Chapter 1 Red Blood Cell and Platelet Preservation PDF

 Chapter 1 Red Blood Cell and Platelet Preservation: Historical Perspectives and Current Trends 3 the pioneer work of Dr. Charles Drew on developing tech- blood drives conducted at their place of work, school, and niques in blood transfusion a...

 Chapter 1 Red Blood Cell and Platelet Preservation: Historical Perspectives and Current Trends 3 the pioneer work of Dr. Charles Drew on developing tech- blood drives conducted at their place of work, school, and niques in blood transfusion and blood preservation led to church, as well as at community and hospital-based blood the establishment of a widespread system of blood banks.1 centers. Volunteer donors are not paid and provide nearly all In February 1941, Dr. Drew was appointed director of of the blood used for transfusion in the United States. the first American Red Cross blood bank at Presbyterian Traditionally, the amount of whole blood in a unit has been Hospital.1 The pilot program Dr. Drew established became 450 mL ± 10% of blood (1 pint). More recently, 500 mL ± 10% the model for the national volunteer blood donor program of blood is being collected.5 These units are collected from of the American Red Cross.1 donors with a minimum hematocrit of 38%.5 Modified plastic In 1943, Loutit and Mollison of England introduced the for- collection systems are used when collecting 500 mL of blood, mula for the preservative acid-citrate-dextrose (ACD). Efforts with the volume of anticoagulant preservative solution being in several countries resulted in the landmark publication of the increased from 63 to 70 mL. The total blood volume of most July 1947 issue of the Journal of Clinical Investigation, which adults is 10 to 12 pints, and donors can replenish the fluid lost devoted nearly a dozen papers to the topic of blood preserva- from the 1-pint donation in 24 hours. The donor’s red blood tion. Hospitals responded immediately, and in 1947 blood cells are replaced within 1 to 2 months after donation.4 A banks were established in many major cities of the United volunteer donor can donate whole blood every 8 weeks. (Refer States; subsequently, transfusion became commonplace. to Chapter 13 on Donor Selection.) The daily occurrence of transfusions led to the discovery Units of the whole blood collected can be separated into of numerous blood group systems. Antibody identification three components: packed RBCs, platelets, and plasma. In surged to the forefront as sophisticated techniques were recent years, less whole blood has been used to prepare developed. The interested student can review historic events platelets because of the increased utilization of apheresis during World War II in Kendrick’s Blood Program in World platelets. Hence, many units are converted only into RBCs War II, Historical Note.2 In 1957, Gibson introduced an im- and plasma. The plasma can be converted by cryoprecipita- proved preservative solution called citrate-phosphate-dextrose tion to a clotting factor concentrate that is rich in fibrinogen. (CPD), which was less acidic and eventually replaced ACD A unit of whole blood–prepared RBCs may be stored for as the standard preservative used for blood storage. 21 to 42 days, depending on the anticoagulant preservative Frequent transfusions and the massive use of blood soon solution used when the whole blood unit is collected and resulted in new problems, such as circulatory overload. whether a preserving solution is added to the separated Component therapy has helped these problems. In the past, RBCs. Donated blood is free. However, there is a cost asso- a single unit of whole blood could serve only one patient. ciated with collection, testing, processing, storing, and ship- With component therapy, however, one unit may be used for ping of the blood components. The donation process multiple transfusions. Today, health-care providers can select consists of three predonation steps. Donors receive the the specific component for their patient’s particular needs following (Box 1–1): without risking the inherent hazards of whole blood trans- 1. Educational reading materials fusions. Health-care providers can transfuse only the re- 2. A donor health history questionnaire quired fraction in the concentrated form, decreasing the 3. An abbreviated physical examination possibility of overloading the circulatory system. Appropriate blood component therapy now provides more effective treat- ment and more complete use of blood products. Extensive use of blood during this period, coupled with component BOX 1–1 separation, led to increased comprehension of erythrocyte The Donation