hematology and the analysis of body fluids
COVER STORY
hematology and the analysis of body fluids
By Jeri Walters, SH(ASCP)
A basic understanding of where body fluids come from, why they are there and what’s normal is essential for every laboratorian who shoulders the responsibility for testing them.
The fact that body fluids are not as accessible as the customary peripheral blood sample, and are often “one time only” specimens requires a somewhat tolerant and discretionary guideline of laboratory acceptance.
Body fluid taps are not performed as a matter of screening–the fact that the fluid is in the lab means something is wrong. Clotted samples should be tested as diligently as possible. These specimens do not preclude the important finding of malignant cells or infectious organisms. Body fluids are one of the unique specimens received in the lab that require multidisciplinary testing. A limited volume of fluid must often be shared with hematology, chemistry, immunology and histo/cytopathology. When necessary, the physician should be asked to prioritize the tests needed so those of greatest clinical importance are performed first.
The types of fluids most commonly examined in hematology are cerebrospinal (CSF), serous (pleural and peritoneal) and synovial.
Cerebral Spinal Fluid
CSF is normally a clear, colorless fluid which circulates around the brain and spinal cord, providing a protective cushion for the delicate tissues. This cushion is more chemical than physical in nature. The CSF protects the brain and central nervous system from sudden changes in pressure and pH, maintains a stable chemical environment, supplies nutrients and removes metabolic waste products. The total volume of CSF present is 90-150 mL in adults and 10-60 mL in neonates. An adult produces 0.35 mL of fluid per minute.1 Since this equates to a total production of 500 mL of fluid per day, it is obvious the CSF is a dynamic and constantly changing fluid. Laboratory findings can certainly vary accordingly in the presence of disease.
There are four legitimate reasons, or categories of disease, which warrant the examination of CSF.2-4
* suspected meningitis
* detection of subarachnoid hemorrhage
* detection of central nervous system (CNS) malignancy/leukemia
* diagnosis of demyelinating disease
The first three reasons to test CSF intimately involve the hematology laboratory, the fourth does not. Gross evaluation is of greater importance in CSF than in most other fluids since appearance may indicate the difference between pathology and traumatic sample collection. Blood due to trauma may be present in as many as 20 percent of the CSF samples received in the laboratory.5
The usual protocol for proper sample collection requires that aliquots of CSF be placed in sequentially numbered sterile tubes as the fluid is collected. Thus, a traumatic “tap” may be identified by observing decreasing amounts of blood in the sequentially numbered tubes. However, it is important to remember that this is not a foolproof method. On rare occasions, the tubes may be filled in non-sequential order. In extremely rare instances, the amount of blood may increase as the sequential tubes are collected, if the physician is forced to probe or re-direct the needle to maintain flow. A combination of methods and observations will usually differentiate a traumatic “tap” from a subarachnoid hemorrhage (Table 1).
Of course the possibility of a traumatic tap superimposed on a subarachnoid hemorrhage always exists. Erythrophagocytosis and a positive D-dimer are clear evidence of a subarachnoid bleed if the fluid is tested in a timely manner.
Serous Fluids
Serous fluids include both pleural and peritoneal fluids. The pleura and peritoneum are thin, double-layered membranes that surround the lungs and the abdominal/pelvic organs respectively. The space between these membranes forms the pleural and peritoneal cavities which are lined by a single layer of mesothelial cells. Normally, there is just enough fluid between the two membranes to provide lubrication. The normal adult has just 5-15 mL of pleural fluid and less than 50 mL of peritoneal fluid. Thus, these are potential cavities and become true cavities only in the presence of diseases which cause an accumulation of fluid.
