Patient-reported outcome measures obtained via E-Health tools ease the assessment burden and encourage patient participation in cancer care (PaCC Study).

Patient-reported outcome measures obtained via E-Health tools ease the assessment burden and encourage patient participation in cancer care (PaCC Study) BACKGROUND: E-health based patient-reported outcome measures (PROMs) have the potential to automate early identification of both nutrition status and distress status in cancer patients while facilitating treatment and encouraging patient participation. This cross-sectional study assessed the acceptability, accuracy, and clinical utility of PROMs collected via E-Health tools among patients undergoing treatment for stomach, colorectal, and pancreatic tumors. RESULTS: Eight-nine percent mostly, or completely, agreed that PROMs via tablets should be integrated in routine clinical care. Men were significantly more likely to require help completing the questionnaires than women (inv.OR= 0.51, 95% CI=(0.27, 0.95), p = 0.035). The level of help needed increased by 3% with each 1-year increase in age (inv. OR=1.03, 95% CI=(1.01, 1.06), p = 0.013). On average, a patient tended to declare weight which was 0.84 kg inferior to their true weight (Bland and Altman 95 % CI=(-3.9, 5.6); SD: 2.41) and a height which was 0.95 cm superior to their true height (Bland and Altman 95 % CI=(-5, 3.1); SD 2.08). Patient-reported nutrition status was significantly associated with the professionally generated assessment (95% CI=(2.27, 4.15), p < 0.001). As nutrition status declined, the distress score increased (95%CI=(0.88, 1.68), p < 0.001). Of the patients, 48.8% who were both distressed and malnourished requested supportive care to address their problems. CONCLUSION: Patient-reported assessments utilizing E-health tools are an accurate and efficient method to encourage patient participation in cancer care while simultaneously ensuring that regular assessment of psycho-social and nutritional aspects of care are efficiently integrated in the daily clinical routine.

Gestational weight gain and the risk of offspring obesity at 10 and 16 years: a prospective cohort study in low-income women.

OBJECTIVE: To study the association between gestational weight gain (GWG) and offspring obesity risk at ages chosen to approximate prepuberty (10 years) and postpuberty (16 years). DESIGN: Prospective pregnancy cohort. SETTING: Pittsburgh, PA, USA. SAMPLE: Low-income pregnant women (n = 514) receiving prenatal care at an obstetric residency clinic and their singleton offspring. METHODS: Gestational weight gain was classified based on maternal GWG-for-gestational-age Z-score charts and was modelled using flexible spline terms in modified multivariable Poisson regression models. MAIN OUTCOME MEASURES: Obesity at 10 or 16 years, defined as body mass index (BMI) Z-scores ≥95th centile of the 2000 CDC references, based on measured height and weight. RESULTS: The prevalence of offspring obesity was 20% at 10 years and 22% at 16 years. In the overall sample, the risk of offspring obesity at 10 and 16 years increased when GWG exceeded a GWG Z-score of 0 SD (equivalent to 30 kg at 40 weeks); but for gains below a Z-score of 0 SD there was no relationship with child obesity risk. The association between GWG and offspring obesity varied by prepregnancy BMI. Among mothers with a pregravid BMI <25 kg/m(2) , the risk of offspring obesity increased when GWG Z-score exceeded 0 SD, yet among overweight women (BMI ≥25 kg/m(2) ), there was no association between GWG Z-scores and offspring obesity risk. CONCLUSIONS: Among lean women, higher GWG may have lasting effects on offspring obesity risk.

Staphylococcal alpha toxin promotes blood coagulation via attack on human platelets.

