Download - Ix. Csf Analysis

Denielle Genesis B. Camato

IX. CSF ANALYSIS ANALYSIS OF URINALYSIS AND BODY FLUIDS | REVIEWER

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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� Specimen is usually collected in three sterile tubes labelled 1, 2, 3 in the order to which they are withdrawn TUBE 1- CHEMICAL & SEROLOGICAL TESTS TUBE 2– MICROBIOLOGY TUBE 3- CELL COUNT- least likely to contain cells introduced by spinal tap procedure (other methods: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



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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



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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)