Optimized processing for pathogen inactivation of double-dose buffy-coat platelet concentrates: maintained in vitro quality over 7-day storage.

BACKGROUND AND OBJECTIVE: Efficient pathogen inactivation (PI) offers the possibility of increasing the number of buffy coats per pool without the concurrent increased risk of pathogen transmission. Here, we describe the findings of in vitro analyses of platelets from pools of eight buffy coats treated with amotosalen and UVA light (INTERCEPT Blood System for Platelets) using INTERCEPT disposable processing sets with plastic materials sourced from alternate suppliers and split afterwards to obtain two therapeutic transfusion doses. METHODS: Double-dose platelet concentrates were prepared from pools of eight buffy coats in additive solution (SSP+) using either previous 6-lead or new 8-lead pooling sets and PI processing sets in previous or alternate supplier sourced plastics (AS). Platelets were treated with the INTERCEPT Blood System then stored for up to 7 days and tested for in vitro quality. RESULTS: All tested units (n = 30) were in conformity with European guidelines. Using AS sets more effectively maintained glucose reserves (P < 0·01), reduced lactate production (P < 0·01), reduced CD62P expression (P < 0·01) and downregulated levels of surface CD42b (P < 0·01) overtime. AS set maintained JC-positive cells (NS) between day 2 and day 7 and sustained platelet integrin activation (PAC-1) between day 2 and day 7 (NS). Overall sCD40L and PGDF accumulated in an equivalent way (P < 0·01) within series. SUMMARY/CONCLUSIONS: In summary, our data demonstrate that PI treatment using AS sets, in combination with the new pooling set for double-dose platelet preparation, maintained the platelet in vitro quality over 7 days of storage.

Pathogen inactivation of double-dose buffy-coat platelet concentrates photochemically treated with amotosalen and UVA light: preservation of in vitro function.

BACKGROUND: The INTERCEPT Blood System for Platelets (PLT) utilizes amotosalen (S-59) in combination with ultraviolet A (UVA) light to inactivate viruses, bacteria, protozoa and leucocytes that may contaminate PLT concentrates. However, limited data are available on the quality of INTERCEPT-treated double-dose (DD) buffy-coat (BC) PLT units allowing a single treatment procedure to produce two pathogen-inactivated PLT units for transfusion. STUDY DESIGN AND METHODS: The objective of this study was to evaluate potential in vitro effects of the INTERCEPT treatment on pools of 7 BCs as compared to untreated units. Functional, phenotypic and mitochondrial properties of DD BC PLTs during storage over 7 days were studied. RESULTS: For some parameters measured, small yet significant differences were observed including PLT count (P < 0·05), pH, pCO2 and glucose concentration. Throughout storage, no significant differences were observed in ATP levels, ESC, HSR reactivity and CD62P expression. Similarly, no differences were observed in the expression of PAC-1, CD42b and PECAM-1 at any time-points. The mitochondrial membrane potential (MMP) determined by JC-1-labelling was well maintained until day 7 in all treated and untreated units (>90%). The release of sCD40L increased over time (P < 0·01) in all units but without any significant differences between treated and untreated PLTs. CONCLUSION: Our data demonstrate that photochemical pathogen inactivation of DD-BC PLT concentrates with the INTERCEPT Blood System had no influence on the PLT in vitro quality over the 7 day of storage. However, whether in vivo efficacy of INTERCEPT-treated PLTs is affected may require clinical evaluation.

High-yield platelet units revealed immediate pH decline and delayed mitochondrial dysfunction during storage in 100% plasma as compared with storage in SSP+.

BACKGROUND AND OBJECTIVES: Initial elevated and prolonged high carbon dioxide levels related to mitochondrial dysfunction are recently being suggested as a contributing factor to rapid pH decline in platelet (PLT) units. The use of different storage environments may influence this phenomenon. This study has two objectives (i) to investigate the relationship of mitochondrial function and apoptotic events with different storage environments capability of pH control and (ii) to examine the cause and relationship between pH decline in PLT units, carbon dioxide levels and mitochondrial function. MATERIALS AND METHODS: Platelet units were prepared for storage in (A) 70% SSP+, 300-400 × 10(9) /unit, (B) 70% SSP+, 550-600 × 10(9) /unit, (C) 100% plasma, 550-600 × 10(9) /unit, and (D) additional 100% plasma, >600 × 10(9) /unit. In vitro variables including mitochondrial function (JC-1), reactive oxygen species (ROS) and caspase 3 activity were analysed on days 2, 5 and 7. RESULTS: Glucose/lactate was higher, pH, ATP, Hypotonic shock response (HSR) and extent of shape change (ESC) decreased (P < 0·001 on day 7), CD62P (P < 0·001 on day 7) increased, the JC-1-positive PLTs were lower (P < 0·001 on day 7), and ROS was higher (P < 0·001 days 2-7) in the plasma (C) units as compared with the SSP+ (A) and (B) units. All plasma (D) units showed rapid pH and pCO(2) decline from day 2 but by means of >80% maintenance of mitochondrial function until day 7. CONCLUSIONS: The use of SSP+ instead of plasma may reduce the risk of triggering pro-apoptotic events in high-yield PLT units. A rapid decline in pH in PLT units cannot be explained with initial elevated and prolonged high carbon dioxide levels and mitochondrial dysfunction.

