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Operational Medicine 2001
United States Naval Hospital Corpsman 3 & 2 Training Manual
NAVEDTRA 10669-C June 1989

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Hospital Corpsman 3 & 2: June 1989

Chapter 6: Clinical Laboratory

Naval Education and Training Command


Introduction

Blood Collection

The Microscope

Complete Blood Count

Urinalysis

The Hospital Corpsman and Clinical Laboratory Techniques

Administrative Responsibilities in the Laboratory

Ethics in the Laboratory

References

Introduction

A basic knowledge of clinical laboratory procedures is required of all hospital corpsmen, particularly those working at small dispensaries and isolated duty stations without the supervision of a medical officer. The patient's complaint may be of little value by itself, but coupled with the findings of a few easily completed laboratory studies, a diagnosis can usually be surmised and treatment initiated.

Hospital corpsmen who can perform blood and urine tests and interpret the results are better equipped to determine the cause of illness or to request assistance, since they can give a more complete clinical picture. Consequently, their patients can get treated sooner.

In this chapter we will discuss blood collection, the microscope, and step-by-step procedures for the complete blood count and basic urinalysis.

 Blood Collection

The two principal methods of obtaining blood samples are finger puncture and venipuncture. Both methods have their advantages and disadvantages, but for most clinical examinations, blood is best obtained from a vein.

Finger Puncture

The finger puncture is used when a patient is burned severely or is bandaged so that the veins are either covered or inaccessible. It is also used when only a small amount of blood is needed.

Equipment Required

  • Sterile gauze pads (2 x 2)

  • 70 percent isopropyl alcohol pad or Povidone-iodine solution

  • Blood lancets

  • Capillary tubes

  • Bandages

Arrange your equipment in an orderly manner and have it within easy reach. As with many other laboratory procedures, wash your hands prior to the procedure.

Procedure

  1. Using the middle or ring finger, massage or "milk" the finger down towards the fingertip. Repeat this "milking" five or six times.

  2. Cleanse the fingertip with an alcohol pad or Povidone-iodine solution and let dry. 3. Take a lancet and make a quick deep stab on the side of the finger (off-center). To obtain a large rounded drop, the puncture should be across the striations of the fingertip (fig. 6-1).

  3. Wipe away the first drop of blood to avoid dilution with tissue fluid. Avoid squeezing the fingertip to accelerate bleeding as this tends to dilute the blood with excess tissue fluid, but gentle pressure some distance above the puncture site may be applied to obtain a free flow of blood.

  4. When the required blood has been obtained, apply a pad of sterile gauze and instruct the patient to apply pressure, then apply a bandage.

When dealing with infants and very small children, the heel or great toe puncture is the best method to obtain a blood specimen. It is performed in much the same way.

Venipuncture (Vacutainer Method)

The collection of blood from a vein is called venipuncture. For the convenience of technician and patient, arm veins are best for obtaining a blood sample. If arm veins cannot be used due to bandages, IV fluid therapy, thrombosed or hardened veins, etc., consult your supervisor for instructions on the use of hand or foot veins. DO NOT DRAW BLOOD FROM AN ARM WITH IV FLUID RUNNING INTO IT. CHOOSE ANOTHER SITE. THE FLUID ALTERS TEST RESULTS.

Equipment required

  • Sterile gauze pads (2 x 2)

  • 70 percent isopropyl alcohol or Povidone-iodine solution

  • Tourniquet

  • Vacutainer needles and holder

  • Vacutainer tubes appropriate for the test to be performed

Position the patient so that the vein is easily accessible and you are able to perform the venipuncture in a comfortable position. Always have the patient either lying in bed or sitting in a chair with the arm propped up. NEVER PERFORM A VENIPUNCTURE WITH THE PATIENT STANDING UP, AND USE CAUTION TO ENSURE THE PATIENT DOES NOT FALL FORWARD FROM HIS OR HER SEAT.

Procedure

  1. Wash hands.

  2. Assemble equipment.

  3. Explain the procedure to the patient.

  4. Apply the tourniquet around the arm approximately 2 to 3 inches above the anticubital fossa with enough tension so that the VEIN is compressed but not the ARTERY. A sphymomanometer may be used instead of a tourniquet if a patient is difficult to draw.

  5. Position the patient's arm extended with little or no flexion at the elbow.

  6. Locate a prominent vein by palpation (feeling). If the vein is difficult to find, it may be made more prominent by massaging the arm with an upward motion to force blood into the vein.

  7. Cleanse the puncture site with a 70 percent alcohol pad or Povidone-iodine solution and allow to dry. 
    CAUTION: After cleaning the puncture site, only the sterile needle should be allowed to touch it.

  8. "Fix" or hold the vein taut. This is best accomplished by placing the thumb under the puncture site and exerting a slight downward pressure on the skin or placing the thumb to the side of the site and pulling the skin taut laterally. (See figure 6-2). 9. Using a smooth continuous motion, introduce the needle into the side of the vein at about a 15 degree angle with the skin (fig. 6-2). (Bevel of the needle should be up.)

  9. Holding the vacutainer barrel with one hand, push the tube into the holder with the other hand and watch for the flow of blood into the tube until filling is completed.

  10. While holding the vacutainer with one hand, release the tourniquet with the other.

  11. Place a sterile gauze over the puncture site and remove the needle with a quick, smooth motion.

  12. Apply pressure to the puncture site and instruct the patient to keep the arm in a straight position. Have the patient hold pressure for at least 3 minutes.

  13. Take this time to invert any tubes that need to have anticoagulant mixed with the blood, then label the specimens.

  14. Reinspect the puncture site and apply a bandage.

The Microscope

Before any attempts are made to view blood smears, urinary sediments, bacteria, parasites, etc., it is absolutely essential that the beginner know the instrument with which he or she will be spending considerable time-the microscope (fig. 6-3). The microscope is a precision instrument used repeatedly in many areas of the medical laboratory to make visible those objects that are too small to be seen by the unaided eye. This is accomplished by means of a system of lenses of sufficient magnification and resolving power (ability to show, separate, and distinguish) so that small elements lying close together in a specimen appear larger and distinctly separated. Most laboratories are equipped with binocular (two-eye-piece) microscopes, but monocular microscopes are also commonly used. The microscope most often used in the laboratory is a compound microscope that consists of the various pieces identified and discussed briefly below:

Framework:

Base-structure on which the microscope rests.

Arm-structure that supports the magnification and adjustment system; it is the handle by which the microscope is carried.

Stage-platform on which a preparation is placed for examination. In the center of the stage is the aperture or hole to allow passage of light from the condenser. Mechanical stage-means by which the preparation may be moved about on the stage.

Illumination System:

Mirror-usually double, a flat surface on one side, and a concave surface on the other side. The concave surface is used in the absence of a condenser. Many microscopes have a built-in light source instead of a lamp and mirror. Internal light source-built into the base of the microscope, and provides a more precise steady source of light into the microscope.

Condenser-composed of a compact lens system located between the mirror and stage. The condenser (usually an Abbe condenser) concentrates (condenses) the light through the aperture in the stage to the objective lens.

Iris diaphragm-controls the amount of light reaching the condenser. The size of the iris diaphragm opening should approximate that of the face of the objective lens. Thus, as a general rule, the diaphragm is completely closed when liquid.preparations are observed with the low-power objective, and wide open when stained preparations are observed with the oil-immersion lens using natural light.

