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Cerebrospinal fluid (CSF) is a clear, plasma-like fluid which circulates around the outside of the brain, in cavities within the brain (ventricles) and in the space surrounding the spinal cord.

Most cerebrospinal fluid originates in the choroid plexus. The choroid plexus is composed of a mass of tiny blood vessels which are located in the third lateral and fourth ventricles. The remaining CSF, about 30%, is formed in other sites such as the subarachnoid space and the ependymal lining of the ventricles.

The composition of CSF has some similarities to plasma. However, the protein level of normal CSF is dramatically lower than that of plasma.

In addition to chemical components, a few cells are also found in normal CSF. In an adult, 0 - 5 WBC/µl is considered normal. Children will have slightly higher cell counts. Up to 30 WBC/µl is within normal limits for newborns. Lymphocytes account for 60 - 100% of these cells.

A syringe is used to remove 6 - 15 ml of spinal fluid. Less fluid is removed in babies and small children. The CSF sample is divided among 3 - 4 tubes, with 2 - 4 ml in each tube. Glass tubes should be avoided due to cell adhesion which may affect the cell counts or differential. The tubes are numbered in the order in which the CSF is obtained.

The technologist is responsible for examining CSF samples as they are received. If any of the following conditions are present, the results of testing could be uninterpretable: Tubes are not labeled.Tubes are not numbered.Specimen contains a blood clot.Specimen contains less than 0.5 ml CSF.

Important safety precautions must be observed when handling cerebrospinal fluid. The following guidelines apply:Semi-automatic micropipettes and disposable plastic chambers are the safest option for CSF testing. Many laboratories still use the hemacytometer with disposable pipets.If disposable materials are not used, soak contaminated reusable pipets, hemacytometer and coverslip in 70% alcohol or Wexide.All disposable items should be placed in a biohazard container for appropriate disposal.Wash hands thoroughly when the examination is completed.Spinal fluids which are to be discarded must be placed in biohazard containers for appropriate disposal.Careful attention to specimen processing and handling will help ensure that accurate results are obtained.

Spinal fluid samples are either clear or turbid. Some sources use the following rating system for turbid CSF specimens: 0 = crystal clear fluid 1+ = faintly cloudy, smoky, or hazy 2+ = turbidity clearly visible but newsprint read easily through tube 3+ = newsprint not easily read through tube 4+ = newsprint cannot be seen through the tubeTurbidity may be caused by leukocytes, erythrocytes, fungi, bacteria, amoebae, contrast media, or aspiration of epidural fat during puncture.200 leukocytes/mm3 will cause slight turbidity (1+); increased numbers of WBCs will cause increased turbidity. At least 400 erythrocytes/mm3 are needed to produce 1+ turbidity.Occasionally CSF will have an oily appearance due to the presence of substances remaining in the CSF after radiologic (x-ray) procedures have been performed.

When blood is present in a CSF specimen, it is necessary to determine whether the blood is due to a traumatic puncture or to a pathologic condition. There are several clues to help make this distinction: Traumatic tap:More blood is present in tube 1 than in tubes 2, 3, or 4.When sample is centrifuged within one hour, supernatant is clear.Blood clots on standing.Subarachnoid or cerebral hemorrhage:Blood is evenly distributed in all tubes.When sample is centrifuged within one hour, supernatant is pink or yellow.Blood does not clot on standing.

Examples of sources of pigment other than oxyhemoglobin and bilirubin that can cause xanthochromia include: methemoglobinincreased CSF protein (> 150 mg/dL)contamination by skin antiseptic (iodine or merthiolate)

It is important to recognize that xanthochromia is the appearance of color in the supernatant of a fresh, centrifuged CSF sample. There are a variety of causes for the appearance of this color. Therefore, it is important that xanthochromia be reported. The physician is responsible for determining the reason for its presence.

Specimens that are clear may be counted undiluted as long as there is no overlapping of the cells. Examining an undiluted CSF involves the following steps: Mix the CSF manually 6 - 10 times or place it in a mechanical mixer for 5 minutes.Using a Pasteur pipet or Dispo® pipet, fill both sides of the hemacytometer and allow the cells to settle for 5 minutes. To prevent the fluid in the chamber from evaporating, place it in a Petri dish containing moist filter paper. A disposable chamber similar to a hemacytometer is preferred, if one is available.Focus on low power (10x) and scan for the presence of cells. If cells are located, switch to high power (40x) to determine whether the cells are leukocytes or erythrocytes. Erythrocytes will be smooth refractile discs or spheres. Some red cells may appear crenated. Keep in mind that some red cells may be folded or in a vertical position rather than flat. In this situation only a small portion of the cell will be visible.

