Cerebrospinal fluid analysis Acknowledgements Addisa Ababa University Jimma University Hawassa University Haramaya University University of Gondar American Society for Clinical Pathology ID: 1038116
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1. Body fluid analysis
2. Chapter One Cerebrospinal fluid analysis
3. AcknowledgementsAddisa Ababa UniversityJimma UniversityHawassa UniversityHaramaya UniversityUniversity of GondarAmerican Society for Clinical Pathology Center for Disease Control and Prevention-Ethiopia
4. Chapter outlineIntroduction to Cerebrospinal fluidRoutine laboratory assaysCollection of sampleGross appearanceCell countsChemical analysisMorphological ExaminationMicrobiological ExaminationSerological Examination
5. Cerebral Spinal Fluids
6. Learning ObjectivesUpon completion of this chapter the student will be able to:1.Describe the formation of CSF from blood. 2. Describe the appearance of normal CSF. 3. Define xanthochromia and state its significance. 4. Differentiate between CSF specimens caused by intracranial hemorrhage and a traumatic tap. 5 Differentiate between CSF specimens caused by intracranial hemorrhage and a traumatic tap.
7. Learning Objectives (continued)Given the laboratory observations of a bloody CSF, evaluate the supernatant and propose the type of pathological condition associated with a clear supernatant versus a xanthochromic supernatant. Compare the difference of pathological conditions associated with the types of cells observed in a CSF. List the normal range of glucose, protein, and cell count for a CSF. Evaluate abnormal laboratory results with a pathological condition related to CSF. Discuss appropriate collection requirements for CSF following a lumbar puncture.
8. CSF Formation and PhysiologyFirst recognized by Cotugno in 1764, cerebrospinal fluid(CSF) is a major fluid of the body. CSF provides a physiologic system to supply nutrients to the nervous tissue, remove metabolic wastes, and produce a mechanical barrier to cushion the brain and spinal cord against trauma.CSF is produced in the choroid plexuses of the twolumbar ventricles and the third and fourth venticles. Inadults, approximately 20 mL of fluid is produced every hour..
9. Formation and PhysiologyThe fluid flows through the subarachnoid space located between the arachnoid and pia mater. To maintain a volume of 90 to 150 mL in adults and 10 to 60 mL in neonates. the circulating fluid is reabsorbed back into theblood capillaries in the arachnoid granulations/villae at a rate equal to its production
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11. Cerebrospinal fluid (CSF)Fluid in the space called sub-arachnoid space between the arachnoid mater and pia materProtects the underlying tissues of the central nervous system (CNS)Serve as mechanical buffer to prevent trauma,regulate the volume of intracranial pressurecirculate nutrientsremove metabolic waste products from the CNS Act as lubricantHas composition similar to plasma except that it has less protein, less glucose and more chloride ion
12. Cerebrospinal fluid analysisCollection of CSF sampleRoutine Laboratory assays of CSFGross appearanceRBC &WBC countsMorphological ExaminationMicrobiological ExaminationSerological Examination
13. CSF cont’dMaximum volume of CSFAdults 150 mL Neonates 60 mLRate of formation in adult is 450-750 mL per day or 20 ml per hour reabsorbed at the same rate to maintain constant volumeCollection by lumbar puncture done by experienced medical personnelAbout 1-2ml of CSF is collected for examination lumbar puncture is made from the space between the 4th and 5th lumbar vertebrae under sterile conditions.
14. Collecting CSF specimen Location of CSFCollected in three sequentially labeled tubesTube 1 Chemical and immunologic testsTube 2 MicrobiologyTube 3 Hematology (gross examination, total WBC & Diff)This is the list likely to contain cells introduced by the puncture procedure
15. Report the appearance of the c.s.f.As soon as the c.s.f. reaches the laboratory, note its appearance. Report whether the fluid:– is clear, slightly turbid, cloudy or definitely purulent (looking like pus),– contains blood,– contains clots.Normal c.s.f. Appears clear and colourless.
