Collection, Processing, Storage and Shipment

Table of Contents

1. Anticoagulants and Preservatives
2. Blood Collection Systems
3. Blood Product Overview
4. Blood Collection Systems and Pre-Processing Guidelines
5. Preparation of Fresh Whole Blood
6. Preparation of Red Cells and Fresh Frozen Plasma
7. Preparation of Cryoprecipitated Anti-Hemophilic Factor and Cryo-Poor Plasm
8. Preparation of Platelet Rich Plasma
9. Short Draw and Decreasing the Amount of Anticoagulant Preservative
10. Purchasing and Receiving Blood Products from Outside Sources
11. Preparing Blood Products for Shipment

Helpful Hints:

Scattered throughout this text are the following symbols to help you focus on what is really important.

♣  This is a routine feature of the subject being discussed. We've tried to narrow them down.

♠  This is an important feature. You should remember this.

♥  Something serious will happen if you do not remember this.

1. Anticoagulants and Preservatives

♣  The following questions should be answered prior to blood collection and will assist in selecting the proper anticoagulant-preservative and blood collection system best suited to the product need.

• What blood product is needed?
• Will the product be used for immediate transfusion?
• Will components be made?
• Will the blood be stored?

Anticoagulant-Preservatives

♣  Obviously, an anticoagulant should prevent coagulation of the unit of blood, thereby maintaining the unit in a liquid, transfusible state. However, the anticoagulant should also ensure that the blood product maintains its integrity so that the product will provide optimal benefit to the patient. Modern blood collection devices utilize a liquid solution that contains both anticoagulant and preservatives. The anticoagulant is citrate; the preservatives are phosphate-dextrose solutions. These solutions enhance preservation of the red cells and prevent detrimental changes to the product by maintaining pH and promoting adenosine triphosphate (ATP) production in order to maintain red cell viability. Citrate-phosphate-dextrose (CPD) and citrate-phosphate-double-dextrose (CP2D) contain phosphate and dextrose. Citrate-phosphate-dextrose-adenine (CPDA1) has the addition of adenine to support red cell survival (Table 2-1).

♣  This combination of anticoagulant and preservatives provide an environment safe to store blood products: red cell products should be stored between 1 - 6 °C, plasma products should be stored at -18 °C or lower. Platelets should be stored between 22 - 25 °C.

♣  Anticoagulant-preservative solutions do not inhibit the growth of microbial contaminants. Refrigeration of red cells and freezing plasma products assists in inhibiting microbial growth within the blood product.

Additives

♣  Like preservatives, additives are a combination of chemicals used to extend the life of the red cell. There are three commercially available additive solutions: Adsol® (AS-1, Fenwal), Nutricel® (AS-3, Haemonetics) and Optisol® (AS-5, Terumo). The constituents of these additives vary by manufacturer, but all contain dextrose, adenine, and sodium chloride. Other constituents that may be included are sodium phosphate, mannitol, sodium citrate, and citric acid.

♣  Additives are used in addition to anticoagulant-preservatives. The additive serves to increase red cell survival for a period of time longer than an anticoagulant-preservative alone. The additive allows for removal of maximal amounts of plasma from the unit of red cells. Since the additive solution is directly added to the red cells, the hematocrit of the unit of red cells is decreased, thereby creating a less viscous, more transfusible unit of blood. The additive must be added to the red blood cells within 72 hours of collection.

Shelf Life

♣  Remember that a unit of blood is a biosystem and death is innate. The stored or "banked" product has a specific "shelf life." In general, shelf life is based on functional considerations in regard to blood components.

♠  The shelf life of a blood product is the maximum allowable storage time.

♣  The length of storage time is also affected by the nature of the blood collection device used: the device is "closed" or "open."

♣  A closed collection system does not allow its contents to be exposed to air or outside elements during collection, processing or storage.

♣  Closed systems have integrally attached needles and satellite bags. The manufacturer places anticoagulant-preservative and additives in the system; these systems are available commercially. Use of a closed system promotes longer shelf life, which is dependent on the type of anticoagulant-preservative-additive used.

♣  An open system does expose its contents to air or outside elements at some point during collection, processing or storage. Use of an open system mandates the blood product be used within 4 hours if the product is stored between 22 - 25 °C or 24 hours if stored at 1 - 6 °C. Open systems include syringes and bags or bottles without integrally attached collection needles or satellite bags.

2. Blood Collection Systems

Canine Blood Collection Systems

Open Systems

♣  As previously stated, open systems used in blood collection allows for blood to be exposed to air or outside elements at some point during collection, processing, or storage. This mandates that the product be used within 4 hours if stored at room temperature or 24 hours if stored at 1 - 6 °C. Although many anticoagulants are available, some are better suited for blood collection and transfusion than others. Heparin and sodium citrate are two anticoagulants frequently used in open systems.

