Simon Panzer

Rapid typing for human platelet antigen systems −1, −2, −3 and −5 by PCR amplification with sequence-specific primers

Typing for human platelet antigens (HPA) is useful in a variety of clinical situations. We developed a method for genotyping for HPA‐1, ‐2, ‐3 and ‐5 by means of the PCR amplification with sequence‐specific primers (PCR‐SSP) technique. Primer sets were designed to allow PCR amplification for all systems using the same assay conditions. Specificity and sensitivity of the method were assessed in a blind quality control study (n = 112). In 111 cases, results obtained by PCR‐SSP were identical as compared with PCR‐restriction fragment length polymorphism technique. One discrepancy was found to be due to a typing error in the data sheet. The results of the PCR‐SSP technique were available within 3 h. We conclude that genotyping based on PCR‐SSP enables rapid typing for HPA systems, which makes this technique feasible in most clinical settings where urgent HPA typing is required.

Comparison of methods to evaluate clopidogrel-mediated platelet inhibition after percutaneous intervention with stent implantation

A high on-treatment residual ADP-inducible platelet reactivity in light transmission aggregometry (LTA) has been associated with an increased risk of adverse outcomes after percutaneous coronary intervention (PCI). However, LTA is weakly standardized, and results obtained in one laboratory may not be comparable to those obtained in another one. We therefore sought to determine the test correlating best with LTA to estimate clopidogrel-mediated platelet inhibition in 80 patients on dual antiplatelet therapy after elective percutaneous intervention with stent implantation. We selected the VerifyNow P2Y12 assay, the vasodilator-stimulated phosphoprotein phosphorylation assay, multiple electrode platelet aggregometry and the Impact-R for comparisons with LTA. Cut-off values for residual ADP-inducible platelet reactivity were defined according to quartiles of each assay. Sensitivities and specificities of the different platelet function tests were based on the results from LTA. The results from all four assays correlated significantly with those from LTA. The VerifyNow P2Y12 assay revealed the strongest correlation (r = 0.61, p < 0.001). Sensitivities and specificities ranged from 35% to 55%, and from 78.3% to 85%, respectively. In conclusion, although all assays correlated significantly with LTA, they need to be improved to become clinically used diagnostic tests. Further, it may be too early to define the gold standard method for assessing residual ADP-inducible platelet reactivity and generally acceptable cut-off values.

Calcium-channel blockers decrease clopidogrel-mediated platelet inhibition

Background The extent of clopidogrel-mediated platelet inhibition varies considerably from one person to the next. Numerous studies have shown that low responders have significantly more adverse events after coronary stenting than patients who respond well to antithrombotic treatment with clopidogrel. Dihydropyridine calcium-channel blockers (CCBs) inhibit the cytochrome P450 3A4 enzyme, which metabolises clopidogrel to its active form. Objective To investigate the influence of CCBs on clopidogrel-mediated platelet inhibition. Methods Adenosine-5-diphosphate (ADP)-inducible platelet reactivity was assessed by light transmission aggregometry (LTA) and the VerifyNow P2Y12 assay in 162 patients after percutaneous intervention with stent implantation. Results in the fourth quartiles of both assays were considered as high on-treatment residual ADP-inducible platelet reactivity. Results Patients with concomitant CCB therapy showed a significantly higher on-treatment platelet reactivity than patients without CCB medication (p=0.001 for both assays). Further, high on-treatment residual ADP-inducible platelet reactivity was significantly more common among patients currently taking CCBs (p=0.001 for LTA and p=0.004 for the VerifyNow P2Y12 assay). A multivariate regression analysis confirmed CCB therapy as an independent predictor of reduced clopidogrel-mediated platelet inhibition (p=0.006 for LTA and p=0.004 for the VerifyNow P2Y12 assay). Conclusion CCBs decrease clopidogrel-mediated platelet inhibition in patients undergoing angioplasty and stenting for cardiovascular disease.

Maternal alloimmunization against fetal platelet antigens: a prospective study.

Summary. Neonatal alloimmune thrombocytopenia (NAIT) is induced by maternal alloantibodies to fetal platelet antigens. This prospective study was carried out to evaluate the incidence of anti‐platelet antibodies in 933 mother‐child pairs where the mother and child were typed for the human platelet antigens (HPA)‐l, ‐2,‐3,‐5. Sera from mismatched mother‐child pairs were screened for anti‐platelet antibodies, anti‐HLA class I and blood group ABO IgG antibodies. Platelet‐specific antibodies were anti‐HPA‐3a in one and anti‐HP A‐5b in 17 neonates, respectively. All these neonates had normal platelet counts. One woman had autoreactive antibodies. Anti‐HLA class I and anti‐blood group A IgG antibodies were detected in five and four neonates, respectively, born with a platelet count <150×109/l. None of the 11 homozygous HP A‐lb mothers became immunized against their heterozygous offspring. The maternal HLA‐allotypes HLA‐DR52 and ‐DR6, typically found in individuals immunized against HPA‐la and ‐5b, respectively, were found in three of 11 HPA‐b/b non‐responders and eight of the anti‐HPA‐5b responders. The results indicate that a risk for NAIT due to HPA‐2 and ‐3 alloimmunization is low. The HLA allotypes do not predict the risk for NAIT due to HPA‐1 or ‐5 alloimmunization. Maternal anti‐HPA‐5b antibodies do not correlate with the platelet count in the neonate.

