Effects of storage on quality and function of acid-treated platelets with reduced HLA Class I surface expression.

BACKGROUND: Refractory patients need to be provided with HLA-matched platelets (PLTs), which require time-consuming cross-matching. Treatment of PLTs with citric acid leads to denaturation of the HLA Class I complexes without significant damage to the PLTs. HLA Class I depleted PLTs could alternatively be used to HLA-matched PLTs for transfusion. These PLTs have verified normal function up to 4-6 h after acid treatment. MATERIALS AND METHODS: Buffy coat (BC) PLT concentrates were depleted of HLA Class I complexes by incubation in citric acid. The days after acid-treatment, surface expression of HLA Class I complexes, CD62P and CD63 were determined by flow cytometry, in addition to viability and mitochondrial membrane potential (MMP). Thromboelastography (TEG) tested PLT functionality. RESULTS: Expression of HLA Class I complexes was reduced by 70%-75% in acid-treated PLTs compared to untreated PLTs from day 1 through day 7. Controls and acid-treated PLTs showed insignificant loss of MMP stored for 4 days. Analysis of the residual PLT activation and viability showed no significant differences for 4 days of storage. However, the residual PLT activation potential and viability were significantly decreased in acid-treated PLTs and control PLTs after 7 days of storage. Acid treatment caused a significant decrease in the TEG variable, reaction time (R time), for acid-treated PLTs as compared to control PLTs from days 1 through day 3. CONCLUSION: Our data suggest that extended storage of acid-treated PLTs is possible and will improve flexibility when planning for transfusion of patients with alloimmune PLT refractoriness caused by anti-HLA-antibodies.

Vaccine associated benign headache and cutaneous hemorrhage after ChAdOx1 nCoV-19 vaccine: A cohort study.

OBJECTIVES: Fatal complications have occurred after vaccination with ChAdOx1 nCoV-19, a vaccine against Covid-19. Vaccine-induced immune thrombotic thrombocytopenia (VITT) with severe outcome is characterized by venous thrombosis, predominantly in cerebral veins, thrombocytopenia and anti-PF4/polyanion antibodies. Prolonged headaches and cutaneous hemorrhages, frequently observed after the ChAdOx1 nCoV-19 vaccine, have therefore caused anxiety among vaccinees. We investigated whether these symptoms represent a mild form of VITT, with a potential for aggravation, e.g. in case of a second vaccination dose, or a different entity of vaccine complications MATERIALS AND METHODS: We included previously healthy individuals who had a combination of headache and spontaneous severe cutaneous hemorrhages emerging after the 1st dose of the ChAdOx1 nCoV-19 vaccine. Twelve individuals were found to meet the inclusion criteria, and a phone interview, cerebral MRI, assessment of platelet counts, anti PF4/polyanion antibodies and other laboratory tests were performed. RESULTS: None of the symptomatic vaccinees had cerebral vein thrombosis, hemorrhage or other pathology on MRI. Platelet counts were within normal range and no anti-PF4/polyanion platelet activating antibodies were found. Moreover, vasculitis markers, platelet activation markers and thrombin generation were normal. Furthermore, almost all symptoms resolved, and none had recurrence of symptoms after further vaccination with mRNA vaccines against Covid-19. CONCLUSIONS: The combination of headaches and subcutaneous hemorrhage did not represent VITT and no other specific coagulation disorder or intracranial pathology was found. However, symptoms initially mimicking VITT demand vigilance and low threshold for a clinical evaluation combined with platelet counts and D-dimer.

HLA class I depletion by citric acid, and irradiation of apheresis platelets for transfusion of refractory patients.