Process metabolism and a new awareness of the problems associated with RBC storage. Step 1: Educational Materials Educational material (such as the AABB pamphlet “An Important Current Status Message to All Blood Donors”) that contains information on the risks of infectious diseases transmitted by blood transfusion, including the symptoms and signs of AIDS, is given to each prospective donor AABB, formerly the American Association of Blood Banks, to read. estimates that 6.8 million volunteers donate blood each year. Based on the 2015 National Blood Collection and Utilization Step 2: The Donor Health History Questionnaire Survey (NBCUS) approximately 12.6 million units of red A uniform donor history questionnaire, designed to ask questions blood cells (RBCs) were collected, and around 11.4 million that protect the health of both the donor and the recipient, is given to every donor. The health history questionnaire is used to identify were transfused.3 This represents a decline of 11.6% and donors who have been exposed to diseases that can be transmitted 13.9%, respectively since 2013.4 With an aging population in blood (e.g., variant Creutzfeldt-Jakob, West Nile virus, malaria, and advances in medical treatments requiring transfusions, babesiosis, or Chagas disease). the demand for blood and blood components is expected to Step 3: The Abbreviated Physical Examination continue to be high. It is estimated that one in three people The abbreviated physical examination for donors includes blood will need blood at some point in their lifetime.4 These units pressure, pulse, and temperature readings; hemoglobin or hemat- are donated by fewer than 10% of healthy Americans who ocrit level; and the inspection of the arms for skin lesions. are eligible to donate each year.4 Volunteers can donate at 12 PART I Fundamental Concepts cells has been reported as well.32 However, this has not Table 1–9 Phases of Testing proven practical for routine transfusion. The challenges associated with blood pharming are scalability or large-scale Phase Description of Testing production and cost-effectiveness. Preclinical In vivo and animal testing. RBC Substitutes Phase I Researchers test drug in a small group of people (20 to 80) for the first time to evaluate its safety, Scientists have been searching for a substitute for blood for determine a safe dosage range, and identify side effects. over 150 years. Blood substitutes continue to be of interest because of their potential to alleviate shortages of donated Phase II The drug is given to a larger group of people blood. In the 1980s, safety concerns about HIV led to re- (100 to 300) to see if it is effective and to further newed interest in finding a substitute for human blood; and evaluate its safety. more recently, the need for blood on remote battlefields has Phase III The drug is given to large groups of people heightened that interest.33 The U.S. military is one of the (1,000 to 3,000) to confirm its effectiveness, monitor strongest advocates for the development of blood substitutes, side effects, compare it to commonly used treat- which it supports through its own research and partnerships ments, and collect information that will allow the drug to be used safely. with private-sector companies.33 Today the search continues for a safe and effective oxygen carrier that could eliminate Phase IV Postmarketing studies to gather additional informa- many of the problems associated with blood transfusion, such tion about the drug’s risks, benefits, and optimal use. as the need for refrigeration, limited shelf-life, compatibility, immunogenicity, transmission of infectious agents, and short- ages. Box 1–3 lists the potential benefits of artificial oxygen carriers. Since RBC substitutes are drugs, they must go Hemoglobin-Based Oxygen Carriers through extensive testing in order to obtain FDA approval. HBOC commercial development focused on “oxygen thera- Safety and efficacy must be demonstrated through clinical peutic” indications to provide immediate oxygenation until trials. Table 1–9 outlines the different phases of testing. medical or surgical interventions could be initiated. Early Current research on blood substitutes is focused on two trauma trials with HemAssist® (BAXTER), Hemopure® areas: hemoglobin-based oxygen carriers (HBOCs) and per- (HbO2Therapeutics), and PolyHeme® (NORTHFIELD fluorocarbons (PFCs).34,35 Originally developed to be used Laboratories) for resuscitating hypotensive shock all failed in trauma situations such as accidents, combat, and surgery, due to the safety concerns of cardiac issues and increased RBC substitutes have, until recently, fallen