Platelet concentrates from fresh or overnight-stored blood, an international study.

BACKGROUND: Whole blood and also buffy coats (BCs) can be held for a few hours or overnight before processing into blood components or platelet concentrates (PCs). Individual studies have reported a range of outcomes regarding in vitro variables for PCs prepared from fresh and stored whole blood. In this multicenter study, effects of storage of whole blood or BCs on the in vitro quality of PCs were studied. STUDY DESIGN AND METHODS: The leukoreduced BC PCs were prepared from fresh BCs (2-8 hr after collection; fresh/fresh), from BCs at 20 to 24 hours after collection (fresh/stored), or from BCs prepared from whole blood stored for 20 to 24 hours (stored/fresh). PCs were stored on a flat-bed shaker at 20 to 24°C for 7 days. PCs were tested on Days 0 (only fresh/fresh), 1, 5, and 7 for in vitro quality. There were six participating centers that tested all three conditions with n = 6 per condition. RESULTS: In comparison to fresh/stored and stored/fresh PCs, fresh/fresh PCs exhibited a lower platelet (PLT) count (Day 1-220 × 10(9) ± 70 × 10(9) vs. 324 × 10(9) ± 50 × 10(9) and 368 × 10(9) ± 56 × 10(9) PLTs/PC), lactate, pCO(2), and hypotonic shock response (HSR; Days 5 and 7; Day 7-50 ± 13% vs. 57 ± 12 and 63 ± 11%) and a higher pH, glucose, pO(2), and CD62P expression (than stored/fresh PCs only; Day 7-33 ± 10% vs. 28 ± 12 and 24 ± 11%; p < 0.05). No differences were observed for volume, swirling effect, white blood cell count, annexin V binding, or aggregation between these conditions. CONCLUSION: Based on PLT count, HSR, and PLT activation, PCs are best prepared after 20 to 24 hours hold of the whole blood or BCs.

In vitro effects on platelets irradiated with short-wave ultraviolet light without any additional photoactive reagent using the THERAFLEX UV-Platelets method.

BACKGROUND: A novel short-wave ultraviolet light (UVC) pathogen reduction technology (THERAFLEX UV-Platelets; MacoPharma, Mouvaux, France) without the need of any additional photoactive reagent has recently been evaluated for various bacteria and virus infectivity assays. The use of UVC alone has on the one hand been shown to reduce pathogens but may, on the other hand, have some impact on the platelet (PLT) quality. The purpose of this study was to determine the potential effects on PLT quality of pathogen inactivation treatment using the novel UVC method for PLT concentrates. STUDY DESIGN AND METHODS: Buffy-coat-derived PLTs suspended in SSP+ were irradiated with UVC light in plastic bags (MacoPharma) made of ethyl vinyl acetate, considered to be highly permeable to UVC light. The UVC-treated (test, n=8) as well as the untreated (reference, n=8) PLT units were stored in PLT storage bags composed of n-butyryl, tri n-hexyl citrate-plasticized polyvinyl chloride (MacoPharma) on a flat bed agitator for in vitro testing during 7 days of storage. RESULTS: No significant difference in PLT counts and lactate dehydrogenase between the groups was detected. During storage, glucose decreased more and lactate increased more in the test units. Statistically significant differences were found for glucose (P<0·01) and lactate (P<0·05) on day 7. ATP levels were higher (P<0·01 from day 5) in the reference units. With exception of day 7 (P<0·01 reference vs. test), hypotonic shock response reactivity was not different between groups. Extent of shape change was lower (P<0·01), and CD62P (P<0·05 day 5) was higher in the test units. CD42b and CD41/61 showed similar trends throughout storage, without any significant difference between the units. pH was maintained at >6·8 (day 7) and swirling remained at the highest level (score = 2) for all units throughout storage. CONCLUSION: Our results suggest that irradiation with UVC light has a slight impact on PLT in vitro quality and appears to be insignificant with regard to current in vitro standards.

