Csf composition and significance by Dr. Ashok KUmar J

CSF analysis

1. CSF Composition and significance Dr. Ashok Kumar .J. International Medical School Management and Science University Malaysia

2. CSF Pressure CSF pressure changes with Posture Blood pressure Venous return factors that increase cerebral blood flow Normal opening pressure is 70 to 150 mm of water in left lateral decubitus position Slightly higher in sitting up and varies 10 mm water with respiration In infants and young children the normal range is 10 to 100 mm of water Attain adult value by 6 to 8 years of age Pressure may be as high as 250 mm of water in obese individuals

3. Pressure above 250 mm of water are diagnostic intracranial hypertension Meningitis Intracranial hemorrhage Tumors Thrombosis of venous sinuses Cerebral edema Conditions inhibiting absorption of CSF Opening pressure elevated may be the only abnormality in cryptococcal meningitis Decreased pressure Spinal subarachnoid block Dehydration Circulatory collapse (shock) CSF leakage Significant drop in CSF pressure after taking 1 to 2 ml of CSF suggests herniation or spinal block above the site of puncture

4. CSF specimen should be sent to the lab immediately Delay might initiate cellular degradation (which begins within 1 hour of collection) Indication for CSF analysis (Can be divided in to 4 categories) 1. Meningeal infection 2. Subarachnoid hemorrhage 3. Primary or metastatic malignancy 4. Demylinating disease Identification of infectious meningitis especially bacterial is important indication

5. CSF CSF collected into three sterile tubes for 1. Chemical and immunological studies 2. Microbiological examination 3. Cell count and differentiation An additional tube may be inserted for cytology if malignancy is suspected Glass tubes should be avoided since cells adhere to glass affect the cell count First tube should never be used for microbiological examination ( it may be contaminated with skin bacteria)

6. Gross examination Normal CSF Crystal clear Has viscosity similar to water Abnormal Cloudy Frankly purulent Pigmented or tinted Turbidity or cloudiness begins to appear with Leucocyte count over 200 cells / μ L or Red cell count of 400 cell/ μ L Grossly bloody CSF have RBC count greater than 6000 / μ L Turbidity Coagulum Color

7. Gross examination Experienced observer may be able to detect cell count less than 50 cells / μ L with unaided eye by observing for Tyndll’s effect Turbidity or cloudiness Microorganisms bacteria, fungi, amoeba Radiographic contrast material Aspirated epidural fat Protein level greater than 150 mgs/dl Turbidity Coagulum Color

8. Clot formation may be seen in traumatic tap Not seen in subarachnoid hemorrhage Gross examination Fine surface pellicles may be seen after refrigeration for 12 to 24 hours Clot may interfere with cell count accuracy by entrapping inflammatory cells Turbidity Coagulum Color

9. Gross examination Turbidity Coagulum Color Viscous CSF may be encountered in Metastatic mucin producing adenocarcinoma Cryptococcal meningitis Color : Pink red CSF - indicates presence of blood May be derived from: Subarachnoid hemorrhage Intracerebral hemorrhage cerebral infract Traumatic tap

10. Xanthochromia A pale pink to yellow color in the supernatant of centrifuged CSF, although other colors may be present Pale pink to orange xanthochromia from released oxyhemoglobin usually detected 2–4 hours after the onset of subarachnoid hemorrhage (although it may take as long as 12 hours) Peak intensity occurs in about 24-36 hours and then gradually disappears over the next 4-8 days Yellow xanthochromia is derived from bilirubin develops about 12 hours after a subarachnoid bleed peaks at 2-4 days, but may persist for 2-4 weeks

11. To detect xanthochromia The CSF should be centrifuged and the supernatant fluid compared with a tube of distilled water Pink RBC lysis/hemoglobin breakdown products Yellow Hyperbilirubinemia CSF protein > 150 mg/dL (1.5 g/L) Orange RBC lysis/hemoglobin breakdown products Hypervitaminosis A (carotenoids) Yellow-green Hyperbilirubinemia (biliverdin) Brown Meningeal metastatic melanoma CSF supernatant color Associated diseases/disorders

12. CSF xanthochromia may also be due to the following : Oxyhemoglobin resulting from artifactual red cell lysis caused by detergent contamination of the needle or collecting tube delay of more than 1 hour without refrigeration before examination Bloody traumatic taps : A traumatic tap occurs in about 20% of lumbar punctures Rifampin therapy (red-orange)