short of meeting mortality. requirements for these applications.34 Despite years of re- Although several HBOCs have progressed to phase II and search, RBC substitutes are still not in routine use today. III clinical trials, currently none have been approved for South Africa, Mexico, and Russia are the only countries clinical use in humans in the United States.36,37 A 2008 meta- in which blood substitutes are approved for clinical use. analysis of 16 clinical trials involving 3,711 patients and five None have received FDA approval for clinical use in the different HBOCs found a significantly increased risk of death United States, although specific products are still in phase and myocardial infarction associated with the use of III clinical trials. HBOCs.37 As a result, in 2008, the Food and Drug Adminis- tration (FDA) put all HBOC trials in the United States on clinical hold due to the unfavorable outcomes.35 However, BOX 1–3 Hemopure (HBOC-201) and PolyHeme are still in phase III Potential Benefits of Artificial Oxygen Carriers clinical trials in the United States and Europe.38 Hemopure was approved for clinical use in South Africa in 2001 to treat Abundant supply adult surgical patients who are anemic, and in Russia for Readily available for use in prehospital settings, battlefields, and remote locations acute anemia.34 Many HBOCs have been researched; how- Can be stockpiled for emergencies and warfare ever, the majority have been discontinued due to complica- No need for typing and crossmatching tions of cardiac toxicity, gastrointestinal distress, neurotoxicity, Available for immediate infusion renal failure, and increased mortality.34 Table 1–10 summa- Extended shelf-life (1 to 3 years) rizes some of the many HBOCs developed. Can be stored at room temperature However, some experts believe that HBOCs hold more Free of bloodborne pathogens promise than PFCs.33,39 At full oxygen capacity immediately Table 1–11 lists the advantages and disadvantages of Do not prime circulating neutrophils, reducing the incidence of HBOCs. multiorgan failure Perfluorocarbons Can deliver oxygen to tissue that is inaccessible to RBCs Have been accepted by Jehovah’s Witnesses Perfluorocarbons are synthetic hydrocarbon structures in Could eventually cost less than units of blood which all hydrogen atoms have been replaced with fluorine. They are chemically inert, are excellent gas solvents, and  Chapter 1 Red Blood Cell and Platelet Preservation: Historical Perspectives and Current Trends 13 Table 1–10 Hemoglobin-Based Oxygen Carriers Product Manufacturer Chemistry/Source History/Status HemAssist (DCLHb) Baxter Diaspirin cross-linked Hgb from First HBOC to advance to phase III clini- outdated human RBCs cal trials in United States. Removed from production because of increased mortality rates. PolyHeme (SFH-P) Northfield Laboratories Polymerized and pyridoxalated Underwent phase II/III clinical trials in human Hgb United States. Did not obtain FDA approval. Hemopure (HBOC-201) HbO2 Polymerized bovine Hgb Still in phase II/III clinical trials in [hemoglobin glutamer – 250 (bovine)] United States and Europe. Approved Therapeutics for use in South Africa (2001) to treat adult surgical patients who are anemic, and in Russia for acute anemia. Oxyglobin HbO2 Polymerized bovine Hgb Approved by the FDA and the European Medicines Agency (EMA) to treat Therapeutics canine anemia in veterinary use. MP4OX Sangart Polyethylene glycol (PEG) attached to In phase II trials in United States; the surface of Hgb from human RBCs phase III in Europe. Hemospan (MP4) Terminated development and opera- tions in December 2013. HemoLink Hemosol Purified human Hgb from outdated Abandoned due to cardiac toxicity. RBCs, cross-linked and polymerized HemoTech HemoBioTech Derived from bovine Hgb Limited clinical trial outside the United States. Table 1–11 Advantages and Disadvantages treatment of traumatic brain injury in Switzerand and Israel.39 Refer to Table 1–12 for further details and review of of Hemoglobin-Based Oxygen PFCs, and Table 1–13 for the advantages and disadvantages Carriers of perfluorochemicals. Advantages Disadvantages Tissue Engineering of RBCs Long shelf-life Short intravascular half-life Very stable Possible toxicity Research into large-scale production of RBCs from stem cells (blood pharming) seems to have more promise and is receiving No antigenicity (unless bovine) Increased O2 affinity more attention and funding than are blood substitutes. RBCs No requirement for blood typing Increased oncotic effect have been cultured in-vitro for many years and have been suc- procedures cessfully tested in animal