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Lesson 3-1
Collection of Blood Specimens


Hematological laboratory procedures are based upon the examination of blood specimens. To obtain valid test results, specimens must be properly collected, processed, and recorded. Blood specimens are usually obtained by either venous or capillary puncture. The source of the specimen is determined chiefly by the quantity of blood required to perform the laboratory procedures and the age and condition of the patient.

There is generally little difference in blood counts performed on venous or capillary blood if a free-flowing capillary blood specimen is obtained. Valid blood counts cannot be made when capillary specimens are not taken from a free-flowing sample or when they are obtained from cyanotic or calloused areas or areas of local stasis. White blood cell counts made on blood obtained from such sources can vary as much as 1000 to 1500 cells per cu mm from their real value. For general purposes, however, venous samples are preferable since they allow for multiple and repeated hematological examinations and provide a sufficient quantity of blood for performing any other required laboratory procedure. Further, with venous blood the chances of error are reduced because operations are made under ideal conditions and repeat operations are possible. In situations where there are limitations on the quantity of blood that can be obtained, that is, in small infants or extensive burn cases, microquantitative methods are satisfactory for performing an analysis on a specimen obtained by capillary puncture.


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.

Figure 3-1. Site of venipuncture.

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 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.

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 are necessary to perform a venipuncture.

  • Isopropyl alcohol, 70 percent, prep pads.
  • Tourniquet.
  • Sterile syringes or vacuum blood sample devices.
  • Gauze pad, 2 x 2 inches.
  • Needle, 1 to 1 ½ inches long, 19–23 gauge.
  • Suitable blood collection tubes and labels.
  • Gloves, latex.


  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 plastic holder. Always leave the cap over the needle when not in use.
  4. Check to make 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.

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

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

  1. Instruct the patient to make a tight fist.
  2. 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).
  3. Release tourniquet.
  4. Cleanse the skin over the selected vein with prep pads in 70 percent isopropyl alcohol in a circular motion starting from the center and working your way out. Allow the area to air dry for 30 seconds to 1 minute. Do not contaminate the area after cleaning (see figure 3-2b).

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

  1. Put on gloves.
  2. Replace tourniquet on arm and have the patient straighten out the arm and make a fist.
  3. 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).

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

  1. 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 3-2d. Venipuncture procedure: Insert needle into the vein.

  1. 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.

  1. 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 (Pooling of blood under the skin).
  1. 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.

  1. 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.

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 1-8 in syringe procedure.

(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.

Cleanliness is essential when performing a venipuncture.

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.

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

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.

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.

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

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.

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 anticoagulated 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 count must be done within 3 hours of collection. Under no circumstances should blood taken for hematological examinations be stored overnight.


Site. Several different sites are suitable for capillary puncture. Because it is the most accessible, the palmer or lateral surface of the tip of the finger (preferably ring finger) is the most common site in adults. However, certain problems can be encountered such as heavy calloused areas or excessive tissue fluids (edema) that tend to result in non-representative samples. The lobe of the ear can be used for capillary puncture. However, differences in cell concentration do occur when blood is obtained from this site, primarily because of higher lymphocyte concentrations in the ear lobe. Because of the small amount of tissue on the fingers of infants, preferred site is the heel or big toe. A modification of the normal technique that has proven quite satisfactory when working with the heel of infants is to make two incisions in a crisscross fashion or “T”.

NOTE: To be a valid report, work done on capillary blood must be from a FREE-FLOWING puncture wound.


(1) Gauze pads 2 x 2 inches.

(2) Blood lancet.

(3) Glass slides, heparinized capillary tubes, and other devices to receive the specimen.

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


(1) The puncture site should be warm to assure good circulation of blood. If it is cold, apply warm water (38º to 40º C) for a few minutes. If blood is to be drawn from the ear, the edge of the lobe, not the flat side, should be punctured.

(2) The site to be punctured is first rubbed with alcohol prep pads to remove dirt and epithelial debris, increase circulation, and render the area reasonably disinfected (see figure 3-4 capillary puncture procedure, a through d).

Figure 3-4a. Capillary puncture procedure: Clean the puncture site.

(3) Allow sufficient time for the circulation to equalize.

