The allele frequencies of HPA 1‐16 determined by PCR‐SSP in Chinese Cantonese donors

Background: Typing of human platelet antigens (HPA) has proven to be useful in some clinical situations related to platelet alloimmunization. Objective: The objective of this study was to investigate HPA 1–16 and to determine genotype and allele frequencies by polymerase chain reaction‐sequence specific primer (PCR‐SSP) in apheresis platelet donors in Guangzhou Blood Center. Methods: A total of 200 random samples from donors were involved in the study. Genotype and allele frequencies of HPA 1 to 16 were detected by PCR‐SSP method. Results: The frequencies obtained from these donors were 99·50 and 0·50% for HPA‐1a and ‐1b; 96·25 and 3·75% for HPA‐2a and ‐2b; 54·25 and 45·75% for HPA‐3a and ‐3b; 99·50 and 0·50% for HPA‐4a and ‐4b; 99·00 and 1·00% for HPA‐5a and ‐5b; 97·00 and 3·00% for HPA‐6a and ‐6b and 42·25 and 57·75% for HPA‐15a and ‐15b. There is only a/a homozygosis detected in HPA‐7, ‐8, ‐9, ‐10, ‐11, ‐12, ‐13, ‐14 and ‐16. In this study, none of HPA‐1b/ 1b, ‐2b/2b, ‐5b/5b homozygosis were detected which were found in other racial groups. One homozygosis of HPA‐6b/6b in 200 individuals was detected which was not found in a study involving 1000 Chinese ( Feng et al., 2006 ). Conclusion: The HPA‐3 and ‐15 appear to the highest priority and HPA‐2, ‐6, ‐5 ‐1 and ‐4 to be the second priority in Chinese Cantonese when it comes to the diagnosis of neonatal alloimmune thrombocytopenia and to provide the HPA‐matched platelet for patients with platelet transfusion refractoriness. The PCR‐SSP method makes it possible to detect genotype of HPA‐1 to ‐16 in less than 4 h and to establish a donor database for HPA genotype in a blood bank.     

Gene frequencies of platelet‐specific antigens in Croatian population

The human platelet antigens (HPA) are genetically defined polymorphisms expressed on platelet membrane glycoproteins. As platelet antigens are very important in several clinical situations and in population genetics, we used the polymerase chain reaction with sequence‐specific primers (PCR‐SSP) to investigate HPA‐1, ‐2, ‐3 and ‐5 allele frequencies in the Croatian population. The HPA frequencies obtained in 219 Croatians were: 1a–0·854, 1b–0·146, 2a–0·890, 2b–0·110, 3a–0·575, 3b–0·425, 5a–0·895 and 5b–0·105. These data are similar to the frequencies reported in most European studies with some significant differences in HPA‐2 when compared with the Dutch and German population, in HPA‐3 when compared with the Swiss population and in HPA‐5 when compared with the Finnish population. The three most common condensed HPA genotypes in the Croatian population were: HPA‐1a/a, ‐2a/a, ‐3a/b, ‐5‐a/a (0·283), HPA‐1a/a, ‐2a/a, ‐3a/a, ‐5‐a/a (0·137) and HPA‐1a/b, ‐2a/a, ‐3a/b, ‐5‐a/a (0·087). Data obtained in this study can be used for better understanding and treatment of immune‐mediated platelet disorders in our population.     

Mass-scale high-throughput multiplex polymerase chain reaction for human platelet antigen single-nucleotide polymorphisms screening of apheresis platelet donors

BACKGROUND: Treatment with human platelet antigen (HPA)‐matched platelets (PLTs) is the optimal therapy for bleeding secondary to neonatal alloimmune thrombocytopenia. Recent advances in high‐throughput DNA‐based blood group and PLT antigen genotyping have made it possible to screen plateletpheresis donors for potential HPA‐matched PLT transfusion. STUDY DESIGN AND METHODS: This prospective study evaluated genomic DNA from plateletpheresis donors for single‐nucleotide polymorphisms (SNPs) associated with HPA‐1, ‐2, ‐3, ‐4, ‐5, and ‐15 to determine whether high‐throughput multiplex genomic DNA PCR and oligonucleotide extension technology can be used for mass‐scale PLT antigen genotyping. Genotyping using SNP technology was confirmed using sequence‐specific polymerase chain reaction (SSP‐PCR). RESULTS: Of the 748 donors screened, 277 were found to be negative for antigens implicated in alloimmune thrombocytopenia. In addition, two donors were homozygous for HPA‐1b/b and ‐2b/b, six donors for HPA‐1b/b and ‐3b/b, one for HPA‐2b/b and ‐3b/b, one for HPA‐1b/b and ‐5b/b, 10 for HPA‐1b/b and ‐15 b/b, four for HPA‐5b/b and ‐15b/b, and one for HPA‐2b/b and ‐15b/b. Retesting using SSP‐PCR was conducted for 60 donors. Discrepant results occurred between SNP and SSP‐PCR in less than 20% of samples for HPA‐1b/1b/HPA‐3b/3b, HPA‐5b/5b, and HPA‐15b/b. DISCUSSION: High‐throughput multiplex PCR SNP and confirmatory molecular genotyping are useful for mass‐scale screening of apheresis PLT donors to provide antigen‐negative genotypes. Refinements to mass‐scale multiplex analysis technology would reduce further the confirmatory testing needed.     