♣  Heparin acts as an anticoagulant by accelerating the action of antithrombin III, neutralizing thrombin, and preventing the formation of fibrin from fibrinogen. Heparin also deactivates platelets, making its use prohibitive for collection of blood for treatment of coagulation disorders. Heparin does not provide nutrients to facilitate red cell survival.

♣  Sodium citrate acts as an anticoagulant by chelating calcium. When used alone, sodium citrate is not suitable for collection of blood for transfusion due to its pH. Citrate used in conjunction with other chemicals is the anticoagulant of choice for collection of blood for transfusion because it has low toxicity and is easily metabolized. Glass bottles containing citrate dextrose (anticoagulant-citrate dextrose, ACD) have been used in the veterinary field for many years. Since glass bottles are an open system, this product should be used within 24 hours of collection.

♥  Glass inactivates platelets, coagulation factor XII and factor VIII. Use of other blood collection systems may provide a superior blood product.

Closed Systems

♣  A closed blood collection system does not allow blood to be exposed to air or outside elements during collection, processing or storage. Closed systems have integrally attached needles and satellite bags. In human blood collection systems (used for dogs), the manufacturer places anticoagulant-preservative and additives in the system (listed in Appendix 1). Anticoagulant must be added to the feline blood collection system (listed in Appendix 1).

♣  Anticoagulant-preservatives include ACD, CPD, CPDA-1, and CP2D. CPD and CPDA-1 are the most widely used in closed systems.

♣  ACD, CPD, or CP2D used in a closed system allows red cell shelf life of 21 days when stored at 1 - 6 degrees C, CPDA-1allows red cell shelf life for up to 35 days when stored at 1 - 6 degrees C.

♠  Remember that blood bags purchased commercially have an expiration date independent of product collection. This expiration date guarantees anticoagulant-preservative activity and bag sterility.

Additives

♣  Adsol® (AS-1, Fenwal), Nutricel® (AS-3, Haemonetics), and Optisol® (AS-5, Terumo) are three commercially available additives. These additives allow red cell shelf life of 42 days when stored at 1 - 6 °C for 42 days. In human blood collection systems, these additives are used in conjunction with the anticoagulant CPD.

Feline Blood Collection Systems

Open Systems

♣  Remember that the volume of blood drawn from a feline donor is considerably less than that of its canine counterpart. A typical feline donation is approximately 50 milliliters, making 60 cc syringes a popular blood collection device for cats. Since this is an open system, blood stored at 1 - 6 °C should be used within 24 hours of collection.

♣  The anticoagulant of choice is citrate phosphate dextrose adenine. The amount of blood taken into any citrate based anticoagulant is critical. Too little blood with too much free citrate is contraindicated for use in cats. Citrate not consumed in anticoagulation is a notable chelator of patient calcium, so much so that severe and often delayed hypocalcemia can occur. CPDA anticoagulant is far superior than any other commonly used anticoagulant as red cell viability is maintained and plasma proteins remain functional. This anticoagulant may be purchased from a commercial veterinary blood bank in multiuse vials.

Closed Systems

♣  Blood collection systems used for canine blood collection are not suitable for collection of feline blood since the volume of anticoagulant in the blood collection bag used for canine is intended for a 450-ml blood draw. Although the amount of anticoagulant may be reduced in order to perform a smaller volume blood draw, the integrally attached 16-gage collection needle is too large for the feline jugular vein used to collect blood. For this reason, blood bags used for the collection of canine blood are not used with cats.

♣  A specialized blood collection system for cats (Figure 2-1) is available through Animal Blood Bank (Appendix 1).

Figure 2-1. Small animal double syringe collection set (Photo used with permission of Animal Blood Bank).

3. Blood Product Overview

Blood Products

♣  While the properties of blood products are similar in dogs and cats, this discussion is limited to closed systems and 450 ml blood draws.

♠  Remember - blood products collected in open systems should be transfused within 24 hours of collection. Closed systems that have been opened should be used within 24 hours of opening.

♣  The following information is summarized in Table 2-2.

Whole Blood and Red Cell Products

Fresh Whole Blood

♣  Fresh whole blood provides blood volume expansion and increased oxygen carrying capacity to the recipient. It also delivers viable platelets and coagulation factors. It is often used for actively bleeding patients with acute blood volume loss of greater than 25%.

♣  A unit of whole blood is called fresh whole blood for a time period of 24 hours after phlebotomy.

♣  Fresh whole blood contains all blood elements: red cells, platelets, clotting factors and plasma proteins.

♣  Fresh whole blood is stored at 1 - 6 °C.

