Reduction of biological response modifiers in the supernatant of washed paediatric red blood cells.

BACKGROUND: Washing of red blood cells (RBC) can reduce unwanted biological response modifiers (BRMs) that can mediate transfusion complications in infants. The aim of this study was to examine the in vitro quality and the changes in BRMs following washing in paediatric RBC units. MATERIALS AND METHODS: A pool and split design was used to prepare RBC (either 1 or 4 days old; n = 26 pairs). One unit was washed with 0·9% saline by centrifugation and then resuspended in SAG-M, while the other remained unwashed. Each RBC unit was divided to produce four units of paediatric-sized components. Samples were taken after 3 h and subsequently on days 1, 2, 7 and 14 post-wash. RESULTS: Washing of RBC resulted in some red cell loss, with a minor increase in haemolysis. Washing effectively reduced supernatant potassium and IgA, as well as cytokines and complement proteins. RBC microparticles were significantly reduced in RBC washed at 1, but not 4 days post-collection. Incubation with supernatant from unwashed but not washed RBC led to endothelial cell activation, with increased cell surface expression of CD62E (E-selectin) and CD106 (VCAM). CONCLUSION: Although washing affected some aspects of the in vitro quality of RBC, it effectively reduced the concentration and activity of BRMs in the supernatant of RBC. Such a reduction may be clinically beneficial in selected patient groups.

Treatment of platelets with riboflavin and ultraviolet light mediates complement activation and suppresses monocyte interleukin-12 production in whole blood.

BACKGROUND AND OBJECTIVES: Pathogen inactivation (PI) and storage may alter the immunomodulatory capacity of platelets (PLTs). The aim of this study was to examine the effect of PI (Riboflavin and ultraviolet light treatment) and storage on the capacity of PLTs to induce cytokine responses in recipient inflammatory cells. MATERIALS AND METHODS: A pool and split design was used to prepare untreated and PI-treated buffy coat-derived platelet concentrates (PCs). Samples were taken on days 2 and 7 postcollection and incubated with ABO/RhD-matched fresh whole blood for 6 h with or without lipopolysaccharide (LPS). The intracellular production of IP-10, MCP-1, MIP-1α, IL-8, IL-6, IL-10, IL-12, TNF-α and MIP-1ß in monocytes and neutrophils was assessed using flow cytometry. Complement proteins in PLT supernatants were measured using a cytometric bead array. RESULTS: PLTs and PLT supernatant (both untreated and PI-treated) resulted in modulation of intracellular MIP-1ß and IL-12 production in monocytes. Compared to untreated PLTs, PI-treated PLTs resulted in significantly lower LPS-induced monocyte IL-12 production (day 7). The concentration of C3a and C5a (and their desArg forms) was significantly increased in PLT supernatants following PI. CONCLUSION: PI results in decreased LPS-induced monocyte IL-12 production and increased complement activation. The association between platelet-induced complement activation and IL-12 production warrants further investigation.

In vitro assessment of buffy-coat derived platelet components suspended in SSP+ treated with the INTERCEPT Blood system.

BACKGROUND: The INTERCEPT Blood System uses amotosalen-HCl and UVA light to cross-link DNA and RNA, thereby inhibiting pathogen replication. Although previous studies have shown that this treatment alters in vitro platelet quality, most studies have assessed apheresis platelets or platelets pooled from 5 or 6 donors. In Australia, platelets are prepared using buffy-coats from 4 donors, with SSP+ and have lower plasma carryover than recommended by the manufacturer (32-47%). As such, it is currently unknown whether these platelet concentrates are suitable for INTERCEPT treatment. MATERIALS AND METHODS: Platelet concentrates were prepared by pooling four buffy-coats with SSP+, resulting in 30% plasma carryover. Two platelet units were pooled and split to generate paired units, with one unit treated with the INTERCEPT System (n = 6), whilst the other remained untreated (n = 6). All units were stored for seven days at 22 °C with agitation. RESULTS: INTERCEPT treatment resulted in 10·4 ± 4·3% loss of platelets, but did not significantly affect the functional integrity of mitochondria. INTERCEPT-treated platelets demonstrated a decreased pH, accelerated lactate production and glucose consumption, as well as higher surface expression and increased secretion of P-selectin and reduced collagen-induced aggregation. These changes were particularly evident from day 5 of storage. CONCLUSION: The observed increase in platelet glycolysis following INTERCEPT treatment is consistent with previous literature reports. Importantly, the in vitro changes were less marked than previously reported indicating that the platelets suspended in SSP+ with reduced plasma carryover are of suitable in vitro quality following INTERCEPT treatment and storage.