Magnification System:

Revolving nosepiece-contains openings into which objective lenses may be fitted and that may be revolved to bring an objective into the desired position.

Objective lenses-usually a set of three consisting of a low-power lens (approximate focus 16 mm, magnification 10X), a highpower lens (approximate focus 4 mm, magnification 45X), and an oil-immersion lens (approximate focus 1.8 mm, magnification 100X). Numerical aperture (NA) refers to the angle of the maximum cone of light that may enter the objective. The greater the numerical aperture, the greater the resolution, or ability of the microscope to separate small details clearly.

The body tube-through which light passes from the objective to the ocular lens. The ocular lenses (eyepieces)-usually a 10X is provided: the number indicates the magnification (in diameters) produces by the ocular of the image formed by the objectives. Magnification is determined by the ratio between the size of the virtual image and the real size of the object. It is expressed in diameter multiples, for example 100X. By multiplying the magnification engraved on the objective by that engraved on the eyepiece, one can determine the total magnification. The total magnification resulting from the systems of lenses is determined by the combination of objectives and oculars:

 

Objective lens

Color Code

l0X Ocular

Total Magnification

16 mm-l0X

green

l0x

lOOX

4 mm-45X

yellow

l0x

450X

1.8 mm-100X

red

l0X

1OOOX

Adjustment System 

Composed of two parts, both of which raise or lower the body tube together with the lens system.

Coarse adjustment-the larger and innermost knob; by rotating the control knob, the image appears and is in approximate focus.

Fine adjustment-the smaller and outermost knob; by rotating this control knob, it renders the image clear and well-defined.

Focusing the Microscope

The process of focusing consists of adjusting the relations between the optical system of the microscope and the object to be examined so that a clear image of the object is obtained. The distance between the upper surface of a glass slide on the microscope stage and the faces of the objective lens varies according to which of the three objectives is in focusing position. Thus, the intervening distance with the low-power objective (l0X) is the greatest (16 mm), that for the oil-immersion lens (l00X) is the smallest (1.8 mm), and that for the high-power objective (45X) is intermediate (4 mm). As a result, the focusing operation must be conducted with skill to avoid damage to the objective lens, the specimen, or both. It is good practice to obtain a focus with the low-power objective first, then change to the higher objective required. Most modern microscopes are equipped with parfocal objectives, which means that if one objective is in focus, the others will be in approximate focus when the nosepiece is revolved. With the low-power objective in focusing position, observe the following steps in focusing.

  1. Seated behind the microscope, lower your head to one side of the microscope until your eyes are approximately at the level of the stage.

  2. Using the coarse adjustment knob, lower the body tube until the face of the objective is within 1/4 inch of the object. Most microscopes are constructed in such a manner that the low-power (green) objective cannot be lowered to make contact with the object on the stage.

  3. While looking through the ocular, use the coarse adjustment knob to elevate the body tube until the image becomes visible. Then use the fine adjustment knob to obtain a clear and distinct image. Do not move the focusing knob while changing lenses.

  4. If the high-power objective (yellow) is to be used next, bring it into position by revolving the nosepiece (a distinct "click" indicates it is in proper alignment with the body tube). Use the fine adjustment knob only to bring the object into exact focus. Of course, light adjustment must be made; open the iris diaphragm of the condenser to accommodate more light.

  5. The oil-immersion objective (red) is used for detailed study of stained blood and bacterial smears. Remember that the distance between objective lens and object is very short, and great care must be employed. After focusing with the highpower objective 'and scanning for well defined cells, raise the objective, place a small drop of immersion oil, free of bubbles, on the slide, centering the drop in the circle of light coming through the condenser. Next, revolve the nosepiece to bring the oil-immersion objective into place, and by means of the coarse adjustment knob, slowly lower the body tube until the lens just makes contact with the drop of oil on the slide. The instant of contact is indicated by a flash of light illuminating the oil. The final step in focusing is done with the fine adjustment knob. It is with this lens in particular that lighting is important; the final focus, clear and well-defined, will be obtained only when proper light adjustment is made.

Care of the Microscope

The microscope is an expensive and delicate instrument that should be given proper care.

Moving or transporting the microscope should be done by grasping the arm of the scope in one hand and supporting the weight of the scope with the other hand. Avoid sudden jolts and jars.

Make sure the microscope is kept clean at all times; when not in use, enclose it in a dustproof cover or store in its case. Remove dust with a camel hair brush. Lenses may be wiped carefully with lens tissue.

When the oil immersion lens is not being used, remove the oil with lens tissue. Use oil solvents, such as xylol, on lenses only when required to remove dried oil and only in the minimal amount necessary. Never use alcohol or similar solvent to clean lenses.

Complete Blood Count

The complete blood count consists of:

  • Total red blood cell count (RBC)

  • Hemoglobin determination

  • Hematocrit reading

  • Total white blood cell count (WBC)

  • Differential white blood cell count

Red Blood Cell (Erythrocyte) Count

The red blood cell count is made to determine the number of red cells in one cubic millimeter (mm3) of blood. The normal red blood cell count is as follows:

adult male 5.4 ± .8 million/mm3

adult female 4.8 ± .6 million/mm3

newborn 5.1 ± .9 million/mm3

A lower count is usually a sign of anemia.

A lower count is usually a sign of anemia.

Manual Sahli Pipette Method

The materials listed below are required to perform a red blood cell count using the manual Sahli pipette method:

  • Hemacytometer set

  • Microscope with lamp

  • Red cell pipette

  • Suction tube

  • Laboratory chits

  • Hand held counter

  • Red cell diluting fluid. 0.85 percent sodium chloride (normal saline) is preferred because of its ready availability and its ability to prevent clumping of red cells.

Procedure

  1. Using well mixed anticoagulated blood or blood directly from a fingerstick, fill the clean, dry, red cell pipette (fig. 6-4) exactly to the 0.5 mark with the aid of the suction tube. Hold the pipette in a nearly horizontal position so that the exact level of the blood can be seen. The curve of the tip may rest on the skin, but the orifice must be free to immerse in the drop of blood to avoid air bubbles. If the blood level exceeds the 0.5 mark, withdraw excess blood by touching the tip to the skin surface. Do not touch it to gauze or cotton since these material absorb the fluid portion of the blood, leaving behind a much higher concentration of cells.

     

  2. Wipe the blood from the outside of the pipette, taking care not to touch the very tip. Immerse the tip in the RBC diluting fluid and aspirate fluid exactly to the 101 mark, slightly rotate the pipette while doing so. It is best to hold the pipette in an almost vertical position to avoid formation of air bubbles in the bulb. DO NOT DELAY BETWEEN STEPS 1 AND 2. IF THE BLOOD IS NOT DILUTED PROMPTLY, IT WILL DRY IN THE PIPETTE. Start to draw diluting fluid into the pipette as soon an the tip of the pipette is immersed in fluid to avoid loss of blood cells. Wipe the excess diluting fluid from the pipette, taking care not to touch the very tip. Filter the diluting fluid regularly to remove accidentally introduced blood cells.

     

  3. Remove the suction tube and shake the pipette vigorously for 3 minutes. DO NOT SHAKE IN THE DIRECTION OF THE LONG AXIS. (See figure 6-5.)

     

  4. Discard the clear fluid (about three drops) from the stem of the pipette. The counting chamber (figure 6-6) must be loaded with fluid from the pipette's bulb.

