CSF fluid Analysis, CSF final

Specific gravity of CSF

1.006-1.008

pH of CSF

7.3-7.45

A CSF WBC count is diluted with: A.Distilled water B.Normal saline C.Acetic acid D.Hypotonic saline

C.Acetic acid

Alpha-globulins found in CSF

Haptoglobin and Ceruloplasmin

Uneven distribution of blood

blood from a cerebral hemorrhage will be evenly distributed throughout the 3 CSF tubes, whereas a traumatic tap will have the HEAVIEST in 1, with gradually decreasing in 2 and 3

Immunological tests are available for

syphilis (VDRL), cryptococcus and other infectious agent - most have low sensitivity

How to compute for cells/uL when Neubauer counting chamber is used

# of cells counted x dilution ____________________________ # of squares counted x volume of 1 square

Traumatic Tap (blood vessel punctured during tap) vs sub arachnoid hemorrhage (SAH)

*Uneven blood distribution* (decreasing count) in tubes 1 through 3 often indicates a traumatic tap; SAH will have an even distribution in all tubes *Clot formation* indicates a traumatic tap (plasma), SAH does not have enough fibrinogen Erythrophagocytosis, hemosiderin granules and xanthrochromia are indicative of SAH since these processes *take some time to occur* and are not present in a recent traumatic tap

Methods of collection of CSF

1) Lumbar/spinal tap 2) Ventricular Puncture (directly from ventricles) 3) Cisternal Puncture (or suboccipital puncture) 4) Lateral cervical puncture

Functions of the CSF

1.) *Physical Support* 2.) Protective effect against *sudden changes in BP and Impact pressure* 3.) *Excretory waste* function for metabolic products 4.) Maintains *CNS ionic homeostasis* 5.) Medium for *exchange and transfer of substances *between bloodstream and tissue of the brain and spinal cord. 6.) Supplies *nutrients* to nervous tissues 7.) *Pathway* - Hormone Releasing Factor

Normal value of CSF lactate

10-22mg/dL Can be valuable aid in diagnosing and managing meningitis cases. Bacterial, tubercular, fungal meningitis, CSF lactate levels greater than 25 mg/dL occurs more consistently than does decreased glucose and provides more reliable information when initial diagnosis is difficult. Greater than 35 mg/dL bacterial meningitis Viral meningitis lactate remain lower than 25 mg/dL Levels remain elevated during initial treatment but fall rapidly when treatment is successful Tissue destruction w/in the CSF owing to oxygen deprivation (hypoxia) increases CSF lactic acid levels. Therefore, elevated CSF lactate is not limited to meningitis and can result from any condition that decreased oxygen glow to the tissues.

Normal protein in CSF

15-45 mg/dL (Blood protein is 6.5-8 mg/dL)

Increased levels of total protein is seen in

1contamination with peripheral blood from traumatic tap 2Obstruction of CSF circulation (hematoma) 3Tissue degradation 4Increased permeability of the blood-barrier membrane caused by toxic factors or infection 5Chronic bleeding and hemolysis

Macrophages appears when in CSF

2-4 hours after introduction of RBCs into fluid (frequently seen in repeated taps)

CSF volume produced by choroid plexuses.

20 ml/hour (in adults) or 500ml/Day *CSF is produced in the choroid plexuses of the two lumbar ventricles and the third and fourth ventricles. In adults 20 mL produced every hour

NL protein (mostly albumin) value

20-50 mg/dL with albumin representing 50-70%

Diluting fluid for CSF WBC count that lyses RBCs

3% acetic acid

How many tubes used for CSF, test performed, and storage.

4. Tube 1 - Chemistry and Serology - Freezer Tube 2 - Microbiology (culture and sensitivity) - RT Tube 3 - Hematology (RBC and WBC count) - Ref Tube 4 - Additional tests for *better exclusion of skin contamination* or for additional serologic tests Specimen are collected in sterile tubes Tube 1- chemical and serological tests, these tubes are least affected by blood or bacteria introduced as result of tap = frozen Tube 2- designated for the microbiology lab = RT Tube 3- cell count, because is the least likely to contain cells introduced by the spinal tap = refrigerated **A fourth tube may be drawn for microbiology laboratory to better exclude skin contamination Test are STAT Hematology tubes are refrigerated Microbiology tubes remain at room temp Chemistry and serology tubes are frozen

NL glucose

50-80 mg/dL or approx two thirds of the plasma glucose

In CSF, which of the following findings indicates a traumatic puncture? A. The presence of erythrophagocytic cells in the CSF B. Hemosiderin granules within macrophages in the CSF sediment C. An uneven distribution of blood in the CSF collection tubes D. A xanthochromic supernatant following CSF centrifugation

A uneven distribution of blood in the CSF collection tubes

Measurement of which of the following can be replaced by CSF glutamine analysis in children with Reye syndrome? A.Ammonia B.Lactate C.Glucose D.α-Ketoglutarate

A.Ammonia

CSF lactate will be more consistently decreased in: A.Bacterial meningitis B.Viral meningitis C.Fungal meningitis D.Tubercular meningitis

A.Bacterial meningitis

Test not routinely perfomed on CSF specimens unless TB meningitis is suspected.

Acid-fast or Fluorescent antibody stain

Type of electrophoresis most frequently performed in the lab (regarding quantitative protein testing of CSF)

Agarose Electrophoresis followed by Coomasie Blue brilliant staining

Major CSF Protein

Albumin

Cerebrospinal fluid is found between the:

Arachnoid & pia mater

Cells from primary CNS tumors

Astrocytomas, retinoblastomas, and medulloblastomas which usually appear in clusters. The cells found in normal CSF Lymphocytes Monocytes- Adults usually have a predominance of lymphocytes to monocytes (70:30), whereas the ratio is essentially reversed in children The presence of increased numbers of these normal cells- pleocytosis is considered abnormal as is the finding of Immature leukocytes, Eosinophils Plasma cells Macrophages Increased tissue cells Malignant cells A high CSF WBC count of which the majority of cells are neutrophils is considered- bacterial meningitis

Cerebrospinal fluid is found between the A. arachnoid and dura mater. B. arachnoid and pia mater. C. pia mater and dura mater. D. pia mater and choroid plexus.

B) arachnoid and pia mater

The reference range for CSF protein is: A.6 to 8 g/dL B.15 to 45 g/dL C.6 to 8 mg/dL D.15 to 45 mg/dL

B.15 to 45 g/dL

A patient with a blood glucose of 120 mg/dL would havea normal CSF glucose of: A.20 mg/dL B.60 mg/dL C.80 mg/dL D.120 mg/dL

B.60 mg/dL

Why is measurement of CSF glutamine preferred over direct measurement of CSF ammonia or blood ammonia.

Because concentration of glutamine remains moe stable than that of the volatile ammonia in collected specimen. CSF glutamine level also correlates with clinical symptoms much better than does the blood ammonia.

Positive result and reagent in Pandy's test

Bluish white cloud. Phenol

Nucleated RBC are seen in CSF as a result of this. Found in approximately % of specimens.

Bone Marrow Contamination during spinal tap. 1%

Nucleated RBCs (NRBC) Seen from tap in 1% of specimens Neutrophils with pyknotic nuclei may resemble NRBCs Capillary structures and epithelial cells from traumatic taps

Bone marrow contamination

Lipophages may be seen in CSF following what

Brain Infarct

Melanin will cause this appearance/color in CSF and is probably due to this.

Brown or Xanthochromic. Melanoma (Meningeal Metastatic Melanosarcoma)

Methemoglobin causes this appearance/color in CSF

Brown or Xanthocromic

A hematoma will cause this appearance/color in CSF and is probably due to this.

Brown. Subdural or intracerebral hematoma.

How CSF collection is done.

By a physician *usually by lumbar puncture* in the fetal position. CSF is collected by lumbar puncture b/w the third, fourth, or fifth lumbar vertebra THE volume is CSF that can be removed is based on volume available and the opening pressure of the CSF, measured when the needle first enters the subarachnoid space.

