An autologous blood-derived patch as a hemostatic agent: evidence from thromboelastography experiments and a porcine liver punch biopsy model.

Perioperative bleeding is a common complication in surgeries that increases morbidity, risk of mortality, and leads to increased socioeconomic costs. In this study we investigated a blood-derived autologous combined leukocyte, platelet, and fibrin patch as a new means of activating coagulation and maintaining hemostasis in a surgical setting. We evaluated the effects of an extract derived from the patch on the clotting of human blood in vitro, using thromboelastography (TEG). The autologous blood-derived patch activated hemostasis, seen as a reduced mean activation time compared to both non-activated controls, kaolin-activated samples, and fibrinogen/thrombin-patch-activated samples. The accelerated clotting was reproducible and did not compromise the quality or stability of the resulting blood clot. We also evaluated the patch in vivo in a porcine liver punch biopsy model. In this surgical model we saw 100% effective hemostasis and a significant reduction of the time-to-hemostasis, when compared to controls. These results were comparable to the hemostatic properties of a commercially available, xenogeneic fibrinogen/thrombin patch. Our findings suggest clinical potential for the autologous blood-derived patch as a hemostatic agent.

Autologous Blood-Derived Patches Used as Anti-adhesives in a Rat Uterine Horn Damage Model.

BACKGROUND: Intra-abdominal adhesions are frequent side effects of surgery, associated with risks of serious complications such as abdominal pain, infertility, and small bowel obstruction. This study investigated a new autologous blood-based approach to adhesion prophylaxis. MATERIALS AND METHOD: Two autologous blood-derived patches (whole-blood-derived, n = 20, and plasma-derived, n = 20) were evaluated as anti-adhesives. The patches were tested in a rat uterine horn damage model. We simulated an intraabdominal surgery by cauterizing and suturing the uterine horns and created an opposing damage by denuding a part of the abdominal wall. Each rat served as its own control with one treated uterine horn and one untreated. After 14 d of post-surgical recovery, the adhesions were assessed and graded macroscopically and microscopically. Statistical analyses were performed with Wilcoxon signed rank and Mann-Whitney U tests. RESULTS: Both whole-blood and plasma-derived patches resulted in significantly less macroscopic adhesions than were found in untreated uterine horns (P = 0.001 and P = 0.002, respectively). Unpaired analysis found no significant differences between the whole-blood and plasma-derived patch outcomes in this study design. Histopathological evaluation of inflammation and fibrosis did not reveal significant differences between the patches and their matched controls. CONCLUSIONS: The autologous blood-derived patches reduced macroscopic adhesion formation significantly compared with no treatment. There were no adverse events and no histological differences between treatment and control, suggesting that the treatments were feasible and safe. In summary, this study confirms the potential of autologous anti-adhesives for the use in intraabdominal surgery.

Novel human in vitro vegetation simulation model for infective endocarditis.

Infective endocarditis (IE) is a heart valve infection with high mortality rates. IE results from epithelial lesions, inducing sterile healing vegetations consisting of platelets, leucocytes, and fibrin that are susceptible for colonization by temporary bacteremia. Clinical testing of new treatments for IE is difficult and fast models sparse. The present study aimed at establishing an in vitro vegetation simulation IE model for fast screening of novel treatment strategies. A healing promoting platelet and leucocyte-rich fibrin patch was used to establish an IE organoid-like model by colonization with IE-associated bacterial isolates Staphylococcus aureus, Streptococcus spp (S. mitis group), and Enterococcus faecalis. The patch was subsequently exposed to tobramycin, ciprofloxacin, or penicillin. Bacterial colonization was evaluated by microscopy and quantitative bacteriology. We achieved stable bacterial colonization on the patch, comparable to clinical IE vegetations. Microscopy revealed uneven, biofilm-like colonization of the patch. The surface-associated bacteria displayed increased tolerance to antibiotics compared to planktonic bacteria. The present study succeeded in establishing an IE simulation model with the relevant pathogens S. aureus, S. mitis group, and E. faecalis. The findings indicate that the IE model mirrors the natural IE process and has the potential for fast screening of treatment candidates.

Characteristics of an autologous leukocyte and platelet-rich fibrin patch intended for the treatment of recalcitrant wounds.