RHD sequencing: a new tool for decision making on transfusion therapy and provision of Rh prophylaxis

. The serological differentiation of weak D from partial D, D‐negative and D‐positive is not always unequivocal. Therefore, sequencing of the RHD gene is required in some cases. Very recently, several new differences between RHD and RHCE have been identified which permitted us to design primers close to the exon/intron boundaries of the RHD‐exons. We evaluated these primers in 83 D‐positive and 18 D‐negative blood donors and applied the new method for the characterization of the RHD gene in six individuals with weak D phenotype. The amplification reactions were concordant with serological findings in 100 of 101 donors (99·0%). In one D‐positive donor the PCR for exons 2 and 5 gave a negative result, while the sequence of the remaining eight exons was unchanged. By sequencing samples with very weak D serological reactions, we identified weak D type 4.2.2 and weak D type 15, both previously reported to be associated with anti‐D‐alloimmunization. Consequently, we recommended the selection of D‐negative blood in the weak D type 4.2.2 patient, and the provision of Rh prophylaxis for pregnant women with weak D type 15. In summary, a new RHD sequencing method was developed which can be applied if serological reactions are inconclusive.

Adenosine diphosphate‐inducible platelet reactivity shows a pronounced age dependency in the initial phase of antiplatelet therapy with clopidogrel

Summary.  Background: Until recently, there were hardly any data on the antiplatelet effect of clopidogrel in advanced age. Like other metabolic processes, the conversion of clopidogrel to its active metabolite may be impaired in older patients, leading to high on‐treatment residual ADP‐inducible platelet reactivity. Objective: To investigate the age dependency of clopidogrel‐mediated platelet inhibition. Patients and methods: This was a prospective observational study. We determined adenosine 5′‐diphosphate (ADP)‐inducible platelet reactivity using light transmission aggregometry (LTA) and the VerifyNow P2Y12 assay in 191 patients on dual antiplatelet therapy after angioplasty and stenting for cardiovascular disease. Results: ADP‐inducible platelet reactivity increased linearly with age after adjustment for cardiovascular risk factors, type of intervention, medication, C‐reactive protein (CRP) and renal function [using LTA 0.36% of maximal aggregation per year, 95% CI 0.08–0.64%, P = 0.013; using the VerifyNow P2Y12 assay 3.2 P2Y12 reaction units (PRU) per year, 95% CI 1.98–4.41 PRU, P < 0.001]. ADP‐inducible platelet reactivity was significantly higher in patients aged 75 years or older compared with younger patients (P = 0.003 for LTA and P < 0.001 for the VerifyNow P2Y12 assay). Further, high on‐treatment residual ADP‐inducible platelet reactivity was significantly more common among patients aged 75 years or older (P = 0.02 for LTA and P < 0.001 for the VerifyNow P2Y12 assay). Conclusion: ADP‐inducible platelet reactivity shows a pronounced age dependency in the initial phase of antiplatelet therapy with clopidogrel. The clinical implications of these findings need to be addressed in future trials.

Functional Asplenia after Bone Marrow Transplantation: A Late Complication Related to Extensive Chronic Graft-Versus-Host Disease

STUDY OBJECTIVE To evaluate splenic function in bone marrow transplant recipients, with relation to chronic graft-versus-host disease and infections. DESIGN Survey, outpatients geographically accessible for voluntary participation. SETTING Bone marrow transplantation referral center. PATIENTS Fifteen bone marrow graft recipients (13 allogeneic, 2 autologous), out of a total of 33 patients who received transplants at the center and survived more than 6 months after grafting. MEASUREMENTS AND MAIN RESULTS In 6 of 15 patients impaired splenic function (functional asplenia) was indicated by the presence of Howell-Jolly bodies in peripheral blood smears, reduced spleen size (P less than 0.001), higher platelet counts (P less than 0.01), higher indium-111 labeled autologous platelet recovery (P less than 0.005), reduced splenic blood flow (P less than 0.001), and reduced accumulation of radioactivity at the splenic site (P less than 0.001). All patients with functional asplenia but only 2 patients without functional asplenia had extensive chronic graft-versus-host disease. The incidence of bacterial infections was four times higher in patients with impaired splenic function. CONCLUSIONS Functional asplenia is a late complication after allogeneic bone marrow transplantation and contributes to the high susceptibility to bacterial infections in patients with extensive chronic graft-versus-host disease.