Given the following results, calculate the IgG index: CSF IgG,50 mg/dL; serum IgG, 2 g/dL; CSF albumin, 70 mg/dL;serum albumin, 5 g/dL. A.0.6 B.6.0 C.1.8 D.2.8

C.1.8

Given the following information, calculate the CSF WBC count: cells counted, 80; dilution, 1:10; large Neubauer squares counted, 10. A.8 B.80 C.800 D.8000

C.800

The presence of xanthochromia can be caused by all of the following except: A.Immature liver function B.RBC degradation C.A recent hemorrhage D.Elevated CSF protein

C.A recent hemorrhage

The CSF flows through the: A.Choroid plexus B.Pia mater C.Arachnoid space D.Dura mater

C.Arachnoid space

Before performing a Gram stain on CSF, the specimen must be: A.Filtered B.Warmed to 37°C C.Centrifuged D.Mixed

C.Centrifuged

The presence of which of the following cells is increased in a parasitic infection? A.Neutrophils B.Macrophages C.Eosinophils D.Lymphocytes

C.Eosinophils

Elevated CSF protein values can be caused by all of the following except: A.Meningitis B.Multiple sclerosis C.Fluid leakage D.CNS malignancy

C.Fluid leakage

The finding of oligoclonal bands in the CSF and not in the serum is seen with: A.Multiple myeloma B.CNS malignancy C.Multiple sclerosis D.Viral infections

C.Multiple sclerosis

All of the following statements are true about cryptococcal meningitis except: A.An India ink preparation is positive B.A starburst pattern is seen on Gram stain C.The WBC count is over 2000 D.A confirmatory immunology test is available

C.The WBC count is over 2000

Normal values of CSF/serum albumin ratio and index

CSF/Serum albumin ratio - 1:230 CSF/Serum albumin index - <9

Tests performed to determine whether IgG is increased because its being produced within CNS or is elevated as a result of defect in BBB

CSF/serum albumin index and ratio (to evaluate integrity of BBB) CSF IgG index (measures IgG synthesis within CNS) To accurately determine whether IgG is increased because of it being produced w/in the CNS or elevated as a result of a defect in the blood-brain barrier, comparison b/w serum and CSF levels of albumin and IgG must be made CSF/serum albumin index- integrity of the blood brain barrier. Calculated after determining the concentration of CSF albumin in milligrams. An index values less the 9 represents an intact blood brain barrier. The index increases relative to the damage CSF IgG index- measure synthesis w/in the CNS Values greater than 0.70 indicate IgG production w/in the CNS

When chroid plexus cells and ependymal cells are present in CSF, they

Can closely resemble clusters of malignant cells

Where does IgG in CSF come from

Can originate both from production in CSF or from Blood.

Primary use of electrophoresis in quantitative protein testing for CSF

Detection of Oligoclonal bands in the gamma region representing inflammation within CNS. Electrophoresis and Immunophoretic Techniques- purpose is perfroming CSF protein electrophoresis to detect oligoclonal bands, which represent inflammation w/in the CNS. bands located in the gamma region , indicating immunoglobulin production. To ensure that oligoclonal bands are present as a result of neurological inflammation, simultaneous serum electrophoresis is performed. leukemia, lymphoma, and viral infections may produce serum banding, which can appear in the CSF as a result of blood-brain barrier leakage or traumatic introduction of blood into the CSF specimen. The presence of two or more oligoclonal bands in the CSF that are not present in the serum can be- diagnosis of multiple sclerosis, particularly when accompanied by an increased IgG index. Other diseases: encephalitis, neurosyphilis, Guillain-Barre syndrome, neoplastic disorders Low protein levels in the CSF make concentration of the fluid before performing electrophoresis essential for most electrophoretic techniques. Better resolution can be made using CSF immunofixation electrophoresis and isoelectric focusing followed by silver staining. CSF can be identified by extra isoform of tau transferrin Myelin Basic protein- indicates recent destruction of the myelin sheath that protects the axons of the neurons (demyelination). The course of multiple sclerosis can be monitored by measuring the amount of MBP in the CSF

WBC count-Diluted and undiluted

Dilute with 3% acetic acid; methylene blue helps to see cells; To count undiluted: rinse transfer pipette with acetic acid, gently rotate pipette

Outer layer that lines the skull and vertebral canal

Dura Mater

All of the following proteins are normally present in the CSF except:

Fibrinogen

Appearance

Initial appearance of normal crystal clear CSF can provide valuable diagnostic information. First examined at bedside, then sent to lab for results.

elevated AST value

Intracerebral *hemorrhage*, subarachnoid hemorrhage, *bacterial meningitis*

Detects Cryptococcus neoformans antigen in serum and CSF

Latex Agglutination Test

Indications for Lumbar Puncture

Meningeal Infection, Subarachnoid Hemorrhage, Primary or Metastatic Injury, Demyelinating Diseases

Brown appearance

Methemoglobin Presence of melanin caused by melanoma Subdural or intracerebral hematoma

Bacterial antigen test for CSF does not appear to be as sensitive to the detection of this as it is to other organisms.

N. Meningitidis

How are nonpathologically significant cells distinguished from malignant cells

Often appear in clusters and has a uniform appearance.

Pink appearance

Oxyhemoglobin

Oily appearance

Radiographic contrast media

Formula for CSF/Serum albumin ratio and index

Ratio - CSF albumin g/dL / Serum albumin g/dL Index - CSF albumin mg/dL / Serum Albumin g/dL

What does presence of Myelin Basic Protein indicate

Recent destruction of Myelin Sheath

Purpose of macrophages in CSF

Remove cellular debris and foreign objects

Bacterial antigen test should be used in combination with what

Results from hematology and clin chem for diagnosing meningitis.

Cell Count

Routinely performed on CSF is the WBC count. RBC counts are usually only to determine when a traumatic tap has occurred and a correction for leukocytes or protein is desired.

Organisms frequently encountered in CSF

S pneumoniae, H. Influenzae, L. monocytogenes, E. coli, N. meningitidis, S. agalactiae

CSF is produced primarily from:

Secretions by the chroid plexus

Used to detect active cases of syphilis within CNS

Serology

Failure to isolate CSF organisms should/should not be interpreted as absent.

Should not be interpreted to mean that organisms are absent because there must be 10^5/mL in order for them to be demonstrated by gram stain.

contain RBCs degraded to HEMOSIDERIN granules and further degraded to HEMATOIDIN crystals representing unconjugated bilirubin

Siderophage

elevated CPK value

Stroke, multiple sclerosis, degenerative disorders, primary brain tumors, viral and bacterial meningitis, and epileptic seizures

Which of the following is not an analytical concern when the processing and testing of CSF are delayed?

The liability of the immunoglobulin

Quantitative Protein tests for CSF

Turbidimetric, Nephelometric, Dye-Binding Technique, Biuret Method, Electrophoresis.

Increased glucose

in hyperglycemia and traumatic taps

Standard Neubauer formula is used for counts; know this formula!

# cells counted × dilution ____________________________ = cells/µL # of sqs × vl of 1 sq (0.1)

Clot formation

*Fluid collected from a traumatic tap may form clots owing to the introduction of plasma fibrinogen into specimen *Bloody CSF caused by intracranial hemorrhage does not contain enough fibrinogen to clot *Clot formation that do not produce a bloody fluid: meningitis, Froin syndrome, blocked CSF circulation tru subarachnoid space. *A classic web like pellicle is seen with tubercular meningitis after overnight stay in the refrigeration.

How many Leukocytes are normally present in the CSF obtained from an adult:

0 to 5 cells

How many leukocytes are normally present in the CSF obtained from an adult? A. 0 to 5 cells/mL B. 0 to 10 cells/mL C. 0 to 20 cells/mL D. 0 to 30 cells/mL

0 to 5 cells / mL

Normal value of CSF WBCs

0-5 wbc/uL (adult) higher in children 0-30 mononuclear cells/uL (newborn)

Traumatic tap occurs in how many % of lumar punctures

20%

How any percent of Plasma glucose is CSF glucose

60-70%

Normal values of CSF glutamine

8-18mg/dL Produced from ammonia and alpha-ketoglutarate by the brain cells. This process serves to remove the toxic metabolic waste product ammonia from the CNS. Normal 8 to 18 mg/dL Elevated levels- liver disorders that result in increased blood and CSF ammonia As the concentration of ammonia in the CSF increases, the supply of alpha-ketoglutarate becomes depleted; glutamine can no longer be produced to remove to toxic ammonia and coma ensues CSF for patients with coma of unknown origin

Volume of CSF maintained and how.

90-150 mL in adults and 10-60mL in neonates. Due to arachnoid granulations that allow for reabsorption. They are reabsorbed at the same rate they are produced.

