A comparison of frozen and fresh platelet concentrates in the support of thrombocytopenic patients

Ten patients scheduled to receive intensive chemotherapy were plateletapheresed and the platelet-rich plasma was frozen with 5 percent dimethyl sulfoxide at -80 to -95 degrees C until needed. Paired comparisons of frozen autologous platelets with fresh single-donor platelets were made in seven patients using corrected platelet increments at 1 and 24 hours, and pre- and posttransfusion bleeding times. In vitro tests of 12 units of platelet-rich plasma before and after freezing included platelet factor 4 (PF4) secretion, malondialdehyde production, and electron microscopic evaluation of morphology. Fresh platelets provided significantly better 1- and 24-hour corrected increments compared with frozen autologous platelets. In only one case of alloimmunization did frozen autologous platelets provide a better increment than fresh platelets. Bleeding times after transfusion showed no consistent improvement regardless of type of transfusion or platelet count. Secretable PF4 remained constant after freezing, but malondialdehyde production fell significantly. Platelets showed considerable structural damage with 33 percent balloon forms counted after thawing, compared to less than 1 percent before freezing. Except in the case of alloimmunization, frozen autologous platelets are inferior to single-donor fresh platelets, and are significantly damaged in the freezing process.     

Effectiveness of Platelet Transfusion in Leukemia and Aplastic Anemia

Hemorrhage resulting from thrombocytopenia in patients with acute leukemia and aplastic anemia can be controlled by platelet transfusions. Severe gross hemorrhage was rarely observed when platelet counts were higher than 20,000 per cu. mm. Transfusion of 1 × 10¹¹ platelets produced an average increment of 12–14,000 platelets per cu. mm./square meter (m²) of body surface in acute leukemia. One unit of platelet rich plasma (PRP) contains an average of 1 × 10¹¹ platelets and 4 PRP/m² twice weekly will maintain the platelets above 20,000 per cu. mm. most of the time. When very large doses of platelets are required in a small volume then platelet concentrates (PC), prepared by centrifuging PRP and removing most of the plasma, are used. PC are 80 to 90 per cent as effective as PRP in elevating the platelet count if prepared from plasma with a p H of 6.8 or less, achieved by the addition of citric acid.
The major hazard of platelet transfusion is posttransfusion hepatitis. This can be minimized by the use of plasmapheresis thus using the same donors repeatedly. In the presence of anemia platelets can be given effectively in fresh whole blood transfusions until the patient's hematocrit is raised.     

Viability and function of platelet concentrates stored in CPD-adenine (CPDA-1)

The effective use of CPDA-1 as an anticoagulant in routine blood banking practice requires demonstration that platelet concentrates prepared in this solution meet both in vitro quality control standards and maintain posttransfusion viability and function after storage. In this study of 138 units of CPDA-1 platelet concentrates, the average platelet count was 8.0 +/− 0.2 × 10(10) with 81 per cent of the units having greater than 5.5 × 10(10) platelets. The mean poststorage pH was 6.68 +/− 0.03 and only four of the units had a pH of less than 6.0 (3%). Residual plasma volume averaged 75 +/− 1 ml. Platelet viability was determined in 16 normal volunteers by measuring survival of 51Cr- labeled autologous platelets after storage for 72 hours at 22 +/− 2 C. Platelet recovery averaged 50 +/− 4 per cent, while survival was 7.3 +/− 0.4 days for the 15 units with a pH above 6.0. Measurements of posttransfusion platelet viability and function were made in 12 paients with thrombocytopenia secondary to marrow failure. Their mean pretransfusion platelet count was 17,000 +/− 2,000/microliter, and their standardized template bleeding times were all greater than 30 minutes. Platelet recovery averaged 44 +/− 5 per cent and survival 3.3 +/− 0.5 days. In seven of the patients with the best posttransfusion increments, bleeding time was improved. Five patients with poor posttranfusion platelet increments showed no improvement in bleeding time with CPDA-1; two of these patients were also transfused with CPD platelets and had no response. Our studies indicate that platelet concentrates prepared in CPDA-1 meet in vitro quality control standards and after transfusion, maintain viability and function comparable to that of CPD collected platelets.     

