Blood Collection Techniques

By | 2013-07-26

In most instances, a 3- to 5-mL sample of anticoagulated whole blood is adequate for routine hematology; some laboratories will accept as little as 1 mL. For routine biochemical analyses, the volume of serum requested can vary from 1 to 2 mL, depending on the number and type of tests requested. Plan ahead which samples are required to prevent the need for further venipuncture at a later time. In small dogs and cats, using the jugular veins facilitates collection of an adequate volume of blood. If smaller samples are required, the cephalic, lateral saphenous, or medial saphenous vein can be used for sample collection. Do not use the jugular vein if a coagulopathy is suspected, as hemorrhage may be difficult to control after venipuncture.

Patient Preparation

For successful venipuncture, proper restraint of the animal is important. Details for the proper restraint for various venipuncture locations are discussed with each specific topic throughout this text. The patient must remain comfortable yet relatively motionless to avoid iatrogenic vessel laceration. Stretch the skin tightly over the selected vessel without causing vascular occlusion to help anchor the vessel in place during penetration by the needle.

Technique

The specific venipuncture will vary somewhat depending on the specific vein selected. The following sections describe venipuncture technique for each of four commonly accessed veins: the cephalic vein, jugular vein, lateral saphenous vein, and medial saphenous vein.

Cephalic Venipuncture

To restrain a dog or cat for venipuncture of the cephalic vein, place the dog or cat on the table, sitting or in sternal recumbency. If the right vein is to be tapped or catheterized, the assistant should stand on the left side of the animal and place the left arm or hand under the animal’s chin to immobilize the head and neck. The assistant should reach across the animal and grasp the leg just behind and distal to the right elbow joint. The assistant should use the thumb to occlude and rotate the cephalic vein laterally while the palm of the hand holds the elbow in an immobilized and extended position. Make sure that the animal stays on the table if struggling occurs. The person performing the venipuncture then grasps the leg at the metacarpal region and begins the venipuncture on the medial aspect of the leg, just adjacent to the cephalic vein proximal to the carpus.

Jugular Venipuncture

For a jugular venipuncture in the dog, place the patient in sternal recumbency, with the hands of the assistant placed around the patients muzzle to extend the neck and nose dorsally toward the ceiling. In short-coated dogs, the jugular vein usually can be seen coursing from the ramus of the mandible to the thoracic inlet in the jugular furrow. The vessel may be more difficult to visualize in dogs with long hair coats or if excessive subcutaneous fat or skin is present. The person performing the venipuncture should place the thumb of the nondominant hand across the jugular vein in the thoracic inlet or proximal to the thoracic inlet to occlude venous drainage from the vessel and allow it to fill. With the dominant hand, the person performing the venipuncture should insert the needle and syringe or Vacutainer (BD, Franklin Lakes, New Jersey) into the vessel at a 15- to 30-degree angle to perform the venipuncture.

For smaller and very large animals, the jugular vein also can be tapped by placing the patient in lateral recumbency. The assistant should pull the animal’s front legs caudally and extend the head and neck so that the jugular vein can be visualized. The venipuncture then can be performed as previously described. A jugular venipuncture is contraindicated in patients with thrombocytopenia or vitamin K-antagonist rodenticide intoxication.

Place cats in sternal recumbency. The assistant should stand behind the patient so that the patient cannot back away from the needle during the venipuncture. The assistant should extend the cat’s head and neck dorsally while restraining the cat’s front legs with the other hand. The cat’s fur can be clipped or moistened with isopropyl alcohol to aid in visualization of the jugular vein as it stands up in the jugular furrow. The person performing the venipuncture should occlude the vessel at the thoracic inlet and insert the needle or Vacutainer apparatus into the vessel as previously described to withdraw the blood sample. Alternately, place the cat in lateral recumbency as described in the previous paragraph.