BACKGROUND: Patients can form antibodies to foreign human leukocyte antigen (HLA) Class I antigens after exposure to allogeneic cells. These anti-HLA class I antibodies can bind transfused platelets (PLTs) and mediate their destruction, thus leading to PLT refractoriness. Patients with PLT refractoriness need HLA-matched PLTs, which require expensive HLA typing of donors, antibody analyses of patient sera and/or crossmatching. An alternative approach is to reduce PLT HLA Class I expression using a brief incubation in citric acid on ice at low pH. METHODS AND MATERIALS: Apheresis PLT concentrates were depleted of HLA Class I complexes by 5 minutes incubation in ice-cold citric acid, at pH 3.0. Surface expression of HLA Class I complexes, CD62P, CD63, phosphatidylserine, and complement factor C3c was analyzed by flow cytometry. PLT functionality was tested by thromboelastography (TEG). RESULTS: Acid treatment reduced the expression of HLA Class I complexes by 71% and potential for C3c binding by 11.5-fold compared to untreated PLTs. Acid-treated PLTs were significantly more activated than untreated PLTs, but irrespective of this increase in steady-state activation, CD62P and CD63 were strongly upregulated on both acid-treated and untreated PLTs after stimulation with thrombin receptor agonist peptide. Acid treatment did not induce apoptosis over time. X-ray irradiation did not significantly influence the expression of HLA Class I complexes, CD62P, CD63, and TEG variables on acid treated PLTs. CONCLUSION: The relatively simple acid stripping method can be used with irradiated apheresis PLTs and may prevent transfusion-associated HLA sensitization and overcome PLT refractoriness.

Biological response modifiers in photochemically pathogen-reduced versus untreated apheresis platelet concentrates.

BACKGROUND: Lipids and other biologically active substances accumulate in platelet concentrates (PCs) during storage. Some of these substances have been suggested to modulate immune responses and to play a pathogenic role in the development of transfusion-related acute lung injury. This study compared the content and impact of some biological response modifiers in PCs treated with pathogen reduction (PR) technology and nontreated PCs. STUDY DESIGN AND METHODS: Apheresis PCs (n = 12) were split in two: one split was subjected to PR treatment (INTERCEPT, Cerus Corp.) and the other split was left untreated. Basic characterization and content of vascular endothelial growth factor (VEGF) and sCD154 were measured. Lipopolysaccharide (LPS)-induced secretion of interleukin-10 (IL-10) and tumor necrosis factor-α (TNF-α) was measured after incubation of heparinized whole blood with platelet (PLT) supernatants. The supernatants' neutrophil (PMN)-priming capacity, and thereby activation of the NADPH oxidase, was measured as the rate of superoxide anion production after formyl-Met-Leu-Phe activation. Lipids were extracted from the supernatants on Day 6 and tested for PMN-priming activity. RESULTS: Supernatants from PR-treated PCs demonstrated significantly higher mean PLT volume (MPV) and O(2) , lower pH, CO(2) , and HCO(3-) , and significantly less LPS-induced TNF-α secretion compared to untreated PCs. No differences in swirling, PLT count, potassium levels, glucose consumption, lactate production, IL-10, VEGF, sCD154, or PMN-priming activity were found between the groups over time. CONCLUSION: INTERCEPT PR treatment caused no substantial differences in PCs, except for minor changes in MPV and metabolic variables. Further studies are needed to explain the differences in the LPS-induced TNF-α secretion.

Recovery, survival, and function of transfused platelets and detection of platelet engraftment after allogeneic stem cell transplantation.

BACKGROUND: Recovery and survival of transfused platelets (PLTs) are usually assessed by radioisotope labeling methods for evaluation of transfusion efficacy and new progress in the processing of PLT concentrates. Alternative, nonradioactive methods are warranted. STUDY DESIGN AND METHODS: A multicolor flow cytometry method was developed for simultaneous studies of recovery, survival, and function of transfused PLTs. Eight consecutive patients undergoing allogeneic stem cell transplantation (TX) were transfused with apheresis PLTs of nonself human leukocyte antigen (HLA) Class I types, and HLA Class I discrepancy between donor and recipient was used to identify transfused PLTs. Hematologic status and HLA Class I surface expression were analyzed immediately before transfusion, 1 and 6 hours after transfusion, and daily during the subsequent week. PLT activation was assessed by surface expression of CD63, CD62P, or CD42a, before and after stimulation with thrombin receptor agonist peptide. RESULTS: PLT recovery was 43, 41, and 31% for fresh (5-72 hr old) and 30, 27, and 17% for stored (73-148 hr old) PLTs, after 1, 6, and 15 to 28 hours, respectively. Survival of fresh versus stored PLTs were 160 and 105 hours, respectively. Spontaneous PLT activation and residual activation potential were almost equal for fresh and stored PLTs. PLT engraftment was detected between Day 7 and Day 9, which was significantly earlier than first sign of neutrophil engraftment (Days 11-19; p=0.01). CONCLUSION: Flow cytometry is an attractive alternative to radiolabeling of PLTs for simultaneous studies of survival, recovery, and function of transfused PLTs and early detection of PLT engraftment after allogeneic stem cell TX.

