Introduction to Body Fluids, Graff's Textbook of Urinalysis and Body Fluids, BODY FLUID COMPOSITION, TYPES OF BODY FLUIDS , ACCUMULATION OF EXCESS BODY FLUIDS, BODY FLUID COLLECTION, CELL COUNTS IN BODY FLUIDS , CELLULAR MORPHOLOGIES AND DIFFERENTIALS, CRYSTAL ANALYSIS MICROSCOPY,
The study of body fluids presents challenges to the laboratory. Analysis involves multiple departments of the laboratory and specialized knowledge of each type of body fluid. Hematology is important in examining the cells and crystals found, chemical analyses are required to assess significant physiologic changes in the patient, microbiology can help detect infectious agents in a nearby body cavity or membrane, and immunological tests and other miscellaneous tests can also provide the physician with critical information. Further consultation with pathology may be required for the identification of tumor cells and other abnormal cells.
BODY FLUID COMPOSITION
While body fluids vary in composition, they share some elements in common. The critical roles of water and electrolytes are important determinants of any fluid composition and movement in the body. Water and electrolytes play crucial roles in many metabolic processes. Water enters the system through consumption of either water or food and also through cellular metabolic processes. For example, the water of oxidation can yield about 300 mL of water per day.
Fluids of the body can be intracellular or extracellular, with about 55% of the water being intracellular and about 45% being extracellular. Extracellular fluid can be further divided into interstitial fluid, transcellular fluids in various body cavities, and plasma. Fluids typically move around in body because of various forces and body conditions. The electrolyte and enzyme composition of intracellular fluid differs from extracellular fluids and knowledge of these differences can aid in understanding disease processes. For example, potassium levels are higher inside the cell than outside and sodium concentrations also vary between the intracellular fluid and the extracellular fluid. Depending upon the local conditions of various adjacent membranes and tissues, other fluid constituent concentrations can be altered as well. Examining these biochemical differences, along with examination of cellular elements, can assist in diagnosing and monitoring the patient’s condition.
TYPES OF BODY FLUIDS
Body fluids are diverse, with variation in physical appearance, properties, cell types, and cell counts. In general, studies of body fluids are most helpful to assess inflammation,infection, malignancy, and hemorrhage. Body fluids can be divided into categories such as cerebrospinal fluid, various serous fluids from cavities lined with serous membranes, synovial fluid, semen, vaginal secretions, respiratory secretions such as from bronchoalveolar lavage, amniotic fluid, and even feces, which is considered in this category, although there are more body fluids not covered in this list.Technically, urine is also in this category, but we have covered it in the beginning of this text.
ACCUMULATION OF EXCESS BODY FLUIDS
The amount of serous fluids found in the space between an organ and the membrane sac that encompasses the organ varies according to body site. Normally, only a small amount of fluid is present: <30 mL pleural fluid, <50 mL pericardial fluid, and <100 mL ascites. Body fluids are necessary for lubrication of the body cavity/organ interface during movement. A delicate equilibrium is maintained by the capillaries and the lymphatic vessels. Any obstruction or altered pressure in these vessels can affect the amount of fluid and its constituents.3
Several forces, within and outside of the capillaries, work together to maintain fluid equilibrium. The tissue’s colloidal osmotic pressure (interstitial fluid pressure), along with the capillary’s hydrostatic pressure (filtration pressure), regulates the outward flow of fluid from the capillary.
The colloidal osmotic pressure of the capillary and the tissue’s hydrostatic pressure regulate the inward flow of fluid into the capillary from the tissue. Figure 8-1 illustrates the direction of these forces. Normal removal of fluids entering into the interstitial space is handled by the lymphatic system. Figure 8-2 shows the normal flow of fluids among the bloodstream, tissues, and lymphatic vessels. However, an imbalance in pressures causes excess egress of fluid into tissue spaces and can lead to accumulation of fluid in the body cavity. This accumulation of fluid is called an effusion. The causes and types of effusions are explained in Chapter 10, Serous Fluids.
Figure 8-1. Forces governing the exchange of fluid at the capillary level.
Figure 8-2. Exchanges through capillary membranes in the formation
and removal of interstitial fluid.
BODY FLUID COLLECTION
The procedure for collecting body fluid specimens involves a minor surgical procedure that is usually named for the site of collection. A pleural fluid collection is termed thoracentesis, while cerebral spinal fluid (CSF) is obtained by a spinal tap. Table 8-1 lists the body fluids commonly examined and the procedure used to collect each.