Lateral Saphenous Venipuncture

To perform a lateral saphenous venipuncture, place the patient in lateral recumbency. The lateral saphenous vein can be visualized on the lateral portion of the stifle, just proximal to the tarsus. The assistant should extend the hindlimb and occlude the lateral saphenous vein just proximal and caudal to the tarsus. The person performing the venipuncture should grasp the distal portion of the patient’s limb with the nondominant hand and insert the needle or Vacutainer apparatus with the dominant hand to withdraw the blood sample.

Medial Saphenous Venipuncture

To perform a medial saphenous venipuncture, place the patient in lateral recumbency. Move the top hindlimb cranially or caudally to allow visualization of the medial saphenous vein on the medial aspect of the tibia and fibula. The assistant should scruff the patient, if the patient is small, or should place the forearm over the patient’s neck to prevent the patient from getting up during the procedure. With the other hand, the assistant should occlude the medial saphenous vein in the inguinal region. The person performing the medial saphenous venipuncture should grasp the paw or hock of the limb and pull the skin taut to prevent the vessel from rolling away from the needle. The fur may be clipped or moistened with isopropyl alcohol to aid in visualization of the vessel. The needle or Vacutainer apparatus can be inserted into the vessel at a 15- to 30-degree angle to withdraw the blood sample.

Special Considerations

Incorrect proportions of blood to anticoagulant may result in water shifts between plasma and red blood cells (RBCs). Such shifts may alter the packed cell volume (PCV), especially when small amounts of blood are added to tubes prepared with volumes of anticoagulant sufficient for much larger volumes of blood. Erroneous laboratory results also may be obtained when small volumes of blood are placed in a relatively large container. Evaporation of plasma water and adherence of the cells to the surface of the container can produce arti-factual changes in hematologic results.

Refrigerate liquid blood mixed with anticoagulant after collection if the sample is to be held before being transported to a laboratory. White blood cell (WBC) and RBC counts, PCV, and hemoglobin level can be measured within 24 hours of sample collection. Platelet counts, however, should be done within 1 hour of collection. Dried, unfixed blood smears can be stained with most conventional stains 24 to 48 hours after being made. If a considerable delay is anticipated between the time that the blood smear is made and the staining process, the blood smear should be fixed by immersion in absolute methanol for at least 5 minutes. Blood smears fixed by this method are stable indefinitely.

Never place unfixed blood smears in a refrigerator because condensation forming after the smear is removed from the refrigerator will ruin the blood smear and make it unusable for cytologic evaluation. Take care to leave unfixed blood smears face down on a countertop or in a closed box. Special stains, such as peroxidase, may require fresh blood films.

Routine Hematologic Testing

The anticoagulant of choice for hematologic testing is EDTA. Heparin is especially to be avoided if blood films are to be made from blood mixed with anticoagulant because contact with whole blood will distort the morphology of cells significantly. Heparin is acceptable for most procedures requiring blood plasma. The anticoagulant effect of heparin is transitory. Specimens still may clot after 2 to 3 days.

Make blood films immediately after collection because cell morphology rapidly deteriorates after sample collection. Although blood films can be made after introducing blood to EDTA, a better practice is to make blood smears (films) immediately from the collection needle before the blood comes in contact with any anticoagulant. Never use blood exposed to heparin to make blood smears.

Incorrect proportions of blood to anticoagulant may result in water shifts between plasma and RBCs. Such shifts may alter the PCV, especially when small amounts of blood are added to tubes prepared with volumes of anticoagulant sufficient for much larger volumes of blood. Erroneous laboratory results also may be obtained when small volumes of blood are placed in a relatively large container. Evaporation of plasma water and adherence of the cells to the surface of the container can produce artifactual changes in hematologic results.

