Angle-stable interlocking nailing in a canine critical-sized femoral defect model for bone regeneration studies: In pursuit of the principle of the 3R's.

Introduction: Critical-sized long bone defects represent a major therapeutic challenge and current treatment strategies are not without complication. Tissue engineering holds much promise for these debilitating injuries; however, these strategies often fail to successfully translate from rodent studies to the clinical setting. The dog represents a strong model for translational orthopedic studies, however such studies should be optimized in pursuit of the Principle of the 3R's of animal research (replace, reduce, refine). The objective of this study was to refine a canine critical-sized femoral defect model using an angle-stable interlocking nail (AS-ILN) and reduce total animal numbers by performing imaging, biomechanics, and histology on the same cohort of dogs. Methods: Six skeletally mature hounds underwent a 4 cm mid-diaphyseal femoral ostectomy followed by stabilization with an AS-ILN. Dogs were assigned to autograft (n = 3) or negative control (n = 3) treatment groups. At 6, 12, and 18 weeks, healing was quantified by ordinal radiographic scoring and quantified CT. After euthanasia, femurs from the autograft group were mechanically evaluated using an established torsional loading protocol. Femurs were subsequently assessed histologically. Results: Surgery was performed without complication and the AS-ILN provided appropriate fixation for the duration of the study. Dogs assigned to the autograft group achieved radiographic union by 12 weeks, whereas the negative control group experienced non-union. At 18 weeks, median bone and soft tissue callus volume were 9,001 mm3 (range: 4,939-10,061) for the autograft group and 3,469 mm3 (range: 3,085-3,854) for the negative control group. Median torsional stiffness for the operated, autograft treatment group was 0.19 Nm/° (range: 0.19-1.67) and torque at failure was 12.0 Nm (range: 1.7-14.0). Histologically, callus formation and associated endochondral ossification were identified in the autograft treatment group, whereas fibrovascular tissue occupied the critical-sized defect in negative controls. Conclusion: In a canine critical-sized defect model, the AS-ILN and described outcome measures allowed refinement and reduction consistent with the Principle of the 3R's of ethical animal research. This model is well-suited for future canine translational bone tissue engineering studies.

Quantifying dog meniscal volume at 1.5T and 3.0T MRI.

The dog has been used extensively as an experimental model to study meniscal treatments such as meniscectomy, meniscal repair and regeneration. Accurate quantification of meniscal size and morphology are a crucial step for developing models of the meniscus. 3.0T magnetic resonance imaging (MRI) has been found to be highly accurate in analyzing the meniscus in both clinical and research fields. However, 3.0T MRI systems are still uncommonly used in veterinary medicine. The goal of the study was to compare meniscal volume measurements from 1.5T MRI system with 3.0T MRI system using proton density sequence, a clinically relevant protocol. The MR images were segmented to reconstruct 3D surface representations of both medial and lateral menisci to compare the meniscal volumes measurements. Average volume differences were 8.8% (P=0.42) and 8.9% (P=0.535) for medial and lateral meniscus, respectively. No significant volume differences were found between 1.5T and 3.0T magnetic resonance (MR) measurements, with high Pearson's correlation coefficient of r > 0.8 and the intraclass correlation coefficient (ICC) of 0.899. For inter- and intra-observer reproducibility, high correlation (ICC = 0.942 and 0.814) was observed, but with high variability for intra-observer reproducibility (lower bound 0.478, upper bound 0.949). We have shown that common clinical MR scanners and pulse sequences can be used to quantify dogs' meniscal volumes with good reproducibility. We believe that repeatable measurements of meniscal volumes using MR may provide a useful capability for assessment of postoperative results following meniscal treatments such as meniscectomy and meniscal regeneration.

PO-63 - Exhausted platelets in cancer patients with high risk of venous thromboembolism and poor prognosis.

