Correction of poor platelet transfusion responses with leukocyte-poor HL-A-matched platelet concentrates.

Matching donor-recipient pairs for HL-A antigens provides a logical starting point for selecting donors for recipients with extensive prior transfusion histories. However, during the course of continued exposure to even HL-A-matched platelet concentrates, further sensitization occurs, as indicated by progressively poorer post-transfusion increments and transfusion reactions. There is evidence that such sensitization may be due to non-HL-A antigens. Finally, it is postulated that the poor post-transfusion platelet increments obtained when standard platelet concentrates are used result from the leukoagglutinin antigen-antibody reaction involving the platelet as an "innocent bystander." The standard platelet concentrate can be purified by a simple method of centrifugation (178 g times 3 min), removing about 96% of the contaminating white blood cells with concomitant loss of about 21% of the platelets. The use of these leukocyte-poor platelet concentrates can restore compatible transfusion increments in highly alloimmunized thrombocytopenic recipients. The luekocyte-poor concentrates can diminish undesirable transfusion reactions following imcompatible platelet transfusions.     

Prevention of HLA immunization with leukocyte-poor packed red cells and platelet concentrates obtained by filtration

HLA immunization is a common complication of transfusion therapy in 30% to 60% of oncohematologic patients. Evidence shows that leukocytes present in cellular blood products are the main component involved in the occurrence of HLA immunization, and several studies showed that leukocyte-poor blood products are less able to induce it. However, leukocyte-poor platelet concentrates obtained by conventional techniques, ie, centrifugation, frequently have a high level of remaining leukocytes. Cotton wool filter Imugard IG 500 can be used to obtain leukocyte-poor cellular blood products. The technique is easy to perform, even in an emergency, and can be used with either packed RBCs or platelet concentrates. Means of 97%, 92%, and 76% elimination of leukocytes are obtained for packed RBCs, pooled standard platelet concentrates, and single-donor platelet concentrates, respectively. Patients were randomized to receive either standard (control group) or filtered (leukocyte-poor group) blood products. Of 112 randomized patients, 69 were evaluable, 35 in the control group and 34 in the leukocyte-poor group. Both groups are comparable according to age, diagnosis, sex ratio, previous transfusions, and pregnancies. There is a significant difference in regard to the HLA immunization rate (31.4% in the control v 11.7% in the leukocyte-poor group, P less than .05) and frequency of refractoriness to platelet transfusions (46.6% v 11.7%, P less than .05). We conclude that this filtration technique can be an efficient means to reduce the HLA immunization rate in polytransfused oncohematologic patients.     

Preservation of platelet function in cryopreserved platelet concentrates with prostacyclin

Prostacyclin (Epoprostenol) or a stable prostacyclin analogue (ZK 36,374) were added to platelet concentrates prior to cryopreservation. This resulted in significantly better preserved function of the thawed platelet concentrate, assessed by platelet aggregation to various concentrations of ADP, collagen and ristocetin, compared to control cryopreserved platelet concentrates. The use of prostacyclin or one of its stable analogues should be considered to reduce platelet activation and subsequent loss of function during the various manipulative procedures when preparing standard and cryopreserved platelet concentrates.     

Evaluation of leukocyte-depleted platelet concentrates obtained by in-line filtration

BACKGROUND AND OBJECTIVES: The use of leukocyte-depleted platelet concentrates (PCs) is justified, yet questions remain regarding their properties. We have assessed the in vitro quality of WBC-reduced PCs obtained by using a new in-line filter. MATERIALS AND METHODS: Twenty blood units were randomized for standard component preparation, or for processing including in-line leukodepletion of platelet-rich plasma using an ATS(TM) PL Pall filter. The resultant PCs were compared during storage for several in vitro platelet quality parameters, content of cytokines and anaphylatoxins, and coagulation markers. RESULTS: In-line filtration of platelet-rich plasma through ATS PL was highly efficient rendering PCs with 99.93% less white blood cells (WBCs) than standard PCs. During storage for up to 9 days, leukocyte-depleted units displayed platelet content, biochemical parameters, and platelet surface expression of glycoproteins Ibalpha, IIb/IIa, CD62 and CD63, similar to those of standard units. However, in-line leukocyte-reduced PCs displayed no significant accumulation of IL-6 and IL-8 during storage in contrast to standard units. Moreover, while storage promoted complement activation with C3a and C4a liberation in both WBC-reduced PCs and standard units, the concentration of these anaphylatoxins after the 9-day storage period was higher in the former group. Finally, all units, standard and leukocyte-reduced, displayed a similar storage-promoted rise in TGF-beta(1), and a mild prolongation of activated partial thromboplastin and prothrombin times. CONCLUSION: In-line filtration during component preparation appears as an easy and effective procedure for obtaining prestorage leukocyte-depleted PCs. During subsequent blood bank storage, these WBC-reduced PCs display, in comparison with standard PCs, normal in vitro platelet properties, decreased accumulation of cytokines IL-6 and IL-8, and increased complement activation.     

