Treatment of recalcitrant ulcers with allogeneic platelet gel from pooled platelets in aged hypomobile patients.

We evaluated growth factor contents and clinical efficacy of allogeneic platelet gel (PG) prepared with standard blood banking procedures from routine platelet concentrates (PCs) obtained from buffy coats. The PGs were used to treat 11 hypomobile very elderly patients unable to undergo autologous blood processing and previously ineffectively treated with expensive advanced medications for 8-275 weeks. PGs were prepared by platelet activation with human thrombin or commercial batroxobin. Median and range growth factor contents (ng/mL) were: platelet derived growth factor (PDGF-AB/-BB) 112 (31-157) and 20 (3.8-34); transforming growth factor (TGF-ß1/-ß2) 214 (48-289) and 0.087 (0.03-0.28); basic-fibroblast growth factor (b-FGF) 0.03 (0.006-0.214); vascular endothelial growth factor (VEGF) 1.15 (0.18-2.46); epidermal growth factor (EGF) 4.50 (0.87-6.64); insulin-like growth factor (IGF-l) 116 (72-156). In the clinical study, 222 PGs were used within 2 h of activation to treat 14 chronic skin ulcers in the 11 patients. No improvement was seen in 3 patients with 24, 27 and 30 cm(3) ulcers who could be treated for no more than 4, 7 and 8 weeks due to progressively worsening clinical conditions, while 11 ulcers with 3.2 cm(3) median size (range 0.2-3.6) in the remaining 8 patients showed 91 ± 14 % reduction after a median of 12 weeks (range 1-20). Cost of PG treatment (19,976 euro) amounted to about 10% of the ineffective advanced medication hospital reimbursement fees (191,236 euro). This study supports efficacy and feasibility of allogeneic PG to treat recalcitrant ulcers in very elderly hypomobile patients for whom autologous blood processing may be difficult.

Not every PRP-gel is born equal. Evaluation of growth factor availability for tissues through four PRP-gel preparations: Fibrinet, RegenPRP-Kit, Plateltex and one manual procedure.

BACKGROUND: The rationale for using topical platelet gel therapy is to provide the healing tissues with concentrated platelet-derived factors. Several systems are available to prepare platelet-rich plasma (PRP) and from these, the platelet gel. These systems produce two- to six-fold platelet and growth factor-enriched concentrations. The bioavailability of growth factors in tissue healing depends on the amount of growth factors stored in platelets but a portion of these is lost during platelet manipulation. Very few data have been reported on the kinetics of growth factor release from PRP-gels. The aim of this study is to assess the growth factor recovery and its bioavailability to tissues in four different PRP and PRP-gel preparation techniques. MATERIALS AND METHODS: Three commercially available devices (Fibrinet, RegenPRP-Kit, Plateltex) and one manual procedure (home made, HM) were evaluated with reference to resulting platelet concentration, growth factor content and the kinetics of growth factor release from gel. RESULTS: Platelet concentration increased from 1.65- to 4.4-fold in comparison with whole blood initially used. The final platelet concentration (x 10(3)/microl) was: Fibrinet 1358 +/- 419, Regen 430 +/- 109, HM 1196 +/- 188, and Plateltex 1160 +/- 164. A high variation (5- to 27-fold) was found in growth factor concentration in relation to the method used and also a high variation in the kinetics of growth factor release from gels. CONCLUSIONS: Similar methods for platelet gel preparation revealed different performances concerning growth factor recovery and the kinetics of its release from the gel. It is unclear whether these noticeable differences are important for clinical management.

Platelet-rich plasma and platelet gel preparation using Plateltex.