13. Distinction of a traumatic puncture from pathologic hemorrhage is of vital importance Traumatic tap : Hemorrhagic fluid usually clears between the first and third collected tubes S ubarachnoid hemorrhage : Remains relatively uniform Traumatic tap : microscopic evidence of erythrophagocytosis , or hemosiderin-laden macrophages indicate a subarachnoid bleed in the absence of a prior traumatic tap Hemosiderin-laden macrophages ( siderophages ) from the CSF of a patient with subarachnoid hemorrhage. Hemosiderin crystals (golden-yellow) are also present

14. Chemical Analysis Analyte   Conventional units Protein 15–45 mg/dL   Pre-albumin 2–7 %     Albumin 56–76%     Alpha-1 - globulin 2–7%     Alpha-2-globulin 4–12%     Beta-globulin 8–18%     Gamma-globulin 3–12%  

15. Total Protein. Over 80% of the CSF protein content is derived from blood plasma, in concentrations of less than 1% of the plasma level Protein CSF (mg/L) Prealbumin 17.3 Albumin 155.0 Transferrin 14.4 Ceruloplasmin 1.0 IgG 12.3 IgA 1.3 Alpha-2-microglobulin 2.0 Fibrinogen 0.6 IgM 0.6 Beta-lipoprotein 0.6

16. CSF protein levels of 15-45 mg/dl accepted as the ‘normal’ reference range infants have significantly higher CSF protein levels than older children and adults CSF protein concentration fall rapidly from birth to 6 months of age (40 mg/ dL ) Plateaued between 3 and 10 years (32 mg/ dL ) Then rose slightly from 10-16 years (41 mg/ dL ) for term infants and for preterm infants the upper levels were 150 mg/dl and 170 mg/dl

17. Increased CSF Total Protein May be caused by Increased permeability of the blood–brain barrier Decreased resorption at the arachnoid villi Mechanical obstruction of CSF flow due to spinal block above the puncture site An increase in intrathecal immunoglobulin synthesis

18. Conditions Associated Traumatic spinal puncture Increased blood–CSF permeability Arachnoiditis (e.g., following methotrexate therap ) Meningitis (bacterial, viral, fungal, tuberculous ) Hemorrhage (subarachnoid, intracerebral ) 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 )

19. Qualitative tests for globulins Pandy’s test : One drop of CSF is added to one ml of Pandy’s reagent (clear 7% solution of phenol in water) A turbidity indicates increased globulin in CSF Nonne-Apelt test : One ml of CSF is slowly layered over one ml of ammonium sulphate solution A white ring at the junction of the two liquids indicates the increased globulins

20. Turbidimetric methods Based on trichloroacetic acid (TCA) or sulfosalicylic acid (SSA) and sodium sulfate for protein precipitation Simple , rapid, and require no special instrumentation Quantitative test

21. Albumin and IgG Measurements Permeability of the blood–brain barrier may be assessed by immunochemical quantification of the CSF albumin-to-serum albumin ratio in grams per deciliter (g/ dL ) The normal ratio of 1:230 ( 0.004) CSF/serum albumin index Arbitrarily calculated as follows CSF/ Serum albumin index = CSF albumin (mg/dl) Serum albumin (g/dl)

22. An index value less than 9 is consistent with an intact barrier Slight impairment is considered with index values of 9-14 Moderate impairment with values of 14-30 Severe impairment at values greater than 30 Traumatic tap invalidates the index calculation

23. CSF IgG index Elevated “ IgG index” indicates increased production of IgG within the CNS : e.g Multiple sclerosis CSF IgG index = CSF IgG mg/ dl X Serum IgG g/ dl X CSF albumin mg/ dl Serum albumin g/ dl Normal upper limit is 0.8

24. Protein Major diseases/disorders Alpha-2-macroglobulin Subdural hemorrhage, bacterial meningitis Beta-amyloid and tau proteins Alzheimer's disease Beta-2-microglobulin Leukemia/lymphoma C-reactive protein Bacterial and viral meningitis Fibronectin Lymphoblastic leukemia, AIDS, meningitis Methemoglobin Mild subarachnoid/subdural hemorrhage Myelin basic protein Multiple sclerosis, tumors, others Protein 14-3-3 Creutzfeldt–Jakob disease Transferrin CSF leakage (otorrhea, rhinorrhea) Approximately 300 different proteins have been identified in CSF

25. Cerebrospinal fluid leakage otorrhea rhinorrhea usually presents as otorrhea or rhinorrhea following head trauma, in some cases beginning months to years after the injury Recurrent meningitis is a serious complication making accurate identification of the leaking fluid very important Transferrin- an iron-binding glycoprotein synthesized primarily in the liver Two transferrin isoforms are present in the CSF Major isoform (beta-1-transferrin ) is present in all body fluids The second isoform (beta-2-transferrin), present only in the central nervous system - is produced in the central nervous system by the catalytic conversion of beta-1-transferrin by neuraminidase