models. However, there are limita- tions in the number of RBCs that can be cultured from one unit of blood and the associated costs of these expensive cultures. By culturing stem cells in the presence of the essential carry O2 and CO2 by dissolving them. Because of their small cytokines, stem cell factor, and erythropoietin, unilineage size (about 0.2 µm in diameter), they are able to pass production of erythroblasts has been achieved.39 Culture of through areas of vasoconstriction and deliver oxygen to tis- cells in expansion medium and subsequently in maturation sues that are inaccessible to RBCs.39 PFCs have been under medium has shown progress of in-vitro erythroid expansion.39 investigation as possible RBC substitutes since the 1970s. The general consensus for producing RBCs in-vitro is a precul- Fluosol (Green Cross Corp.) was approved by the FDA in ture of hematopoietic stem cells (HSC) for erythroid progenitor 1989 but was removed from the market in 1994 due to clini- cells, with a subsequent generation of high numbers of ery- cal shortcomings and poor sales. Other PFCs have proceeded throblasts. This is followed by a erythroid maturation phase in to clinical trials. Perftoran West Ltd is in clinical use in the presence of a feeder layer to facilitate progression to a ma- Russia and Mexico.39 Two others are no longer under devel- ture RBC.39 Maturation without a feeder layer has also been opment, and one (Oxycyte, Oxygen Biotherapeutics Inc.) is reported and this development is necessary if tissue engineer- currently being investigated as an oxygen therapeutic for ing RBCs is to become used for transfusion therapy.39 14 PART I Fundamental Concepts Table 1–12 Perfluorocarbons Fluosol-DA Green Cross Corporation of Japan The first and only oxygen-carrying blood substitute ever to receive approval from the FDA for human clinical use in the United States. Approved in 1989; discontinued in 1994 because of clinical shortcomings and poor sales. Oxygent Alliance Pharmaceutical Corporation Phase III trial in Europe completed; phase III trial in United States terminated due to adverse effects. Development stopped due to lack of funding. Oxycyte Originally Synthetic Blood International; Shift in research from use as RBC substitute to other medical applications. Cur- name changed to Oxygen Biotherapeutics rently in phase II trials in Switzerland for treatment of traumatic brain injury. in 2008 Perftoran Perftoran Approved for use in Russia and Mexico. PHER-O2 Sanguine Corporation Under evaluation for transfusion, therapy for heart attack and stroke. Table 1–13 Advantages and Disadvantages Maintaining pH was determined to be a key parameter for retaining platelet viability in vivo when platelets were stored of Perfluorochemicals at 20°C to 24°C.41 The loss of platelet quality during storage Advantages Disadvantages is known as the platelet storage lesion. During storage, a varying degree of platelet activation occurs that results in Biological inertness Adverse clinical effects release of some intracellular granules and a decline in ATP Lack of immunogenicity High O2 affinity and ADP. 41 The reduced oxygen tension (pO2) in the plastic platelet Easily synthesized Retention in tissues storage container results in an increase in the rate of glycol- Requirement for O2 administration ysis by platelets to compensate for the decrease in ATP re- when infused generation from the oxidative (TCA) metabolism. This Deep-freeze storage temperatures increases glucose consumption and causes an increase in lac- tic acid that must be buffered. This results in a fall in pH. During the storage of platelet concentrates (PCs) in plasma, the principal buffer is bicarbonate. When the bicarbonate Platelet Preservation buffers are depleted during platelet concentrate storage, the Approximately 2.4 million platelet units are distributed and pH rapidly falls to less than 6.2, which is associated with a 2.2 million platelet transfusions are administered yearly in loss of platelet viability. In addition, when pH falls below 6.2, the United States.3 Platelets are involved in the blood coag- the platelets swell and there is a disk-to-sphere transforma- ulation process and are given to treat or prevent bleeding. tion in morphology that is associated with a loss of mem- They are given either therapeutically to stop bleeding or pro- brane integrity.40 The platelets then become irreversibly phylactically to prevent bleeding. Better availability and swollen, aggregate together, or lyse, and when infused, will management of platelet inventory has been a goal of blood not circulate or function. This change is irreversible when banks