Genotyping of human neutrophil antigens 1, 3, 4 and 5 using a novel multiplex polymerase chain reaction.

OBJECTIVES: Our study aimed to establish a novel multiplex polymerase chain reaction (PCR) for rapid simultaneous detection of all relevant human neutrophil antigen (HNA)-1, -3, -4 and -5 alleles. BACKGROUND: Granulocyte-reactive antibodies are involved in several diseases, such as neonatal alloimmune neutropenia, autoimmune neutropenia and transfusion-related acute lung injury. A panel of well-defined test granulocytes is required for diagnostic antibody detection and prospective blood donor screening. Several genotyping methods for the detection of HNA alleles have been described, but most approaches require separate amplification of each HNA allele or at least a separate amplification of the HNA-1 alleles. METHODS: The new method is based on simultaneous detection in one reaction tube, where each HNA-1 allele is amplified by two allele-specific primers, one primer of which is labelled with a fluorescent dye (HEX, FAM). Allelic polymorphisms for HNA-3, -4 and -5 were amplified with one common unlabelled primer and two fluorescence-labelled (HEX, FAM) allele-specific primers. DNA fragments of HNA alleles are analysed on a Genetic Analyser 3130xl by amplicon size and fluorescent dye. A total of 110 blood donors with known genotypes were studied. RESULTS: In the 110 DNA samples studied, all HNA-1, -3, -4 and -5 alleles could be detected precisely. All results matched perfectly with those from reference typing by PCR-sequence-specific primer. Amplification performed well even at low DNA concentrations (10 ng µL-1 ). CONCLUSION: Our method enables fast and easy genotyping of all relevant HNA-alleles in one PCR reaction. Results are easy to analyse due to different amplicon sizes and fluorescent dyes. Furthermore, the method is suitable for high sample throughput.

Low incidence of heparin-induced skin lesions in orthopedic surgery patients with low-molecular-weight heparins.

BACKGROUND: Heparins are widely prescribed for prevention and therapy of arterial and venous thromboembolic diseases. Heparin-induced skin lesions are the most frequent adverse effect of subcutaneous heparin treatment in non-surgical patients (7.5%-39.8%); no data exist on surgical patients. Commonly, they are due to a delayed-type hypersensitivity reaction (DTH), but may also be a manifestation of life-threatening heparin-induced thrombocytopenia (HIT). Lesions of both entities resemble initially. The risk of HIT is highest among heparin-anticoagulated orthopedic surgery patients. OBJECTIVE: To determine incidence and causes of heparin-induced skin lesions in major orthopedic surgery patients. METHODS: In a prospective cohort study, consecutive patients with subcutaneous low-molecular-weight heparin (LMWH) treatment were examined for cutaneous adverse effects. Further diagnostics (skin biopsy, clinical/laboratory assessment for thrombosis, bleeding, HIT, cross-allergies) were performed. RESULTS: Six of 316 enrolled patients (1.9%; 95% CI: 0.4%-3.4%) developed heparin-induced skin lesions. All were caused by a DTH reaction, and none was due to HIT or other rare heparin-associated skin diseases. Therapeutic use (dosage) of LMWH was identified as only risk factor (odds ratio: 3.1, 95% CI: 1.4-4.9; P = .00141). In addition to DTH, 5 thromboembolic, 4 major bleeding complications but no cases of HIT or cross-allergies were observed. CONCLUSIONS AND CLINICAL RELEVANCE: Orthopedic surgery patients have-unlike non-surgical patients-a low risk for heparin-induced skin lesions during LMWH treatment; all lesions were due to a DTH reaction. The risk for DTH differs considerably between individual patient cohorts. No association with HIT was observed. These data help to tailor anticoagulatory treatment individually and to increase patient safety.

Same-Day Sinus and Brain CT Imaging in the Medicare Population: Are Practice Patterns Changing in Association with Medicare Policy Initiatives?