     

  5. Place the coverglass on the counting chamber, making sure both are clean and grease-free. (Fingerprints must be completely removed.) Load the counting chamber by touching the tip of the pipette against the edge of the coverglass and the surface of the counting chamber (figure 6-7). A properly loaded counting chamber should have a thin, even film of fluid under the coverglass. Allow 3 minutes for cells to settle. If fluid flows into the grooves (moats) at the edges of the chamber or if air bubbles are seen in the field, the chamber is flooded and must be cleaned with distilled water, dried with lens tissue, and reloaded. If the chamber is underloaded, carefully add additional fluid until properly loaded.

     

  6. Place the hemacytometer (figure 6-8) on the microscope. Use the low-power lens to locate the five small fields (1, 2, 3, 4, and 5) in the large center square bounded by the double or triple lines. Each field measures 1/25 mm2, 1/10 mm in depth, and is divided into 16 smaller squares. These smaller squares form a grid that makes accurate counting possible.

     

  7. Switch to the high-power lens and count the number of cells in field 1. Move the hemacytometer until field 2 is in focus and repeat the counting procedure. Continue until the cells in all five fields have been counted. Note that the fields are numbered clockwise around the chamber, with field 5 being in the center. Count the fields in this order. To count the cells in each field, start in the upper left small square and follow the pattern indicated by the arrow in figure 6-8. Count all of the cells within each square, including cells touching the lines at the top and on the left. DO NOT COUNT ANY OF THE CELLS TOUCHING THE LINES ON THE RIGHT AND AT THE BOTTOM.

     

  8. Total the number of cells counted in all five fields and multiply by 10,000 to arrive at the number of red cells per cubic millimeter of blood. The number of cells counted in each field should not vary by more than 20. A greater variation may indicate poor distribution of the cells in the fluid, resulting in an inaccurate count.

     

  9. Immediately after completing the count, clean the counting chambers with distilled water and dry it with lens tissue. Rinse pipettes first with cold water, then with acetone. Draw air through the pipette until it is dry. The pellet should move freely in the bulb if the pipette is dry.

Some common sources of error are:

  • Improper dilution-not drawing blood exactly to the 0.5 mark or using too much diluting fluid

  • Dirty equipment-diluting fluid unfiltered; greasy glassware; dirty microscope; wet pipettes

  • Poor mixing or not discarding first few drops of fluid

  • Poorly loaded counting chamber

  • Chipped pipettes. Discard pipettes with chipped or broken tips.

  • Use of gauze, cotton, or filter paper to remove excess blood from the pipette.

Uniopette Method

The Uniopette disposable diluting pipette system for the red blood cell count provides a convenient, precise, and accurate method for obtaining a red blood cell count. The disposable kit consists of a shielded capillary pipette (10 microliter (ul) capacity) and a plastic reservoir containing a premeasured volume of diluent (1:200 dilution).

Procedure

  1. Using the shield on the capillary pipette, puncture the diaphragm in the neck of the reservoir with the tip of the capillary shield.

  2. After obtaining free-flowing blood from a lancet puncture of the finger, remove the protective plastic shield from the capillary. Holding the capillary slightly above the horizontal, touch the tip to the blood source. Capillary action will fill the tube until blood collection stops automatically, e.g., when the proper amount (10 ul) has been obtained. Wipe blood off the outside of the capillary tube, making sure none is removed from inside the capillary tube. An alternative source of blood is a thoroughly mixed fresh venous blood sample obtained by venipuncture.

  3. Squeezing the reservoir slightly, cover the upper opening of the capillary overflow chamber with the index finger and seat the capillary tube holder in the reservoir neck. Release pressure on the reservoir and remove the finger from the overflow chamber opening. Suction will draw the blood into the diluent in the reservoir.

  4. Squeeze the reservoir gently two or three times to rinse the capillary tube, forcing diluent into-but not out of-the overflow chamber, releasing pressure each time to return diluent to the reservoir. Close the upper opening with your index finger and invert the unit several times to mix the blood sample and the diluent.

  5. For specimen storage, cover the overflow chamber of the capillary tube with the capillary shield.

  6. Immediately prior to cell counting, mix adequately again by gentle inversion, taking care to cover the hole with your index finger.

  7. Remove the pipette from the reservoir. Squeeze the reservoir and reseat the pipette in the reverse position, releasing pressure to draw any fluid in the capillary tube into the reservoir. Invert and fill the capillary tube by gentle pressure on the reservoir. After discarding the first 3 drops, charge the counting chamber of the hemacytometer by gently squeezing the reservoir.

  8. Using the high power objective, count the red blood cells in the red blood cell counting areas.

  9. Calculations: Multiply the number of cells counted by 10,000 to obtain the red cell count.

    Example:

    The number of cell in the 5 counting squares was 423.

     

    The cell count

    =

    423 x 10,000

     

     

    =

    4,230,000

 

Hemoglobin Determination

Of the many methods of hemoglobin estimation, the most accurate is reading of hemoglobin as oxyhemoglobin in the photometer, after dilution of the blood with a weak alkali. The Haden-Hausse, Sahli-Hellige, and Newcomer tests, based on acid hematin formed by the action of hydrochloric acid on hemoglobin, are sufficiently accurate for routine examination, provided they are properly done. Since relatively few ships and stations are equipped with a photometer, we will discuss the Sahli-Hellige method.

Materials Required for Sahli-Hellige Test

  • Distilled water

  • Sahli-hellige hemoglobinometer kit containing:

    • Small bottle of dilute (approx. 0. 1N) hydrochloric acid. Prepare this solution by adding 1 ml of concentrated HCl to 99 ml of distilled water. POUR ACID INTO WATER. Replenish this periodically-it must be of proper strength.

    • Graduated tube, with a scale on two sides. On one side is the percentage scale, and on the opposite side is the gram scale. The percentage scale reads from 0 to 170. The gram scale reads from 0 to,24.

    • Pipette, marked at the 20 mm3 level

    • Stirring rod

    • Color comparator, with a window in the side. On the right and left sides of this opening is the color standard for comparison. The center has an open slot to hold the graduated tube.

Procedure

  1. With a medicine dropper, place 5 drops of the 0.1N HCl in the bottom of the graduated tube. Place the tube in the color comparator.

  2. Using well-mixed venous blood or fingertip blood, fill the pipette to the 20 mm3 mark.

  3. Wipe blood from the outside of the pipette. Transfer blood to the Sahli tube. Note the time.

  4. Aspirate distilled water into the pipette two or three times and transfer these washings to the tube.

  5. Shake until the blood is well mixed and the tube is a uniform color.

  6. Add distilled water, drop by drop, each time mixing the solution with the stirring rod. Keep adding water and mixing until the color of the solution matches the standards on either side. Remove the stirring rod from the tube each time before comparing. Natural light makes more accurate readings possible.

  7. Five minutes after time noted, read the result from the scale on the tube by noting the graduation mark at the lower edge of the meniscus. Read and report both scales.

Reporting. Findings are reported both in grams per 100 ml of whole blood and in percentages of normal values. There are a number of modifications of the Sahli-Hellige method, and 100 percent may be equal to from 13.8 g to 17.3 g. In the sets usually used in the Navy, however, 100 percent is equal to 14.5 g of hemoglobin per 100 ml of whole blood. After reading the percentage on the scale, turn the tube and read from the other side to get the equivalent reading in grams.