(4) While making a finger puncture, apply gentle pressure to the finger to hold the skin taut. Hold the finger in one hand and the lancet in the other. The puncture is made perpendicular to the lines of the fingerprints, which results in a more free- flowing wound (see figure 3-4b).

Figure 3-4b. Capillary puncture procedure: Puncture the finger.

(5) The first drop of blood that appears is wiped away before specimens are taken (see figure 3-4c).


Figure 3-4c. Capillary puncture procedure: Wipe away first drop of blood.

(6) The blood must not be squeezed out since this dilutes it with fluid from the tissues, thus altering the ratio of cellular elements to fluid, as well as the ratio of cellular elements to each other.

(7) After the desired specimens have been collected, have the patient hold a sterile dry gauze pad over the wound until bleeding stops (see figure 3-4d).

Figure 3-4d. Capillary puncture procedure: Apply pressure to the site.


(1) Us a disposable lancet for puncture of the skin.

(2) Do not use the finger on a hand which has been hanging over the side of the bed as it is likely to be congested. Edematous or cyanotic areas should not be used.

(3) The finger should be thoroughly dry prior to puncture; blood will not well up on a finger that is moist. Furthermore, the alcohol or other antiseptic used can coagulate the blood proteins causing cell clumping and erroneous values as well as dilute cell volumes. This will result in incorrect counts and differentials.

(4) Finger punctures should be made along the lateral aspect of the fingertip. More nerve endings are located on the fingerprint area of the fingers; therefore, more pain results from punctures in this area. Scars can also form in these sensitive areas, and difficulty may be encountered in puncturing a callous. All of these difficulties are eliminated by drawing the blood from the lateral rather than the ventral aspect of the finger.


Anticoagulants are used to prevent the clotting of the blood specimens and the reagent employed should not bring about alteration of blood components. Unfortunately, many anticoagulants can alter cell structures as well as coagulation. The anticoagulants most often used are ethylene-diamine-tetra-acetate (EDTA), ammonium- potassium oxalate and heparin.

The choice of anticoagulant will depend on the analysis to be made. Ethylene-diamine-tetra-acetate (EDTA) is the anticoagulant of choice for most hematological analyses. This anticoagulant causes a minimum of distortion to the cells and platelets. It does not dissolve quickly in blood, however, so the tube must be inverted four or five times after blood is added. The dipotassium salt is prepared as a 1 percent solution in distilled water, and a final concentration of 0.5 ml of anticoagulant for each 5 ml blood is used. Another common anticoagulant is arnmonium-potassium oxalate. This combination of oxalates does not shrink or enlarge the red blood cells appreciably. It is essential, however, to add an optimal volume of blood to the oxalate, no less than 3.5 nor more than 6.0 mI.

Heparin does not alter the size of cellular components. It is, in fact, the standard for comparison of anticoagulant distortion. Heparin is more expensive and dissolves less readily than double oxalate salts. Approximately 0.5 to 1.0 mg is required to anticoagulate 5 ml of blood for 72 hours. The quantity of anticoagulant noted above in each case is sufficient to prevent clotting of the blood specimen. On the other hand, an excess of anticoagulant should be avoided because too much will result in distortion of cells and hemolysis. Ideally, differential blood smears should not be prepared from blood that contains an anticoagulant.

If oxalate is added to vials and dried in an oven, take great care to avoid temperatures above 80oC. Oxalates are converted to carbonates by prolonged exposure to elevated temperatures. Under normal circumstances, it should not be necessary to prepare your own oxalate solutions since prepared anticoagulant vacuum tubes are available from Federal medical supply sources.

Sodium citrate is the anticoagulant of choice for coagulation studies. It is used in a concentration of 1 part 0.11 M sodium citrate to 9 parts whole blood. It prevents coagulation by binding the calcium of the blood in a soluble complex.

Sodium oxalate is another anticoagulant widely used in coagulation studies. It is used in a concentration of 1 part 0.1 M sodium oxalate to 9 parts whole blood. The sodium oxalate combines with calcium in the blood to form insoluble calcium oxalate, thereby, preventing coagulation.

A correctly anticoagulated blood sample is essential to the proper performance of a blood cell count. The cellular constituents must remain free in the plasma and should be as similar as is possible to those remaining in the patient's circulation.


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