Storage of Buffy-coat-derived platelets in additive solutions: in vitro effects on platelets prepared by the novel TACSI system and stored in plastic containers with different gas permeability.

BACKGROUND: The novel TACSI system is designed for automated preparation of platelets (PLTs) from pooled buffy coats (BCs). One TACSI device will handle 6 units at the same time. The aim of our in vitro study is to investigate the effects of using this automated equipment with subsequent storage in two different plastic containers and to compare these results with PLTs prepared by the OrbiSac system. STUDY DESIGN AND METHODS: Buffy-coat-derived PLTs (n=8) were prepared by using the TACSI system, including storage in polyvinyl chloride (PVC)-based plastic containers with di, n-decyl phthalate (DnDP) (TACSI R) and BTHC (TACSI T)-based plasticizers. As a reference, the OrbiSac System was used to prepare PLTs (n=8) with subsequent storage in a PVC plastic container with a citrate-based plasticizer (BTHC). In total, 16 TACSI and eight reference units, supplied by approximately 30% plasma and 70% SSP+, were analysed for various in vitro variables during the 7-day storage period. RESULTS: No significant difference in PLT counts, LDH, mean platelet volume (MPV) and adenosine triphosphate between the groups was detected. Glucose was lower (P<0·05) and lactate was higher (P<0·05) in TACSI R vs. OrbiSac. With exception of day 7 (P<0·05 TACSI R vs. OrbiSac), HSR reactivity were not different between groups. Extent of shape change was lower and CD62P higher in TACSI T when compared with TACSI R and OrbiSac units (P<0·05). pH was maintained at >6·8 (day 7) and swirling remained at the highest level (score=2) for all units throughout storage. CONCLUSION: Platelets prepared by the TACSI system with subsequent storage in two different PVC-based plastic containers were equivalent to reference PLTs with regard to in vitro characteristics during 7 days of storage.

Storage of buffy-coat-derived platelets in additive solutions: in vitro effects on platelets stored in reformulated PAS supplied by a 20% plasma carry-over.

BACKGROUND AND OBJECTIVES: Platelet additive solutions (PAS) have been shown to be suitable for extended platelet (PLT) storage. Depending on the PAS formulation, the percentage of plasma carry-over contributes to success. Improving PLT quality by optimizing the composition of PAS may allow a reduction to be made in the amount of plasma carried over to the final unit. Reducing the proportion of plasma carried over would probably decrease transfusion of unwanted antibodies and make greater amounts of plasma available for other needs. STUDY DESIGN AND METHODS: Platelets from eight pools of 25 buffy coats were aliquoted and prepared for storage in plasma and different PAS units: InterSol and three alternate PAS named PSM1, PSM2 and PSM3. All PAS units were supplied with a 20% plasma carry-over and stored at room temperature with agitation for 9 days with in vitro testing for metabolic, cellular and activation parameters. Results During storage, PLTs stored in InterSol displayed significantly lower glucose concentration (P < 0.01), lower adenosine triphosphate levels (P < 0.01), a higher mean PLT volume (P < 0.01), a lower response to hypotonic shock response activity (P < 0.01) and a higher CD62P expression (P < 0.01) when compared with PLTs stored in plasma and PSM1-3 solutions. pH was maintained at > 6.8 (day 9) and swirling remained at the highest level (score = 2) for all units throughout storage. CONCLUSION: Our results suggest that PLTs stored in PAS with addition of magnesium, potassium and glucose (PSM2 and PSM3) and 20% plasma carry-over maintained metabolic and cellular characteristics, equivalent to PLTs stored in 100% plasma during 9 days of storage. Our results also suggest that presence of potassium in addition to magnesium or alternatively the concentration of phosphate as well as the supply of additional glucose to normal plasma levels improve in vitro data of PLTs stored in PAS.

In vitro and in vivo effects of potassium and magnesium on storage up to 7 days of apheresis platelet concentrates in platelet additive solution.

