Leukemia and lymphoma cells can be found in cerebrospinal, pleural, peritoneal and pericardial fluids at the time of diagnosis as well as anytime during the course of the disease. They can be found in any of these fluids, even when there are not abnormal cells present in the peripheral blood. Just as there are many morphological variations of leukemia and lymphoma in the peripheral blood and bone marrow, there are as many variations of leukemia/lymphoma in cytospins from fluids.Once the diagnosis is made from the initial tissue samples, there may be frequent spinal taps performed to monitor disease status and to instill intrathechal chemotherapy to prevent the development of central nervous system involvement by the leukemia or lymphoma.Cytospins are made for each sample and must be meticulously scrutinized. If any display abnormalities indicative of a disease state, or if there are any suspicious cells, then they should be sent for hematology or pathology review in compliance with your hospital's policy.
|Medulloblastoma in Cerebrospinal Fluid|
Medulloblastoma is a malignant brain tumor that originates in the cerebellum. It is included in the family of primitive neuroectodermal tumors, or PNETs. It is a highly invasive brain tumor which is known to spread through the brain and cerebrospinal fluid.Patients with meduloblastoma will typically have spinal taps performed after resection to evaluate for the presence of metastasis. These samples are then evaluated in the laboratory.This cytospin shows multiple large bizarre tumor clumps. Note that there are also several tumor cells that appear to have ingested other tumor cells (see arrows).
|Bone Marrow Contamination in Cerebrospinal Fluid|
When a spinal tap is performed correctly, the cell types found in the cytospin present a "snapshot" of the cerebrospinal fluid cell content. When there is difficulty obtaining a clean tap, the cell types present will reflect the type of fluids that are co-mingled with the cerebrospinal fluid. In traumatic taps, the cell types and proportions of red and white blood cells present will mirror that found in the patient's peripheral blood, as there has been a contamination of peripheral blood (from the trauma) mixed into the cerebrospinal fluid during the sample collection.In patients with softer bones (infants and osteopenic patients), it is not uncommon for the contamination to come, not from peripheral bleeding, but from bone marrow found inside the softened spinous process that was penetrated inadvertently during the procedure. When this occurs, any or all of cell types found within the bone marrow can be present in the cytospin.In the image shown here there are early nucleated RBC and WBC precursors, typical of contamination with bone marrow.
Ependymal cells (see arrow), are part of the lining of the brain and ventricles. They produce cerebrospinal fluid (CSF) and are involved in circulating the CSF over the brain, ventricles and the spinal cord. These cells are not a common finding in CSF cytospins but may be present in cases of hydrocephalus or chronic ventricular infection. They usually appear as very large multinucleated clumps.In the image on the right, notice how large this clump is in comparison to the red blood cells in the background.
|A patient with an infectious mononucleosis infection presents in the emergency room. Physicians order a spinal tap which is immediately sent to the laboratory for review. Please identify the cell in the image below from this patient's cerebrospinal fluid sample.||View Page|
|Which of the following are functions of CSF?||View Page|
|What is the most common site for a spinal puncture?||View Page|
|What is Cerebrospinal Fluid?|
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.
|Amount of CSF|
The volume of spinal fluid in an adult is about one ml per pound, or approximately 150 ml. In babies up to 4 weeks, there is an average of 10 - 60 ml of fluid.
|Three Main Functions of CSF|
Cerebrospinal fluid has three main functions:CSF protects brain and spinal cord from trauma.CSF supplies nutrients to nervous system tissue.CSF removes waste products from cerebral metabolism.
|CSF Evaluation and Diagnosis|
Examination of CSF provides vital information which aids in the diagnosis of a wide variety of disorders:
benign disordersmeningitisencephalitisbrain abscesssubarachnoid hemorrhagecerebral infract vs. intracerebral hemorrhagemultiple sclerosisGuillian-Barre's syndromespinal cord tumormalignant disordersleukemia CNS involvementmalignant tumors of the brain or spinal cordmetastasis of malignant tumors
|Where does spinal fluid circulate?||View Page|
|What is the volume of spinal fluid in an adult (approximately)?||View Page|
|How many cells may be seen in a 1 µl spinal fluid sample in a normal adult?||View Page|
|Specimen Collection (continued)|
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.