Human platelet antigen allele frequencies and new mutations on platelet glycoprotein genes in the Chinese Han population

Background and Objectives: The frequencies of human platelet antigens (HPAs) vary between different populations. In this study, we determined the HPA allele frequencies in the Chinese Han population and identified situation of incompatibility possibly leading to alloimmunisation. Methods: A total of 750 volunteer blood donors of the Chinese Han population were genotyped for HPA‐1 to ‐17w systems. HPA genotyping was determined by polymerase chain reaction sequence‐based typing. Results: Among the 17 HPA systems, the allele frequency is different from other populations. We noted the absence of HPA‐7bw to HPA‐14bw, HPA‐16bw and HPA‐17bw alleles in the population. The estimated incompatibility probabilities regarding platelet antigens 1 to 6w and 15 systems after transfusion of random donor platelet were from 0·004 to 0·373. Thirteen glycoprotein alleles were observed in the population. In addition, we identified 16 novel mutations on the glycoprotein genes separated from HPA polymorphisms, including GP1BA (517‐525delAAC), ITGA2B (2722C>T and IVS26+85T>C), ITGA2 (1521C>T, 2474T>G and IVS20+10 G>C), ITGB3 (1476G>A, IVS10+19C>A, 1813G>A, IVS11+21G>A, IVS11+152A>G and IVS11‐104T>C), GP1BB (IVS1‐79G>A, IVS1‐27C>T and 129G>A) and CD109 (2139A>G). Five of them could lead to amino acid deletion, substitution or premature stop codon in corresponding glycoprotein. Conclusions: There was a high degree of polymorphism of the membrane glycoprotein genes related to human platelet alloantigen‐1 to ‐17w systems in the Chinese Han population. These data could have some impact on the diagnosis, prevention and treatment of alloimmune thrombocytopenia.     

Genotyping of HPA-1 to -7 and -15 in the Thai population using multiplex PCR

Background : Different polymerase chain reaction (PCR) techniques for human platelet antigens (HPA) genotyping have been implemented, in order to diagnose the clinical syndromes of patients with thrombocytopaenia and provide effective HPA‐matched platelet donors. Objectives : The aim of this study is to develop an in‐house multiplex PCR for HPA‐1 to ‐7 and ‐15 genotyping in the Thai population. Methods : One hundred DNA samples of known HPA genotyping by the PCR with sequence‐specific primers (PCR‐SSP), as previously described, were tested with the multiplex PCR. Additionally, 300 DNA samples of group O donors were tested for HPA‐1 to ‐7 and ‐15 genotyping using multiplex PCR. Results : The comparison of HPA‐1 to ‐7 and ‐15 genotype results between multiplex PCR and PCR‐SSP technique was in 100% concordance. Interestingly, HPA‐2b2b genotype was found in two samples; however, other low‐incidence genotypes such as HPA‐1b1b, HPA‐5b5b, HPA‐6b6b and HPA‐7b7b were not found in this study. Moreover, 30 samples were randomly tested twice for HPA genotyping using the multiplex PCR and demonstrated reproducible results. Conclusions : This study shows that the in‐house multiplex PCR is simple, cost‐effective and suitable for HPA genotyping for routine laboratories in other developing countries. Nevertheless, a large‐scale evaluation of this technique through multicentre analysis is suggested.     

