Cerebrospinal Fluid Analysis PDF

Email: [email protected] 1 CHAPTER TWO Cerebrospinal Fluid Analysis  Email: [email protected] 2 Course outline  Introduction to Cerebrospinal fluid  Routine laboratory assays  Collection of sample  Gross appearance  Cell cou...

Email: [email protected] 1 CHAPTER TWO Cerebrospinal Fluid Analysis  Email: [email protected] 2 Course outline  Introduction to Cerebrospinal fluid  Routine laboratory assays  Collection of sample  Gross appearance  Cell counts  Chemical analysis  Morphological Examination  Microbiological Examination  Serological Examination  Email: [email protected] 3 Introduction  Cerebrospinal fluid (CSF) is the 3rd major fluid of the body after blood and urine.  CSF:- provides a physiologic system to:-  supply nutrients to the nervous tissue,  remove metabolic wastes, and  produce a mechanical barrier to cushion/protect the brain and spinal cord against trauma.  CSF is produced in the choroid plexuses of the two lumbar ventricles between 3rd & 4th, or 4th & 5th lumbar vertebrae.  In adults, approximately 20 mL of fluid is produced every hour.  Email: [email protected] 4 Formation and Physiology  The 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 the blood capillaries in the arachnoid granulations/villae at a rate equal to its production. Email: [email protected] 5 Email: [email protected] 6  Email: [email protected] 7 Cerebrospinal fluid  Fluid in the space called sub-arachnoid space between the arachnoid mater and pia mater.  Protects the underlying tissues of the central nervous system (CNS).  Serve as mechanical barrier to:-  prevent trauma,  regulate the volume of intracranial pressure  circulate nutrients  remove metabolic waste products from the CNS  act as lubricant  Has composition similar to plasma except that it has less protein, less glucose and more chloride ion.  Email: [email protected] 8 CSF…  Maximum volume of CSF:-  Adults 150 mL  Neonates 60 mL  Rate of formation in adult is 450-750 mL per day or 20 ml per hour  reabsorbed at the same rate to maintain constant volume  Collection by lumbar puncture done by experienced medical personnel.  About 1-2ml of CSF is collected for examination  lumbar puncture is made from the space between the 3rd and 4th or 4th and 5th lumbar vertebrae under sterile conditions.  Email: [email protected] 9 CSF…  Blood Brain Barrier  There is a secure barrier between the blood and the CSF fluids that surround the brain called the Blood Brain Barrier.  Occurs due to tight fitting endothelial cells that prevent filtration of larger molecules.  Controls / restricts / filters blood components  Restricts entry of large molecules, cells, etc.  Therefore, CSF composition is unlike blood.  ** CSF is NOT considered an ultrafiltrate of plasma.  Email: [email protected] 10 CSF… Blood Brain Barrier  Essential to protect the brain  Blocks chemicals, harmful substances  Antibodies and medications also blocked  Tests for those substances normally blocked can indicate level of disruption by diseases: i.e. meningitis and multiple sclerosis.  Email: [email protected] 11 CSF… Four major categories of disease:-  Meningeal infections  Subarachnoid hemorrhage (stroke)  CNS malignancy  Demyelinating disease:- any disease affecting the nervous system where the myelin sheath surrounding neurons is damaged.  Email: [email protected] 12 CSF… Indications for analysis CSF:-  To confirm diagnosis of meningitis  Evaluate for intracranial hemorrhage  Diagnose malignancies, leukemia  Investigate central nervous system disorders  Email: [email protected] 13 Specimen collection and handling  Routinely collected via lumbar puncture between 3rd & 4th, or 4th & 5th lumbar vertebrae under sterile conditions.  Intracranial pressure measurement taken before fluid is withdrawn.  Email: [email protected] 14 Collecting CSF specimen Collected in three sequentially labeled tubes:-  Tube 1: Chemistry and immunologic or serological tests  Tube 2: Microbiology  Tube 3: Hematology (gross examination, total WBC & Diff)  This is the list likely to contain cells introduced by the puncture procedure. Email: [email protected] 15  Email: [email protected] 16 Report the appearance of the CSF  As soon as the CSF 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 CSF appears clear and colourless.  If immediate processing not possible:-  Tube 1 (chem-sero) frozen  Tube 2 (micro) room temp  Tube 3 (hema) refrigerated  Email: [email protected] 17 Color & Clarity  Normal CSF appearance is  Pathological: cloudy, turbid, crystal clear and colorless bloody, viscous, or clotted.  Normal CSF Levels:-  Pleocytosis:- increased CSF Protein (10 - 45 mg/dL) Glucose (40 - 70 mg/dL) cell numbers  Physical Appearance:-  WBC > 200 cells/Ml Clear/colorless RBC 400 cells/mL WBC 200WBC/mm3  Email: [email protected] 19 Report the appearance of the CSF…  Purulent or cloudy CSF:-  Indicates presence of pus cells, suggestive of acute pyogenic bacterial meningitis.  Blood in CSF:-  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.  