Methods of
Hematology

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3-2. VENIPUNCTURE

a. Site. To obtain blood by venipuncture, draw the specimen directly from a patient's vein with a sterile hypodermic needle and syringe or a vacuum blood sample device. In adults use the veins located in the proximal forearm or antecubital space as illustrated in figure 3-1. In infants employ the jugular or femoral vein for the venipuncture. The vein selected should be large, readily accessible, and sufficiently close to the surface to be seen and palpated. If venipuncture poses a problem due to the age of the patient, sclerotization due to repeated venipuncture, or any other unusual circumstance, the technician should consult a physician concerning the procedure. UNDER NO CIRCUMSTANCES SHOULD A TECHNICIAN WITHDRAW BLOOD FROM A SAGITTAL SINUS, JUGULAR VEIN, OR FEMORAL VEIN. This should be left to the discretion of the physician in charge. Occasionally, the best vein is found on the hand, leg, or foot. These areas are more sensitive, and the veins are not as firmly anchored as those of the arm. Veins can become distended and easier to enter by allowing the arm to hang down for 2 or 3 minutes, by massaging the blood vessel

Figure 3-1. Site of venipuncture.

toward the body, or by gently slapping the site of puncture. Young and vigorous persons usually have elastic veins well filled with blood. Elderly or debilitated persons can have sclerosed or fragile veins, which are hard to enter or which collapse easily.

b. Equipment. All syringes, needles, lancets, and other instruments used for the collection of blood specimens must be sterile. Disposable syringes or blood collection sets with vacuum tubes are available through normal supply channels. These should be used whenever possible. Aseptic technique is necessary to prevent the possible transmission of homologous serum hepatitis. The following equipment is necessary to perform a venipuncture:

(1) Isopropyl alcohol, 70 percent, prep pads.

(2) Tourniquet.

(3) Sterile syringes or vacuum blood sample devices.

(4) Gauze pad, 2 x 2 inches.

(5) Needle, 1 to 1 ½ inches long, 19–23 gauge.

(6) Suitable blood collection tubes and labels.

(7) Gloves, latex.

c. Preparation.

(1) Cleanse hands thoroughly with soap and water.

(2) Place an identifying label on the blood collecting tube.

(3) Assemble the sterile needle and syringe. If a vacuum system is used, screw the needle into the plastic holder. Always leave the cap over the needle when not in use.

(4) Check to be sure that the syringe works smoothly. The syringe must be dry to avoid hemolysis of the red cells. The plunger must match the syringe and must be pushed firmly to the bottom of the cylinder to prevent injection of air into the vein. This can be fatal.

d. Syringe Procedure.

(1) Place a tourniquet around the patient’s arm above the elbow tightly enough to check venous circulation, but not so tightly as to stop arterial flow. (If latex tubing is used, place it approximately 2 inches above he proposed venipuncture site). Form a loop with the longer end and draw the loop under the shorter end so that the tails of the tubing are turned away from the proposed site (see figure 3-2a).

CAUTION: Do not allow the tourniquet to remain in place for more than 2 minutes.

Check the pulse at the wrist to make sure that arterial circulation is not cut off.

Figure 3-2a. Venipuncture procedure: Locate the vein.

(2) Instruct the patient to make a tight fist.

(3) By inspection and palpation locate the desired vein, determine the direction of its course, and estimate its size and depth (see figure 3-2a venipuncture procedure, a through h).

(4) Release tourniquet.

(5) Cleanse the skin over the selected vein with prep pads in 70 percent isopropyl alcohol. Wipe off excess alcohol with a sterile dry gauze. Do not contaminate the area after cleaning (see figure 3-2b).

Figure 3-2b. Venipuncture procedure: Clean the puncture site.

(6) Put on gloves.

(7) Replace tourniquet on arm and have the patient straighten out the arm and make a fist.

(8) Grasp the syringe in the right hand and place forefinger on the hub of the needle to guide it. Grasp the forearm with the left hand about 2 inches below the area to be punctured and hold the skin taut with the thumb (see figure 3-2c).

3-2c. Venipuncture procedure: Guide needle toward the vein.

(9) With the needle bevel up, parallel to, and alongside the vein, insert the needle quickly under the skin and then into the vein. The insertion into the skin and vein can be performed in one complete motion (see figure 3-2d). After entry into the vein, blood will appear in the needle hub. Do not probe or move the needle horizontally, as discomfort and possible nerve damage may result.

Figure 2-3d. Venipuncture procedure: Insert needle into the vein.

(10) Aspiration of the blood is accomplished by gently pulling upon the syringe plunger (see figure 3-2e). The syringe barrel should be held steady during this process. Withdraw the desired quantity.

Figure 3-2e. Venipuncture procedure: Aspirate the blood.

(11) Remove the tourniquet by pulling on the long, looped end of the tubing only after blood is drawn into the syringe (see figure 3-2f).

Figure 3-2f. Venipuncture procedure: Remove the tourniquet.

CAUTION: Do not remove the needle now. If the needle were remove prior to the Tourniquet being removed, blood would be forced out of the venipuncture site, resulting in blood loss and/or hematoma formation (tumor-like cluster of blood forming under the skin).

(12) Place a sterile gauze pad over the point where the needle entered the skin and deftly withdraw the needle simultaneously putting pressure on the site (see figure 3-2g).

Figure 3-2g. Venipuncture procedure: Place a sterile pad over the site and withdraw the needle.