Recovery, lifespan, and function of CPD-Adenine (CPDA-1) platelet concentrates stored for up to 72 hours at 4 C

We studied the in vivo recovery, lifespan, and hemostatic effectiveness of CPDA-1 platelet concentrates stored for up to 72 hours at 4 C. A total of 120 CPDA-1 concentrates containing an average (+/− 1 S.D.) of 6.6 +/− 2.0 × 10(10) platelets were prepared. The pH of the units following storage at 4 C was 6.8 +/− 0.2; no unit had a pH below 6.3. Autologous transfusion of six normal volunteers showed that platelets stored at 4 C for 72 hours had an in vivo recovery of 40 +/− 18 per cent and a lifespan of 5.1 +/− 1.5 days. The hemostatic effectiveness of CPDA-1 platelets was determined by platelet counts and template bleeding time measurements in 10 thrombocytopenic patients. Patients receiving 48-hour-stored platelets had a four- to six-hour posttranfusion corrected platelet increment averaging 15,300 +/− 3,200/microliter which was 67 +/− 34 per cent of expected recovery. Four of the five patients transfused with this preparation showed an improved bleeding time. In contrast, three patients receiving 72-hour- stored platelets had a four- to six-hour posttransfusion increment of 5,800 +/− 2,400/microliter that was only 26 +/− 13 per cent of the expected recovery; furthermore, only one of these patients showed any correction of the bleeding time. These data indicate that CPDA-1 platelets are hemostatically effective when stored at 4 C for up to 48 hours.     

Treatment of refractoriness to platelet transfusion by protein A column therapy

Ten thrombocytopenic patients (platelets < 10–24 × 10(9)/L) who were refractory to platelet transfusion were investigated for their responsiveness to staphylococcal protein A column therapy. Nine patients had previously been treated with steroids, intravenous immune globulin, and/or other forms of immunosuppressive therapy without improvement in their transfusion response. All patients were receiving multiple platelet transfusions without achieving 1-hour corrected count increments (CCIs) > or = 7500. Eight patients had antibodies that reacted with platelets and were directed against HLA class I antigens, ABO antigens, and/or platelet-specific alloantigens. Plasma (500-2000 mL) from each patient was passed over a protein A silica gel column and then returned to the patient. Patients received from 1 to 14 treatments. A positive response to protein A therapy was defined as at least a doubling of the pretreatment platelet count and/or two successive 10- to 120-minute posttransfusion CCIs > or = 7500. Following plasma treatments, 6 of 10 patients responded with daily platelet counts that averaged 48 +/− 11 × 10(9) per L as compared with counts of 16 +/− 7 × 10(9) per L (p < 0.0005) before treatment. Posttransfusion CCI values determined in four of these patients averaged 2480 +/− 810 and 10,010 +/− 3540 (p < 0.005) before and after treatment, respectively. In contrast, among the four unresponsive patients, platelet counts averaged 10 +/− 9 and 13 +/− 10 × 10(9) per L (p = NS), respectively, while posttransfusion CCIs were 700 +/− 1410 and 1520 +/− 2460 (p = NS), respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

Platelet preservation by freezing. Use of dimethylsulfoxide as cryoprotective agent

Variables important in the preservation of platelets by freezing with dimethylsulfoxide (DMSO) as cryoprotective agent were studied in normal volunteers and thrombocytopenic patients. Use of 5 per cent DMSO and a freezing rate of 1-3 degrees C/minute yielded optimal preservation of platelet viability. The addition of 5 per cent Dextrose did not improve results. In vivo yield using 5 per cent DMSO was superior to previous results in which glycerol was used as the cryoprotective agent. Viability after freezing was equivalent when platelets were frozen in small (10 ml) and large (60 ml) volumes of plasma. The larger volume had the advantage that a smaller percentage of the platelets was lost during transfer from one plastic container to another.     