BACKGROUND AND OBJECTIVE: Prolonged storage of platelets up to 7 days provides improved availability, logistical management and decreased wastage. Beside methods of bacterial detection, addition of magnesium and potassium to the platelet storage solution (SSP+) may further improve the quality of platelets with extended storage. MATERIALS AND METHODS: Apheresis platelets from 10 donors were divided and stored in two different platelet additive solutions (PAS) (Intersol and SSP+) for a paired comparison. A variety of in vitro platelet function and metabolic assays were performed both on day 1 and after 7 days of storage. For in vivo study, platelets were labelled with either (111)Indium or (51)Chromium after 7 days of storage and were injected into the corresponding donor. Serial blood samples were drawn for recovery and survival measurements. RESULTS: In vitro parameters for SSP+ showed significantly reduced glycolysis (lower glucose consumption and decreased production of lactate), a higher hypotonic shock response (HSR) and the extent of shape change reactivity and a lower degree of platelet activation by means of RANTES (regulated on activation, normal, T cell-expressed, and secreted), CD62p and CD63 expression. Platelet recovery on day 7 was higher for Intersol as compared to SSP+, 65 +/- 11 vs. 53 +/- 13% (P = 0.023), and survival showed no difference 4.2 +/- 1.9 vs. 3.6 +/- 1.4 days. CONCLUSION: In vitro characteristics of platelets stored in PAS with addition of potassium and magnesium indicated higher quality, but this could not be verified by the in vivo parameters by means of recovery and survival.

Storage of buffy-coat-derived platelets in additive solutions at 4 degrees C and 22 degrees C: flow cytometry analysis of platelet glycoprotein expression.

BACKGROUND: The aim of our in vitro study is to compare the effects on platelet membrane glycoproteins that play an important role in the main functions of platelets, when platelets are stored for a period of 21 days at 4 degrees C or 22 degrees C. STUDY DESIGN AND METHODS: Platelet concentrates (PC) were prepared from pooled buffy-coats (BC) for paired studies (total eight pools from 80 BCs) by using the OrbiSac system. We divided each pool into two PCs and stored them at 4 degrees C or 22 degrees C. RESULTS: The activation marker CD62 remained almost unchanged during storage in all units. The expression of CD63 was higher in PCs stored at 22 degrees C than in those stored at 4 degrees C. No significant difference in CD41 expression was detected over time. The expression of CD42b declined during storage and even more in PCs stored at 4 degrees C until day 21 [day 14: mean flourscence intensity: 32.5 +/- 13.1 vs. 46.5 +/- 19.1], but the percentage of platelets expressing CD42b remained high in platelets stored at 4 degrees C, but gradually decreased at 22 degrees C (day 14: 95.0 +/- 1.5 vs. 59.0 +/- 9.9). Storage at 4 degrees C reduced the rate of glycolysis and maintained the pH better after day 10 than in PCs stored at 22 degrees C (day 14: 7.009 +/- 0.067 vs. 7.233 +/- 0.125). The concentration of regulated upon activation of normal T-cells expressed and secreted was higher in PCs stored at 22 degrees C than at 4 degrees C (day 7: 414.7 +/- 32.3 vs. 49.6 +/- 19.0). No response to extent of shape change and no swirling were detected at 4 degrees C. CONCLUSION: Platelets stored at 4 degrees C retain their in vitro characteristics better than those stored at 22 degrees C, except for parameters that reflect changes in shape. Storage at 4 degrees C is not associated with an increased expression of glycoprotein (GpIb, GpIIb/IIIa) and platelet activation markers (CD62p and CD63) as compared with storage at 22 degrees C.

Automated preparation of platelet concentrates from pooled buffy coats: in vitro studies and experiences with the OrbiSac system.

BACKGROUND: The aim was to evaluate platelet concentrates (PCs) prepared by the automated OrbiSac system, from pooled buffy coats (BCs) stored in a platelet (PLT) additive solution. STUDY DESIGN AND METHODS: Experiment 1 was a paired in vitro study of PCs (from six BCs), prepared by automated and manual procedures. Experiments 2 and 3 evaluated PCs from OrbiSac (from six BCs); Experiment 3 included selection of BCs based on donor data. Experiment 4 was a paired in vitro study of PCs (from six BCs) with an integrated white blood cell (WBC) filter and two different storage containers. Experiment 5 evaluated PCs (from six BCs) from the OrbiSac with an integrated WBC filter. Experiment 6 was similar to Experiment 5 with computer-selected pools of 5 BCs. The in vitro studies evaluated the effects of 7-day storage of PLTs regarding PLT metabolism and disintegration. RESULTS: Experiments 1 and 4 had similar in vitro results. In Experiment 2, PLT content was 370 x 10(9) +/- 70 x 10(9) per PC and recovery from BCs was 76 +/- 6 percent. In Experiment 3, the PLT content was 380 x 10(9) +/- 50 x 10(9) per PC and variation was reduced compared with randomly pooled BCs. In Experiment 5, increased PLT content was found (420 x 10(9) +/- 70 x 10(9) per PC and recovery from BCs of 80 +/- 5%). In Experiment 6, five rather than six BCs gave 340 x 10(9) +/- 60 x 10(9) PLTs per PC and recovery was 79 +/- 5 percent. CONCLUSION: These in vitro studies suggest that the OrbiSac technique is equivalent to the standard manual method regarding the PLT in vitro characteristics during storage for 7 days. The results of standardizing the PLT count in PCs by selecting the BCs pools on the basis of the blood donor PLT concentration were encouraging.

