CSF

• Cerebrospinal fluid (CSF)


  Fluid produced by the choroid plexuses that line the ventricular system of the brain
• CSF production


  1. Choroid plexuses filter plasma through selective filtration and under influence of hydrostatic pressure and active transport

  2. Rate: 20mL/hour
• Functions of CSF


  • Provides physical support to the brain
  • Protects against sudden changes in acute venous and arterial pressure as well as impact pressure
  • Provides an excretory waste function for the brain
  • Maintains CNS ionic homeostasis, especially during acute disturbances of plasma components
• The chemical composition of CSF is NOT the same as that of plasma or it is NOT considered an ultrafiltrate of plasma
• CSF flow


  1. Flows through the subarachnoid space

  2. Flows through the ventricles of the brain and the spinal cord

  3. Reabsorbed in the cauliflower-shaped arachnoid granulations and recirculates back to the subarachnoid spaces
• Meninges


  Layers lining the brain and spinal cord (dura mater, arachnoid, pia mater)
• The subarachnoid space of the cranium is continuous with the subarachnoid space of the spinal cord
• The spinal needle is inserted for CSF collection in the subarachnoid space
• Blood-brain barrier


  The endothelial cells lining the choroid plexus capillary network are uniquely very tight-fitting junctures, preventing the passage of many molecules
• Functions of the blood-brain barrier

  • Acts as a selective filter of certain substances
  • Protects the brain from injurious substances and chemicals present in the blood
  • Can prevent the passage of helpful substances such as antibodies and certain medications
• Many electrolytes and proteins can cross the blood-brain barrier normally but these transports are regulated
• Passage of electrolytes and proteins through the blood-brain barrier

  • H+, K+, CA2+, HCO3- can be transported across the barrier using specific transport systems
  • Glucose, Urea, Creatinine diffuse freely but require 2 hours or longer to equilibrate
  • Proteins cross by passive diffusion at a rate dependent on the plasma-to-CSF concentration gradient
• If the proteins are very large, they are not able to pass through the blood-brain barrier as readily via simple passive diffusion
• Lumbar puncture procedure


  1. Patients need to be in the left, side-lying position

  2. Performed by trained doctors under sterile or aseptic conditions

  3. A specialized needle is inserted into the subarachnoid space between the third, fourth, or fifth lumbar vertebrae (L3-L5)

  4. Intracranial pressure must be measured before CSF can be aspirated
• Normal opening pressure


  • 90 to 180 mm H2O in adults
  • 10 to 100 mm H2O in infants and young children
• In many cases of meningitis, intracranial hemorrhage, and large CNS tumors, opening pressures can be as high as 250 mm H2O

  
• CSF collection


  • CSF is collected in 3-4 sterile tubes labeled 1, 2, 3, and/or 4 in chronological order
  • Glass tubes should NOT be used especially for cell counts and differentials
  • Tube 1 is used for chemical and serologic tests, Tube 2 for microbiologic tests, Tube 3 for cell counts and differentials
• If only very little CSF was obtained, the physician and receiving medical technologist must communicate effectively to prioritize tests
• Ideally, any CSF specimens received in the laboratory should be run on a STAT basis because cellular degradation occurs quickly
• Handling of CSF specimens

  Tube 1 should be frozen, Tube 2 kept at room temperature, Tube 3 refrigerated, Tube 4 depends on the tests to be done
• Excess fluid should not be discarded, it should be kept frozen until there is no further use
• Appearance of CSF


  • Normal CSF is crystal clear and colorless
  • Blood CSF can be due to traumatic taps or subarachnoid hemorrhage
  • Turbid or milky CSF can be due to increased protein and WBC counts
  • Oily-appearing CSF can be due to radiographic contrast material

  
• Xanthochromia


  • Term used to describe CSF supernatant that is pink, orange or yellow
  • Most common cause is RBC degradation products from subarachnoid hemorrhage
  • Detectable 2-4 hours after onset, peaks 24-36 hours, and disappears over 4-8 days

  
• Traumatic tap


  • Uneven distribution of blood among the tubes or streaks of blood in the CSF
  • First tube (Tube 1) will contain the heaviest concentration of blood and this will gradually decrease in the succeeding tubes
• Heavy hemolysis


  Coinciding with the conversion of oxyhemoglobin to unconjugated bilirubin, can persist for up to 4 weeks
• Other causes of Xanthochromia

  • Oxyhemoglobin release from RBC lysis due to delayed CSF
  • Examination or contamination with detergents or disinfectants
  • High CSF bilirubin in jaundiced patients/patients with severe liver disease
  • Carotenoids (orange) in persons with dietary hypervitaminosis A
  • Metastatic spread of melanoma to the meninges (brownish xanthochromia)
• Traumatic tap


