II. Blood and Blood Components Terry Kotrla, MS, MT(ASCP)BB Spring 2010

Goals Of Blood Collection Maintain viability and function Maintain viability and function Prevent physical changes Prevent physical changes Minimize bacterial contamination Minimize bacterial contamination

Anticoagulants Preservative Solutions Anticoagulants prevent blood clotting Anticoagulants prevent blood clotting Preservatives provide nutrients for cells Preservatives provide nutrients for cells Heparin Heparin –Rarely if ever used anymore –Anticoagulant ONLY –Transfuse within 48 hours, preferably 8

Anticoagulants CPDCPD-A1 Storage time 21 days 35 days Temperature 1-6 C Slows glycolytic activity AdenineNone Substrate for ATP synthesis Volume 450 +/- 10% Dextrose Supports ATP generation by glycolytic pathway Citrate Prevents coagulation by binding calcium

Additive Solution Primary bag with satellite bags attached. Primary bag with satellite bags attached. One bag has additive solution (AS) One bag has additive solution (AS) Unit drawn into CPD anticoagulant Unit drawn into CPD anticoagulant

Additive Solution Remove platelet rich plasma within 72 hours Remove platelet rich plasma within 72 hours Add additive solution to RBCs, ADSOL, which consists of: Add additive solution to RBCs, ADSOL, which consists of: –Saline –Adenine –Glucose –Mannitol Extends storage to 42 days Extends storage to 42 days Final hematocrit approximately 66% Final hematocrit approximately 66%

Changes Occur During Storage Shelf life = expiration date Shelf life = expiration date –At end of expiration must have 75% recovery –At least 75% of transfused cells remain in circulation 24 hours AFTER transfusion

Storage Lesion Biochemical changes which occur at 1-6C Biochemical changes which occur at 1-6C Affects oxygen dissociation curve, increased affinity of hemoglobin for oxygen. Affects oxygen dissociation curve, increased affinity of hemoglobin for oxygen. –Low 2,3-DPG, increased O 2 affinity, less O 2 released. –pH drops causes 2,3-DPG levels to fall –Once transfused RBCs regenerate ATP and 2,3-DPG Few functional platelets present Few functional platelets present Viable (living) RBCs decrease Viable (living) RBCs decrease

Plasma hemoglobin Plasma K + Viable cells pH ATP 2,3-DPG Plasma Na + Helps release oxygen from hemoglobin (once transfused, ATP & 2,3- DPG return to normal) K+K+ Na +

Storage Lesion Significant for infants and massive transfusion. Significant for infants and massive transfusion. Other biochemical changes Other biochemical changes –ATP decreases –Potassium increases –Sodium decreases –Plasma hemoglobin increases

Preparation of Components Collect unit within 15 minutes to prevent activation of coagulation system Collect unit within 15 minutes to prevent activation of coagulation system Draw into closed system – primary bag with satellite bags with hermetic seal between. Draw into closed system – primary bag with satellite bags with hermetic seal between. If hermetic seal broken transfuse within 24 hours if stored at 1-4C, 4 hours if stored at C If hermetic seal broken transfuse within 24 hours if stored at 1-4C, 4 hours if stored at C

Preparation of Components Centrifuge – light spin, platelets suspended Centrifuge – light spin, platelets suspended Remove platelet rich plasma (PRP) Remove platelet rich plasma (PRP) Centrifuge PRP heavy spin Centrifuge PRP heavy spin Remove platelet poor plasma Remove platelet poor plasma Freeze plasma solid within 8 hours Freeze plasma solid within 8 hours Thaw plasma at 1-4C – precipitate forms Thaw plasma at 1-4C – precipitate forms Centrifuge, express plasma leaving cryoprecipitate. Store both at -18C Centrifuge, express plasma leaving cryoprecipitate. Store both at -18C RBCs – CPD – 21 days, ADSOL – 42 days – 1-6C RBCs – CPD – 21 days, ADSOL – 42 days – 1-6C

Preparation of Components Summary – One unit of whole blood can produce: Summary – One unit of whole blood can produce: –Packed RBCs –Fresh frozen plasma (FFP) –Cryoprecipitate (CRYO) –Single donor plasma (SDP) – cyro removed –Platelets – terms PC (platelet concentrate) OR RD PC (random donor platelet concentrate)

Preparation of Components Sterile docking device joins tubing Sterile docking device joins tubing –Used to add satellite bags to maintain original expiration of component –May be used to pool components