Pleural and peritoneal fluids also are dynamic. There is constant filtration (of plasma) across one membrane and reabsorption by the other. This constant filtration and reabsorption is a function of differences in pressure on either side of the membranes and the integrity of the membrane itself. Diseases which disrupt this balance or damage the membrane cause an effusion or accumulation of fluid. A peritoneal effusion is sometimes referred to as ascites. Common causes of a pleural effusion are congestive heart failure, infection and malignancy. Alcoholic cirrhosis is the most common cause of ascites.6
Pleural and peritoneal effusions are traditionally separated into transudates and exudates for diagnostic purposes. These determinations no longer involve the hematology laboratory, but are based on chemical analyses such as LDH, cholesterol and protein. Criteria such as the WBC count and specific gravity are no longer used due to their lack of sensitivity and specificity.7-9
Synovial Fluid
Synovial fluid is an ultrafiltrate of plasma combined with a hyaluronate-protein complex produced by the lining cells of the synovial membrane. The complex is responsible for the thick and gooey nature of synovial fluid and makes it an excellent lubricant. Synovial fluid not only lubricates the joint space, but also provides nutrients to the cartilage. Inflammatory joint diseases cause depolymerization of the hyaluronate-protein complex and a resulting decrease in lubricating ability.
Examination of synovial fluid is usually performed to:
* detect sepsis
* detect a hemorrhage
* diagnose crystal induced
inflammation
Abnormal synovial fluids are separated into four groups based on the cause of the effusion and laboratory findings. These groups are:
Group I–noninflammatory (causes include osteoarthritis)
Group II–inflammatory
(crystal induced arthritis, rheumatoid arthritis)
Group III–infectious (bacterial, fungal, viral, etc.)
Group IV–hemorrhagic (trauma, hemorrhagic diathesis, tumor)
Some authors report five groups, placing the crystal induced arthritis separately in Group IV, and the hemorrhagic fluids in a Group V.
Cell Identification
The most important aspect of cell identification is sample preparation. Body fluid samples should be prepared for microscopic observation using a cytocentrifugation method. If a cytocentrifuge is not available, beg, borrow, steal or hold a bake sale, but get one. The cytocentrifuge method is superior at preserving cellular morphology and capable of harvesting sufficient cells to examine even in extremely low fluid cell counts. This is especially important in CSF analysis, since the identification of even a few leukemic blasts may impact both the patient’s treatment and prognosis.
All types of peripheral blood cells may be seen in the various body fluids. Additionally, there are cell types that are seen only in specific types of fluid or have clinical significance when seen in that fluid (see photos A-E, page 11).
Cell Counting
Body fluid cell counts are usually performed in a hemocytometer–a labor intensive and costly test. Procedures for the performance of hemocytometer cell counts as well as normal values for various fluids can be found in most hematology text books.
Cell counting should be performed immediately since cells in body fluids will degenerate rapidly. Studies have shown that up to 30 percent of the neutrophils in CSF may be lost within 1 hour of collection.10 Because of this increased fragility, cell counts (including the RBC) should be performed on all CSF samples. It is not a test that can be added later. Cell survival is improved in the serous and synovial fluids but is limited to hours, especially in septic fluids.
Most authors agree the RBC count is of limited value in body fluids other than CSF. In the current climate of cost containment, RBC counts should not be routinely performed on all serous and synovial fluids. If a hemorrhagic condition is suspected a hematocrit may be used for confirmation. A hematocrit greater than 1 percent in pleural fluid is typically associated with malignancy. When the hematocrit is greater than 50 percent of the peripheral blood hematocrit, a true hemorrhagic condition is indicated. Of course, a traumatic tap must be ruled out. The nucleated cell count, or WBC, is diagnostically important in CSF and synovial fluid, but has limited value in serous fluids. The type of cells seen in serous fluids has greater significance than the count itself.
In the past, automated cell counts on body fluids were considered risky at best, due to the limitations of available instrumentation. These limitations included:
* unacceptable background counts and poor precision for the low cell count ranges typically seen in body fluids;
* restrictive linearity limits;
* volume of sample required;
* lack of distribution curves and/or scattergrams to detect debris or microclots and determine the acceptability or accuracy of results;
* anticoagulant interference, specifically heparin on some instruments, was a significant problem. In many laboratories, body fluids arrive in a variety of packaging and it is difficult to determine when heparin is present.
