A new automated technique for platelet aggregation measurement

A Multistat III Centrifugal Analyser (MCA) was used to measure platelet aggregation in vitro. It has a capacity of about 40 samples per h. In the analyser platelet-rich plasma and collagen-reagent were mixed, and the turbidity was measured as a function of time. The results were presented in arbitrary units ( arb . units), i.e. change in turbidity per min X 1000. The estimated 0.95 reference range was 50-95 arb . units, (n = 46) and the coefficient of correlation between MCA results and results obtained by conventional aggregometry ( Fibromat ) was 0.85 (P less than 0.001). The MCA method registered 50-75% reduction of platelet aggregation after intake of low dose acetylsalicylic acid (ASA) (1.2-4.0 mg/kg) during 3 days in 19 subjects. The MCA method is suitable to monitor ASA treatment routinely in order to establish an individual appropriate ASA dose during prophylactic treatment of arterial thromboembolic disease.     

The Lui elution technique. A simple and efficient method for eluting ABO antibodies

The Lui elution and the heat elution techniques are the most simple techniques available for eluting antibodies from red cells. Both are very effective for eluting ABO antibodies. We compared the two techniques and found the Lui technique superior in that it more efficiently eluted ABO antibodies, required less hands-on time, and allowed more flexibility in the procedure. We therefore recommend the Lui elution technique as the method of choice for eluting ABO antibodies, especially for the evaluation of hemolytic disease of the newborn.     

A microtiter plate technique for the detection of platelet antibodies and platelet antigen typing

A microtiter plate assay is described for platelet serologic studies. The assay is based on an indirect radiolabeled antiglobulin test. The test was performed in microtiter wells of 400 microliter capacity manufactured to form strips that fit into a standard 96-well carrier. The strips were broken apart and placed into tubes for counting in a gamma counter. The technique does not require fixation of the platelets to the wells. Freshly collected platelets or platelets that have been stored frozen in 5 percent dimethylsulfoxide can be used. Results are presented using the technique for platelet alloantibody identification, platelet antigen typing, and platelet crossmatching.     

PCR-SSP与血清学技术用于ABO血型分型的比较

目的 探讨ABO血型基因分型技术应用于临床检验的可行性.方法 随机抽样100例江西健康汉族个体,并收集20例正反定型不符的标本,采用血清学和序列特异性引物PCR法(PCR-SSP)鉴定血型.结果 100例健康个体的基因分型结果与血清学分型结果一致,19例正反定型不符标本的基因分型结果准确.分型结果表明江西地区以O型为多,100例健康个体中,O型36例(36%),A型32例(32%),B型24例(24%),AB型8例(8%),基因频率:p(A)基因为0.225 3,q(B)基因为0.175 3,r(O)基因为0.599 8,且符合Hardy-Weinberg平衡.结论 基因分型技术应用于临床检验具有可行性,在鉴定疑难血型方面可以作为血清学分型的补充.     

Selective elution of HLA antigens and beta 2-microglobulin from human platelets by chloroquine diphosphate

To determine whether chloroquine can specifically elute HLA antigens and beta 2-microglobulin (beta 2-M) from the platelet surface, quantitative immunofluorescence flow cytometry and monoclonal antibodies were used to show that HLA antigens and beta 2-M were proportionally eluted from the platelet surface without affecting the membrane glycoproteins IIb and IIIa. Second, an autoradiogram of electrophoresed I125-labeled platelets showed that only beta 2-M but not other I125-labeled membrane proteins could be eluted. Although HLA antigens were poorly labeled by I125 and could not be detected on the autoradiogram, the eluted HLA antigens could be detected by anti-HLA monoclonal antibody and immunoblotting techniques. No loss of plasma membrane integrity was observed by transmission electron microscopy after chloroquine treatment of platelets. The results indicate that chloroquine selectively elutes HLA antigens and their noncovalently associated beta 2-M without affecting other integral platelet membrane proteins.     