Email: [email protected] 20 Report the appearance of the CSF…  Following a subarachnoid haemorrhage:-  the fluid may appear xanthrochromic, i.e. yellow-red (seen after centrifuging).  Xanthrochromia is a yellowing discoloration of the CSF supernatent (may be pinkish, or orange). Due to presence of ‘old’ blood, increased bilirubin, carotene, proteins, melanoma.  Clots in CSF:-  Indicates a high protein concentration with increased fibrinogen, as can occur with pyogenic meningitis or when there is spinal constriction.  Email: [email protected] 21 CSF…  Email: [email protected] 22 Recording:- Label each tube with:-  Patient’s first and last name  Date and time of collection  Specimen source (CSF and i.e. lumbar, shunt)  Tube identification number (1, 2, 3, 4) indicating order of collection  Record immediate results:-  Appearance of CSF  Pressure of CSF  Email: [email protected] 23 Handling &Transport  CSF must be processed within 1 hour of collection  Do not refrigerate!  Only put CSF in an incubator if the temperature is < 150C, if can’t be taken to the lab quickly.  Most of CSF pathogens are very fastidious  Email: [email protected] 24 Handling &Transport · A delay in examining CSF:-  Reduces the chances of isolating pathogens  Lower cell count due to WBCs being lysed  Falsely low glucose value due to glycolysis · Criteria for rejection:-  Sample improperly labeled  Not freshly collected  Traumatic sample Email: [email protected] 25  Email: [email protected] 26 Possible pathogens according to age groups · Neonates  Group B Streptococci, E.coli, L.monocytogenes. · Children  H.influenzae, N.meningitidis, S. pneumoniae · Adults  S. pneumoniae, N.meningitidis, Mycobacteria, Cryptococci · Elderly  As in neonates, L.monocytogenes. Email: [email protected] 27  Email: [email protected] 28 Processing of CSF samples  Priority! specimen that requires prompt attention by the laboratory staff.  Laboratory tests on CSF:-  Gross exam  Cell Counts + Diffs  Chemical test  Microscopy  Cultures  Serology  PCR  Email: [email protected] 29 Physical Examination  Color:- Xanthochromia  Hyperbilirubinemia  Increased Protein  Turbidity:-  Increased White Blood Cells (Pleocytosis) Bloody CSF:-  When the CSF is pinkish red, this usually indicates the presence of blood, which may have resulted from:-  Sub arachnoid hemorrhage  Intra cerebral hemorrhage  traumatic tap  Email: [email protected] 30 CSF Supernatant  A traumatic tap shows progressively decreasing RBC in serial samples.  Generally, in subarachnoid hemorrhage, the RBC would be consistent from one tube to the next.  After the CSF is centrifuged, the supernatant fluid is clear in a traumatic tap, but it is xanthochromic in a subarachnoid hemorrhage.  Xanthochromia is a pink, orange, or yellow color of the supernatant after the CSF has been centrifuged.  Email: [email protected] 31 Cell Count  The white cell count is increased when there is inflammation of the central nervous system, particularly the meninges.  Bacterial infections are usually associated with the presence of neutrophils in the CSF.  Viral infections are associated with an increase in mononuclear cells.  An increase in mononuclear cells may also be seen with:- cerebral abscess intracranial vein thrombosis acute leukemia cerebral tumor Lymphoma multiple sclerosis  Email: [email protected] 32 Cell Count cont’d  To identify whether white cells in the CSF are polymorphonuclear neutrophils (pus cells) or lymphocytes, dilute the CSF in a fluid which stains the cells.  Isotonic 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.  Email: [email protected] 33 CSF Cell Count procedures 1 Mix 1 drop of the CSF (sample No. 3 uncentrifuged CSF) 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 CSF.  Email: [email protected] 34 CSF Cell Count procedures… 3 Using a fine bore Pasteur pipette or capillary tube, carefully fill the counting chamber with the well-mixed diluted CSF. 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.  Email: [email protected] 35 CSF Cell Count procedures… Modified Fuchs-Rosenthal ruled Improved Neubauer ruled chamber chamber  Email: [email protected] 36 Calculation of CSF Cell Counts  Email: [email protected] 37 Differential WBC  Performed on a stained smear made from CSF.  It is recommended that stained smears be made even when the total cell count is within normal limits.  Count 100 cells in consecutive oil-power fields.  Report percentage of each type of cell present.  Email: [email protected] 38 Differential cotn’d…. · Predominant Neutrophils-  Meningitis(bacterial, early viral ,early tubercular and fungal.  Sub arachnoid hemorrhage  Email: [email protected] 39 Differential cotn’d…. · Predominant Lymphocytes:-  Meningitis (viral or tubercular)  Incompletely treated bacterial meningitis.  Toxoplasmosis  Email: [email protected] 40 Reference range WBC count · “Normal” CSF:-  0-5 cells/ mm3 in adult  20 cells/ mm3 in newborns  Mononuclears · 87% of patients with bacterial meningitis will have >1,000/mm3 · 99% will have >100/ mm3. · 0.7 = CNS sourced < 0.3 = compromised BBB Email: [email protected] 53 Email: [email protected] 54  Email: [email protected] 55 Microscopic examination  Preparation of specimen:-  The CSF is purulent (very cloudy), examined immediately without centrifugation.  