[Phlebotomy] Veterinary Supplies for Drawing Blood

There are several basics and veterinary phlebotomy supplies you will need when drawing blood on pet patients. It is wise to gather all your supplies before you enter the exam room.

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1) First you will choose the appropriate size syringe for the quantity of blood you will require for your test.

Second, you will choose the correct size needle based on the size of the sample you require and the size of the patient. For a heartworm test or FeLV/FIV test that only requires 3 drops of blood, you may just use a tuberculin syringe.

For a full blood panel including specialized tests, you may use a 6 cc syringe. For basic CBC and Chemistry, 3 cc is usually sufficient. For most pets, we use 22g needles. For large dogs when you need higher quantity samples, a 20g is often used. If drawing blood from a large dog who is donating blood, an 18g is used.

If you are drawing blood from a cat, you may choose to use a butterfly catheter when drawing from the medial saphenous vein if the cat won't cooperate for a jugular stick. 

You will need alcohol to wet and disinfect the patient's fur/skin prior to drawing your sample.

2) Know what blood tubes you will need for the test.

Here is a list of the most common tubes we use and what they contain:

Purple/Lavender-Top Tube

• Contains EDTA as the anti-coagulant. It prevents coagulation by binding to calcium ions (calcium is required for clotting).
• Cell constituents are stable for up to 24 hours in a purple-top tube.
• This is the tube used for running a complete blood count (CBC), hematocrit/packed cell volume, reticulocyte count (immature erythrocytes), or any counts of the aforementioned cells.
• Purple tops are also used for collecting fluid that may clot from body cavities in which cell counts need to be performed. This may include cerebrospinal fluid, peritoneal or pleural effusions, and synovial fluid.
• These samples are not centrifuged because there is no clot to be separated.

Speckled Red-Top Tube (Tiger Top)

• Serum separator tubes - contains a clot activator and gel for serum separation.
• Used for chemistry panels and are used to check any laboratory value seen on these panels and other tests requiring a serum sample. This is the majority of tests (BUN, creatinine, ALT, ALP, triglycerides, cholesterol, electrolytes, etc.)

Plain Red-Top Tube

• Glass tubes have no additive; plastic tubes have clot activators.
• Red-Top Tubes are used in immunology and in some specialized tests such as phenobarbital levels
• These can also be used as a sterile tube for collecting samples for culture such as urine.

Light Blue-Top Tube

• Contains Sodium citrate - citrate is an anti-coagulant that binds calcium in the blood.
• For accuracy, these tubes must be completely filled to recommended level. The appropriate ratio is 9 parts blood to 1 part citrate.
• Used to check for coagulation disorders which may include testing of Prothrombin time (PT), Partial Thromboplastin Time (PTT), Fibrin Degradation Products (FDP), D-dimer, von Willebrand Factor, and other factor and fibrinolysis assays.

Green-Top Tube

• Green tops are plasma separator tubes (PST).
• These tubes contain heparin - heparin activates antithrombins, which block coagulation.
• Produces a whole blood/plasma sample and is used for collecting a plasma sample.

Gray-Top Tube

• These tubes contain sodium fluoride, which is best known as a glucose preservative, and some contain potassium oxalate
• Used most often if an accurate glucose measurement is needed and sample processing will be delayed. A blood glucose tested from serum in a red- or tiger-top tube in a delayed sample will be falsely low due to ongoing glucose metabolism by red blood cells in the tube.

3) After collecting your samples, make sure you label all tubes with the patient's full name and date.

This is critical, especially when you have multiple patients that are having blood draws. I recommend that you remove the needle and open the tube to put the blood in.

The vacuum when putting the needle in the tube can sometimes cause lysis of the cells. If you do use the vacuum, let it flow in naturally, don't push the blood in at a high speed.

Contact us to discuss your requirements of PET Blood Collection Tube factory. Our experienced sales team can help you identify the options that best suit your needs.

All of this information is part of obtaining a patient history.

