CEREBROSPINAL FLUID EVALUATION IN DISEASESpptx

A vast array of clinical conditions with their respective CSF findings. Also includes a practical approach to sampling and evaluation of CSF samples in the clinical setting.

1. DIAGNOSTIC EVALUATION OF CEREBROSPINAL FLUID Dr Innocent Agaba (MBBS) Department of Paediatrics , Modibbo Adama University Teaching Hospital Yola 14/02/24

2. “… I punctured the subarachnoid space in the lumbar area, passing a very fine cannula two centimeter deep between the third and fourth lumbar spinal arches and drop by drop I drained a few cubic centimeters of watery clear fluid.” Quincke , April 1891

3. OUTLINE Introduction Historical Background Relevant anatomy and physiology Evaluation - Obtaining the CSF - Specimen Processing - Normal CSF - Investigative modalities CSF findings in Diseases Future perspectives Conclusion References

4. INTRODUCTION Cerebrospinal Fluid (CSF) is a dynamic metabolically active fluid surrounding the brain and the spinal cord. It is a sterile, crystal clear, colourless and odourless body fluid produced in the choroid plexus of the brain. CSF obtained by lumbar puncture is the main diagnostic tool for neurological disorders. CSF analysis is not necessarily diagnostic but can be useful in the evaluation of several neurologic conditions. In the last 30 years, great progress has been achieved in diseases like Dementia & Alzheimer’s Disease using biological markers

5. HISTORICAL PERSPECTIVES The first description of the meningis , ventricles, and CSF appeared in the Edwin Smith Surgical Papyrus believed to be written in the 17th century BC. In 1664, Galen described “a liquid” in the aqueduct of Sylvius that connects the ventricles, noting that in “epidemic fever,” the consistency of the “liquid” is altered In 1902 the term “ xanthochromia ” was first used to describe a yellowish pigmentation of CSF from a patient with SAH. Two years later, Henri Dufour (1904) turned diagnostic cytology of the CSF possible with the development of technology to identify neoplastic cells. In 1912, William Mestrezat made the first detailed description of the chemical composition of CSF.

6. HISTORICAL PERSPECTIVES

7. RELEVANT ANATOMY & PHYSIOLOGY

8. RELEVANT ANATOMY & PHYSIOLOGY

9. RELEVANT ANATOMY & PHYSIOLOGY

10. RELEVANT ANATOMY & PHYSIOLOGY

11. RELEVANT ANATOMY & PHYSIOLOGY

12. EVALUATION Aim: Diagnosis, Therapy, Monitoring, prognostication Parameters : Quantity, pressure and components. Invasive - Lumbar puncture (LP) -Subdural puncture - Cisternal puncture - Lateral cervical puncture - ICP monitoring devices Non-invasive - Transfontannel USS -CT scanning - Neuroimaging -Radionuclide tracers -Phase-contrast MR -FLAIR sequences

13. Obtaining the CSF – Lumbar puncture Proper understanding of the underlying anatomy and careful positioning of the patient are the two main keys to performing a successful LP. Positioning 1. Lateral recumbent -The neck, hips, and knees should be flexed, -the entire spine should be parallel to the table, -the coronal plane of the trunk should form a right angle to the floor, and -the patient’s shoulders should be perpendicular to the table 2. Sitting - Preferable for sick neonates - Should be returned to lateral decubitus before measurement of opening pressure

14. Obtaining the CSF – Lumbar puncture Because the conus medullaris extends below L1–L2 in infants and children, the lowest possible interspace should always be used in young patients, with needle insertion preferably no higher than at L4–L5. Once the appropriate interspace has been identified and marked, the overlying skin should be sterilized and draped using standard aseptic technique. The bevel of the needle is inserted in the midline, parallel to the longitudinal fibers of the Dura, and directed toward the umbilicus. Once fluid appears at the needle hub, a manometer with a three-way stopcock should be gently connected to measure the opening pressure.

