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.

An immunoenzymatic assay for the detection and quantitation of platelet antibodies: the platelet beta-galactosidase test (PGT).

An immunoenzyme assay for detection of platelet antibodies and suitable for routine use is described. Purified rabbit IgG anti-human IgG antibodies are conjugated to beta-galactosidase with meta-maleimidobenzoyl-hydroxysuccinimide ester as a bifunctional reagent and o-nitrophenyl-beta-galactopyranoside as a substrate to evaluate the enzymatic activity of the labeled antiglobulin. The sera of 26 patients suffering from various diseases (acute leukemia, aplastic anemia and systemic lupus erythematosus) and 40 control subjects were assayed with the enzyme-labeled reagent and, for comparison, with an indirect immunofluorescence technique. Half of these patients had never been transfused. Platelet antibodies were detected by both assays in all the transfused patients except one, and in 3 out of 13 non-transfused patients. The sera of all the control subjects were negative. Quantitation of platelet antibodies was obtained by a sensitive antiglobulin absorption technique. A method for standardization of the reagents allowing comparison of results obtained in the same patient at different times and suitable for long-term follow-up studies is also described.

Acute Intravascular Haemolysis Associated with High Dose Immunoglobulin after Bone Marrow Transplantation for Acute Myelogenous Leukemia.

A 12-year-old boy developed persistent thrombocytopenia after undergoing bone marrow transplantation for acute myelogenous leukemia. High doses of intravenous immunoglobulins were used to treat overt hemorrhage and a sudden intravascular hemolysis occurred. The serologic findings point to an acute hemolysis secondary to the presence of isohemoagglutinins in the intravenous immunoglobulin preparations.

Blood transfusion and kidney transplantation: effect of small doses of blood on kidney graft function and survival.

A controlled clinical trial was started to evaluate whether small doses of blood given pretransplant determine a transfusion effect while reducing the risk of antibody production. For this purpose, 65 consecutive never transfused patients suffering from end-stage renal failure were assigned to one of two groups: the first group was transfused with 1 unit of packed red cells (containing a mean of 2350 x 10(6) leukocytes, 900 x 10(6) mononuclear cells) 3 times at 15-day intervals. The second group received one transfusion of about 30 ml of blood adjusted to contain 100 x 10(6) mononuclear cells. While no definitive conclusions are still possible, preliminary data indicate the following: (1) three small transfusions are capable of immunizing the recipient, but lymphocytotoxic antibodies tend to disappear rapidly; (2) in vitro lymphocyte response to lectins of patients receiving small transfusions is not significantly different from that of patients receiving standard transfusions; (3) the two groups of patients differ significantly as far as the T4+ /T8+ cell ratio is concerned: in fact while a decrease of the ratio is observed after standard transfusions, small transfusions determine an increase of the ratio, mainly due to a decrease in the number of T8+ cells; and (4) the clinical course and survival of the graft is worse in patients treated with small transfusions than in those treated with standard transfusions.

ELISA procedures for the characterization of platelet specific antibodies. Evaluation of some variables.

BACKGROUND: Characterization of antiplatelet antibodies is often difficult. METHODS: Four similar ELISA procedures for the characterization of the glycoprotein specificity of antiplatelet antibodies were compared. RESULTS: The four techniques gave overlapping results when tested on thirty-five patients' sera containing antiplatelet antibodies. With ten sera the four procedures gave slightly dissimilar results. All the discrepancies consisted of incomplete glycoprotein identification with partial loss of complete glycoprotein complex recognition. CONCLUSIONS: The analysis of the main technical variables led to the following conclusions: a) in some case antiplatelet antibodies recognize non-glycoproteic moieties of the platelet membrane (glyco- and phospholipids): the negative results found when investigating glycoprotein targets were the correct ones; b) the solubilization of unsensitized glycoproteins possibly leads to conformational changes able to mask the antigenic sites, while previous sensitization confers more stability to the molecule; c) best results were obtained using anti-mouse IgG as capturing agent for sensitized glycoproteins: it is likely that this antibody let a larger number of binding sites available for the second antibody.

Colloidal gold immunostaining for the detection of platelet reactive allo- and autoantibodies. Use on fresh and frozen cells.

BACKGROUND: Fresh washed platelets are commonly used as target cells for the detection of platelet reactive antibodies. The use of stored platelets would allow a faster execution of the tests and a rapid selection of phenotyped cells. METHODS: Glass-adherent platelet monolayers were freeze stored. The antigen-antibody reaction between platelet antigens and platelet reactive allo- or autoantibodies was revealed by a modified antiglobulin test, using colloidal gold-labeled anti-human IgG. Immunogold staining (IGS) was compared with the platelet suspension immunofluorescence test (PSIFT) and with the solid-phase red cell adherence assay (SPRCA). The reactivity of fresh and freeze-stored platelets was compared. RESULTS: IGS proved to be slightly less sensitive (10%) than PSIFT and SPRCA: The results obtained with fresh and freeze-stored cells were scored identically. CONCLUSIONS: The immunogold staining assay on freeze-stored platelets seems to be a convenient technical approach for platelet serology.

Granulocyte serology findings in juvenile symptomatic idiopathic autoimmune neutropenia using a multiassay procedure. Report on 21 cases.