Blood Component General Information Blood separated into components to specifically treat patients with product needed Blood separated into components to specifically treat patients with product needed Advantages of component separation Advantages of component separation –Allow optimum survival of each component –Transfuse only component needed

Blood Component General Information Transfusion practice Transfusion practice –Transfusion requires doctor’s prescription –All components MUST be administered through a filter –Infuse quickly, within 4 hours –D (Rh) neg require D neg cellular products –ABO identical preferred, ABO compatible OK –“Universal donor” – RBCs group O, plasma AB

Blood Component General Information Fresh Whole Blood Fresh Whole Blood –Blood not usually available until hours –Candidates  Newborns needing exchange transfusion  Patients requiring leukoreduced products

Blood Component General Information Summary of storage temperatures: Summary of storage temperatures: –Liquid RBCs 1-6C –Platelets, Cryo (thawed) and granulocytes C (room temperature) –ANY frozen plasma product ≤ -18C –ANY liquid plasma product EXCEPT Cryo 1-6C

Blood Components Cellular Cellular –Red blood cell products –Platelets –Granulocytes Plasma Plasma –FFP –Cryoprecipitate

Products With Red Cells

Whole Blood Clinical indications for use of WB are extremely limited. Used for massive transfusion to correct acute hypovolemia such as in trauma and shock, exchange transfusion. RARELY used today, platelets non-functional, labile coagulation factors gone. Must be ABO identical.

Changes in Stored Blood

Red Blood Cells, Packed (PRBC) Used to treat symptomatic anemia and routine blood loss during surgery Hematocrit is approximately 80% for non- additive (CPD), 60% for additive (ADSOL). Allow WB to sediment or centrifuge WB, remove supernatant plasma.

Leukocyte Reduced Red Cells (LR-RBC) Leukocytes can induce adverse affects during transfusion, primarily febrile, non-hemolytic reactions. Reactions to cytokines produced by leukocytes in transfused units. Other explanations to reactions include: immunization of recipient to transfused HLA or granulocyte antigens, micro aggregates and fragmentation of granulocytes. Historically, indicated only for patients who had 2 or more febrile transfusion reactions, now a commonly ordered, popular component. “CMV” safe blood, since CMV lives in WBCs. Most blood centers now leukoreduce blood immediately after collection. Bed side filters are available to leukoreduce products during transfusion.

Leukocyte Reduction

Washed Red Blood Cells (W-RBCs) Washing removes plasma proteins, platelets, WBCs and micro aggregates which may cause febrile or urticarial reactions. Patient requiring this product is the IgA deficient patient with anti-IgA antibodies. Prepared by using a machine which washes the cells 3 times with saline to remove and WBCs. Two types of labels: – –Washed RBCs - do not need to QC for WBCs. – –Leukocyte Poor WRBCs, QC must be done to guarantee removal of 85% of WBCs. No longer considered effective method for leukoreduction. e.Expires 24 hours after unit is entered.

Cell Washer to Prepare Washed Cells

Frozen Blood

Red Blood Cells Frozen; Red Blood Cells Deglycerolized (D-RBC) Blood is frozen to preserve: rare types, for autologous transfusion, stock piling blood for military mobilization and/or civilian natural disasters. Blood is drawn into an anticoagulant preservative. – –Plasma is removed and glycerol is added. – –After equilibration unit is centrifuged to remove excess glycerol and frozen. Expiration – –If frozen, 10 years. – –After deglycerolization, 24 hours. Storage temperature – –high glycerol -65 C. – –low glycerol -120 C, liquid nitrogen.

Red Blood Cells Frozen; Red Blood Cells Deglycerolized (D-RBC) Thaw unit at 37C, thawed RBCs will have high concentration of glycerol. A solution of glycerol of lesser concentration of the original glycerol is added. This causes glycerol to come out of the red blood cells slowly to prevent hemolysis of the RBCs. After a period of equilibration the unit is spun, the solution is removed and a solution with a lower glycerol concentration is added. This procedure is repeated until all glycerol is removed, more steps are required for the high glycerol stored units. The unit is then washed.

Rejuvenated Red Blood Cells A special solution is added to expired RBCs up to 3 days after expiration to restore 2,3-DPG and ATP levels to prestorage values. Rejuvenated RBCs regain normal characteristics of oxygen transport and delivery and improved post transfusion survival. Expiration is 24 hours or, if frozen, 10 years

Platelet Products

Platelets (PLTS), Platelet Concentrate (PC) or Random Donor Platelet Concentrate (RD-PC) Used to prevent spontaneous bleeding or stop established bleeding in thrombocytopenic patients. Prepared from a single unit of whole blood. Due to storage at RT it is the most likely component to be contaminated with bacteria. Therapeutic dose for adults is 6 to 10 units. Some patients become "refractory" to platelet therapy. Expiration is 5 days as a single unit, 4 hours if pooled. Store at C (RT) with constant agitation. D negative patients should be transfused with D negative platelets due to the presence of a small number of RBCs.