Proteomic study of apheresis platelets made HLA class I deficient for transfusion of refractory patients.

PURPOSE: Refractoriness can occur after repeated platelet (PLT) transfusions because of alloimmunization to HLA class I antigens on transfused PLTs and generation of anti-HLA antibodies that bind to the foreign PLTs and initiate their destruction. Such refractoriness can be overcome by provision of HLA-matched PLTs from HLA typed donors. However, since the procedure is both expensive and time-consuming, an alternative approach is to deplete PLTs of HLA class I molecules by a brief treatment with citric acid, on ice. This is shown to be feasible without damaging PLT function. We used label free quantitative mass spectrometry (MS)-based proteomics to investigate the effect of acid treatment on apheresis PLTs for combatting immunologic PLT refractoriness. EXPERIMENTAL DESIGN: Proteomic analyses are undertaken using PLTs from seven apheresis concentrates, which were split in two with or without acid treatment. RESULTS: In total 1717 proteins in apheresis PLTs were quantified using proteomics. Data are available via ProteomeXchange with identifier PXD027893 . Of these, the amount of 80 proteins changed significantly after acid treatment, but overall there were not any major differences in proteomes between samples with and without acid treatment. CONCLUSIONS AND CLINICAL RELEVANCE: In general, the changes of PLT proteins after treatment with citric acid were quite small and functionally safe. Hence, this result taken together with our previously published data indicates that acid treated PLTs can be used for treatment of patients with PLT refractoriness and opens up for a clinical trial.

The effect of pre-storage cooling on 2,3-DPG levels in red cells stored in SAG-M.

BACKGROUND: The concentration of red cell 2,3-DPG (2,3-diphosphoglycerate) rapidly decreases during storage. A favourable effect on red cell 2,3-DPG has been demonstrated by rapid cooling of whole blood prior to storage. In our study we have investigated how different methods of cooling whole blood immediately after donation effect 2,3-DPG levels during storage. STUDY DESIGN AND METHODS: Thirty-six whole blood units (in 6 groups) of 450 ml were collected in 63 ml CPD. SAG-M was used as preservative solution for red cell concentrates (RCC). The units in one group were cooled down at ambient temperature, while units in the other groups were cooled down rapidly by different ways immediately after bleeding. Samples from the whole blood units were collected at various days during storage for 2,3-DPG measurements. RESULTS: The decline in 2,3-DPG during the first two weeks of storage was significantly slower in the groups which were cooled down rapidly to 17-18 degrees C within 1h after bleeding (all p

Evaluation of platelet activation and cytokine release during storage of platelet concentrates processed from buffy coats either manually or by the automated OrbiSac system.

BACKGROUND: This study aimed to compare platelet (PLT) quality during storage of buffy coat (BC) PLT concentrates (PCs), prepared either manually or by the automated OrbiSac system (Gambro BCT). STUDY DESIGN AND METHODS: Following overnight storage at 20 to 22 degrees C, five BCs were pooled with 300 mL of PLT additive solution. Twenty-one PCs were produced manually (M-PCs) and 21 by the automated OrbiSac system (A-PCs). Swirling, PLT count, mean PLT volume, blood gas analyses, potassium, glucose, and lactate were assessed. Expression of the activation markers CD42a, CD62P, and PAC-1 was analyzed by flow cytometry on resting PLTs and PLTs stimulated with thrombin receptor agonist peptide (TRAP). Levels of CCL5 and transforming growth factor-beta1 (TGF-beta1) were measured by enzyme-linked immunosorbent assay. RESULTS: The A-PCs had significantly larger volume and higher PLT yield, PLT recovery, and white blood cell concentration than the M-PCs, whereas the red blood cell content was significantly highest in the M-PCs. pH levels were between 6.9 and 7.2 in all PCs. Neither glucose consumption nor lactate production differed significantly over time. A-PCs had, compared to M-PCs, significantly higher expression of CD62P on resting PLTs, lower capacity for up regulating CD62P on TRAP-stimulated PLTs, and higher levels of CCL5 during storage. TRAP-stimulated A-PCs had a significantly higher potential for down regulation of CD42a than M-PCs. No difference was found in TGF-beta1 levels or TRAP-induced up regulation of PAC-1. CONCLUSION: The levels of CCL5 and the expression of CD62P in resting and stimulated PLTs indicate that PLTs in A-PCs are slightly more activated than in M-PCs, but the clinical importance of this finding is yet unknown.