Staphylococcus aureus plays a major role as a bacterial pathogen in human medicine, causing diseases that range from superficial skin and wound to systemic nosocomial infections . The majority of S. aureus strains produces a toxin, a proteinaceous exotoxin whose hemolytic, dermonecrotic, and lethal properties have long been known (1-6). The toxin is secreted as a single- chained, nonglycosylated polypeptide with a M(r) of 3.4 x 10(4) (7, 8). The protein spontaneously binds to lipid monolayers and bilayers (9-14), producing functional transmembrane pores that have been sized to 1.5-2.0-nm diameters (15-18). The majority of pores formed at high toxin concentrations (20 mug/ml) is visible in the electron microscope as circularized rings with central pores of approximately 2 nm in diameter. The rings have been isolated, and molecular weight determinations indicate that they represent hexamers of the native toxin (7). We have proposed that transmembrane leakiness is due to embedment of these ring structures in the bilayer, with molecular flux occurring through the central channels (15, 19). Pore formation is dissectable into two steps (20, 21). Toxin monomers first bind to the bilayer without invoking bilayer leakiness . Membrane-bound monomers then laterally diffuse and associate to form non-covalently bonded oligomers that generate the pores. When toxin pores form in membranes of nucleated cells, they may elicit detrimental secondary effects by serving as nonphysiologic calcium channels, influx of this cation triggering diverse reactions, including release of potent lipid mediators originating from the arachidonate cascade (22-24). That alpha toxin represents an important factor of staphylococcal pathogenicity has been clearly established in several models of animal infections through the use of genetically engineered bacterial strains deleted of an active alpha toxin gene (25-27). Whether the toxin is pathogenetically relevant in human disease, however, is a matter of continuing debate. Doubts surrounding this issue originate from two main findings. First, whereas 60 percent hemolysis of washed rabbit erythrocytes is effected by approximately 75 ng/ml alpha toxin, approximately 100-fold concentrations are required to effect similar lysis of human cells (4-6, 13). The general consensus is that human cells display a natural resistance towards toxin attack. The reason for the wide inter-species variations in susceptibility towards alpha toxin is unknown but does not seem to be due to the presence or absence of high-affinity binding sites on the respective target cells (20, 21). Second, low-density lipoprotein (28) and neutralizing antibodies present in plasma of all healthy human individuals inactivate a substantial fraction of alpha toxin in vitro. These inactivating mechanisms presumably further raise the concentration threshold required for effective toxin attack, and it is most unlikely that such high toxin levels will ever be encountered during infections in the human organism. The aforegoing arguments rest on the validity of two general assumptions. First, the noted natural resistance of human erythrocytes to alpha toxin must be exhibited by other human cells. Second, toxin neutralization by plasma components, usually tested and quantified after their preincubation with toxin in vitro, must be similarly effective under natural conditions, and protection afforded by these components must not be restricted to specific cell species.

The overlap of overweight and anaemia among women in three countries undergoing the nutrition transition.

OBJECTIVE: To compare the odds of anaemia in overweight and obese (OVWT) (body mass index (BMI) > or =25) versus non-overweight (non-OVWT) (BMI<25) women in three countries at different stages of the nutrition transition. DESIGN: Analysis of cross-sectional data. SETTING: Nationally representative data from Mexico (1998 National Nutrition Survey), Peru and Egypt (2000 Demographic and Health Surveys) were analyzed. SUBJECTS: Data from non-pregnant women ages 18-49 years were used. ANALYSIS: Logistic regression was used to test whether the odds of anaemia differed by BMI category, controlling for sociodemographic factors. RESULTS: More than half of the women were OVWT in all three countries and the prevalence of OVWT reached 77% in Egypt. Anaemia prevalence was similar across countries (28, 31 and 23% in Egypt, Peru and Mexico respectively). In Egypt, OVWT women had significantly lower odds of anaemia than non-OVWT women (OR=0.78, 95% CI: 0.68, 0.90). Similar results were found in Peru, but the difference was smaller in magnitude (OR=0.83, 95% CI: 0.71, 0.96). In Mexico, there were no differences in the odds of anaemia by BMI group. CONCLUSIONS: These findings show that the iron needs of OVWT women in developing countries are not necessarily being met. The intakes of other micronutrients might also be insufficient. Diet quality remains an important issue even among women with sufficient energy intakes.

The human platelet alloantigens Br(a) and Brb are associated with a single amino acid polymorphism on glycoprotein Ia (integrin subunit alpha 2).