Many newer generation instruments no longer have these limitations. Backgrounds are essentially zero, precision is excellent in low ranges, and a variety of histograms and scattergrams are available to interpret results. However, none of the commercially available hematology analyzers are FDA approved for body fluid cell counts–and probably never will be. Leading manufacturers frankly admit the FDA approval process is lengthy and costly, and the low volume body fluid applications are not worth the expense.
Thus, fear has been a great deterrent to automating body fluid cell counts in many laboratories. This does not preclude automated body fluid cell counts, but does place the burden on the laboratory for documenting the performance of a new application. The key word is document…document…document!
Laboratory regulating agencies, such as CAP, approve automated body fluid cell counts if the laboratory has documented linearity limits, reportable range, background limits and correlation studies. This process is not difficult since many laboratories are already doing the difficult part–the manual hemocytometer count. It must be noted, when automated cell counting techniques are used, that a total nucleated cell count is performed rather than a specific WBC count. This should be reflected in the differential report. All nucleated cell types should be identified and included in the differential.
It is imperative to treat synovial fluids with hyaluronidase, an enzyme which depolymerizes the hyaluronic acid, before performing automated cell counts, and advantageous to do so before hemocytometer counts. It is difficult, if not impossible, to obtain an even dispersion of cells in extremely viscous fluids and some automated instruments may not be able to aspirate the fluid. A simple method is to add about 5 mg hyaluronidase (Sigma Chemical Co., St. Louis, MO) to an aliquot (up to 1ml) of synovial fluid. Mix the aliquot gently for a few minutes before performing cell counts or smear preparation.
Where to Start
1. Access the background count on the instrument. Since normal cell counts on body fluids are very low, it is imperative to have a low, preferably 0.00 background. A higher background will limit the reportable range and lower linearity limit. Differences in instrument sensitivities also affect the reportable range and linearity. Some instrument WBC channels can detect and enumerate cells to two decimal points, others are limited to one decimal point. A background count guideline follows:
0.00–indicates 5 or fewer particles present
0.01–indicates 6-15 particles
0.0 –indicates approximately 50 or fewer particles
0.1–indicates anywhere from 50-150 particles
2. Analyze the instrument-generated histogram and/or scattergram on all body fluid samples. The same rules used for the acceptability of results for peripheral blood also apply to body fluids. The “high take-off” seen on three-part differential analyzers when things such as platelet clumps, fibrin debris or NRBC’s interfere with the peripheral blood WBC will also indicate interference and a potentially inaccurate nucleated cell count in body fluids. The same is true on five-part differential analyzers with scattergram interpretation. The discriminator used to indicate interference for peripheral blood samples should be used to interpret body fluid cell counts. Figs. 1 and 2 show both acceptable and unacceptable body fluid cell counts using SysmexTM three-part and five-part analyzers.
In addition to observing the portion of the histogram or scattergram that indicates interference, the far right side of both displays should be observed for the presence of clumps or large clusters of cells. These are often clusters of mesothelial cells or malignant cells. The nucleated cell count should be reported as approximate due to the clusters of cells, as should any body fluid sample with small clots present. Manual counting does not provide better accuracy on these fluids since the clusters do not disperse in the hemocytometer.
Although platelets are not an issue in body fluid analysis, the platelet histogram can provide additional information. The probability of very few RBCs and several thousand platelets is quite low. Significant particles in the platelet histogram are more likely debris or fragments from disintegrating cells if the RBC count is very low. Extremely high particle counts in the platelet channel and few RBCs are often seen when fluids take the scenic route to the lab or are collected in bedside drainage receptacles, in which case the fluid is already old and cells have disintegrated before the sample reaches the lab.
3. Perform precision studies. The automated cell count must be reproducible (as if the hemocytometer was!) especially in the low ranges.