15. Obtaining the CSF – Lumbar puncture

16. Obtaining the CSF – Lumbar puncture Peri -procedural considerations Cranial CT or magnetic resonance imaging (MRI) scan is obligatory for any patient who presents with focal neurological deficits, new-onset seizures, altered level of consciousness, and/or papilledema before an LP can safely be performed. The above must include a fundoscopic exam to search for any evidence of papilledema Anticoagulation therapy should be stopped or reversed prior to performance of an LP whenever possible Finally, careful thought should always be given to the sample volume that will be needed to perform all the desired assays For patient with an uncertain diagnosis, it is advised to set aside a few extra milliliters of CSF in a clinical refrigerator whenever possible

17. Obtaining CSF – Lumbar Puncture CSF ASSAY SAMPLE VOLUME NEEDED Cell count & Differential 1ml each in tube #1 and #4 Glucose & Protein concentration 1ml Bacterial Gram stain & Culture Minimum of 1ml Viral PCRs 1ml per viral PCR AFB smear & Culture 5ml Flow Cytometry & Cytopathology Minimum of 5ml, preferably 10 – 15ml Oligoclonal bands, Myelin Basic proteins,IgG index 0.5ml per test

18. Contraindications Localised infection – such as cellulitis , sacral decubitis ulcers, or known epidural abscess – overlying the lumbar spine Intracranial space-occupying brain lesions that result in midline shift or pressure on the contents of the posterior fossa . Known coagulopathy related to anticoagulant medications, liver failure, or thrombocytopenia INR greater than 1.4 or a platelet count of less than 20 x 10 9 /l Critical illness with cardiopulmonary compromise

19. Complications Brain herniation Bleeding Spinal (subarachnoid, subdural, epidural) Intracranial (subarachnoid, subdural) Traumatic puncture (“bloody tap”) Infection Headache Backache/ radicular symptoms Other Cranial neuropathies ( diplopia , hearing loss) Implantation of an epidermoid tumor Spurious laboratory data Unique complications of LP in infants include hypoxemia due to improper patient positioning

20. Specimen processing Should be transported without delay as cells can lyse leading to falsely abnormal biochemical analytes . At room temp, cell count depreciate by 32% in 1 hr, 50% in 2 hrs. 1 st tube collected should be the one sent for culture. Preferably 10 – 15ml. Minimum should be 2ml esp for Fungi & mycobacterium. Gram staining less sensitive than Acridine orange for detecting bacteria.

21. Normal CSF macroscopy Under normal circumstances, CSF should appear clear and colorless. An aliquot of CSF should be visually inspected in direct comparison to an equal volume of water at the time of acquisition, preferably using clear glass tubes. Normal opening pressure : 10 – 100mmH 2 0 in newborns,60 – 180mmH 2 0 in young children & 12 – 120mmH 2 0 in older children. Turbidity resulting from increased cellularity can be seen under standard lighting conditions at counts starting around 400 cells/mm 3 CSF will appear pink at counts of 500–6,000 RBCs/mm 3 . The fluid becomes frankly bloody above 6,000 RBC/mm 3 .

22. Normal CSF Cellularity Techniques : - Direct microscopic examination. - Automated cell-counting methodologies. - Immunofluorescent staining of cells and analysis by flow cytometry . There is general consensus that normal CSF should not contain more than 5 WBCs/mm3 and 5 RBCs per ul . Neonates in contrast may have up to 20wbcs. Neutrophils are always abnormal in a child but 1-2/mm 3 may be present in healthy neonates .

23. Normal CSF Cellularity Normal CSF leukocytes are about 80% T cells (CD4+ > CD8+ T cells). Monocytes constitute only about 5% of normal CSF cells, and fewer than 1% are B cells. Normal CSF may also rarely contain cells that derive from tissues in physical contact with the subarachnoid space, including the choroid plexus, the ependymal lining of the ventricles, and the arachnoid membrane

24. Normal CSF proteins

25. Normal CSF proteins Measured using a spectrophotometer About 10% of the total protein is IgG With a traumatic tap, CSF protein is increased by approximately 1mg/dl for every 1000 RBCs/mm 3.

26. Normal CSF glucose The CSF should be tested for glucose as quickly as possible as glucose levels begin to fall after 20 minutes. Measured by Colorimetry Approximately two – thirds of serum levels CSF glucose levels of 40–45 mg/dl are often abnormal, and that values below 40 mg/dl always reflect a pathological state. Thus, it is advisable to simultaneously monitor the CSF:serum glucose ratio in addition to absolute glucose concentrations.