If either scale is hard to read, remember that 100% / 14.5 g = 6.9, so one gram of hemoglobin is equal to 6.9 percent. If only one scale can be read, the other reading can be computed.

Caution: Equipment must be clean and dry before determination is started. Wipe all blood from the outside of the pipette before you insert it into the tube. Twenty cubic millimeters is a small volume, and a few blood cells clinging to the outside of the pipette can cause a significant error in findings.

 Hematocrit (Packed Cell Volume) Determination

Hematocrit is the volume of erythrocytes expressed as a percentage of the volume of whole blood in a sample. The venous hematocrit agrees closely with the hematocrit obtained from a skin puncture; both are greater than the total body hematocrit. Dried heparin, balanced oxalate, or EDTA is satisfactory as an anticoagulant.

Although the microhematocrit method is not available at all duty stations, it is the most accurate means of determining blood volume and should be used whenever feasible. This test is rapidly replacing the red cell count for general purposes since it is easier, quicker, and more accurate. The method described here is the microhematocrit method.

Normal Values. The normal hematocrit for males is 42 to 50 percent, for females, 40 to 48 percent. A value below an individual's normal range for sex and age indicates anemia.

Materials Required

  • Capillary tubes, plain or heparinized

  • Modeling clay sealant or microburner

  • Microhematocrit centrifuge

  • Microhematocrit reader

Procedures

  1. Fill the capillary tube two-thirds to three- quarters full with well-mixed, oxalated venous blood or fingertip blood (for fingertip blood use heparinized tubes, and invert several times to mix).

  2. Seal one end of the tube with clay or seal the empty end of the tube in a small flame of a microburner.

  3. Place the filled tube in the microhematocrit centrifuge, with the plugged end away from the center of the centrifuge.

  4. Centrifuge at a preset speed of 10,000 to 12,000 rpm for 5 minutes. If the hematocrit exceeds 50 percent, centrifuge for an additional 3 minutes.

  5. Place the tube in the microhematocrit reader. Read the hematocrit by following the manufacturer's instruction on the microhematocrit reading device.

 White Blood Cell (Leukocyte) Count

The total white cell count determines the number of white cells per cubic millimeter of blood. A great deal of information can be derived from white cell studies. The white cell count and the differential count are common laboratory tests and almost a necessity in determining the nature and severity of systemic infections.

Normal Values. The normal range is 4,000 to 11,000 cells per cubic millimeter of whole blood.

Abnormal White Blood Cell Count

  1. Leukocytosis (abnormally high count). This may be caused by:

    1. Systemic or local infections (usually due to bacteria). These counts are highly variable and not diagnostic. Some infections and representative white cell counts are:

      1. pneumonia-20,000 to 30,000/mm3

      2. meningitis-20,000 to 30,000/mm3

      3. appendicitis-10,000 to 30,000/ mm3

    2. Dyscrasia of blood-forming tissues. This is not caused by any known bacteria, but is due to a malfunctioning of the marrow and lymph tissues, resulting in extremely high white cell counts, which sometimes exceed 1,000,000/mm3. This is commonly known as leukemia, or blood cancer.

    3. Physiologic conditions, with counts as high as 15,000/mm3. Some of these may occur as follows:

      1. in the newborn

      2. during late pregnancy

      3. during labor

      4. accompanying severe pain

      5. after exercise or meals

      6. after cold baths

      7. during severe emotional upset

  2. Leukopenia (abnormally low count). This may be caused by:

    1. Severe or advanced bacterial infections, such as typhoid, paratyphoid, and sometimes tularemia, or when the bacterial infection has been undetected for a period of time as with chronic beta streptococcal infections of the throat.

    2. Infections caused by viruses and rickettsiae, such as measles, rubella, smallpox (until the 4th day), infectious hepatitis, psittacosis, dengue, tsusugamushi fever, and influenza (when it may fall to 1,5000/mm3, or shift to leukocytosis if complications develop).

    3. Protozoal infections (such as malaria) and helminthic infections (such as Trichinosis). In malaria, slight leukocytosis may develop for a short time during paroxysm, but shortly after its onset leukopenia ensues. With trichinosis there may be leukocytosis with an increase in eosinophils (as high as 50 to 70 percent).

    4. Overwhelming infections when the body's defense mechanism breaks down

    5. Anaphylactic shock

    6. Radiation

Manual Sahli Pipette Method

The materials listed below are required to perform a white blood cell count using the manual Sahli pipette method:

  • Hemacytometer set

  • Microscope with lamp

  • White cell pipette

  • Suction tube

  • Laboratory chits

  • White cell diluting fluid. This may be either of two acids. The acid ruptures the red cells, leaving the white cells intact. These acids are:

    • Dilute hydrochloric acid. Prepare this by mixing 1 ml of concentrated hydrochloric acid with 99 ml of water. POUR ACID INTO WATER.

  • Glacial acetic acid

    2 ml

    1 percent aqueous solution of gentian violet

    1 ml

    Distilled water

    97 ml

The gentian violet is not necessary, but by staining the nucleus, it makes the cells more refractile and helps to make an accurate count.

  • Four plain glass slides to prepare smears for differential count

  • hand held counter

Procedure

  1. Draw well-mixed anticoagulated venous blood or fingertip blood to the 0.5 mark on the white cell pipette.

  2. Observing the same precautions as for red cell count, draw diluting fluid to the 11.0 mark.

  3. Shake the pipette for 3 minutes. Do not shake along the long axis. (See figure 6-5.)

  4. Load the counting chamber, using the same technique as for the red cell count.

  5. Count the white cells with the low-power lens in each of the four large corner fields (A, B, C, and D, in figure 6-8). Use subdued lighting. Go clockwise around the counting chamber; that is, from field A to field B to field C to field D. For convenience each field is divided into 16 smaller squares. Starting with the small square in the upper left corner of the field, count the cells in each square in the top row, moving across the field to the right, then drop down one row of squares and work back to the left, as indicated by the arrow in figure 6-8. Remember the rule for counting cells: COUNT THE CELLS TOUCHING THE BORDER LINES AT THE TOP AND ON THE LEFT. DO NOT COUNT THE CELLS TOUCHING THE LINES ON THE RIGHT AND AT THE BOTTOM.

  6. When all the cells in the four fields have been counted, multiply the count by 50 for the total white cell count.

  7. Immediately after completing the count, clean the counting chamber with distilled water and dry it with lens tissue. Rinse pipettes first with cold water, then with acetone. Draw air through the pipette until it is dry. The pellet should move freely in the bulb if the pipette is dry. Sources of error. The errors are generally caused by the same mistakes as described for red cell counts.

Uniopette Method

The Uniopette disposable kit for doing a white blood cell count consists of a shielded capillary pipette (20 ul capacity) and a plastic reservoir containing a premeasured volume of diluent (1:100 dilution).

Procedure

  1. Using the shield on the capillary pipette, puncture the diaphragm in the neck of the reservoir with the tip of the capillary shield.

  2. After obtaining free-flowing blood from a lancet puncture of the finger, remove the protective plastic shield from the capillary. Holding the capillary slightly above the horizontal, touch the tip to the blood source. Capillary action will fill the tube until the blood collection stops automatically, e.g., when the proper amount (20 ul) has been obtained. Wipe blood off the outside of the capillary tube, making sure none is removed from inside the capillary tube. An alternative source of blood is a thoroughly mixed fresh venous blood sample obtained by venipuncture.