Preparation of platelet concentrate RBCsPRP Plasma Platelet concentrate

Platelets (PLTS), Platelet Concentrate (PC) or Random Donor Platelet Concentrate (RD-PC) One bag from ONE donor One bag from ONE donor Need 6-10 for therapeutic dose Need 6-10 for therapeutic dose

Pooling Platelets 6-10 units transferred into one bag 6-10 units transferred into one bag Expiration = 4 hours Expiration = 4 hours

Platelets Pheresis, Apheresis Platelet Concentrate, Single Donor Platelet Concentrate (SD-PC) Used to decrease donor exposure, obtain HLA matched platelets for patients who are refractory to RD-PC or prevent platelet refractoriness from occurring. Prepared by hemapheresis, stored in two connected bags to maintain viability. One pheresed unit is equivalent to 6-8 RD-PC. Store at C (RT) with agitation for 5 days, after combining, 24 hours D negative patients should be transfused with D negative platelets due to the presence of a small number of RBCs

Apheresis

Apheresis

Platelets Pheresis One bag (unit) from one donor One bag (unit) from one donor One unit is a therapeutic dose One unit is a therapeutic dose Volume approximately 250 ccs Volume approximately 250 ccs

Granulocytes Granulocytes LymphocyteMonocyte NeutrophilsEosinophilsBasophils

Granulocytes Primary use is for patients with neutropenia who have gram negative infections documented by culture, but are unresponsive to antibiotics. Therapeutic efficacy and indications for granulocyte transfusions are not well defined. Better antimicrobial agents and use of granulocyte and macrophage colony stimulating factors best for adults, best success with this component has been with babies Daily transfusions are necessary. Prepared by hemapheresis. Expiration time is 24 hours but best to infuse ASAP. Store at C.

Plasma Components

Fresh Frozen Plasma – Volume cc

Fresh Frozen Plasma (FFP) Used to replace labile and non-labile coagulation factors in massively bleeding patients OR treat bleeding associated with clotting factor deficiencies when factor concentrate is not available. Must be frozen within 8 hours of collection. Expiration – –frozen - 1 year stored at <-18 C. – –frozen - 7 years stored at <-65 C.thawed - 24 hours

Fresh Frozen Plasma (FFP) Storage temperature – –frozen -18 C, preferably -30 C or lower – –thawed C Thawed in 30-37C water bath or FDA approved microwave Must have mechanism to detect units which have thawed and refrozen due to improper storage. Must be ABO compatible

Plasma, Liquid Plasma, Recovered Plasma and Source Plasma Used to treat patients with stable clotting factor deficiencies for which no concentrate is available or for patients undergoing therapeutic plasmapheresis. Prepared by separating the plasma from the RBCs on or before the 5th day after expiration of the whole blood. Once separated can: – –Freeze, store at -18 C for 5 years – –If not frozen, called liquid plasma, store at 1-6 C for up to 5 days after expiration of WB. Once FFP is one year old can redesignate as Plasma, expiration is 5 years.

Pooled Plasma/Solvent Detergent Treated Most recently licensed product. Prepared from pools of no more than 2500 units of ABO specific plasma frozen to preserve labile coagulation factors. Treated with chemicals to inactivate lipid-enveloped viruses. Contains labile and non-labile coagulation factors but lacks largest Von Willebrand’s factor multimers. Used same as FFP.Safety concerns – –Decreases disease transmission for diseases tested for. – –Doesn’t inactivate viruses with non-lipid envelopes: parvo virus B19, hepatitis A, and unrecognized pathogens

Cryoprecipitate (CRYO), Factor VIII or Anti-Hemophilic Factor (AHF) Cold insoluble portion of plasma that precipitates when FFP is thawed at 1-6C. Cryoprecipitate contains high levels of Factor VIII and Fibrinogen, used for treatment of hemophiliacs and Von Willebrands when concentrates are not available. Used most commonly for patients with DIC or low fibrinogen levels. A therapeutic dose for an adult is 6 to 10 units. Can be prepared from WB which is then designated as "Whole Blood Cryoprecipitate Removed" or from FFP – –Plasma is frozen. – –Plasma is then thawed at 1-6 C, a precipitate forms. – –Plasma is centrifuged, cryoprecipitate will go to bottom. – –Remove plasma, freeze within 1 hour of preparation