Efficacy of the Latham Blood Processor To Perform Plateletpheresis

Platelets were separated during extracorporeal circulation by the method of Tullis et al . in the Latham Blood Processor equipped with a 375 ml disposable centrifuge bowl. The average yield of platelets recovered from each 710 ml unit of blood was 0.665 × 10¹¹ ± 0.244 × 10¹¹ (± 1 S. D.) N = 105, corresponding to an efficiency of 45.8 ± 12.4 per cent.
Platelets were also separated during extracorporeal circulation by a modification of the method. In this technic the final platelet concentrate contained about 50 ml of red blood cells that were removed by an additional centrifugation. The average quantity of platelets recovered from each 710 ml of blood was 1.23 × 10¹¹ ± 0.285 × 10¹¹ (± I S. D.) corresponding to an efficiency of removal of 69.5 ± 14.6 per cent (± I S. D.). The modified method permitted a collection of large numbers of platelets from single donors in a relatively short time.
Donor effects of the procedure were insignificant. Mild to moderate donor reaction consisting of chilling and hypotensive reactions occurred in three of 145 donors. Transient decreases occurred in hematocrit and platelet count. No untoward effects were observed following repeated procedures.     

Viability and function of platelets frozen at 2 to 3 C per minute with 4 or 5 per cent DMSO and stored at −80 C for 8 months

Each of 15 healthy male volunteers was treated with 650 mg of aspirin 24 hours before the autologous transfusion of one unit of freeze- preserved platelets. Freeze-thaw-wash recovery values in vitro, viability and function in vivo, and the bleeding time and platelet aggregation response were measured. The platelets were frozen with 4 or 5 per cent dimethylsulfoxide (DMSO) at an overall rate of 2 to 3 C per minute and were stored at −80 C in a mechanical freezer for up to eight months. They were washed by dilution/centrifugation. The mean recovery in vitro of platelets frozen with 4 per cent DMSO was 76+/−16%; the value was 64+/−16% for platelets frozen with 5% DMSO. The mean in vivo 51Cr recovery of autologous platelets frozen with 4% DMSO was 34+/−6%, and for platelets frozen with 5% DMSO it was 33+/−7%. In both groups the platelet lifespan was normal. There was a significant reduction in bleeding time after the transfusion of a single unit of autologous platelets preserved with either 4 or 5% DMSO, but no improvement in the aspirin-induced platelet aggregation pattern.     

Platelet collection and transfusion using the fenwal CS-3000 cell separator

A prototype model of the Fenwal CS-3000 Blood Cell Separator (Deerfield, IL) was studied for plateletpheresis in 63 donors and 5 transfusions in patients. Donor effects were consistent with platelet removal and mild hemodilution. The incidence of reactions (9 of 63) was low and all were mild "citrate" type. A two-hour collection yielded 4.0 ± 0.72 × 10(11) platelets at an efficiency of 45 ± 6.9 per cent. The product had little contamination with leukocytes (0.26 ± 1.2 × 10(9) and red blood cells (hematocrit less than 1%). Morphology and pH were well preserved during 24 hours of storage. Four patients with uncomplicated aregenerative thrombocytopenia were transfused on five occasions, with a mean of 4.5 ± 0.87 × 10(11) platelets resulting in a mean platelet count increment of 55,000/microliter and dramatic reduction in template bleeding times.     

Statewide support of thrombocytopenic patients with ABO matched single donor platelets

Use of ABO matched, HLA nonmatched platelet units obtained from single donors by pheresis, using the Haemonetics 30 Cell Separator, has a lowered hepatitis risk, and possibly delays the onset of the refractory state. Pheresis also offers a method of obtaining HLA matched platelets for the already refractory patient. A mean of 4.2 × 10(11) platelets are collected in about 1 1/2 hours. ABO matched, HLA nonmatched platelets produced corrected increments of more than 2500/mm3 per 7 × 10(10) platelets infused in 67 per cent of transfusions to nonselected recipients. The procedure is simple, safe, and requires little time. It is a worthwhile large-scale program for a regional blood center to undertake in order to provide optimal therapy for thrombocytopenic patients.     