♣  ACD, CPD, CP2D, and CPDA-1 are suitable anticoagulant-preservatives. The use of heparin is discouraged.

♣  Red cell additives are NOT mixed with whole blood.

♣  Due to its limited vitality, fresh whole blood is generally not available unless it is drawn immediately prior to its need. Since obtaining a blood donor on an emergency basis may not be practical, it may not be feasible to transfuse fresh whole blood. Instead, fresh whole blood is generally used for component preparation.

Whole Blood and Stored Whole Blood

♣  Whole blood provides for volume expansion, increased oxygen carrying capacity, protein source, and stable coagulation factors.

♣  Once fresh whole blood is stored at 1 - 6 °C for a period of time longer than 24 hours, it is called whole blood.

♣  Whole blood contains red cells and plasma proteins, but platelets and coagulation factors are diminished. Stable coagulation factors II, VII, IX, X, and fibrinogen are preserved.

♣  The anticoagulant-preservatives ACD, CPD, and CP2D maintain this product for 21 days. The use of CPDA-1 maintains whole blood for 35 days.

♣  Red cell additives are NOT mixed with whole blood.

♣  Storage of whole blood is an option for extending the use of a unit of fresh whole blood once the 24 hour shelf life is exceeded.

Short draw

♣  Most commercially available blood bags are intended for a 450 ml + 45 ml blood volume. These bags contain 63 mls of anticoagulant-preservative for a final blood to anticoagulant ratio of 1:10.

♣  If 300 - 404 mls of blood is drawn into a bag intended for a 450 ml blood draw, the unit should be maintained as whole blood; components should not be made from this unit. The unit may be used for transfusion, but it should be labeled as a low volume unit. Underfilled units may cause citrate intoxication when transfused.

♣  If a blood collection of less than 300 mls of blood is needed, the amount of anticoagulant-preservative may be aseptically reduced prior to blood collection.

Red Blood Cells or Packed Cells

♣  Red blood cells assist in restoring oxygen carrying capacity to the recipient with less expansion of blood volume in comparison to whole blood. Red blood cells are used to treat anemia in normovolemic patients or pharmacologically untreatable anemia.

♣  Red blood cells are prepared from a unit of fresh whole blood or whole blood. To prepare this product, plasma is extracted from the unit of whole blood and the cells remaining in the bag are called red blood cells. This process may be expedited by centrifugation, or the red cells may be allowed to settle to the bottom of an undisturbed unit of whole blood. ACD, CPD, and CP2D provide a 21 day shelf life at 1 - 6 °C. CPDA-1 provides for 35 day shelf life.

♣  Red cell additives will extend the life of a unit of red cells to 42 days at 1 - 6 °C. Red blood cell additives must be mixed with red cells within 72 hours of phlebotomy.

Plasma Products

Fresh Frozen Plasma

♣  Fresh frozen plasma (FFP) contains all the coagulation factors, including the liable Factors V and VIII. It is a source of plasma proteins and volume expander. Fresh frozen plasma is used for treatment of patients with inadequate clotting factors for any reason. Fresh frozen plasma used in conjunction with red blood cells provide most of the benefits of fresh whole blood.

♣  Fresh frozen plasma is prepared from a unit of fresh whole blood. The plasma is separated from the red cells by centrifugation and removed. It is then completely frozen at -18 °C or lower. Complete freezing of the plasma must occur within 8 hours of phlebotomy if the anticoagulant-preservative is CPD, CP2D, or CPDA-1. If ACD is used, separation and freezing must occur within 6 hours of phlebotomy. Once frozen, the product has a shelf life of 1 year from its original phlebotomy date.

Plasma

♣  Plasma is used to treat stable clotting factor deficiencies. This product contains vitamin K dependent factors, albumin and immunoglobulins. It may be used as a volume expander, in warfarin/coumarin toxicity and canine parvovirus.

♣  This product is derived from extending the shelf life of a unit of fresh frozen plasma. When a unit of fresh frozen plasma is stored at -18 °C in excess of one year, it is relabeled as Plasma. This is done to reflect the loss of clotting factors that occurs during the one-year storage.

♣  Plasma has a shelf life of 5 years from the original phlebotomy date. Plasma not separated from red blood cells within 6 - 8 hours after phlebotomy is labeled Plasma (not FFP.)

Cryoprecipated Antihemophilic Factor

♣  Cryoprecipitated Antihemophilic Factor (AHF, also known as Cryoprecipitate or Cryo) is a rich source of VWF, Factor VIII, fibrinogen Factor XIII, and fibronectin. It is useful in the treatment of von Willebrand’s Disease, Hemophilia A, hypofibrinogenenemia, disseminated intravascular coagulation, and septicemia.