16. Report the appearance of the c.s.f.Purulent or cloudy c.s.f. Indicates presence of pus cells, suggestive of acute pyogenic bacterial meningitis.Blood in c.s.f. This may be due to a traumatic (bloody) lumbar puncture or less commonly to haemorrhage in the central nervous system. When due to a traumatic lumbar puncture, sample No. 1 will usually contain more blood than sample No. 2.
17. Report the appearance of the c.s.f.Following a subarachnoid haemorrhage, the fluid may appear xanthrochromic, i.e. yellow-red (seen after centrifuging).Clots in c.s.f. Indicates a high protein concentrationwith increased fibrinogen, as can occur withpyogenic meningitis or when there is spinal constriction.
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19. Clinical SignificanceDiagnosis of meningitis of bacterial, fungal, mycobacterial and amoebic origin or differential diagnosis of other infectious diseasessubarachnoid hemorrhage or intracerebral hemorrhage
20. Principle of the testCSF specimen examined visually and microscopically and total number of cells can be counted and identifiedSpecimen: the third tube in the sequentially collected tubesmust be counted within 1 hour of collection (cells disintegrate rapidly). If delay is unavoidable store 2-8oC.All specimens should be handled as biologically hazardous
21. Uptake and Utilization of GlucoseGlucose is major energy substrate for brain as well as a major carbon source for many molecules. Brain uses 20-25% of total oxygen and 15% of cardiac output is directed to CNS.
22. Glucose UtilizationWhen body glucose supply is decreased, other organs decrease glucose utilization to maintain adequate supply of glucose to brain. Other organs can readily switch to oxidation of another substrate for energy production. Under certain conditions, such as chronic starvation, the brain can oxidize other substances but maintains a minimal obligatory requirement for glucose.
23. Brain Utilization of GlucoseGlycolysis--conversion to lactic acid Hexokinase has high activity in brainServes to trap glucose and maintain concentration gradient for diffusion
24. Brain Utilization of Glucose2-deoxyglucose is also taken up by brain and phosphorylated by hexokinase, but then becomes trappedMarker to correlate changes in neural activity with changes in glucose utilization. Enolase, an enzyme in glycolytic pathway, exists in nerve cells in unique isoform (neuron specific enolase, NSE)Used as a specific marker for neurons.
25. Brain Utilization of GlucosePentose shunt Provides source of D-ribose for synthesis of DNA and RNA Produces NADPH required for lipid syntheses Most active during development
26. Utilization of Amino AcidsConcept: Amino acids serve many functions in CNS Peptide and Protein synthesis Precursors for transmitters Neurotransmitters
27. Proteins in the CNSConcept: Neurons must produce those proteins essential for their special functions: conduction of action potentialssynaptic transmissionaxoplasmic transportestablishment of specific connections
28. Specific Neural/GlialPeptides/Proteins Structual-cytoskeletal Cell Surface proteins play a role during development in directing neural connections Contractile proteinsfunction in axoplasmic movement Neurotubular protein Glial proteins (glial fibrillary protein)
29. Specific Neural/Glial Peptides/ProteinsEnzymes for transmitter synthesis and degradation Transmitter receptors Membrane transporters Ion Channels Growth Factors Synaptic Vesicle
30. Microscopic Observations of Cerebral Spinal Fluid
31. Neutrophils and BandsMorphologically identical to neutrophils and bands in bloodOccasionally granulation disappears and pseudo-hypersegmentation is observed.
32. LymphocytesAlmost identical morphology to lymphocytes in the bloodDue to "flattening-out" of the lymphs during cytocentrifugation, nucleoli may be visible.Found in all fluid
33. MacrophagesLeukophages:Macrophagescontaining phagocytized WBC. WBCs are often pyknotic and easily confused with NRBC's. Found in all fluids.Erythrophages: Macrophages containing phagocytized RBC or RBC fragments. May contain several RBC. Found in all fluids.Siderophages: Macrophages containing phagocytized particles of hemosiderin, which stain a blue-black color.
34. Hematoidin CrystalsThese are bright-yellow diamond-shaped crystals of hemosiderinintracellular or extracellular on the slide.They are iron-negative on the Prussian blue stain and thereforeCan be noted on the patient report without performing an iron stain.