for many years. The financial impact of outdated and the pH falls to less than 6.2.40 During storage of platelet con- returned platelet units is the primary reason to find a way to centrates, the pH will remain stable as long as the production improve inventory management. Increasing the storage time of lactic acid does not exceed the buffering capacity of the during platelet preservation is one way to reduce the number plasma or other storage solution. Table 1–14 summarizes of outdated platelet units. With the limit of five days of stor- platelet changes during storage (the platelet storage lesion). age for platelet concentrates, approximately 20% to 30% of It should be noted that except for change in pH, the effect of the platelet inventory is discarded either by the blood sup- in vitro changes on post-transfusion platelet survival and plier or the hospital blood bank.4 function is unknown, and some of the changes may be re- versible upon transfusion.42 The Platelet Storage Lesion Generally, the quality-control measurements required by various accreditation organizations for platelet concentrates Platelet storage still presents one of the major challenges to include platelet concentrate volume, platelet count, pH of the the blood bank because of the limitations of storing platelets. unit, and residual leukocyte count if claims of leukoreduction In the United States, platelets are stored at 20°C to 24°C with are made.43 In addition, immediately before distribution to maintaining continuous gentle agitation throughout the stor- hospitals, a visual inspection is made that often includes an age period of 5 days. Agitation has been shown to facilitate assessment of platelet swirl (no visible aggregation).43 The oxygen transfer into the platelet bag and oxygen consump- absence of platelet swirling is associated with the loss of tion by the platelets. The positive role for oxygen has been membrane integrity during storage, resulting in the loss of associated with the maintenance of platelet component pH.40 discoid shape with irreversible sphering.44 Box 1–4 lists the  Chapter 1 Red Blood Cell and Platelet Preservation: Historical Perspectives and Current Trends 15 Table 1–14 The Platelet Storage Lesion apheresis (apheresis platelets). Currently, greater than 92% of platelet transfusions are from apheresed platelets and Characteristic Change Observed about 8% are pools of whole blood-derived platelets (WBD).3 Lactate Increased Platelets still remain the primary means of treating throm- bocytopenia, even though therapeutic responsiveness varies pH Decreased according to patient status and platelet storage conditions.43 ATP Decreased (See Chapter 15 for the methods for preparing platelet con- centrates.) One unit of whole blood-derived platelet concen- Morphology scores change Decreased trate contains ≥5.5 × 1010 platelets suspended in 40 to from discoid to spherical 70 mL of plasma.45 These platelets may be provided as a (loss of swirling effect) single unit or as pooled units; however, pooled units only Degranulation Increased have a shelf life of 4 hours. Apheresis platelets contain ((β-thromboglobulin, ≥3.0 × 1011 in one unit which is the therapeutic equivalent platelet factor 4) of 4 to 6 units of whole blood-derived platelets.45 There are Platelet activation markers Increased a number of containers used for 5-day storage of whole (P-selectin [CD62P] or CD63) blood–derived (WBD) and apheresis platelets. Box 1-5 lists the factors to be considered when using 5-day plastic Platelet aggregation Drop in responses to some agonists storage bags. Additive solutions may be used for storage of apheresis platelets. In the United States, platelets are being stored in a 100% plasma medium, unless a platelet additive solution is BOX 1–4 used. Two platelet additive solutions (PAS) are FDA ap- In Vitro Platelet Assays Correlated With proved, PAS-C (Intersol) and PAS-F (Isoplate), for the storage In Vivo Survival of apheresis platelets for 5 days.46 The PASs are designed to support platelets during storage in reduced amounts of resid- pH ual plasma. With the addition of a PAS, residual plasma is Shape change reduced to 35% with both InterSol and Isoplate.46 One Hypotonic shock response advantage is that this approach provides more plasma for Lactate production fractionation. In addition, there are data indicating that pO2 optimal additive solutions may improve the quality of platelets during storage, reduce adverse effects associated with transfusion of plasma, and promote earlier detection of in vitro platelet assays that have been correlated with in vivo bacteria.46 Box 1–6 lists the advantages of