♣  Cryoprecipitate is made from a unit of fresh frozen plasma. FFP contains high molecular weight plasma proteins which precipitate in the cold. When FFP is thawed at 1 - 6 °C, the resulting precipitate is Cryoprecipitated AHF. After approximately 90% of the cryo poor plasma is removed, Cryoprecipitated AHF is refrozen and maintained at -18 °C or lower. Its shelf life is for one year from the phlebotomy date.

Cryoprecipitate Poor Plasma

♣  Cryoprecipitate Poor Plasma (Cryo-Poor Plasma) contains very low amounts of fibrinogen, fibronectin, Factors XI, XIII, VIII, and von Willebrand factor. It contains no Factor V. Cryo-poor plasma contains the vitamin K dependent factors, albumin and globulins. It is suitable treatment of canine parvovirus or coumarin/warfrin toxicity.

♣  Cryo-poor plasma is the residual plasma left from making cryo. When maintained at -18 °C or lower, it has a shelf life of one year from the original phlebotomy date.

Platelet Rich Plasma and Platelet Concentrates

♣  Platelets stop hemorrhage, as they are the first cellular element of the peripheral blood to react when blood vessels are damaged.

♣  Platelet Rich Plasma (PRP) is prepared by differential centrifugation from fresh whole blood that has been maintained between 22 - 25 °C.

♣  In order to provide viable platelets to the recipient, platelet rich plasma should be transfused as soon as it is prepared.

♣  Platelet Concentrates are prepared by centrifugation of Platelet Rich Plasma. Excess plasma is removed and the remaining platelets are viable for at least 5 days from the phlebotomy date when stored between 22 - 25 °C. In order to prevent platelet aggregation and to provide adequate oxygen and carbon dioxide exchange, platelet concentrates should be stored with gentle, continuous agitation.

4. Blood Collection Systems and Pre-Processing Guidelines

About Blood Collection Systems

♣  Blood Collection Systems are available in a variety of configurations. They are composed of the primary bag and satellite bags (Figure 2-2).

Figure 2-2. Blood collection system (Photo used with permission of Baxter Healthcare Corporation.)

♣  The optimum blood draw volume is dependent on the Blood Collection System used. The draw volume is determined by the size of the bag and the amount of anticoagulant contained in the primary bag.

♣  The needle is attached to the primary bag. The primary bag contains anticoagulant; blood will flow directly into the primary bag during phlebotomy. Other smaller bags, which are integrally attached to the primary bag through sealed ports, are the satellite bags. There may be one or more satellite bags depending on the configuration of the blood collection system selected.

♣  Satellite bags intended for storage of red cells, plasma or platelets are labeled as such by the manufacturer.

Pre-processing Guidelines

♠  All blood products should be labeled with the phlebotomy date, product name and expiration date. Additional information such as the blood type and donor may also be helpful. Permanent markers should be used for labeling so that this information is not washed off during storage, thawing or warming of the product.

♣  Before processing begins, the needle should be removed from the blood collection system. This is accomplished by heat seal (Hematron Dielectric Sealer, Baxter Healthcare, Fenwal Division) or metal clips (Hand Sealer, Hand Sealer Aluminum Clips, Baxter Healthcare, Fenwal Division.)

♣  Once the needle is removed, the phlebotomy line should be cleared using a tube stripper (Donor Tube Stripper, Baxter Healthcare, Fenwal Division). This will assure that the line is properly anticoagulated (Figure 2-3). This is important because this line will now be separated into segments (Figure 2-4) that will be used as the donor blood sample for compatibility testing.

♣  Each blood bag has a set of identification numbers repeated the length of the blood collection line. Place the first seal after the first identification number that is located at the top of the blood bag and repeat sealing segments for the length of the phlebotomy line. Fold segments end to end and rubber band them together. This will prevent the segments from getting tangled in the centrifuge head should the unit be processed for components (Figure 2-5).

♣  If segments are inadvertently separated from the blood bag during storage, the identification number on the segments can be compared to the identification number on the blood bag.

Figure 2-3. Clearing the phlebotomy line.

Figure 2-4. Sealing the segments.

Figure 2-5. Segments, secured.

5. Preparation of Fresh Whole Blood

Canine

♣  Once a unit of whole blood has been collected, it should be stored at 1 - 6 °C until processing is possible unless it will be used to produce platelet preparations. A unit of whole blood is considered Fresh Whole Blood for a time period of 24 hours after phlebotomy. Fresh Whole Blood contains all blood elements.

♣  If absolutely no components will be processed from the unit of Fresh Whole Blood, the satellite bags may be sealed from the primary bag, detached and discarded.

♣  This procedure is repeated in a step-by-step format in the Methods Section.