FFP Frozen within 8 hours Thawed FFP Cryoprecipitate (VIII, vW) Plasma cryoprecipitate, reduced (TTP, FII, V, Vii, IX, X, XI) Thaw at 30-37°C Store at RT 4 hrs Refrozen with 24 hrs of separation Store at ≤18°C 1 yr 5 day expiration at 1-6°C

Cryoprecipitate (CRYO), Factor VIII or Anti-Hemophilic Factor (AHF) Storage Temperature – –Frozen -18 C or lower – –Thawed - room temperature Expiration: – –Frozen 1 year – –Thawed 6 hours – –Pooled 4 hours Best to be ABO compatible but not important due to small volume

Cryoprecipitate – volume 15ccs

Irradiation of Blood Components

Cellular blood components are irradiated to destroy viable T- lymphocytes which may cause Graft Versus Host Disease (GVHD). GVHD is a disease that results when immunocompetent, viable lymphocytes in donor blood engraft in an immunocompromised host, recognize the patient tissues as foreign and produce antibodies against patient tissues, primarily skin, liver and GI tract. The resulting disease has serious consequences including death. GVHD may be chronic or acute

Irradiation of Blood Components Patients at greatest risk are: – – severely immunosuppressed, – –immunocompromised, – –receive blood donated by relatives, or – –fetuses receiving intrauterine transfusions Irradiation inactivates lymphocytes, leaving platelets, RBCs and granulocytes relatively undamaged. Must be labeled "irradiated". Expiration date of Red Blood Cell donor unit changes to 28 days. May be transfused to "normal" patients if not used by intended recipient.

Irradiation of Blood Components

Donor Blood Inspection and Disposition It is required that donor units be inspected periodically during storage and prior to issuing to patient. The following may indicate an unacceptable unit: – –Red cell mass looks purple or clots are visible. – –Zone of hemolysis observed just above RBC mass, look for hemolysis in sprigs, especially those closest to the unit. – –Plasma or supernatant plasma appears murky, purple, brown or red. – –A greenish hue need not cause a unit to be rejected. – –Inspect platelets for aggregates. Inspect FFP and CRYO for signs of thawing, evidence of cracks in bag, or unusual turbidity in CRYO or FFP (i.e., extreme lipemia).

Inspection of Donor Blood Segment closest to unit is hemolyzed. Segment closest to unit is hemolyzed. May indicate bacterial contamination May indicate bacterial contamination

Donor Blood Inspection and Disposition If a unit's appearance looks questionable do the following: – –Quarantine unit until disposition is decided. – –Gently mix, allow to settle and observe appearance. If bacterial contamination is suspected the unit should be cultured and a gram stain performed. Positive blood cultures usually indicative of: – –Inadequate donor arm preparation – –Improper pooling technique – –Health of donor - bacteremia in donor If one component is contaminated, other components prepared from the same donor unit may be contaminated.

Inspection of Donor Blood Reissuing blood cannot be done unless the following criteria is met: – –Container closure must not have been penetrated or entered in any manner. – –Most facilities set 30" time limit for accepting units back, warming above 6-10C even with subsequent cooling increases RBC metabolism producing hemolysis and permitting bacterial growth. – –Blood must have been kept at the appropriate temperature. – –One sealed segment must remain attached to container. – –Records must indicate that blood has been reissued and inspected prior to reissue.

Transportation of Blood and Blood Components WB and RBC – –Sturdy well insulated cardboard and/or styrofoam container, wet ice in ziplock bag to cool, temperature must be monitored. – –Mobile collection units should transport blood ASAP and leave at RT if platelets are to be made. – –In-house transport place in cooler with wet ice and thermometer, monitor temperature every 30 minutes.

Safe-T-Vue Temperature Monitor

Transportation of Blood and Blood Components Frozen components – –Temperature must be maintained at or below required storage temperature. – –Use dry ice in well insulated container. Platelets and granulocytes – –Maintain at C. – –Transport in well insulated containers without ice. Commercial coolers available to maintain at C.

Transportation of Blood and Blood Components Handling donor units – –Should not remain at RT unnecessarily, when blood is issued it should be transfused as soon as possible. – –When numerous units are removed from fridge, remove fluid filled container with a thermometer at same time as blood, when temperature reaches 6 C return to fridge.

Records Must be made concurrently with each step of component preparation, being as detailed as possible for clear understanding. Must be legible and indelible. Must include dates of various steps and person responsible.

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