General Information

In general, hematology testing is performed on EDTA- (purple top tube) anticoagulated blood. This is the only type of anticoagulant that can be assayed with our hematology analyzer, therefore all hematology tests performed with this analyzer (routine hemograms, red and white cell counts, etc) will only be done from EDTA tubes. Heparin (green top tube) is not recommended as an anticoagulant for cell counts, because the cells clump in heparin, invalidating counts. Citrate (blue top tube) is not recommended due to the dilution of the blood by the liquid citrate. These guidelines should be followed for collecting blood for hematology tests:

• A full EDTA tube should be submitted. Partially filled EDTA tubes affect the cells because EDTA is hypertonic (e.g. echinocytes will form in underfilled EDTA tubes and red cells shrink, decreasing the mean cell volume and increasing the mean cell corpuscular hemoglobin concentration). EDTA tubes should ideally be more than half full.
• Ensure that the blood is mixed promptly with the EDTA to avoid sample clotting. This is especially pertinent with Microtainer® tubes. This should be done by rolling the tube between your palms or gentle inversion several times. Do not shake the tube!
• Microtainer® tubes should be avoided. If only a small amount of blood can be collected, e.g. from a young dog or cat, or very sick animals in which multiple, sequential samples are going to be collected, the blood should be collected into a Microtainer® tube. The tube should be full. Full Microtainer® tubes are required, because if we have too little blood, we may not be able to perform other tests that may be required, e.g. diluting the sample, checking counts etc.
• The tubes should be labeled with the patient identification and owner name at the minimum. A request form with pertinent history details should be submitted concurrently with the sample, e.g. dog administered oxyglobin.
• If there is going to be a delay between sample collection and submission, always make 2-3 blood smears from the sample and submit with the EDTA tube (see making a blood smear below).
  • Smears should be submitted unfixed, unstained, with the EDTA blood for any hemogram or test involving counts or blood smear examination. We do not charge any extra for these blood smears, and we always (provided smear quality is sufficient) do our blood smear examination from the submitted smears.
  • We request these smears because changes occur in cells when they are stored for more than a few hours. Platelets begin to clump, white cells become pyknotic and undergo nuclear swelling so that many neutrophils look like bands when they actually are not. The red cells may lyse. Red cells also consistently swell in vitro, such that old samples (usually > 24 hours) have macrocytic hypochromic red blood cells.
• Some hematology samples, e.g. packed cell volume and total protein by refractometer, can be performed on heparin or citrate anticoagulants. We can also perform cell counts on these anticoagulants, however this will only be done on specific research samples or on individual patients, after consultation with the Clinical pathologist on duty. In these cases, our automated hematology analyzer will not be used for counts; instead we will use bench methods, including an impedance-based cell counter for white and red cell counts and a hemocytometer for manual leukocyte or platelet counts. Note that for a fecal occult blood, we need feces, not blood!
• EDTA blood should be kept refrigerated until submission or mailing and should be mailed on a cold pack, but should be kept out of direct contact with the pack (insert paper towels between the blood and the icepack). Direct contact may cause freezing of red cells, with subsequent hemolysis. Furthermore, blood smears should not be refrigerated (causes cell lysis) or exposed to formalin (alters staining characteristics).

Making a blood smear

When there is going to be a delay between sample collection and submission, e.g. samples shipped to the laboratory or collected after hours, always make 2-3 peripheral blood smears. We have provided tips and an illustration for making a good blood smear below.

Tips for making a good blood smear

• Clean slides: It is imperative to use clean high-quality glass slides with clean edges. Touching the edges of the spreader slide will affect the quality of the smear.
• The size of the drop: If the drop is too large, the smear will be too long and thick. A small drop may not be fully representative of the blood.
• Speed of spreading action: The speed at which the spreader slide is moved is very important. If you move it too fast, the smear is too short and all the cells are at the feathered edge. If you go too slow, the smear is too long (lacks a feathered edge).
• Angle of the spreader slide: The angle determines the length of the smear. An angle of approximately 30-40° is optimal. If you use a larger angle (45°), the smear is very short. If you use a lower angle, the smear will be too long. Maintain this angle through the duration of the spreading action.
• Even contact: Even contact between the two slides is essential throughout the smear preparation process &#; do not add much downward pressure onto the spreader (top) slide.

Illustration on how to make a peripheral blood smear (wedge smear).