27. Normal CSF

28. Other constituents of Normal CSF Electrolytes Amino acids Neurotransmitters Enzymes and enzyme inhibitors Lipids Cytokines Hormones Trace Metals Vitamins Metabolic by products

29. Investigative modalities A “standard” profile of : Opening pressure, Cell count and diff Glucose Protein concentrations Bacterial Gram stain and culture status. Acid-fast bacilli (AFB) using a smear Is there a place for these? Urinalysis strips Glucometer strips

30. Investigative modalities As indicated : Countercurrent Immunoelectrophoresis (CIE) - Almost as sensitive as culture Coagglutination Test (COAG) Latex agglutination test (LAT) ELISA Polymerase chain reaction 4-3-3 protein Oligoclonal bands, Elevated immunoglobulin ( Ig ) G levels ( IgG index) Flow cytometric and cytopathologic analyses Paraneoplastic antibodies

31. CSF FINDINGS IN DISEASES

32. Infections of the nervous system Neoplastic & Paraneoplastic disorders Inflammatory & Demyelinating disorders Cerebrovascular disorders Isolated seizures & epileptic disorders Others are Neurodegenerative and movement disorders Parasitic, spirochetal disorders Transmissible spongiform Encephalopathies Nutritional and metabolic disorders Developmental disorders Headache syndromes Psychiatric disorders Neuromuscular disorders

33. 1. Bacterial Meningitis Opening pressure Raised i.e >200 mmH 2 Macroscopy Clear in early disease Cloudy or purulent Occasionally, pus gets encapsulated , as in pneumococcal meningitis in the frontal lobe area. In such cases, even though the CSF may remain clear the patient does not respond adequately to therapy and may eventually succumb to the illnes Cellularity Pleocytosis is the rule with WBC 100 – 10,000/mm 3 PMNs dominate (>90% of total). WBC > 50,000/mm 3 – suspect Brain abscess. Pleocytosis may be absent in patients with overwhelming sepsis & meningitis.

34. CSF microscopy - Neisseria meningitides

35. CSF microscopy – Haemophilus influenzae

36. CSF microscopy – Streptococcus pneumonia

37. Bacterial Meningitis cont… Biochemistry Protein is usually Glucose is less than 2/3 rd random serum glucose. May be absent. Microscopy Gram stain is positive in 70 – 90% of patients with untreated bacterial meningitis Culture Ideally inoculated on sheep blood agar & enriched chocolate agar and incubated for 4 days at 35 – 37% in 5% Co2. NAATS

38. 2. Partially treated (Bacterial) Meningitis 25-50% of children being evaluated for bacterial meningitis are receiving oral antibiotics when their CSF is obtained Cellularity Pleocytosis with neutrophil predominance. Biochemistry Elevated protein levels and hypoglycorrhachia usually persists for several days after administration of appropriate IV antibiotics. Microscopy Gram staining may be negative . Culture May yield no growth. Bacterial Antigen testing is indicated .

39. 3. Viral Meningoencephalitis Macroscopy Clear Ocassionally slightly turbid Cellularity PMNs- predominate ;Later - Mononuclear cells Biochemistry : Protein is normal or slightly elevated. Concentrations may be very high if brain destruction is extensive e.g HSV Encephalitis Glucose is usually normal (In mumps, hypoglycorrhacia may be observed) Culture Isolating a virus is most likely early in the illness. If not isolated, Serology is advised 2 -3 wks later to demonstate rising titers Take concomitant samples from Nasopharyngeal swabs, faeces & urine to increase yield Antibody or PCR studies

40. Viral meningoencephalitis cont… COVID 19 WITH CNS INVOLVMENT Has been reported to affect – directly & indirectly –the nervous system in a substantial number of cases. Biochemistry : Total protein is elevated An elevated CSF/serum ratio for albumin Cytoalbumin dissociation was found in half of patients involved in a large scale multicenter study in 2022 Important to avoid misinterpretation of treatable coexisting neurological disorders as complications of COVID 19

41. 4. Mycobacterial Meningitis Opening pressure Usually raised Macroscopy Turbid Cobweb appearance after 10 – 20min Cellularity During early stage 1, the CSF can resemble that of viral aseptic meningitis only to progress to the more-severe CSF profile over several weeks. Biochemistry: The CSF glucose is typically less than 40mg/dl but rarely <20mg/dl The protein level is elevated and may be markedly high (400-5,000 mg/ dL ) i.e 100 – 300 in early stage. Often >1000 in Late stages.

42. Mycobacterial meningitis cont… Microscopy Staining with Ziehl-Neelsen stain yields acid fast bacilli in up to 30% of cases. Culture: Culture in Lowenstein Jensen medium is positive in 40-60% of cases. The success of the microscopic examination of acid-fast–stained CSF and mycobacterial culture is related directly to the volume of the CSF sample. Examinations or culture of small amounts of CSF are unlikely to demonstrate M. tuberculosis. When 5-10 mL of lumbar CSF can be obtained, the acid-fast stain of the CSF sediment is positive in up to 30% of cases and the culture is positive in 50-70% of cases. Polymerase chain reaction (PCR) testing of the CSF can improve diagnosis. Cultures of other body fluids can help confirm the diagnosis

43. 4 . Fungal infection of the nervous system Cellularity CSF WBC count in Cryptococcal meningitis can range from 0 to 3,700 cells/mm3. Levels tend to be significantly lower in HIV- seropositive individuals, where up to 50% of patients with proven CM can actually have normal CSF WBC counts at the time of diagnosis. Biochemistry Total protein levels are usually elevated while CSF glucose concentrations can either be normal or depressed.

