Ix. Csf Analysis
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Transcript of Ix. Csf Analysis
Denielle Genesis B. Camato
IX. CSF ANALYSIS ANALYSIS OF URINALYSIS AND BODY FLUIDS | REVIEWER
1
FORMATION & PHYSIOLOGY
� First recognized by COTUGNO, 1974 � Third major body fluid of the body � Provides nutrients to the nervous tissue to remove
metabolic wastes � Produce mechanical barrier to cushion brain and
spinal cord against trauma MENINGES¢those that lined the brain and spinal cord THREE LAYERS:
� Dura mater � Arachnoid mater � Pia mater
SUBARACHNOID SPACE- where CSF flows; located between arachnoid & pia mater
� Approximately 20mL /hr in choroid plexuses & reabsorbed by the arachnoid villi to maintain total volume of 140-170 mL in adults & 10-60mL in neonates
BLOOD-BRAIN BARRIER- used to represent the control and filtration of blood components to the CSF and then to the brain SPECIMEN COLLECTION & HANDLING
� CSF routinely collected by LUMBAR PUNCTURE between third, fourth, fifth lumbar vertebrae
� Specimen is usually collected in three sterile tubes labelled 1, 2, 3 in the order to which they are withdrawn TUBE 1- CHEMICAL & SEROLOGICAL TESTS TUBE 2– MICROBIOLOGY TUBE 3- CELL COUNT- least likely to contain cells introduced by spinal tap procedure (other methods: Systernal and Ventricular puncture)
If specimen cannot be performed on STAT basis; specimens should be maintained in the ffg manner:
� Hematology tubes are refrigerated � Microbiology tubes remain at room temperature � Chemistry and Serology tubes are frozen
APPEARANCE
� Normally crystal clear CSF � Crystal Clear, Cloudy/ Turbid, Milky, Xanthochromic,
hemolyzed/ Bloody � XANTHOCHROMIA
ê term used to described CSF supernatant that is pink, orange or yellow- pink (very slight amount of oxyhemoglobin) to orange (heavy hemolysis) to yellow (conversion of oxyhemoglobin to unconjugated bilirubin
� Other causes of xanthochromia: elevated serum bilirubin, presence of pigment carotene, increase protein concentrations and melanoma pigment
� Xanthochromia is due to immature liver function seen commonly in infants particularly those premature
TRAUMATIC COLLECTION 1. UNEVEN DISTRIBUTION OF BLOOD
ê blood from intracranial haemorrhage will be evenly distributed throughout the three CSF specimen tubes whereas the traumatic tap will have the heaviest concentration ¢ tube 1, gradually diminishing amounts in tube 2, and tube 3; traumatic procedure
2. CLOT FORMATION
ê traumatic tap may form clots owing to the introduction of plasma fibrinogen into specimen; intracranial haemorrhage will not contain fibrinogen to clot
TUBERCULAR MENINGITIS- classic web-like pellicle is seen after overnight refrigeration of the fluid
3. XANTHOCHROMIC SUPERNATANT ê results of blood that has been present longer
than that introduced by the traumatic tap; care should be taken because a very recent haemorrhage would produce a clear supernatant & introduction of serum protein from traumatic tap could also cause the fluid to appear xanthochromic.