using a platelet survival. additive solution for platelet storage. Clinical Use of Platelets Platelet Testing and Quality Control Monitoring Platelet components are effectively used to treat bleeding as- For component testing, the FDA Guidance document rec- sociated with thrombocytopenia, a marked decrease in ommends: 1) Actual platelet yield (volume × platelet count) platelet number, or dysfunctional platelets. That is, platelets must be determined after each platelet collection; 2) Weight/ are transfused when there is a quantitative or qualitative de- volume conversion is necessary to determine the volume fect with the patient’s platelets. The efficacy of the platelet of each platelet collection; and 3) Bacterial contamination transfusion is usually estimated from the corrected count increment (CCI) of platelets measured after transfusion.22 The corrected count increment (CCI) is a calculated measure of patient response to platelet transfusion that adjusts for the BOX 1–5 number of platelets infused and the size of the recipient, based upon body surface area (BSA).22 Factors to Be Considered When Using 5-Day Plastic Storage Bags CCI = (postcount – precount) × BSA/platelets transfused Temperature control of 20°C to 24°C is critical during platelet where postcount and precount are platelet counts (/µL) after preparation and storage. and before transfusion, respectively. BSA is the patient body Careful handling of plastic bags during expression of platelet-poor surface area (meter2) and platelets transfused is the number plasma helps prevent the platelet button from being distributed of administered platelets (× 1011).22 The CCI is usually de- and prevents removal of excess platelets with the platelet-poor plasma. termined 10 to 60 minutes after transfusion. It should be Residual plasma volumes recommended for the storage of platelet noted that the CCI does not evaluate or assess function of concentrates from whole blood (45 to 65 mL). the transfused platelets.23 For apheresis platelets, the surface area of the storage bags needs Today, platelets are prepared as concentrates from whole to allow for the number of platelets that will be stored. blood (whole blood-derived platelet concentrates) and by 16 PART I Fundamental Concepts BOX 1–6 Table 1–15 Performance Criteria for Platelet Concentrate Collections Advantages of Using Platelet Additive Solutions Test Recommended Result Optimizes platelet storage in vitro Saves plasma for other purposes (e.g., transfusion or fractionation) Actual platelet yield of ≥ 3.0 × 1011 Facilitates ABO-incompatible platelet transfusions transfusable component Reduces plasma-associated transfusion side effects, such as febrile pH ≥ 6.2 and allergic reactions, and may reduce risk of transfusion-related acute lung injury (TRALI) Percent component ≥ 85% component retention if Improves effectiveness of photochemical pathogen reduction retention performed* technologies Residual WBC Count** Single collection: < 5.0 × 106 Potentially improves bacterial detection Double collection: Collection: < 8.0 × 106 or Components: < 5.0 × 106 Triple collection: Collection: < 1.2 × 107 or Components: < 5.0 × 106 testing as specified by the storage container manufacturer.45 In terms of Quality Control (QC), the FDA recommends *Or per the container/automated blood cell separator device manufacturer’s the following as a part of your QC: 1) “define a plan for non- specifications selectively identifying collections to be tested. This should **The stratified recommended results should ensure that the individual transfusable units will be < 5.0 × 106 even with a 25% error in equilibration of the volume for ensure testing of components collected on each individual double and triple collections. automated blood cell separator device, each collection type, Modified from: Guidance for Industry and FDA Review Staff: Collection of Platelets and each location.”45 2) “define sampling schemes for actual by Automated Methods (December 2007), available at: https://www.fda.gov/ platelet yield (including volume determination) and pH, downloads/BiologicsBloodVaccines/GuidanceComplianceRegulatoryIn formation/ Guidances/Blood/UCM062946.pdf and residual WBC.” (The platelet yield of the collection and designation of single, double, or triple PC should be made prior to performing the residual WBC count QC.) 3) “test actual platelet yield (platelet count times the volume) and to 24°C has been associated with the retention of post- pH at the maximum allowable storage time for the container transfusion platelet viability and has been the key issue that system used (or representing the dating period).” “In addi- has been addressed to improve conditions for storage at this tion, actual platelet yield and pH testing may be conducted temperature.47 on one storage container of a double or triple collection.” Retaining platelet function during storage is also an issue. 