BACKGROUND AND PURPOSE: Monitoring the frequency of same-day sinus and brain CT (Outpatient Measure 14, "OP-14") is part of a recent large Centers for Medicare and Medicaid Services hospital outpatient quality initiative to improve imaging efficiency. This study investigates patient-level claims data in the Medicare population focusing on where same-day sinus and brain CT imaging is performed and how the frequency of same-day studies changed with time before and during OP-14 measure program implementation. MATERIALS AND METHODS: Research Identifiable Files were used to identify all sinus and brain CT examinations from 2004 through 2012 for a 5% random patient sample of Medicare fee-for-service beneficiaries. Overall and site of service use rates were calculated for same- and non-same-day examinations. Changes were mapped to policy initiative timetables. RESULTS: The number of same-day sinus and brain CT studies from 2004 to 2012 increased 67% from 1.85 (95% CI, 1.78-1.91) per 1000 Medicare beneficiaries in 2004 to 3.08 (95% CI, 3.00-3.15) in 2012. The biggest driver of increased same-day studies was the emergency department setting, from 0.56 (95% CI, 0.53-0.60) per 1000 to 1.78 (95% CI, 1.72-1.84; +215.7%). Overall use of brain CT from 146.0 (95% CI, 145.1-146.9) per 1000 to 176.3 (95% CI, 175.4-177.2; +21%) and sinus CT from 12.6 (95% CI, 12.4-12.8) per 1000 to 15.4 (95% CI, 15.2-15.6; +22%) increased until 2009 and remained stable through 2012. CONCLUSIONS: Previously increasing same-day sinus and brain CT in Medicare beneficiaries plateaued in 2009, coinciding with the implementation of targeted measures by the Centers for Medicare and Medicaid Services. Same-day imaging continues to increase in the emergency department setting.

High sensitivity and specificity of a new functional flow cytometry assay for clinically significant heparin-induced thrombocytopenia antibodies.

INTRODUCTION: Heparin-induced thrombocytopenia (HIT) is a life-threatening condition, in which the anticoagulant heparin, platelet factor 4 (PF4), and platelet-activating antibodies form complexes with prothrombotic properties. Laboratory tests to support clinical diagnosis are subdivided into functional, platelet activation assays, which lack standardization, or immunological assays, which have moderate specificity toward HIT. In this study, clinical performance of HITAlert, a novel in vitro diagnostic (IVD) registered platelet activation assay, was tested in a large cohort of HIT-suspected patients and compared with immunological assays. METHODS: From 346 HIT-suspected patients (single center), clinical data including 4T pretest probability results, citrated platelet-poor plasmas, and sera were collected, allowing direct comparison of clinical observations with HITAlert results. HITAlert performance was compared with PF4 IgG ELISA (246 patients, three centers) and PF4 PaGIA (298 patients, single center). RESULTS: HITAlert showed high sensitivity (88.2%) and specificity (99.1%) when compared with clinical diagnosis. Agreement of HITAlert with PF4 ELISA- and PF4 PaGIA-positive patients is low (52.7 and 23.2%, respectively), while agreement with PF4 IgG ELISA- and PF4 PaGIA-negative patients is very high (98.1 and 99.1%, respectively). CONCLUSION: HITAlert performance is excellent when compared with clinical HIT diagnosis, making it a suitable assay for rapid testing of platelet activation due to anticoagulant therapy.

Report on the 15th International Society of Blood Transfusion platelet immunology workshop.

BACKGROUND AND OBJECTIVES: The aim of the 15th ISBT Platelet Immunology Workshop was to evaluate the detection of free platelet-reactive autoantibodies from ITP patients by the use of a standardized MAIPA protocol, to compare sensitivity and specificity of antibody detection for anti-HPA-1a and serologically difficult-to-assess antibodies against HPA-3, to identify whether anti-HPA-1a titration results can be compared between laboratories, and to evaluate HPA genotyping methods. MATERIALS AND METHODS: Workshop materials were shipped from the organizing laboratory in Giessen, Germany. Thirty laboratories from 19 countries participated. RESULTS: Results for the detection of autoantibodies differed greatly between the laboratories and no consensus was reached for one of the two sera. Detection and titration of antibodies against HPA-1a, in contrast, gave largely congruent results. Serologically difficult-to-assess antibodies recognizing HPA-3a and HPA-3b were not detected by many laboratories. For genotyping, good agreement was achieved. CONCLUSIONS: Detection of HPA-1a antibodies, titration of anti-HPA-1a, and HPA genotyping are well performed in most participating laboratories. The workshop has identified two specific areas with room and need for improvement: the detection of autoantibodies and the detection of HPA-3 alloantibodies. Recommendations of the Working Party on techniques that can help to overcome these problems are desirable.