BODY FLUID VOLUME
The volume of body fluid also varies greatly depending upon the source of the body fluid. In addition, disease can tremendously alter the amount of body fluid present. This is especially true in serous fluid where the normal amount of fluid is quite small, only the amount between the two adjacent serous membrane layers. In disease, this fluid level can increase from a few milliliters to a few liters of fluid.
Table 8-1 Commonly Analyzed Body Fluids
BODY FLUID APPEARANCE
The normal color and turbidity of body fluids is dependent on the body cavity from which they are obtained. Cerebro- spinal fluid and synovial fluid are normally colorless and clear, whereas serous fluids are usually slightly yellow and clear. Terms used to describe the appearance of body fluids are listed in Table 8-2.
Table 8-2 Commonly Used Terms in the Description of Body Fluid Appearance
Abnormal color or turbidity may indicate a disease process’s physiological changes in the body cavity from which the fluid is obtained. These body fluid abnormalities are detailed in each corresponding chapter.
CELL COUNTS IN BODY FLUIDS
Although automated cell counters are continually improving (see Chapter 14), most cell counts are still performedmanually using the hemocytometer. The most common hemocytometer used is the Neubauer hemocytometer. As shown in Figure 8-3, this hemocytometer has a platform on each of two sides. Each platform contains an etched grid that is scored with markings for ease of counting. The largest sections on the grid are each one square millimeter. The grid is laid out as three millimeters by three millimeters for a total of nine square millimeters. Each square millimeter is divided further with varying degrees of detail.
These grids are etched into thick glass plates and have a moat that isolates them. The outer wall of the moat is 0.1 mm taller than the platform on which the grids are etched. A special optically corrected coverslip is placed over the grid area with edges resting on the moat walls. This depth must be considered when calculating cell counts performed on the hemocytometer. Both sides of the hemocytometer are loaded with well-mixed undiluted specimen.
Once the specimen settles to the grid lines, both sides are counted and averaged if within 20%.3 If 20% or better precision is not obtained, the specimen is mixed again, reloaded, and recounted.
Normally, the cells in the entire nine square millimeters are counted and a cell count per square millimeter is calculated. However, if the concentration of cells is high, adjustments can be made to the procedure. Fewer square millimeters may be counted, dilutions may be made, or a combination of both. If many red blood cells (RBCs) are present, counting the entire center square millimeter may be as accurate as counting all nine. If the RBC concentration
Figure 8-3. Neubauer hemocytometer diagrams
is extremely high, making a dilution and counting five (four corners and the center) of the medium areas in the center square millimeter may be acceptable. Laboratory professionals must use judgment in establishing criteria for when to employ adjusted cell counting techniques for body fluid cell counts.
If many nucleated cells are present, counting these cells in the four corner square millimeters is often sufficient.
A simple formula to remember when performing hemocytometer count calculations is as follows:
Cell Count = Nx Dx 10/A. N is the number of cells counted. D is the dilution factor.
Multiplying by 10 is necessary to bring the depth up to 1 mm. The product is then divided by A (area in square millimeters counted). It is important to note that square millimeters is NOT the same as number of squares counted. This calculation results in number of cells per cubic millimeter.
For example, if an undiluted body fluid is counted and 35 white blood cells (WBCs) are counted in a total of all nine square millimeters, the calculation is 35x1x10/9 = 39 per cubic millimeter. If a specimen is rather bloody, it may be diluted and less area may be counted. For example, if a 1:10 dilution is made using saline and 127 RBCs are counted using the center of the middle square millimeter and its four corners (one fifth of a square millimeter), the calculation is 127x10 x10/(1/5) OR 127x100x10 x5, because to divide by a fraction is to multiply by its reciprocal. Therefore, multiplying by 5 is the same as dividing by 1/5. This example results in 63,500 RBCs per cubic millimeter.
In order to accurately perform these counts, some body fluids require addition of substances to the sample to facilitate counting by reducing viscosity or preventing coagulation of the sample. Acetic acid is often used in cell count diluents to lyse RBCs and enhance the nuclei of WBCs. Acetic acid cannot be used for synovial fluid because it precipitates the hyaluronic acid present in synovial fluid. A solution of hyaluronidase may need to be added to perform the synovial fluid count accurately. For CSF and serous fluids, hyptonic saline or 1% ammonium oxylate can lyse RBCs, while keeping WBCs intact for counting. Stains may also be added to differentiate cells, such as methylene blue for differentiating RBCs from WBCs in cell counts.
