What are key things to remember when collecting a light-blue top tube for coagulation testing?

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	PRC-PALM-SPC-5.51-pro1-prs3: COAGULATION SPECIMEN COLLECTION AND TRANSPORT Specimen Collection Collect specimen in blue top (3.2% sodium citrate) tube. If multiple tests are being drawn, draw coagulation studies before additive-containing tubes such as the EDTA, heparin, or clot activator (SST) tubes. If the coagulation tube is being drawn using a winged collection device with variable tubing length so air in the tubing is not introduced into the blood collection tubes leading to under-filling, draw 1-2 mL into another blue top (3.2% sodium citrate) Vacutainer®, discard, and then collect the specimen. When using the hypodermic needle/syringe, it is important the blood is added to the appropriate volume of anticoagulant within one minute of completion of draw. Once obtained, the tube should be gently inverted three or six times end over end inversions; do not over mix as excessive mixing will affect the test result. Insufficient mixing may have a greater effect on specialized hemostasis assays. Citrate tubes must be adequately filled (to the mark noted on the tube if provided) or to not less than 90 % of this total volume. Inadequate filling of the collection tube will lead to inaccurate results. The final citrate concentration in the blood should be adjusted in patients who have hematocrit values above 0.55L/L (55%). For hematocrits below 02.0L/L (20%), there are no current data available to support a recommendation for adjusting the citrate concentration. PRC-HFH-COA-5.51-stw3: VISUAL AID OF VOLUME REQUIREMNTS FOR COAGULATION SAMPLES STANDARD WORK When drawing the specimen, avoid contaminating the sample with tissue thromboplastin as this may affect results. Venipuncture must be clean with no trauma, and the application of the tourniquet should be limited to 1 minute. Collection of the blood through lines that have been previously flushed with heparin should be avoided. If the blood must be drawn through a VAD (vascular access device), possible heparin contamination and specimen dilution should be considered.  In this case the line should be flushed with 5 mL of saline and the first 5 mL of blood or six dead space volumes of the VAD discarded. Blood should never be transferred from 1 collection tube to another in effort to provide the required fill volume. This is true even if two sodium citrate tubes are combined as this may lead to doubling up of anticoagulant citrate levels and further dilution of the plasma sample.   Transport of Whole Blood Specimens Store and transport whole blood specimens at room temperature. Avoid exposure of whole blood samples to ice or freezing temperatures. Unless otherwise directed, transport the specimen to the laboratory at room temperature within two hours of collection. PT specimens from the Medical Centers may be transported to the laboratory at room temperature for up to 24 hours after collection. APTT from the HFHS Medical Centers:If patient is not receiving heparin, specimen may be sent in the native tube at room temperature or in a common cooler for up to 12 hours after collection. If delivery to the laboratory will be delayed more than 12 hours, centrifuge specimen at 1500xg (at least 3000 rpm) for 15 minutes or at a centrifuge speed to consistently produce platelet poor plasma. Transfer platelet poor plasma to a polypropylene tube and cap. Avoid contamination of plasma with platelets from the buffy coat layer. Freeze plasma rapidly. Transport specimen to laboratory on dry ice in a suitable container. Specimens for other assays (e.g. thrombin time, protein C, Factor V and other Factors) kept at 2°- 4°C or 18 to 24°, should be centrifuged and tested within four hours from time of specimen collection. Specimens for tests other than PT/APTT that are collected at Medical centers will require special processing (please refer to the specific tests page for additional instructions as needed). Centrifuge the tube at 1500 x g (3000 RPM) for 15 minutes to separate the plasma. Transfer the platelet poor plasma to a 12x75 mm polypropylene tube. Avoid contamination of plasma with platelets from the buffy layer. Securely cap the tube. Properly label the tube and freeze rapidly at -20°C or below. Specimens can be frozen up to two weeks at -20° or for six months at -70°. Transport specimen to the laboratory on dry ice in a suitable container. DO NOT POOL SPECIMENS FOR COAGULATION. EACH TUBE SHOULD BE ALIQUOTED INTO AN INDEPENDENT TUBE PRIOR TO FREEZING. The number of tests that can be done depends on the combination of tests requested, condition of the sample(s), and potential need for reflexive testing. Specimens that are improperly filled (under filled/QNS or overfilled), clotted, hemolyzed, received after prolonged delay, collected in an improper container (wrong tube/anticoagulant), collected with the wrong sodium citrate concentration, or collected in an expired collection tube will be rejected. For technical information, contact the Special Coagulation Laboratory at (313) 916-1825.   Last Modified: Friday, March 25, 2022 11:05 AM
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So, you’re designing a project using human blood samples. Maybe you’re studying blood cells using hematology microscopy, or maybe you’re after genetic material or circulating biomarkers. For many basic scientists, the process of getting that blood out of a person and into your project can be intimidating.