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.
|WBC Correction for Traumatic Tap|
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.
|Which of the following is the safest and most accurate tool for diluting spinal fluids?||View Page|
|If the spinal fluid is slightly hazy, what is the best dilution?||View Page|
|Which of the following diluting fluids are suitable for spinal fluid counts?||View Page|
|Pia Arachnoid Mesothelial Cells (continued)|
A reactive pia arachnoid mesothelial cell as noted by the darker cytoplasm is present in this field. Reactive cells are a common finding in cytospin smears from spinal fluid samples and are sometimes difficult to distinguish from tumor cells. Mesothelial cells are usually interspersed among the other cells, rather than appearing in clumps. They have a single distinct nuclei that may be eccentric.
The macrophages (histiocytes) are seen next to the mesothelial cell. Macrophages are distinguished from circulating monocytes by the irregular appearing cytoplasm. Bacteria, red cells or other debris can often be seen in the cytoplasm of macrophages.
|More Blast Cells|
Four blast cells are seen in this field. Notice the smooth chromatin pattern, nucleoli, high NC ratio and irregularly shaped nuclei. These blasts were observed in a spinal fluid sample from a patient with acute lymphocytic leukemia.
|Mature Peripheral Blood Cells|
In normal spinal fluid from an adult, 60% of cells are lymphocytes and up to 30% are monocytes. Up to 2% neutrophils 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.
Tissue cells that are never seen in peripheral blood but are often seen in spinal fluid samples are presented in the table below: Cells Causes macrophages RBC's in CSF viral meningitis tubercular meningitis ependymal normal - due to shedding of cells that line the ventricles pia arachnoid mesothelial cells (PAM) normal - due to shedding of cells lining the arachnoid space These cells are important because they must be differentiated from tumor cells and blast cells.
|Immature Hematopoietic Cells|
The presence of blast cells or immature stages of hematopoietic cell lines are significant abnormal findings in any spinal fluid sample. However, the presence of nucleated red blood cells is the result of bone marrow contamination during the spinal tap.
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.
|Which of the following statements are true regarding spinal fluid differentials?||View Page|
Four small mature lymphocytes are seen in this picture. Sixty percent of the cells found in normal adult spinal fluid are lymphs.
Many neutrophils are present in this slide from a spinal fluid sample from a patient with bacterial meningitis. Several macrophages (histiocytes) which have engulfed some of the bacteria can also be seen among the neutrophils.
Blast cells may be seen in the spinal fluid when cell proliferation in acute leukemia or lymphoma spreads to the central nervous system. The arrows indicate the two blasts in this field. Notice the smooth chromatin pattern in the nucleus and prominent nucleoli in both cells. Notice that an Auer rod is present in the cytoplasm in the blast to the right. The Auer rod indicates that these blasts are myeloblasts rather than lymphoblasts. A segmented neutrophil and several red cells can also be seen.
|Spinal Fluid Sample|
The arrow in this slide indicates the location of another example of a blast that was seen in a spinal fluid sample. Notice the two prominent nucleoli in the nucleus of this blast. The other three cells in the field are mature lymphs. Notice that the chromatin pattern in the mature lymphs is more clumped than the chromatin in the blast cells.
Malignant cells that have broken away from tumors located in other areas of the body may be seen in spinal fluid. All of the cells in this field are tumor cells. The cells in this slide are characterized by an open, loose chromatin pattern, nucleoli and vacuoles. Notice that the vacuoles are present in both the nucleus and the cytoplasm. Vacuoles in the nucleus are an unusual finding even in tumor cells. Tumor cells are often found in clumps and may have more than one nucleus due to their erratic mitotic patterns. Malignant cells sometimes have an irregular nuclear shape. Bizarre granules may be found in malignant cells but are absent in mesothelial cells.
|Pia Arachnoid Mesothelial Cells|
Pia arachnoid mesothelial (PAM) cells are often found in spinal fluid because they line the arachnoid space between the skull and the cerebrum. Ependymal cells which line the ventricles may also be present. Since ependymal and mesothelial cells are normal findings, the term mesothelial is sometimes used as a general term for both. It is essential to differentiate these tissue cells from blast cells or tumor cells which have diagnostic significance. An example of a pia arachnoid mesothelial cell, as indicated by the arrow, is present in this slide. Differentiating characteristics of this type of cell include an off-center, single, round nucleus and irregular cytoplasm that exhibits pseudopods.