Tuberculosis (TB) is one of the leading causes of death by an infectious disease. It remains a major health burden worldwide, in part due to misdiagnosis. Therefore, improved diagnostic tests allowing the faster and more reliable diagnosis of patients with active TB are urgently needed. This prospective study examined the performance of the new molecular whole-blood test T-Track® TB, which relies on the combined evaluation of IFNG and CXCL10 mRNA levels, and compared it to that of the QuantiFERON®-TB Gold Plus (QFT-Plus) enzyme-linked immunosorbent assay (ELISA). Diagnostic accuracy and agreement analyses were conducted on the whole blood of 181 active TB patients and 163 non-TB controls. T-Track® TB presented sensitivity of 94.9% and specificity of 93.8% for the detection of active TB vs. non-TB controls. In comparison, the QFT-Plus ELISA showed sensitivity of 84.3%. The sensitivity of T-Track® TB was significantly higher (p < 0.001) than that of QFT-Plus. The overall agreement of T-Track® TB with QFT-Plus to diagnose active TB was 87.9%. Out of 21 samples with discordant results, 19 were correctly classified by T-Track® TB while misclassified by QFT-Plus (T-Track® TB-positive/QFT-Plus-negative), and two samples were misclassified by T-Track® TB while correctly classified by QFT-Plus (T-Track® TB-negative/QFT-Plus-positive). Our results demonstrate the excellent performance of the T-Track® TB molecular assay and its suitability to accurately detect TB infection and discriminate active TB patients from non-infected controls.

Neutralizing antibodies against SARS-CoV-2 are important to protect against infection and/or disease. Using an assay to detect antibodies directed against the receptor binding domain (RBD) of SARS-CoV-2 Spike, we identified individuals with SARS-CoV-2 infection after an outbreak at a local health institution. All but one COVID-19 patient developed detectable anti-RBD antibodies and 77% had virus neutralizing antibody titers of >1:25. Antibody levels declined slightly over time. However, we still detected virus neutralizing antibody titers in 64% of the COVID-19 patients at >300 days after infection, demonstrating durability of neutralizing antibody levels after infection. Importantly, full COVID-19 vaccination of these individuals resulted in higher antibody titers compared to fully vaccinated individuals in the absence of prior infection. These data demonstrate long-lived antibody-mediated immunity after SARS-CoV-2 infection, and a clear benefit of two vaccine doses for recovered individuals.

Background. qRT-PCR investigations on patient specimen seem to be the only probable laboratory technology to validate the current individual SARS-CoV-2 status correctly. This kind of nucleic acid amplification technology is available in most countries, but missing in resource limited areas with poor laboratory infrastructure. The most feasible way to build up PCR test capacities for SARS-CoV-2 in these areas is the use of conventional PCR. PCR-thermocycler and gel-electrophoresis systems could be shipped easily by parcel service to the whole world, because they are almost maintenance free and need no engineers for their installation and service. Methods. In order to reduce the complexity of sensitive reagent kits we have developed a freeze dried test kit for the traditional RT-PCR for the detection of SARS-CoV-2 in single ready to use tubes. Transport and storage of the reagents are now free of any cooling chain.Results. We have analysed 244 patient samples and compared the results to the SEEGENE Allplex™ 2019-nCoV assay. The sensitivity and specificity were 95.5% and 98.5%, respectively. Next to these performance parameters we could implement a workflow for processing 96 patient samples in parallel, handled by only 1 laboratory technician in an 8 hours working day.Conclusion. The use of conventional PCR in combination with gel-electrophoresis for test analysis is a feasible approach to make SARS-CoV-2 diagnostic available in the whole world. This is needed urgently, because SARS-CoV-2 is a question of global health and not only of the well-equipped countries.