The human GPIa/IIa complex, also known as integrin alpha 2 beta 1, serves as a major receptor for collagen in platelets and other cell types. In addition to its role in platelet adhesion to extracellular matrix, GPIa/IIa is also known to bear the clinically important Br(a) and Brb alloantigenic determinants, which can result in antibody-mediated platelet destruction. Immunochemical studies showed that the Br antigenic epitopes reside solely on the GP Ia subunit and do not depend on sialic acid residues. To define the polymorphism responsible for the Br alloantigen system platelet RNA PCR technique, was used to amplify GPIa mRNA transcripts. Nucleotide sequence analysis of the amplified platelet GPIa cDNA from Br(a/a) and Brb/b individuals revealed a single A<-->G polymorphism at base 1648. MnlI RFLP analysis of cDNA from serologically determined individuals confirmed that this polymorphism segregates with Br phenotype. This single base change results in a substitution of Lys (AAG) in Br(a) to Glu (GAG) in Brb at amino acid residue 505 In spite of the reversal in charge at this position, however, we found no difference in the ability of Bra and Brb homozygous platelets to adhere to collagens types I, III, or V, nor did anti-Bra or anti-Brb alloantibodies interfere with platelet adhesion to any of these fibrillar collagens. The identification of the nucleotide substitution that defines the Bra/Brb alloantigen system will now permit both pre- and postnatal diagnosis for Br phenotype.

Factors affecting the stability of screws in human cortical osteoporotic bone: a cadaver study.

We investigated several factors which affect the stability of cortical screws in osteoporotic bone using 18 femora from cadavers of women aged between 45 and 96 years (mean 76). We performed bone densitometry to measure the bone mineral density of the cortical and cancellous bone of the shaft and head of the femur, respectively. The thickness and overall bone mass of the cortical layer of the shaft of the femur were measured using a microCT scanner. The force required to pull-out a 3.5 mm titanium cortical bone screw was determined after standardised insertion into specimens of the cortex of the femoral shaft. A significant correlation was found between the pull-out strength and the overall bone mass of the cortical layer (r(2) = 0.867, p < 0.01) and also between its thickness (r(2) = 0.826, p < 0.01) and bone mineral density (r(2) = 0.861, p < 0.01). There was no statistically significant correlation between the age of the donor and the pull-out force (p = 0.246), the cortical thickness (p = 0.199), the bone mineral density (p = 0.697) or the level of osteoporosis (p = 0.378). We conclude that the overall bone mass, the thickness and the bone mineral density of the cortical layer, are the main factors which affect the stability of a screw in human female osteoporotic cortical bone.

Optimization of corticosteroid induced osteoporosis in ovariectomized sheep. A bone histomorphometric study.

During osteoporosis induction in sheep, side effects of the steroids were observed in previous studies. The aim of this study was to improve the induction regimen consisting of ovariectomy, calcium/vitamin D- restricted diet and methylprednisolone (-MP)- medication with respect to the bone metabolism and to reduce the adverse side effects. Thirty-six ewes (age 6.5 +/- 0.6 years) were divided into four MP-administration groups (n = 9) with a total dose of 1800 mg MP: group 1: 20 mg/day, group 2: 60 mg/every third day, group 3: 3 x 500 mg and 1 x 300 mg at intervals of three weeks, group 4: weekly administration, starting at 70 mg and weekly reduction by 10 mg. After double-labelling with Calcein Green and Xylenol Orange, bone biopsy specimens were taken from the iliac crest (IC) at the beginning and four weeks after the last MP injection, and additionally from the vertebral body (VB) at the end of the experiment. Bone samples were processed into stained and fluorescent sections, static and dynamic measurements were performed. There were no significant differences for static parameters between the groups initially. The bone perimeter and the bone area values were significantly higher in the VB than in the IC (Pm: 26%, p < 0.0001, Ar: 11%, p < 0.0166). A significant decrease (20%) of the bone area was observed after corticosteroid-induced osteoporosis (p < 0.0004). For the dynamic parameters, no significant difference between the groups was found. Presence of Calcein Green and Xylenol Orange labels were noted in 50% of the biopsies in the IC, 100% in the VB. Group 3 showed the lowest prevalence of adverse side effects. The bone metabolism changes were observed in all four groups, and the VB bone metabolism was higher when compared to the IC. In conclusion, when using equal amounts of steroids adverse side effects can be reduced by decreasing the number of administrations without reducing the effect regarding corticosteroid-induced osteoporosis. This information is useful to reduce the discomfort of the animals in this sheep model of corticosteroid-induced osteoporosis.