Preparation of leukocyte-poor platelet concentrates from buffy coats by a new type of quadruple bag

Summary A new quadruple blood bag system for collection of platelet concentrates is described. The principle modification consists of a smaller (150 ml) buffy coat bag in conjunction with SAG-mannitol for red cell preservation. Platelet yield and function were comparable to conventional techniques, leukocyte contamination was lower (1.7 ± 1.3×10⁷ per unit). The system is recommended for specialized institutions with a high demand of blood components.     

Stabilization of standard platelet concentrates and minimization of the platelet storage lesion by a prostacyclin analogue

Summary Platelet concentrates were pretreated with a stable synthetic prostacyclin analogue (Iloprost) at two different concentrations before the second centrifugation step (pelleting step) of preparation. This resulted in loss of platelet sensitivity to aggregating agents. To mimic the situation after transfusion and to assess the reversibility of platelet inhibition, platelets were washed during and after storage and resuspended in fresh-frozen autologous plasma. The Iloprost-treated and washed platelets exhibited an increased sensitivity to the aggregating agents, compared with the control platelets (p< 0.01). Post-storage recovery of the synergistic aggregation was more than 80% of prestorage aggregation, -thromboglobulin (TG) release and thromboxane B2 (TXB2) formation were significantly inhibited in Iloprost-treated platelets (p< 0.01). After the second centrifugation step, TG release was 0.7%±0.3%, compared with 2.7%±0.9% for the controls. TXB2 was 99±91 pg/ml, compared with 495±356 pg/ml for the controls. Platelet morphology and ultrastructure were well preserved during 5-day storage. In addition, Iloprost exerted a cytoprotective effect, as evidenced by the significant reduction in lactate dehydrogenase leakage. Post-storage LDH was 378±159 and 415±239 U/l respectively by the two Iloprost concentrations, compared with 1180±937 U/l for the control platelets. The inhibitory and cytoprotective effects of Iloprost were sustained throughout storage, in contrast to the effect of PGE1 (Prostin) which was limited to the early phase of storage.     

In vitro function of platelet concentrates prepared after filtration of whole blood or buffy coat pools

BACKGROUND/METHOD: Data on the quality of platelet concentrates (PC) produced by the buffy coat method and stored beyond 5 days in plasma are limited. We therefore evaluated the quality of PCs prepared by leucocyte depletion of whole blood (Terumo WBSP, n = 10) or a buffy coat pool (Pall Autostop, n = 10), and stored for 7 days in plasma by assessing platelet parameters and markers of platelet activation. RESULTS: In both types of PC, levels of glucose decreased during storage but were not totally depleted (> 11 mM on day 7). In contrast, lactate levels increased on storage and was consistently < 20 mM throughout, with pH maintained at > 6.8 in all units. Hypotonic shock response scores were > 47% in all units at day 7. On day 1, markers of platelet activation were significantly higher in WBSP PC, but by day 7 were similar for percentage CD63+ and CD62P + (40%) with levels of platelet microparticles and annexin V binding two-fold higher in WBSP. The expression of CD61 did not alter during storage and the percentage of platelets expressing CD42b was > 88% in all units on day 7. RANTES (Regulated on activation, normal, T-cell expressed and secreted) and TGFbeta released from platelets by day 7 was < 800 ng/ml and 90 ng/ml, respectively. C3a(desarg) increased throughout storage in both types of PC, but without a commensurate increase in the terminal complex SC5b-9 or activation of factor XII. CONCLUSION: Our data indicates that the in vitro characteristics of PCs prepared using these methods is maintained over storage for 7 days in plasma and is not associated with significant deterioration of platelet function. ONE SENTENCE SUMMARY: In vitro function of platelet concentrates prepared by either filtration of whole blood, or pooled buffy coats.     