35. Immature GranulocytesMetamyelocytes, myelocytes, and promyelocytes may be found in fluids, though they are rarely seen. They are morphologically identical to those in the blood May be due to bone marrow contamination in CSF
36. BlastsMorphologically similar to blasts found in the bloodThere may be some clover-leaf shaped nuclei due to cytocentrifugal distortion.May be found in all fluidsSeen in association with leukemias, lymphomasBone marrow contamination of CSF
37. Nucleated Red Blood CellsNRBC are rarely seen body fluids. If observed, they should be reported as the number of NRBC per number of WBC countedThey must be differentiated from pyknotic WBCsNRBC’s are commonly due to peripheral blood or bone marrow contamination of CSF
38. Abnormal LymphocytesPlasmacytoid lymphs: Identical in morphology to plasmacytoid lymphs in bloodFound in all fluids.Mott cells: Plasma cells with numerous clear cytoplasmic vacuoles containing immunoglobulins
39. Reactive MacrophagesThese are most common in CSF from small children with subarachnoid hemorrhage but may be found in all body fluidsMay be very difficult to distinguish morphologically from large atypical lymphocytes
40. Malignant CellsMalignant cells may be shed from solid tissue (non-hematopoietic) neoplasms into CSF or body cavity fluid submitted for cell countsFluid will be turbid or bloodyMalignant cells are usually seen in clusters of 3-5 or more, but may occur singly
41. MicroorganismsIntracellular bacteria or yeast can be observed in acute bacterial or fungal infectionsIt is important to coordinate your findings with those of the Microbiology Section of the laboratory
42. Bloody CSFWhen the CSF is pinkish red, this usually indicates the presence of blood, which may have resulted from:Sub arachnoid hemorrhageIntra cerebral hemorrhageInfarcttraumatic tap
43. Order of Draw of Lumbar Puncture1st - Chemistry2nd - Microbiology3rd - Hematology
44. Physical ExaminationColor – XanthochromiaHyperbilirubinemiaIncreased ProteinTurbidityIncreased White Blood Cells (Pleocytosis)
45. CSF SupernatantA traumatic tap shows progressively decreasing RBC in serial samplesGenerally, in subarachnoid hemorrhage, the RBC would be consistent from one tube to the next
46. CSF SupernatantAfter the CSF is centrifuged, the supernatant fluid is clear in a traumatic tap, but it is xanthochromic in a subarachnoid hemorrhage Xanthochromia of the CSF refers to a pink, orange, or yellow color of the supernatant after the CSF has been centrifuged
47. Cell CountThe white cell count is increased when there is inflammation of the central nervous system, particularly the meningesBacterial infections are usually associated with the presence of neutrophils in the CSF
48. Cell CountViral infections are associated with an increase in mononuclear cells An increase in mononuclear cells may also be seen with:cerebral abscessacute leukemiaLymphomaintracranial vein thrombosiscerebral tumormultiple sclerosis
49. Cell Count cont’dA white cell count with an indication whether the cells are pus cells or lymphocytes, is required when the c.s.f. appears slightly cloudy or clear or when theGram smear does not indicate pyogenic bacterial meningitis
50. Cell Count cont’dMethodTo identify whether white cells in the c.s.f. are polymorphonuclear neutrophils (pus cells) or lymphocytes, dilute the c.s.f. in a fluid which stains the cells. Istonic 0.1% toluidine blue is recommended because it stains lymphocytes and the nuclei of pus cells blue. C. neoformans yeast cells stain pink. Red cells remain unstained. The motility of trypanosomes is not affected by the dye. When toluidine blue is unavailable, isotonic methylene blue can be used which will also stain the nuclei of leucocytes.
51. CSF Cell Count proceduresMix 1 drop of the c.s.f. (sample No. 2 uncentrifuged c.s.f.) with 1 drop of toluidine blue diluting fluid, 2 Assemble a modified Fuchs-Rosenthal ruled counting chamber, making sure the chamber and cover glass are completely clean.When unavailable, an improved Neubauer (preferably Bright-Line) chamber can be used. A Fuchs-Rosenthal chamber is recommended because it has twice the depth (0.2 mm) and is more suitable for counting WBCs in c.s.f.