INTRODUCTION: Platelets play a crucial role in cancer development, progression and metastatic spread of malignancy. In vitro data show that cancer cells have the ability to activate platelets, and clinical studies found increased levels of platelet activation markers in cancer patients. Moreover, platelets are thought to be involved in the development of venous thromboembolism (VTE) in cancer patients, a frequent complication of malignant disease associated with high morbidity and mortality. AIM: In this study, we aimed to examine the activation status of platelets in cancer patients and investigate the association with risk of future venous thromboembolism (VTE) and mortality. MATERIALS AND METHODS: In a prospective observational cohort study of cancer patients we measured pre-chemotherapy platelet P-selectin and glycoprotein (GP) IIb/IIIa expression and monocyte-platelet aggregates (MPA) in vivo and in response to ex vivo stimulation of the platelet activation receptors protease-activated receptor (PAR) -1, -4, and GPVI by whole blood flow cytometry. Primary and secondary endpoints of the study were occurrence of objectively confirmed VTE and death during 2-year follow-up, respectively. RESULTS: Out of 62 patients (median age [interquartile range, IQR]: 63 [54-70] years, 48% female) with cancers of the pancreas (n=19), lung (n=18), brain (n=14), colon (n=8) and stomach (n=3), 9 (14.5%) developed VTE and 32 (51.6%) died. P-selectin, activated GPIIb/IIIa expression and MPA formation did not significantly differ between tumor sites (Kruskal Wallis test). Reduced platelet responsiveness to PAR-1 and GPVI stimulation was associated with a higher risk of VTE (hazard ratio [HR] per decile increase in %P-selectin positive platelets: 0.73 [95% confidence interval: 0.56-0.92, p=0.007] and 0.77 [0.59-0.98, p=0.034], respectively; Table 1). Further, lower platelet P-selectin and activated GPIIb/IIIa expression in vivo and in response to PAR-1, -4 and GPVI stimulation, but not MPA formation, were associated with a higher risk of death (Table 1). CONCLUSIONS: Cancer patients with a poor prognosis had degranulated platelets, presumably as a consequence of previous activation. Our data suggest that platelets' continuous activation and thus exhaustion is involved in cancer-associated VTE and cancer mortality.

Additive solutions differentially affect metabolic and functional parameters of platelet concentrates.

BACKGROUND: Pathogen inactivation (PI) of platelet concentrates with extension of shelf life to 7 days requires the use of platelet additive solutions (PAS). We examined the quality of platelets resuspended in three different PAS stored for up to 7 days. MATERIALS AND METHODS: Twelve triple adult dose platelet concentrates (PC) were collected using the TrimaAccel® collection system. Each highly concentrated product was divided into three equal parts, and the additive solutions (Composol® or SSP+® or Intersol™) were added to a final concentration of 56% PAS and 44% plasma. Samples were drawn on days 1, 5 and 7 to measure pH, glucose, lactate dehydrogenase (LDH), lactate, mean platelet volume (MPV) and the aggregation response to collagen and the thrombin receptor agonist peptide-6. Further, p-selectin expression on platelets was assessed. RESULTS: No statistically significant changes were observed for pH and MPV during 7 days of storage in all PAS containing PCs, whereas glucose decreased and LDH and lactate increased over time (P < 0·05). These changes were particularly evident in Intersol PCs on days 5 and 7 compared with Composol® PCs or SSP+® PCs (P < 0·05). Platelets from Intersol PCs exhibited the highest baseline activation of p-selectin and showed reduced collagen- and TRAP-6-induced aggregation. CONCLUSION: Resuspension of platelets in Intersol for 7 days results in increased platelet activation and platelet metabolism compared with SSP+® or Composol®. Further clinical studies are needed to evaluate whether the observed differences in PAS-PCs affect the recovery rate or the life span of transfused platelets.

The effect of plasma removal from apheresis platelet concentrates on platelet function.

The effect of plasma removal on platelet function has scarcely been investigated. Plasma removal from apheresis platelet concentrates was achieved by centrifugation at 5000 g for 6 min or 2000 g for 10 min. After resting for 1 h, platelet concentrates were resuspended in 0·9% NaCl. Platelet function was tested before centrifugation and after resuspension by multiple electrode impedance aggregometry (MEA) and light transmission aggregometry (LTA). Plasma removal resulted in 10-14% lower response to TRAP-6 by MEA using both washing procedures, whereas TRAP-6-inducible aggregation by LTA increased slightly (2-5%). Neither plasma removal method affected collagen-induced aggregation. Thus, platelet function did not deteriorate significantly by either method.

Loss of high-molecular-weight von Willebrand factor multimers mainly affects platelet aggregation in patients with aortic stenosis.