Therapeutic effectiveness of frozen platelet concentrates for transfusion

Six patients received platelet concentrate transfusions from their HLA- identical siblings. Platelet concentrates were administered either fresh, or after being frozen in 10% dimethylsulfoxide, at a slow controlled rate (1 degree C/min) or rapidly (approximately 8 degrees C/min) in the vapor-phase of a liquid nitrogen refrigerator. The median freeze-thaw loss was 13.5%. The mean 1-hr and 20-hr corrected increments in platelet count were calculated for fresh platelet concentrates transfused before and after transfusion with controlled- rate frozen and vapor-phase frozen platelet concentrates. There was no significant difference among the first and second transfusion of fresh platelet concentrates, nor was the difference observed between fresh and controlled-rate frozen platelet concentrates significant. The difference between fresh and vapor-phase frozen platelet concentrates, and between controlled-rate frozen and vapor-phase frozen platelet concentrates were highly significant (p < 0.01). In vitro tests of aggregation using ristocetin and platelet ultrastructural studies paralleled the transfusion experience. Our results indicate that HLA- identical platelet concentrates can be successfully frozen and thawed for transfusion if a slow, controlled rate of freezing is employed. The use of HLA-identical frozen platelet concentrates may be important in emergency situations for the refractory patient and potentially for the establishment of a platelet concentrate bank.     

Quality of pooled platelet concentrates prepared from buffy coats and stored in an additive solution after filtration

Platelet concentrates prepared from buffy coat were pooled and stored for 6 days after removal of leukocytes by filtration. The platelets were stored in plasma or in an additive solution, Plasmalyte-A. In vitro platelet function was better preserved using Plasmalyte-A than plasma with regard to osmotic reversal and aggregation. No significant differences for the release of platelet markers-thromboglobulin, platelet factor 4, or lactate dehydrogenase pre- and post-filtration and storage in plasma or Plasmalyte-A was observed. Expression of the surface membrane glycoproteins Ib, Ia/ IIa, Ilb/IIIa, and IV measured by flow cytometry after binding of monoclonal antibodies did not change during storage. The expression of activation-dependent- granula glycoprotein GMP140, the thrombospondin, and the glycoprotein 53 from the lysosomal granules was not different between platelet pools stored in plasma or in Plasmalyte-A. The in vitro quality of platelets stored as pools is comparable for plasma and the additive solution Plasmalyte-A.Part of this work (R.W.) served as requirement for the degree of M.D. at the University of Ulm     

Lipid composition of freshly prepared and stored platelet concentrates

To evaluate the possibility that changes in lipid composition might be related to the functional lesion that develops when platelets are stored as concentrates for several days, we measured lipid constituents of platelets in freshly prepared concentrates and in concentrates stored for 72 hr at 4 degrees C or at 20 degrees C under standard blood banking conditions. At 20 degrees C, but not at 4 degrees C, platelets lost about 15% of total cholesterol and 7%--11% of total phospholipid. The distribution of individual phospholipids remained unchanged. This was also true of the fatty acid distribution in total phospholipids and in individual phospholipids except for a statistically significant reduction of linoleic acid (18:2) and an increase in oleic acid (18:1) in phosphatidyl inositol (PI). Platelets collected in citrate-phosphate- dextrose (CPD) anticoagulant did not differ significantly in lipid composition from those collected in acid-citrate-dextrose (ACD) anticoagulant during the period of observation. These findings do not provide a basis to suggest that functional abnormalities developing in stored platelets are related to changes in lipid composition.     