未洗脱二甲亚砜冰冻复融血小板的功能及其临床应用研究

目的 探讨未洗脱二甲亚砜(DMSO)冰冻复融血小板的功能及其临床应用的可行性.方法 采用简单抽样法选择2015年1月至12月山东省东营市中心血站制备和保存的60份(每份为200 mL)未洗脱DMSO冰冻复融血小板作为研究对象,纳入复融后血小板原液组(n=60).取复融后血小板原液组血小板10 mL与复融后的新鲜冰冻血浆30 mL,按照体积比为1∶3的比例混匀后,纳入血小板混合液组(n=60).并随机选择同期本血站采集的60份新鲜液态血小板原液,纳入对照组(n=60).采用简单随机抽样法选择同期于东营市第二人民医院接受未洗脱DMSO冰冻复融血小板输注的150例患者作为输血反应的观察对象.3组血小板均进行血栓弹力图及复钙后凝血时间检测,比较各组血小板凝血功能.回顾性分析150例患者输注未洗脱DMSO冰冻复融血小板后输血反应发生情况;并比较150例输注未洗脱DMSO冰冻复融血小板患者输注前及输注后1、24、48和72 h的外周血血小板计数,分析输注未洗脱DMSO冰冻复融血小板的临床应用效果.本研究临床试验遵循的程序符合东营市第二人民医院人体试验委员会所制定的伦理学标准,得到该伦理会批准,征得受试对象本人的知情同意,并与之签订临床研究知情同意书.结果 ①血小板混合液组血栓弹力图凝血指数(CI)为2.5±0.2,高于对照组的1.0±0.1和复融后血小板原液组的0.9±0.2,并且差异均有统计学意义(t=16.69、23.02,P＜0.05).②血小板混合液组、复融后血小板原液组及对照组血小板复钙后凝血时间分别为(6.3±0.5)、(9.0±0.8)、(11.7±1.1)min,3者比较,差异有统计学意义(F=27.38,P＜0.05).复融后血小板原液组血小板复钙后凝血时间较对照组显著缩短,并且差异有统计学意义(q=-7.38,P＜0.05).血小板混合液组血小板复钙后凝血时间较对照组及复融后血小板原液组亦显著缩短,并且差异均有统计学意义(q=-3.36、-2.29,P＜0.05).③本组150例患者共计输注未洗脱DMSO冰冻复融血小板319个治疗量,无1例发生输血不良反应.血小板输注48 h后血小板计数较输注前增加(7.6±1.1)×109/L,较输注1、24 h后的增加值[(16.3±2.5)×109/L和(13.5±2.5)×109/L]低,并且差异有统计学意义(t=-24.87、-25.92,P＜0.05).结论 未洗脱DMSO冰冻复融血小板与新鲜冰冻复融血浆(体积比为1∶3)混合物凝血功能明显提高,快速止血效果好,未洗脱DMSO冰冻复融血小板临床输注无输血不良反应,可满足急性止血需求.     

External quality assessment of platelet serology and human platelet antigen genotyping: a 10-year review

BACKGROUND: In this review, the results of an external quality assessment (EQA) over 10 years of platelet (PLT) serology and of human platelet antigen (HPA) polymorphisms genotyping are shown. The detection and identification of PLT antibodies and the distinction between PLT-specific antibodies and HLA Class I antibodies are evaluated. STUDY DESIGN AND METHODS: Each year, serum samples from five patients and four donor blood samples for DNA typing were distributed. Laboratories could participate as a screening laboratory (SL; n = 7) or as an identification laboratory (IL; n = 8). RESULTS: SLs scored 57 to 100 percent correct positive and 91 to 100 percent correct negative results in the detection of PLT-specific antibodies. SLs only using a PLT immunofluorescence test (PIFT) scored less well than those using a PLT glycoprotein-based antibody detection method. ILs scored 70 to 100 percent correct positive and 87 to 100 percent correct negative results for, respectively, the detection and identification of PLT-specific antibodies. Both the specificity and the sensitivity for the detection and identification of PLT-specific antibodies were not as good in ILs using solid-phase enzyme-linked immunosorbent assay methods as in those using the monoclonal antibody immobilization of PLT antigens (MAIPA) assay. For HPA-1, -2, -3, and -5 genotyping, incorrect results were observed only twice in 280 genotyping assays. CONCLUSION: The data underscore the necessity of using the most accurate methods with a high level of knowledge, experience, and technical training. For screening purposes, it is not sufficient to use only the PIFT, whereas for identification of PLT-specific antibodies, the MAIPA assay is the superior assay.     

A radioimmunoassay for the antimalarial drug chloroquine

We describe a 3H-based RIA for the antimalarial drug chloroquine (CLQ), the most commonly used antimalarial drug. In the assay a monoclonal antibody is used that is directed against 4-amino-7-chloroquinoline conjugated to keyhole limpet hemocyanin by the glutaraldehyde method. Besides CLQ, this antibody also recognizes with good affinity the 4-aminoquinoline homologs, hydroxychloroquine and amodiaquine. No extraction step or sample preparation is required, and the method can detect as little as 10 micrograms/L, the lower concentration in plasma of humans who are taking CLQ as a preventive measure. The between-assay CV is less than or equal to 10%, the within-assay CV less than or equal to 3%. Results correlate with those by liquid chromatography (r2 = 0.96). The speed and simplicity of the RIA method make it useful in evaluating the CLQ concentrations in acutely toxic patients.     