Intensity of factor VIII treatment and the development of inhibitors in non-severe hemophilia A patients: results of the INSIGHT case-control study.

Essentials Research suggests that intensive treatment episodes may increase the risk to develop inhibitors. We performed an international nested case-control study with 298 non-severe hemophilia A patients. Surgery and a high dose of factor VIII concentrate were associated with increased inhibitor risk. Physicians need to review arguments for factor VIII dose and elective surgery extra critically. SUMMARY: Background Inhibitor development is a major complication of treatment with factor VIII concentrates in hemophilia. Findings from studies among severe hemophilia A patients suggest that intensive treatment episodes increase the risk of developing inhibitors. Objectives We set out to assess whether intensive treatment is also associated with an increased risk of inhibitor development among non-severe hemophilia A patients. Patients/Methods We performed a nested case-control study. A total of 75 inhibitor patients (cases) and 223 control patients were selected from 2709 non-severe hemophilia A patients (FVIII:C, 2-40%) of the INSIGHT cohort study. Cases and controls were matched for date of birth and cumulative number of exposure days (EDs) to FVIII concentrates. Conditional logistic regression was used to calculate both unadjusted and adjusted odds ratios (aOR); the latter were adjusted for a priori specified confounders. Results Peak treatment of 5 or 10 consecutive EDs did not increase inhibitor risk (aOR, 1.0; 95% confidence interval (CI), 0.4-2.5; and aOR, 1.8; CI, 0.6-5.5, respectively). Both surgical intervention (aOR, 4.2; CI, 1.7-10.3) and a high mean dose (> 45 IU kg-1 /ED) of FVIII concentrate (aOR, 7.5; CI, 1.6-35.6) were associated with an increased inhibitor risk. Conclusions Our findings suggest that high-dose FVIII treatment and surgery increase the risk of inhibitor development in non-severe hemophilia A. Together with the notion that non-severe hemophilia A patients are at a lifelong risk of inhibitor development, we suggest that in the future physicians will review the arguments for the FVIII dose and elective surgery extra critically.

Variation in baseline factor VIII concentration in a retrospective cohort of mild/moderate hemophilia A patients carrying identical F8 mutations.

Essentials Factor VIII levels vary in mild and moderate hemophilia A (MHA) patients with the same mutation. We aimed to estimate the variation and determinants of factor VIII levels among MHA patients. Age and genotype explain 59% of the observed inter-individual variation in factor VIII levels. Intra-individual variation accounted for 45% of the variation in the three largest mutation groups. SUMMARY: Background The bleeding phenotype in patients with mild/moderate hemophilia A (MHA) is inversely associated with the residual plasma concentration of factor VIII (FVIII:C). Within a group of patients with the same F8 missense mutation, baseline FVIII:C may vary, because, in healthy individuals, von Willebrand factor (VWF) levels, ABO blood group and age are also known to influence baseline FVIII:C. Our understanding of the pathophysiologic process of the causative genetic event leading to reduced baseline FVIII:C in MHA patients is still limited. Objectives To estimate the variation and determinants of baseline FVIII:C among MHA patients with the same F8 missense mutation. Methods Three hundred and forty-six patients carrying mutations that were present in at least 10 patients in the cohort were selected from the INSIGHT and the RISE studies, which are cohort studies including data of 3534 MHA patients from Europe, Canada, and Australia. Baseline FVIII:C was measured with a one-stage clotting assay. We used Levene's test, univariate and multivariate linear regression, and mixed-model analyses. Results For 59% of patients, the observed variation in baseline FVIII:C was explained by age and genotype. Compared to FVIII:C in patients with Arg612Cys, FVIII:C was significantly different in patients with eight other F8 missense mutations. Intra-individual variation explained 45% of the observed variance in baseline FVIII:C among patients with the same mutation. Conclusion Our results indicate that baseline FVIII:C levels are not exclusively determined by F8 genotype in MHA patients. Insights into other factors may provide potential novel targets for the treatment of MHA.

Mortality caused by intracranial bleeding in non-severe hemophilia A patients.