Values for CSF IgG index

<0.6 - normal >0.77 - IgG production within CNS / MS

Normal value for Myelin Basic Protein in CSF

<4ng/mL Myelin Basic protein- indicates recent destruction of the myelin sheath that protects the axons of the neurons (demyelination). The course of multiple sclerosis can be monitored by measuring the amount of MBP in the CSF

Index values for CSF/Serum regarding BBB

<9 - Intact >9 - damage 9-14 - slight impairment 15-30 - moderate impairment >30 - severe impairment

Bloody appearance

>600 RBC/uL: Hemorrhage or traumatic tap

Which of the following procedures frequently provides a rapid presumptive diagnosis of bacterial meningitis? A. A blood culture B. A CSF culture C. A CSF Gram stain D. Immunologic tests on CSF for microbial antigens

A CSF Gram stain

What is Latex Agglutination test for regarding CSF

A serologic test that detects Cryptococcus neoformans antigen in Serum and CSF **Latex agglutination detects the presence of C. neoformans antigen in serum and CSF provide a more sensitive method than the India Ink preparation, but immunologic testing results should be confirmed by culture and demonstration of the organism by India ink preparation. Interference of rheumatoid factor is the most common cause of false positive reactions. he lateral flow assays (LAF) can provide rapid method for detecting C.neoformans. Procedure utilizes a reagent strip coated with monoclonal antibodies that react with the cryptococcal polysaccharide capsule Latex agglutination and enzyme-linked immunosorbent assay provides rapid means for detecting and identifying microorganisms in CSF The bacterial antigen test (BAT) does not appear to be as sensitive to N. meningitidis as it is to the other organisms The amoeba Naegleria fowleri- opportunistic parasite that enters the nasal passages and migrates along the olfactory nerve to invade the brain- use a wet mount*

Which of the following proteins in the CSF is used to monitor the integrity of the blood-brain barrier?

Albumin

Which of the following proteins in the CSF is used to monitor the integrity of the blood-brain barrier? A. Albumin B. Transthyretin C. Transferrin D. Immunoglobulin G

Albumin

Proteins found in CSF

Albumin, Prealbumin (transthyretin), haptoglobin, ceruloplasmin, transferrin, tau, gamma globulins

Where is CSF Albumin derived

All CSF Albumin is derived from transport across BBB because CNS does not synthesize Albumin.

During a lumbar puncture procedure, the first collection tube of CSF removed should be used for A. chemistry tests. B. cytologic studies. C. hematologic tests. D. microbiological studies.

Chemistry studies

During a lumbar puncture procedure, the first collection tube of CSF removed should be used for

Chemistry tests

Xanthochromic appearance of CSF is caused by this/these.

Hemoglobin, Oxyhemoglobin, Bilirubin, Methemoglobin, Merthiolate, Carotene, Protein, Melanin, rifampin therapy. **Xanthochromia- CSF supernatant is pink/ orange/ or yellow. **The most common cause is RBC degradation Other causes- elevated serum bilirubin, presence of pigment carotene, markedly increased protein concentrations and melanoma pigment. Xanthochromia caused by serum bilirubin due to immature liver function

Neutrophil pleocytosis

Primary in bacterial meningitis Often contain phagocytized bacteria Increased early viral, fungal, tubercular, parasitic Vacuoles may be present

What is the CSF function?

Protects and cushions the central nervous system and provides nutrients to neural and removes metabolic waste

Most frequently performed chemical test on CSF

Protein

CSF Test that is more sensitive than india ink.

Latex Agglutination Test The lateral flow assays (LAF) can provide rapid method for detecting C.neoformans. Procedure utilizes a reagent strip coated with monoclonal antibodies that react with the cryptococcal polysaccharide capsule Latex agglutination and enzyme-linked immunosorbent assay provides rapid means for detecting and identifying microorganisms in CSF

Macrophage with ingested fats

Lipophage

When is CSF Glutamine increased

Liver disorders that result in increased blood and CSF ammonia, Reye's syndrome, coma of unknown origin

Metastatic carcinoma cells of nonhematologic origin are primarily what cancers.

Lung, breast, renal and gastrointestinal malignancies

Cells frequently seen in CSF that are a serious complication of acute leukemias

Lymphoblasts, myeloblasts, and monoblasts

Which of the following cell types predominate in CSF during a classic case of viral meningitis

Lymphocytes

Which of the following cell types predominate in CSF during a classic case of viral meningitis? A. Lymphocytes B. Macrophages C. Monocytes D. Neutrophils

Lymphocytes

Which of the following cells can be present in small numbers in normal CSF? A. Erythrocytes B. Lymphocytes C. Macrophages D. Plasma cells

Lymphocytes

Which of the following cells can be present in small numbers in normal CSF:

Lymphocytes

Predominant cells found in a normal CSF

Lymphocytes (predominant in adults 70:30), Monocytes (predominant in children 30:70)

A thin membrane lining the surfaces of the brain and spinal cord.

Pia Mater

Xanthochromia colors and indications

Pink - very slight amount of oxyhemoglobin is present Orange - Heavy hemolysis Yellow - conversion of oxyhemoglobin to conjugated bilirubin

Presence of increased numbers of normal cells in CSF and is considered abnormal

Pleocytosis

Nonpathologically significant cells are most frequently seen following diagnostic procedures such as these.

Pneumoencephalography, Fluid obtained from ventricular taps, during neurosurgery.

2nd most prevalent protein in CSF. and why.

Prealbumin or Transthyretin. Dual synthesis by liver and choroid plexuses

Xanthochromia indicates what and occurs when and clears when.

Presence of RBC degradation products. Within 4 to 5 hours after subarachonoid hemorrhage and usually clears approximately 3 weeks after the event.

An unknown fluid can be positively identified as CSF by determining the

Presence of carbohydrate-deficient transferrin on electrophoresis.

CSF Glutamine provides an indirect test for what.

Presence of excess ammonia in CSF

Describe Fuchs-Rosenthal counting chamber

Undiluted sample, Phase Microscopy. 16 Large squares, 0.2mm depth.

How is CSF glucose analyzed

Using same procedures employed for blood glucose

Recommended by CDC to diagnose neurosyphilis

VDRL

Gamma Globulins in CSF

Primary IgG and small amounts of IgA

Uses of CSF lactate measurements

Valuable aid in diagnosis and management of meningitis cases Sensitive method for evaluating effectiveness of antibiotic therapy *because it remains elevated during initial treatment and falls rapidly when treatment is successful* Elevated in any condition that *decreases flow of oxygen to tissues* To monitor severe head injuries

Oxyhemoglobin cause this appearance/color in CSF

Xanthochromic

Rifampin Therapy causes this appearance/color in CSF

Xanthochromic.

Carotene cause this appearance/color in CSF and correlates to this

Xanthochromic. Carotinoids. Increased serum levels

Merthiolate cause this appearance/color in CSF and correlates to this

Xanthochromic. Disinfectant contamination.

Bilirubin cause this appearance/color in CSF and correlates to this.

Xanthochromic. RBC degradation and elevated serum bilirubin level.

Which of the following procedures frequently provides a rapid presumptive diagnosis of bacterial meningitis?

a CSF Gram stain

Total protein represents

a combination of prealbumin, albumin, transferrin and trace amounts of IgG Provides information as to the integrity of the blood-brain barrier

Pleocytosis is a term used to describe A. an increased number of cells in the CSF. B. a pink, orange, or yellow CSF specimen. C. an increased protein content in the CSF caused by cellular lysis. D. inflammation and sloughing of cells from the choroid plexus.

an increased number of cells in the CSF

Decreased CSF glucose indications

Determining causative agents of meningitis. Hypoglycemia, pyogenic meningitis, fungal meningitis, subarachnoid block, toxoplasmosis, primary tumor of brain, disorders affecting BBB, increased CNS glycolytic activity, metastatic carcinoma. A markedly decreased CSF glucose level accompanied by an increased WBC count and a large percentage of neutrophils indicates- bacterial meningitis. WBC are lymphocytes instead of neutrophils- tubercular meningitis Decreased CSF glucose are caused by primarily by the alteration in the mechanism of glucose transport across the blood brain barrier and by increased use of glucose by brain cells.

Increased CSF glucose level indications

Diabetes, Encephalitis, Conditions associated with intracranial pressure

VDRL is recommended by CDC to do what regarding CSF.