BACKGROUND: The platelet gel is made by embedding concentrate platelets within a semisolid (gel) network of polymerized fibrin. It is believed that this blood component will be used more and more in the treatment of several clinical conditions and as an adjunctive material in tissue engineering. Several systems are available to produce platelet-rich plasma (PRP) for topical therapy. Recently, a new system became commercially available, Plateltex. Here we report the technical performance of this system in comparison with the performance of other commercially available systems: PRGF, PRP-Landesber, Curasan, PCCS, Harvest, Vivostat, Regen and Fibrinet. MATERIAL AND METHODS: Both the PRP and the gel were prepared according to the manufacturer's directions. The blood samples of 20 donors were used. The yield, the efficiency, and the amount of platelet-derived growth factor AB (PDGF-AB), transforming growth factor beta, vascular endothelial growth factor and fibroblast growth factor were measured in the resulting PRP. The feature of the batroxobin-induced gelation was evaluated. RESULTS: The yield, the collection efficiency and the growth factor content of Plateltex were comparable to those of most of the other available systems. The gelation time was not dependent on the fibrinogen concentration; however, it was strongly influenced by the contact surface area of the container where the clotting reaction took place (P < 0.0001). CONCLUSIONS: Plateltex provided platelet recovery, collection efficiency and PDGF-AB availability close to those provided by other systems marketed with the same intended use. Batroxobin, the enzyme provided to induce gelation, acts differently from thrombin, which is used by most other systems. Platelets treated with thrombin become activated; they release their growth factors quickly. Furthermore, thrombin-platelet interaction is a physiological mechanism that hastens the clot-retraction rate. On the contrary, platelets treated with batroxobin do not become activated; they are passively entrapped within the fibrin network, and their growth factor release occurs slowly. In these conditions, the clot retraction takes longer to occur. According to these differences between thrombin and batroxobin, it is expected that batroxobin-induced PRP activation will tailor slow release of the platelet content, thus, providing longer in loco availability of trophic factors. In selected clinical conditions, this durable anabolic factor availability might be preferable to quick thrombin-induced growth factor release.

Collagen I-coated titanium surfaces: mesenchymal cell adhesion and in vivo evaluation in trabecular bone implants.

The goal of the study was the evaluation of the effect of modification of titanium implants by acrylic acid surface grafting-collagen I coupling. Tests were performed on titanium samples treated by galvanostatic anodization to create a porous surface topography. Surface characterization by X-ray photoelectron spectroscopy (XPS) and scanning electron microscopy (SEM) confirms the biochemical modification of the surface and shows a surface topography characterized by pores mostly below 1 mum diameter. In vitro evaluation involving human mesenchymal cells shows enhanced cell growth on collagen coated surfaces as compared to titanium ones. Four weeks in vivo evaluation of implants in rabbit femur trabecular bone shows improvements of bone-to-implant contact, while improvement of bone ingrowth is slightly not significant (p = 0.056), when compared to the control. Overall, these data indicate that integration in trabecular, or cancellous, bone can be enhanced by the surface collagen layer, confirming previous findings obtained by modification of machined surfaces by the same approach in cortical bone implants.

Evaluation of the hemostatic function of stored platelet concentrates using the platelet function analyzer (PFA-100 ).

BACKGROUND AND OBJECTIVE: Progressive functional impairment is known to occur in platelet concentrates through the storage period. Standardized methods providing direct measurement of residual platelet function in stored platelets are lacking. The purpose of this study was to determine whether a new platelet function analyzer (PFA-100 ) could provide standardized methods for assessing the hemostatic capacity of stored platelets. DESIGN AND METHODS: The PFA-100 was used to evaluate platelet function in stored platelets. The instrument can process citrated whole blood but it is unable to process platelet suspensions. Accordingly, the function of platelet concentrates should be measured following reconstitution of pseudo-whole blood. The analysis of the results included the closure time (sec) and a predictive index, an arithmetical index computed on the basis of the instrument's output data: the flow rate, the flow volume, the closure time. RESULTS: A final hematocrit of 58+/-2 and a final platelet concentration of 230+/-20x10(9)/L were used as standardized operative conditions to measure the function of stored platelet concentrates. The closure time (PFA-CT) and the predictive index (PFA-PI) both resulted to be capable of discriminating platelet concentrates with maintained or impaired function. PFA-PI was more informative than PFA-CT in terms of description of the residual platelet function. Of the two agonists used, epinephrine (EPI) resulted to be particularly sensitive for the detection of initial platelet hyporeactivity, whereas adenosine 5'-diphosphate (ADP) was particularly useful for measuring the residual platelet reactivity. INTERPRETATION AND CONCLUSIONS: PFA-CT and PFA-PI can be standardized; they provide new information about the hemostatic function of stored platelet concentrates and can be used to assess the quality of platelet concentrates.

Microdroplet fluorochromatic assay for the enumeration of white cells (WBCs) in WBC-reduced blood components: validation and application for evaluating newly developed WBC-reduction filters.