Severe aortic stenosis is associated with a haemostatic abnormality that resembles acquired von Willebrand syndrome type 2. It is assumed that high shear conditions render large von Willebrand factor (VWF) multimers accessible to cleavage by ADAMTS-13. However, whether loss of these large multimers affects platelet function by impairing adhesion, aggregate formation, or both has not been evaluated in clinical studies. We prospectively enrolled 47 patients with severe aortic stenosis, and studied them prior to aortic valve surgery and at a median of six months after valve replacement. We investigated levels of large VWF multimers, platelet function under high shear conditions, and residual response to suboptimal concentrations of ADP to express P-selectin. As expected, there was a significant reduction of VWF large multimers before surgery that resolved thereafter in most patients (p<0.0001). The closure time of the ADP cartridge of the PFA-100 was also corrected in most patients after the operation (p<0.0001). We used the cone and plate(let) analyser Impact-R to differentiate between adhesion and aggregation. Both adhesion (p=0.03) and ADP-inducible platelet aggregation (p=0.002) improved considerably after valve replacement. Consequently, ADP-inducible expression of P-selectin was higher after valve replacement (p=0.001). We conclude that reduced levels of large VWF multimers associated with aortic stenosis lead to impairment of both adhesion and, especially, ADP-inducible platelet aggregation.

Platelet function to estimate the bleeding risk in autoimmune thrombocytopenia.

BACKGROUND: Knowledge of platelet function may assist in patient care in chronic autoimmune thrombocytopenia (cAITP). MATERIALS AND METHODS: We evaluated the association of platelet function with haemorrhage in 41 patients, median age 41 years (range 14-82 years, 24 females) with chronic autoimmune thrombocytopenia (cAITP). Samples were investigated for platelet P-selectin, and adhesion and aggregate formation under high shear conditions. Data were compared to those from 28 healthy controls (median age 39 years, range 23-70 years, 17 females) and correlated with a bleeding score of 0 (no bleeding) to 3 (overt mucosal bleedings). RESULTS: P-selectin levels were higher in patients than in controls (P < 0.0004). Compared to controls, the patients' samples responded to high shear with decreased adhesion to the polystyrene surface (P < 0.0001), but formed aggregates of normal size. P-selectin expression was neither correlated with platelet counts, nor platelet adhesion, nor the bleeding score. Only the size of formed aggregates correlated with P-selectin (P = 0.01). Platelet counts (odds ratio 0.5, 95% confidence interval 0.22-0.88; P = 0.04) and adhesion (odds ratio 0.45, 95% confidence interval 0.17-0.87; P = 0.04) were independently inversely correlated with bleeding symptoms. CONCLUSION: Platelet adhesion correlates with bleeding symptoms, while the size of aggregates that are formed under high shear correlates with in vivo platelet activation. The determination of these parameters may assist in estimating an individual bleeding risk and thus a decision for treatment.

Monitoring survival and function of transfused platelets in Glanzmann thrombasthenia by flow cytometry and thrombelastography.

Patients with Glanzmann thrombasthenia (GT) may form isoantibodies which induce refractoriness or inhibition of function of transfused platelets. We monitored the survival and function of transfused platelets by flow cytometry and thrombelastography in a patient with GT. Gating on CD42a+ allowed identification of even a few transfused platelets. Only by gating on these CD41+ CD42a+ cells were we able to demonstrate their capability to bind fibrinogen and PAC-1 upon activation. Platelets were rapidly cleared from the circulation as a result of boosted isoantibodies. The contribution of transfused platelets to clot formation was also demonstrated by thrombelastography by blocking their function with abciximab.

Light chain phenotypes of HLA antibodies in cases with suspected neonatal alloimmune thrombocytopenia.

BACKGROUND AND OBJECTIVES: Platelet-specific antibodies are detectable in only about 30% in suspected neonatal alloimmune thrombocytopenia (NAIT). Human leucocyte antigen (HLA) class I antibodies are often detectable, and as platelets express the corresponding antigens, it has been suggested that these antibodies can be responsible for NAIT. Light chain phenotyping may assist in the diagnosis of HLA antibodies-induced NAIT. MATERIALS AND METHODS: We determined light chain phenotypes of platelet reactive HLA antibodies in 17 sera from mothers who delivered offspring with suspected NAIT. Ten sera also contained platelet-specific antibodies: HPA-1a (n = 5, one with HPA-15b), HPA-5b (n = 4) and autoantibodies (n = 1). Sera were tested for kappa or lambda restriction by monoclonal antibody-specific immobilization of platelet antigens (MAIPA). RESULTS: The cross-match with paternal platelets was positive in all cases due to HLA antibodies. We identified 5, 3 and 3 sera to contain lymphocytotoxic antibodies of single, two or multiple specificities, respectively. In six cases, HLA antibodies were only detectable by MAIPA. Light chain restriction (n = 9) was not associated with HPA containing antibodies or to any pattern of the HLA antibodies. Similarly, polyclonal antibodies (n = 8) were seen in all categories. CONCLUSION: We show that pregnancy-associated HLA antibodies can be clonal or polyclonal, irrespective of a diagnosis of NAIT. ONE-SENTENCE SUMMARY: Pregnancy associated HLA antibodies can be clonal or polyclonal, irrespective a diagnosis of NAIT.