Diagnose neurosyphilis

Lymphoma cells in CSF indicate

Dissemination from the lymphoid tissue which resembles large and small lymphocytes and usually appears in clusters of large, small, or mixed cells based on classification of lymphoma

CSF Glucose

Glucose enters the CSF by selective transport across the blood brain barrier. 60-70% of that of plasma glucose. The blood glucose should be drawn about 2 hours before the spinal tap to allow time for equilibration b/w the blood and the fluid Tested immediately before glycolysis occurs Diagnostic significance- finding values that are decreased relative to plasma values Elevated CSF glucose values are always as a result of plasma elevations. Low CSF can be of considerable value determining the causative agents in meningitis. A markedly decreased CSF glucose level accompanied by an increased WBC count and a large percentage of neutrophils indicates- bacterial meningitis. WBC are lymphocytes instead of neutrophils- tubercular meningitis Normal CSF glucose value is found with an increased number of lymphocytes- viral meningitis. Decreased CSF glucose are caused by primarily by the alteration in the mechanism of glucose transport across the blood brain barrier and by increased use of glucose by brain cells.

A chemical test frequently performed in CSF and not in blood

Glutamine

Routinely performed on CSF from all suspected cases of meningitis

Gram Stain

Traumatic Collection (Tap)

Grossly bloody CSF can be an indication of intracranial hemorrhage, but it may also be due to the puncture of a blood vessel during the spinal tap procedure.

Increased proteins and lipids in CSF may cause this appearance and correlates to this.

Hazy,cloudy,turbid,or milky. Disorders that affect BBB. Proteins are probably due to production of IgG within CNS.

3 layers of meninges that line brain and spinal cord. In order from outer to inner.

* Dura mater "hard mother"- outer layer that lines the skull and vertebral canal * Arachnoid "spider web-like"-filamentous inner membrane * Pia mater "gentle mother"-thin membrane lining surfaces of the brain

CSF Formation:

*Hydrostatic pressure* and active transport CSF is *not an ultra-filtrate* Very tight-fitting endothelial cells, prevent filtration of large molecules - called the blood-brain barrier

Explain the BBB or Blood-brain barrier

*In the choroid plexuses, the endothelial cells have very tight-fitting junctures that prevent the passage of many molecules. This tight fitting structure of the endothelial cells in the choroid plexuses is termed the blood-brain- barrier Blood brain barrier essential to protect the brain The junctures prevent the passage of helpful substances (antibodies, medication) Disruption of the blood-brain barrier by disease such as meningitis, and multiple sclerosis allows leukocytes, proteins, and additional chemicals to enter CSF

All of the following can cause xanthochromia in CSF except A. high concentrations of protein. B. high concentrations of bilirubin. C. increased numbers of leukocytes. D. erythrocytes from a traumatic tap.

increased numbers of leukocytes

An immunoglobulin G index greater than 0.70 indicates A. intrathecal synthesis of immunoglobulin G. B. a compromised blood-brain barrier. C. active demyelination of neural proteins. D. increased transport of immunoglobulin G from plasma into the CSF.

intrathecal synthesis of immunoglobulin G.

Four labeled sterile collection tube:

Tube1: *Chemical* Tube2:Microbiology Tube3:Cell counts Tube4:Cytology studies

An immunoglobulin G index greater than 0.70 indicates

intrathecal synthesis of immunoglobulin G.

Ependymal cells

lining ventricles and neural canal; less defined cell membranes in clumps

Cerebrospinal Fluid (CSF)

major fluid of the body. Provides a physiologic system to supply nutrients to the nervous tissue, remove metabolic waste, and produce a mechanical barrier to cushion the brain and spinal cord against trauma. Produced in the choroid plexuses of the 2 lumbar ventricles and the 3rd and 4th ventricles.

Clinical Significance of elevated protein values

most frequently seen in pathologic conditions. Abnormally low values present when fluid is leaking from CNS. Elevated caused by damage to blood brain barrier, production of immunoglobulins within CNS, decreased clearance of normal protein from fluid, and degeneration of neural tissue. Meningitis and hemorrhage conditions that damage the blood brain barrier are most common causes of elevated CSF protein Clinical significance of elevated protein values- seen in pathologic conditions. Low values are seen when fluid is leaking from CNS. the causes of elevated CSF protein: Damage to the blood-brain barrier, immunoglobulin production w/in the CNS, decreased normal protein clearance from fluid, and neural tissue degradation. Meningitis and hemorrhage conditions that damage the blood-brain barrier are the most common cause of elevated CSF protein

High CSF WBC count

of which majority of the cells are NEUTROPHISL-is considered indicative of bacterial meningitis. An elevated CSF WBC count with a high percentage of LYMPHOCYTES AND MONOCYTES- suggests meningitis of viral, tubercular, fungal, or parasitic origin.

CSF albumin is increased when the

permeability of the blood-barrier is compromised

An unknown fluid can be positively identified as CSF by determining the A. lactate concentration. B. albumin concentration. C. presence of oligoclonal banding on electrophoresis. D. presence of carbohydrate-deficient transferrin on electrophoresis.

presence of carbohydrate-deficient transferrin on electrophoresis.

WBC Count

prior to WBC count, must LYSE the RBCs. Specimens that require dilution can be diluted, substituting 3% glacial acetic acid to lyse the RBCs. You may also add Methylene blue to diluting fluid, staining WBCs, providing better differentiation between neutrophils and mononuclear cells. The number is multiplied by dilution factor to obtain the # of WBCs per microliter.

Cerebrospinal Fluid is produced primarily from A. secretions by the choroid plexus. B. diffusion from plasma into the central nervous system. C. ultrafiltration of plasma in the ventricles of the brain. D. excretions from ependymal cells lining the brain and spinal cord.

secretions by the choroid plexus.

Xanthochromic

term used to describe CSF supernatant that is pink, orange, or yellow. The most common cause is presence of RBC degradation products. Depending on amount of blood and length of time it has been present, color will vary from pink (slight oxyhemoglobin) to orange (heavy hemolysis) to yellow (conversion of oxyhemoglobin to unconjugated bilirubin). Other causes are: elevated serum bilirubin, presence of pigment carotene, marked increased protein concentrations, and melanoma pigment. Caused by bilirubin due to immature liver function is also common in infants, premature.

CSF cellular constituents

the cells found in normal CSF are primarily lymphocytes and monocytes. Adults have 70:30 lymphocytes:monocytes and in children, reversed. The presence of increased of these normal cells is abnormal, as well as finding of immature leukocytes, eosinophils, plasma cells, macrophages, increased tissue cells, and malignant cells.

Normal CSF lactate levels (less than 25 mg/dL) are commonly found in patients with A. bacterial meningitis. B. fungal meningitis. C. tuberculous meningitis. D. viral meningitis.

viral meningitis.

Pleocytosis

with this, involving neutrophils, lymphocytes, or monocytes is present, the CSF differential count is most frequently associated with its role in providing diagnostic information about the type of microorganism that is causing an infection of the meninges (meningitis)

Protein- methodology

*Methodology- Principles of turbidity or dye binding ability. The turbidity method has been adapted to automated instrumentation in the form of nephelometry *Protein fractions- Routine CSF procedures are designed to measure total protein concentration but abnormal CSF proteins requires measurement of individual protein fractions. Proteins that appear in the CSF as a result of damage to the integrity of the blood-brain barrier contains fractions proportional to those in plasma while albumin is highest Multiple sclerosis that stimulate the immunocompetent cells in the CNS show a higher proportion of IgG To accurately determine whether IgG is increased because of it being produced w/in the CNS or elevated as a result of a defect in the blood-brain barrier, comparison b/w serum and CSF levels of albumin and IgG must be made CSF/serum albumin index- integrity of the blood brain barrier. Calculated after determining the concentration of CSF albumin in milligrams. An index values less the 9 represents an intact blood brain barrier. The index increases relative to the damage CSF IgG index- measure synthesis w/in the CNS Values greater than 0.70 indicate IgG production w/in the CNS

Xanthochromic Supernatant

*RBCs most usually remain in the CSF for approximately 2 hrs before noticeable hemolysis begins; therefore, a xanthochromic supernatant would be the result of blood that has been present longer than the introduced by the traumatic tap *To examine: centrifuged and supernatant examined against white background *Additional testing for differentiation: microscopic examination and the D-dimer test. The microscopic finding of macrophages containing ingested RBCs or hemosiderin granules indicates intracranial hemorrhage *Detection of fibrin degradation product D-dimer by latex agglutination immunoassay indicates fibrin formation at a hemorrhage site

The integrity of the blood-brain barrier is measured usingthe: A.CSF/serum albumin index B.CSF/serum globulin ratio C.CSF albumin index D.CSF IgG index

A.CSF/serum albumin index

What department is the CSF tube labeled 3 routinely sent to? A.Hematology B.Chemistry C.Microbiology D.Serology

A.Hematology

Cells resembling large and small lymphocytes with cleaved nuclei represent: A.Lymphoma cells B.Choroid cells C.Melanoma cells D.Medulloblastoma cells

A.Lymphoma cells

The primary concern when pleocytosis of neutrophils and lymphocytes is found in the CSF is: A.Meningitis B.CNS malignancy C.Multiple sclerosis D.Hemorrhage

A.Meningitis

A web-like pellicle in a refrigerated CSF specimen indicates: A.Tubercular meningitis B.Multiple sclerosis C.Primary CNS malignancy D.Viral meningitis

A.Tubercular meningitis

Pleocytosis is a term used to describe:

An increased number of cells in the CSF.