The serological findings on 21 children with idiopathic autoimmune neutropenia are reported. A multiassay procedure was adopted including agglutination, immunofluorescence and cytotoxicity tests. Beside a cause-effect correlation between granulocyte antibodies and clinical course, a serologic polymorphism was found. As the sensitivity of each assay seemed to be related to the characteristics of the involved antigens and antibodies, as well as to the intrinsic sensitivity of the tests, the performance of a multiassay procedure appears to be advisable for the diagnosis and the follow-up of autoimmune neutropenia.

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.

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.

Evidence for the co-existence of immunocomplexes and platelet specific antibodies in HIV infection-related thrombocytopenia in narcotic drug addicts. Experimental results and immunopathogenetic discussion.

BACKGROUND: The development of symptomatic or asymptomatic thrombocytopenia is not uncommon in HIV seropositive drug addicts. METHODS: Platelet binding immunoglobulins, as immune complexes or as platelet specific antibodies, were studied in eleven patients. Whole sera anche serum factions obtained by PEG sedimentation and gel filtration were investigated using methods able to characterize the IgG and IgM immunoglobulin classes. Immune complexes-containing fractions were used in competition assays with some monoclonal antibodies with receptor specificities. RESULTS: Immune complexes able to bind both autologous and allogenic platelets were detected in all patients, while platelet specific IgG or IgM autoantibodies were detected in most, but not in all, patients. The results obtained with the various individual tests were sometimes discrepant, owing to the low avidity binding of platelet-reacting immunoglobulins. Anti-low-affinity FcRII monoclonal antibody moderately reduced the platelet binding of IgG containing immune-complex fractions. CONCLUSIONS: In thrombocytopenic, HIV seropositive drug addicts immunecomplexes and platelet specific antibodies usually coexist and possibly have different pathogenetic potential in the development of thrombocytopenia.

Complement receptors (C3b, C4b/C3d) unbalance on CLL lymphocytes.

A study of lymphocytes bearing C3b, C4b and C3d complement receptor (CRL) was performed on human peripheral blood from 16 healthy donors and 12 patients affected with Chronic Lymphatic Leukemia (CLL). In the latter group a clear rise of C3dCRL was demonstrated, when compared with immunoadherence receptors bearing lymphocytes. However, when compared with controls, also these latter were slightly augmented. Furthermore in CLL under treatment CRL populations showed the same profile as CLL: so it was suggested that the treatment reduced not selectively all the three types of CRL, within the population of sIg bearing lymphocytes. Here we discuss the hypothesis that, in CLL, the proliferating lymphocytes population is chiefly sIg+, C3d+/C3b-, C4b-, but also sIg+, C3d+/C3b+, C4b+.

IgG and C3 receptors on polymorphonuclear leukocytes in phagocytosis. Role of C3-receptors in the attachment and ingestion phases.

The role of IgG and complement (C3) receptors in the adhesion and ingestion phases of immune complexes by normal human polymorphonuclear leukocytes (PMN) were examined. The immune complexes were sheep red cells (E) sensitized with IgG (EA) or IgM antibodies plus complement (EAC'). Both types of receptor sites are involved in the adhesion phase. Moreover IgG receptors are primarily involved in the ingestion phase. Nevertheless even C3-receptors may be sufficient for complete phagocytosis. Even if EAC' adhesion was still high, C3-receptor specific phagocytosis decreased parallel with the amount of the complement used for EA19S sensibilization. The role of receptor sites on human PMN in adhesion and ingestion phases is discussed.

Complement profile and anticomplementary potential in mixed cryoglobulinemia.

In six mixed cryoglobulinemias we have found the indications of a complement classic pathway activation and the fall of the total hemolytic activity (CH50): cryoprecipitate anticomplementary power was always proportional to the respective serum CH50 fall, while no correlation with its own immunoglobulin constitution was found.

A long-term follow-up study in essential cryoglobulinemia.

In a case series of 56 patients with essential cryoglobulinemia, 35 were followed-up for 4-13 years (mean 7 years). A membranous proliferative glomerulonephritis, which in about half the cases showed a progression to renal insufficiency, was the commonest complication, observed in more than one third of the patients. In 2 patients hepatic cirrhosis became manifest after a completely asymptomatic period and in 2 others a lymphoproliferative disease appeared 2 and 8 years after the onset of purpura. In 51% of patients the intial clinical pattern did not change. In searching for a correlation between the development of nephropathy and cryoglobulin characteristics, none was demonstrated studying the cryoglobulin level, the presence of autoantibody and the complement components.

Allo and auto-antibodies against platelet antigens: a new immunoenzymatic assay for their detection and quantitation.

A new immunoenzymatic assay for the detection of serum-free and platelet-associated (PA) antibodies is described. This method evidences the platelet bound IgG employing the biotin-avidin system. Following a common sensitization step, the PA-IgG can be directly detected on the cell membranes, and their amount can be evaluated by an anti-globulin consumption assay. In comparison with the platelet suspension immunofluorescence test (PSIFT), the ELISA assay is a little less specific but it is more sensitive: less than 1 ng/ml of IgG can be detected. The number of IgG molecules/platelet detected investigating normal and pathological sera is higher than that reported by others: such results are probably related to the nonhomogeneity of immunoglobulins implicated in the assay.