  3. Squeezing the reservoir slightly, cover the upper opening of the capillary overflow chamber with your index finger and seat the capillary tube holder in the reservoir neck. Release pressure on the reservoir and remove your finger from the overflow chamber opening. Suction will draw blood into the diluent in the reservoir.

  4. Squeeze the reservoir gently two or three times to rinse the capillary tube, forcing diluent into-but not out of-the overflow chamber, releasing pressure each time to return diluent to the reservoir. Close the upper opening with your index finger and invert the unit several times to mix the blood sample and diluent.

  5. For specimen storage, cover the overflow chamber of the capillary tube with the capillary shield.

  6. Immediately prior to cell counting, mix adequately again by gentle inversion, taking care to cover the hole with your index finger.

  7. Remove the pipette from the reservoir. Squeeze the reservoir and reseat the pipette in the reverse position, releasing pressure to draw any fluid in the capillary tube into the reservoir. Invert and fill the capillary tube by gentle pressure on the reservoir. After discarding the first 3 drops, charge the counting chamber of the hemacytometer by gently squeezing the reservoir.

  8. Using the high power objective, count the white blood cells in the nine large squares

  9. Calculation: Add 10 percent to the number of cells counted in the nine large squares and multiply by 100 to obtain the white cell count.

    Example: The number of cells in 9 large squares was 90.

     

    The cell count

    =

    [90 + (0.1 x 90)] x 100

     

     

    =

    [90 + 9] x 100

     

     

    =

    99 X 100 = 9900

 

Differential White Blood Cell Count

The total white cell count is not necessarily indicative of the severity of a disease, since some serious ailments may show a low white cell count. However, the percentage distribution of the different types of leukocytes in the blood often provides more helpful information in determining the severity and extent of the infection than any other single procedure used in the examination of the blood. The differential count gives these percentages.

Normal Values. The normal percentages of the different leukocytes are:

Eosinophils

(Eos)

2-4 percent

Basophils

(Basos)

0-2 percent

Lymphocytes

(Lymphs)

21-35 percent

Monocytes

(Monos)

4-8 percent

Neutrophils

(Neuts)

 

Metamyelocytes

(Metas)

0 percent

Bands or Stab forms

(Bands)

0-10 percent

Segmented

(Segs or Polys)

51-67 percent

Most hospital corpsmen have heard the expression "shift to the left" and "shift to the right." These terms are often loosely used in refer best be explained by the following diagram:

Percentage Distribution of the Different Leukocytes

 

EOS

BASOS

METAS

BANDS

SEGS

LYMPHS

MONOS

 

Normal Percent

2-4

0-2

0

0-10

51-67

21-35

4-8

The metamyelocytes, bands, and segmented neutrophils constitute the neutrophilic cells. When the cells to the left of the segs are increases, it is a "shift to the left." If the segmented neutrophils increase, it is a "shift to the right." The true "right shift" will show numerous hypersegmented (having six or more lobes) neutrophils.

General Interpretations of Leukocytic Changes

The severity of an infection may be determined by the total white cell count and the differential count.

  • Leukocytosis (abnormally high white cell count) with an increase in the percentage of neutrophil indicates a severe infection with a good response of the bone marrow. The primary phagocytes (bacteria-destroying cells) are the neutrophils, and the bone marrow should supply large numbers of these to combat the infection. The greater the "shift to the left" (increase in immature neutrophils), the more severe the infection. The appearance of numerous juvenile cells (metamyelocytes) indicates irritation of the bone marrow with regeneration. If the infection continues and the patient's resistance declines, the shift advances further to the left. If improvement ensues, the shift declines and recedes to normal.

  • A falling white cell count with the number and maturity of neutrophils progressing toward normal indicates recovery.

  • A continued "shift to the left" with a falling total white cell count indicates a breakdown of the body's defense mechanism and is a poor prognosis.

  • The percentage of eosinophils, lymphocytes, and monocytes generally decreases in acute infections.

  • In tuberculosis an increase in monocytes (monocytosis) indicates activity in the infected area. An increase in lymphocytes (lymphocytosis) indicates healing.

  • Eosinophils increase in parasitic infections and allergic conditions.

Materials Required for Differential Count

  • Four plain glass microscope slides, clean and dry.

  • Wright's stain, powder or tablet form. Prepare this stain as follows.

    • Tablets: Use one tablet for each 10 ml of reagent grade methyl alcohol. Dissolve tablets after crushing them with the aid of a clean, dry mortar and pestle. Pour the solution in a stopper bottle and store for 30 days in a dark place, shaking it periodically during this period. Filter before use. Fill a dropper bottle for use at the lab bench.

    • Powder (preferred): Use 0.1 g of powder for each 60 ml of reagent grade methyl alcohol. Prepare in the same manner as the tablets. Fill a dropper bottle.

    • Buffering solution. This may be either:

      • Distilled water with a pH of 7. Use from a dropper bottle.

      • A prepared buffer solution with a pH of 6.4. Prepare this by dissolving 6.63 g monobasic potassium phosphate and 3.20 g of dibasic sodium phosphate in 1,000 ml of distilled water.

    • Staining rack

    • Mouthpiece and tube as used in blood counting pipettes

    • Microscope and lamp

    • Immersion oil

    • Blood cell counter

Technique for Making Smears

  1. Cleanse finger with 70 percent isopropyl alcohol and puncture it as described previously.

  2. Wipe off the first drop of blood with dry, sterile gauze.

  3. When a second drop forms, touch it lightly with a clean, grease-free slide. Place the slide on a flat surface with the drop of blood up. A smear can also be made with a drop of blood from a needle or collecting tube.

  4. Hold a second slide between thumb and forefinger and place the edge at a 45 degree angle against the top of the slide, holding the drop of blood. Back the second slide down until it touches the drop of blood. The blood will distribute itself along the edge of the slide in a formed angle.

  5. Push the second slide along the surface of the other slide, drawing the blood across the surface in a thin, even smear. If this is done with uniform rapidity and without wobbling the slide, a good smear will result. Try to keep the blood from reaching the extreme edges of the slides. Large cells have a tendency to stack up on the perimeter of the smear, and letting the smear reach the edges of the slide will aggravate this tendency. The smear should show no wavy lines or blank spots (see figure 6-9).

  6. Let the smear air-dry.

Technique for Staining Smears

  1. Place the smear on a staining rack. Flood it with about 1 ml of Wright's stain and allow it to stand for 2 minutes.

  2. Add an equal quantity of buffer. There should be no run-off of fluid from the slide. A little experimentation will show just how much stain and buffer the slide will hold. Mix the buffer and stain by blowing air through the rubber pipette tube and directing the current of air about the surface of the slide. Mix until a metallic (copper looking) film appears. Let stand for 3 minutes.

  3. Wash with tap water, provided pH testing has shown the water to be neutral. If the tap water is not neutral, wash the slide with distilled water. The stain and the metallic film must be floated off to prevent streaking, so keep the slide flat and horizontal in the stream of water. If the slide is tilted, the metallic film will settle to the surface of the smear and remain there.

  4. Wipe the stain from the bottom of the slide. Let it air-dry. A good smear should be thin and evenly distributed, and it must be dry before staining. Always make at least two smears for each patient, as the additional smear should be examined to verify any abnormal findings.