A: Use clean slides with a frosted end. Place a drop of blood on this slide as follows (we recommend the use of a microhematocrit or capillary tube rather than the pipette shown in the image). Fill a capillary tube at least 3/4 full with well-mixed blood; then hold your finger over one end to prevent it flowing out. Holding the tube horizontally over the slide, release the pressure of your finger from the end, and tilt the tube slightly toward the vertical to allow a controlled amount of blood to flow out of the tube and onto the slide. Place a drop of blood approximately 4 mm in diameter on the slide, approximately 0.5 cm from the frosted area.

B: Pick up a second clean slide and hold it by placing your first two or three fingers on one edge of the slide and your thumb on the opposite edge; the slide in your hand is the spreader slide. Do not touch the spreading edge (short non-frosted end) with your hands. Place the spreading end of the spreader slide at a 30&#;40 degree angle on the slide in front of the blood droplet. The entire short edge of the spreader slide should be in complete even contact with the lower slide. Using your other hand, pin the lower slide to the countertop to prevent it moving. In one smooth motion, draw the spreader slide back through the entire drop of blood (C).

C and D: Once the blood spreads along the edge of the spreader slide (this occurs quickly), push the blood forward along the length of the lower slide. It is very important to relax your wrist and maintain a constant smooth motion and the same angle for the spreader slide when spreading the drop of blood as well as consistently even contact (with very slight downward pressure) between the two slides.  

E: If the drop size and speed/angle of the spreader slide are correct, you will run out of blood before reaching the end of the slide, thus producing a &#;feathered edge&#; and a smear that is no more extends no more than ¾ along the length of the slide. If your smears do not look like the example shown above, look at the table below to identify the fault(s) and the cure(s).

Common blood smear faults and their cures

Clotted samples

If blood has clotted in the EDTA tube, the sample will not be analyzed. Clotting affects our automated hematology analyzer adversely and also invalidates cell counts in an unpredictable fashion. For CUHA, we make every effort to notify the clinician/technician/student that a sample has clotted so that a new sample can be drawn from that patient. Furthermore, as soon as we know the sample is clotted, the test is cancelled in the computer. For samples submitted through the Samples for Hematology, we cancel hemograms or tests involving counts if the sample is clotted. However, if a blood smear is provided with the sample, we will add on a blood smear examination, which can provide valuable information.

Non-mammalian samples

Only small amounts of blood can be collected from these species, necessitating the use of Microtainer® tubes. Similar to mammals, EDTA is the preferred anticoagulant for non-mammalian hematology. However, there are certain species of birds, e.g. cranes, and reptiles, e.g. turtles, whose blood hemolyzes on contact with EDTA. This hemolysis invalidates the PCV and affects assessment of red blood cell morphology during blood smear examination. For these species, blood can be collected directly from the needle into citrate anticoagulant. However, the correct citrate to blood ratio must be maintained, i.e. 1 part citrate to 9 parts blood. Ideally, the citrate should be placed into the syringe and the appropriate volume of blood withdrawn directly into anticoagulant. For example, to collect 1 ml blood, 0.1 ml citrate is placed into a syringe and 0.9 ml of blood is taken from the patient (collect blood up to the 1 ml mark). If less blood is collected, you will have to resample, hence make sure you can obtain the correct amount of blood. We require at least 500 µL of blood for performing a hemogram, hence you can collect only this amount of blood, which is achievable in most non-mammalian patients. The correct amount of citrate to blood must be maintained because citrate dilutes the blood; this dilution must be corrected for when evaluating the hemogram (i.e. each value should be multiplied by 1.1 for a 1:9 citrate:blood ratio). We do not make this correction in our reports. Heparin is not recommended as an anticoagulant because leukocytes and thrombocytes clump, invalidating WBC counts and differential cell counts.

Samples for Coagulation Tests

As of September 1, , the Clinical Pathology Lab will no longer be performing coagulation tests; these tests will instead be offered exclusively by the Comparative Coagulation Laboratory in the Diagnostic Lab at Cornell University. The Clinical Pathology lab will continue to offer the Fibrinogen by Heat Precipitation test.

Test Components Specimen Comments Fibrinogen by Heat Precipitation FIB EDTA tube (>1/2 full) Large animals only

This is performed on EDTA blood only (lavender top tube). Fibrinogen is quite stable, although hemolysis and lipemia will interfere with the test.

Microtainer® is a registered trademark of Becton, Dickinson and Company.

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