  • Uneven distribution of blood among the tubes or streaks of blood in the CSF within the tubes
  • Grossly bloody CSF: either intracranial hemorrhage or puncture of a blood vessel during the spinal tap procedure
  • If blood is truly coming from the subarachnoid and intracranial space, it will be evenly distributed throughout the tubes
  • In traumatic taps, the first tube (Tube 1) will contain the heaviest concentration of blood and this will gradually decrease in the succeeding tubes (Tubes 2, 3 and 4 if available)
• Clot formation


  • Because a blood vessel may have been inadvertently punctured during traumatic taps, fibrinogen can contaminate the CSF therefore inducing the formation of a clot
  • Bloody CSF coming from intracranial hemorrhage does not contain enough fibrinogen to form a clot
  • Meningitis/Froin syndrome (diseases that can disrupt the blood-brain barrier) causing leakage of various proteins including coagulation factors into the subarachnoid space can cause clot formation but with little to no blood
  • Be sure to exclude the possibility of prior refrigeration of the CSF sample because overnight refrigeration can also induce the formation of a clot-like substance
  • In tuberculous meningitis, a classic web-like pellicle can be seen
  • Traumatic tap: may form clots owing to introduction of fibrinogen

  
• Subarachnoid hemorrhage


  • The brains are invested with a rich network of blood vessels
  • These blood vessels can rupture such as in aneurysms
  • Characteristic Finding: Uniform distribution of blood among all the tubes
  • Xanthochromic supernatant does not favor a traumatic tap
  • Xanthochromia is a discoloration of the CSF supernatant most commonly caused by the presence of RBC degradation products after a bleeding episode. For these products to be present in the CSF, the RBCs must have been present in the CSF for at least 2 hours
  • Thus, a xanthochromic supernatant would likely result from blood that has been present in the subarachnoid space much longer than the time period it would take for blood to be introduced via a traumatic tap
  • In some cases of very recent intracranial bleeding however, the supernatant may remain clear (hemolysis has not occurred yet)
  • Aside from xanthochromia, the microscopic demonstration of macrophages that have ingested RBCs (termed erythrophagocytosis) or macrophages containing hemosiderin granules favors intracranial hemorrhage over traumatic taps
  • D-dimer (measured by latex agglutination immunoassay), if present in the CSF sample, is specific for fibrin degradation. It is not seen in traumatic taps

  
• Hydrocephalus


  • A condition in which the heads appear enlarged because of too much CSF in the ventricles
  • Many causes are genetic. There are genetic problems in the CSF drainage or reabsorption
  • In some cases in adults with hydrocephalus, it is probably an acquired case of hydrocephalus. It can be brought about by a trauma in the head that disrupts the normal circulation of CSF

  
• Installment of Ventricular Shunts

  1. Ventriculoperitoneal shunt: It diverts some of the accumulated CSF in the ventricles to the peritoneal space

  2. Ventriculoatrial shunt: It diverts accumulated CSF to aorta or the atria of the heart

  3. This is the usual therapy offered for infants or babies with hydrocephalus. This is done so that eventually, the accumulated CSF will become part of the blood circulation and will be drained from the ventricles from the brain

  4. If the device is improperly placed or there's problems with the device and equipment that was used, that can be associated with an increased number of eosinophils in the CSF
• Cell count


  • The cell count that is routinely performed on CSF is the WBC count
  • RBC counts are often not done anymore unless it is to confirm that a traumatic tap has occurred and there has to be a correction for leukocytes or for protein
  • Traumatic taps can introduce into the CSF leukocytes and proteins (aside from erythrocytes), thus if no corrections are done, many of the leukocytes seen in a CSF sample may actually just have originated from blood vessels injured during a traumatic tap
  • To perform a correction, the peripheral blood RBC and WBC counts must be determined beforehand in order to establish the ratio of WBCs to RBCs in the peripheral blood and this can be compared to the number of contaminating RBCs in the CSF
  • WBC counts should be performed immediately, because WBCs, especially granulocytes start to lyse and disintegrate within one hour; if examination really cannot be done right away, the sample should be refrigerated
  • Normally, in adults there are about 0 to 5 WBCs per microliter but in children and newborns, it can be higher
  • Even though the CSF may appear clear, it may contain many WBCs already hence all CSF samples should at least be examined microscopically
  • Routine cell counts have been traditionally performed manually using improved Neubauer counting chambers; electronic cell counters have not been popular for CSF cell counts because of poor reproducibility especially if counts are low
  • Nine squares measuring 1 mm2 are counted and the chamber has a depth of 0.1 mm
  • Using the improved Neubauer counting chamber, the same calculation formula is used (as in manual blood counts)
  • CSF that appears clear can undergo counting undiluted, but if a dilution is necessary, make sure to adjust the values in the formula
  • Diluents that may be used include normal saline solution
  • If the CSF is bloody and the RBCs need to be lysed to be able to visualize the WBCs better for counting, 3% glacial acetic can be used instead
  • Methylene blue can be added to diluents to stain the WBCs, enhancing differentiation between neutrophils and mononuclear leukocytes
  • Undiluted specimen: 4 drops of mixed specimen in a clean tube, rinse Pasteur pipette with 3% glacial acetic acid draining thoroughly, draw the 4 drops of CSF into the pipette, allow pipette to sit for 1 minute, mix solution, discard 1st drop and load the hemocytometer
  • WBCs are counted in the four corner squares and the center square on both sides of the hemocytometer
  • A different kind of hemocytometer known as the Fuchs-Rosenthal hemocytometer has also been used in CSF cell counting. It has a depth of 0.2 mm