Feline

When a syringe or bag of feline whole blood is collected in an open system, it should be transfused as soon as possible. If significant delays are imminent, store the product at 1 - 6 °C until transfusion is possible.

6. Preparation of Red Cells and Fresh Frozen Plasma

♣  When the blood collection process is complete, the unit of whole blood should be stored at 1 - 6 °C until component preparation is possible.

♣  This discussion is limited to 450 ml blood collection for dogs. For preparation of feline blood components, Animal Blood Bank (listed in Appendix 1) provides blood component preparation techniques with the purchase of the "Small Animal Double Syringe Collection Set."

♣  If the component Fresh Frozen Plasma is to be made, the plasma must be separated from the red cells and completely frozen within 8 hours of collection if the anticoagulant-preservative is CPD, CP2D, or CPDA-1. If the anticoagulant-preservative is ACD, separation and freezing must occur within 6 hours of phlebotomy.

♣  Whole blood designated for platelet preparations should remain at room temperature until the platelets are removed.

♣  This procedure is repeated in a step-by-step format in the Methods Section.

Preparation for Centrifugation

♣  The entire unit of blood should be weighed. This weight is used exclusively for balancing the centrifuge.

♣  Proper centrifuge balance is important to prevent wear of the centrifuge rotor; total weight in opposing cups should be equal (Figure 2-6).

♣  An empty blood bag may be filled with 10% glycerin in order to provide an equally weighted balance bag for centrifugation. Rubber bands and weighted plastic discs may also be used to achieve balance (Figure 2-7).

Figure 2-6. A balanced centrifuge. (Photo used with permission of Kendro Laboratory Products.)

Figure 2-7. Balancing devices.

Centrifugation

♣  Blood bags should be placed in the centrifuge bucket with the label facing out. This reduces the centrifugal force on sealed margins of the blood bag. Centrifuges with swinging cups provide for easier separation of plasma from the red cells (Figure 2-6).

♣  The unit of whole blood should be centrifuged using a "heavy spin" in a refrigerated centrifuge between 1 and 6 degrees centigrade. A heavy spin is defined as 5000 g for 5 minutes. (See "Centrifuge Calibration" in the Methods Section for further information regarding centrifuge speed and time.)

♣  Once centrifugation has ceased, it is important to allow the centrifuge to stop spinning without assistance, as brake use or an acute stop of the rotor will disturb the red cell/plasma separation, thus contaminating the plasma with red cells.

Figure 2-8. Extracting plasma from centrifuged whole blood. (Image used with permission of Acculab and Baxter Healthcare Corporation.)

Component Separation

♣  The unit of blood should be removed from the centrifuge without agitation so as not to disturb red cells and plasma. The unit should be placed on a plasma extractor (Fenwal; Plasma Separation Stand, Terumo®) This piece of equipment provides a rigid stand in which to place units of whole blood. A hinged plate is attached to the stand and may be released to apply pressure to the unit of whole blood in order to express the plasma into a satellite bag (see Figure 2-8).

♠  For this discussion, there are two satellite bags: one contains Adsol®, one is empty. However, the number of integrally attached satellite bags depends on the blood collection system being used.

♣  The plasma will be expressed into the empty satellite bag. The empty satellite bag should be placed on a balance. The weight should be tared to zero.

♣  Remove 230 - 256 grams of plasma.

About this calculation:

The specific gravity of plasma is 1.023, so 1 gram of plasma is approximately equal to 1 milliliter of plasma. Removing 230 - 256 grams of plasma will leave the unit of red cells with a final packed cell volume of 70 - 80%.

Note: This guideline should be followed using blood collection systems in which red cells will be supplemented with Adsol®. However, red cells may be prepared without Adsol® of varying packed cell volumes. See Table 2-3 for guidelines.

♣  Using hemostats, clamp off the line of the bag containing the harvested plasma. Then, break the seal from the Adsol® bag and let the Adsol® flow into the red cells. Seal the bag containing the red cells and Adsol® and remove the red cell bag from the plasma bags. Gently mix the red cells and Adsol®.

♣  Two satellite bags remain; one contains 230 - 256 grams of plasma with a volume of 225 - 250 mls. The plasma may be left in one bag, or divided equally between the two bags. Seal plasma bag(s) appropriately.

♠  The choice to divide the plasma should be made based on typical recipient size and plasma demand. Blood collection systems may be purchased with one to four satellite bags and should be selected accordingly.

♣  Tare the weight of the balance to zero and weigh each of the filled blood bags. The weight of the empty bag should be subtracted from the final weight of the bag. The specific gravity of red cells is 1.080 - 1.090; the specific gravity of plasma is 1.023. By dividing the final weight of the product by the appropriate specific gravity, the volume of product in milliliters can be calculated.