[Prophylactic intraaortic balloon pumping in high-risk cardiac surgery patients]. / Prophylaktische intraaortale Ballongegenpulsation bei herzchirurgischen Hochrisikopatienten.

BACKGROUND: We examined the impact of prophylactic IABP insertion in EuroSCORE-stratified high-risk cardiac surgery patients with a score ≥8. MATERIAL AND METHODS: A randomized trial with 104 patients either without prophylactic IABP insertion (group A, n=52) or with IABP (group B, n=52) was conducted. The primary endpoint was 30-day mortality. RESULTS: The median age of the patients was 74 years and 43% of participants were females. The 30-day mortality did not differ between group A (17.3%) and group B (13.4%; p=0.78). The median hospital stay was 14 days in both groups. Intra- and postoperative IABP support was required by 13 patients (21%) in group A. The median ventilation time (14 hours versus 13 hours), median catecholamine dose, frequency of dialysis-dependent acute renal failure (28% versus 18%), cardiac indices, and frequency of a low cardiac output syndrome (26% versus 25%) did not significantly differ between groups. CONCLUSION: Prophylactic preoperative IABP insertion in EuroSCORE-stratified high-risk patients is not associated with decreased 30-day mortality.

Heparin-induced non-necrotizing skin lesions: rarely associated with heparin-induced thrombocytopenia.

SUMMARY BACKGROUND: Recently, there has been an increasing number of reports regarding adverse skin reactions to subcutaneous heparin administration. Case series have implied that heparin-induced skin lesions are predominantly associated with life-threatening heparin-induced thrombocytopenia (HIT) in at least 22% of patients. Skin lesions, therefore, have been included in clinical scores for HIT. OBJECTIVES: To determine the association of heparin-induced skin lesions with HIT. This would have a pivotal impact on further anticoagulatory management in patients with heparin-induced skin lesions. PATIENTS/METHODS: In our observational cohort study, 87 consecutive patients with heparin-induced skin lesions (85 occurring during low-molecular-weight heparin administration) were evaluated using a standardized internal protocol, including HIT diagnostics (heparin-platelet factor 4-ELISA, heparin-induced platelet activation assay), platelet count monitoring, clinical/sonographical screening for thrombosis, skin allergy testing and, if necessary, histology. RESULTS: None of the observed heparin-induced skin lesions was due to HIT; all lesions were caused by delayed-type IV-hypersensitivity reactions (DTH) instead. Even the cutaneous reaction in one patient with concomitant HIT could be classified histologically as DTH reaction, amounting to an association of heparin-induced skin lesions and HIT in 1.2% (1/87; 95% confidence interval, 0.00-0.06). CONCLUSION: Heparin-induced skin lesions associated with use of low-molecular-weight heparins do not seem to be strongly associated with a systemic immunologic reaction in terms of HIT and might rather be due to DTH reactions than due to microvascular thrombosis. Hence, we propose refining existing pretest probability scores for HIT, unless underlying causes have been clarified.

Perfusion-CT of developmental venous anomalies: typical and atypical hemodynamic patterns.

This article reports perfusion-CT patterns that can be observed in patients with DVAs. In atypical DVAs, an abnormal venous congestion pattern with increased CBV, CBF and MTT can be observed in the vicinity of a DVA, and needs to be recognized and differentiated from other entities such as cerebral neoplasms or stroke. This pattern might help to stratify risks of associated complications such as hemorrhage.

Ribosomal alterations contribute to bacterial resistance against the dipeptide antibiotic TAN 1057.

TAN 1057-resistant Staphylococcus aureus and Escherichia coli strains were selected to elucidate the mechanism of resistance and the mode of action of this dipeptide antibiotic. Cell-free translation with isolated ribosomes and S150 fractions from sensitive and resistant S. aureus strains demonstrated that alterations in the ribosomes contribute to the resistance of the bacteria.

Danaparoid sodium (Orgaran) in four children with heparin-induced thrombocytopenia type II.