Staining times will vary with different batches of Wright's stain. One batch may give best results with 3 minutes of staining and 2 minutes of buffering, another may give better results with 2 minutes of staining and 3 minutes of buffering. The only way to determine the best time interval for a particular stain is by experimentation.

Next to incorrect time intervals, the most common cause of poor results with Wright's stain is incorrect pH of the staining fluid. If the stain is too acid, the red cells in the smear will stain a bright pink or may even be colorless, while an alkaline stain will cause the red cells to appear blue-gray, with poor color definition. In either case, the pH of the buffering solution should be checked.

Technique for Differential Count

  1. Place the stained slide on the microscope with a drop of immersion oil and adjust the ocular lenses.

  2. Using the oil immersion lens (red) (highest power), scan the fields for areas where the red cells just touch. In this area the blood smear is thinner, and consequently, the white cells are easier to identify.

  3. Count 100 consecutive white cells (see figure 6-10 for identification), pressing the correct key on the cell counter for each type of white cell identified.

  4. If you count 20 lymphocytes among the 100 cells, then the patient has 20 percent lymphocytes. If an absolute count has been requested, the total leukocyte count is multiplied by individual percentages.

    Example:

    Patient has a white count of 8,000.

    Differential count shows 20 percent lymphocytes.

    20 percent x 8,000 = 1,600

    The patient has 1,600 lymphocytes per cubic millimeter of blood.

Cell Identification

The ability to identify the different types of white cells is not difficult to develop, but it does require a thorough knowledge of staining characteristics and morphology that can be gained only by extensive, supervised practice. The beginner is likely to encounter some difficulty in learning to differentiate between monocytes and large lymphocytes, or between monocytes and atypical (not typical) lymphocytes. It is possible also to confuse eosinophils with basophils, since an alkaline stain may cause the orange granules in an eosinophil to appear deep blue or purple. If this happens, the reddish cast in the granules can often be detected by judicious use of the minor focusing knob. Also, the granulations of an eosinophil are generally much finer than those of a basophil, and they tend to concentrate in the cytoplasm.

The neutrophils are subclassified according to age, and the age is indicated by the nucleus. If there is any doubt as to the identity of the neutrophil, always classify it with the older stage. For instance, if a single irregularity in a horseshoe-shaped nucleus appears as a break or a concentration of color, classify it as a segmented neutrophil, not as a neutrophilic band. Practice is the key. And remember-even experienced technicians often disagree as to the identity of a particular cell.

If it is desirable to save a smear for reexamination, remove the immersion oil by placing a piece of lens tissue over the slide and moistening the tissue with xylol. Draw the damp tissue across the slide and dry the smear with another piece of lens paper.

A good smear (thin and evenly distributed) is essential for accurate identification and counting.

The cytoplasm of an eosinophil contains numerous coarse, reddish-orange granules, which are lighter colored than the nucleus.

Scattered large, dark-blue granules, which are darker than the nucleus, characterize the cell as a basophil. Granules may overlay the nucleus as well as the cytoplasm.

The cytoplasm of a lymphocyte is clear sky blue, scanty, with few unevenly distributed, azurophilic granules with a halo around them. The nucleus is generally round or oval or slightly indented, and the chromatin is lumpy and condensed at the periphery.

The largest of the normal white blood cells is the monocyte. Its color resembles that of a lymphocyte, but its cytoplasm is a muddy gray-blue. The nucleus is lobulated, deeply indented or horseshoe-shaped, and has a relatively fine chromatin structure. Occasionally the cytoplasm is more abundant than in the lymphocyte.

The cytoplasm of a neutrophil has numerous fine lilac-colored granules, which sometimes are hardly visible. The nucleus is dark purple or reddish purple, and it may be oval, horseshoe- or S- shaped, or segmented (lobulated). The neutrophil is further subclassified according to age as:

  1. Metamyelocyte (also called "juvenile"). This is the youngest neutrophil generally reported. The nucleus is fat, indented, and is usually "bean" shaped or "cashew nut" shaped.

  2. Band (sometimes called stab). This is the older or intermediate neutrophil. The nucleus has started to elongate and has curved itself into a horseshoe- or S-shape. As the band ages, it progresses to:

  3. Segmented. The nucleus is separated into two, three, four, or five segments or lobes.

  4. Hypersegmented. The nucleus is divided into six or more segments or lobes.

 Urinalysis

The physical and chemical properties of normal urine are markedly constant; any abnormalities are easily detected. The use of simple tests provides the physician with helpful information concerning the diagnosis and management of many diseases.

This section will deal with the routine and microscopic examination of urine specimens, some of the principles involved, and some of the simpler interpretations of the findings.

Urine Specimens

Urine specimens for routine examinations must be collected in aseptically clean containers. Unless circumstances warrant, catheterization should be avoided because it may cause urinary tract infections. Specimens of female patients are likely to be contaminated with albumin and blood from menstrual discharge, or with albumin and pus from vaginal discharge. For bacteriologic studies, care must be taken to ensure that the external genitalia have been thoroughly cleansed with soap and water. The patient must then void the initial stream of urine into the toilet or a suitable container and the remainder directly into a sterile container. All urine specimens should be examined when freshly voided or should be refrigerated to prevent decomposition of urinary constituents and to limit bacterial growth.

Random Specimen

This is a sample of urine voided without regard to the time of day or fasting state. This sample is satisfactory for most routine urinalyses. It is the least valid specimen, since tests results may reflect a particular meal or fluid intake.

First Morning Specimen

This is the first specimen of urine voided upon rising. It is the best sample for routine urinalysis because it is usually concentrated and more likely to reveal abnormalities. If positive results are obtained from the first morning specimen, the physician may order a 24-hour specimen for quantitative studies.

Twenty-four Hour Specimen

This specimen measures the exact output of a specific substance over a 24-hour period. To collect this specimen:

  1. Have the patient empty his or her bladder at 0800. Discard this urine.

  2. Collect all urine voided during the next 24 hours.

  3. At 0800 the following day (end of 24-hour period), instruct the patient to empty his or her bladder. Add this urine to the pooled specimen.

  4. Specimen must be refrigerated during collection.

  5. Preservatives will be added to the first specimen voided, according to the types of tests being ordered.

 Preservation of Specimens

To delay decomposition, use

  1. Refrigeration. All specimens not being examined immediately should be refrigerated.

  2. Toluene. Simply add enough toluene to form a thin film on the surface of the specimen. This film will prevent air from reaching the urine. False positives are seldom encountered with this preservative. Remember that the toluene is on the surface, and all test samples must be pipetted from BENEATH the surface.

  3. Thymol. A small lump, floating on the surface, will preserve a urine specimen for several days. Enough thymol may dissolve to produce false positives for albumin. Do not use more that 0.1g of thymol per 100 ml of urine.

Other common preservatives are formaldehyde, boric acid, hydrochloric acid, and chloroform. The preservative used must be identified on the label of the container. If no preservative is used, it should be so stated.

 Routine Examination

Volume (For 24-Hour Specimen or When Requested)

The normal daily urine volume for adults ranges from 800 to 2000 ml, averaging about 1,500 ml. The amount of urine excreted in 24 hours varies with fluid intake and the amount of water lost through perspiration, respiration, and bowel activity. Diarrhea or profuse sweating will reduce urinary output; a high protein diet tends to increase it. Daytime urine output is normally two to four times greater than nighttime output.