26. Methemoglobin and Bilirubin Subarachnoid and intracerebral hemorrhage are readily identified by computed tomography (CT) M ild subarachnoid hemorrhage Small subdural or cerebral hematomas Blood seepage from aneurysm or neoplasm from small cerebral infarcts are often not identified by this technique CSF spectrophotometric analysis has been shown to detect methemoglobin in colorless CSF (< 0.3 μmol /L) Increase in CSF bilirubin is now recognized as the key finding supporting the diagnosis of subarachnoid hemorrhage

27. Glucose Derived from blood glucose fasting CSF glucose levels are normally 50-80 mg/ dL (about 60% of plasma values) Results should be compared with plasma levels, ideally following a 4-hour fast, for adequate clinical interpretation The normal CSF/plasma glucose ratio varies from 0.3 - 0.9 CSF values below 40 mg/ dL are considered to be abnormal Hypoglycorrhachia is a characteristic finding of bacterial, tuberculous , and fungal meningitis

28. Decreased CSF glucose results from Increased anaerobic glycolysis in brain tissue and leukocytes Impaired transport into the 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.

29. Lactate CSF and blood lactate levels are largely independent of each other Reference interval for older children and adults is 9.0-26 mg/ dL Newborns have higher levels, ranging from about 10-60 mg/ dL for the first 2 days, and 10-40 mg/ dL for days 3 to 10 Elevated CSF lactate levels reflect CNS anaerobic metabolism due to tissue hypoxia.

30. Lactate measurement has been used as an adjunctive test in differentiating viral meningitis from bacterial, mycoplasma, fungal, and tuberculous meningitis in which routine parameters yield equivocal results . Viral meningitis, lactate levels are usually below 25 mg/ dL (almost always less than 35 mg/ dL ) Bacterial meningitis typically has levels >35 mg/ dL Persistently elevated ventricular CSF lactate levels are associated with a poor prognosis in patients with severe head injury

31. F2-isoprostanes F2-isoprostanes are increased in diseased regions of the brain in patients with Alzheimer's disease (AD) CSF F2-isoprostanes are also elevated in patients with probable AD In conjunction with CSF tau and beta-amyloid protein, the measurement of CSF F2-isoprostanes appear to enhance the accuracy of the laboratory diagnosis of AD

32. Enzymes A wide variety of enzymes derived from brain tissue, blood, or cellular elements have been described in the CSF. Although CSF enzyme assays are not commonly used in the diagnosis of CNS diseases, there are diseases/disorders whereby they may prove useful .

33. Adenosine deaminase (ADA). ADA catalyzes the irreversible hydrolytic deamination of adenosine to produce inosine . ADA is particularly abundant in T lymphocytes Which are increased in tuberculosis Higher ADA levels are present in tuberculous infections than in viral, bacterial, and malignant diseases ADA levels greater than 15 U/L were found to be a strong indication of tuberculous meningitis Non- tuberculous meningitis consistently had levels less than 15 U/L

34. Creatine kinase (CK). Brain tissue is rich in CK Increased CSF CK activity has been reported in disorders Hydrocephalus C erebral infarction Primary brain tumors Subarachnoid hemorrhage Head trauma, CSF CK levels correlate directly with the severity of the concussion CK-BB isoenzyme comprises about 90% of brain CK activity and mitochondrial CK ( CKmt ) the other 10%, CK isoenzyme measurements are more specific for CNS disorders

35. CSF CK-BB is increased about 6 hours following an ischemic or anoxic insult Global brain ischemia following respiratory or cardiac arrest results in diffuse cerebral injury with peak CK-BB levels in about 48 hours CSF CK-BB activity less than 5 U/L (upper normal level) indicates minimal neurologic damage 5-20 U/L indicates mild to moderate CNS injury Levels between 21-50 U/L are commonly correlated with death. Death occurs in essentially all patients with levels above 50 U/L.