Establishment of a cell line panel as an alternative source of platelet antigens for a screening assay of anti-human platelet antibodies

Background: A panel of platelets expressing various human platelet antigens (HPAs) for a platelet antibody screening assay is difficult to prepare because some antigens are rarely expressed. Therefore, an alternative method without using platelets would be helpful in detecting HPA antibodies. This study describes the establishment of cell lines that stably express specific HPAs and their application for detecting specific antibodies. Methods: Wild‐type β3, HPA‐1b, ‐6b, ‐7b and ‐7 variant cDNA as well as wild‐type αIIb and HPA‐3b cDNA were individually co‐transduced with wild‐type αIIb and β3 cDNA in the K562 cell line. We performed an immunobead monoclonal antibody immobilisation of platelet antigens (MAIPA) assay to evaluate this cell line panel for antibody detection using identified sera containing HPA antibodies, whose specificities had been determined by the mixed passive haemagglutination test. Results and Conclusion: Of the 12 sera containing HPA‐1a (n = 2), HPA‐3a (n = 6), HPA‐6b (n = 3) or HPA‐7 variant (n = 1) antibodies, all antibodies were detected and determined by our new method, except for two HPA‐3a antibodies. One of the two antibodies was also negative for conventional platelet MAIPA, suggesting that the cell line panel might be used as an alternative source of platelet antigens in the MAIPA assay.     

Low-avidity anti-HPA-1a alloantibodies are capable of antigen-positive platelet destruction in the NOD/SCID mouse model of alloimmune thrombocytopenia

BACKGROUND: Neonatal alloimmune thrombocytopenia (NAIT) is mostly caused by maternal antibodies against human platelet antigen 1a (HPA‐1a) expressed on glycoprotein (GP) IIb/IIIa. Accumulated evidence indicated that anti‐HPA‐1a could be overlooked by standard methods due to low avidity. Low‐avidity HPA‐1a antibodies were shown to be detectable by surface plasmon resonance (SPR). We sought to investigate the frequency and in vivo relevance of low‐avidity anti‐HPA‐1a. STUDY DESIGN AND METHODS: A retrospective cohort consisting of 82 HPA‐1bb mothers of HPA‐1ab newborns with thrombocytopenia was analyzed using standard serologic methods. Maternal immunoglobulin (Ig)G fractions were investigated for low‐avidity antibodies in SPR using purified GPIIb/IIIa (HPA‐1a or ‐1b). The capability of HPA‐1a antibodies to clear platelets (PLTs) in vivo was analyzed using the NOD/SCID mouse model of alloimmune thrombocytopenia. RESULTS: HPA antibodies were detectable in sera from 68 of 82 (83%) mothers using standard serologic methods and undetectable in 14 of 82 sera. In SPR, IgG fractions of sera reacting positive in monoclonal antibody immobilization of PLT antigen (MAIPA) assay showed specific binding to an HPA‐1a flow cell (mean, 87 ± 21 resonance units [RU]). When MAIPA‐negative sera were tested in SPR, binding with low avidity was observed in 7 of 14 to HPA‐1a (mean, 31 ± 5 RU), but not to HPA‐1b flow cell (mean, 5 ± 2 RU). In vivo, low‐avidity antibodies were capable of clearing HPA‐1ab PLTs but not HPA‐1bb PLTs in a NOD/SCID mouse model. Elimination kinetics were slower than observed with MAIPA‐positive antibodies. CONCLUSIONS: Low‐avidity HPA‐1a antibodies are present in a significant number of NAIT cases and, although they can escape detection by standard serology, they harbor the capability of PLT destruction in vivo.     

The first case of alloantibody against human platelet antigen‐15b in Japan: possible alloimmunization by a hydatidiform mole

BACKGROUND: The involvement of the human platelet antigen (HPA)‐15 system in neonatal alloimmune thrombocytopenia (NAIT) has been reported in various populations, but not in the Japanese population. In Japan, the mixed passive hemagglutination assay (MPHA) is used for detection of HPA alloantibodies. However, most of the reported cases of HPA‐15 incompatibility are based on the monoclonal antibody immobilization of platelet antigen (MAIPA) assay or immunoprecipitation; thus there is a possibility that HPA‐15 alloantibodies are not efficiently detected by the MPHA, and currently, the causative antibody is not detectable in approximately half of the suspected NAIT cases in Japan. STUDY DESIGN AND METHODS: We examined the sera of mothers from NAIT cases, previously with undetected HPA antibodies by MPHA, using the MAIPA technique. Sera from 90 mothers of suspected NAIT were tested by MAIPA for the presence of anti‐HPA‐15 alloantibodies. RESULTS: Anti‐HPA‐15b was detected in one case. This case was a mother in the first pregnancy diagnosed as hydatid mole–coexisting fetus, and the baby was born with suspected NAIT. The familial analysis revealed compatibility of HPA‐15 genotype between the mother and the baby (both HPA‐15a/a), but incompatibility with the paternal one (HPA‐15a/b). The hydatid mole's tissue was genotyped as HPA‐15b positive. Besides anti‐HPA‐15b, maternal sera contain strong HLA Class I antibody CONCLUSIONS: Here we reported the first case of anti‐HPA‐15 in Japan. Alloimmunization against the hydatid mole seems to be responsible for the production of HPA‐15b alloantibody. This antibody, however, did not apparently involve in the development of NAIT of the newborn, the coexisting anti‐HLA Class I being the possible cause.     