Color

The normal color of urine varies from straw to light amber. Diluted urines are generally pale; concentrated urines tend to be darker. The terms used to describe the color of urine are:

  1. Colorless

  2. Light straw

  3. Straw

  4. Dark straw

  5. Light amber

  6. Amber

  7. Dark amber

  8. Red

The color of urine may be changed by the presence of blood, drugs, or diagnostic dyes. Examples are:

  1. Red or red-brown (smokey appearance), due to the presence of blood.

  2. Yellow or brown (turning greenish with yellow foam when shaken), due to the presence of bile.

  3. Olive green to brown-black, caused by phenols.

  4. Milky appearance, caused by chyle.

  5. Dark orange, due to treatment with Pyridium.

  6. Blue-green, due to methylene blue.

Transparency

Urine may be reported as clear, hazy, slightly cloudy, cloudy, or very cloudy. Some physicians prefer the term "turbidity" to "transparency," but both terms are acceptable.

Freshly passed urine is usually clear or transparent. In certain conditions it may be cloudy due to the presence of blood, phosphates, crystals, pus, bacteria, etc. A report of transparency is of value only if the specimen is fresh. After standing, all urine becomes cloudy due to decomposition, salts, and the action of bacteria. Upon standing and cooling, all urine specimens will develop a faint cloud composed of mucus, leukocytes, and epithelial cells. This cloud settles to the bottom and is of no significance.

Reaction

Normal urine is slightly acid but will become more alkaline upon standing. The pH ranges from 4.6 to 8.0. The acidity of urine is influenced by many factors, such as a diet high in protein or fat, fasting and starvation, and acid therapy. Alkaline urine may be produced by cystitis, pyelonephritis, and sulfonamide therapy.

It is essential that an alkaline urine be maintained during treatment with sulfonamides, since these compounds are precipitated as crystals in acid solution. The crystals will cause damage to the uriniferous tubules. Sodium bicarbonate is generally used as an alkalizer.

Reaction to pH, protein, glucose, ketones, bilirubin, blood, nitrite, and urobilinogen in urine may be determined by the use of the Multistix and Color Chart. This is a specially prepared multitest strip that is simply dipped into the urine specimen and then compared with the color values for the various tests on the accompanying chart. The color chart indicates pH values, and the numerical value should be reported.

Specific Gravity

The specific gravity of the specimen is the weight of the specimen as compared to an equal volume of distilled water. The specific gravity varies directly with the amount of solids dissolved in the urine and normally is from 1.015 to 1.030 during a 24-hour period.

The first morning specimen of urine is more concentrated and will have a higher specific gravity than a specimen passed during the day. A high fluid intake may reduce the specific gravity to below 1.010. In disease the specific gravity of a 24-hour specimen may vary from as low as 1.001 to as high as 1.060.

The specific gravity may be measured with the urinometer or the index refractometer, available as standard equipment at most duty stations. The refractometer may be held manually (fig. 6-11) or mounted on a stand like a microscope. The specific gravity is determined by the index of light refraction through solid material.

Measurement with Urinometer

  1. Pour urine into the cylinder until it reaches a point approximately 1 1/2 inches below the top of the cylinder. Insert the urinometer, making sure that it is floating freely. The cylinder should not overflow when the urinometer is immersed. Read the bottom of the meniscus.

  2. If the specimen is cloudy, the urine should be centrifuged before specific gravity readings are taken. Cloudy urine tends to show low (and invalid) specific gravity.

Measurement with Index Refractometer

  1. Hold the index refractometer in one hand, and with the other hand and two applicator sticks, place a drop of urine on the glass section beneath the coverglass.

  2. Hold the refractometer so that the light reflects on the glass section and look into the ocular end. Read the number that appears where the light and dark lines meet. This is the specific gravity.

 Glucosuria

Glucosuria is the presence of an abnormal amount of glucose in the urine. Traces of sugar are often found in normal urine, but are not enough to react to any of the routine tests for glucose. However, different sugars in the urine are of various clinical significance. When performing routine urinalysis, we can test for glucose, and any trace of glucose in the test suggests that something is wrong with the patient's carbohydrate metabolism.

Methods for Measuring Glucose

  1. Test Strip, Glucose and Blood. This test is specific for glucose. Other sugars do not interfere.

    1. Remove strip from container.

    2. Dip into specimen. Remove and wait 30 seconds.

    3. Immediately compare the test strip against the color chart.

    4. Record results. Normal urine is negative for glucose.

    5. If there is a positive reaction for glucose, a Clinitest is done for both confirmation and quantitation.

  2. Urine-Sugar Test Tablets (Clinitest Tablets)

    1. Place 5 drops of urine in a large test tube.

    2. Add 10 drops of water and mix well.

    3. Add one Clinitest tablet.

    4. Put the tube in a rack. Let stand until the reaction is completed (foaming action has stopped).

    5. Compare the color with the color chart and record the results.

    6. If an orange color appears and then disappears, run the test again, diluted in half.

    7. Be careful not to touch the end of the tube as it is extremely hot.

 Albuminuria

This is the presence of albumin in the urine. Albumin is a protein, consisting of serum albumin and serum globulin that has been eliminated from the blood plasma. It contains carbon, hydrogen, nitrogen, oxygen, and sulfur. Its exact composition has not been determined.

Urinary albumin does not necessarily indicate diseased kidneys; it may reflect reactions to toxic and nontoxic substances originating within the body. Albuminuria is frequently found in young men who have no other signs of disease. This condition is usually transitory. However, albuminuria usually is of clinical significance and generally requires further examination.

Methods for Measuring Albumin in Urine

The test is accomplished by means of test strips. Since practically all urine is tested for both glucose and albumin, the tests are combined in the multitest strips. If the strips are unavailable, or positive for albumin, the sulfosalicylic acid method of albumin determination may be used.

Sulfosalicylic Acid Method of Albumin Determination

  1. If the fresh specimen is clear, the test may be run without centrifuging. If the specimen is cloudy, centrifuge about 15 ml at 2,000 rpm for 5 minutes.

  2. Pour 2.5 ml of clear urine into a test tube measuring 16 mm x 150 mm.

  3. Add 7.5 ml of 3 percent sulfosalicylic acid to the urine.

  4. Mix by inversion and let stand 10 minutes before reading

  5. A white turbidity indicates albuminuria. Compare the specimen with the standards and report as indicated, i.e., 1, 5, etc.

CAUTION: The centrifuge is a carefully balanced machine, and efforts should be made to maintain that balance. Specimens should be placed directly opposite each other in the machine. If only one urine specimen is being centrifuged, place a tube containing an equivalent weight of water directly opposite the urine.

 Microscopic Examination of Urine Sediment

Usually performed in addition to routine procedures, this examination requires a degree of skill that can be acquired only through practice under the immediate supervision of a competent technician. The specimen should be as fresh as possible, since red cells and many formed solids tend to disintegrate upon standing, particularly if the specimen is warm or alkaline.

Procedure

  1. Mix the specimen well.

  2. Pour 15 ml of urine into a conical centrifuge tube and centrifuge at 1,500 rpm for 5 minutes.

  3. Invert the centrifuge tube and allow all of the excess urine to drain out. DO NOT SHAKE THE TUBE WHILE INVERTED. Enough urine will remain in the tube to resuspend the sediment. Too much urine remaining will cause diluting of the sediment and difficulty in reading.