Frequencies of maternal platelet alloantibodies and autoantibodies in suspected fetal/neonatal alloimmune thrombocytopenia, with emphasis on human platelet antigen-15 alloimmunization.

BACKGROUND AND OBJECTIVES: Serological evaluation of maternal sera for platelet antibodies in suspected fetal/neonatal alloimmune thrombocytopenia (FNAITP) discloses in only approximately 30% of individuals a platelet-specific antibody. Transfusion-induced alloimmunization against human platelet antigen-15 (HPA-15) has been reported to be about as common as against HPA-5, the second most common platelet antibody. Thus, anti-HPA-15 may also contribute significantly to yet-unclear cases of FNAITP. MATERIALS AND METHODS: In this retrospective analysis, we provide data on maternal platelet antibodies from 309 mothers who delivered an offspring with suspected FNAITP. RESULTS: Genotyping maternal and paternal samples (together n = 573) revealed a gene frequency of 0.496 for HPA-15a and a gene frequency of 0.504 for HPA-15b. HPA-15 antibodies were detected in 2% of all samples. Anti-HPA-15a and -15b were detected in two and three samples, respectively. One serum reacted equally with HPA-15a and -15b platelets. The most frequent platelet-specific antibodies were anti-HPA-1a (22%), but anti-HPA-5b (8.4%) were more frequent than anti-HPA-15. In addition, panreactive (5.5%) or autoreactive (5.2%) anti-GPIIb/IIIa or anti-GPIb/IX were detectable in maternal samples. CONCLUSIONS: These data indicate that HPA-15 alloimmunization needs only to be considered in subjects with suspected FNAITP if no other platelet-specific antibody is detectable. The presence of panreactive or autoreactive antibodies should also be considered in neonatal thrombocytopenia.

Inhibitory activity of aspirin on von Willebrand factor-induced platelet aggregation.

The effect of aspirin (ASA) on vWF induced platelet - platelet interaction is unknown. We therefore tested the response of platelets to von Willebrand factor (vWF) coated beads induced platelet aggregation before and after i.v. and oral ASA. 1000 mg ASA was infused to 10 healthy individuals and after a wash-out period 7 volunteers received 100 mg ASA orally over a period of 11 days. Prior to ASA and in regular intervals thereafter we tested the reactivity to vWF-coated beads to assess platelet adhesion/aggregation and the fade-out time of ASA effects on platelets. Considerable interindividual variability in response to vWF-coated beads was observed, both before ASA and after treatment with ASA. The maximal response to vWF-coated beads (Tmax), the time lag, and the slope of the curve were significantly affected by i.v. ASA, whereas 100 mg of ASA had only inconstant effect on Tmax and slope. The absolute reduction of Tmax after ASA depended on the pre-ASA level, while the percentage of the reduction was similar in all individuals. Thus, platelet aggregation induced by vWF-coated beads is impaired by ASA. Furthermore, our data indicate a large interindividual variability of the response to ASA shortly after treatment induction, which becomes more constant after prolonged treatment.

IgG titer, subclass, and light-chain phenotype of pregnancy-induced HPA-5b antibodies that cause or do not cause neonatal alloimmune thrombocytopenia.