4. Perform correlation studies. Analyze a minimum of 40-50 body fluid samples on automated instrumentation after the hemocytometer cell count is done. Include all types of fluids–serous, synovial and cerebral spinal–customarily analyzed in the hematology laboratory. At least 10 of the samples should have cell counts in the very low range, i.e., between 0 and the lowest reportable number on the instrument, either 0.1 or 0.01.
Plot the results and visually look for a positive or negative bias. A significant bias means the instrument count will always be higher (probably due to background) or lower (maybe missing some cells) than it should be and automated cell counts are not advisable. Of course, the unacceptable results identified by histogram or scattergram analysis are not plotted, but all histogram and scattergram printouts from the comparison samples must be saved. Documented evidence that the instrument will detect unacceptable results is imperative.
Perform correlation statistics on the comparison data. Be aware that correlation coefficients are limited when dealing with very low numbers.
5. Determine and record the lower linearity limits and reportable range for body fluid cell counts from the data obtained. And…document…document…document!
A Note on Body Fluid
Automated Differentials
Automated differentials have limited accuracy on most body fluid samples. The increased fragility and rapid degeneration of cells suspended in various body fluids, as opposed to their normal plasma medium, causes inaccurate measurements and misclassification. This is especially true of neutrophils, which can rapidly blend with other cell populations on current hematology instrumentation.
* About the author: Jeri Walters is hematology supervisor at Sinai Samaritan Medical Center in Milwaukee. She also presents seminars, makes educational videotapes and occasionally lectures at Marquette University.
References
1. Albright, R.E. Management of cerebral spinal fluid and other body fluids. In Koepke, J.A., ed: Practical Laboratory Hematology, New York, Churchill Livingstone, 1991, pp. 295-310.
2. Brailas Ventura, C.D., Derwinko, B. Laboratory evaluation of body fluids. In Lotspeich-Steininger, C.A., Steine-Martin, E.A., Koepke, J.A., eds: Clinical Hematology, New York, J.B. Lippincott, 1992, pp. 394-406.
3. Kjeldsberg, C.R., Knight, J.A. Body Fluids, 3rd Ed., Chicago, ASCP Press, 1993.
4. Martin, K.I., Gean, A.D. The spinal tap: A new look at an old test. Ann. of Intern. Med., 1986, Vol. 104, No. 6, pp. 840-848.
5. Health and Public Policy Committee: The diagnostic spinal tap. Ann. of Intern. Med., 1986, Vol. 104, pp. 880-885.
6. Rocco, V.K., Ware, A.J. Cirrhotic ascites: pathophysiology, diagnosis and management. Ann. of Intern. Med., 1986, Vol. 105, No. 4, pp. 573-585.
7. Health and Public Policy Committee. Diagnostic thorocentesis and pleural biopsy in pleural effusions. Ann. of Intern. Med., 1985, Vol. 103, pp. 799-802.
8. Valdes, L., et al. Cholesterol: a useful parameter for distinguishing between pleural exudates and transudates. Chest, 1991, Vol. 99, No. 5, pp. 1097-1102.
9. Hamm, H., Brohan, U., Bohmer, R., et al. Cholesterol in effusions. Chest, 1987, Vol. 92, No. 2, pp. 296-302.
10. Steele, R. Leukocyte survival in cerebral spinal fluid. J. of Microbiol., 1986, Vol. 23, No. 5, pp. 965-966.
Table 1
Indications of a
Subarachnoid
Hemorrhage
* Visible amount of blood or RBC count is consistent in first and last tube
* Supernatant is xanthochromic–due to breakdown of hemoglobin
* Erythrophagocytosis is present
* Positive D-dimer–due to breakdown products of cross-linked fibrin
Indications of a
Traumatic ‘Tap’
* Decreasing amounts of blood or RBC count in sequential tubes
* Clot formation
* Clear supernatant
* Negative D-dimer test
(table/courtesy Jeri Walters)
