Erythrocytes-Part 3 Ms. Nelson, lvt

Blood collection, PCV, Total Proteins & blood films Let’s put it together!

Blood collection Determine what tests are needed Determine the needles you will need and the type of tubes you will use Preferred blood source should almost always be fresh blood, not dated. Use the appropriate needle that your patient can tolerate Choose the best size syringe to best match the amount of blood you will need to collect. If treatments have been given, they must be noted on the blood sample collection record. Some test can not be performed correctly after medication has been given.

Needle & Syringe varieties The preferred blood source is almost always venous blood. Jugular blood collection is appropriate in most species. Exotics you might have to draw from a peripheral. Use the size syringe closest to the amount of sample you need. Some large syringes could collapse the vein so if possible a vacuum system is best for larger amounts, especially in tiny guys.

Vacutainer collection Sets Butterfly Catheter Blood Draw (DOVE) 1min Butterfly catheters have a vacutainer in them. They are less likely to collapse a vein.

Blood Collection The amount of blood collected from an animal depends on the amount of tests or blood needed, as well as the size of the animal Enough blood should be taken to run the required tests three times. Canine Jugular Venipuncture (DOVE) 2mins Dorsal Pedal Blood Draw (DOVE) 1min Arterial Blood Draw (DOVE) 3mins

When collecting whole blood… Choose the correct container with the proper anticoagulant to prevent clotting Once the blood is collected, gently mix the blood with a gentle rocking motion Shaking the sample vigorously can cause hemolysis of the cells Order of draw: Tubes need to be collected in a specific order to avoid potential contamination from additives. Tubes that contain citrate are drawn first, then the plain red or gray top can be used. Then green top and purple top. Gray with additive will be last.

How do I know which tube to use? Each tube contains chemicals that prevent or delay the clotting process of blood. Depending on the test, you will choose the appropriate collection tube. Blood Collection Tubes (DOVE) 2 mins Anticoagulants are required when whole blood or plasma samples are needed (Whole blood and plasma contain clotting factors). When an anticoagulant is added to whole blood or plasma, it delays or prevents it from clotting.

Contains no anticoagulant Used for serum or clotted whole blood Red topped tube Contains no anticoagulant Used for serum or clotted whole blood Chemistries: These substances include electrolytes (such as sodium, potassium, and chloride), fats, proteins, glucose (sugar), and enzymes (needs to be ran immediately) will usually clot within 30 mins Immunology: immune mediated testing Serology: serum test (immune deficiencies) Blood will clot in this tube, there is just no additive. We use this a lot for drug screening. Since there is not additive, it won’t interfere with the results We can also put urine in these tubes

Tiger topped (Striped) Tube/Serum Separator Contains no anticoagulant. Has a yellowish ‘plug’ of clot activation gel that separates serum from plasma when spun. Used for serum samples. Clot will fall to the bottom of the tube, especially after you spin it down. We can use serum for chemistry test (BUN, CREATINE, ALT, ALP, ELECTROLYTES)

Wait, what’s the difference between plasma & serum?? Plasma is the fluid portion of whole blood, in which the cells are suspended. It is composed of approx. 90% water and 10% dissolved constituents (vitamins, hormones, enzymes, lipids, etc.) Serum is plasma from which fibrinogen is removed. During the clotting process, the soluble fibrinogen in plasma is converted to an insoluble fibrin clot matrix. When blood clots, the fluid that is squeezed out around the cellular clot is serum.

Lavender topped tube Contains the anticoagulant EDTA or Ethlenediamine tetracetic acid Used for whole blood samples or plasma samples Used for complete blood counts because it does not alter cellular morphology. HOWEVER, an excess of anticoagulant in a sample may cause cells to shrink and invalidate cell counts done on automated analyzers. These are used for hematologic studies, because it does not alter cell morphology when it is used in the proper ratio for samples to be analyzed soon after collection You need to fill the appropriate amount We can also put body fluids in here if we pull fluid from a body cavity and there is blood in it (CSF) Available in liquid or powder, but most places use liquid (liquid does cause some dilution)

Contains the anticoagulant Sodium Fluoride. Grey topped tube Contains the anticoagulant Sodium Fluoride. Best for glucose preservation Interferes with many other tests performed on serum Activated Clotting Time (DOVE) 5mins Glucose testing Can be used for activated clotting times.