BACKGROUND: Sensitive and accurate counting methods are required to assess the residual white cells (WBCs) in WBC-reduced blood components. The Nageotte hemocytometer, widely used for this purpose, is cumbersome, and its efficacy is dependent upon the skill of the operator. The performance of a simple fluorochromatic assay using tissue-typing microdroplet trays is presented here. STUDY DESIGN AND METHODS: Undiluted samples of blood components were mixed with a fluorochromatic dye in trays. WBCs were counted under an epifluorescence microscope. The accuracy and sensitivity of this method were compared with those of the reference Nageotte hemocytometer method by using serial dilution of samples of platelets and red cells containing known concentrations of WBCs and by calculating the standard curves. The Nageotte hemocytometer and the microdroplet fluorochromatic assay (MFA) were also compared in terms of count correlation and reproducibility in 320 paired counts of plateletpheresis samples. MFA was used to evaluate a newly developed WBC-reduction red cell filter. RESULTS: The MFA for platelets and red cells was linear to 0.1 and 0.03 WBCs per microL, respectively. The linear regression line of log10 MFA versus log10 Nageotte method had a slope of 0.963, intercept of -0.04, and r2 of 0.968. The Nageotte method gave an estimation of WBC content 12 to 20 percent greater than that of the MFA. The MFA, with a larger neat sample volume, showed precision comparable to that of the Nageotte method. The filters demonstrated a median WBC reduction of 4.8 log10. CONCLUSION: The MFA is a sensitive and accurate method for quality control processes to assess the residual WBCs in WBC-reduced blood components.

CD36 autoantibodies and thrombotic diathesis, thrombocytopenia and repeated early fetal losses.

BACKGROUND AND OBJECTIVES: Autoantibodies to CD36, a platelet glycoprotein, have been found in patients with thrombotic thrombocytopenic purpura, and in those with lupus-like anticoagulant with thrombotic complications. MATERIALS AND METHODS: Conventional hematologic and laboratory methods were used. The patient was a pregnant woman, who had had two early fetal losses separated by a normal offspring. Despite severe thrombocytopenia, she was asymptomatic. RESULTS: Serological investigations were strongly suggestive of CD36 autoantibodies. Neither clinical nor laboratory data were typical of those usually associated with cd36 autoantibodies, namely thrombotic thrombocytopenic purpura (TTP), systemic lupus erythematosis (SLE), or prothrombotic compliance. Prophylaxis with salicylates and prednisone was started at the 8th week of gestation, and an offspring with mild thrombocytopenia was delivered by cesarean section at the 32nd week of gestation because of abruptio placentae. CONCLUSIONS: There may be a cause-and-effect relationship between early fetal losses and CD36 autoantibodies.

Specific antiplatelet autoantibodies in patients with antiphospholipid antibodies and thrombocytopenia.

By means of immunoblotting and monoclonal antibody immobilization of platelet antigens (MAIPA) we have studied the specificity of antiplatelet antibodies in patients with antiphospholipid antibodies and thrombocytopenia defined as presence of anticardiolipin IgG and a platelet count below 100 x 10(9)/l. The study group consisted of 10 patients with systemic lupus erythematosus (SLE), 8 patients with primary anti-phospholipid syndrome (PAPS) and 16 patients with idiopathic thrombocytopenic purpura (ITP). The comparison group was formed by 17 patients with classical chronic ITP without anticardiolipin IgG. We identified the 80-100, 130-150 and 150-170 KD surface proteins that comigrate with GPIIIa, GPIIb and GPIb and a 50-70 KD cytoplasm band by immunoblot. In patients with classical chronic ITP, the prevalence of the antiplatelet antibodies against GPIIIa was 53% on immunoblot assay and 47% on MAIPA. In ITP patients who had also anti-phospholipid antibodies in serum, the percentage of reactivity to GPIIIa declined to 37% on immunoblot and 21% on MAIPA but it was not statistically different from the percentage observed in patients with classical ITP. Autoantibodies to platelet surface glycoproteins were almost absent in SLE and PAPS patients, who showed a significant prevalence (78%) of IgG reactivity to the 50-70 KD internal platelet protein which was frequently encountered also in patients with ITP and aPL (56%). Our study provides additional evidence that platelet antigens in patients with phospholipid-associated secondary immune thrombocytopenia are different from those of primary ITP, and that surface glycoproteins were not involved.