BACKGROUND: The most common pregnancy-induced platelet-specific antibody is HPA-5b. Neonatal alloimmune thrombocytopenia that results from anti-HPA-5b may cause severe hemorrhage in only a few infants, but the sequelae for the affected children can be severe. It is therefore essential that infants at risk for neonatal alloimmune thrombocytopenia are identified. STUDY DESIGN AND METHODS: The IgG titer, subclass, and light-chain composition of pregnancy-induced anti-HPA-5b were determined by the monoclonal antibody-specific immobilization of platelet antigens assay. Sera were from 12 mothers, who among them had 16 pregnancies that resulted in an HPA-5-mismatched fetus (positive for HPA-5b). Eight mothers gave birth to an infant with a normal platelet count. Three maternal sera were obtained after delivery of a severely thrombocytopenic infant. Three alloimmunized women were followed repeatedly during the course of a subsequent pregnancy, again with an HPA-5-mismatched infant. RESULTS: There was no difference in the antibody titer or its subclass in mothers who had a thrombocytopenic child and the titer or subclass in mothers compared with those who had a child with a normal platelet count. All pregnancy-induced HPA-5b antibodies were of a predominant, lambda, light-chain type. The IgG subclass did not change during pregnancy. CONCLUSION: Neither the antibody titer nor the subclass composition predict the occurrence of thrombocytopenia in a newborn whose mother is alloimmunized against HPA-5b.

Screening for platelet auto-antibodies by flow cytometry and their evaluation by the MAIPA technique.

The determination of platelet antibodies assists in the diagnosis of immune thrombocytopenia. Among the various techniques which have been used two major ways for the determination of these antibodies have entered the routine use, determination of in vivo platelet bound total IgG, termed platelet-associated IgG, PAIgG, and that of specifically to particular platelet glycoproteins bound IgG, GP-IgG. The former has been found to be non-specific, and the evaluation of the latter is rather laborious. Furthermore, both require a large number of platelets. By flowcytometry, however, PAIgG can be determined even if platelet counts are very low. We therefore evaluated in 30 patients' samples if the flowcytometric determination of PAIgG can serve to screen for platelet antibodies. Positive samples subsequently are evaluated by a glycoprotein-specific detection technique (MAIPA). We show that in patients with suspected autoimmune thrombocytopenia (AITP) and in secondary AITP PAIgG is elevated in 83%. However, only 30% of patients' samples have detectable antibodies by the MAIPA technique. Based on the findings that by the MAIPA technique antibodies were only detectable in samples which had also elevated levels of PAIgG we consider the flowcytometric determination of PAIgG useful for screening, prior to the more laborious investigation by the MAIPA assay.

Spontaneous rise of fetal platelet counts despite increasing anti-HPA-5b antibodies.

BACKGROUND AND OBJECTIVES: Maternal anti-HPA alloantibodies are a rare cause of severe fetal thrombocytopenia. So far there have been no reports on the dynamics of maternal anti-HPA-5 during gestation and its effect on the fetus. MATERIALS AND METHODS: We monitored maternal anti-HPA-5b antibody titers and fetal platelet counts during gestation in a woman with known anti-HPA-5b alloimmunization. The patient was a 32-year-old woman in her third pregnancy. The 1st pregnancy and delivery of a healthy child had been uneventful. At delivery, anti-HPA-5b was detectable in the maternal serum. A 2nd pregnancy ended in early miscarriage. RESULTS: A steady rise in the alloantibody titer was recorded throughout the 3rd pregnancy. Therefore, cordocentesis was performed at 28 weeks of gestation (platelet count 76 x 10(9)/l). Serial platelet transfusions were administered to the fetus at 28, 32, and 37 weeks of gestation. The platelet counts rose spontaneously thereafter and were 220 x 10(9)/l at delivery, despite an increase in the anti-HPA-5b antibody titer. The child developed normally during the first year of life. CONCLUSIONS: This case illustrates the spontaneous recovery of fetal platelet counts in late pregnancy despite a rise in maternal alloantibody titer.

Estrogen prescribing practices scrutinized at NIH conference.

PIP: Despite reports of studies linking menopausal estrogen therapy with endometrial cancer in 1975, physicians have persisted in considering menopause a condition needing medication--with estrogens. To assess the risks and benefits, NIH held a conference on estrogen usage in postmenopausal women. A panel of physicians heard the therapeutic, epidemiologic, and sociologic evidence and summarized the findings in a final report. It was agreed that estrogen therapy will be effective in combating vasomotor flushes (hot flashes) and vaginal atrophy and dryness. Evidence to justify estrogen use for coronary vascular disease and depression does not exist. Further evidence is needed to deterine whether estrogen therapy will decrease osteoporosis-related fractures. The risk of endometrial cancer increases 4-8-fold following 2-4 years of continuous estrogen usage. It was concluded that estrogens should be prescribed in the lowest effective dosages and for short terms, not exceedng 2-4 years. Cyclic progestin administration and yearly curettage sampling for endometrial cancer might reduce the risk of developing endometrial cancer while on estrogen therapy.^ieng