Examination of all fluids should include observation of color and turbidity, determination of cell counts, and white blood cell (WBC) evaluation. Blood cell counts should be performed and cytocentrifuge slides should be prepared as quickly as possible after collection of the specimen, because WBCs begin to deteriorate within 30 minutes after collection.¹ It is important to mix the specimen gently but thoroughly before every manipulation (i.e., counting cells, preparing any dilution, and preparing cytocentrifuge slides). Cell counts on fluids usually are performed using a hemacytometer (see Chapter 14); however, some automated instruments now are capable of performing blood cell counts on fluids. Automated cell counts are limited by the poor sensitivity of automated methods for specimens with low counts. Each instrument manufacturer should provide a statement of intended use that defines which body fluids have been approved by a regulatory agency for testing on the instrument.^2,3 Care should be taken to observe the operating limits of these instruments and the volume limits for the fluid received. Red blood cell (RBC) counts on serous and synovial fluids have little clinical value⁴; relevant clinical information is obtained merely from the appearance of the fluid (grossly bloody, bloody, slightly bloody).

Cell counts are performed with undiluted fluid if the fluid is clear. If the fluid is hazy or bloody, appropriate dilutions should be made to permit accurate counts of WBCs and RBCs. The smallest reasonable dilution should be made. The diluting fluid for RBCs is isotonic saline. Diluting fluids for WBCs include glacial acetic acid to lyse the RBCs, or Türk solution, which contains glacial acetic acid and methylene blue to stain the nuclei of the WBCs. Acetic acid cannot be used for synovial fluids, because synovial fluid contains hyaluronic acid, which coagulates in acetic acid. A small amount of hyaluronidase powder (a pinch, or what can be picked up between two wooden sticks) or one drop of 0.05% hyaluronidase in phosphate buffer per milliliter of fluid should be added to the synovial fluid sample to liquefy it before performing cell counts or preparing cytocentrifuge slides. Dilutions should be based on the turbidity of the fluid or on the number of cells seen on the hemacytometer when using an undiluted sample.

A WBC count of approximately 200/mm³ or an RBC count of approximately 400/mm³ causes a fluid to be slightly hazy. If the fluid is blood-tinged to slightly bloody, the RBCs can be counted using undiluted fluid, but for WBCs or nucleated cells it is advisable to use a small (1 : 2) dilution with Türk solution (or similar) to lyse the RBCs and allow an accurate count. If the fluid is blood-tinged to slightly bloody, the RBCs can be counted using undiluted fluid, but it is advisable to use a small (1 : 2) dilution with Türk solution (or similar) to lyse the RBCs and provide an accurate WBC or nucleated cell count. If the fluid is bloody, a 1 : 200 dilution with isotonic saline for RBCs and either a 1 : 2 or a 1 : 20 dilution with Türk solution for nucleated cells should be used to obtain an accurate count. When performing dilutions for blood cell counts, a calibrated pipette should be used, such as MLA pipettes or the Ovation BioNatural pipettes (VistaLab Technologies, Inc., Mt. Kisco, N.Y.).

The number of squares to be counted on the hemacytometer should be determined on the basis of the number of cells present. In general, all nine squares on both sides of the hemacytometer should be counted. If the number of cells is high, however, fewer squares may be counted.⁵ Each square equals 1 mm². The formula for calculating the number of cells (see Chapter 14) is:

Cells counted×depth factor×dilution factorArea counted(mm2)

Guidelines for counting are summarized in Table 17-1.

TABLE 17-1

Guidelines for Counting Fluids

	GROSS APPEARANCE
Test	Clear	Hazy	Blood-Tinged	Cloudy	Bloody
WBCs	0-200/mm³	>200/mm³	Unknown	High	Unknown
Dilution for counting cells	None	1 : 2 Türk	1 : 2 Türk	1 : 20 in Türk solution	1 : 2 in Türk solution
No. squares to count on hemacytometer	9	9	9	9 or 4	9 or 4
RBCs	0-400/mm³	Unknown	>400/mm³	Unknown	>6000/mm³
Dilution for counting cells	None	None	None	None	1 : 200 in isotonic saline
No. squares to count on hemacytometer	9 large	9 large	9 or 4 large	4 large or 5 small	5 small
Cytospin dilution (0.25 mL [5 drops] of fluid)*	Undiluted	Dilute with saline to 100-200/mm³ nucleated cell count	Straight or by nucleated cell count	Dilute with saline to 100-200/mm³ nucleated cell count	Dilute by nucleated cell count; if RBC count >1 million/mm³, make a push smear and differentiate cells that are pushed out on the end

RBC, Red blood cell; WBC, white blood cell.

^*Expected cell yield (WBC count for no. of cells recovered on slide): 0/mm³ for 0-70, 1-2/mm³ for 12-100, >3/mm³ for >100.