In CSF which of the following finding indicates a traumatic puncture?

An uneven distribution of blood in the CSF collection tubes

A filamentous (*spider-like*) inner membrane

Arachnoid Mater

Serology test not recommended for use on CSF

RPR (Rapid plasma reagin)

Substances present in the CSF are controlled by the: A.Arachnoid granulations B.Blood-brain barrier C.Presence of one-way valves D.Blood-CSF barrier

B.Blood-brain barrier

CSF can be differentiated from serum by the presence of: A.Albumin B.Globulin C.Prealbumin D.Tau transferrin

B.Globulin

Place the appropriate letter in front of the statement that best describes CSF specimens in these two conditions: A.Traumatic tap B.Intracranial hemorrhage ____ Even distribution of blood in all tubes ____ Xanthochromic supernatant ____ Concentration of blood in tube 1 is greater than in tube 3 ____ Specimen contains clots

B.Intracranial hemorrhage _B___Even distribution of blood in all tubes _B___Xanthochromic supernatant _A__ Concentration of blood in tube 1 is greater than in tube 3 __A _Specimen contains clots

Hemosiderin granules and hematoidin crystals are seen in: A.Lymphocytes B.Macrophages C.Ependymal cells D.Neutrophils

B.Macrophages

Neutrophils with pyknotic nuclei may be mistaken for: A.Lymphocytes B.Nucleated RBCs C.Malignant cells D.Spindle-shaped cells

B.Nucleated RBCs

In serum, the second most prevalent protein is IgG; inCSF, the second most prevalent protein is: A.Transferrin B.Prealbumin C.IgA D.Ceruloplasmin

B.Prealbumin

The functions of the CSF include all of the following except: A.Removing metabolic wastes B.Producing an ultrafiltrate of plasma C.Supplying nutrients to the CNS D.Protecting the brain and spinal cord

B.Producing an ultrafiltrate of plasma

The CSF tube that should be kept at room temperature is: A.Tube 1 B.Tube 2 C.Tube 3 D.Tube 4

B.Tube 2

Differentiate kinds of Meningitis with regards to Predominating cells and Glucose level

Bacterial - PMNs, Marked decreased Tubercular - Lymphocytes, Decreased Viral - Lymphocyte, Normal

elevated LDH value

Bacterial and viral meningitis, subarachnoid hemorrhage, lymphomas, leukemias and metastatic tumors

Presence of neutrophils in CSF is associated with these.

Bacterial meningits (may contain phagocytized bacteria), CNS Hemorrhage, Repeated lumbar punctures, and injection of medications or radiographic dye.

An oily appearance or color of CSF is caused by this/these

Radiographic Contrast Media

Malignant cells is present as a result

CNS tumor or leukemic process

Formula for CSF IgG index

CSF IgG (mg/dL) / Serum IgG (g/dL) _____________________________________ CSF Albumin (mg/dL) / Serum Albumin (g/dL)

Take NOTE!

CSF culture is actually a *confirmatory* rather than a diagnostic procedure

Chemistry tests

CSF is formed by filtration of plasma, one would expect to find the same low molecular weight chemicals in the CSF that are found in plasma , however because the filtration process is selective and the chemical composition is controlled by the blood brain barrier, reference values for CSF chemicals are not the same as the plasma values. Abnormal values- alteration in the permeability of the blood brain barrier or increased production or metabolism by the neural calls response to a pathologic condition because CSF is formed by filtration of plasma, same low molecular weight chemicals in CSF are found in plasma. Because the filtration process is selective and the chemical composition is controlled by the blood brain barrier, normal values for CSF chemicals NOT the same as plasma values.

Specimen Collecton

CSF is usually collected by lumbar puncture between the 3rd, 4th, or 5th vertebrae.

Naegleria fowleri

Causes primary amebic meningoencephalitis (PAM) Heat-loving (thermophilic) microbe Enters body through the nose The infection destroys brain tissue causing brain swelling and death Fatality rate >99% within 5 days

Tests in CSF done immediately and why

Cell Count - deteriorates in time and clumps in presence of *fibrinogen* Glucose - To prevent glycolysis Bacteriologic - Prevent proliferation

Acute bacterial meningitis values: Cell count Protein Glucose other test

Cell count: 1,000-10,000 neutrophil Protein: 100-500 Glucose: <40 other test: Gram stain

Tuberculosis values: Cell count Protein Glucose other test

Cell count: 100-600 Neutrophils early followed by lymphocytes Protein: 50-300 Glucose: decreased <45 other test: Pellicle formation

Fungal values: Cell count Protein Glucose other test

Cell count: 40-400 lymphocytes and monocytes Protein: 50-300 Glucose: NL to decrease other test: India Ink prep

Viral Meningitis values: Cell count Protein Glucose other test

Cell count: 5-300 lymphocytes Protein: <100 Glucose: Normal other test: NL lactase

Cause of turbidity of CSF

Cellular elements, Microorganisms, and contrast media.

Which of the following statements regarding CSF is true? A. Cerebrospinal fluid is constantly produced. B. Cerebrospinal fluid is reabsorbed into the blood at the choroid plexus. C. Cerebrospinal fluid is essentially composed of diluted plasma. D. Cerebrospinal fluid circulates through the brain and spinal cord because of active and passive diffusion processes.

Cerebrospinal fluid is constantly produced

Which of the following statements regarding CSF is true?

Cerebrospinal fluid is essentially composed of diluted plasma.

Sources of CSF.

Choroid Plexuses of the Ventricles of the Brain 70% and Other sites 30% such as ependymal lining of ventricles and cerebral subarachnoid space. CSF is produced in the choroid plexuses of the two lumbar ventricles and the third and fourth ventricles. In adults 20 mL produced every hour * The choroid plexuses are capillary networks that form under the CSF from plasma by mechanisms of selective filtration under hydrostatic pressure and active transport secretion * The chemical composition of the CSF does not resemble an ultrafiltrate of plasma * In the choroid plexuses, the endothelial cells have very tight-fitting junctures that prevent the passage of many molecules. This tight fitting structure of the endothelial cells in the choroid plexuses is termed the blood-brain- barrier

Cells formed from the epithelial lining of the choroid plexus. Seen singularly and in clumps where nucleoli are usually absent and nuclei have a uniform appearance.

Choroidal Cells

Examples of nonpathologically significant cells and describe each

Choroidal cells - epithelial lining of choroid plexus. seen singularly and in clumps where nucleoli are usually absent and nuclei have a uniform appearance Ependymal cells - Linings of the ventricles and neural canal. Have less defined cell membranes and are frequently seen in clusters. Nucleoli are often present. Spindle-shaped cells - lining cells from the arachnoid. Usually seen in clusters and may be seen with systemic malignancies.

Regarding Cryptococcus Neoformans, a fungus, what does G/S reveal.

Classic starburst pattern produced by it which may be seen more often positive than india ink.

Describe CSF

Clear and Colorless Fluid: Supply nutrients to the nervous tissue, remove metabolic wastes, and produce a mechanical barrier to cushion the brain and spinal cord against trauma

Total cell count

Clear specimens may be counted undiluted, provided no overlapping of cells is seen. When dilutions are required, calibrated automatic pipettes are used. Dilutions are made with normal saline, mixed inversion, and loaded into hemocytometer with Pasteur pipette.

Proteins cause this appearance/color in CSF and correlates to this.

Clotted or Pellicle OR Xanthocromic (>150mg/dL). Disorders affecting BBB.