UNLABELLED: We report on four children with heparin-induced thrombocytopenia type II. In three patients, therapy with unfractionated heparin was associated with development of cardiac thrombi or with thrombosis progression up to the inferior vena cava or with aggravation of peripheral arterial occlusion. In the fourth child, the disease was recognized early on, and no complication occurred. Heparin-induced thrombocytopenia type II was confirmed by heparin-induced platelet activation assay and/or heparin/platelet factor 4-ELISA. Concomitant elevated antiphospholipid antibodies were seen in all patients. Danaparoid sodium applied at a dosage of between 1.2 and 7.1 U/kg/h stopped the disease progression in each patient. Three children had a clinical recovery with partial recanalization, but for the child with peripheral arterial occlusion disease, amputation of some of the toes became necessary. CONCLUSION: Our data indicate that heparin-induced thrombocytopenia type II is a potential life-threatening disease in children and danaparoid sodium is beneficial in this age group.

A comparison of danaparoid and lepirudin in heparin-induced thrombocytopenia.

Heparin-induced thrombocytopenia (HIT) is a hypercoagulable syndrome strongly associated with thrombosis that is usually treated with drugs that inhibit factor Xa (danaparoid) or thrombin (lepirudin). In the present study the outcome of HIT-patients treated with danaparoid or lepirudin was compared using the single or combined endpoints of new thromboembolic complications (new TECs), amputations and/or death, and major bleeding. HIT-patients treated with lepirudin were enrolled in two prospective trials and patients, who were identified in the same two laboratories during the same time period, who were not enrolled into these studies but treated with danaparoid, were assessed retrospectively according to a standardized questionnaire. 126 danaparoid (60.3% female) and 175 lepirudin treated patients (58.3% female) fulfilled the same inclusion and exclusion criteria. In a time-to-event-analysis the cumulative risk of combined endpoint was higher in HIT-patients without thromboembolic complication at baseline treated with danaparoid (usually in prophylactic dose 750 anti-factor Xa units b.i.d. or t.i.d.s.c.) as compared to lepirudin (aPTT adjusted) (P = 0.020). Whereas HIT-patients with TEC at baseline who were usually treated with therapeutic dose had a similar outcome in both treatment groups (P = 0.913). Major bleeding occurred in 2.5% (95% CI 0.5-7.0%) of danaparoid treated patients as compared to 10.4% (95% CI 6.3-15.9%) of lepirudin treated patients until day 42 (P = 0.009). This indicates that the efficacies of therapeutic doses of danaparoid or lepirudin in preventing death, amputation or new TEC in HIT-patients do not differ largely, but the risk of bleeding seems to be higher in lepirudin treated patients. The prophylactic dose of danaparoid approved in the European Union for HIT without TEC at baseline seems suboptimal. A prospective comparative trial is required to verify these preliminary conclusions.

Workshop report on the genotyping of blood cell alloantigens.

The immunization against alloantigens present on platelets, granulocytes and red blood cells (RBCs) is responsible for various clinical syndromes. Since the molecular basis of these antigens has become clear during the last decade, genotyping is nowadays used in several laboratories. However, many DNA-based techniques still have to be evaluated. We therefore organized a workshop on the genotyping of the most relevant alloantigens on platelets and granulocytes as well as on selected RBC alleles. DNA was isolated from peripheral blood lymphocytes or from B-lymphoblastoid cell lines (B-LCL). We distributed samples for the identification of platelet (n = 7), granulocyte (n = 6) and RBC (n = 4) polymorphisms, respectively. There were 33 institutions in Germany, Austria and Switzerland, which participated in at least one part of the workshop. Twenty-four laboratories reported results on HPA-1, and 23 laboratories on HPA-2, -3, and -5 typing. In addition, five laboratories typed for HPA-4 and -6. The HNA-1a/b (NA1/NA2) alleles were identified by eight laboratories, one of which also typed for HNA-1c (SH). The most frequent genes of the ABO (A1, B, O) and Rh (D, C, c, E, e) systems were typed by 12 participating laboratories, and an additional four laboratories restricted their RBC typing to the RHD gene. The typing technique mainly used for all three cell lineages was the polymerase chain reaction with sequence-specific primers. Other techniques were restriction fragment length analysis, oligonucleotide ligation assay, enzyme-linked mini-sequence assay or direct sequence analysis. The following typing errors were observed: HPA: 15/1442 (1.0%), HNA: 4/108 (3.7%), ABO: 5/96 (5.2%) RH 1/320 (0.3%). Our workshop demonstrated the existence of a number of reliable techniques for the genotyping of blood cell alloantigens and a high standard in the participating laboratories. In addition, we could show the usefulness of B-LCL as a source of reference DNA. However, the 5.2% rate of mistyping in the ABO system demonstrated that further efforts are needed to improve the precision of the genotyping techniques. Future workshops will have to challenge methods and participants with rare variants of RBC genes to guarantee reliable genotyping, e.g. in prenatal diagnosis of fetomaternal incompatibility.