In an undiluted specimen, count and differentiate red cells and white cells at the same time. You can count red cells on a hand counter and use the differential counter for white cells. If you cannot differentiate white cells from red cells in the undiluted specimen, a plain capillary tube may be filled with crystal violet acetic acid diluent which is subsequently expelled from the tube. A very thin coating of the diluent will remain on the inside of the tube. CSF is drawn halfway up into the tube, which is then rocked back and forth to mix. The hemacytometer is then filled with the fluid containing stained white blood cells and lysed red cells. If cells are numerous and overlapping and it is necessary to focus through several planes in order to see all of the cells, a dilution must be made. When macroscopic appearance is turbid, milky or bloody, a significant dilution is usually necessary.

Examining a diluted CSF specimen involves the following steps: Mix the CSF sample manually 6 - 10 times or place it on a mechanical mixer for 5 - 10 minutes.Use a calibrated automatic pipet and place the appropriate volume of sample and diluent in a tube. Mix the diluted sample well.Use a Pasteur pipet and fill both sides of the hemacytometer. Allow the cells to settle for 5 minutes in a moist environment.Count cells in the four corner squares and the center square on both sides of the chamber. The number of cells counted times the dilution factor is then equal to the number of cells per microliter.

A calculation is used to correct CSF WBC counts which are falsely increased due to a traumatic tap: WBCs added = WBC(blood) x RBC(CSF) / RBC(blood)The blood WBC count is multiplied by the ratio of the cerebrospinal fluid RBC count to blood RBC count.The result is the number of artificially introduced WBCs. The true CSF white cell count is then calculated by subtracting the artificially introduced WBCs from the actual CSF WBC count. If the patient's peripheral WBC and RBC counts are within normal limits, some laboratories use the following formula: Subtract one white cell from the CSF WBC count for each 750 RBC counted in the spinal fluid.

White cells are less refractile and appear somewhat granular in appearance. In general, white cells will be larger than red cells. The segmented nucleus in neutrophils can be seen on high power. Lymphocytes and monocytes may be more difficult to differentiate in an undiluted, unstained specimen.Cells are counted in the four corner squares and the center square on both sides of the hemacytometer. The number of cells counted equals the number of cells/microliter.The ruled area of one side of a hemacytometer is shown on the right, with routine counting squares for red and white cell counts. Each large square is 1 mm wide by 0.1 mm in depth. The area for counting an undiluted specimen is 10 square millimeters, or 5 large squares on each side.

The following table lists the various cell types and macroscopic descriptions of CSF, and the patient conditions that could cause those properties to be present in the patient's CSF: Predominant Cell Appearance Conditions lymphs variable; clear - turbid viral meningitis tubercular meningitis multiple sclerosis drug abuse lymphoma leukemia Guillain-Barré syndrome chronic alcoholism neutrophils variable; clear - turbid bacterial meningitis mycotic meningitis early tuberculosis hemorrhage cerebral abscess tumors monocytes variable chronic bacterial meningitis partial treatment of meningitis tumors macrophages clear - turbid or clear - xanthochromic bloody tuberculosis fungal meningitis following hemorrhage blood contamination eosinophils variable parasitic meningitis fungal meningitis allergic reaction medications shunts dyes tumor cells variable metastatic carcinoma blast cells variable leukemia lymphoma normal to increased lymphs clear - xanthochromic benign tumor spinal cord brain ependymal or orchoid cells (often clumped) variable; may be xanthochromic bloody trauma spinal tap Adapted from Saunders Manual of Clinical Laboratory Science. Craig A. Lehrmann, Ed. WB Saunders, 1998.

In normal spinal fluid from an adult, 60% of cells are lymphocytes and up to 30% are monocytes. Neutrophils abundance up to 2% is also considered within normal limits when a cytospin smear is used for the differential. In children, normal CSF cells are 70% monocytes, up to 20% lymphocytes and up to 4% neutrophils. When any of these normal cell abundances are increased, the term pleocytosis is used. Neutrophil pleocytosis is an increase in neutrophils and usually indicates the presence of a bacterial infection.

Malignant cells that have broken away from a tumor within the brain or meninges may also be present in spinal fluid. Tumor cells may be difficult to distinguish from macrophages or pia arachnoid mesothelial cells. While blasts in the CSF also indicate malignancy, in particular leukemia, for the purposes of this discussion, they are considered separately.