Clotting factors cause this appearance/color in CSF and correlates to this

Clotted or Pellicle. Traumatic tap (clotted)/Tubercular meningitis (pellicle)

CSF NL physical exam: Color Clarity

Colorless Clear

Dye used in Dye-binding technique for quantitative protein testing of CSF

Coomasi Brilliant Blue G250, Ponceau S

After a CNS diagnostic procedure, which of the following might be seen in the CSF? A.Choroidal cells B.Ependymal cells C.Spindle-shaped cells D.All of the above

D.All of the above

Macrophages appear in the CSF after: A.Hemorrhage B.Repeated spinal taps C.Diagnostic procedures D.All of the above

D.All of the above

Myeloblasts are seen in the CSF: A.In bacterial infections B.In conjunction with CNS malignancy C.After cerebral hemorrhage D.As a complication of acute leukemia

D.As a complication of acute leukemia

A CSF glucose of 15 mg/dL, WBC count of 5000, 90% neutrophils, and protein of 80 mg/dL suggests: A.Fungal meningitis B.Viral meningitis C.Tubercular meningitis D.Bacterial meningitis

D.Bacterial meningitis

Nucleated RBCs are seen in the CSF as a result of: A.Elevated blood RBCs B.Treatment of anemia C.Severe hemorrhage D.Bone marrow contamination

D.Bone marrow contamination

The purpose of adding albumin to CSF before cytocentrifugation is to: A.Increase the cell yield B.Decrease the cellular distortion C.Improve the cellular staining D.Both A and B

D.Both A and B

A total CSF cell count on a clear fluid should be: A.Reported as normal B.Not reported C.Diluted with normal saline D.Counted undiluted

D.Counted undiluted

The test of choice to detect neurosyphilis is the: A.RPR B.VDRL C.FAB D.FTA-ABS

D.FTA-ABS

Quality Control

Daily control of 0.2 mL saline and 2 drops of albumin stained for bacterial contamination

How to differentiate Traumatic tap from subarachnoid hemorrhage.

Distribution of blood - TT (uneven) . SH (even) Clot Formation - TT (+ or clumped RBC) . SH ( - ) Supernatant - TT (clear anc colorless) . SH (Xanthoc..) Micro exam - SH (presence of erythrophagocytes) D-dimer test - TT (+ detection of fibrin degradation product (D-dimer) by latex agglutination immunoassay) Others - TT (Ratio of >500 RBCs for every WBCs)

Quantitative protein test for CSF that uses smaller sample size and has less interference from external sources.

Dye-Binding Technique

Cells from the lining of the ventricles and neural canal. Less defined cell membranes and are frequently seen in clusters. Nucleoli are often present.

Ependymal Cells

Blood-brain barrier

Essential to protect brain Chemicals and harmful substances do not pass Antibodies and medications are excluded Meningitis, multiple sclerosis disrupt membrane

Serology test that is more sensitive in detecting syphilis in CSF

FTA-ABS (Fluorescent Treponemal Antibody Absorbance)

India ink preparations and microbial antigen tests on CSF can aid in the diagnosis of

Fungal meningitis.

Which of the following substances does not normally pass through the blood-brain barrier? A. PO2 B. Albumin C. Glucose D. Fibrinogen

Fibrinogen

Concentration methods for CSF before differential count and describe the last two.

Filtration Sedimentation Cytocentrifugation - 0.1 mL of CSF combined with one drop of 30% albumin produces adequate cell yield when cytocentrifuged. As soecien inside conical tube is centrifuged, cells present in the fluid are forced into a monolayer within a 6mm diameter circle on the slide. Routine Centrifugation (*wright's after*) - centrifuge for 5-10mins, supernatant fluid saved for other tests, slides are air dried.

Why are the normal values for CSF chemicals not the same with plasma values.

Filtration process is selective and chemical composition is controlled by the BBB

Normal eletrophoretic pattern for CSP proteins

First band - prealbumin Second band - albumin Followed by 2 transferrin bands. Faint bands of alpha1-antitrypsin and IgG may also be present.

Cryptococcus neoformans

Frequent complication of AIDS Gram stain produces a starburst pattern India ink stain on CSF to detect the capsule and budding yeast; beware of air bubbles

Counting chamber that uses undiluted samples and phase microscopy

Fuchs-Rosenthal Counting Chamber

Ways of CSF cell count

Fuchs-Rosenthal Counting Chamber Neubauer Counting Chamber

Role of microbiology test

Identification of the causative agent in meningitis

A classic web-like pellicle is associated with this and can be seen in this

Tuberculous Meningitis. After overnight refrigeration of fluid.

Microbiology Tests

Identify the causative agent of meningitis. The micrograms must be recovered from the fluid by growing it on the appropriate culture medium. CSF culture is confirmatory rather than a diagnostic procedure Gram stain- performed on CSF from all suspected of meningitis. Concentrated specimens should be used. The use of cytocentrifuge provides a highly concentrated specimen for gram stains. Blood cultures should be taken A gram stain is one of the most difficult slides to interpret because the number of organisms present is usually small. Organisms- Strep pneumoniae gram + cocci H. influenza- pleomorphic gram - rods E.coli gram - rods N. meningitidis- gram - cocci S. agalactiae- gram + cocci in newborns L. monocytogenes- gram + rods in newborns Acid fast/ fluorescent antibody when tubercular meningitis is suspected. Fungal meningitis- gram stain and india ink preparation to detect Cryptococcus neoformans.

Differential Count on CSF

Identifying the types of cells present in CSF is valuable diagnostic aid. Should be performed on a stained smear and NOT from the cells in Neubauer counting chamber. CSF is added to conical chamber, and as specimen centrifuged, cells present in fluid are forced into a monolayer within a 6mm diameter circle. Fluid is absorbed by filter paper blotter, producing more concentrated area of cells. As little as 0.1 mL CSF combined with 1 drop of 30% albumin produces adequate cell yield when processed with cytocentrifuge. It increases cell yield and decreases cellular distortion.

Is a faint band in normal CSF but is present as oligoclonal bands in multiple sclerosis and in some lymphoproliferative diseases

IgG gamma globulin -

Indication of Lymphoblasts, myeloblasts, and monoblasts in CSF

In complication of acute leukemias

CSF collection is usually perfomed best when and why.

In the morning. Patient's blood and CSF glucose levels will be equilibrated.

Which of the following statements about CSF glucose is false?

Increase CSF glucose values are diagnostically significant.

Increase Neutrophil and Lymphocytes

Increase Neutrophils indicates *bacterial infection*; Increase Lymphocytes = viral, tubercular, fungal, parasitic

Which of the following statements about CSF glucose is false? A. Increased CSF glucose values are diagnostically significant. B. Glucose enters the CSF by active transport and simple diffusion. C. Decreased CSF glucose values reflect a defective blood-brain barrier and increased glycolysis. D. CSF glucose values reflect the plasma glucose concentration 30 to 90 minutes preceding collection.

Increased CSF glucose values are diagnostically significant.

This may indicate MS regarding CSF

Increased IgG. Oligoclonal banding which remains positive during remission of MS but disappears in other disorder.

In cases of both asymptomatic HIV infection and AIDS, this is seen regarding CSF differential count

Increased Lymphocytes

Describe reactive lymphocytes

Increased dark blue cytoplasm and clumped chromatin

Clotted appearance

Increased fibrinogen seen with traumatic tap and *not subarachnoid hemorrhage*

All of the following can cause xanthochromia in CSF except:

Increased number of leukocytes

Test Performed to detect the presence of thickly encapsulated Cryptococcus neoformans, a possible complication of AIDS

India Ink

Moderately elevated WBC count (<25 cells/uL) with increased normal and reactive lymphocyte and plasma cell indicates what.

Indicative of multiple sclerosis/other degenerative neurologic disorders

Macrophages in CSF indicate what. What may be contained as well in this indication.

Intracranial hemorrhage. Hemosiderin or hematoidin crystals.

Purpose in CSF is to remove cellular debris and foreign objects

Macrophages

What are pellicles. When are they seen.

Macroscopically small fine clots which may be seen on the surface of CSF after 12-24 hours.

Plasma cells may be found in

May be the only CSF abnormality in multiple sclerosis Can be seen in the spinal fluid of patients with multiple myeloma

Take NOTE!

Measurement of the amount of MBP can be used to monitor the course of MS or provide a valuable measure of the effectiveness of current and future treatments.

Diagnosis of Multiple Sclerosis is best accomplished by.