Evaluation of nonleukoreduced red blood cell transfusion units collected at delivery from the placenta.

BACKGROUND: The objective of this study was to evaluate the suitability of cord blood (CB) as a source of red blood cells (RBCs) for autologous transfusion. STUDY DESIGN AND METHODS: CB was collected in 150-mL storage containers with citrate phosphate dextrose (CPD) as anticoagulant and stored in either saline, adenine, glucose, and mannitol (SAG-M; n = 18) or phosphate, adenine, glucose, guanosine, saline, and mannitol (PAGGS-M; n = 18) for 35 days at 4 degrees C. Hematologic status and hemolysis were studied. The lipopolysaccharide (LPS)-induced production of tumor necrosis factor (TNF)-alpha and transforming growth factor (TGF)-beta1 from CB monocytes was analyzed after incubation with addition of weekly sampled supernatants from the CB RBC units. Five additional units (PAGGS-M) were leukoreduced and thereafter analyzed as indicated above. RESULTS: Hemolysis increased significantly over time, in SAG-M more than in PAGGS-M. During storage in both media, the number of white blood cells (WBCs) decreased, and the LPS-induced production of TNF-alpha and TGF-beta1 decreased and increased, respectively. There were no significant changes in the LPS-induced production of TNF-alpha and TGF-beta1 in the leukoreduced CB RBC units. CONCLUSION: Hemolysis in CB RBC units increased significantly over time, and PAGGS-M appears to be superior to SAG-M as a preservation solution for CB RBC. The changes in LPS-induced TNF-alpha and TGF-beta1 production over time were probably caused by substances released from apoptotic and/or necrotic WBCs. Further studies are needed to identify both which substances are responsible for the changes in LPS-induced cytokine release and the clinical significance hereof.

Platelet activation and residual activation potential during storage of hyperconcentrated platelet products in two different platelet additive solutions.

BACKGROUND: To improve platelet (PLT) quality, hyperconcentrated PLT concentrates (hcPCs) were compared to standard PLT concentrates (stdPCs) in two different PLT additive solutions, T-Sol and PAS-27a. PAS-27a differs from T-Sol by containing glucose, phosphate, potassium, magnesium, and bicarbonate. STUDY DESIGN AND METHODS: PLTs were harvested by apheresis twice from 14 donors; each unit was divided into two. Four units from each donor were produced: hcPCs, 2000 x 10(9) per L in T-Sol or PAS-27a; and stdPCs, 1400 x 10(9) per L in 65 percent T-Sol or PAS-27a and 35 percent acid citrate dextrose-plasma. On Days 1 through 4, swirling was scored and PLT count, mean PLT volume, pH, blood gas, glucose, and lactate were measured. Expression of CD42a, CD62P, CD63, and PAC-1 was analyzed by flow cytometry on resting PLTs and PLTs stimulated with thrombin receptor agonist peptide (TRAP). RESULTS: Glucose consumption and lactate production were significantly higher in hcPCs stored in PAS-27a than in T-Sol. Both stdPC and hcPC PLTs in T-Sol expressed CD62P and PAC-1 significantly higher than in PAS-27a. Over time the T-Sol hcPCs revealed highest expression of CD62P and CD63. A significantly higher capacity for up regulation of CD62P, CD63, and PAC-1 upon TRAP stimulation was found for stdPCs and hcPCs in PAS-27a compared to PLTs in T-Sol. TRAP-stimulated PLTs in stdPCs and hcPCs suspended in PAS-27a showed significantly higher potential for down regulation of CD42a than the T-Sol concentrates. CONCLUSIONS: PLTs appear better preserved in vitro in PAS-27a than in T-Sol, and this suggests that storage of hcPCs in PAS-27a could be extended beyond 24 hours.