♣  The final product should be labeled with the product name and volume in milliliters. If Adsol® has been added to the red cells, this should be noted on the bag.

Storage

♣  Red cells should be refrigerated at 1 - 6 °C. While a refrigerator dedicated to blood storage is ideal, there are a variety of refrigerators available in a wide price range that maintain adequate temperature. The blood product should be stored in an organized fashion. It is advantageous to place the shortest date (the unit which expires first) at the front of the refrigerator so that it will be used first. Other products or supplies that may be stored in the same refrigerator should be segregated from the units of red cells (Figure 2-9).

Figure 2-9. Storage of red cells.

♣  Plasma products should be stored at -18 °C or below. A freezer dedicated to plasma storage is optimal. Freezers used to store plasma products should not have a defrost cycle, since the defrost cycle may temporarily increase the temperature in the freezer and may cause inadvertent product thawing. Inadvertent product thawing may also occur during power outages.

♣  To monitor stored frozen plasma for inadvertent thawing, one of the two following techniques may be utilized (Figure 2-10).

♣  A rubber band may be placed around the middle of a unit of plasma before it is frozen. Once the product freezes, the rubber band is removed, forming a "waist" indentation on the unit of plasma. If the unit thaws, the waist disappears. Thus, a unit of plasma removed from the freezer without the waist indention has likely been warmed above freezing during storage.

♣  Another technique that may be used involves initially freezing the unit of plasma inverted. This creates a "bubble" in the bottom of the plasma bag. Once frozen, the bag should be placed upright. Thus, if a unit of plasma is removed from the freezer without the bubble in the bottom of the bag, it has likely been warmed above freezing during storage.

♣  Additionally, an external temperature monitor on the plasma storage freezer(s) is helpful in monitoring freezer temperature. These monitors are available with electronic or chart readouts.

♣  Plastic blood storage bags may break if mishandled at temperatures below zero. To protect the frozen plasma, wrap the product in plastic bubble wrap prior to freezing. Frozen plasma products should be handled with care!

Figure 2-10. Monitoring frozen product storage: The product on the right has a frozen "waist" indentation, while the product on the left contains dead space frozen at the bottom of the bag. The product in the center represents a product that has thawed and refrozen.

Product Records

♣  A product log is helpful in tracking product use. Donor identification number, donation date, expiration date, volume of product, and final disposition of unit may be useful information to maintain.

7. Preparation of Cryoprecipitated Anti-Hemophilic Factor and Cryo-Poor Plasma

♣  Cryoprecipitated Anti-Hemophilic Factor and Cryo-Poor Plasma are made from one unit of Fresh Frozen Plasma (FFP).

♣  To process Cryoprecipitated Anti-Hemophilic Factor from Fresh Frozen Plasma, a full unit (225 - 250 mL) of Fresh Frozen Plasma with at least one integrally attached satellite bag is needed.

♣  Fresh Frozen Plasma should be processed as outlined in the above procedure except:

♣  The entire unit (225 - 250 mL) of FFP should remain in one satellite bag.

It is important to document the total volume of the unit of FFP as this value will be used in a subsequent calculation.

♣  The line between the two satellite bags should be temporarily occluded in order to prevent transfer of plasma in to the second satellite bag.

♣  The FFP should be frozen solid before preparing Cryoprecipitated Anti-Hemophilic Factor.

♣  Allow the unit of Fresh Frozen Plasma to thaw at 1 - 6 °C. This process takes approximately 8 hours. When the plasma has a slushy consistency, harvest the Cryoprecipitated Anti-Hemophilic Factor using one of the two following methods.

1. Place the thawing plasma in a plasma extractor. Express the liquid plasma into the empty integrally attached satellite bag. The newly filled satellite bag should contain 90% of the original volume of FFP. Seal both bags.
2. Centrifuge the plasma using a heavy spin. The Cryoprecipitated Anti-Hemophilic Factor will precipitate and adhere to the sides of the bag (Figure 2-11). Express 90% of the supernatant into the empty satellite bag. Seal both bags.

♣  The newly filled satellite bag containing 90% of the FFP is now Cryo-Poor Plasma. The satellite bag containing the residual 10% of the FFP is Cryoprecipitated Anti-Hemophilic Factor

♣  Freeze the Cryoprecipitated Anti-Hemophilic Factor and Cryo Poor Plasma within 1 hour of completion of preparation. Both products should be stored at -18 °C or lower.

♣  Product expiration is one year from the date of phlebotomy (not the date of preparation.)

♣  The product should be labeled with product name, total volume and expiration date.

♣  This procedure is repeated in a step-by-step format in the Methods Section.

Figure 2-11. Cryoprecipitate.