Measuring amount of myelin basic protein

A brown appearance of CSF is caused by this/these

Methemoglobin, Hematoma, Melanin

If volume of CSF collected is inadequate to fill more than one tube what test should be done first.

Microbiology procedures should be performed first and then any remaining specimen may be used for cell counts and chemistries.

In cases of MS/other degenerative neurologic disorders, this is seen regarding CSF differential count

Moderately elevated WBC count (<25 cells/uL) with increased normal and reactive lymphocyte and plasma cell

Diluting fluids for total CSF cell count

NSS

Diluting fluids used in Cell count for CSF

NSS, 3% Acetic Acid, Toluidine blue O, Saponin Sol'n, Glacial acetic acid, methylene blue, Crystal Violet, gentian violet

Counting chamber that uses both diluted and undiluted samples

Neubauer Counting Chamber

Increased numbers of these are seen in CSF in *early* stages of viral, fungal, tubercular, and parasitic meningitis

Neutrophils

Which of the following cell types predominate in CSF during a classic case of Bacterial meningitis:

Neutrophils

Which of the following cell types predominate in CSF during a classic case of bacterial meningitis? A. Lymphocytes B. Macrophages C. Monocytes D. Neutrophils

Neutrophils

Choroidal cells, Ependymal cells Spindle cells Are seen after encephalography procedures

Nonclinically Significant Cells

Methodology of Cell Count

Normal adult CSF: 0-5 WBCs/ul Higher in children, up to 30 considered normal in newborns. Specimens that contain up to 200 WBCs or 400 RBCs may be clear, so all specimens examined microscopically. An improved Neubauer counting chamber is routinely used for CSF cell counts. The standard calculation for blood cell is also CSF to determine # of cells per microliter. This formulat can be used for diluted and undiluted offers flexibility. Elimnate the need to correct for volume by counting 4 large corner and large center square on each side of chamber.

NL Cellular constituent

Normal lymphocytes and monocytes Adults: 70% lymphocytes, 30% monocytes Children ratio is reversed

Opening pressure criteria when taking CSF

Normal opening pressure is between 50-200 mmH2O (3.67-15.74 mmHg). If initial pressure is 200 mmHg (2719 mmH2O), only 1-2mL is collected. If pressure is not elevated, 10-20mL

Acid-fast or Fluorescent antibody stain is performed on CSF when.

Not routinely performed on specimens unless TB meningitis is suspected.

Differential diagnosis of Meningitis regarding Opening Pressure, WBC count, Cells, Protein Levels, Glucose Levels, Lactate Levels, Microbiologic Tests.

Opening pressure - B (elevated) V (usually normal) T & F (Variable) WBC Count - All are elevated Cells - B (Neutrophils) V (Lymphocytes. neutrophils early) T & F (Lymphocytes and monocytes) Protein Levels - B (Markedly elevated) V (Moderately elevated) T & F (Moderately to markedly elevated) Glucose Levels - B (Usually < or equal to 40mg/dL) V (Normal) T (Decreased. May be <45 mg/dL) F (Normal to decreased) Lactate Level - B (>35 mg/dL) V (normal) T & F (>25mg/dL) Micro - B (Positive gram stain and BAT). F (Positive india ink and immunologic test for C. neoformans)

Eosinophils pleocytosis

Parasitic and fungal infections Medications and *SHUNTS* into the central nervous system

Eosinophils in CSF are increased in these cases

Parasitic infections, Fungal infections primarily *Coccidoides Immitis*, introduction of foreign material (medications and shunts into the CNS

A clotted appearance of CSF is caused by this/these.

Protein and Clotting Factors * Fluid collected from a traumatic tap may form clots owing to the introduction of plasma fibrinogen into specimen Bloody CSF caused by intracranial hemorrhage does not contain enough fibrinogen to clot Clot formation that do not produce a bloody fluid: meningitis, Froin syndrome, blocked CSF circulation tru subarachnoid space. A classic web like pellicle is seen with tubercular meningitis after overnight stay in the refrigeration.

A pellicle appearance of CSF is caused by this/these

Protein and clotting factors

Take NOTE!

Proteins > Amino acids > Ammonia > Urea (liver) if sira ang liver, > alpha ketoglutarate (certain number) > glutamine (brain)

Purpose of macrophages in CSF

Purpose is to remove cellular and other debris May be seen after repeated taps Hemorrhage: enter CSF within 2 hours to phagocytize RBCs (erythrophagocytic macrophages) Macrophages that phagocytose lipids are lipophages

A hazy, cloudy, turbid, or milky color of CSF is caused by this/these.

RBC (>400/uL), WBC (>200/uL), microorganisms, increased protein and lipids **Appearance Normal- Crystal clear A cloudy, turbid, milky- increased protein or lipid concentration but also indicative of infection

Myelin Basic protein is a protein not normally found in CSF but is detected by

RID (radial Immunodiffusion)

Take NOTE!

The blood-brain barrier, therefore, represents the control and filtration of blood components to the CSF and then to the brain. NEED TO KNOW: RBC+WBC in CSF: Number of cells counted X dilution ------------------------------------------------- =cells/microL Number of cells counted X volume of 1 sq

Popular quantitative protein tests because they are simple, rapid, and require no special instrumentation.

Turbidimetric

Specimen collection

Spinal tap, lumbar puncture in third or fourth lumbar space 1-2 mL 4 labeled sterile collection tubes

Lining cells from the arachnoid. Usually seen in clusters and may be seen with

Spindle-shaped Cells

Reagent for Turbidimetry that precipitates only Albumin.

Sulfosalicylic acid

Presence of clot formation but not bloody indicates these.

Suppurative meningitis Tubercuolous Meningitis Froin Syndrome Bloackage in CSF circulation Neurosyphilis

Protein only seen in CSF and what it is. Referred to as these.

Tau. *Carbohydrate-deficient transferrin fraction*. Beta-2-transferrin or tau protein

Which of the following statements about oligoclonal bands is false?

The Bands usually correlate with the stage of the disease and can be used to predict disease progression.

Which of the following statements about oligoclonal bands is false? A. In the CSF, these bands indicate increased intra- thecal concentrations of immunoglobulin G. B. The bands usually correlate with the stage of disease and can be used to predict disease progression. C. The bands are often present in the CSF and serum of individuals with a lymphoproliferative disease. D. The bands are often present in the CSF but not in the serum of individuals with multiple sclerosis.

The bands usually correlate with the stage of disease and can be used to predict disease progression

Formation and Physiology

The brain and spinal cord are lined by the meninges which consists of three layers: Dura mater , Arachnoid,Pia mater *CSF is produced in the choroid plexuses of the two lumbar ventricles and the third and fourth ventricles. In adults 20 mL produced every hour *The choroid plexuses are capillary networks that form under the CSF from plasma by mechanisms of selective filtration under hydrostatic pressure and active transport secretion *The chemical composition of the CSF does not resemble an ultrafiltrate of plasma *In the choroid plexuses, the endothelial cells have very tight-fitting junctures that prevent the passage of many molecules. This tight fitting structure of the endothelial cells in the choroid plexuses is termed the blood-brain- barrier *Blood brain barrier essential to protect the brain The junctures prevent the passage of helpful substances (antibodies, medication) *Disruption of the blood-brain barrier by disease such as meningitis, and multiple sclerosis allows leukocytes, proteins, and additional chemicals to enter CSF *The fluid flows through the subarachnoid space located b/w the arachnoid and pia mater Adults- 90 to 150 mL Neonates- 10-60 mL *The circulating fluid is reabsorbed back into the blood capillaries in the arachnoid granulations/villaw at a rate equal to its production *The cells of the arachnoid granulations act as one way valves that respond to pressure within the central nervous system and prevent reflux of the fluid

Meninges

The brain and spinal cord are lined by this, which consist of 3 layers, the dura mater, arachnoid, and pia mater.

Which of the following is not an analytical concern when the processing and testing of CSF are delayed? A. The viability of microorganisms B. The lability of the immunoglobulins C. The lysis of leukocytes and erythrocytes D. Alterations in the chemical composition

The lability of the immunoglobulins

Why does CSF not resemble an ultrafiltrate of Plasma.