Rapid enzyme‐linked immunosorbent assay for the detection of antibodies against human neutrophil antigens ‐1a, ‐1b,and ‐1c

BACKGROUND: Several methods exist for the detection of neutrophil antibodies; most of them, however, require fresh neutrophils. In this study, an enzyme‐linked immunosorbent assay (ELISA) using recombinant HNA‐1 antigens (rHNAs) was developed to detect HNA‐1a, ‐1b, and ‐1c alloantibodies in serum samples. STUDY DESIGN AND METHODS: Soluble rHNA‐1a, ‐1b, and ‐1c were isolated from culture supernatant of transfected insect cells. Purified rHNA antigens were immobilized on microtiter wells using antibody against V5‐Tag protein. Sera were added, and bound antibodies were detected by enzyme‐labeled secondary antibodies. In parallel, monoclonal antibody–immobilized granulocyte antigen (MAIGA) was performed with two different monoclonal antibodies (MoAbs) against FcγRIIIb (3G8 and BW209). RESULTS: Fifteen MAIGA‐positive sera containing HNA‐1a alloantibodies were tested in ELISA. Thirteen of 15 (86.7%) MAIGA‐positive sera captured by MoAbs 3G8 and/or BW209 reacted specifically with rHNA‐1a. Four (26.7%) HNA‐1a sera showed additional reaction with rHNA‐1c. When anti‐HNA‐1b alloantibodies were analyzed in ELISA, 13 of 15 (86.7%) showed specific positive reaction with rHNA‐1b, and 12 of 15 (80.0%) cross‐reacted with rHNA‐1c. Two HNA‐1c sera reacted specifically with rHNA‐1c. Immunoprecipitation analysis of all ELISA‐negative HNA‐1a and ‐1b sera did not show any specific band indicating false‐positive reaction of these sera in MAIGA assay. CONCLUSIONS: These results suggested that rapid ELISA using recombinant neutrophil antigens may provide a valuable method for rapid screening of human alloantibodies against HNA‐1a, ‐1b, and ‐1c in patients with neutropenia and in blood donors.     

Measurement of maximal isometric torque and muscle quality of the knee extensors and flexors in healthy 50‐ to 70‐year‐old women

Muscle quality is defined as strength per unit muscle mass. The aim of this study was to measure the maximal voluntary isometric torque of the knee extensor and flexor muscle groups in healthy older women and to develop an index of muscle quality based on the combined knee extensor and flexor torque per unit lean tissue mass (LTM) of the upper leg. One hundred and thirty‐six healthy 50‐ to 70‐year‐old women completed an initial measurement of isometric peak torque of the knee extensors and flexors (Con‐Trex MJ; CMV AG, Dubendorf, Switzerland) that was repeated 7 days later. Subsequently, 131 women returned for whole‐ and regional‐body composition analysis (iDXA^?; GE Healthcare, Chalfont St Giles, Buckinghamshire, UK). Isometric peak torque demonstrated excellent within‐assessment reliability for both the knee extensors and flexors (ICC range: 0·991–1·000). Test–retest reliability was lower (ICC range: 0·777–0·828) with an observed mean increase of 5% in peak torque [6·2 (17·2) N m] on the second day of assessment (P<0·001). The relative mean decrease in combined isometric peak torque (?12·2%; P = 0·001) was double that of the relative, non‐significant, median difference in upper leg LTM (?5·3%; P = 0·102) between those in the 5th and 6th decade. The majority of difference in peak isometric torque came from the knee extensors (15·1 N m, P<0·001 versus 2·4 N m, P = 0·234). Isometric peak torque normalized for upper leg LTM (muscle quality) was 8% lower between decades (P = 0·029). These findings suggest strength per unit tissue may provide a better indication of age‐related differences in muscle quality prior to change in LTM.     