8. Preparation of Platelet Rich Plasma

♣  Platelet Rich Plasma (PRP) is made from one unit of Fresh Whole Blood (450 ml draw).

♣  To prepare Platelet Rich Plasma, a unit of Fresh Whole Blood with at least one integrally attached satellite bag is needed. The unit of Fresh Whole Blood should be maintained at 22 - 25 °C and processed immediately in order to harvest viable platelets.

Preparation for Centrifugation

♣  The entire unit of blood should be weighed. This weight is used exclusively for balancing the centrifuge.

♣  Proper centrifuge balance is important to prevent wear of the centrifuge rotor; total weight in opposing cups should be equal (see Figure 2-6).

♣  An empty blood bag may be filled with 10% glycerin in order to provide an equally weighted balance bag for centrifugation. Rubber bands and weighted plastic discs may also be used to achieve balance (see Figure 2-7).

♣  The unit of whole blood should be centrifuged using a "light spin" in a refrigerated centrifuge between 22 - 25 °C.

A light spin is defined as 2000 g for 3 minutes. (See "Centrifuge Calibration" in the Methods Section for further information regarding centrifuge speed and time.)

♣  Once centrifugation has ceased, it is important to allow the centrifuge to stop spinning without assistance, as brake use or an acute stop of the rotor will disturb the red cell/plasma separation, thus contaminating the plasma with red cells.

Component Separation

♣  The unit of blood should be removed from the centrifuge without agitation so as not to disturb red cells and plasma. The unit should be placed on a plasma extractor (Fenwal; Plasma Separation Stand, Terumo®) This piece of equipment provides a rigid stand in which to place units of whole blood. A hinged plate is attached to the stand and may be released to apply pressure to the unit of whole blood in order to express the plasma into a satellite bag (see Figure 2-8).

♣  The plasma will be expressed into the empty satellite bag. The empty satellite bag should be placed on a balance. The weight should be tared to zero.

♣  Remove plasma.

♥  The task of extracting platelets from centrifuged whole blood can be challenging, as red cells lie just below the platelet layer (Figure 2-12). Platelet Rich Plasma should be light yellow in color and should not contain visible red cell contamination.

Figure 2-12. Centrifuged fresh whole blood with platelet layer.

♣  Using hemostats, clamp off the line of the bag containing the harvested plasma and seal. Process the Red Cells as described on Section "Preparation of Red Cells and Fresh Frozen Plasma".

♣  Calculate the volume of Platelet Rich Plasma.

About this calculation:

The specific gravity of plasma is 1.023, so 1 gram of plasma is approximately equal to 1 milliliter of plasma.

♣  Tare the weight of the balance to zero and weigh the Platelet Rich Plasma. The weight of the empty bag should be subtracted from the final weight of the bag. By dividing the final weight of the product by the appropriate specific gravity, the volume of product in milliliters can be calculated.

♣  The final product should be labeled with the product name and volume in milliliters.

Storage

♣  In order to preserve platelet viability, Platelet Rich Plasma should be allowed to "rest" at room temperature, label side down for 1 - 2 hours and transfused as soon as possible thereafter.

♣  This procedure is repeated in a step-by-step format in the Methods Section.

9. Short Draw and Decreasing the Amount of Anticoagulant-Preservative

♣  Commercially available blood bags are designed to anticoagulate and preserve a specific amount of blood. Deviations from the expected blood volume must be handled appropriately in order to assure product viability. This discussion will be limited to those bags designed for a 450 mL draw, although the principles may be applied to bags with a different expected draw volume.

Short Draw

♣  Blood collection bags intended for a 450 mL draw contain approximately 63 mL of anticoagulant-preservative. This amount of anticoagulant-preservative is sufficient to support 405 - 495 mL of whole blood. If 300 - 404 mL of whole blood is drawn into a bag intended for a 450 mL draw, the unit of blood should not be processed into components and should remain as whole blood. The unit of blood should be labeled as "Short Draw" and the volume of the draw should be documented on the bag.

♥  Be aware that these units contain an increased amount of anticoagulant and citrate toxicity to the recipient is a concern. This may prohibit the use of short draw units of blood for some recipients.

Decreasing the Amount of Anticoagulant-Preservative

♣  If a phlebotomy of less than 300 mL is planned, the amount of anticoagulant-preservative in the blood bag should be decreased prior to blood collection. Anticoagulant-preservative may be removed by expressing the excess anticoagulant-preservative solution into one of the integrally attached satellite bags.

♣  The calculation for the amount of anticoagulant-preservative to be removed:

• Amount of Anticoagulant-Preservative needed in mL = (mL of Blood to be Drawn/100) X 14 mL
• Anticoagulant-Preservative to Remove from Primary Blood Bag = 63 mL - Amount of Anticoagulant-Preservative needed in mL.