CELLULAR MORPHOLOGIES AND DIFFERENTIALS
Morphologies of both abnormal cells and cells that are normal for that body fluid need to be learned to assess body fluids. A cytologist or pathologist can assist in identifying these
Figure 8-4. Cytocentrifuge
cells, especially in malignancies. For body fluid cell examination, cytocentrifuged preparations are preferred. Cytocentrifugation requires relatively little sample, is fast, requires little skill, and provides good cell recovery with much less cell distortion. Cytocentrifuged body fluid preparations are suitable for a variety of staining techniques. A special centrifuge and accompanying chambers need to be purchased for this technique. A cytocentrifuge is pictured in Figure 8-4.
A cuvette and slide are assembled and placed in the centrifuge head as shown in Figure 8-5. The cuvette top is removed and one to two drops of body fluid are pipetted into the cuvette. The cuvette top is replaced, the cover of the centrifuge head is secured, and the centrifuge lid is closed.
The time and speed are set (usually 5 minutes at 500 rpm) and the start button depressed. Cuvettes are removed after centrifugation and a circle is drawn on the backside of the slide to indicate the area of cell deposit. This is done because not all body fluids have a sufficient amount of cells present
Figure 8-5. Cytocentrifugation method of body fluid concentration.
An assembly of a special cuvette, with attached filter paper, and a glass
slide are clipped into place in the cytocentrifuge. One to two drops of well-
mixed fluid are pipetted into the cuvette. The lids are closed and the
sample is centrifuged at 500 rpm for 5 minutes
Figure 8-6. Cytocentrifuge-prepared smears are marked with
a wax pencil to outline the area of cell deposit. Smears are
stained with Wright stain for microscopic evaluation.
to be visible macroscopically. The slides are then stained with Wright stain. Figure 8-6 shows both unstained and stained slides.
If a cytocentrifuge is not available, a sedimentation method may be used. A small plastic specimen cup is affixed to a glass slide that is wrapped with filter paper containing a hole that is aligned with the specimen cup. The assembly is inverted and the cells are gently deposited onto the slide as the liquid in the body fluid is absorbed by the filter paper.
A 22% solution of albumin is added to cytocentrifuged body fluids with lower protein content to increase cell yield and decrease cell distortion. Addition of albumin is not needed with synovial fluid, however, if the viscosity or the protein content is high. Care must be taken to avoid contamination of this albumin solution because contamination can alter results.
CRYSTAL ANALYSIS MICROSCOPY
Some body fluids, especially synovial fluid and urine, contain crystals that can be distinguished by microscopy. Special techniques such as polarized microscopy and compensated polarized microscopy may be needed to distinguish different crystals. A polarizing lens permits the passage of light that is vibrating in only one direction, blocking the rest. If a second polarizing lens is placed in this path and it is parallel to the first lens the light will continue on as for the first lens. If the second lens is placed in the path in a perpendicular direction to the first lens (known as the “crossed position”) all light will be blocked. The first lens is referred to as the “polarizing filter” and it is nearest the light source, below the condenser. The second filtering lens is called the “analyzer” and is located between the objective and the eyepiece lenses.
Some crystals, when placed between crossed polarized lenses, are capable of rotating the plane of the polarized light, enabling the light to then pass through the second perpendicular lens. These crystals are said to “polarize” the light and they are said to be optically active.
Another property that certain crystals possess is birefringence. Birefringence is detected with compensated polarized light. When compensation is used, a red compensator is added between the crystal and the analyzer. Birefringence is a double refraction (or bending) of the light into two rays, one parallel to the light axis and one at right angles to it. These two rays have two different speeds and indexes of refraction. This birefringence has either a positive or a negative designation. The crystals show differing colors in negative and in positive birefringence. Polarized light and compensated polarized light are helpful to detect and identify many urine and synovial crystals. In synovial fluid two similar looking crystals, monosodium urate and calcium pyrophosphate can be differentiated using birefringence. The monosodium urate crystals polarize strongly with negative birefringence and calcium pyrophosphate crystals have weak polarization with positive birefringence with compensated polarized light.
REFERENCES
Lillian A. Mundt and Kristy Shanahan, Graff's Textbook of Urinalysis and Body Fluids, Second Edition 2011
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