(13) Have the patient extend the arm and maintain light pressure on the gauze pad over venipuncture site (see figure 3-2h).

Figure 3-2h. Venipuncture procedure: Have the patient extend the arm and maintain light pressure on the site.

e. Vacutainer Procedure.

(1) Place the Vacutainer tube in the holder until the rubber stopper reaches the guideline. The short needle should be embedded in the stopper, but the needle must not break the vacuum (see figure 3-3).

Figure 3-3. Vacutainer system.

(2) Follow steps 1-6 in paragraph 3-2d.

(3) Enter the vein with the needle parallel to and alongside the vein. Probing or horizontal movement of the needle while under the skin must be avoided.

(4) After entry into the vein push the tube all the way into the holder; vacuum is broken, and blood flows freely into the tube. Release the tourniquet at this time by pulling the long, looped end of the tube.

(5) If the multiple needle is used or more than one tube is required, release the tourniquet after the first tube is filled; remove the filled tube and insert the next one.

CAUTION: Ensure the needle is not moved while tubes are being changed.

(6) Place a sterile gauze pad over the point where the needle enters the skin and deftly withdraw the needle, placing pressure on the site.

(7) Have the patient extend the arm and maintain light pressure on the gauze pad over the venipuncture site.

f. Discussion.

(1) Cleanliness is essential when performing a venipuncture.

(2) It is most important that correct technique be practiced in order to avoid unnecessary pain to the patient, prevent tissue damage, secure a good representative blood specimen, and prevent contamination of the specimen or infection of the patient.

(3) Syringes and needles must be thoroughly inspected for damage or malfunction.

(4) If difficulty is experienced in entering the vein or a hematoma begins to form, release the tourniquet and promptly withdraw the needle and apply pressure to the wound.

(5) Vigorous pulling on the plunger of the syringe can collapse the vein, produce hemolysis of the blood specimen, or cause air to enter the syringe.

(6) When repeated venipunctures have to be performed on one patient, it is advisable to select different sites for blood withdrawal.

(7) Remove the tourniquet as early as possible once a good flow of blood has been established. Prolonged application of the tourniquet results in partial stasis of blood and changes many quantitative values of blood components.

(8) Blood drawn by venipuncture is often stored for a period of time before it is analyzed. For this reason, certain general precautions must be followed in order to ensure a valid analysis. Before withdrawing blood from its container, make sure the blood sample is thoroughly but gently mixed. Blood containers should Be tightly stoppered at all times to prevent drying or contamination. Store the blood specimen in the refrigerator. Blood counts should be done within 3 hours after collection. Under no circumstances should blood taken for hematological examinations be stored overnight.

3-5. INTRODUCTION

a. The type of blood cells found in the peripheral blood smears may be of diagnostic and prognostic importance. For this reason proper preparation and staining of blood films is essential for the identification and study of different kinds of leukocytes. The appearance of erythrocytes and thrombocytes will often give important clues that help distinguish between different types of diseases or other physical changes.

b. There are two basic methods for the preparation of blood smears: the cover slip and the slide methods. The cover slip method has certain advantages over the slide method; distribution of cells is like that of the in vivo circulation. The principal disadvantage of the latter method is that covers lips are very fragile and easily broken during processing.

c. The slides and cover glasses must be chemically clean and dry. New slides must first be washed with soapy water and rinsed thoroughly with distilled water. The slides are then placed in a beaker of 95 percent ethyl alcohol. As the slides are needed, dry them with a soft, lint free cloth. The slides may be reused by properly cleaning them and making sure they are not chipped or scratched.

d. The foundation for the morphological study of blood was based on Ehrlich's investigations of the aniline dyes, dating back to 1877, while he was still a student. Originally, simple dyes were used in the clinical laboratory and tissues were stained successively if more than one color was desired. The majority of the aniline dyes are in the form of salts of acids and bases. During the process of staining, compounds are probably formed between the basic dyes and the acid nuclear substances of cells and between the acid dyes (so called "neutral" dyes). In this way, the staining principles of the original components were preserved; and, in addition, new staining properties dependent upon the union of the component dyes were developed. These were, therefore, termed polychromic dyes.

e. One modification of these polychromic stains is Wright's stain. This is the stain most used in Army laboratories today. Wright's stain is a methyl alcohol solution of an acid dye and a basic dye. The acid dye is known as eosin, which is red in color. The basic dye, methylene blue, is blue in color. The white cells are mostly identified by their preference for these dyes. In some cases the cells are even named for the dye that they prefer. For example, cells that prefer a mixture of the acid and basic dye are called neutrophils. In the staining process, a buffer solution is used to control the acid-base balance of the stain. This is a most important function. If the buffer solution is too acid it makes the acid dye too bright and the basic dye too faint. On the other hand, if the buffer solution is too basic it makes the basic dye too bright and the acid dye too faint. In either case, the result is a poorly stained slide. The acid-base balance of a solution is measured by its pH value. A buffer solution should have a pH value between 6.4 and 6.8. This allows the best color contrast between acid and basic dyes.

f. When optimal staining conditions exist, Wright’s stain is very satisfactory and easily differentiates cells. The eosin component stains cell cytoplasm, and the methylene blue component stains nuclear material, granules, and inclusions. Both stains oxidize rapidly because they are in alkaline solution. Giemsa, a purified polychrome stain, is added to compensate for this defect by maintaining the azurophilic staining property of the mixture.

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