♥  This calculation is valid only for anticoagulant-preservatives in which the ratio of anticoagulant to blood is 1.4:10. This includes most blood collection systems containing CPD or CPDA-1.

♣  Procedure for Removal of Anticoagulant-Preservative from Primary Blood Bag:

• Obtain the specific gravity and amount of the anticoagulant-preservative in the primary bag. Calculate the weight of the anticoagulant-preservative to be removed from the primary bag.
• Weigh both the primary bag and the satellite bag designated for collection of excess anticoagulant-preservative.
• Open the seal from the primary bag. Express the calculated amount of anticoagulant-preservative to be removed into the empty satellite bag. Seal and remove the satellite bag.
• The unit may be processed for components.

10. Purchasing and Receiving Blood Products from Outside Sources

Purchasing Blood Products

♣  It may be necessary for a veterinary practice to order blood products from outside sources.

• This could be due to an emergency case that consumes all stored blood products during a time period of which donors are not readily available for blood donation.
• Some veterinary practices may order blood products from outside sources exclusively.

♣  A veterinary blood bank should be identified.

• Some veterinary blood banks that provide blood products for purchase are listed in Appendix 2.
• It is advisable to establish a client relationship with the blood supplier before the need for blood products arises.

♣  Communication regarding anticipated need of blood products provides the supplier with valuable information; the supplier can adjust blood collection so that adequate blood supplies may be maintained.

• This also provides the purchaser an opportunity to inquire as to methods of collection and quality of the donor pool.
• Additionally, the purchaser may obtain information regarding the suppliers policies and procedures regarding ordering blood products. Caveat emptor!

♣  The purchaser might inquire:

• What is the purchase price of the product?
• How long does it usually take to get product on a routine basis?
• How long will it take to obtain the product on an emergency basis?
• How will the product be packed?
• Once packed, how long does the packing permit the product optimal storage temperatures?
• Will overnight shipping be used? What are the associated costs?
• Does the delivery route incur extremely hot or extremely cold outside temperatures? Will the packing materials compensate for this?
• Are temperature monitoring devices used for the shipping process? Time temperature tags, high-low thermometers (Taylor Instruments, Rochester, NY, USA) or R&D Temperature Indicators (Chek Lab Inc, Aurora, IL, USA) may be used.
• What is the policy if units are received that have exceeded acceptable shipping temperatures?
• What is the policy if the unit is mishandled or lost during shipping?
• What will the expiration date(s) of the unit(s) be?

Receiving Blood Products

♣  Upon receipt of the product, immediately unpack the product.

♣  Perform a visual inspection. Is the product damaged in any way? Is there any leaking?

♣  Does it appear that the product has been maintained at the proper temperature?

• If no temperature monitoring device is used, the temperature of Red Cells may be checked by folding the blood bag in half (labels facing out) and wedge a thermometer within the "sandwich" of the bag (Figure 2-13). Read the temperature; it should not exceed 10 °C.
• Frozen products should arrive frozen!

♣  The product should be placed at appropriate storage temperature and documented as received in the product log.

Figure 2-13. Acquiring red cell temperature by "sandwich" technique.

11. Preparing Blood Products for Shipment

♣  Blood products should be packaged and shipped in a manner that preserves product integrity.

♥  Federal, state, and local guidelines should be reviewed. In some instances, it is necessary to obtain licensure from federal or state agencies prior to shipping blood products.

Product Packaging

♣  Transport containers and packaging procedures should be validated prior to use. These protocols should be periodically monitored to ensure the proper product temperature is maintained throughout the shipping process.

♣  Transport containers should have tight fitting covers and withstand leakage and pressure. Most shippers require that a cooler be shipped with a cardboard box cover. Address labels should be inscribed legibly with indelible ink.

♣  To package blood products for shipping:

1. Obtain a suitable transport container.
2. Within the container, starting from the bottom, layer:

1. Coolant
2. Absorbent paper
3. Product
4. Absorbent paper
5. Coolant
6. Container lid

3. Secure the coolant lid and exterior box.
4. Attach label.
5. Deliver to shipper.

♣  For Whole Blood and Red Cells, the coolant of choice is ice, which may be confined in a zippered closure plastic bag.

♣  For frozen products, dry ice is the coolant of choice.

♥  Dry ice is considered a hazardous material as it causes skin burns and releases carbon dioxide as it volatizes. Be sure to notify the shipper that the package contains this hazardous material.

♣  Absorbent paper should be capable of containing any leakage within the package. Paper towels, newsprint or disposable diapers are suitable.

♣  The blood product should also be secured in a zippered closure plastic bag in case of accidental damage resulting in subsequent product leakage.