Evaluation of the in vitro Function of Platelet Concentrates from Pooled Buffy Coats or Apheresis.

BACKGROUND: Platelet concentrates play an important role in transfusion medicine. Their short lifespan and lack of robustness require efforts to ensure adequate product quality. In this study, we compared the in vitro quality of the main concentrate types, pooled platelet concentrate (PPC) from whole blood donations, and platelet concentrate from single-donor apheresis (APC). METHODS: Twenty PPCs and 20 APCs prepared in plasma were analyzed on days 2, 4, and 7 of storage. Variables related to metabolism, degranulation, platelet aggregation, P-selectin expression, and annexin V binding were analyzed. Morphology was assessed by transmission electron microscopy of ultrathin sections. A microfluidic device was applied to test the effects of shear stress on platelet function. RESULTS: The metabolic parameters indicated stable storage conditions throughout the 7-day period. The resting discoid form was the prevailing morphology on days 2 and 4 in the PPCs and APCs. Chemokine release and receptor shedding of soluble P-selectin and soluble CD40L equally increased in PPCs and APCs. Aggregation responses to ADP and collagen were heterogeneous, with marked losses in collagen responsiveness on day 4 in individual concentrates. Baseline expression of P-selectin in PPCs and APCs was low, and inducibility of P-selectin was well preserved until day 4. Under shear stress, equal adhesiveness and stability were found with platelets from PPCs and APCs. CONCLUSIONS: Platelets from PPCs and APCs showed similar in vitro function and stability parameters. However, platelet concentrates presented a high variability and individual concentrates an impaired functional capability. Identifying the factors contributing to this would help increase product reliability.

Significance of platelet and monocyte activation for therapeutic immunoglobulin-induced thromboembolism.

OBJECTIVES: Thromboembolic events (TEE) in patients receiving infusions of intravenous immunoglobulin (IVIG) products have recently been associated with contaminating factor XIa. We studied whether platelet and monocyte activation could also be involved. METHODS: Twenty IVIG samples from five manufacturers were tested for the induction of visible whole blood clot formation. A selection of TEE-associated and not associated lots was further analyzed for effects on thromboelastometry, platelet activation and adhesion, as well as monocyte tissue factor surface expression. Pure factor XIa was included for comparison. Western blotting was applied to analyze anti-CD154-reactive proteins in IVIG. RESULTS: In whole blood, IVIG enhanced macroscopic clotting additively with factor XIa. In monocytes, all IVIG products induced the FcγRII-dependent tissue factor expression to a similar extent, which was not affected by addition of factor XIa. Testing platelet aggregation, IVIG strengthened the ADP and TRAP-6-elicited response. Furthermore, IVIG increased platelet-monocyte adhesion and annexin V binding to platelet microvesicles, and promoted platelet adhesion to IVIG-coated surfaces. The strongest effects were observed with TEE-associated lots. CD154-related proteins were detected in all IVIG products. CD154-related high molecular weight complexes were particularly found in the TEE-associated IVIG. In platelet aggregation, recombinant soluble CD154 enhanced aggregate formation and stability. CONCLUSION: Our data demonstrate that IVIG modulate platelet and monocyte activation and can thereby affect the hemostatic balance. These effects are either additive to or independent from factor XIa. CD154-related proteins are assumed to be involved in these interactions, the mechanism of which needs to be elucidated in further studies.

Inducible expression of tissue factor in small-cell lung cancer: impact on morphology and matrix metalloproteinase secretion.

PURPOSE: Tissue factor (TF), the transmembrane receptor for factor VIIa (FVIIa), has key regulatory functions in coagulation as well as in tumour progression and metastasis. Small-cell lung cancer (SCLC) metastasises more aggressively than non-small-cell lung cancer (NSCLC). Previously, we described the transition of SCLC cell line H69 to adherent growth and TF expression. Here, we explored the differential expression of TF and its functional impact on morphology and matrix metalloproteinase (MMP) secretion. METHODS: The constitutional TF expression was evaluated in a panel of established NSCLC and SCLC cell lines. Furthermore, in three stress-selected adherent SCLC H69 cells, TF and MMP expressions were determined by mRNA, protein, and activity measurements. RNA interference-mediated TF down-regulation and FVIIa stimulation were used to study the impact of TF on cellular functions. RESULTS: NSCLC cells expressed high TF antigen (median 3.75 ng/mg; range 0.31-65.2 ng/mg protein, n = 8), while SCLC expressed none or low TF (median 0.07 ng/mg; range 0-0.39 ng/mg protein, n = 6). However, selected H69 adherent cells markedly expressed TF (range: 4.8-44.3 ng/mg protein, n = 3) and secreted MMP-2 and MMP-9. FVIIa stimulated MMP-2 and MMP-9 secretion in H69adh cells, whereas TF down-regulation diminished MMP-2 and MMP-9 expression and promoted reversion to suspension growth. CONCLUSIONS: Our data show the significance of TF expression in the reversible growth phenotype of H69. Because TF, MMP expression, and adherence are highly relevant to cancer metastasis, this study suggests a novel mechanism of adaptation, thereby adding to the understanding of SCLC biology and its aggressiveness.

Evaluation of Quality Parameters for Cord Blood Donations.

SUMMARY: BACKGROUND: Umbilical cord blood (CB) is widely used for hematopoietic stem cell transplantation and holds promise for the development of innovative medicinal products. In order to find out whether the conditions for collection and storage before processing might have an impact on the quality of CB preparations, viability and the clonogenic potential were assessed. METHODS: CB was collected under field conditions. Flow cytometry was used to determine leukocytes, CD34/CD45+ cells, viability, and nucleated red blood cells (NRBC). Clonogenic activity was determined using isolated mononuclear cells (MNC). RESULTS: Neither plasma citrate concentrations nor storage temperature (within 24 h) affected cell viability or colony formation. After storage for 49-80 h, leukocyte viability declined by about 16% compared to CB stored up to 24 h. In contrast, the clonogenic activity and CD34/CD45+ cell content were not affected. A higher gestational age was associated with a lower yield of clonogenic activity compared to midterm deliveries. NRBC varied widely (median 7.3%; range 0.63-17.3%) without relation to gestational age or colony formation. There was a close correlation between the percentage of viable CD34/CD45+ cells and colony formation (r = 0.77 for CFU-GM; r = 0.75 for CFU-C). CONCLUSIONS: The content of viable CD34/CD45+ cells represents the clonogenic activity of CB preparations. Therefore, determination of viable CD34/CD45+ cells should be generally performed as a routine quality control assay.