A platelet-derived hydrogel improves neovascularisation in full thickness wounds.

Platelets are a reservoir of growth factors, cytokines and chemokines involved in spontaneous wound repair. In this study, a platelet-rich and fibrin-rich hydrogel was generated from expired platelet components that would have otherwise been transfused. The material contained physiological concentrations of transforming growth factor ß1 (TGF-ß1, platelet-derived growth factor AB (PDGF-AB), PDGF-BB, insulin-like growth factor-1 (IGF-1), fibroblast growth factor 2 (FGF-2), and epidermal growth factor (EGF). The effect of the hydrogel on wound repair was investigated in SKH-1 mice. Full thickness dorsal wounds were created on the mice and treated with the hydrogel at various concentrations. Immunohistochemical staining with CD31 (endothelial cell marker) revealed that wounds treated with the hydrogel showed significantly enhanced vascularisation in the wound bed. Moreover, high levels of interleukin-6 (IL-6) and KC (IL-8 functional homologue) in treated wounds were sustained over a longer period of time, compared to untreated wounds. We postulate that sustained IL-6 is a driver, at least partly, of enhanced vascularisation in full thickness wounds treated with the hydrogel. Future work is needed to explore whether this hydrogel can be utilised as a treatment option when vascularisation is a critical limitation. STATEMENT OF SIGNIFICANCE: The economic cost of wound repair is estimated in billions of dollars each year. In many cases time required to vascularise wounds is a major contributor to slow wound repair. In this study, we developed a blood-derived platelet- and fibrin-rich hydrogel. It contains a number of growth factors actively involved in spontaneous wound healing. This hydrogel significantly improved dermal repair and vascularisation in a full-thickness wound mouse model. This study provides an action mechanism for modulation of localised inflammation.

The human leucocyte antigen complex: its role in colorectal cancer.

The human leucocyte antigen (HLA) is frequently altered in human tumours compared to the tissue from which they originate. The ability of the tumour to invade requires a change in the phenotype of the cell allowing it to advance and expand without restraint from adjacent tissue and the host immune system. One important regulator of tumour growth is cellular immunity. Antigen recognition by "T" cells occurs in the context of the HLA products. Changes in the HLA expression of a malignant tissue may directly influence immunosurveillance mechanisms, as these molecules play a central role in presenting potentially immunogenic peptides to "T" cells. To assess whether alteration in HLA expression is a critical step in the multistep origin of tumours, it is important to examine the HLA expression in premalignant precursor lesions of tumours and in tumours themselves. Our studies have recently reported a downregulation of HLA class I expression in colorectal cancers and adenomatous polyps when compared to normal appearing mucosa adjacent to the cancer. Our study on the class I antigen expression showed the presence of the antigen on all samples of control colonic epithelium. In sporadic adenomatous polyps of the colorectum, 21% (n = 7) of adenomas expressed levels of HLA that were comparable to controls, 51% (n = 17) expressed low levels, and in 28% (n = 9) the antigen was undetectable. Similarly, in colorectal adenocarcinomas the antigen was reduced even further with levels comparable to controls in 5% (n = 2) of tumours, reduced in 30% (n = 6) and undetectable in 65% (n = 13). This abnormality may result in the loss of immunosurveillance which could be one mechanism by which cancer cells can evade "T" cell control, thus resulting in invasion and metastasis.

Characterization of hemopoietic engraftment kinetics and development of secondary cytopenia in AML post auto-SCT and its correlation with survival outcome.

We performed a single center retrospective analysis of 84 autologous hemopoietic stem cell transplants done for AML to characterize the pattern of hemopoietic engraftment, post-transplant cytopenia and their impact on survival outcome. Following autologous transplant and engraftment, 30 patients (35.7%) had a transient secondary decline in their plt counts, which was not associated with graft rejection, relapse or infection. The median time to onset of thrombocytopenia was 59 days post transplant, with spontaneous recovery after a median period of 41 days. A secondary decline in ANC also occurred in eight patients. Patients with secondary plt decline had a significantly earlier primary plt engraftment (median 15 days) and a trend towards earlier neutrophil engraftment compared with patients who maintained steady plt counts (median 21 days). There was a trend towards a lower incidence of secondary plt decline in patients who received BM stem cells compared with those who received PBSC. No cause was evident for the occurrence of a secondary cytopenia, and it did not adversely affect survival. We conclude that secondary cytopenia is a common and harmless occurrence after autologous transplant especially from PBSC graft.