Additional tests for differentiation: microscopic examination & D-dimer test
m Microscopic: macrophages ingesting RBCS (erythrophagocytosis or hemosiderin granules)= intracranial hemorrhage
m D-Dimer: detection of fibrin degradation product by
Dimer, latex aggln immunoassay indicates formation of fibrin at a haemorrhage site
CELL COUNT
� Routinely performed on CSF specimens is the WBC count
� RBC counts are usually determined only when a traumatic tap has occurred and a correction for WBC count is needed
� Cell count should be performed immediately because WBCs (particularly the granulocytes) and RBCs will begin to lyse within 1 hour with 40% of leukocytes disintegrating after 2 hours
Denielle Genesis B. Camato
IX. CSF ANALYSIS ANALYSIS OF URINALYSIS AND BODY FLUIDS | REVIEWER
2
METHODOLOGY � Normal adult CSF contains 0-5 WBC/uL; higher in
children (as many as 30 mononuclear cells/uL) consider as normal in newborns
� Improved Neubauer counting chamber is traditionally used
� Calculation formula: Number of cel ls counted x di lution =cel ls.uL Number of squares x volume of 1 square
� This formula can be used in both diluted and undiluted specimens
TOTAL CELL COUNT
� Clear specimens may be counted undiluted, provided no overlapping of cells is seen during the microscopic examination
� When dilutions are required, calibrated automatic pipettes are used
� Dilutions for total cell counts are made with normal saline mixed by inversion and loaded in the hemocytometer by a Pasteur pipette
� Cells are counted in the four corner square on both sides of the haemocytometer
Clarity Di lution Amount of Sample
Amount of Di luent
Sl ightly hazy 1 : 10 30μL 270 μL
Hazy 1 : 20 30μL 570 μL Sl ightly cloudy
1 : 100 30μL 2970 μL
Sl ightly bloody
1 : 200 30μL 5970 μL
Cloudy Bloody Turbid
1 : 10,000
0.1 mL of a 1:100 dilution
9.9 mL
WHITE BLOOD CELL COUNT
� Specimens requiring dilution can be diluted, substituting 3% acetic acid to lyse the RBCs
� Addition of methylene blue to the diluting fluid will stain the WBCs providing better differentiation
� To prepare a clear specimen that does not require dilution for counting, place four drops of mixed specimen in a clean tube.
� Rinse a Pasteur pipette with glacial acetic acid, draining thoroughly and draw the four drops of CSF into the rinse pipette.
� Allow the pipette to sit for 1 minute, mix the solution in the pipette, discard first drop and load the hemocytometer
CORRECTIONS for CONTAMINATIONS WBC (added)= WBC (blood) X RBC (CSF)
RBC (blood)
� When peripheral blood RBC and WBC counts are in the normal range, many laboratories choose to simply subtract 1 WBC for every 700 RBCs present in the CSF
� Differential count should be performed on a stained smear and not from the cells in the counting chamber
� When performing diff count, 100 cells should be counted, classified, and reported in terms of percentage
� If the cell count is low and finding 100 cells is not possible, report only the numbers of the cell types seen
CYTOCENTRIFUGATION
� As little as 0.1mL of CSF combined with one drop of 30% albumin produces an adequate cell yield when processed with the cytocentrifuged.
� Addition of albumin increases the cell yield and decrease the cellular distortion frequently seen on cytocentrifuged specimens
CSF CELLULAR CONSTIUTENTS
� Adults have usually predominance of lymphocytes to monocytes (70:30) ratio whereas monocytes are more prevalent in children.
� Pleocytosis- increased numbers of these normal cells is considered abnormal, as the finding of immature leukocytes, eosinophils, plasma cells and macrophages, increased tissue cells & malignant cells
� High WBC count of which the majority of the cells are neutrophils is indicative of bacterial meningitis
� High percentage of lymphocytes & monocytes suggests: viral, tubercular, fungal, or parasitic origin
� Cell forms differing those found in blood include macrophages, choroid plexus, and ependymal cells, spindle-shaped cells, and malignant cells
Denielle Genesis B. Camato
IX. CSF ANALYSIS ANALYSIS OF URINALYSIS AND BODY FLUIDS | REVIEWER
3
Formation and Composition of CSF
• Blood brain barrier maintains the relative homeostasis of CNS
environment by tightly regulating the concentration of substances by specific transport systems for H+, K+, Ca2+, Mg2+, HCO3-.
• Glucose, urea and creatinine diffuse freely between blood and the CSF.
• Proteins cross freely by passive diffusion along the concentration
gradient and is also influenced by molecular weight.