BACKGROUNDBlood or plasma samples from rural areas are often transported under suboptimal conditions to central laboratories. The negative influence of different storage temperatures during transportation as well as long transportation times on the stability of unprotected HIV RNA is well known. Therefore, the correct and reliable quantification of HIV RNA might be very difficult. A stabilization solution for the storage and transportation of plasma samples was developed which stabilizes RNA for seven days up to 45°C without viral load changes.METHODSBlood samples from HIV positive individuals were collected into EDTA containing tubes. The isolated plasma samples in Germany were pipetted into pre-prepared RNA stabilization tubes and incubated for seven days at 45°C. HIV-1 RNA quantification was performed on a HIV-1 LCx m 2000 system from Abbott and a Qiagen/Artus HI Virus-1 RG RT-PCR Kit on a Rotor-Gene Q PCR machine. In addition, plasma samples were collected and tested using existing SOP for storage and transportation in Nigeria. Plasma samples were treated with and without stabilization solution and the AMPLICOR HIV-1 MONITORTM test was used to determine viral load.RESULTSSeventy-four stabilized plasma samples were tested in Germany and results were compared to those tested unprotected within two hours. No significant changes of viral load were detected up to seven days and 45°C in case of stabilized samples. In contrast RNA of the same unprotected samples was no longer detectable after one day at 45°C. Additionally, 22 plasma samples were investigated on day zero and under field conditions in Nigeria without changes of the viral load after seven days under given temperature conditions.CONCLUSIONSNo cooling chain is necessary for the storage and/or transportation of plasma samples treated with the new RNA stabilization solution for up to seven days. The use of this solution to preserve plasma RNA will be very helpful in countries where the environmental temperature is higher than 30°C, thus addressing the problem of unreliable viral load results due to suboptimal storage or transportation conditions. Further, the costs of storage and transportation of samples for viral load quantification could be significantly reduced.

BACKGROUND:A new screening method was developed for the detection and identification of methicillin resistant staphylococcus aureus (MRSA) from sterile sites or mixed flora samples (inguinal or nose swabs).METHODS:After rapid treatment of samples, the method consists of two steps, template DNA preparation by a simple and rapid boiling procedure and a multiplex real time PCR. The triplex PCR system simultaneously detects the following targets, (i) the integration site for the open reading frame X (orfX) of the staphylococcal cassette chromosome mec type I - V (SCCmec), (ii) the mecA gene which codes for the penicillin-binding protein PBP-2a, and (iii) an internal control (IC) which can be amplified with the SCCmec primer system. A new buffer system, which contains the fluorescent dye SYTO 9, allows a reproducible real time PCR for the discrimination of the above mentioned PCR products by means of a high resolution melting point analysis (HRM).RESULTS:This new PCR system distinguishes between MRSA, MSSA, and mecA positive but coagulase-negative staphylococci (CoNS) strains. An internal control confirms the integrity of the PCR run. The HRM shows three melting points specific for each amplification product. 78.75 degrees C for the mecA gene, 83.15 degrees C for the SCCmec/orfX fragment and 88.25 degrees C for the internal control.CONCLUSIONS:This new multilocus MRSA PCR system is a fast and inexpensive alternative to commercially available test systems and costs only five to six euros.

Proviral DNAs are being measured increasingly as a marker of the efficacy of highly active anti-retroviral therapy (HAART) and is accepted for the early diagnosis of perinatal HIV-1 infections. This requires a standardized test which enables the detection of a wide range of subtypes worldwide including O, N and circulating recombinant forms (CRFs). Based on a previous publication, a PCR - Test for HIV-1 provirus detection in peripheral blood mononuclear cells (PBMCs) was developed. Blood samples from 80 individuals infected with HIV-1 and 20 persons negative for HIV-1&2 from Africa and Germany were tested for the presence of HIV-1 provirus DNA. The primer system used enables the detection of proviral DNA despite the high concentrations of human DNA. The limit of detection was determined to be 5 copies per 10(5) cells. All 20 samples from persons negative for HIV were negative for HIV-1 proviral DNA while provirus DNA was amplified from 76 of the 80 (95%) samples from persons infected with HIV. The amplified products were detected by gel-electrophoresis, flow cytometry and real-time PCR. All three detection systems provided the same results.