Getting Started With Blood Collection Tubes

Whether you’re collecting your samples in-house or through a clinic, hospital, or pathology center, you’ll need to have a good idea of what kind of blood collection tubes suit your purposes. The first thing to check is your protocol—for example, some ELISAs will specify the types of samples you can and can’t use.

But what if your protocol doesn’t specify, or you’re adapting a method from another system, or you just want to make sure you’re storing the best type of sample for future not-yet-defined analyses? Hopefully, I can help you start to find your way around all those differently colored tubes.

A quick note about those cap colors before we begin: I’ve listed them below, and the color-coding system is generally pretty consistent, but I can’t promise the colors are the same in every company producing blood collection tubes, so always check first!

Serum Tubes

The first thing to figure out is whether you are after serum, or whether you’ll need to stop the blood from clotting.

Don’t get serum confused with plasma—while they’re both the liquid, cell-free part of the blood that can be obtained by centrifugation, the key difference is that serum is the product of blood that has been allowed to clot, while in a plasma sample, the dense cells are simply spun to the bottom.

Serum is, in simple terms, what remains in the blood after it clots: a cell-free liquid that is also depleted of coagulation factors. It can be a good, stable way of measuring the blood’s proteins, lipids, hormones, electrolytes, and so on. Many of these markers can be stored for days in the fridge, or frozen down and measured in batches later.

• Serum (clot activator) tubes (color dependent on brand: BD is commonly gold but also red, Greiner is red). These tubes have silica particles, which activate clotting. Some also have a gel to separate the serum. Those without the separating gel are potentially more useful in sensitive diagnostic testing. If you’re looking for a protein that isn’t involved in coagulation, this is a good place to start.
• Thrombin-based clot activator tubes (orange). Although the silica-coated tubes clot within about 30 minutes, the orange tubes clot within 5 minutes. They’re mainly used clinically for tests that are needed especially quickly. However, some of the serum components are a little less stable in these tubes.

Anticoagulant Tubes

This is the category to consider if you need cells or plasma (a cell-free liquid that still contains coagulation factors).

EDTA (Purple)

EDTA prevents clotting by chelating calcium, an essential component of coagulation. This is your basic hematology tube (by which I mean identifying and counting blood cells, blood typing, etc).

Plasma stored from EDTA-treated blood can also be used to measure most proteins, and genetic material can easily be stored from EDTA buffy coats (the interface between the red cells and the plasma after centrifugation, containing white cells and platelets). Note: these tubes contain either K2EDTA or K3EDTA.

Sodium Citrate (Light Blue)

For coagulation and platelet function tests. Like EDTA, citrate acts by removing calcium from the blood. Unlike EDTA, it’s reversible—so calcium can be added back to study coagulation under controlled conditions. Citrated plasma is also used to measure coagulation-relevant factors.

It’s worth noting that a citrate tube should not be the first type of tube filled after venepuncture—the first few mL of blood drawn will be a bit activated. If you only need samples collected in citrate blood collection tubes for your project, then you should collect a discard tube first.

Also, note that different concentrations of citrate are available from different companies.

CTAD (Also Light Blue)

CTAD stands for citrate, theophylline, adenosine, and dipyridamole. These aren’t very commonly used but are worth knowing about—they prevent ex vivo activation of your platelets, making them useful for some more sensitive platelet function and coagulation studies. Note that CTAD is light-sensitive, so keep these guys in the dark.

Lithium/Sodium Heparin (Green)

Similar in use to serum clot activator tubes, but suitable for tests in plasma rather than serum. Like the serum tubes, heparin tubes can also come with a separating gel. Heparin acts by inhibiting thrombin formation. Note: if your endgame is PCR, you should know that heparin is particularly known to interfere with PCR reactions.

However, whichever anticoagulant you choose, you may need to allow for it in your reaction mix.

Sodium Fluoride (Color Dependent on Brand: BD is Grey, Greiner is Black)

Sodium fluoride is an antiglycolytic agent, so these tubes are used for glucose and lactate testing. They also contain an anticoagulant (there are different types available).

Acid Citrate Dextrose—ACD (Yellow)

These ones are not common, but they are used for blood and tissue typing and DNA analysis.

Sodium Polyanethol Sulfonate—SPS (Also Yellow)

SPS stabilizes bacterial growth. Useful for microbiology.

For more specific purposes, there are more blood collection tubes out there, but, hopefully, this has given you a handle on where to start. Good luck, and welcome to the world of—let’s be honest—feeling just a little bit like a vampire.

Originally published May 4, 2015. Reviewed and updated April 2021.

Image Credit: Caroline Reddel