Essentials Data on bleeding-related causes of death in non-severe hemophilia A (HA) patients are scarce. Such data may provide new insights into areas of care that can be improved. Non-severe HA patients have an increased risk of dying from intracranial bleeding. This demonstrates the need for specialized care for non-severe HA patients. SUMMARY: Background Non-severe hemophilia (factor VIII concentration [FVIII:C] of 2-40 IU dL-1 ) is characterized by a milder bleeding phenotype than severe hemophilia A. However, some patients with non-severe hemophilia A suffer from severe bleeding complications that may result in death. Data on bleeding-related causes of death, such as fatal intracranial bleeding, in non-severe patients are scarce. Such data may provide new insights into areas of care that can be improved. Aims To describe mortality rates, risk factors and comorbidities associated with fatal intracranial bleeding in non-severe hemophilia A patients. Methods We analyzed data from the INSIGHT study, an international cohort study of all non-severe hemophilia A patients treated with FVIII concentrates during the observation period between 1980 and 2010 in 34 participating centers across Europe and Australia. Clinical data and vital status were collected from 2709 patients. We report the standardized mortality rate for patients who suffered from fatal intracranial bleeding, using a general European male population as a control population. Results Twelve per cent of the 148 deceased patients in our cohort of 2709 patients died from intracranial bleeding. The mortality rate between 1996 and 2010 for all ages was 3.5-fold higher than that in the general population (95% confidence interval [CI] 2.0-5.8). Patients who died from intracranial bleeding mostly presented with mild hemophilia without clear comorbidities. Conclusion Non-severe hemophilia A patients have an increased risk of dying from intracranial bleeding in comparison with the general population. This demonstrates the need for specialized care for non-severe hemophilia A patients.

Is gestational weight gain associated with offspring obesity at 36 months?

OBJECTIVE: We examined the association between gestational weight gain (GWG) and offspring obesity at age 36 months. METHODS: Mother-infant dyads (n = 609) were followed from a first study visit (mean [standard deviation]: 18.8 [2.7] weeks gestation) to 36 months postpartum. Total GWG over the entire pregnancy was defined as excessive or non-excessive according to the 2009 Institute of Medicine guidelines. Four mutually exclusive categories of excessive or non-excessive GWG across early (conception to first study visit) and late (first study visit to delivery) pregnancy defined GWG pattern. Body mass index (BMI) z-scores ≥95th percentile of the 2000 Centers for Disease Control (CDC) references defined offspring obesity at 36 months. Multivariable log-binomial models adjusted for pre-pregnancy BMI and breastfeeding were used to estimate the association between GWG and childhood obesity risk. RESULTS: Nearly half of the women had total excessive GWG. Of these, 46% gained excessively during both early and late pregnancy while 22% gained excessively early and non-excessively late, and the remaining 32% gained non-excess weight early and excessively later. Thirteen per cent of all children were obese at 36 months. Excessive total GWG was associated with more than twice the risk of child obesity (adjusted risk ratio [95% confidence interval]: 2.20 [1.35, 3.61]) compared with overall non-excessive GWG. Compared with a pattern of non-excessive GWG in both early and late pregnancy, excessive GWG in both periods was associated with an increased risk of obesity (2.39 [1.13, 5.08]). CONCLUSIONS: Excessive GWG is a potentially modifiable factor that may influence obesity development in early childhood.

Inhibitor development and mortality in non-severe hemophilia A.

BACKGROUND: The life expectancy of non-severe hemophilia A (HA) patients equals the life expectancy of the non-hemophilic population. However, data on the effect of inhibitor development on mortality and on hemophilia-related causes of death are scarce. The development of neutralizing factor VIII antibodies in non-severe HA patients may dramatically change their clinical outcome due to severe bleeding complications. OBJECTIVES: We assessed the association between the occurrence of inhibitors and mortality in patients with non-severe HA. METHODS: In this retrospective cohort study, clinical data and vital status were collected for 2709 non-severe HA patients (107 with inhibitors) who were treated between 1980 and 2011 in 34 European and Australian centers. Mortality rates for patients with and without inhibitors were compared. RESULTS: During 64,200 patient-years of follow-up, 148 patients died (mortality rate, 2.30 per 1000 person-years; 95% confidence interval (CI), 1.96-2.70) at a median age of 64 years (interquartile range [IQR], 49-76). In 62 patients (42%) the cause of death was hemophilia related. Sixteen inhibitor patients died at a median age of 71 years (IQR, 60-81). In ten patients the inhibitor was present at time of death; seven of them died of severe bleeding complications. The all-cause mortality rate in inhibitor patients was > 5 times increased compared with that for those without inhibitors (age-adjusted mortality rate ratio, 5.6). CONCLUSION: Inhibitor development in non-severe hemophilia is associated with increased mortality. High rates of hemophilia-related mortality in this study indicate that non-severe hemophilia is not mild at all and stress the importance of close follow-up for these patients.