Cryopreservation of platelet concentrates using glycerol-glucose

Two to five units of platelet concentrate were frozen together in single bags using glycerol-glucose as cryoprotective agents and a special freezing plate which produced a reproducible, rapid freezing rate when immersed in liquid nitrogen. After thawing there was no loss in platelets compared to prefreezing controls. Phase microscopic evaluation after thawing, however, demonstrated large numbers of severely damaged platelets and a significant decrease in morphology score. Total ATP levels fell to 37–52% of controls, and thawed platelets did not aggregate with adenosine diphosphate or collagen or undergo release of nucleotides following incubation with thrombin. In vivo recovery one hour after transfusion of autologous platelets administered to patients with leukemia was significantly inferior (p less than 0.025) to results achieved in the same patients with autologous platelets frozen with dimethylsulfoxide. These results indicate that significant damage occurs following freezing and thawing utilizing glycerol-glucose as cryoprotective agents for frozen platelets and that further investigation is required prior to their clinical use. Blood banks currently interested in platelet cryopreservation for clinical use should utilize dimethylsulfoxide as a cryoprotective agent.     

Therapeutic transfusions of previously frozen washed human platelets

The platelets used in this study were collected by serial centrifugation, and within four hours of collection were frozen with 5% dimethylsulfoxide (DMSO) at an overall rate of 2 to 3 C per minute by storage in a mechanical refrigerator at −80 C. The frozen platelets were stored for four to ten weeks before thawing and washing. After washing, the units were kept at room temperature for six to eight hours before transfusion. The units were pooled, and an average of eight units was given to each of four patients, with a range of three to 14 units per transfusion. In vitro recovery after washing was about 65 per cent and in vivo recovery of the 51chromium labeled (51Cr) platelets was about 35 per cent. The infusion of these previously frozen washed platelets corrected prolonged bleeding times in patients. The homologous platelets were transfused along with other blood products to treat patients with hematologic disorders. The circulation and function of the donor platelets were influenced by compatibility of the platelets, the quality of platelet preservation and the patient's disease state.     

Dimethylacetamide, a New Cryoprotective Agent for Platelets

Dimethylacetamide (DMAC) was used as cryoprotective agent for the preservation of platelets in the frozen state. The addition of dextrose to the platelet suspension greatly increased the cryoprotective effects of this agent. Rat platelets preserved with 5 per cent DMAC and 5 per cent dextrose showed a higher percentage of morphologically intact cells and higher clot retraction-promoting activity than platelets frozen in other concentrations of these two agents. The infusion of these platelets into thrombocytopenic rats resulted in platelet-count increments greater than those observed after administration of platelets frozen in 5 per cent dimethylsulfoxide (DMSO) and 5 per cent dextrose. At room temperatures, DMAC appeared to be less damaging to platelets than DMSO. Transfusions of human platelets frozen with DMAC and dextrose to thrombocytopenic patients with acute leukemia were associated with temporary increases of their platelet counts.     

High-yield platelet concentrates attainable by continuous quality improvement reduce platelet transfusion cost and donor exposure

BACKGROUND: Donor exposure risk and cost in platelet transfusion practice can be limited by increasing the recovery of platelets from donor units. STUDY DESIGN AND METHODS: This study presents results of continuous quality improvement efforts in platelet production and compares the in vivo therapeutic efficacy of currently produced platelet concentrates (PCs) with that of apheresis platelets. Production quality improvement measures included optimization of instrument performance (rotor speed trials), process (massaging whole- blood units, using cup liners, limiting spin-expression time, and refining plasma expression technique), and staff (intensive training with observation and ongoing quality control data feedback). Corrected count increments and increments per kg were calculated for transfusions of 4 pooled PCs and apheresis platelets over a 30-day period. RESULTS: The mean number of platelets per PC increased from 5.5 × 10(10) in 1975 to 9.69 × 10(10) in 1994. The mean platelet dose was 3.78 × 10(11) for 4 PCs and 4.17 × 10(11) for apheresis platelets. A total of 34 pooled PCs and 17 apheresis platelets was transfused to 21 patients. The mean increment, the increment per kg, and the corrected count increment were, respectively, 31 × 10(3) per microL, 4.8 × 10(2) per microL, and 14,700 for 4 PCs and 35.4 × 10(3) per microL, 5.4 × 10(2) per microL, and 14,700 for apheresis platelets. Differences were not significant. CONCLUSION: Therapeutic efficacy comparable to that of apheresis platelets can be obtained with 4 high-yield PCs.     