Storage of platelets in additive solutions: a pilot in vitro study of the effects of potassium and magnesium.

BACKGROUND AND OBJECTIVES: Platelet additive solutions (PAS) have been shown to be suitable for extended platelet storage but have required the carryover of substantial (30%) amounts of plasma for success. Improving platelet quality by optimizing the composition of PAS may allow a reduction to be made in the amount of plasma carried over. Reducing the proportion of plasma carried over would facilitate some methods of viral inactivation and make available greater amounts of plasma for other needs. MATERIALS AND METHODS: Platelets from six pools of 25 buffy coat platelet units and five apheresis platelet units were aliquoted for storage in plasma, or converted to PAS units in either a specific additive solution (PAS-III), with 30% or 20% plasma, or a modification of PAS-III containing 5.0 mm potassium and 1.5 mm magnesium (PAS-IIIM), with 30% or 20% plasma. Units were stored at room temperature with agitation for 7 days with in vitro testing for biochemical, haematological and functional parameters. RESULTS: Storage of platelets in PAS-IIIM resulted in a reduced rate of glycolysis and better retention of pH, morphology score and ATP levels. Platelets initially showed less evidence of activation when stored in PAS-IIIM, with reduced P-selectin expression. Storage in PAS-IIIM with 20% (rather than the standard 30%) plasma appeared to result in the retention of in vitro properties, similarly to storage in standard PAS-III with 30% plasma. CONCLUSIONS: Storing platelets in an additive solution containing magnesium and potassium improves the functionality of the platelets, as measured by in vitro testing, and may allow a reduction of the amount of plasma required to be carried over to the final unit.

Leukocyte counts and macrophage phenotypes in induced sputum and bronchoalveolar lavage fluid from normal subjects.

It is unclear whether leukocytes in induced sputum (IS) and bronchoalveolar lavage (BAL) represent the same cell populations. To compare leukocyte counts and macrophage phenotypes and investigate any measurable dithiothreitol (DTT)-mediated effect on macrophage immunocytochemical staining results, IS and BAL samples from nine healthy smokers and seven nonsmokers were examined. BAL and IS samples were processed and cell viability and cell counts were assessed. The macrophages were characterized by seven monoclonal antibodies (RFD1, RFD7, CD11b, CD54, CD68, CD71 and HLA-DR) using an indirect immunoalkaline phosphatase method. Intraindividual comparison of IS and BAL showed that IS samples from smokers and nonsmokers contained a lower total cell count (p<0.01 smokers, p<0.05 nonsmokers), a lower percentage of macrophages (both p<0.05) and a higher percentage of neutrophils (both p<0.05) than BAL samples. In addition, nonsmokers sputum samples contained a lower proportion of lymphocytes (p<0.05) than BAL. The macrophage expression of RFD7 and CD71 was higher in smokers sputum samples (both p<0.05) than in BAL, while nonsmokers sputum macrophages showed a higher expression of CD54 and CD71 (both p<0.05) than BAL macrophages. DTT-incubated BAL samples showed no difference in macrophage antigen expression from BAL samples not exposed to DTT. In conclusion, the relative proportions of leukocytes and the macrophage phenotypes differed between induced sputum and bronchoalveolar lavage suggesting that the methods provide samples from different lung compartments, inhabited by cells with different phenotypes.

Onchocerciasis in Minnesota cattle.

Umbilical hides from 536 dairy cattle in Minnesota were tested for the microfilarial stage of Onchocerca species to determine the distribution of onchocerciasis in the state. The infection was widespread as microfilariae were obtained from 214 (40%) of the animals, representing nearly all areas of the state. Adult Onchocerca parasites were collected primarily from nodules associated with tibial bones but also were found to a lesser extent within the gastrosplenic ligament. Specific identity of these organisms is unclear as they exhibit certain morphological features previously described as being characteristic of either Onchocerca gutturosa, Onchocerca lienalis, or Onchocerca stilesi.