44. Fungal infections of Nervous system cont… Microscopy An India ink examination of the CSF shows yeast forms in 70–90% of untreated AIDS patients with CM due to higher pathogen loads, but may be positive in only 50% of the non-AIDS population Serology & antigen detection Immunological detection of cryptococcal polysaccharide antigen in serum and CSF by rapid latex agglutination tests or enzyme immunoassays has a sensitivity in excess of 90%. Culture studies Cryptococcus neoformans isolated from blood or CSF produces white mucoid colonies on many types of culture media, often within 72 h after plating

45. 5 . Parasitic infestation of the nervous system Causes eosinophilic meningitis i.e eosinophil count >10/mm 3 a) CEREBRAL MALARIA Cellularity Nearly all of the cells present in the CSF during cerebral malaria are lymphocytes, and the presence of neutrophils should suggest an alternative diagnosis. Biochemistry The total protein content may be slightly elevated, sometimes reaching a level of 150 mg/dl but rarely more than that. The CSF:serum glucose ratio is invariably normal.

46. Parasitic infestations of the Nervous system cont… b) CEREBRAL TOXOPLASMOSIS Cellularity CSF shows a mild mononuclear cell pleocytosis , Biochemistry Elevated protein content Normal glucose level. Serology The diagnosis is made by showing rising serum and CSF anti- Toxoplasma

47. 6 . Spirochetal infection of nervous system Symptomatic involvement of the CNS by Treponema pallidum can occur any time beyond the primary stage of infection. Various forms : Asymptomatic Neurosyphilis , Syphilitic meningitis, Meningovascular syphilis, Parenchymatous neurosyphilis , Gummatous neurosyphiliis,Neurosyphilis in HIV infected individuals. Current standards advocate that patients with any two of the following features be treated for presumptive neurosyphilis : (a) CSF pleocytosis of >8 WBC/mm3, (b) CSF protein level >50 mg/dl, and (c) reactive CSF VDRL.

48. 7. Drug and chemical induced Meningitis NSAIDS, Antibiotics, IV immunoglobulins are known causes of aseptic meningitis The usual clinical scenario is that signs and symptoms suspicious of such an event begin within hours after drug ingestion, but cases have been reported as far out as several weeks following exposure. Cellularity Predominance of either PMNs or mononuclear cells, and not infrequently has a measurable proportion of eosinophils . Total cell counts can range from 10 to 10,000 WBC/mm3 although most cases have 200–400 WBC/mm3 detected Biochemistry Protein content ranges from normal up to as high as 500 mg/dl

49. 8. Neoplastic disorders Cartilage cells and cells derived from the bone marrow (presumably due to trauma from the spinal needle) have also been rarely identified in CSF samples and these cells are commonly mistaken for malignant cells. Presence of malignant cells aid in the diagnosis and staging of Burkit Lymphoma, Leukemias e.t.c CARCINOMATOUS MENINGITIS Biochemistry Low CSF glucose Increased CSF protein Cellularity Slightly elevated to very high 0-100 or more; mononuclear or blast cells 50-1,000 Cytology May be positive

50. 9. Transmissible spongiform encephalopathies Cellularity Normal Biochemistry Normal Measurement of CSF NSE levels could be useful as a confirmatory test in the setting of a suspected sporadic CJD diagnosis Elevated CSF levels of 14-3-3 protein.

51. 10. Cerebrovascular disorders SUBARACHNOID HAEMORRHAGE Indicated if only if CT scan is normal. Typical Xanthochromic appearance Xanthochromia may be absent in in bleeds May not be seen<12hrs after the incident particularly when laboratories rely on visual inspection rather than spectroscopy.