Preparation of leukocyte-poor platelet concentrates via a short, hard spin of a pool of buffy coats

BACKGROUND AND OBJECTIVES: A new method for the preparation of leukocyte-poor platelet concentrates was developed, based on a short, hard spin of a pool of 5 buffy coats (BCs) combined with automated collection of the platelets. MATERIALS AND METHODS: The characteristics of platelet concentrates (PCs) were studied as a function of the total g force applied to a pool of 5 BCs. Pools of BCs were centrifuged for 1 min with a total g force ranging from about 3,300 to 5,000 gmin (n = 7-9 per applied g force). Deceleration took place without the means of a brake. The total centrifugation time was about 11 min. The platelet-rich plasma (PRP) fraction above the cell layer was separated by an automated component preparation device. RESULTS: A short, hard spin with a total g force of between 3,400 and 4,600 gmin resulted in PCs that contained on average more than 290x10(9) platelets and less than 5x10(6) leukocytes without the use of a leukocyte filter, provided that the transfer of PRP was electronically checked and terminated. The cell concentrations in the PCs are a function of the total g force. Both the platelet and leukocyte levels in the concentrate decreased with an increase in the total g force applied to the pool. CONCLUSION: The preparation of PCs via a short hard, spin of BCs, combined with automated collection of the PRP, may be an alternative method for the preparation of leukocyte-poor PCs.     

Low leukocyte contamination without filtration by preparation of platelet concentrates in cylindrical bags with the buffy-coat method

BACKGROUND AND OBJECTIVES: The buffy-coat (BC) method for platelet concentrate (PC) preparation was modified in order to obtain leukodepleted PCs from single BCs without filtration. MATERIALS AND METHODS: BCs were centrifuged in cylindrical BC bags and the optimal centrifugation conditions and optimal hematocrit were determined. RESULTS: With optimal conditions, a tenfold lower leukocyte contamination was obtained compared with the conventionally shaped, wide BC bag (0.3 +/- 0.19 versus 3.0 +/- 1.71 x 10(6) leukocytes per unit; 85-ml BCs). The platelet yield obtained with the cylindrical bag did not differ significantly from the yield obtained with the conventional bag (56 +/- 16.4 versus 61 +/- 15 x 10(9) platelets per PC). Furthermore, when PCs were prepared from 100-ml BCs in cylindrical bags, a leukocyte contamination of 0.2 +/- 0.11 x 10(6) and a platelet content of 61 +/- 13.5 x 10(9) per PC were obtained. CONCLUSION: The use of cylindrical BC bags reduced the leukocyte contamination in PCs to a level required for leuko-depletion without affecting platelet recovery.     

The increased effectiveness of platelet concentrates prepared in acidified plasma

Platelet concentrates prepared in acidified plasma (pH 6.5-6.7) are superiorto concentrates prepared by standard methods, and are 80-90 per cent aseffective as platelet rich plasma (PRP). The use of excess citric acid to acidifyplasma promotes resuspension of the concentrate by eliminating clumping,which is a major factor in the decreased effectiveness of standard concentrates.Analysis of posttransfusion recovery and survival of platelets reveals no evidence of platelet injury in an acid medium.

Acidification of PRP inhibits the aggregation of platelets by adenosine diphosphate (ADP). The presence of endogenous ADP may be an importantfactor in clumping during standard concentrate preparation.

A method of acidification of PRP using citric acid is described which allowspreparation of an effective concentrate from fresh whole blood without subjecting the red cells to acid pH. Reconstitution of the acidified platelet poorplasma and its native red cells increases the citrate molarity by less than 6 percent and results in minimal decrease in pH of the whole blood.

Submitted on May 6, 1965 Accepted on July 25, 1965     

Fatal septic shock and rhabdomyolysis following transfusion of platelet concentrates contaminated with Streptococcus pneumoniae

We report on a 58-year-old male diagnosed as having primary myelofibrosis with thrombocytopenia, who died of fatal septic shock and rhabdomyolysis after platelet concentrates (PCs) transfusion. The initial diagnosis of primary myelofibrosis was established by splenomegaly, leukoerythroblastosis and bone marrow fibrosis. PCs were transfused because of thrombocytopenia with marked bleeding tendency. Soon after the PCs transfusion in March 2000, he had attacks of chest pain, back pain and myalgia, then went into shock and died of unknown causes. PCs were suspected as being the cause of death, because Streptococcus pneumoniae was found in the culture of PCs in the WBC-reduction in-line filter and fresh frozen plasma from the same donor preserved in the Japan Red Cross Center. Rhabdomyolysis, neutrophil infiltration and phagocytosed bacteria were found from the autopsy materials, which were identified by DNA analysis as the same species found in the PCs. PCs are kept at room temperature because platelet function is lost in the cold. When PCs are contaminated with bacteria, marked multiplication induces fatal bacteremia. This is a rare report in Japan of fatal septic shock caused by PCs with bacterial contamination. We must pay strict attention to bacterial contamination in blood components.     