36. Lactate dehydrogenase (LD). LD activity is high in brain tissue A total LD activity of 40 U/L is a reasonable upper limit of normal for adults and 70 U/L for neonates LD levels are also increased in patients with CNS leukemia, lymphoma, metastatic carcinoma, bacterial meningitis, and subarachnoid hemorrhage

37. Lysozyme. Normal CSF activity is very low Lysozyme ( muramidase ) catalyzes the depolymerization of mucopolysaccharides . Since the enzyme is particularly rich in neutrophil and macrophage lysosomes, its activity is very low in normal CSF CSF lysozyme activity is significantly increased in patients with both bacterial and tuberculous meningitis

38. Ammonia, Amines, and Amino Acids. CSF ammonia levels vary from 30-50% of the blood values Measurement of CSF ammonia has little, if any, clinical value Cerebral glutamine, synthesized from ammonia and glutamic acid, Serves as the means for CNS ammonia removal CSF glutamine levels reflect the concentration of brain ammonia Values over 35 mg/ dL are usually associated with hepatic encephalopathy Elevated CSF glutamine levels have also been reported in patients with encephalopathy secondary to hypercapnia and sepsis

39.   Osmolality 280–300 mOsm/L   Sodium 135–150 mEq/L   Potassium 2.0–3.5 mEq /L   Chloride 120–130 mEq /L   Carbon dioxide 20–25 mEq/L   Calcium 2.0–2.8 mEq/L   Magnesium 2.4–3.0 mEq /L   Lactate Glutamine Iron Cholesterol Creatinine Urea 10–22 mg/ dL 6 – 11 mg/ dL 1 – 2 mg/ dL 0.2 – 0.6 mg/ dL 0.5 – 1.2 mg/ dL 6 – 16 mg/ dL

40. pH   Lumbar fluid 7.28–7.32   Cisternal fluid CSE bicarbonate 7.32–7.34 18 mmol /L P co 2   Lumbar fluid 44–50 mmHg   Cisternal fluid 40–46 mmHg P o 2 40–44 mmHg

41. CSF chloride CSF chloride level is more compared to plasma chloride May be due to difference in the concentration of protein in plasma and CSF CSF concentration of chloride decreases in meningitis – especially in tubercular meningitis

42. Microscopic Examination Total Cell Count Cell counts are performed on undiluted CSF in a manual counting chamber automated flow cytometry of CSF, using the UF-100 flow cytometer, was found to yield rapid and reliable WBC and RBC counts

43. Cell type Adults (%) Neonates (%) Lymphocytes 62 ± 34 20 ± 18 Monocytes 36 ± 20 72 ± 22 Neutrophils 2 ± 5 3 ± 5 Histiocytes Rare 5 ± 4 Ependymal cells Rare Rare Eosinophils Rare Rare Correction when blood contaminated CSF In the presence of a normal peripheral blood RBC count and serum protein, these corrections amount to about 1 WBC for every 700 RBCs and 8 mg/ dL protein for every 10 000 RBC/ μL CSF Reference Values for Differential Cytocentrifuge Counts

44. 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 Cluster of blast-like cells in CSF from premature newborn

45. Increased CSF neutrophils occur in numerous conditions Early bacterial meningitis - the proportion of PMNs usually exceeds 60 % About one-quarter of cases of early viral meningitis the proportion of PMNs also increases

46. Causes of Increased CSF Neutrophils Meningitis   Bacterial meningitis   Early viral meningoencephalitis   Early tuberculous meningitis   Early mycotic meningitis   Amebic encephalomyelitis Other infections   Cerebral abscess   Subdural empyema Following CNS hemorrhage   Subarachnoid    Intracerebral

47. Lymphocytosis (> 50%) is not uncommon in early acute bacterial meningitis When the CSF leukocyte count is under 1000/ μL Atypical reactive lymphoplasmacytoid and immunoblastic variants may be present. Blast-like lymphocytes may be seen admixed with small and large lymphocytes in the CSF of neonates.

48. Causes of CSF Lymphocytosis Meningitis   Viral meningitis    Tuberculous meningitis   Fungal meningitis   Syphilitic meningoencephalitis    Leptospiral meningitis    Degenerative disorders    Multiple sclerosis    Guillain – Barré syndrome  

49. Causes of CSF Plasmacytosis Acute viral infections Guillain – Barré syndrome Multiple sclerosis Parasitic CNS infestations Sarcoidosis Subacute sclerosing panencephalitis Syphilitic meningoencephalitis Tuberculous meningitis 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

50. Typical Lumbar CSF Findings in Meningitis Test Bacterial Viral Fungal Tuberculous Opening pressure Elevated Usually normal Variable Variable Leukocyte count ≥ 1000/μ L < 100/μ L Variable Variable Cell differential Mainly neutrophils Mainly lymphocytes Mainly lymphocytes Mainly lymphocytes Protein Mild–marked increase Normal–mild increase Increased Increased Glucose Usually ≤ 40 mg/ dL Normal Decreased Decreased: may be < 45 mg/ dL CSF-to-serum glucose ratio Normal–marked decrease Usually normal Low Low Lactic acid Mild–marked increase Normal–mild increase Mild–moderate increase Mild–moderate increase

51. Thank you