  4. Resuspend the sediment by tapping the bottom of the tube.

  5. With a medicine dropper, mount one drop of the suspension on a slide and cover it with a coverslip.

  6. Place the slide under the microscope and scan with the low-power objective and subdued lighting.

  7. Switch to the high-power objective for detailed examination of a minimum of 10 to 15 fields.

Clinically Significant Findings

Leukocytes - Normally, 0 to 3 leukocytes per high-power field will be seen on microscopic examination. More than 3 cells per high-power field probably indicates disease somewhere in the urinary tract. Estimate the number of leukocytes present per high-power field and report it as the "estimated number per high-power field."

Erythrocytes - These cells are not usually present in normal urines. If erythrocytes are found, estimate their number per high-power field and report it. Erythrocytes may be differentiated from white cells in several ways:

  • White cells are larger than red cells.

  • When focusing in with the high-power lens, the red cells show a distinct circle; the white cells tend to appear granular and the nucleus may be visible.

  • The addition of one drop of 5 percent acetic acid to the urine sediment will disintegrate any red cells but will not affect the white cells except to make the nuclei more distinct.

Casts - These urinary sediments are formed by coagulation of albuminous material in the kidney tubules. They are cylindrical and vary in diameter depending on the size of the renal tubule or duct of their origin. The sides are parallel, and the ends are usually rounded. Casts in the urine always indicate some form of kidney disorder and should always be reported. If casts are present in large numbers, the urine is almost sure to be positive for albumin. Casts containing organized structures are:

  • RBC casts

  • WBC casts

  • Epithelial casts

Types of casts:

  • hyaline

  • waxy

  • granular

  • fatty

Cylindroids - Resemble hyaline casts but are more slender and have a slender tail that is often twisted or curled. They frequently are seen along with hyaline casts and have the same significance.

Other microscopic structures found in urine are:

  • various kinds of crystals

  • epithelial cells

  • mucus threads

  • spermatozoa

These are not generally pathologic unless present in very large numbers. Certain types of crystals are pathologic; therefore, all crystals seen should be reported.

The Hospital Corpsman and Clinical Laboratory Techniques

As mentioned in the beginning of this chapter, hospital corpsmen are required to have a basic knowledge of laboratory procedures. It is not expected that all hospital corpsmen be proficient in all phases of this field, but it is essential that they know how to perform the tests mentioned in this chapter, since they are eligible for duty independent of a medical officer.

The hospital corpsman is not expected to make diagnoses from test findings or to institute definitive treatment based upon them; however, with the availability of modern communications facilities, the results of these tests will greatly assist him or her in giving a clearer clinical picture to the supporting medical officer.

Needless to say, accuracy, neatness, and attention to detail are essential to obtain optimum test results. Remember also that these tests are only aids to diagnosis-many other clinical factors must be taken into consideration before treatment can be started.

Administrative Responsibilities in the Laboratory

The ability to perform clinical laboratory tests is a commendable attribute of the hospital corpsman. However, the entire effort can come to naught if proper recording and filing practices are ignored and the test results go astray.

Since the test results are a part of the patient's clinical picture, their accuracy and veracity are vital. Since they have a bearing upon the immediate and future medical history, they are made part of the medical record. Erroneous and inaccurate laboratory results have been known to cause extensive embarrassment and medical complications.

As a hospital corpsman, it is your responsibility to assure effective administration of all laboratory reports in your department and to make sure that they are properly filed.

Patient Identification

When accepting laboratory requests and specimens, make absolutely certain that the patient is adequately identified. Proper identification can prevent a great number of errors.

Specimen Identification

Make sure that the specimen is in fact that of the patient submitting it. You need not stand over the patient while it is being collected; however, keep in mind that there are instances when it would be advantageous for persons to substitute specimens.

Use of Proper Forms

The Armed Forces have gone to great lengths to produce workable and effective laboratory forms to serve their purpose with a minimum of confusion and chance for error. These forms are standard forms of the 500 series. Their primary purpose is to request, report on, and record clinical laboratory tests. With the exception of the SF-545, Laboratory Report Sheet, they are multicopy, precarbonized for convenience. The original eventually is filed in the patient's clinical record, and the carbon becomes part of the laboratory's master file. For a complete listing of these forms and their purposes, refer to chapter 23 of the Manual of the Medical Department.

Use of Laboratory Forms

It goes without saying that a separate form is used for each patient and test. The patient's name, rank, Social Security number, and unit identification will be entered on each request in sufficient detail to assure proper identification.

Since the results of the requested laboratory test are usually closely associated with the patient's health and treatment, the requesting physician's name and location shall be clearly stated. This assures that the report will get back to the physician as expeditiously as possible. There is nothing more aggravating to both patient and physician than a lost or misplaced laboratory report.

Since the data requested, the date reported, and the time of specimen collection are usually important in support of the clinical picture, these facts should be clearly written on the request in the areas provided for them.

The type of tests requested should be clearly marked to eliminate all misunderstanding.

Filing the Laboratory Requests

After the physician has seen the results of the laboratory tests, the forms must be filed in the clinical record of inpatients. SF-545, Laboratory Report Sheet, is provided for this. The originals of the test forms are to be stapled on this sheet IN CHRONOLOGICAL ORDER and neatly spaced, as directed on the form. Each sheet will accommodate a certain number of laboratory reports. DO NOT OVERCROWD with more reports-use additional sheets if necessary.

The results of the laboratory tests performed on active duty outpatients will be placed on the SF-545 in the health (medical) treatment record.

 Ethics in the Laboratory

The nature of laboratory tests and their results must be treated as a confidential matter between the patient, physician, and the performing technician. It is good practice to prevent unauthorized access to these reports, to leave interpretation of the test results to the attending physician, and to refrain from discussing them with the patient.

References

  1. Clinical Diagnosis and Management by Laboratory Methods, ed 16. W. B. Saunders Co, 1979

     

  2. Laboratory Medicine: Hematology, ed 6. C. V. Mosby Co, 1982

     

  3. Hematology for Medical Technologists, ed 5. Lea & Febiger Co, 1983

     

  4. Hematology: Principles and Procedures, ed 2. Lea & Febiger Co, 1976

     

  5. Modern Urine Chemistry, Ames Division, Miles Laboratories, Inc

     

  6. A Handbook of Routine Urinalysis, J. P. Lippincott Co, 1983

 

 


Approved for public release; Distribution is unlimited.

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Operational Medicine 2001

Health Care in Military Settings

Bureau of Medicine and Surgery
Department of the Navy
2300 E Street NW
Washington, D.C
20372-5300

Operational Medicine
 Health Care in Military Settings
CAPT Michael John Hughey, MC, USNR
NAVMED P-5139
  January 1, 2001

United States Special Operations Command
7701 Tampa Point Blvd.
MacDill AFB, Florida
33621-5323

This web version is provided by The Brookside Associates Medical Education Division.  It contains original contents from the official US Navy NAVMED P-5139, but has been reformatted for web access and includes advertising and links that were not present in the original version. This web version has not been approved by the Department of the Navy or the Department of Defense. The presence of any advertising on these pages does not constitute an endorsement of that product or service by either the US Department of Defense or the Brookside Associates. The Brookside Associates is a private organization, not affiliated with the United States Department of Defense.

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