  
• Quality control of CSF and other body fluid cell counts

  • Biweekly basis: Diluents checked for contamination by examination in a counting chamber under 4x magnification, contaminated diluents should be replaced and new solutions prepared
  • Monthly basis: Speed of cytocentrifuge checked with a tachometer and timing should be checked with stopwatch
  • Non disposable counting chambers: Soaked in bactericidal solution for at least 15 minutes then thoroughly rinsed with water and cleaned with isopropyl alcohol
• Differential count


  • Involves the identification of the types of WBCs in the CSF and determining their proportions
  • Must be done on CSF that has been smeared on to a slide and then stained with Wright's stain
  • Differential counting cannot be done on unstained, wet preparations on counting chambers that are used in cell counts. One can only vaguely identify the WBCs in these setups as granulocytes or mononuclears; specific identification (whether neutrophil, eosinophil, monocyte, etcetera) is very difficult if not impossible, thus stained CSF smears must be used for differential counting
  • To ensure that the maximum number of cells are available for identification, the CSF specimen should be concentrated before it is smeared onto a slide
  • 100 cells should be counted, classified and reported in percentage

  
• CSF concentration techniques


  1. Sedimentation, filtration: Less commonly used but do produce less cellular distortion than centrifuging methods

  2. Centrifugation: Specimen is centrifuged for 5-10 minutes > supernatant fluid is removed and saved for additional tests > slides made from sediment allowed to air day and stained with Wright's stain

  3. Cytocentrifugation: Method of choice, 0.1ml of CSF plus 1 drop of 30% albumin produces enough cells if cytocentrifugation is used, a specialized type of centrifugation procedure that involves the use of a device with a conical chamber that funnels the specimen and forces it into a monolayer within a 6-mm diameter circle on a glass slide during centrifugation, although the cell yield is very good with this method (it is the method of choice for differential counts on all body fluids), cellular distortion commonly occurs; this can be decreased with the addition of albumin to the sample. If albumin is added to the CSF however, make sure the albumin used is regularly checked for the presence of bacteria
• Leukocytes in CSF


  • Normally, a small number of lymphocytes and monocytes are present in CSF
  • Most of them are mononuclear (monocytes and lymphocytes)
  • An increased number (pleocytosis) of these cells is not normal
  • Appearance of these cells is still the same as they were seen in blood
  • Adults: More lymphocytes than monocytes (approximately 70:30 ratio)
  • Pediatric: More monocytes than lymphocytes; they can even have up to 80% monocytes normally (30:70 ratio of lymphocytes to monocytes)

  
• Neutrophils


  • If the CSF WBC count is very high and majority (typically more than 60%) of the WBCs are neutrophils, this is indicative of bacterial meningitis
  • Some neutrophils can also be seen in the CSF in the early phases of viral, fungal, tuberculous and parasitic meningitis
  • Neutrophils that have pyknotic nuclei are considered degenerating or degenerated. They are important to identify because they can look similar to nucleated RBCs (cells that can indicate bone marrow contamination during the lumbar puncture procedure)
  • Neutrophil with phagocytized bacteria may be seen in bacterial meningitis, CNS hemorrhage, repeated lumbar puncture, injection of medications / radiographic dye

  
• Lymphocytes, monocytes


  • Primary cells found in CSF
  • If the CSF WBC count is moderately high and there is a high percentage of lymphocytes and monocytes, viral, fungal or tuberculous meningitis are possible causes
  • They can also be seen in increased numbers in cases of HIV infection and AIDS, multiple sclerosis and other Neurodegenerative disorders
  • Plasma cells are NOT normal constituents of CSF and may be seen sometimes in cases of multiple myeloma
  • Reactive lymphocytes – viral infection
  • Multiple sclerosis / other degenerative neurologic disorder – moderately elevated WBC with increased normal and reactive lymphocytes and plasma cells

  
• Eosinophils


  • Abnormal in CSF, even in small numbers
  • Increased numbers can be found in parasitic infections, fungal meningitis (often caused by Coccidiodes immitis) and if foreign material is introduced into the CNS (ex. medications, shunts)
  • High numbers can be seen in ventricular shunts malfunction
  • The most common cause of eosinophilic meningitis worldwide is parasitic invasion

  
• Macrophages and siderophages


  • Phagocytose cellular debris and foreign objects in the CNS and even RBCs from hemorrhagic episod