We have investigated the physical, biochemical, and cellular properties of an autologous leukocyte and platelet-rich fibrin patch. This was generated in an automated device from a sample of a patient's blood at the point of care. Using microscopy, cell counting, enzyme-linked immunosorbent assay, antibody arrays, and cell culture assays, we show that the patch is a three-layered membrane comprising a fibrin sheet, a layer of platelets, and a layer of leukocytes. Mean recovery of platelets from the donated blood was 98% (±95%CI 0.8%). Mean levels of platelet-derived growth factor AB, human transforming growth factor beta 1, and vascular endothelial growth factor extracted from the patch were determined as 127 ng (±95% CI 20), 92 ng (±95%CI 17), and 1.35 ng (±95%CI 0.37), respectively. We showed a continued release of PDGF-AB over several days, the rate of which was increased by the addition of chronic wound fluid. By comparison with traditional platelet-rich plasma, differences in immune components were found. The relevance of these findings was assessed by showing a mitogenic and migratory effect on cultured human dermal fibroblasts. Further, we showed that fibrocytes, a cell type important for acute wound healing, could be grown from the patch. The relevance of these findings in relation to the use of the patch for treating recalcitrant wounds is discussed.

A pilot study to evaluate the safety and clinical performance of Leucopatch, an autologous, additive-free, platelet-rich fibrin for the treatment of recalcitrant chronic wounds.

This prospective, uncontrolled pilot study evaluated the safety and clinical performance of Leucopatch an additive-free, autologous platelet-rich fibrin in the treatment of recalcitrant chronic wounds. Fifteen patients, with 16 lower extremity chronic wounds of varying etiologies were treated weekly with Leucopatch, prepared at the point of care from a donation of the patients' blood, for 6 weeks, or until healing was complete. The wounds had been present for 2 to 108 months (median 24 months) and ranged in size from 0.4 to 15.7 cm(2) (median 2.3 cm(2)) and had not responded to previous treatments. Of the 13 wounds (12 patients) included in the per-protocol efficacy analysis, 4 healed completely (31%). Mean wound area decreased significantly by 65% (95% confidence interval = 45.6% to 83.8%) resulting in a median wound size of 0.9 cm(2) (range = 0-9.6cm(2)). There were no serious adverse events. Two adverse events, one of noncompliance and one infection, were observed; neither was considered to be related to treatment. The results indicate that Leucopatch is easy to prepare and apply in the clinic, is safe, and may be a clinically effective treatment of recalcitrant chronic wounds.

Bioactivity and stability of endogenous fibrogenic factors in platelet-rich fibrin.

Platelet-rich fibrin (PRF) is an autologous fibrin sealant (FS) enriched with a platelet concentrate (> 1,000,000 platelets/microL) produced by the automated Vivostat system and used to enhance wound healing. The effects of PRF were compared with supernatant from thrombin-activated platelet concentrate, recombinant human platelet-derived growth factor (rhPDGF) isoforms, and a homologous FS in cultured normal human dermal fibroblasts. Also, the release of selected endogenous growth factors from PRF and their stability against proteolytic degradation were studied. The proliferative effect of PRF exceeded that of FS and rhPDGF-BB, although it was lower than thrombin-activated platelet concentrate possibly due to sustained growth factor release from platelets in PRF. Anti-PDGF antibody blocked the mitogenic effect of rhPDGF-BB but not that of PRF in growth-arrested fibroblasts. PRF promoted secretion of carboxyterminal propeptide of type I collagen into conditioned medium while rhPDGF-AB had no significant effect on collagen biosynthesis. Limited proteolysis of PDGF-AB and no proteolysis of transforming growth factor-beta1 (TGF-beta1) in PRF were observed with trypsin treatment, whereas rhPDGF-AB and rhTGF-beta1 in bovine serum albumin, matching the total protein concentration of PRF, were almost completely degraded after 24 hours at 37 degrees C. To conclude, PRF provides sustained release and protection against proteolytic degradation of endogenous fibrogenic factors important for wound healing.

A novel antibody-dependent cellular cytotoxicity mechanism involved in defense against malaria requires costimulation of monocytes FcgammaRII and FcgammaRIII.