Simultaneous quantification of myocardial perfusion,oxidative metabolism,cardiac efficiency and pump function at rest and during supine bicycle exercise using 1‐11C‐acetate PET – a pilot study

Background: PET using 1‐¹¹C‐acetate (ACE‐PET) applied at rest is used for measuring absolute myocardial blood flow (MBF) and oxidative metabolic rate (k_mono). We evaluated the feasibility of quantitative ACE‐PET during exercise. Methods: Five endurance athletes underwent dynamic PET scanning at rest and during supine bicycle stress. Exercise was maintained at a workload of 120 Watt for 17 min. The rate‐pressure product (RPP) was recorded repeatedly. MBF, k_mono in left (LV) and right (RV) ventricular wall, cardiac output (CO), cardiac efficiency and a lung uptake value reflecting left heart diastolic pressures were calculated from the PET data using previously validated models. Results: MBF increased from 0·71 ± 0·17 to 2·48 ± 0·25 ml min^?1 per ml, LV‐k_mono from 0·050 ± 0·005 to 0·146 ± 0·021 min^?1, RV‐k_mono from 0·023 + 0·006 to 0·087 + 0·014 min^‐1, RPP from 4·7 ± 0·8 to 13·2 ± 1·4 mmHg × min^?1 × 10³ and Cardiac Output from 5·2 ± 1·1 to 12·3 ± 1·2 l min ^?1 (all P < 0·001). Cardiac efficiency was unchanged (P = 0·99). Lung uptake decreased from 1·1 ± 0·2 to 0·6 ± 0·1 ml g^?1 (P < 0·001). Discussion: A number of important parameters related to cardiac function can be quantified non‐invasively and simultaneously with a short scanning protocol during steady state supine bicycling. This might open up new opportunities for studies of the integrated cardiac physiology in health and early asymptomatic disease.     

Maintained cerebral metabolic ratio during exercise in patients with β‐adrenergic blockade

Background: Decreased cerebral metabolic ratio (CMR) [molar uptake of O₂ versus molar uptake of (glucose + ½ lactate)] during exercise is attenuated by intravenous administration of the non‐selective β‐adrenergic receptor antagonist propranolol. We evaluated to what extent cirrhotic patients in oral treatment with propranolol are able to mobilize brain non‐oxidative carbohydrate metabolism. Methods: Incremental cycle ergometry to exhaustion (86 ± 4·2 W; mean ± SD) was performed in eight cirrhotic patients instrumented with a catheter in the brachial artery and one retrograde in the right internal jugular vein. Healthy subjects form the control group. Results: In β‐blocked cirrhotic patients arterial lactate increased from 1·5 ± 0·3 to 5·1 ± 0·8 mM (P<0·05) and the arterial–jugular venous difference (a–v diff) from ?0·01 ± 0·03 to 0·30 ± 0·05 mM (P<0·05) at rest and during exercise, respectively. During exercise the glucose a–v diff of 0·46 ± 0·06 mM remained at a level similar to rest (0·54 ± 0·03 mM) and at exhaustion the CMR was not significantly changed (5·8 ± 1·1 versus 6·0 ± 0·6). In controls, CMR decreased from 5·6 ± 0·9 at rest to 3·4 ± 0·7 (P<0·05) during maximal exercise and at a lactate level comparable to that achieved by the patients it was 3·8 ± 0·4. Conclusion: During exhaustive exercise in cirrhotic patients the CMR is maintained and a significant cerebral uptake of lactate is demonstrated. The data suggest that oral treatment with a non‐selective β‐adrenergic receptor antagonist attenuates cerebral non‐oxidative metabolism.     

Application of the bactericidal activity of ε‐poly‐l‐lysine to the storage of human platelet concentrates

BACKGROUND: ε‐Poly‐l ‐lysine (ε‐PLL) is a polypeptide comprising approximately 30 l ‐lysine subunits generated by bond formation between α‐carboxy and ε‐amino groups. It is an approved antimicrobial food preservative in Japan. However, the efficacy of ε‐PLL as an antibacterial additive for storage of human platelet concentrates (PCs) is not known. STUDY DESIGN AND METHODS: Staphylococcus aureus, Bacillus cereus, and Klebsiella oxytoca (20 colony‐forming units/mL) were inoculated into 100% plasma PCs or PCs containing 80% platelet (PLT) additive solution with 5.0 mmol/L potassium and 1.5 mmol/L magnesium (PAS‐IIIM) and 20% plasma (PAS‐IIIM PCs). Next, a range of ε‐PLL concentrations up to 200 and 50 µg/mL were added to plasma PCs and PAS‐IIIM PCs, respectively, and the bacterial count was determined on Days 1, 2, 5, and 8. The quality of the PCs was also determined. RESULTS: Bacterial growth was inhibited at ε‐PLL concentrations of 200 and 50 µg/mL in the plasma and PAS‐IIIM PCs after 8 days of incubation. The percentage of CD62P‐positive PLTs was higher in plasma PCs treated with 200 µg/mL ε‐PLL and in PAS‐IIIM PCs treated with 50 µg/mL ε‐PLL than in the respective controls without ε‐PLL. There were no remarkable differences in the other variables, that is, PLT number, mean PLT volume, pH, aggregability, percentage of PAC‐1–positive cells, lactate dehydrogenase release, and plasma K and Na concentrations between the ε‐PLL–treated PCs and the controls. CONCLUSIONS: ε‐PLL inhibited the growth of bacteria in the PCs and did not considerably affect the quality of PCs, except CD62P expression. Further studies are required to estimate the in vivo effectiveness and safety of ε‐PLL–treated PCs.     