Immunofluorescence serology. A tool for prognosis of Q-fever

The indirect immunofluorescence antibody test is currently the method of choice for Q-fever laboratory diagnosis. It permits the detection of IgG-, IgM-, and IgA-specific antibodies against the two phases of Coxiella burnetii. Sera from 20 cases of C. burnetii infection have been examined. Only total IgG against phase II were detected in cryptic infections. In acute Q-fever cases, the appearance of total IgG antibodies against phase I was a sign of aggravation, while IgM titers remained low. In subacute cases of Q-fever, anti-phase-I IgG titers were equal to or higher than anti-phase-II titers, and IgM against both phases were produced over a long time. Particularly high IgM titers were found in cases of granulomatous hepatitis. IgA antibodies against phase I were found in cases of Q-fever endocarditis, although the two cases that died had few or no IgA antibodies, despite very high IgG and IgM titers.     

A standardized technique for efficient platelet and leukocyte collection using the Model 30 Blood Processor

The Model 30 Blood Processor is a safe and simple means of harvesting blood cell components. Presently cell collection depends on a visual assessment by the operator of the indistinct boundaries of cell fractions. To determine when each cell component could best be harvested, serial samples were taken from the output port at fixed intervals anf the results of counts and differentials were graphed and tabulated. Studies in normal donors were done using acid-citrate- dextrose (ACD), 2 per cent sodium citrate in 6 per cent hydroxyethyl starch (HES), or heparin as anticoagulants. There was considerable overlap between the latter part of the platelet band, the leukocyte band and the rising hematocrit with all three anticoagulants. Normally functional lymphocytes could be harvested efficiently (approximately 80%) using ACD or heparin. Platelets could be harvested from ACD very efficiently (approximately 90%). Granulocytes could not be harvested from ACD (less than 10%) since they were dispersed in the red blood cell (RBC) layer. Using HES, granulocytes could be harvested efficiently (approximately 70%) by extending collection into the RBC layer. Based on these data, a standard technique for cell collection has been devised. The flow rate is slowed to 20 ml/min and collection is carried 30 ml (90 seconds at a rate of 20 ml/min) for platelets. The RBC loss is approximately 6 to 8 and 2 to 3 ml/pass respectively. These studies indicate that the Model 30 is a highly efficient apparatus for blood cell separation, but the volume of blood processed is limited by the intermittent blood flow.     

Update on leucodepletion, HLA and platelet serology at Lisbon Regional Blood Centre.

The characteristic performance of some leucodepletion processes at the Lisbon Regional Blood Centre is presented. The current position on HLA and platelet serology is briefly described.     

Removing IgG antibodies from intact red cells: comparison of acid and EDTA, heat, and chloroquine elution methods

BACKGROUND: To accurately phenotype red cell from patients with a positive direct antiglobulin test (DAT), nonlytic elution procedures were assessed for their ability to dissociate IgG from antibody-coated red cells without altering red cell antigen expression. STUDY DESIGN AND METHODS: Antibodies coating red cells that were sensitized in vivo (warm-reactive autoantibodies: 8 patients) or in vitro (42 alloantibodies) were eluted by using glycine-HCl and EDTA (acid/ EDTA), heat (56 degrees C, 10 min), or chloroquine method. RESULTS: Acid/EDTA elution gave the best results, reducing DAT positivity to microscopic levels or rendering the DAT negative in 48 of 50 instances, whereas 4 samples remained resistant to heat elution and 24 to chloroquine. Standard DAT agglutination scores demonstrated that both acid/EDTA and heat elution were superior to the chloroquine method (p < 0.0001). With the gel low-ionic-strength saline indirect antiglobulin test, acid/ EDTA was superior to heat (p < 0.001). Overall, acid/ EDTA elution dissociated more antibodies than heat (p < 0.0001), especially for Kell system (K, k, Kpa, Kpb) alloantibodies. Common red cell antigens, other than Kell system antigens, were unaffected by acid/EDTA elution. In contrast, the expression of most blood group antigens was diminished after heat elution. However, it was possible to type red cell antigens by using gel low-ionic-strength saline indirect antiglobulin tests or tube agglutination methods. CONCLUSION: Although heat elution may be used on a limited basis, the acid/EDTA method appears to be the procedure of choice for typing red cell coated with warm-reactive IgG alloantibodies or autoantibodies.     