Platelet alloantibodies in transfused patients.

BACKGROUND: Patients receiving cellular blood components may form HLA antibodies and platelet-specific alloantibodies. STUDY DESIGN AND METHODS: Serum samples from a cohort of 252 patients with hematologic or oncologic diseases who are receiving cellular blood components were studied for platelet-reactive antibodies. Specificity of platelet alloantibodies was determined with a panel of typed platelets RESULTS: Platelet-reactive antibodies were detected in the sera of 113 patients (44.8% of 252), HLA antibodies in the sera of 108 (42.9%), and platelet-specific antibodies in the sera of 20 (8%). The following platelet-specific antibodies were identified: anti-HPA-5b (n = 10), anti-HPA-1b (n = 4), anti-HPA-5a (n = 2), anti-HPA-1a (n = 1), anti-HPA-2b (n = 1), anti-HPA-1b+5b (n = 1), and anti-HPA-1b+2b (n = 1). Fifteen sera from the 108 patients with anti-HLA (13.9%) contained additional platelet-specific alloantibodies, while in 5 sera, platelet-specific alloantibodies only were detected: anti-HPA-5b (n = 4) and anti-HPA-1a (n = 1). Of the 108 sera with HLA antibodies, 29 (26.9%) showed discordant results when studied with the lymphocytotoxicity test and the glycoprotein-specific immunoassay. Ten sera contained panreactive antibodies against platelet glycoproteins (GP) IIb/IIIa, GPIa/IIa, and/or GPIb/IX. Alloimmunization occurred in 58.3 percent of female patients with previous pregnancies, but in only 23.3 percent of those without previous pregnancies (p = 0.0049). CONCLUSION: Platelet alloantibody specificities in transfused patients (predominantly anti-HPA-5b and -1b with antigen frequencies <30% among whites) differ significantly from those observed in patients with neonatal alloimmune thrombocytopenia or posttransfusion purpura, in whom anti-HPA-1a (antigen frequency >95%) is the most prevalent specificity. HLA antibody detection yields discordant results when the lymphocytotoxicity assay and a glycoprotein-specific immunoglobulin-binding assay are used.

Platelet endothelial cell adhesion molecule-1 (PECAM-1) is a target glycoprotein in drug-induced thrombocytopenia.

Drug-induced immune thrombocytopenia (DITP) is a serious complication of drug treatment. Previous studies demonstrated that most drug-dependent antibodies (DDAbs) react with the platelet membrane glycoprotein (GP) complexes IIb/IIIa and Ib/IX/V. We analyzed the sera from 5 patients who presented with DITP after intake of carbimazole. Notably, thrombocytopenia induced by carbimazole was relatively mild in comparison to patients with DITP induced by quinidine. The sera reacted with platelets in an immunoassay on addition of the drug. In immunoprecipitation experiments with biotin-labeled platelets and endothelial cells, reactivity with the platelet endothelial cell adhesion molecule-1 (PECAM-1, CD31) could be demonstrated, whereas neither GPIIb/IIIa nor GPIb/IX was precipitated in the presence of the drug. These results could be confirmed by GP-specific immunoassay (MAIPA) using monoclonal antibodies (mabs) against PECAM-1. In addition, the binding of DDAbs could be abolished by preincubation with soluble recombinant PECAM-1. Carbimazole-dependent antibodies showed similar reactivity with platelets carrying the Leu(125) and Val(125) PECAM-1 isoforms, indicating that this polymorphic structure, which is located in the first extracellular domain, is not responsible for the epitope formation. Binding studies with biotin-labeled mutants of PECAM-1 and analysis of sera with mabs against different epitopes on PECAM-1 in MAIPA assay suggested that carbimazole-dependent antibodies prominently bound to the second immunoglobulin homology domain of the molecule. Analysis of 20 sera from patients with quinidine-induced thrombocytopenia by MAIPA assay revealed evidence that DDAbs against PECAM-1 are involved in addition to anti-GPIb/IX and anti-GPIIb/IIIa. We conclude that PECAM-1 is an important target GP in DITP. (Blood. 2000;96:1409-1414)

Heparin-induced thrombocytopenia: a critical risk/benefit analysis of patients in intensive care treated with R-hirudin.

