Change of the metabolomic profile during short-term mononuclear cell storage.

BACKGROUND AND OBJECTIVES: Short-term storage of leukapheresis products used for immunotherapeutic mononuclear cell (MNC) products is a frequent event. The analysis of time-related metabolic patterns enables the characterization of storage-related effects in MNCs and the hypothesis-based optimization of the MNC medium. MATERIALS AND METHODS: The MNC products from seven leukapheresis procedures were stored within a closed bag system for 48 h. Concentrations of amino acids, biogenic amines, phospho- and sphingolipids and hexoses in the medium were measured by targeted metabolomics. The viability of MNC subpopulations was assayed by Annexin V (AnV) and JC-1 staining. RESULTS: Glucose depletion and a significant change of the acylcarnitine profile are early events within the first 24 h of storage. In contrast, for most amino acids, the maximum increase was observed at 48 h of storage as mirrored by an increase in the amino acid levels by a mean factor of 1·2 (1·3, 2·0) after 6 h (24 h, 48 h, respectively). This was except for the concentrations of glutamine and lysine, which did not change significantly. The taurine concentration showed a twofold increase within the first 24 h and remained constant thereafter. The steepest increase in AnV+ and 7-AAD+ CD4+ T cells was found between 24 and 48 h. CONCLUSION: The time-course of apoptosis and metabolic patterns in the MNC products demonstrate that 24 h of storage is a decisive time-point, as afterwards key metabolic pathways showed nonlinear detrimental changes. Optimization of storage by supplementation of specific substrates demands therefore an early intervention.

The effects of an overnight holding of whole blood at room temperature on haemoglobin modification and in vitro markers of red blood cell aging.

BACKGROUND: Some effects of the red blood cell (RBC) storage lesion are well documented whereas others are not. Whether a period of room temperature hold (RTH) during RBC production enhances the RBC storage lesion has remained controversial. In this study, we compared whole blood (WB)-derived RBCs produced after 24-h RTH with rapidly cooled (RC) RBCs and tested them for classical metabolic markers and signs of oxidative damage. STUDY DESIGN AND METHODS: SAGM-RBCs were prepared from mixed and split pairs (n = 12) of WB units. RBCs prepared after a 24-h period of RTH on day+1 after collection (RTH-RBCs) were compared with RC-RBCs. All RBCs were stored at 4°C for 42 days with assay of in vitro variables on days+1, +15, +22, +29 and +42. The study examined standard quality parameters, glutathione, catalase and superoxide dismutase (SOD) activities, and indicative markers of oxidative cell damage including post-translational haemoglobin modification, malondialdehyde (MDA), and phosphatidylserine expression. RESULTS: RTH-RBCs exhibited decreased levels of potassium (1·98 ± 0·26 vs. 5·23 ± 0·65 mmol/l) and of 2,3-diphosphoglycerate (2,3-DPG) on day+1 compared with RC-RBCs. Haemolysis rate on day+42 was higher in RTH-RBCs than in RC-RBCs (0·52 ± 0·13 vs. 0·37 ± 0·12%). The phosphatidylserine expression amounted to 0·25 ± 0·20% in RTH-RBCs and 0·07 ± 0·12% in RC-RBCs. Haemoglobin modification was not different between both RBC groups. RTH-RBCs showed slightly higher MDA concentration on days +29 and +42. CONCLUSIONS: RC-RBCs and RTH-RBCs show only small differences of classical in vitro parameters and no relevant differences in antioxidative metabolism and oxidative haemoglobin modification. These findings do not explain the loss observed in in vivo survival studies with RBCs.

Buffy coat volume reduction for optimization of leucapheresis harvests produced by the autoMNC program.

BACKGROUND AND OBJECTIVES: Buffy coat (BC) volume reduction was evaluated in leucapheresis (LA) harvests due to the target monocyte yield and the red blood cell (RBC) content. A packed erythrocyte volume (PEV) of 7.5 ml should not be exceeded to avoid RBC debulking with loss of leucocytes (WBCs) and the monocyte fraction during monocyte counterflow elutriation, a next step of monocyte enrichment prior to cell culture. MATERIALS AND METHODS: Two hundred and fifty-three 5-l leucaphereses (autoMNC program) performed in 102 healthy blood donors (24 female and 78 male donors) were retrospectively analysed. Different categories of BC volumes were compared due to the quality of the LA products measured by blood counts and flow cytometry. RESULTS: Collection of maximum BC volume of 10 ml and more each collection cycle (product volume: 169 ± 21 ml) resulted in 1.58 ± 0·41 × 10e9 CD14(+) monocytes and high volume of packed erythrocyte (18.4 ± 8.8 ml). Low BC volume collection below 6 ml each collection cycle produced only 1.07 ± 0.40 × 10e9 CD14(+) monocytes but reduced PEV significantly by 64% (6.7 ± 4.1 ml). CONCLUSION: By reduction of the BC volume, the PEV in LA products could be reduced, which is a precondition for counterflow elutriation of monocytes. A BC volume between 7 and 8 ml per collection cycle should be adjusted to reduce PEV to 7.5 ml without relevant monocyte loss.

First comparative evaluation of a new leukapheresis technology in non-cytokine-stimulated donors.