Preparing Cytocentrifuge Slides

The cytocentrifuge enhances the ability to identify the types of cells present in a fluid. This centrifuge spins at a low speed, which minimizes distortion of the cellular elements and provides a “button” of cells that are concentrated into a small area. The cytocentrifuge assembly consists of a cytofunnel, filter paper to absorb excess fluid, and a glass slide. These three components are fastened together in a clip assembly, a few drops of well-mixed specimen are dispensed into the cytofunnel, and the entire assembly is centrifuged slowly. The cells are deposited onto the slide, and excess fluid is absorbed into the filter paper, which produces a monolayer of cells in a small button (Figure 17-1).

Figure 17-1 Wright-stained cytocentrifuge slide showing a concentrated button of cells within a marked circle. (From Carr JH, Rodak BF: *Clinical hematology atlas,* ed 3, Philadelphia, 2009, Saunders.)

Although there is some cell loss into the filter paper, this is not selective, and an accurate representation of the types of cells present in a fluid is provided. There also may be some distortion of cells as a result of the centrifugation process or crowding of cells when high cell counts are present. To minimize distortion resulting from overcrowding of cells, appropriate dilutions should be made with normal saline before centrifugation. The basis for this dilution should be the WBC count or the nucleated cell count. A nucleated cell count of 200/mm³ or fewer provides a good basis for the differential. If the RBC count is extremely elevated, a larger dilution may be necessary; however, an RBC count of 5000/mm³ would not cause significant nucleated cell distortion. If a fluid has a nucleated cell count of 2000/mm³ and an RBC count of 10,000/mm³, a 1 : 10 dilution should be made, which produces a nucleated cell count of 200/mm³ and an RBC count of 1000/mm³ for the cytocentrifuge slide. If the RBC count of a fluid is greater than 1 million/mm³, it is best to make a “push” slide to perform the differential. In this case, the differential should be performed on the cells “pushed out” on the end of the smear instead of in the body of the smear, because that is where the larger, and possibly more significant, cells would be deposited.

If a consistent amount of fluid is used when cytocentrifuge slides are prepared, a consistent yield of cells can be expected; this can be used to confirm the WBC or nucleated cell count. For example, if 5 drops of fluid (undiluted or diluted) is always used to prepare cytocentrifuge slides, a 100-cell differential count should be obtainable if the WBC or nucleated cell count is equal to or greater than 3/mm³. In all cases, the entire cell button should be scanned before the differential count is performed to ensure that significant clumps of cells are not overlooked. The area of the cell button that is used for performing the differential count is not important, but if the number of nucleated cells present is small, use of a “systematic meander” starting at one side of the button and working toward the other side is best. In case the number of cells recovered is small, the area around the cell button should be marked on the back of the slide with a wax pencil, or premarked slides should be used to prepare cytocentrifuge slides (see Figure 17-1).

Cerebrospinal Fluid

CSF is the only fluid that exists in quantities sufficient to sample in healthy individuals. CSF is present in volumes of 100 to 150 mL in adults, 60 to 100 mL in children, and 10 to 60 mL in newborns.6,7 This fluid bathes the brain and spinal column and serves as a cushion to protect the brain, as a circulating nutrient medium, as an excretory channel for nervous tissue metabolism, and as lubrication for the central nervous system.

Gross Examination

Normal CSF is nonviscous, clear, and colorless. A cloudy or hazy appearance may indicate the presence of WBCs (greater than 200/mm³), RBCs (greater than 400/mm³), or microorganisms.6,7 Bloody fluid may be caused by a traumatic tap, in which blood is acquired as the puncture is performed, or by a pathologic hemorrhage within the central nervous system. If more than one tube is received, the tubes can be observed for clearing from tube to tube. If the first tube contains blood but the remaining tubes are clear or progressively clearer, the blood is the result of a traumatic puncture. If all tubes are uniformly bloody, the probable cause is a subarachnoid hemorrhage. When a bloody sample is received, an aliquot should be centrifuged, and the color of the supernatant should be observed and reported. A clear, colorless supernatant indicates a traumatic tap, whereas a yellowish or pinkish yellow tinge may indicate a subarachnoid hemorrhage. This yellowish color sometimes is referred to as xanthochromia, but because not all xanthochromia is pathologic, the Clinical and Laboratory Standards Institute recommends avoiding the term and simply reporting the actual color of the supernatant (Figure 17-2 and Table 17-2).⁵

TABLE 17-2

Characteristics of Cerebrospinal Fluid

Traumatic Tap	Pathologic Hemorrhage
Clear supernatant	Colored or hemolyzed supernatant
Clearing from tube to tube	Same appearance in all tubes
Bone marrow contamination	Erythrophages
Cartilage cells	Siderophages (may have bilirubin crystals)