Tube Sequence
The sequence in which the collected sample is added to the specimen tubes is important to avoid introducing significant testing 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 the activated clotting factors from the needle. For the direct syringe method, the BTT must be filled 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, unless using a syringe (see Table).
  
• Trace amounts of sodium fluoride (gray top tube) will affect cell morphology, so fill gray top tube after LTT.

The most desirable sequence depends on whether you are using a vacuum tube or needle with syringe for the phlebotomy or are collecting capillary blood from a skin or other puncture site. See Table for a practical guideline.

Chemistry Profiles
The preferred specimen for blood chemistry testing is a spun green top tube (GRT) containing a separator gel. This tube is often referred to as a plasma separator tube. The tube needs to be filled to the top line (0.6 mL) to ensure adequate sample volume, inverted 6–8 times to ensure proper mixing of blood and heparin anticoagulant, and centrifuged immediately.

Hemolysis
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 (depending on the species), 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.

Common causes of hemolysis during blood drawing can be minimized with the following suggestions:

1. Procure a nonlipemic (fasted) sample, as lipemia can increase red cell fragility.
   
2. During phelbotomy, negative pressure created by the vacuum tube or syringe may collapse 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.
   
3. Excessive negative pressure exerted 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.
   
4. 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 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 the tube and aspirating a small amount of air to re-establish negative pressure helps to avoid caps coming off in transit.

Clots and Platelet Clumps in Anticoagulant Samples
Presence of clots and clumped platelets in anticoagulated blood is most commonly caused by a 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 – larger the better.
  
• 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.

If the syringe method is selected and a difficult draw is anticipated, the chance for clotting can be minimized by first rinsing the needle and syringe with a small quantity of liquid citrate (BTT), heparin (green top), or EDTA (LTT). However, the anticoagulant must be emptied from the syringe before proceeding, and care must be taken to match the anticoagulant chosen with the tests to be performed. Even trace amounts of heparin or EDTA will invalidate coagulation testing, whereas EDTA or citrate will alter the accuracy of several chemistry assays. A small amount of heparin contamination is acceptable for most chemistry assays and CBC parameters. Platelet clumping in samples from cats is very common and is caused by contact aggregation. An effective method to prevent this clumping has not been found. Making a fresh blood smear slide right after collection, and submitting it with the CBC sample, provides the laboratory with another method of assessing platelet numbers.

Small Specimen Volumes
Small volumes of blood or other fluids obtained from Avian, Exotic, and Small Mammal species are best collected and transported in Microtainer tubes. The containers are specially constructed to preserve the small specimen properly and maximize the serum or plasma yield. They are available with all of the common anticoagulants used in standard vacuum tubes, including RTT, SST, LTT, and heparin. Microtainers can be filled from a syringe draw (preferred), 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. Detailed information regarding sample preparation and requirements can be found in our Client Services Directory, Blood Sampling and Quick Test Guide poster (contact your sales representative to obtain the poster), February 2000 Antech Newsletter, and on the website listed below. A selection of Microtainer vessels is available from Antech Diagnostics.

Material Safety Data Sheets (MSDS)
Material Safety Data Sheets are available for all applicable Antech client supplies. Requests for this information can be made as "MSDS" with your next supply order.