Characterization of the alloreactive helper T-cell response to the platelet membrane glycoprotein IIIa (integrin-beta3) in human platelet antigen-1a alloimmunized human platelet antigen-1b1b women

BACKGROUND: The aims were to characterize the helper T‐cell response to platelet (PLT) glycoprotein (GP) IIIa, which stimulates the alloimmune antibody response to human PLT antigen (HPA)‐1a, to identify immunodominant epitopes and to examine the HLA Class II associations. STUDY DESIGN AND METHODS: Peripheral blood mononuclear cells (PBMNCs) were obtained from 21 HPA‐1b1b women who had an HPA‐1a–mismatched pregnancy, 14 of whom developed anti‐HPA‐1a, and 11 control donors. PBMNCs were stimulated with two panels of 15‐mer peptides corresponding to the HPA‐1a/1b polymorphic region, with either Leu³³ (‐1a) or Pro³³ (‐1b) at each possible position, and the proliferative responses were measured. HLA Class II and HPA genotyping was by conventional polymerase chain reaction–sequence‐specific priming. RESULTS: Peptides with Leu³³ at, or near, the C‐terminus contained an immunodominant epitope, stimulating proliferation by helper T cells from all nine women who had anti‐HPA‐1a at the time of testing; peptide L1 (Val¹⁹‐Leu³³) stimulated a response in 50 percent of these women. Their T cells did not respond to the corresponding HPA‐1b Pro³³ peptides, and responses to either peptide panel were rare in unimmunized women and controls. HLA‐DRB3*01+ was significantly overrepresented (p = 0.014) in alloimmunized women whose T cells responded to the major HPA‐1a Leu³³‐containing epitope. Conversely, HLA‐DRB1*15 was negatively associated (p = 0.014) with this response. CONCLUSIONS: The HPA‐1a polymorphic region of GPIIIa contains both the linear T‐cell and the conformational B‐cell epitopes. The immunodominant T‐cell epitope is constrained by HLA‐DRB3*01+, and if presented by a tolerogenic route, a peptide containing this epitope may form the basis for the prevention or reversal of the alloimmune response to HPA‐1a.     

Semi‐quantitative tracking of intra‐airway fluids by computed tomography

Background: Airway secretions are a source of complications for patients with acute and chronic lung diseases, yet lack of techniques to quantitatively track secretions hampers research into clinical measures to reduce their pathologic consequences. Methods: In a preserved swine lung model, we tracked a contrasted mucus simulant (CMS) using sequential computed tomography (CT). Known drivers of secretion movement – gravity and ventilation – were tested. Ten millilitres of CMS were unilaterally introduced (1 ml min^?1) into the airways of 12 lung sets. After instillation, six lung sets were maintained prone and six were rotated 180°. Subsequently, all were mechanically ventilated for 10 min. CTs were obtained before infusion, after infusion and after ventilation ± rotation. For CT analysis, the lungs were partitioned into eight sub‐cuboids using anatomic landmarks. The volumes of two CT number ranges representing CMS and poor aeration/collapse were computed in every sub‐cuboid for each CT acquisition. Volume differences between study time points were used to quantify changes. Results: CMS and poor aeration/collapse volume change distributed gravitationally after infusion. After ventilation without rotation, the CMS and poor aeration/collapse volumes remained within the originally injected sub‐cuboid, although the poor aeration/collapse volume expanded (27·3 ± 6·1→50·5 ± 7·4 ml, P<0·05). After ventilation + rotation, there was a reduction in the CMS and poor/aeration collapse volumes in the originally injected sub‐cuboid (14·4 ± 1·7→4·4 ± 0·6 ml, P<0·05 and 18·3 ± 3·8→11·9 ± 2·7 ml, P<0·05, respectively) accompanied by increases in the gravitationally opposite sub‐cuboid (1·7 ± 0·2→11·1 ± 1·1 ml, P<0·05 and 0·8 ± 0·5→40·6 ± 3·5 ml, P<0·05, respectively). Conclusion: Movement of fluids within the bronchial tree can be semi‐quantitatively tracked with analysis of sequential CT acquisitions. In this isolated swine lung model, gravity had an important and brisk effect on movement of a viscous fluid, whereas ventilation tended to embed it peripherally.     