Predictive value of enzyme-linked immunoassay platelet crossmatching for transfusion of platelet concentrates to alloimmunized recipients

Some evidence has shown that platelet crossmatching is useful in multitransfused patients with hypoplastic bone marrows who are refractory to platelet therapy through alloimmunization. Several immunoglobulin binding assays other than enzyme-linked immunospecific assay (ELISA) have been studied previously. We performed 51 ELISA crossmatches on six patients receiving single donor platelets. One bone marrow transplant patient receiving 33 single donor HLA matched (related and unrelated) was also studied. Effectiveness of transfusion was closely monitored by patient evaluation and corrected platelet count increment (CCI) at 1-2 and 18-24 hours posttransfusion. We found the ELISA method very sensitive, specific, and predictive, 85, 96, and 95.6% respectively in the 51 crossmatches studied in six patients with either leukemia, solid tumors, or aplastic anemia. However, variation existed among individual recipients, with sensitivity ranging from 70-100%. The distribution of true positives and negatives and false positives and negatives in the 33 crossmatches performed in the bone marrow transplant patient differed significantly (chi 2 = 101.2; P less than 0.001) from single donor recipients. The specificity in the 51 crossmatches on the six patients was also significantly different from the 33 crossmatches performed in the bone marrow transplant (96 vs 74%). This suggests individual variation occurs as well as differences in diseases and bone marrow suppressive agents affecting platelet crossmatching.     

Platelet size analysis in the quality assurance of platelet concentrates for transfusion

Summary The platelet distribution width (PDW) as analysed on standard haematology cell counters is an indicator of size dispersion in the platelet population. Using a Sysmex® E-2500 analyser platelet concentrates prepared for transfusion showed an increase in PDW over storage. This increase correlated strongly with in vitro indicators of platelet viability (pH and response to osmotic stress). PDW may thus be useful for clinical haematology laboratories as a predictor of the viability of transfused platelets. The same instrument gave a measure of the largest platelets in the platelet population as a large cell ratio (P-LCR). For platelet concentrates with less than 8 × 10¹⁰ platelets/unit, the P-LCR at preparation was negatively associated with the end of storage pH, indicating that the presence of large platelets increases the production of lactic acid and accelerates the platelets’ metabolic storage lesion. This information may be useful in determining storage conditions for single donor platelets harvested by apheresis.     

Predictive value of cross-matching for transfusion of platelet concentrates to alloimmunized recipients

Compatibility tests in which donor platelets were tested with recipient sera were performed retroactively after 64 transfusions of platelets from 59 unrelated donors to 10 alloimmunized patients. Techniques used were serotonin release, aggregometry, platelet factor 3 release, and lymphocytotoxicity, each of which has been advocated as a means of testing donor-recipient platelet compatibility. Although “false positive” reactions were few (positive crossmatch but satisfactory transfusion response), “false negative” reactions (negative crossmatch but poor transfusion response) were unacceptably high (43% by lymphocytotoxicity, 60% by serotonin release, 76% by platelet factor 3 release, and 83% by aggregometry). We conclude that current methods of detecting isosensitization to platelet alloantigens are less satisfactory than HLA phenotyping in selecting unrelated platelet donors for an alloimmunized patient population.     

Clinical aspects of platelet transfusion.

Several clinical complications of platelet transfusions relate to contaminating donor leucocytes, and a number of strategies have been devised to leucodeplete platelet products before transfusion. Both alloimmunization to class I human leucocyte antigens (HLA), which causes febrile transfusion reactions and refractoriness to transfused platelets, and transmission of cytomegalovirus have been shown to be reduced by 3-log10 leucodepletion by filtration. Lesser degree of leucodepletion, e.g. by platelet preparation from buffy coats, will control febrile transfusion reactions, but will not reliably prevent other complications. The clinical implications and cost-effectiveness of different strategies of platelet production remain a matter of debate.