BACKGROUND AND OBJECTIVES: Leukapheresis is an important source for mononuclear cells (MNCs) used in adoptive immunotherapies. Differences in the apheresis technology concerning physical conditions during cell separation and the optical detection system can affect the product's cellular content. MATERIALS AND METHODS: In a paired analysis, twenty healthy non-cytokine-stimulated donors underwent MNC collection at the Spectra Optia (Terumo BCT, Lakewood, CO, USA) and the COM.TEC (Fresenius Kabi, Bad Homburg, Germany) device. In twelve donors, apheresis was additionally performed with the Amicus (Fenwal Inc., Lake Zurich, IL, USA). Donor response to leukapheresis and product composition was compared. RESULTS: Mean yields of CD14+ (CD3+) cells were 1·64±0·70x10(9) (2·36±0·96×10(9)) in the Spectra Optia, 1·45±0·50×10(9) (3·03±1·04×10(9)) in the COM.TEC and 1·20±0·37×10(9) (2·80±1·00×10(9)) in the Amicus products, respectively. The Spectra Optia collected significantly more CD14+ monocytes than the Amicus and significantly less CD3+ T cells than the COM.TEC (P=0·002 and P=0·021). Apheresis products of the Spectra Optia showed the significantly lowest red blood cell yields while the Amicus generated products with the significantly lowest platelet contents. CONCLUSIONS: Leukaphereses with the three devices resulted in almost equal total MNC yields. MNC products of the Spectra Optia and the Amicus could be used in preference for the monocyte enrichment by the Elutra system and the leukapheresis procedures could be also favourably applied in patients with low platelet counts. The COM.TEC is more efficient in monocyte and T-cell collection with the disadvantage of high residual non-target cell content in the products.

The structure of the von Willebrand factor is not altered in patients with colorectal carcinoma.

AIM: Elevated levels of von Willebrand factor (VWF) are often observed in many diseases including colorectal cancer, but this finding is not definite. The aim of our study was to examine the change in VWF multimer distribution in patients with colorectal cancer. METHOD: We randomly selected nine patients from each of the four Union for International Cancer Control (UICC) stages of colon cancer. VWF antigen (VWF:Ag), VWF-cleaving protease ADAMTS-13 level and factor VIII activity (FVIII:C) were determined. The multimer distribution of VWF was visualized using electrophoretic multimer analysis. RESULTS: The VWF multimer structure was normal with no difference between the four UICC stages. There was no significant increase in VWF:Ag and FVIII:C levels in the more advanced UICC stages. There was no significant difference in the ADAMTS-13 level according to the UICC stage. CONCLUSION: There was no change in the VWF multimer distribution to indicate acquired von Willebrand disease.

Storage induced apoptosis of peripheral blood mononuclear cells obtained from leucoreduction system chambers.

BACKGROUND AND OBJECTIVES: Recently, it was reported that leucocytes obtained from leucoreduction system chambers (LRSCs) after plateletapheresis show excellent quality due to culture of dendritic cells. This study analysed apoptosis of mononuclear cells derived from LRSCs of single platelet units (SPUs) and double platelet units (DPUs) during storage. MATERIALS AND METHODS: This randomized prospective study compared eighteen single and double platelet units produced with the Trima Accel cell separator. Buffy coat was drained from the LRSCs and analysed after 1, 6, 24, 48 and 72 h. CD45+ lymphocytes and CD14+ monocytes cells as well as Annexin-V+ and 7-AAD+ mononuclear cells were measured by flow cytometry. RESULTS: The WBC concentration of LRSCs obtained from SPUs and DPUs differed significantly (SPUs: 0·93 ± 0·32 ×10(5) per µl WBCs; DPUs: 1·71 ± 0·55 ×10(5) per µl WBCs; P<0·001). Processed blood volume (PBV) correlated significantly with WBC concentration (r(2)=0·75, P<0·001). Fifty percent of monocytes were Annexin-V-positive 1 h after production decreasing to 30% during 24 h of storage. Compared to that, the part of late apoptotic or necrotic PBMCs increased later on, after 24 h. After 24 h, Annexin-V- and 7-AAD-positive, late apoptotic and necrotic lymphocytes and monocytes doubled. CONCLUSION: PBMCs stored in autologous plasma in PVC-bags at room temperature did not show an increase of 7-AAD-positive PBMCs during 24 h prior to cell processing but increased significantly thereafter.

Transitory interruption of recommended storage conditions does not cause significant changes in in vitro parameters of leucocyte-depleted red blood cells.

BACKGROUND AND OBJECTIVES: Conventional quality control studies of the shelf life of RBC units do not consider cold chain interruptions that occur during cross-matching or between the release of RBCs from the blood bank and their return from the ward. These interruptions may, however, lead to a considerable loss of quality. On the other hand, differences in the quality of RBCs may derive from the different manufacturing processes employed in various blood centres. MATERIALS AND METHODS: One day after the expiry date of the RBC unit, we analysed complete blood count, blood gas, potassium, LDH, hydroxybutyrate dehydrogenase, glucose, lactate, total and free haemoglobin (Hb) and ATP and compared the results with regard to the frequency of storage interruptions and to two manufacturers of these RBCs. RESULTS: We could not find any correlations between the frequency of interruptions (0-11) and these parameters in any of the data sets. However, we found significant differences when comparing the two suppliers. RBCs of manufacturer A ('A', inline filtration of whole blood) contained 25% more Hb than those of manufacturer B ('B', inline filtration after buffy coat reduction). Sixteen percentage of 'A'-RBC, but none of 'B'-RBC, exceeded a haemolysis of 0.8%. CONCLUSIONS: Transitory interruptions of cold chain do not measurably impair the quality of RBCs. The effect on storage of RBCs in the blood bank is not as significant a factor as the differences that exist between RBC manufacturing procedures.