4) “include the residual WBC count for leukocyte-reduced Function is defined as the ability of viable platelets to collections, if manufacturing leukocyte-reduced products.” respond to vascular damage in promoting hemostasis. 5) “describe the criteria for investigation of failures during QC, and have a method to document all calculations and test Platelet Storage and Bacterial Contamination results.”45 Table 1-15 lists the Performance Criteria for Platelet Con- The major concern associated with storage of platelets at centrate Collections. 20°C to 24°C is the potential for bacterial growth if the pre- pared platelets contain bacteria because of contamination at Measurement of Viability and Functional the phlebotomy site or if the donor has an unrecognized Properties of Stored Platelets bacterial infection.48 Environmental contamination during processing and storage is another potential, though less com- Viability indicates the capacity of platelets to circulate after mon, source of bacteria. Room temperature storage and the infusion without premature removal or destruction. Platelets presence of oxygen provide a good environment for bacterial have a life span of 8 to 10 days after release from megakaryo- proliferation. Sepsis due to contaminated platelets is the cytes.6 Storage causes a reduction in this parameter, even most common infectious complication of transfusion.49 when pH is maintained. Viability of stored platelets is deter- According to the FDA, bacterial contamination was the third mined by measuring pretransfusion and post-transfusion leading cause of transfusion-associated fatality in 2015.50 It platelet counts and expressing the difference based on the is estimated that 1:2,000 to 1:5,000 units of platelets are con- number of platelets transfused (CCI). taminated with bacteria.51 An estimated 10% of patients The observation of the swirling phenomenon (absence transfused with a bacterially contaminated platelet unit of aggregation) caused by discoid platelets when placed in develop life-threatening sepsis which is often fatal.51 In 2002, front of a light source has been used to obtain a semiqual- the College of American Pathologists added a requirement itative evaluation of the retention of platelet viability prop- that laboratories have a method to screen platelets for erties in stored units.43 The extent of shape change and the bacterial contamination, and the AABB introduced a similar hypotonic shock response in in vitro tests appears to pro- requirement in 2004. Blood establishments and transfusion vide some indication about the retention of platelet viability services must assure that the risk of bacterial contamination properties.43 The maintenance of pH during storage at 20°C of platelets is adequately controlled using FDA approved  Chapter 1 Red Blood Cell and Platelet Preservation: Historical Perspectives and Current Trends 17 or cleared devices or other adequate and appropriate meth- transfusion but reduces the shelf-life of the platelets to ods found acceptable for this purpose by the FDA. [21 CFR 4 hours because they are prepared in an open system.45 The 606.145(a)] FDA defines “Prestorage pooled WBD platelets” as single Commercial systems such as BacT/ALERT (bioMérieux) units of WBD platelets pooled and tested ≥24 hours after col- and eBDS (Pall Corp.) have been approved by the FDA for lection, and stored in an FDA-cleared container for extended screening platelets for bacterial contamination. These are pool storage for up to 5 days (consistent with the container both culture-based systems.48 As the level of bacteria in the package insert). “Poststorage pooled WBD platelets” repre- platelets at the time of collection can be low, samples are sent stored single units of WBD platelets that are pooled not taken until after at least 24 hours of storage.48 This pro- within 4 hours prior to transfusion.45 vides time for any bacteria present to replicate to detectable In 2005, the FDA approved the use of prestorage-pooled levels. BacT/ALERT measures bacteria by detecting a platelets prepared by AcrodoseTM PL System.45 Acrodose change in carbon dioxide levels associated with bacterial platelets are pooled ABO-matched, leukoreduced WBD platelets growth.48 This system provides continuous monitoring of that have been cultured and are ready for transfusion.54 Acro- the platelet sample–containing culture