Contains the anticoagulant Sodium Citrate. Blue topped tube Contains the anticoagulant Sodium Citrate. Commonly used for clotting times. Sodium Citrate interferes with Sodium assays and many common serum tests. This is used for clotting times, so if you have a patient that has been bleeding abnormally, we will to send it out to check for clotting times. Some machines you can just use the syringe. -binds to calcium Clotting Times (DOVE) 4mins

Contains the anticoagulant Heparin. Green topped tube Contains the anticoagulant Heparin. Can be used for most tests that require plasma blood samples Should never be used for differential blood film analysis, because the anticoagulant interferes with the staining of WBCs Lithium heparin -Contains a little donut inside -This will be helpful because the sample will not be diluted by any liquid -Blood chemistry test/electrolytes CAN NOT USE FOR DIFFERENTIAL BLOOD FILM ( The anticoagulant will interfere with the staining of WBC’s)

Let’s move on to Packed Cell Volume (PCV)

The PCV will tell you if the animal is anemic or dehydrated. In a CBC, we determine the number of RBC’s in several different ways. The quickest and easiest is called the microhematocrit/hematocrit, also referred to as the packed cell volume (PCV) The PCV will tell you if the animal is anemic or dehydrated. PCV increased would be dehydration PCV decreased would be anemia

Normal PCV Values Canine: 37-55% Feline: 30-45% Equine: 32-52% Bovine: 24-46%

PCV, continued Whole blood is collected in an anticoagulant (usually EDTA) tube, and then placed in a capillary tube Microhematocrit tubes should be filled to the designated line, with one end plugged with clay sealant. Red: heparin (fresh) Blue: no heparin (old sample)

Blood sample should be spun in a hematocrit centrifuge for 2-5 minutes PCV, continued… Blood sample should be spun in a hematocrit centrifuge for 2-5 minutes Lay the tube in the centrifuge with the plugged end facing the outside of the centrifuge. Making sure that a balancing tube is placed opposite or have another sample across from yours Cells are heavier than plasma, and are compacted at the end of the tube that has the clay plug. I usually do about 3 mins

Pack Cell Volume Explained Increased Pack Cell Volume = Dehydrated Ex: From Heat Exhaustion Decreased Pack Cell Volume = Anemic

Pcv, continued… Reading your PCV PCV/TS Reading (3 mins)

Normal color plasma is clear or a pale straw/yellow color Plasma Evaluation Plasma color and transparency may be helpful in determining a diagnosis and should be recorded in your findings Normal color plasma is clear or a pale straw/yellow color Cloudy serum = lipemic Reddish tinge = hemolyzed Yellow = icteric (possible liver disease) Hemolyzed Blood and Hematuria (1 min)

Concentration of total protein/total solids Plasma protein concentrations estimated by refractometry is an important component of the CBC in all species Plasma used to determine the TP/TS is collected by breaking the hematocrit tube just above the buffy coat/plasma interface.

The plasma is allowed to flow onto the refractometer The plasma is allowed to flow onto the refractometer. (blow gently through the open end of the hematocrit tube with the broken end of the tube over the prism of your refractometer) Hold the refractometer up to the light and record your findings Make sure to wipe your refractometer after each use.

Blood films Blood films are used to perform the differential WBC count, estimate platelet numbers, and evaluate the morphological features of WBCs, RBCs and platelets Wedge smears are prepared by placing a small drop of blood on a clean glass microscope slide.

Blood Film Blood Smear Preparation (1 min) https://www.youtube.com/watch?v=rqXy45sRJkw (45 seconds)

Always stain using the lightest to darkest stain Staining a slide Always stain using the lightest to darkest stain Remember which side of your slide is the top Rinse off from back side of slide May heat fix to speed up process Staining a Blood Smear (2 mins)

We Focus on the monolayer..Do not venture anywhere else

Performing the differential count This is where the different white blood cells are tallied separately. This can be done by a blood counting machine, or by hand To manually count the different cells, first you must make a perfect slide and stain Using a cell counter, you will tally a total of 100 cells This will make it easy to turn the numbers into a percent Blood Smear Differential and Morphology (8 mins)

Time to go To the lab!