Theoretical approaches to physical transformations of active pharmaceutical ingredients during manufacturing processes.

Processing-induced transformations (PITs) during pharmaceutical manufacturing are well known but difficult to predict and often difficult to control. This review of the concepts of transformations is couched in terms of the issues associated with identifying rate-controlling events from the materials side and the processing side. Specifically, the approach is reconciling the characteristic time scale of the structural change(s) in the material with the time scale of the processing-induced stress. This is definitely a model (or rather a melding of a group of existing theories) in development. This overview is a 'snapshot' of the authors' attempts to identify the categories of existing theories needed to encompass all of the relevant events for each possible PIT. The ultimate goal is to establish a framework of concepts and theories for consideration, discussion, and modeling of PITs as well as to locate much of the relevant literature in the framework.

Studies on the platelet radioactive anti-immunoglobulin test.

A platelet radioactive antiimmunoglobulin test (PRAT) has recently been introduced. In the present report, a detailed analysis the influence of varying test conditions (i.e. efficiency of iodination of anti-IgG, cell number, degree of sensitization of platelets, times and temperatures of incubation, frequency of washings) on the results of this technique is presented. A standard procedure based on these findings is outlined. It is shown that the PRAT is two to four times more sensitive than platelet complement fixation for the detection of HLA antibodies.

Heparin-associated thrombocytopenia: the antibody is not heparin specific.

In this study the hypothesis was assessed whether heparin-associated thrombocytopenia (HAT) may be caused by an antibody dependent on polysulfated oligosaccharide epitopes, present not only on heparin but also on different polysulfated substances such as dextran sulfate and pentosan polysulfate. We found that the major factor for eliciting platelet activation with sera of HAT type II patients is neither the structure nor the AT III binding capacity of an oligosaccharide, but rather its grade of sulfation. This was shown by in vitro crossreactivity studies with 40 sera of HAT type II patients using unfractionated heparins, LMW heparins (Fragmin, Fraxiparin), enoxaparin, LMW heparinoid (Org 10172 and its subfractions), de-N-sulfated heparin, dermatan sulfate, dextran sulfate, pentosan polysulfate and dextran. Platelet activation was measured by the heparin induced platelet activation (HIPA) assay and the serotonin release assay (SRA). The platelet activating factor was isolated with the IgG fraction, but did not bind to heparin and dextran sulfate fixed to a solid phase. By isoimmune fixation electrophoresis a monoclonal gammopathy was ruled out in the three sera assessed. The in vivo effect of different LMW heparins and the heparinoid Org 10172 was observed in 10 patients with HAT type II. In a prospective study, a compatible heparin-like anticoagulant was selected for 10 HAT patients for whom further parenteral anticoagulation was required. The only substance that showed no crossreactivity in vitro was the LMW heparinoid Org 10172, which differs from heparin and LMW heparins by its low-grade sulfation. Upon treatment with the heparinoid, all 10 patients had a good clinical outcome, even if they had previously developed thromboembolic complications under LMW heparin administration.(ABSTRACT TRUNCATED AT 250 WORDS)

Redistribution of platelet glycoproteins induced by allo- and autoantibodies.

The redistribution of platelet glycoproteins (GP) Ib, IIb and IIIa in response to stimulation with human platelet specific alloantibodies, autoantibodies and a quinidine-dependent antibody was investigated using immunofluorescence and a quantitative radioimmunoassay. The platelet GPs carrying the corresponding epitopes were determined using immunoblot technique or radioimmunoprecipitation. In accordance with previous results obtained with murine monoclonal platelet specific antibodies, redistribution upon stimulation with human platelet reactive antibodies was observed only due to reactions with epitopes on GP IIb, IIIa, respectively, but not on GP Ib. We therefore believe that membrane redistribution of human platelets induced by various antibodies is a function of the GP recognized by those antibodies rather than the source of the platelet reactive antibody.