Autoimmune thrombocytopenic purpura (AITP) is an acquired autoimmune bleeding disorder, characterized by isolated thrombocytopenia because of destruction of auto‐antibody‐coated platelets by Fc‐receptor‐mediated phagocytosis. The destruction of autoantibody‐sensitized platelets by FcγR‐bearing phagocytic cells and the following antigen presentation are considered to play a key role for the pathophysiology of AITP. Although different isotypes of AITP‐mediating autoantibodies, e.g. IgG, IgM and IgA, are frequently found in AITP patients, their role in the pathophysiology of AITP remains unclear. Using a flow cytometric monocyte‐based phagocytosis assay, we investigated the impact of disease‐associated autoantibody isotype in antibody‐mediated phagocytosis of platelets. Platelets, labelled with 5‐chloromethyl fluorescein diacetate (CMFDA), were incubated with AITP patients’ serum characterized by pure IgG or IgM antiplatelet autoantibodies. Labelled platelets were incubated with monocytes. Phagocytosis was defined as the product of percentage of CMFDA‐positive monocytes and mean fluorescence intensity of CMFDA. Adherence of platelets to monocytes was quantified by anti‐CD61‐PerCp in a CMFDA+ CD14+ gate. IgG‐coated platelets showed a significantly higher phagocytic index than IgM‐coated platelets (mean 796 ± 157 versus 539 ± 78, P < 0.01). There were no significant differences regarding platelet adherence to monocytes. The isotype of autoantibodies influences the quantity of in vitro phagocytosis of autologous platelets by monocytes. Therefore, the AITP‐mediating autoantibody isotype should be considered more carefully in pathophysiologic models and furthermore in diagnostic, therapeutic and prognostic approaches in AITP.

Die Transfusion von Blutkomponenten ist im Allgemeinen unbedenklich, es besteht aber ein geringes Risiko für unerwünschte Wirkungen, die immunologische, nichtimmunologische oder infektiologische Ursachen haben können. Obwohl bei Patienten die Angst vor Infektionsübertragungen überwiegt, ist dieses Risiko seit der Einführung verlässlicher serologischer und molekularbiologischer Tests extrem gering. Der vorliegende Beitrag diskutiert die Häufigkeit, die klinischen Bilder und die Ätiologie unerwünschter Wirkungen. Auf aktuelle Erkenntnisse bezüglich der in den letzten Jahren viel diskutierten transfusionsassoziierten akuten Lungeninsuffizienz (TRALI) wird eingegangen. Berücksichtigt sind neben Hepatitis- und HI-Viren auch das Zytomegalievirus, Parvovirus B19, Prionenübertragung und das Risiko der Variante der Creutzfeld-Jakob-Erkrankung.

While transfusion of blood components is usually safe, there are risks of adverse effects that can have immunologic, nonimmunologic, or infectious causes. In patients, the fear of infectious disease transmission predominates, although the risk has been extremely low since the introduction of reliable serologic and molecular biological testing methods. This article addresses the incidence, clinical picture, and etiology of adverse effects of transfusion. It also reports on current knowledge concerning transfusion-associated acute lung injury, which has gained much attention in the last few years. Besides hepatitis and human immunodeficiency viruses, cytomegalovirus, parvovirus B19, prion transmission, and the risk of variant Creutzfeld-Jakob disease are also discussed.

Most commercially available assays for diagnosis of HIV infection have shown shortcomings in the detection and quantification of rare genotypes of the virus. Most of the assays do not detect subtype O (outlier) and/or N (nonmajor, nonoutlier) or new circulating recombinant forms (CRFs), which are becoming more important in sub‐Saharan Africa. Furthermore, the commonly available tests require costly measuring devices and expensive test kits, which are not easily affordable for developing countries. This study was designed to explore solutions to the problem of viral load assays in developing countries. Two forward primers, digoxygenin (DIG) and dinitrophenol (DNP) labeled, and one biotin (BIO) labeled reverse primer were used to amplify both, the HIV‐1‐5′LTR (long terminal repeat) region and an internal standard sequence. The two polymerase chain reaction (PCR)‐products were captured by anti‐DIG and anti‐DNP antibody coated microparticles. Flow cytometric analyses were carried out after labeling with streptavidin‐R‐phycoerythrine. The primer system used recognized all HIV‐1 subtypes. A coamplified internal standard warranted the functionality of the PCR and allows reproducible viral load measurements. Two drawbacks of current viral load measurements are overcome by the flow cytometry based test described hereof. First, all known worldwide relevant HIV‐1 subtypes including subtypes O, N, and new CRFs are quantifiable with high sensitivity (50 to >1 × 106 copies per PCR). Second, the cost per test can be reduced to less than 12 US$ instead of the current 50–100 US$. Additionally, the test described in this report offers the possibility to perform complete monitoring program (CD4 T‐cell count, CD4% and viral load) for the first time, with the same device for HIV‐infected persons. © 2008 International Society for Advancement of Cytometry