Patients in intensive care may be at high risk of in vivo platelet activation because comorbid conditions, such as infections, septicemia, shock, disseminated intravascular coagulation, and cancer represent procoagulant states. Hyperreactivity of platelets with or without a decline of cell count may result in thromboembolic complications potentially associated with the phenomenon of heparin-induced thrombocytopenia. We analyzed the data of 10 patients highly suspected of having heparin-induced thrombocytopenia during their intensive care treatment of 29 plus or minus 22 days. In seven patients, thrombocytopenia coincided with thromboembolic complications. Six patients had additionally undergone fibrinolytic therapy before starting activated partial thromboplastin time-adapted alternative anticoagulation with r-hirudin. In three patients, the platelet count decreased without a clinical manifestation, of heparin-induced thrombocytopenia. R-Hirudin treatment monitored by activated partial thromboplastin time and prothrombin time (PT) was effective and safe. The target value for activated partial thromboplastin time was a twofold prolongation. In four of five patients with deep venous thrombosis, a partial recanalization of the lower extremity could be achieved. Three patients with pulmonary embolism associated with deep venous thrombosis in two cases and in one additional case with an acute myocardial infarction did clinically profit from fibrinolysis with recombinant tissue plasminogen activator (rtPA) and r-hirudin treatment. Two lethal events probably caused by the underlying multimorbidity could not be prevented. No recurrence of thrombosis occurred, and there were no severe bleeding complications attributed to r-hirudin treatment. Platelet counts were significantly reduced on day 9.4 plus or minus 6.4 of heparin administration in all cases (>50% decrease related to the initial values) from 224,000 plus or minus 126,000/microL to 96,000 plus or minus 61,000/microL, and increased during rhirudin treatment to mean values of 224,000 plus or minus 126,000/microL. The heparin-induced platelet activation assay (HIPAA) assay was positive in 8/10 cases, whereas the PF4 enzyme-linked immunosorbent assay showed a positive result in four of eight analyzed cases. In four cases, the assays were concordantly positive. The PF4 enzyme-linked immunosorbent assay was not performed in two cases.

The impact of the glycoprotein Ia collagen receptor subunit A1648G gene polymorphism on coronary artery disease and acute myocardial infarction.

Platelet glycoprotein (GP) Ia is the major receptor for collagen and plays an important role in platelet adhesion and aggregation. Different gene polymorphisms have been identified that induce either various expression levels (C807T) or alterations of the tertiary structure (A1648G) of GPIa. Previously, we could demonstrate an association of the GPIa C807T dimorphism with nonfatal myocardial infarction. We have now analysed the influence of the GPIa A1648G (Br, HPA-5) dimorphism on the risk of coronary artery disease (CAD) and acute myocardial infarction (AMI). DNA samples from 2163 male Caucasian patients who underwent coronary angiography were genotyped by polymerase chain reaction and restriction fragment length analysis. The relation of the GPIa A1648G dimorphism to the extent of CAD was determined by multiple regression analysis with adjustment for coronary risk factors. Odds ratios (OR) as an estimate of relative risk of CAD and AMI and two-tailed p-values were calculated by multiple logistic regression. In the total study sample, no association was detected between the A1648G dimorphism and CAD or AMI. However, upon analysis of low-risk patient subgroups we found an association of the GPIa A1648G polymorphism with the risk and the extent of CAD (patients with high apoAI/apoB ratio: OR 0.59, p = 0.0090; non- and ex-smokers: OR 0.66, p = 0.0131; both inclusion criteria: OR 0.44, p = 0.0003). The relative frequency of the A1648 allele was higher in controls whereas the GG1648 genotype was overrepresented in patients with CAD. This association was also detectable when individuals with low expression levels of GPIa (C807 homozygotes) were analysed (patients with high apoAI/apoB ratio: OR 0.44, p = 0.0045; non- and ex-smokers: OR 0.61, p = 0.0370). Our findings indicate that the A1648G polymorphism of the platelet collagen receptor plays a role in CAD in well defined patient groups.