52. CSF Cell Count procedures3 Using a fine bore Pasteur pipette or capillary tube, carefully fill the counting chamber with the well-mixed diluted c.s.f. The fluid must not overflow into the channels on each side of the chamber.4 Wait 2 minutes for the cells to settle. 5 Count the cells microscopically. Focus the cells and rulings using the 10 objective Count the cells in 5 of the large squares
53. Modified Fuchs-Rosenthal ruled chamberImproved Neubauer ruled chamber
54. Calculation of CSF Cell Counts
55. Chemistry TestsBecause CSF is formed by filtration of the plasma, chemicals in the CSF are that are found in the plasma. chemical composition is controlled by the blood-brain barrier,normal values for CSF chemicals are not the same as the plasma values. Abnormal values result from alterations in the permeability of the blood-brain barrier or increased production or metabolism by the neural cells in response to a pathologic condition. .
56. Measurement of c.s.f total protein Use the supernatant fluid from centrifuged c.s.f. or uncentrifuged c.s.f. when the sample appears clear.Total protein can be measured in c.s.f. using a colorimetric technique or a visual comparative technique,Normal Total c.s.f. protein is normally 0.15–0.40 g/l (15–40 mg%).
57. Measurement of c.s.f. total protein When the total protein exceeds 2.0 g/l (200 mg%), the fibrinogen level is usually increased sufficiently to cause the c.s.f. to clot. This may occur in severe pyogenic meningitis, spinal block, or following haemorrhage.
58. Clinical Significance of Elevated Protein ValuesThe causes of elevated CSF protein include damage to the blood-brain barrier, production of immunoglobulins within the CNS, decreased clearance of normal protein from the fluid, and degeneration of neural tissue. Meningitis and hemorrhage conditions that damage the blood-brain barrier are the most common causes of elevated CSF protein. Many other neurologic disorders can elevate the CSF protein, and finding an abnormal result on clear fluid with a low cell count is not unusual
59. Increased CSF Protein >80mg/dLDiabetes MellitusBrain tumor Meningioma Acoustic neuroma Ependymoma Encapsulated brain abscess Spinal cord tumor Multiple SclerosisAcute purulent Meningitis
60. Increased CSF Protein >80mg/dLGranulomatous MeningitisCarcinomatous MeningitisSyphilis (protein may be normal if longstanding) Guillain-Barre Syndrome (Infectious polyneuritis) Cushing's DiseaseConnective tissue disease Uremia Myxedema Cerebral hemorrhage
61. Cerebrospinal Fluid GlucoseGlucose enters the CSF by selective transport across the bloodbrainbarrier, a normal value 60% to 70% that of the plasma glucose. If the plasma glucose is 100 mg/dL, then a normal CSF glucose would be approximately 65 mg/dL. For an accurate evaluation of CSF glucose, a blood glucose test must be run for comparisonCSF glucose is analyzed using the same procedures employed for blood glucose. Specimens should be tested immediately because glycolysis occurs rapidly in the CSF.
62. Clinical Significance of Elevated glucose ValuesThe diagnostic significance of CSF glucose is confinedto the finding of values that are decreased in relation to plasma values. Elevated CSF glucose values are always a result ofplasma elevations. Low CSF glucose values can be of considerablediagnostic value in determining the causative agentsin meningitis. The finding of a markedly decreased CSF glucoseaccompanied by an increased WBC count and a largepercentage of neutrophils is indicative of bacterial meningitis.
63. Measurement of c.s.f. glucoseGlucose must be measured within 20 minutes of thec.s.f. being withdrawn otherwise a falsely low resultwill be obtained due to glycolysis. Use the supernatant fluid from centrifuged c.s.f. or uncentrifuged c.s.f. if the sample appears clear.Glucose can be measured in c.s.f. using a colorimetrictechnique or a simpler semiquantitative technique using Benedict’s reagent.