Zusammenfassung Ein ständiger, sich beschleunigender Fortschritt in der hämatologischen Diagnostik führte von mehr philosophisch zu betrachtenden Denkansätzen im alten Griechenland zu den logisch geprägten Grundlagen in der Methodik der Untersuchung menschlichen Bluts in den letzten zwei Jahrhunderten. Das 20. Jahrhundert war vor dem Hintergrund industrieller Standardisierung das Zeitalter der Perfektionierung etablierter Verfahren in der Hämatologie, die von Menschen bedient wurden. Im 21. Jahrhundert wurden moderne Computer- und Netzwerktechniken in komplexe Gerätesysteme integriert, die untereinander mechanisch und logisch über algorithmisch geprägte Regelsysteme verbunden sind. Die Automation in der Hämatologie erlaubt heute, weitestgehend ohne menschliche Eingriffe, eine umfassende, hochpräzise Diagnostik, die am Beispiel von vier modernen Analysesystemen vorgestellt wird. Diese Diagnostik ist nicht nur in der Patientenversorgung von hoher Relevanz, sondern auch bei der Untersuchung von Blutspendern und Blutprodukten.

Although the pathogenesis of HIV infection is independent of age, specific considerations have to be taken into account for the treatment of infants, children and adolescents. In addition to more cautious drug therapy, it is proposed to follow up disease progression by repeated measurement of CD4 lymphocyte percentage (CD4%). Unfortunately, this requires complicated, expensive analytical techniques and complex sample preparation protocols. This is one of the reasons why treatment of the increasing number of children suffering from HIV/AIDS in developing countries remains a challenge. We evaluated a new and simplified no lyse no wash protocol to measure CD4% on a volumetric flow cytometer. Eight blood samples of healthy and HIV-infected individuals were sent to five German hospital laboratories (centres B-F) and were measured by their flow cytometric in-house techniques. The results were compared to those of centre A using the volumetric technique with the new sample preparation protocol. Comparative data with established protocols and flow cytometric techniques showed good correlations for the determination of leukocytes and for lymphocytes. Comparing CD4% results of centre A (new protocol and volumetric measuring technique) with the highest and lowest values of centres B-F by Bland-Altman analysis yielded a bias of -1.02% (SD 3.16) and 4.27% (SD 3.72) over the whole range (4.01%-70.56%), respectively. A comparably simple three-step protocol as introduced for absolute CD4+ cell count can be used to calculate CD4% only by the addition of two antibodies (CD4 and CD45). The cost per test is in the range of (sic)2.50 and therefore far below current prices of (sic)5.00-(sic)40.00.

Bacteria show differences in their growth kinetics depending on the type of blood component. On to storage at 22 degrees C, platelet concentrates (PCs) seem to be more prone to bacterial multiplication than red cell concentrates. Knowledge of the potential for bacterial proliferation in blood components, which are stored at a range of temperatures, is essential before considering implementation of a detection strategy. The efficacy of bacterial detection was determined, using real-time reverse transcriptase-polymerase chain reaction (RT-PCR), following bacterial growth in blood components obtained from a deliberately contaminated whole-blood (WB) unit. Cultivation was used as the reference method. WB was spiked with 2 colony-forming units mL(-1)Staphylococcus epidermidis or Klebsiella pneumoniae, kept for 15 h at room temperature and component preparation was processed. Samples were drawn, at intervals throughout the whole separation process, from each blood component. Nucleic acids were extracted using an automated high-volume extraction method. The 15-h storage revealed an insignificant increase in bacterial titre. No bacterial growth was detected in red blood cell or plasma units. K. pneumoniae showed rapid growth in the pooled PC and could be detected immediately after preparation using RT-PCR. S. epidermidis grew slowly and was detected 24 h after separation. These experiments show that sampling is indicative at 24 h after preparation of PCs at the earliest to minimize the sampling error.