Neonatal alloimmune thrombocytopenia due to anti-HPA 5a in a HPA-5a homozygous neonate

The most frequently involved antigen in severe fetal and neonatal alloimmune thrombocytopenia (FNAIT) is the human platelet antigen 1a. Cases of FNAIT caused by HPA-5a antigen are extremely rare, and usually not severe. We report a case of FNAIT caused by anti-HPA antibodies directed to the HPA-5a antigen. The thrombocytopenia was moderate with a minimal platelet count of 36 × 10⁹/L by day 3, and spontaneously resolved by day 10.The pregnancy had been obtained by in vitro fertilization using embryo donation, creating a complete genetic disparity between the HPA 5b5b mother and the HPA 5a5a homozygous neonate.The use of ART with gamete donation can increase the risk and the severity of alloimmune thrombocytopenia and must be considered in new and subsequent pregnancies.     

The detection of platelet antibodies by simultaneous analysis of specific platelet antibodies and the monoclonal antibody–specific immobilization of platelet antigens: an interlaboratory comparison

BACKGROUND: Glycoprotein (GP)‐specific platelet (PLT) antibodies can cause allo‐ or autoimmune thrombocytopenia. Their detection is of high diagnostic value. The simultaneous analysis of specific PLT antibodies (SASPA) assay is based on simultaneous detection of various PLT‐specific antibodies by flow cytometry and has entered routine use in our Mannheim institution. In this study, we performed an interlaboratory comparison investigation of PLT‐specific antibodies using SASPA versus the “gold standard,” the monoclonal antibody–specific immobilization of PLT antigen (MAIPA) assay. STUDY DESIGN AND METHODS: Sera from 194 patients with suspected PLT allo‐ or autoantibodies were tested against GPIIb/IIIa, IX, Ia/IIa, IV, and HLA Class I by SASPA (in Mannheim) and MAIPA (in Vienna). All data were reported blinded to those from the respective other method. Sensitivity studies included dilution studies with known antibodies against HPA‐1a, ‐1b, ‐3b, ‐5b, and ‐15b and HLA Class I. RESULTS: Overall, results were concordant in 78.9%. The specificity and sensitivity of SASPA, based on the MAIPA results, were 97.3 and 86.3%, respectively, for the detection of alloantibodies. The respective results for the detection of autoantibodies were 95.3 and 44.9%. Serial dilution experiments with sera containing anti‐HPA1a, ‐1b, ‐3b, ‐5b, and ‐15b and anti‐HLA Class I revealed a higher sensitivity of the SASPA assay with all alloantibodies. CONCLUSION: In this first blind interlaboratory comparison, SASPA yielded similar results to those of MAIPA. The SASPA assay may be superior to the MAIPA assay for the detection of weak alloantibodies while simultaneous detection of a variety of antibody specificities or immunoglobulin classes and the need of fewer PLTs are obvious advantages.     

Interactions between CYP7A1 A-204C and ABCG8 C1199A polymorphisms on lipid lowering with atorvastatin

What is known and Objective: Cholesterol excretion by ATP binding cassette transporters G5 and G8 (ABCG5/G8) and bile acid biosynthesis by 7a‐hydroxylase (CYP7A1) are major pathways for the removal of cholesterol into bile. This suggests that variations in the CYP7A1 and ABCG8 genes may influence the statin response. We aimed to investigate the effect of CYP7A1 A‐204C and ABCG8 C1199A polymorphisms and their interactions on the lipid‐lowering response to atorvastatin in a Chinese population. Methods: Genotypes were determined by using polymerase chain reaction‐restrict fragment length polymorphism (PCR‐RFLP) in 185 hyperlipidaemic patients treated with atorvastatin, 20 mg once daily for 4 weeks. Serum levels of triglycerides (TGs), total cholesterol (TC), low‐density lipoprotein cholesterol (LDL‐C), and high‐density lipoprotein cholesterol (HDL‐C) were determined before and after treatment. Results and Discussion: For 181 patients (89 males), variant allele frequencies of CYP7A1 ‐204C and ABCG8 1199A were 0·347 and 0·128, respectively. Among all patients, homozygotes for the ‐204A allele showed a slightly significant mean percentage reduction from baseline in TG level after treatment than heterozygotes and homozygotes for the ‐204C allele (?25·49 ± 8·12%vs. ?22·80 ± 8·72%, P = 0·054, and ?25·49 ± 8·12%vs.?22·51 ± 8·82%, P = 0·048, respectively). For patients with the ABCG8 C1199A variant allele, the difference in percentage reduction from baseline in TG level was increased between the CYP7A1 A‐204C wild‐type allele homozygotes and variant allele homozygotes after atorvastatin treatment (?28·35%vs.?19·28%, P = 0·001), and increased differences were found between the CYP7A1 A‐204C wild‐allele homozygotes and variant allele homozygotes (?18·95%vs.?15·61%, P = 0·009) and between the CYP7A1 A‐204C variant allele heterozygotes and homozygotes (?18·69%vs.?15·61%, P = 0·012, respectively). What is new and Conclusion: The CYP7A1 ‐204A and ABCG8 1199A alleles appear to interact to affect lipid‐lowering response to atorvastatin. However, given the relatively small number of subjects with the influential variant allele combinations, and the heterogeneity in response, even in the selected sub‐populations, testing would be of little clinical utility in the Chinese population sampled.     