Effect of storage time on clinical efficacy of single-donor platelet units

Decrements in platelet function and recovery are known to accumulate during the 5-day storage period, and the clinical response to platelets transfused after several days' storage has been suggested by some researchers to be less than that seen with platelets stored for shorter periods. The clinical response to single-donor platelet (SDP) units (as measured by corrected count increments [CCIs] and intertransfusion intervals) was investigated in autologous bone marrow transplant patients. Twenty-seven consecutive autologous bone marrow transplant patients with a variety of hematologic and solid organ malignancies were evaluated for posttransfusion CCIs after 419 SDP transfusions of units stored for 1 to 5 days. Patients were not excluded from the study because of clinical condition, such as fever or sepsis. The mean 15- minute posttransfusion CCI for SDP units stored for only 1 day (11,006 +/− 5,157) was no different than that for units stored for 5 days (10,225 +/− 4,481; p > 0.05); 24-hour posttransfusion CCIs were also not different if the SDP unit had been stored for 1 day or 5 days (6229 +/− 4489 vs. 4786 +/− 2759; p > 0.05) or for any intermediate period. Nor were intertransfusion intervals affected by storage time. While platelets may exhibit a progressive lesion during the 5-day storage period, these changes do not result in a decreased clinical response.     

Cryopreservation of platelets simplified: a modified glycerol-glucose method

Platelets were prepared for freezing in liquid nitrogen by prediluting with 1 ml glycerol-glucose freezing solution, allowing the platelet concentrate to set for ten minutes at 29 C, and then adding an additional 8 ml glycerol-glucose freezing solution. A 3-mm thick cardboard holder was constructed and used to hold the platelet concentrate during immersion and storage in liquid nitrogen. The mean recovery of frozen thawed platelets was 79 per cent. Electron microsopic examination of the frozen thawed platelets showed that the platelets to be structurally intact. In vitro testing showed the platelets to be functional.     

Review of In Vivo Studies of Dimethyl Sulfoxide Cryopreserved Platelets

A literature review was conducted to assess the efficacy and safety of dimethyl sulfoxide (DMSO) cryopreserved platelets for potential military use. In vivo DMSO cryopreserved platelet studies published between 1972 and June of 2013 were reviewed. Assessed were the methods of cryopreservation, posttransfusion platelet responses, prevention or control of bleeding, and adverse events. Using the Department of Defense's preferred 6% DMSO cryopreservation method with centrifugation to remove the DMSO plasma before freezing at ? 65°C and no postthaw wash, mean radiolabeled platelet recoveries in 32 normal subjects were 33% ± 10% (52% ± 12% of the same subject's fresh platelet recoveries), and survivals were 7.5 ± 1.2 days (89% ± 15% of fresh platelet survivals). Using a variety of methods to freeze autologous platelets from 178 normal subjects, mean radiolabeled platelet recoveries were consistently 39% ± 9%, and survivals, 7.4 ± 1.4 days. More than 3000 cryopreserved platelet transfusions were given to 1334 patients. There were 19 hematology/oncology patient studies, and, in 9, mean 1-hour corrected count increments were 11 100 ± 3600 (range, 5700-15 800) after cryopreserved autologous platelet transfusions. In 5 studies, bleeding times improved after transfusion; in 3, there was either no improvement or a variable response. In 4 studies, there was immediate cessation of bleeding after transfusion; in 3 studies, patients being supported only with cryopreserved platelets had no bleeding. In 1 cardiopulmonary bypass study, cryopreserved platelets resulted in significantly less bleeding vs standard platelets. In 3 trauma studies, cryopreserved platelets were hemostatically effective. No significant adverse events were reported in any study. In summary, cryopreserved platelets have platelet recoveries that are about half of fresh platelets, but survivals are only minimally reduced. The platelets appear hemostatically effective and have no significant adverse events.     