64. Measurement of c.s.f. glucoseNormal c.s.f. glucose: This is about half to two thirds that found in blood i.e. 2.5–4.0 mmol/1 (45–72 mg%).Raised c.s.f. glucose: Occurs when the blood glucose level is raised (hyperglycaemia) and sometimes with encephalitis.Low c.s.f. glucose: The c.s.f. glucose concentration is reduced in most forms of meningitis, except viral meningitis.In pyogenic bacterial meningitis it is markedlyreduced and may even be undetectable.
65. GlucoseLow glucose levels, as compared to plasma levels, are seen in:bacterial meningitiscryptococcal meningitismalignant involvement of the meninges and sarcoidosisGlucose levels are usually normal in viral infections of the CNS
66. CSF Normal Adult Lab RangesNormal CSF Levels:Protein (10 - 45 mg/dL)Glucose (40 - 70 mg/dL) Physical Appearance Clear/colorless RBC <5/mL WBC <5/mL
67. LactateIn bacterial and cryptococcal infection, an increased CSF lactate is found earlier than a reduced glucoseIn viral meningitis, lactate levels remain normal, even when neutrophils are present in the CSFRaised levels may also occur with severe cerebral hypoxia or genetic lactic acidosis
68. CSF India ink preparation When cryptococcal meningitis is clinically suspected, e.g. patient with HIV disease, or when yeast cells are detected when performing a cell count or examining a Gram smear, examine an India ink preparation or a wet preparation by dark-field microscopy for encapsulated yeasts.
69. India ink preparation 1 Centrifuge the c.s.f. for 5–10 minutes. Remove the supernatant fluid and mix the sediment.2 Transfer a drop of the sediment to a slide, cover with a cover glass and examine by dark-field Microscopy or add a drop of India ink , use nigrosin 200 g/l (20% w/v solution.3 Examine the preparation using the 40 objective Look for oval or round cells, some showing budding, irregular in size, measuring 2–10 m in diameter and surrounded by a large unstained capsule
70. Wet preparation to detect amoebaeExamine a wet preparation for motile amoebaewhen primary amoebic meningoencephalitis is clinically suspected (rare condition caused by N. fowleri) or the c.s.f. contains pus cells with raised protein and low glucose, but no bacteria are seen in the Gram smear. Red cells may also be present.
71. Wet preparation to detect amoebae1 Transfer a drop of uncentrifuged purulent c.s.f. or a drop of sediment from a centrifuged specimen to a slide and cover with a cover glass.2 Examine the preparation using the 10 and 40objectives, with the condenser closed sufficientlyto give good contrast. Look for small, clear,motile, elongated forms among the pus cells. Usethe 40 objective
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73. cont’d
74. Exercise 1 A CSF WBC count is diluted with: 2 A total CSF cell count on a clear fluid should be: 3 The purpose of adding albumin to CSF beforecytocentrifugation is to:4 The primary concern when pleocytosis of neutrophilsand lymphocytes is found in the CSF is: 5 Neutrophils with pyknotic nuclei may be mistakenfor:6 The presence of which of the following cells isincreased when a CNS shunt malfunctions?
75. SummaryYou should now be able to discuss the formation and collection procedure for cerebral spinal fluid, normal and abnormal findings in CSF and methods of analysis and evaluation of CSF
76. References:Urinalysis and body fluids / Susan King Strasinger, 5th ed. 2008District laboratory practice in tropical countries. 2nd ed. Part I. Monica Cheesbrough, 2005Text book of urinalysis and body fluids. Doris LR, Ann EN, 1983Urinalysis and body fluids: A color text and atlas. Karen MR, Jean JL. 1995Clinical chemistry: Principles, procedures, correlation. 3rd ed. Michael L. Bishop et al. 1996Tietz Text book of clinical chemistry. 3rd ed. Carl AB, Edward RA, 1999Clinical chemistry: Theory, analysis, correlation 4th ed. Lawrence AK. 2003ASCP DocumentUrinalysis lecture note . Mistire W. , Dawite Y.76