A flow cytometric method for platelet counting in platelet concentrates

BACKGROUND: The platelets (PLTs) in PLT concentrates are counted with hematology analyzers, but varying results among different hematology analyzers are observed, making comparisons very difficult. Due to the absence of red blood cells in PLT concentrates, the International Council for Standardization in Hematology (ICSH) reference method was modified to be used for PLT concentrates and validated in an international comparative study. STUDY DESIGN AND METHODS: Five PLT samples were shipped to eight participating centers of the Biomedical Excellence for Safer Transfusion (BEST) Collaborative and counted on the same day. PLTs were stained with fluorescein isothiocyanate–labeled anti‐CD41a in tubes (TruCount, BD Biosciences), measured on a flow cytometer, and analyzed with a uniform template. These samples were also counted on 15 hematology analyzers. RESULTS: The ICSH method and newly developed BEST method yielded PLT counting results with less than 1% difference (not significant). The intercenter coefficient of variation (CV) of the BEST method was on average 6.3% versus 7.6% on average for hematology analyzers. The CV of individual hematology analyzers was on average 0.9%, which was considerably lower than for the flow cytometers with a mean of 3.7%. CONCLUSION: The BEST flow cytometric method has a smaller intercenter CV and a smaller center‐to‐center deviation from the group mean compared to hematology analyzers. Conversely, individual hematology analyzers are more precise than the flow cytometric method. Thus, the flow cytometric method provides a calibration tool to allow comparisons between centers, but there is no need to replace routine counting with hematology analyzers.     

非特异磁性分离法在血小板制品污染菌富集中的应用研究

目的建立非特异磁性分离法富集血小板制品中的大肠杆菌和金黄色葡萄球菌,以提高应用核酸扩增方法检测血小板制品污染菌的效率。方法应用6种磁珠(80 nm羧基、200 nm羧基、2.8μm氨基、2.8μm羧基、2.8μm环氧基、2.8μm甲苯磺酰基磁珠),分别对高浓度(102—103CFUs.ml-1)、低浓度(<102CFUs.ml-1)血小板制品中的大肠杆菌进行非特异性富集,选取富集效率最高的磁珠,进一步对不同稀释浓度的大肠杆菌、金黄色葡萄球菌(10-1、10-2、10-3、10-4、10-5、10-6)进行富集,与直接离心法进行对比,比较不同细菌富集方法对后续聚合酶链式反应(polymerase chain reaction,PCR)检测灵敏度的影响。结果 6种磁珠对高、低浓度的血小板制品中大肠杆菌均有富集作用,其中80 nm羧基磁珠的富集效率最高(P<0.05);应用直接离心法和80 nm羧基磁珠富集样品后,PCR检测大肠杆菌的灵敏度分别为588 CFUs.ml-1、1 CFUs.ml-1,检测金黄色葡萄球菌的灵敏度分别为1 130 CFUs.ml-1、40 CFUs.ml-1。结论应用磁性分离法富集血小板制品中污染菌,其操作简单,所获细菌纯度高,为后续PCR检测方法在血小板制品污染菌检测中的临床应用提供帮助。     

Acute chloroquine and hydroxychloroquine toxicity: A review for emergency clinicians

BackgroundAcute chloroquine and hydroxychloroquine toxicity is characterized by a combination of direct cardiovascular effects and electrolyte derangements with resultant dysrhythmias and is associated with significant morbidity and mortality.ObjectiveThis review describes acute chloroquine and hydroxychloroquine toxicity, outlines the complex pathophysiologic derangements, and addresses the emergency department (ED) management of this patient population.DiscussionChloroquine and hydroxychloroquine are aminoquinoline derivatives widely used in the treatment of rheumatologic diseases including systemic lupus erythematosus and rheumatoid arthritis as well as for malaria prophylaxis. In early 2020, anecdotal reports and preliminary data suggested utility of hydroxychloroquine in attenuating viral loads and symptoms in patients with SARS-CoV-2 infection. Aminoquinoline drugs pose unique and significant toxicological risks, both during their intended use as well as in unsupervised settings by laypersons. The therapeutic range for chloroquine is narrow. Acute severe toxicity is associated with 10–30% mortality owing to a combination of direct cardiovascular effects and electrolyte derangements with resultant dysrhythmias. Treatment in the ED is focused on decontamination, stabilization of cardiac dysrhythmias, hemodynamic support, electrolyte correction, and seizure prevention.ConclusionsAn understanding of the pathophysiology of acute chloroquine and hydroxychloroquine toxicity and available emergency treatments can assist emergency clinicians in reducing the immediate morbidity and mortality associated with this disease.