Clinical experiments have shown that the Ab-dependent cell-mediated inhibition of Plasmodium falciparum is a major mechanism controlling malaria parasitemia and thereby symptoms. In this study, we demonstrate that a single merozoite per monocyte (MN) is sufficient to trigger optimal antiparasitic activity. Using particulate Ag as pseudomerozoites, we show that only Ags, and no other parasite-derived factor, are required to trigger MN activation and that a single Ag is as potent as the complex combination of Ags constituting the merozoite surface. Moreover, we found that soluble Ags binding at least two Abs are as effective as the parasite at stimulating MN and that nonmalarial Ags are as efficient provided they are targeted by cytophilic Abs. Indeed, only cytophilic IgGs are potent and, in agreement with immunoepidemiological findings, IgG3 is superior to IgG1. Very low Ab concentrations (>700 pM), i.e., in the range of molecules having a hormonal effect, are effective, in contrast to Abs having a direct, neutralizing effect. Finally, Ab-dependent cell-mediated inhibition proved to require the synergistic activation of both FcgammaRIIa and FcgammaRIIIa which both distinguish it from other Ab-dependent cellular cytotoxicity and implies that all MN are not equally effective. These findings have both fundamental and practical implications, particularly for vaccine discovery.

[Antenatal determination of fetal RhD-blood type based on fetal DNA in plasma from the RhD-negative mother--secondary publication]. / Antenatal bestemmelse af føtal RhD-blodtype baseret på føtalt DNA i plasma fra den RhD-negative mor--sekundaerpublikation.

We present a reliable test for prenatal prediction of fetal RhD type using maternal plasma from RhD-women. This test is needed for a future antenatal RH prophylaxis. A new real time PCR based assay targeting RHD exon 7 and a published assay for RHD exon 10 were used to determine the fetal RHD status in DNA extracted from plasma from 56 pregnant women in 15th-36th week of gestation. Prediction of fetal RhD type was compared with the phenotype determined after birth, and showed 100% concordance. This setup will be of value in antenatal RH prophylaxis and in the management of immunised women.

Human recombinant antibodies against Plasmodium falciparum merozoite surface protein 3 cloned from peripheral blood leukocytes of individuals with immunity to malaria demonstrate antiparasitic properties.

Immunoglobulins from individuals with immunity to malaria have a strong antiparasitic effect when transferred to Plasmodium falciparum malaria infected patients. One prominent target of antiparasitic antibodies is the merozoite surface antigen 3 (MSP-3). We have investigated the antibody response against MSP-3 residues 194 to 257 (MSP-3(194-257)) on the molecular level. mRNA from peripheral blood leukocytes from clinically immune individuals was used as a source of Fab (fragment antibody) genes. A Fab-phage display library was made, and three distinct antibodies designated RAM1, RAM2, and RAM3 were isolated by panning. Immunoglobulin G1 (IgG1) and IgG3 full-length antibodies have been produced in CHO cells. Reactivity with the native parasite protein was demonstrated by immunofluorescence microscopy, flow cytometry, and immunoblotting. Furthermore, the antiparasitic effect of RAM1 has been tested in vitro in an antibody-dependent cellular inhibition (ADCI) assay. Both the IgG1 and the IgG3 versions of the antibody show an inhibitory effect on parasite growth.

Reliable test for prenatal prediction of fetal RhD type using maternal plasma from RhD negative women.

OBJECTIVES: The objective of this study was to establish a reliable test for prenatal prediction of fetal RhD type using maternal plasma from RhD negative women. This test is needed for future prenatal Rh prophylaxis. METHODS: A novel real-time PCR-based assay targeting RHD exon 7 combined with a published assay for RHD exon 10 were used to determine the fetal RHD status in DNA extracted from plasma, sampled from 56 pregnant RhD negative women in 15th-36th week of gestation. Thirty-eight samples were from ongoing pregnancies of Danish women and 21 samples from 18 pregnant women were stored anonymized samples from the International Blood Group Reference Laboratory, Bristol, United Kingdom. Prediction of fetal RhD type was compared with the serological result obtained after birth. RESULTS: The prediction of the fetal RhD type was in 100% concordance with the serological RhD type from the 16th week of gestation. One sample from the 15th week of gestation was inconclusive. The number of copies of fetal RHD DNA was found to increase with gestational age. Low levels of DNA were found to follow the Poisson distribution (p = 1.0000). CONCLUSION: Our set-up was very reliable for determination of fetal RhD genotype, and thus will be of value in prenatal Rh prophylaxis and in the management of immunized women.