Heart rate recovery improvement in patients following acute myocardial infarction: exercise training, β‐blocker therapy or both

Heart rate recovery (HRR) is a strong mortality predictor. Exercise training (ET) and β‐blocker therapy have significant impact on the HRR of patients following myocardial infarction (MI). However, the combination of ET and β‐blocker therapy, as well as its effectiveness in patients with a more compromised HRR (≤12 bpm), has been under‐studied. Male patients (n = 64) post‐MI were divided: Training + β‐blocker (n = 19), Training (n = 15), β‐blocker (n = 11) and Control (n = 19). Participants performed an ergometric test before and after 3 months of intervention. HRR was obtained during 5 min of recovery and corrected by the cardiac reserve (HRR_corrCR). Compared to pre‐intervention, HRR_corrCR was significantly increased during the 1st and 2nd minutes of recovery in the Training + β‐blocker group (70·5% and 37·5%, respectively; P<0·05). A significant improvement, lasting from the 1st to the 4th minute of recovery, was also observed in the Training group (47%, 50%, 25% and 8·7%, respectively; P<0·05). In contrast, the β‐blocker group showed a reduction in HRR_corrCR during the 2nd and 3rd minutes of recovery (?21·2% and ?16·3%, respectively; P<0·05). In addition, interventions involving ET (Training + βb, Training) were significantly more effective in patients with a pre‐intervention HRR ≤ 12 bpm than for patients with HRR > 12 bpm. Combination of β‐blocker therapy with ET does not compromise the effect of training and instead promotes HRR and aerobic capacity improvement. In addition, this combination is particularly beneficial for individuals presenting with a more compromised HRR. However, chronic administration of β‐blocker therapy alone did not promote improvement in HRR or aerobic capacity.     

Pharmacokinetics of midazolam tablet in different Chinese ethnic groups

What is known and Objectives: Subjects of different ethnic groups may respond differently to drugs. The present study was conducted to compare the oral pharmacokinetics of midazolam among healthy volunteers from five different ethnic groups in China: Han, Mongolian, Uygur, Hui and Korean. Methods: Healthy volunteers (10 Hans, 10 Mongolians, 10 Uygurs, 10 Huis and 9 Koreans) of Chinese nationality received a single oral tablet dose of 15 mg midazolam in an open label, parallel‐group study. Blood samples were collected at intervals and analysed for midazolam by high performance liquid chromatography (HPLC). Ethnic differences in pharmacokinetic parameters of midazolam using non‐compartmental methods and anova and Kruskal–Wallis rank test. Results and Discussion: Midazolam maximum concentration (C_max) was significantly lower in Mongolians than that in Hans, Uygurs, Huis and Koreans (74·9 ± 33·7, 103·1 ± 26·4, 124·8 ± 50·0, 130·0 ± 38·3 and 189·0 ± 82·1 μg/L, respectively). C_max for the Koreans were significantly greater, compared with Hans and Mongolians. The time to attain C_max (t_max) for Hans was significantly longer as compared with Koreans and Uygurs (1·5 ± 0·7, 0·8 ± 0·5, 0·6 ± 0·7 h, respectively). Midazolam terminal half‐life (t_1/2z) were 3·0 ± 0·8, 2·2 ± 0·7, 1·9 ± 0·7, 3·5 ± 1·9, 3·8 ± 2·3 h for Hans, Mongolians, Uygurs, Huis and Koreans, respectively. The differences in half‐life were significant between Koreans and Mongolians, Koreans and Uygurs, Uygurs and Huis, respectively. There were no differences between young males and females for all pharmacokinetic parameters. Double peaks in the concentration–time profiles were observed in some subjects. What is new and Conclusion: There were some significant differences in midazolam pharmacokinetics between the five Chinese ethnic groups. However, the wide intra‐ethnic variability observed in PK parameters makes predictions of midazolam kinetics, using ethnicity as predictor, unreliable.