Blood group A and B determinants are expressed on platelet glycoproteins IIa, IIIa, and Ib.

We report the localization of A, B blood group determinants on intrinsic glycoproteins using anti-A, B IgG antibodies. By radioimmunoprecipitation, anti-A and anti-B precipitated three bands with apparent molecular masses in sodium dodecylsulphate-polyacrylamide gel electrophoresis (SDS-PAGE) of 159, approximately 120 and 85 kDa under non-reduced conditions and 145, approximately 126 and 97 kDa under reduced conditions. In two-dimensional gel electrophoresis (isoelectric focussing/SDS-PAGE) these three bands could be resolved into six spots and fulfilled previously defined criteria for platelet membrane glycoprotein complexes I a/IIa, Ic/IIa, I b/IX and II b/IIIa. The results were supported by data obtained by an assay employing antibody-specific immobilization of platelet antigens (MAIPA). By this technique, blood group A, B determinants were shown to be immobilized by monoclonal antibodies specific for GPIa, Ic', IX, IIb/IIIa and strongly by mab specific GPIIa, but not by mab specific for HLA class I molecules. The more precise localization on platelet glycoproteins was achieved by immunoblotting technique by which blood group A determinants could be assigned to GPs IIa, IIIa and Ib.

Heparin-associated thrombocytopenia: the effects of various intravenous IgG preparations on antibody mediated platelet activation--a possible new indication for high dose i.v. IgG.

The immunologic type of heparin-associated thrombocytopenia (HAT) is caused by antibodies which activate platelets via the Fc-receptor in the presence of polysulfated oligosaccharides. The antigen is formed by a releasable platelet protein (in many cases PF4) complexed to heparin. Since the role of GP IIb/IIIa in platelet activation by HAT antibodies is controversial, we investigated platelet activation by antibodies related to HAT. We used normal platelets and platelets from a patient with Glanzmann's thrombasthenia (GT) lacking GP IIb/IIIa. Heparin and sera from patients with HAT stimulated GT platelets in the same manner as determined by 14C-serotonin release and the changes in phosphorylation of p20 and p47. Platelet activation could be inhibited by an anti FcRII monoclonal antibody (IV. 3, Fab-fragments), and by Fc-fragments, but not by F(ab')2-fragments of human IgG. The effect of four different, commercially available preparations of intact i.v. IgG on the platelet activation by six HAT sera was investigated by 14C-serotonin release. The inhibitory effect was strongly dependent upon the manufacturing process. At a concentration of 20 mg/ml only IgG that had been subjected to low pH and traces of pepsin sufficiently inhibited platelet activation. IgG treated with polyethylenglycol or sulfitolysis was less effective, whereas beta-propiolactone-treated IgG almost completely lost the ability to inhibit platelet activation by antibodies related to HAT. We conclude that inhibition of GP IIb/IIIa-fibrinogen interaction is insufficient for preventing platelet activation in HAT.(ABSTRACT TRUNCATED AT 250 WORDS)

A rapid and sensitive test for diagnosing heparin-associated thrombocytopenia.

Heparin-associated thrombocytopenia (HAT) is a severe complication of heparin therapy. Its diagnosis is difficult. Conventional assays employ platelet aggregometry (PAA) and/or 14C-serotonin release (SRA) which are either insensitive (PAA) or require radioactive tracers (SRA). We here describe a newly developed sensitive and rapid assay based on visual evaluation of heparin-induced platelet activation (HIPA) in microtiter wells. Using sera of 34 patients with clinically suspected HAT we found the HIPA assay to be as sensitive as the SRA and superior to PAA. The HIPA assay allows investigation of crossreactivity with different types of heparins, low molecular weight (LMW) heparins and heparinoids. Three patients who required further parenteral anticoagulation and in whom the HIPA assay was negative before treatment with the LMW heparinoid Org 10172, were treated with this new heparinoid without adverse reactions. We conclude that the HIPA assay may be a useful tool for differential diagnosis and therapy in patients with HAT.