52. Neuromuscular Diseases a) GULLAIN BARRE SYNDROME The classic triad of findings in GBS – weakness, areflexia and elevated CSF protein – were first reported by Guillain , Barré in 1916 The CSF changes in this disorder is termed “ cytoalbuminologic dissociation,” reflecting an elevation of CSF protein, frequently greater than 100 mg/dl, with minimal or no elevation in the number of CSF leukocytes. The initial CSF results should be interpreted cautiously, however, as only 50% of patients with GBS have an elevated CSF protein concentration at presentation. The mean CSF protein level reaches a maximum at 16–30 days from symptom onset, ranging from 140 to 213 mg/dl in one cohort, at which time 90% of patients demonstrate an elevation of CSF protein. An exception to the cytoalbuminologic dissociation occurs in HIV-associated GBS, where there may be up to 50 leukocytes/mm3 in the CSF

53. 11. Neuromuscular diseases b) Poliomyelitis A cause of aseptic meningitis 1-2% of all cases of poliomyelitis Cellularity Lymphocytic pleocytosis Biochemistry Rise in protein Normal sugar content

54. 12 . Nutritional & Metabolic Disorders a) PROTEIN – ENERGY MALNUTRITION Ammonia levels were elevated in the CSF of children with either type of protein-calorie malnutrition Activities of two particular enzymes, glutamic-oxalacetic transaminase and lactate dehydrogenase , were elevated in the CSF of children with marasmus or kwashiorko

55. Nutritional and Metabolic Disorders cont… b) HEPATIC ENCEPHALOPATHY Cases of acute liver failure are commonly associated with an elevated Opening pressure if LP is performed in this setting Elevated levels of ammonia, glutamine, alpha- ketoglutaramate , and lactate. High CSF and brain levels of both glutamine and ammonia cause visible swelling of astrocytes .

56. 13 . Inflammatory & demyelinating disorders a) Multiple Sclerosis The CSF cell count is typically normal or only mildly elevated in most MS patients Diagnosis based on clinical features with - IEF evidence of ≥2 Oligoclonal Bands, - Elevated IgG index

57. 14. Connective tissue disorders SYSTEMIC LUPUS ERYTHEMATOSUS Cellularity Slightly elevated 0-500; PMNs usually predominate; lymphocytes may be present Biochemistry Normal or slightly decreased glucose Culture No organisms on smear or culture. Positive neuronal and ribosomal P protein antibodies in CSF

58. 15. Psychiatric Disorders AUTISTIC SPECTRUM DISORDER Hx : impaired social, behavioral, and communication skills. It can be associated with mental retardation and epilepsy CSF shows high levels of Multiple inflammatory mediators including ; - monocyte chemoattractant protein (MCP)-1 - interferon-gamma - transforming growth factor (TGF)-beta2 -Interleukin (IL)-8)

59. 16. Neurodegenerative and Movement Disorders a) Huntington’s disease Autosomal Dominant Characterised by chorea, behavioral disturbances, dementia, and motor impersistence . A diagnosis is made by genetic testing that reveals an expansion of a CAG repeat in the huntington gene found on chromosome 4 CSF Findings Levels of the dopamine metabolite homovanillic acid (HVA) were reduced compared to normal individuals

60. 17. Developmental disorders b). Pyridoxine dependent Epilepsy Rare autosomal recessive disorder, Presents with seizures in the neonatal period. Unresponsive to the classic anticonvulsants, Rapidly cease when intravenous pyridoxine is administered CSF findings Elevated plasma, urine, and CSF levels of both Pipecolic Acid

61. 18. Disorders of intracranial pressure and cerebrospinal fluid circulation IDIOPATHIC INTRACRANIAL HYPERTENSION (IIH) The hallmark of idiopathic intracranial hypertension – previously known as pseudotumor cerebri – is elevated pressure in the CSF compartment CSF Findings : Pcsf less than 20 cmH2O is considered normal, while readings of greater than 25 cmHO are consistent with IIH. Pressures between 20 and 25 cmH2O are ambiguous; if clinical suspicion is high, treatment can be started with careful follow-up

62. Future perspectives “ Omic ” approaches including : - proteomics - metabolomics - transcriptomics and - micro analysis.

63. Future perspectives

64. REFERENCES Kliegman R. M. et al, Nelson Textbook of Paediatrics . 20 th ed Elsevier 2016 Azubike JC, Nkangineme KE. Paediatrics and Child Health in a tropical region. 3 rd edition, Educational Printing Press 2016 Rudolph M., Rudolph CD, Paller,Hostettter MK,Lister G. & Siegel Nj (2011) Rudolphs Textbook of Paediatrics 22 nd Ed. McGraw Hill Nkangineme KE et al, Pathophysiology of Clinical symptoms, signs and Laboratory parameters, 1 st ed Athena Academic Ltd 2016. Regeniter A, Kuhle J, Mehling M, (2009) . A modern approach to CSF analysis: pathophysiology , clinical application, proof of concept and laboratory reporting. Clin Neurol Neurosurg . May;111(4)

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