Chronic kidney disease is associated with increased platelet activation and poor response to antiplatelet therapy

BACKGROUND Chronic kidney disease (CKD) is a common co-morbidity of patients with atherosclerotic vascular disease, and may influence the response to antiplatelet therapy. We, therefore, sought to investigate its effect on platelet activation and on-treatment residual platelet reactivity. METHODS We assessed platelet activation and the response to clopidogrel and aspirin in 316 patients after percutaneous intervention with stent implantation. CKD was defined as a glomerular filtration rate <60 mL/min/1.73 m(2) according to the Modification of Diet in Renal Disease formula. Surface expression of activated glycoprotein IIb/IIIa without the addition of agonists was determined to assess baseline platelet activation. GPIIb/IIIa in response to adenosine diphosphate (ADP) and arachidonic acid (AA), as well as the VerifyNow assays and light transmission aggregometry (LTA) were used to measure residual platelet reactivity. RESULTS Baseline platelet activation was significantly increased in CKD patients compared with patients without renal insufficiency [3.1 versus 2.7 mean fluorescence intensity (MFI), P = 0.001]. Moreover, patients with CKD exhibited a more pronounced expression of GPIIb/IIIa in response to ADP (13 versus 9.6 MFI) and AA (6 versus 5.1 MFI; both P≤ 0.02) than patients without CKD. In the VerifyNow assays, CKD patients showed significantly higher platelet reactivity than patients without CKD (P2Y12 assay: 239 versus 182 P2Y12 Reaction Units; aspirin assay: 415 versus 399 Aspirin Reaction Units; both P≤ 0.03). Further, patients with CKD had significantly higher platelet reactivity by LTA in response to ADP (49.9 versus 43.2%, P = 0.01). Finally, high on-treatment residual ADP-inducible platelet reactivity by the VerifyNow P2Y12 assay and by LTA occurred significantly more frequent in patients with CKD (VerifyNow: 52.2 versus 26.2%, P < 0.001; LTA: 23.3 versus 12.1%, P = 0.01). CONCLUSIONS Patients with CKD exhibit increased platelet activation, and an attenuated response to dual antiplatelet therapy compared with patients without renal insufficiency.

Inherited Platelet Glycoprotein Polymorphisms and a Risk for Coronary Heart Disease in Young Central Europeans

Among central Europeans polymorphisms of GPIIIa, GPIb, GPIIb, and GPIa named human platelet antigen-1 (HPA-1), -2, -3, and -5 are the clinically most relevant systems in which alloimmunization occurs. These genetically determined polymorphisms of glycoproteins may render platelets sensible for plaque formation and thus could increase risk for coronary artery disease (CAD). We therefore determined gene frequencies of HPA-1, -2, -3, and -5 in European patients suffering from CAD (n = 92; median age, 46 years) or CAD accompanied by diabetes mellitus (DM) (n = 30; median age, 60 years, DM I/II, 5/25) and compared the data obtained with those in DM patients without CAD (n = 80; median age, 43 years; DM I/II, 53/27) and a control group (newborns, n = 906). Triglyceride and cholesterin levels as well as the percentage of smokers was significantly higher in the CAD group compared with the diabetics without DM (p < 0.005). No significant difference of the frequencies of any HPA-type between CAD patients with or without DM, diabetics, or controls could be detected. This was also true when evaluating a subgroup of patients aged 45 years or younger. To include a mutual influence of the described HPA-polymorphisms, we condensed the four HPA genotypes to joint glycoprotein variants. Again the same frequencies were found in patient groups and controls, when analyzing the five most common condensed joint glycoprotein variants. The analysis of the combined published studies shows that the pooled HPA-1 allele frequencies are identical in controls and CAD patients. Thus, no significant association between the polymorphisms of any of the studied HPA systems and the development of CAD can be found in central Europeans.

Specificities of anti‐platelet antibodies in multitransfused patients with haemato‐oncological disorders

The clinical condition and the formation of platelet‐reactive antibodies influence the post‐transfusion platelet increment. We analysed the specificities of platelet‐reactive antibodies in 81 multitransfused patients with haemato‐oncological diseases refractory to platelet transfusions, or prior to a scheduled stem cell transplantation. In 17 additional patients we prospectively determined the development of platelet‐reactive antibodies at the time of chemotherapy in weekly intervals. Sera were tested by the monoclonal antibody‐specific immobilization of platelet antigens (MAIPA)‐technique for antiplatelet antibodies against HLA class I antigens, the human platelet‐specific alloantigens (HPA)‐1, ‐2, ‐3, ‐5, and the platelet membrane glycoproteins (GP) Ia/IIa, Ib/IX, IIb/IIIa (panreactive).