The sequence in which the collected sample is added to the specimen tubes is important to avoid introducing significant variables. Specific concerns include:

• Specimens collected in citrate anticoagulant (blue top tube, BTT> for coagulation testing give the most accurate results with clean venipuncture to minimize contamination with tissue juice. When using the vacuum tube collection system, the SST or RTT is collected first to flush activated clotting factors from the needle. For the direct syringe method, the BTT should be tilled first, unless a blood culture is desired (see Table). All BTT should be collected before the green, gray or lavender tops, as even trace amounts of other anticoagulants can affect the accuracy of coagulation tests.
  
• As trace amounts of EDTA (lavender top tube, LTT) clinging to a vacuum tube needle can affect the calcium, sodium, potassium and iron levels, fill the RTT or green top tubes beforehand (see Table).
  
• Trace amounts of sodium fluoride (gray top tube) will affect cell morphology evaluation of the CBC.

The presence of hemolysis of moderate or greater degree can adversely affect the blood specimen in several ways:

• It colors the blood plasma and serum which will interfere with colorimetric chemistry assays.
  
• It releases intracellular contents, such as proteins and potassium ions, into the serum thereby elevating serum potassium levels of several species and affecting other chemistry values.
  
• As some red blood cells have been damaged, a falsely depressed Packed Cell Volume (PCV) and Red Cell Count (RBC) will likely be obtained. Although the Hemoglobin value will be accurate, the calculated red blood cell indices will be affected by the depressed PCV and RBC.

1. During phlebotomy, negative pressure created by the vacuum tube or syringe collapses the lumen of the vein against the needle, thereby crushing numerous red cells. The flutter of the lumen against the needle can be stopped by reducing the negative pressure exerted during collection and by repositioning the needle with slight rotation or deeper insertion.
   
2. Applying excessive negative pressure as the blood enters the vacuum tube or syringe can create hemolysis. This occurs during a slow or difficult collection as the natural tendency is to use more negative force to enhance blood flow. More patience and "milking" the vein by alternating gentle negative pressure with a short release of all pressure usually solves the problem.
   
3. Hemolysis often occurs during the transfer of blood from a syringe into vacuum or other tubes. If a small gauge needle is used, transfer of blood to specimen tubes is slowed especially if small clots are present. Forcing the blood through a small bore needle contributes further to hemolysis. This problem can be avoided by removing the needle and top of the specimen tube, and transferring the blood directly into the open tube, recapping and aspirating a small amount of air to re-establish negative pressure.

Presence of clots and clumped platelets in anticoagulated blood is most commonly caused by slow blood draw and the resulting delay in mixing it with the appropriate anticoagulant. If the venipuncture was traumatic, tissue juices, activated clotting factors and hemolysis will quickly promote clot formation. The slight transfer delay when using a syringe for collection can also contribute to this problem. The best methods for avoiding clots are:

• Select a vein with good blood flow.
  
• Minimize the trauma of venipuncture.
  
• Collect blood directly into anticoagulated vacuum tubes (BTT, LTT).
  
• Mix the tube well by inverting several times immediately after filling.

Small volumes of blood or other fluids obtained from Avian, Exotic, and Small Mammal species are best collected and transported in Microtainer vessels. The containers are specially constructed to preserve the small specirnen properly and maximize the serum or plasma yield. They are available with all the common anticoagulants used in standard vacuum tubes, including RTT, SST, LTT, and heparin. Microtainers can be filled from a syringe draw, vessel catheterization/drip, or a capillary/skin puncture. However, Microtainers are not vacuum tubes. Please note that capillary hematocrit tubes are not recommended for collection of small specimen volumes.

Their high glass-surface to blood-volume ratio makes the specimen very difficult to recover and wastes precious volume. A green top, heparinized Microtainer is the most versatile for collecting a small volume, and is the container of choice for Avian and Exotic specimens. Most CBC parameters and chemistry analytes can be tested accurately from this type of specimen, along with a well-made glass slide. A selection of Microtainer vessels is available from ANTECH Diagnostics.