Refrigerate liquid blood mixed with anticoagulant after collection if there is a delay in making the laboratory determinations. WBC and RBC counts, PCV, and hemoglobin level can be measured within 24 hours of sample collection. Platelet counts, however, should be done within 1 hour of collection. Dried, unfixed blood smears can be stained with most conventional stains 24 to 48 hours after being made. If a considerable delay is anticipated between the time that the blood smear is made and the staining process, the blood smear should be fixed by immersion in absolute methanol for at least 5 minutes. Blood smears fixed by this method are stable indefinitely. Neverplace unfixed blood smears in a refrigerator because condensation forming after the smear is removed from the refrigerator will ruin the blood smear and make it unusable for cytologic evaluation. Take care to leave unfixed blood smears face down on a countertop or in a closed box. Special stains, such as peroxidase, may require fresh blood films.

Routine Biochemistry Testing

Patient Preparation

Prepare the selected vein as described earlier.

Technique

Most clinical chemistry procedures are performed on serum. The serum is obtained by collecting blood without any anticoagulant and allowing the blood to clot in a clean, dry tube. Separate serum from cells within 45 minutes of sample collection (venipuncture). Special vacuum vials are available that produce a gel barrier between the clot and the serum (serum separator tubes) which avoid the need to draw off the serum into a separate vial. Clotting of the blood and retraction of the clot occur best and maximum yields of serum are obtained at room or body temperature. Refrigeration of the sample delays clot retraction. Some samples clot and retract faster than others.

Special Considerations

If a serum separator tube is not used, it is recommended to free the clot from the walls of the container by rimming with an applicator stick. After the clot is freed, allow clot retraction to occur, and then centrifuge and draw off the clear supernatant serum using a pipette or suction bulb. Allow whole blood samples to completely clot before attempting to remove serum. Failing to do so may result in a mixture of plasma and serum in the submitted sample. Serum yield is usually one third of the whole blood volume. Patients that are hypovolemic or dehydrated can have a significantly lower serum yield.

Many clinical chemistry procedures can be performed on plasma and on serum. The advantage of using plasma is that separation of cells can be accomplished immediately after centrifugation or sedimentation, without the need to wait for clot formation and retraction. The disadvantage of plasma is that the presence of the anticoagulant interferes with many of the chemistry assay procedures. Plasma is less clear than serum, which may be an additional disadvantage for colorimetric assays. Plasma and serum are virtually identical in chemical composition except that plasma has fibrinogen and the anticoagulant. For many procedures in which plasma or whole blood is to be used, heparin is the anticoagulant of choice. Heparinized blood is the only acceptable specimen for blood pH and blood gas analyses. Although blood containing EDTA is acceptable for certain chemical procedures, it cannot be used for determination of plasma electrolytes because it contributes to and sequesters them from the specimen. In addition, EDTA can interfere with alkaline phosphatase levels, decrease total carbon dioxide, and elevate blood nonprotein nitrogen.

Refer to the Tube Selection Guide in Section 5 to assure use the proper collection tube is used for the appropriate test requested.

Separate serum or plasma and remove it from the cells as soon as possible after blood is collected, because many of the constituents of plasma exist in higher concentrations in RBCs. With time, these substances leak into the plasma and cause falsely elevated values (positive interference) and falsely lower values (negative interference) (Table Examples of Positive and Negative Interference on Biochemistry Analytes Induced by Sample Hemolysis). Under no circumstances should whole blood be sent via the mail; serum derived from such specimens usually is hemolyzed, and results are often inaccurate. Separate serum and transfer it to a clean, dry tube for shipment.

TABLE Examples of Positive and Negative Interference on Biochemistry Analytes Induced by Sample Hemolysis

Analyte Effect of Hemolysis*
Alanine transaminase Minimal effect
Alkaline phosphatase Increased
Bilirubin Increased
Chloride Decreased
Creatinine Increased
Inorganic phosphate Increased
Lipase Decreased
pH Decreased
Potassium No detectable effect
Total calcium Increased
Total protein Increased
Urea nitrogen Increased

*Type and degree of interference vary among different testing modalities unique to individual laboratories or in-hospital biochemistry analyzers.