Centrifugation, remove the supernatant and transfer it to another tube for chemical and/or serological tests.  Use the sediment for further microbiological tests.  Email: [email protected] 56 Gram staining  Email: [email protected] 57 Gram staining…  Email: [email protected] 58 Gram staining…  Email: [email protected] 59 Gram staining…  Email: [email protected] 60 Positive in 60 to 80 % of bacterial meningitis N.meningitidis S. pneumoniae  Email: [email protected] 61 Acid-fast stain  TB is suspected or the CSF contains lymphocytes and the glucose concentration is low and the protein raised.  Only 37 % positive for acid-fast bacilli.  87 % if four smears are done.  Sensitivity also can be increased by examining the CSF sediment.  Auramine stained smear is a more sensitive  Email: [email protected] 62 India ink preparation  When cryptococcal meningitis is suspected.  HIV disease  Yeast cells are detected when performing a cell count or examining a Gram smear C. neoformans, by  Cryptococcus = identified up to India ink staining 50 % of the time.  Email: [email protected] 63 India ink procedure 1. Centrifuge the CSF 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.  Email: [email protected] 64 Wet preparation to detect amoebae  Examine a wet preparation for motile amoebae  When primary amoebic meningoencephalitis is clinically suspected (rare condition caused by N. fowleri) or the CSF contains pus cells with raised protein and low glucose, but no bacteria are seen in the Gram smear.  Red cells may also be present.  Email: [email protected] 65 Wet preparation to detect amoebae 1. Transfer a drop of uncentrifuged purulent CSF 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 40 objectives, with the condenser closed sufficiently to give good contrast.  Look for small, clear, motile, elongated forms among the pus cells.  Use the 40 objective.  Email: [email protected] 66 Culture  Cultures done on:-  Blood agar  Chocolate agar  MacConkey agar  Centrifugation of the CSF and the removal of some of the deposit for smear.  Remainder of the deposit should be seeded heavily into culture media.  All media should be incubated for 3 days, with daily inspections.  Email: [email protected] 67 Culture…  Email: [email protected] 68 Culture…  Email: [email protected] 69 Culture…  Email: [email protected] 70 Culture…  When MTB is suspected, at least 3 tubes of LJ medium should be inoculated, incubated for 6 weeks.  Large volume samples of CSF, at least 15 mL and preferably 40 to 50 mL of CSF are recommended.  Culture is positive 56 % of the time on the first sample.  83 % of the time if four separate samples are cultured.  Email: [email protected] 71 Culture…  When C.neoformans is suspected:-  India ink preparation  Inoculate on SDA (Sabouraud dextrose agar)  Positive in more than 95 % of C.neoformans cases  66 % of Candidal meningitis cases.  Other fungi are less likely to be culture positive.  Email: [email protected] 72 Serologic testing  Latex agglutination (LA) allows rapid detection of bacterial antigens in CSF.  Tests are available to detect:-  N. meningitidis groups A, B, C..  H. influenzae type b  S. pneumoniae  S. agalactiae  Sensitivity varies greatly between bacteria, 60 to 100 %.  Email: [email protected] 73 Serologic testing…  The specificity for LA is very low.  Partially treated meningitis cases.  False positives, not routinely used today.  Crypto antigen detects C.neoformans, in 90-95% of pts with crypto meningitis.  Email: [email protected] 74 Lactate  In bacterial and cryptococcal infection, an increased CSF lactate is found earlier than a reduced glucose.  In viral meningitis, lactate levels remain normal, even when neutrophils are present in the CSF.  Raised levels may also occur with severe cerebral hypoxia or genetic lactic acidosis.  Email: [email protected] 75 Polymerase Chain Reaction(PCR)  Advance in the diagnosis of meningitis.  PCR has high sensitivity and specificity  Fast, and can be done with small volumes of CSF.  Testing is expensive  Useful in the diagnosis of viral meningitis.  Sensitivity of 95 to 100 %, and a specificity of 100 % for HSV type 2, EBV, and Enterovirus.  Email: [email protected] 76 PCR…  PCR has a sensitivity of 54 to 100 % and a specificity of 94 to 100 % for MTB.  Could replace AFB smear and culture as the test of choice.  Email: [email protected] 77 Differential Diagnosis of Meningitis by Laboratory Results Bacterial Viral Tubercular Fungal Increased WBC Increased WBC Increased WBC Increased WBC count count count count Neutrophils Lymphs Lymps & Monos Lymphs & Monos Marked ↑ protein Mod. ↑ protein Mod-Marked ↑ Mod-Marked ↑ protein protein Marked ↓ glucose ↔ normal glucose ↓ glucose Normal to ↓ glucose Lactate > 35 mg/dL Lactate normal Lactate > 25 mg/dL Lactate > 25 mg/dL + gram stains Pellicle formation + India ink with Cryptococcus neoformans + bacterial antigen + immunological tests test for C. neo. Email: [email protected] 78 Email: [email protected] 79 Email: [email protected] 80