Composition of Normal CSF
Protein - 15 - 45 mg/dL
Glucose - 50 - 80 mg/dL
Urea - 6.0 - 16 mg/dL
Uric acid - 0.5 - 3.0 mg/dL
Creatinine - 0.6 - 1.2 mg/dL
Cholesterol - 0.2 - 0.6 mg/dL
Ammonia - 10 – 35 μg/dL Sodium - 135 – 150 mEq/L
Potassium - 2.6 – 3.0 mEq/L
Chloride - 115 – 130 mEq/L
Magnesium - 2.4 – 3.0 mEq/L
Cells - 0 – 5 Lymph/μL
CSF CELLULAR CONSTITUENTS � Nucleated Red Blood Cells (NRBCs)- result of bone
marrow contamination during spinal tap � Eosinophils- Parasitic infection, fungal infection,
introduction of foreign material in CNS Eg: Rat Lung Worm (Angiostrongylus cantonesis) Entamoeba histolytica- causing amebic encephalitis N. Fowleri, Acanthamoeba
� Macrophages- appear 2-4 hours after RBCs enter the CSF; indicative of previous haemorrhage
� Choroidal Cells- from epithelial lining of the choroid plexuses
� Ependymal Cells- from linings of ventricles and neural canal
� Spindle-shaped cells- from lining cells of arachnoid � Malignant cells of hematologic origin (blasts from
leukaemia, lymphoma cells) � Malignant cells of non-hematologic origin (metastatic
carcinoma cells) eg: breast cancer CSF ANALYSIS – CHEMICAL EXAMINATION � CSF - PROTEIN
ê Most frequently performed chemical test on CSF; Normally between 15-45 mg/dL
ê CSF protein is <1% compared to the total serum protein
ê ALBUMIN is the major protein in the CSF ê PRE-ALBUMIN (Transthyretin) second
most predominant protein in the CSF ê IgM, Fibrinogen, Beta-Lipoprotein are not
found in normal CSF ê Tau transferrin is found only in CSF; not
found in blood ê Elevated total protein values are most
frequently seen in pathologic conditions. Abnormally low values are most frequently seen in pathologic conditions
ê Methods of CSF protein measurement: Turbidimetric Mtd (eg: SSA/TCA) Dye-binding Method (BCG/BCP)
ê CSF/Serum-Albumin index is used to assess the integrity of the blood brain barrier
ê IgG index used to assess if there is intrathecal IgG production within the CSF
ê OLIGOCLONAL Bands- for assessment of Multiple Sclerosis; these band are also present in disorders such as encephalitis, neurosyphilis, Guillain-Barre syndrome and neoplastic syndrome
ê Myel in-Basic Protein is indicative of recent destruction of myelin sheath that protects the axon of neurons (demyelination) can be used to monitor the course of multiple sclerosis
� CSF – GLUCOSE ê CSF glucose is 60-70% that of plasma glucose ê Eg: Viral Meningitis ¢ slightly decrease glucose ê Bacterial Meningitis ¢ decrease glucose ê Blood glucose should be drawn in conjunction with
CSF values; blood glucose should be drawn 2 hours before the spinal tap
� CSF - LACTATE
ê Levels greater than 35 mg/dL indicate bacterial meningitis thus increase in lactate
ê Viral Meningitis- produces lactate levels lower than 25 mg/dL thus decrease in lactate
ê Elevated in destruction of CNS tissue owing to oxygen deprivation
ê Falsely elevated results: bloody/hemolyzed/ xanthochromic specimen
� CSF - GLUTAMINE
ê produced from ammonia and alpha-ketoglutarate by the brain cells
ê serves to remove toxic waste product ammonia from the CNS
ê Normal levels of glutamine is between 8-18 mg/dL ê Elevated levels are associated with liver
disorders/ hepatic coma & Reye’s Syndrome CSF ANALYSIS - MICROBIOLOGIC EXAMINATION
ê Used to identify the cause of meningitis ê CSF CULTURE is confirmatory test ê Gram stain, acid fast stain, India ink and latex
agglutination test serve for preliminary diagnosis ê India Ink ¢ for Cryptococcus neoformans; look
for capsules (fungal meningitis) ê Most frequent agents include: Streptococcus
pneumonia, Haemophilus influenza, Escherichia coli, Strep agalactiae & Neisseria meningitis (all of these are encapsulated organisms)
CSF ANALYSIS - SEROLOGIC TESTS
ê For assessment of neurosyphilis using VDRL or FTS-Abs (Treponema pallidum)