bottles, which are dose systems are the only platelet pooling systems available held for the shelf-life of the platelet unit or until a positive in the United States that provide a clinically equivalent reaction is detected. The eBDS system measures the oxygen alternative to apheresis platelets.54 Because they are pro- content of the air within the sample pouch for 18 to duced in a closed system, they can be stored for 5 days from 30 hours following incubation.52 A decrease in oxygen level collection. They provide a therapeutic dose equivalent to indicates the presence of bacteria. BacT/ALERT and eBDS, apheresis platelets and at a lower cost, but they expose the which are used for screening platelets in the United States, recipient to multiple donors. A recent study comparing have documented good sensitivity and specificity; however, transfusion reactions from prestorage-pooled platelets, false-negative test results have been documented.52 With apheresis platelets, and poststorage-pooled WBD platelets both culture systems, the need to delay sampling and the found no difference in reaction rates among the different requirement for incubation delay entry of the platelet prod- products.55 Prestorage-pooled platelets may prove to be a ucts into inventory. Box 1–7 lists the disadvantages associ- useful adjunct to apheresis platelets, which are often in short ated with the use of culture methods for the detection of supply, and may lead to improved utilization of WBD bacterial contamination of platelets. platelets.54 The practice of screening platelets for bacterial contami- In November 2009, the FDA approved the first rapid test nation has reduced, but not eliminated, the transfusion of to detect bacteria in platelets—the Pan Genera Detection contaminated platelet products. False-negative cultures can (PGD) test (Verax Biomedical).56 The PGD test, which was occur when bacteria are present in low numbers and when previously approved by the FDA for testing leukocyte- the pathogen is a slow-growing organism. reduced platelets as an adjunct to culture, is an immunoas- Because current bacterial screening methods are not 100% say that detects lipoteichoic acids on gram-positive bacteria sensitive, they must be supplemented by other precautions, and lipopolysaccharides on gram-negative bacteria. Both such as the donor interview and proper donor arm disinfec- aerobes and anaerobes are detected. A sample of only tion. Another precaution is the diversion of the first aliquot 500 µL is required.56 Following pretreatment, the sample (about 20 to 30 mL) of collected blood into a separate but is loaded into a disposable plastic cartridge with built-in connected diversion pouch.53 This procedure minimizes the controls that turn from yellow to blue-violet when the test placement of skin plugs, the most common source of bacte- is ready to be read, in approximately 20 minutes. A pink rial contamination, into the WBD platelet products. bar in either the gram-positive or gram-negative test In view of the ability to test for bacterial contamination window indicates a positive result. The PGD test can be and the use of diversion pouches and sterile docking instru- performed by transfusion services just prior to release of ments, there is now interest in storing pools of platelets up platelet products.52 The optimum time for sampling is at to the outdate of the individual concentrates. Traditionally, least 72 hours after collection.56 four to six WBD platelets are pooled into a single bag by Transfusion services must either obtain their platelets the transfusion service just prior to issue. This facilitates from a collection facility that performs an approved test for bacterial contamination or they must perform an approved test themselves.52 At this time, the approved tests are bacte- BOX 1–7 rial culture or the Verax PGD test.48 The PGD rapid test has a sensitivity of 99.3% and a speci- Disadvantages of Culture Methods for Detection ficity of 60%.56 While it is essential that the rapid test is sen- of Bacterial Contamination of Platelets sitive enough to detect the majority of platelets containing Product loss due to sampling bacteria, a high false-positive rate could lead to discarding Delay in product release, further reducing already short shelf-life units of platelet that would be suitable for transfusion. The False-negative results false positive rate of 0.51% with the PGD test is a valid con- Cost cern because platelets are a blood product in short supply.52 Logistical problems of culturing WBD platelets Despite sensitive methods to detect bacteria in platelets, septic transfusion reactions still occur.