR nucleic acid amplification for detecting Mycobacterium tuberculosis DNA-specific sequences - yields rapid and accurate diagnosis of tuberculous meningitis.DOT enzyme linked immunosorbent assay (DOT ELISA) has been standardized to detect tuberculosis antigens and antibodies against M. tuberculosis in CSF.Other tests,e.g-ligase chain reaction amplification is reportedly a rapid method for the early diagnosis of tuberculous meningitis.Moreover, adenosine deaminase (ADA) levels are significantly higher in tuberculous meningitis than in other types of meningitis and CNS disorders.Indeed, a level greater than 15 U/L is a strong indicator of tuberculous meningitis ( Choi, 2002 ).
  • 63. PRIMARY AMEBIC MENINGOENCEPHALITISThis rare disease is caused by the free-living ameba Naegleria fowleri or Acanthamoeba species.Naegleria is more likely to cause an acute inflammatory response with a neutrophilic pleocytosis, decreased glucose level, an elevated protein concentration, and the presence of erythrocytes.Gram stain is always negative.Acanthamoeba more often produces a granulomatous meningitis.Motile Naegleria trophozoites may be visualized by light or phase-contrast microscopy in direct wet mounts, allowing rapid diagnosis.Can also be identified on Wrights or Giemsa-stained cytospins, but must be distinguished from macrophages. Acridine orange stain is useful to differentiate ameba (brick red) from leukocytes (bright green).
  • 64. LUMBAR CSF FINDINGS IN MENINGITIS TEST BACTERIAL VIRAL FUNGAL TUBERCULOUSPRESSURE INCREASE NORMAL VARIABLE VARIABLECOUNT >1000 <100 VARIABLE VARIABLEDIFFERENTI NEUTROHIL LYMPHOCYTE LYMPHOCYTE LYMPHOCYTEAL COUNTPROTEIN Mild↑ NORMAL ↑ ↑GLUCOSE <40mg% NORMAL ↓ ↓LACTATE Mild↑ N-Mild↑ Mild-Mod ↑ Mild-Mod ↑
  • 65. REFERENCE Henry’s Clinical Diagnosis & Management by Laboratory Methods, 21st edition, 426-427, Year 2007. Todd-Sanford Clinical Diagnosis by laboratory methods, 15th Edition, 1254-1265, Year 1969. Medical lab manual for tropical countries, Vol II, 1st edition, 160-173, Year 1984. www.google.com images.google.com images.yahoo.com www.answers.com www.wikipedia.org
  • 66. THANK YOU