The Purification of Red Cells for Transfusion by Freeze Preservation and Washing

In the study of 125 units of washed, previously frozen red blood tells approximately 96.6 per cent of the leukocytes and 98.9 per cent of the platelets were removed. Less than 0.5 mg of plasma protein remained per unit. The length of storage as whole blood at 4 C prior to glycerolization of the red blood cells influenced the number of leukocytes in the washed, previously frozen red blood cells. Red blood cells frozen on the day of collection had 7.2 per cent residual white cells, whereas those stored at 4 C for 10 days or longer prior to glycerolization, freezing, and washing had 1.1 per cent residual leukocytes. When red blood cells were stored at 4 C for longer than one week, glycerolized, and washed without freezing, they had about 2 per cent residual leukocytes and platelets. The washing procedure removed at least 95 per cent of the platelets from nonfrozen and previously frozen red blood cells. Because of the removal of large numbers of leukocytes and platelets during the freeze-preserva-tion procedure and the excellent recovery of the red blood cells, washed, previously frozen glycerolized red cells are recommended for patients requiring red blood cells depleted of leukocytes, platelets, and plasma protein.     

ABO compatibility can influence the results of platelet transfusion. Results of a randomized trial

Sixty consecutive patients with untreated acute leukemia alternately received either ABO-matched or ABO-mismatched random-donor platelet transfusions prepared from pooled platelet concentrate stored for 1 to 3 days. Patients were assigned randomly to receive matched or mismatched platelets as their first transfusion, and the first four transfusions were analyzed. In 40 evaluable patients, there was no significant difference (paired t test) between the 10-minute posttransfusion corrected count increments (CCI) of the initial transfusions of matched and mismatched platelets. In contrast, the second matched transfusion was significantly better than the second mismatched transfusion. This effect of ABO compatibility was particularly pronounced in a subset of patients. Six patients in whom mismatched transfusions were consistently inferior to matched transfusions had either a significant increase in anti-A or -B isoagglutinin titers following the first transfusion or elevated titers before or at the conclusion of the study. Conversely, in five patients in whom there was no apparent effect of ABO mismatching, only one had an increase in isoagglutinin titer. Platelet survival was not altered as the ratio of 18-hour to 10-minute posttransfusion CCl was 0.6 for both matched and mismatched platelet transfusions. These data demonstrate that ABO compatibility can affect the results of random-donor platelet transfusions and that patients who experience poor increments from ABO-mismatched platelets may benefit from a trial of ABO-compatible platelets before the initiation of HLA-matched platelet transfusion.     

Description of a closed plastic bag system for the collection and cryopreservation of leukapheresis-derived blood mononuclear leukocytes and CFUc from human donors

Hemopoietic stem cells were collected from blood by means of continuous- flow centrifugation. The therapeutic use of large quantities of autologous blood stem cells requires a suitable, reliable and easily practicable cryopreservation technique which prevents loss of cell number and viability. This paper describes a closed plastic bag system consisting of one part for the collection and freezing of blood-derived mononuclear cells (MNC), among them granulocyte/macrophage progenitor cells (CFUc), and of a second part for thawing the cryopreserved cells and washing them free of DMSO before transfusion to a patient. In a series of 20 leukaphereses, the average number of collected MNC and CFUc was about 11 × 10(9) and 8 × 10(5) respectively. The recovery rate of the leukapheresis derived MNC and CFUc after cryopreservation, thawing, and washing was demonstrated to be 90 per cent of better. Sequential leukaphereses in the same donor showed little effect on red and white blood cell concentration. However, there was a significant decrease in the donor's blood platelet concentration prior to the third leukapheresis.