This is due to the bidirectional active transport between CSF, interstitial brain fluid, brain cells, and blood in the brain capillaries. *The choroid plexuses are capillary networks that form under the CSF from plasma by mechanisms of selective filtration under hydrostatic pressure and active transport secretion *The chemical composition of the CSF does not resemble an ultrafiltrate of plasma

CSF Protein

This is most frequently performed chemical test on CSF. Normal CSF contains very small amounts of protein. Reported in mg/dL, as are plasma protein. As in serum, albumin makes up majority of CSF protein. Reference values for total CSF protein are usually listed as 15 to 45 mg/dL Higher values in infants and people over age 40 CSF contains protein fractions similar to serum, but the ratio of CSF proteins to serum proteins varies In serum albumin makes up most of the CSF protein but prealbumin is the second most prevalent fraction in CSF Transferrin major beta globin present CSF gamma globulin IgG with only small IgA IgM, fibrinogen, beta lipoprotein are not found in normal CSF

Where CSF flows.

Through the subarachnoid space between arachnoid mater and pia mater. *The fluid flows through the subarachnoid space located b/w the arachnoid and pia mater Adults- 90 to 150 mL Neonates- 10-60 mL The circulating fluid is reabsorbed back into the blood capillaries in the arachnoid granulations/villaw at a rate equal to its production The cells of the arachnoid granulations act as one way valves that respond to pressure within the central nervous system and prevent reflux of the fluid

Blood Brain Barrier

Tight-fitting structure of endothelial cells in the choroid plexus. Maintaining this barrier is essential to protect the brain from chemicals and other substances circulating in the blood that could harm brain tissue. Also prevents passage of helpful substances including antibodies and medications. Disruption of the barrier by diseases such as meningitis and multiple sclerosis allows leukocytes, proteins, and additional chemicals to the CSF.

What is CSF serology for

To detect active cases of syphilis within CNS Serologic testing for the CSF is performed to detect the presence of neurosyphilis. Detecting the antibodies associated with syphilis in CSF still remains a necessary diagnostic procedure Procedure recommended- Venereal Disease Research Laboratories it's not as sensitive as fluorescent treponemal antibody- absorption test for syphilis. The rapid plasma reagin test is less sensitive

Major beta-globulin in CSF

Transferrin

Which of the following events does not result in an increased CSF total protein? A. A traumatic puncture procedure B. Alterations in the blood-brain barrier C. Trauma to the central nervous system, resulting in fluid loss D. Decreased reabsorption of CSF into the periph- eral blood

Trauma, to the central nervous system, resulting in fluid loss.

A bloody appearance of CSF is caused by this/these

Traumatic spinal tap, Subarachnoid hemorrhage. Traumatic collection tap Grossly bloody CSF- intracranial hemorrhage, but it may also be due to the puncture of a blood vessel during the spinal tap procedure. Uneven Blood distribution Blood from the cerebral hemorrhage will be evenly distributed throughout the 3 CSF tubes, whereas a traumatic tap will leave the heaviest concentration of blood in tube 1 and gradually diminishing amounts in tube 2 and 3.

Reagent of choice for Turbidimetry because it precipitates both Albumin and Globulin

Trichloroacetic acid

Differentiate different types of meningitis with regards to Lactate level

Viral - <25mg/dL Bacterial, tubercular, fungal - >25mg/dL Bacterial - >35 mg/dL

Reactive lymphocytes indicate this

Viral infections in conjunction with normal cells *Lymphocytes and monocytes- viral, tubercular, fungal meningitis. Reactive lymphocytes containing increasing dark blue cytoplasm and clumped chromatin are present during viral conjunction with normal cells. Increased lymphocytes- HIV infection and AIDS Multiple sclerosis- elevated WBC count with increased normal and reactive lymphocytes and plasma cell

Normal CSF lactate levels (less than 25 mg/dL) are commonly found in patients with

Viral meningitis

Moderately increased CSF WBC with increased Lymphocytes and monocytes indicates what

Viral, Tuberculous, Fungal, or parasitic origin

How to determine the volume of CSF that can be removed.

Volume available in the patient (adult vs neonate) Opening pressure of CSF when needle first enters the subarachnoid space.

Normal CSF: WBC RBC Protein Glucose

WBC: <5 RBC: 0 Protein: 15-45 Glucose: 50-80

When should one perform a differential count for CSF

When increased cells are present Cell count The cell count performed on CSF is leukocyte count. RBC count= total cell count - WBC count Any cell count should be performed immediately because WBC and RBC begin to lyse w/in an hour and 40% of leukocytes disintegrate after 2 hours Differential count on a CSF specimen *The differential count should be performed on a stained smear and not from the cells in the counting chamber *To ensure that the maximum number of cells is available for examination, the specimen should be concentrated before preparing the smear. *Specimen concentration: sedimentation, filtration, centrifugation, cytocentrifugation

When is Electrophoresis the method of choice for quantitative protein testing of CSF

When it is necessary to determine if fluid is actually CSF and identification is made based on the appearance of the protein Tau. Electrophoresis and Immunophoretic Techniques- purpose is performing CSF protein electrophoresis to detect oligoclonal bands, which represent inflammation w/in the CNS. Bands located in the gamma region , indicating immunoglobulin production. To ensure that oligoclonal bands are present as a result of neurological inflammation, simultaneous serum electrophoresis is performed. leukemia, lymphoma, and viral infections may produce serum banding, which can appear in the CSF as a result of blood-brain barrier leakage or traumatic introduction of blood into the CSF specimen. The presence of two or more oligoclonal bands in the CSF that are not present in the serum can be- diagnosis of multiple sclerosis, particularly when accompanied by an increased IgG index. Other diseases: encephalitis, neurosyphilis, Guillain-Barre syndrome, neoplastic disorders Low protein levels in the CSF make concentration of the fluid before performing electrophoresis essential for most electrophoretic techniques. Better resolution can be made using CSF immunofixation electrophoresis and isoelectric focusing followed by silver staining. CSF can be identified by extra isoform of tau transferrin

Positive result and reagent in Ross Jones test

Whtie ring. 2mL ammonium sulfate.

Classic web-like pellicle

associated with tubercular meningitis and can be seen after overnight refrigeration of the fluid

Decreased glucose

bacterial meningitis and fungal infections

A cloudy, turbid, or milky

can be result of increased protein or lipid concentration, but it may also indicate WBCs present.

When choroid plexus cells and ependymal cells are present in CSF, they A. are often clinically significant. B. represent the demyelination of nerve tissue. C. can closely resemble clusters of malignant cells. D. indicate breakdown of the blood-brain barrier.

can closely resemble clusters of malignant cells.

Choroid Plexuses

capillary networks that form the CSF from plasma by mechanisms of selective filtration under hydrostatic pressure and active transport secretion. The endothelial cells in the choroid plexuses are tight-fitting and prevent the passage of many molecules.

Clot formation

fluid collected from a traumatic tap may form clots due to plasma fibrinogen. Bloody CSF caused by intracranial hemorrhage does NOT contain enough fibrinogen to clot. Diseases (meningitis, Froin syndrom, and blockage of CSF circulation through subarachnoid space) in which damage to the blood brain barrier allows increased filtration of protein and coagulation factors also cause clot formation but will NOT produce a bloody CSF.

India ink preparations and microbial antigen tests on CSF can aid in the diagnosis of A. bacterial meningitis. B. fungal meningitis. C. tuberculous meningitis. D. viral meningitis.

fungal meningitis.

Cellular elements in CSF, turbidity causes.

hazy/slightly turbid - 200-500 wbcs/uL (one uL = one mm3) slightly turbid - >400 rbcs/uL distinctly turbid - >500 wbcs/mm3 May appear cleaar - <200 WBCs & 400 RBCs/uL

CSF/serum IgG index <0.77 vs >0.77

less than 0.77 is normal greater than 0.77 is highly indicative of *multiple sclerosis*

Xanthochromic appearance could mean

Pink, orange, yellow Caused by RBC/Hgb degradation and bilirubin formation in CSF Also jaundice, ↑ ↑ protein, carotene Pathologic = cerebral hemorrhage

RBC should be absent, presence of RBC indicates

either cerebral hemorrhage or traumatic tap

Choroidal cells

epithelial lining of choroid plexus, singular and in clumps, uniform cells

Chemical analysis for CSF includes

glucose, proteins, lactate and immunoglobulins, myelin basic protein

Microbiological tests include

gram stain, India ink and culture

Pleocytosis

increased amounts of normal cells Pleocytosis of normal cells is valuable in determining the cause of *meningitis*

Cerebrospinal fluid (CSF) is produced in

choroid plexuses of the four ventricles from plasma Turnover is about 20 mL/hour Volume adults 90-150 mL, neonates 10-60 mL