The current issue of TRANSFUSION MEDICINE AND HEMOTHERAPY focuses on automation in transfusion medicine. Therefore, several reviews and articles in this issue describe historic and current developments in automation of diagnostics in different fields of transfusion medicine. In 1901, Karl Landsteiner’s elucidation of the ABO blood group system was published and established the basis for numberless discoveries of new human blood group antigens and antibodies during the past hundred years. The discovery and identification of human blood groups represented a milestone for a successful administration of compatible blood transfusions and therefore founded the introduction of immunohematology as new scientific subject. Immunohematology covers the analysis of blood group antigens and antibodies and their interactions in health and disease [1]. Nevertheless, for nearly the whole last century, the manual procedures were the only techniques for determination of blood groups and antibodies in the vast majority of laboratories. For many years, the manually performed tube tests were the only standard in immunohematologic laboratories for all routine and scientific questions. These tube tests were later followed and supplemented by more sensitive and specific manual methods, such as solid phase or gel centrifugation techniques. Only since the past few years, partly or fully automated systems for blood group typing and cross-matching were introduced in immunohematology to a greater extent. Nowadays, an increasing amount of immunohematologic laboratories uses automated systems for blood group typing and cross-matching in addition to the manual work strategies. Depending on the magnitude of blood donation centers and immunohematologic laboratories, a far developed automation in immunohematology is nowadays realized for many fields in daily routine work. In the current issue, the contributions of A. Dada et al. [2] and G. Wittmann et al. [3] reflect on the automation in immunohematology for compatibility testing and determination of blood groups using different systems of automation. Another important issue in transfusion medicine is the transmission of infections by transfusions. This problem may still be serious and sometimes causes severe or fatal courses. Although the frequencies of transfusion transmitted infections caused by known pathogens (such as HIV, HBV, HCV) distinctly decreased during the past years, the risks of transmission until now cannot totally be excluded. A high throughput of blood donors to be tested, the necessity to screen all donors for different infections, and always new pathogens require the continuous deployment of fast and reliable methods. For a safe exclusion of infectious diseases, automated serologic techniques as well as automated systems for genetic determinations of viral nucleic acids are established. The review of A. Schuller and W.D. Roth [4] gives a good overview on different automated systems for serological and genetic blood donor testings for infectious diseases. In addition, the correct quantification and characterization of blood cells in blood donors, blood components and transfused patients is of high relevance. Both the investigation of circulating cells in blood donors according to current guidelines and the quality control of blood products require efficient systems for determination of cell numbers and types. New hematology counters enable the contemporaneous determination of numerous cells and attributes. Therefore, many features of automated systems for diagnostics in hematology are not only relevant for clinicians but are also essential in transfusion medicine. The review of J. Lehner et al. [5] reflects the historic and current developments in automation of hematologic diagnostics that are nowadays supported and performed by modern and sophisticated hematology cell counters. In summary, the following reviews and articles focus on automated systems ensuring fast and reliable laboratory analyses. All laboratory units in the field of transfusion medicine need

With increasing self-driven momentum, hematological diagnosis has developed from rather philosophical approaches in ancient Greece to the application of logical principles to investigating human blood within the last two centuries. The 20th century was a time of industrial standardization and thus a period in which established procedures operated by humans were perfected in hematology. In the 21st century, modern computers and network techniques are being integrated into a complex instrument, linked to each other mechanically and logically by control systems based on algorithms. Automation in hematology now permits comprehensive and highly precise diagnosis, with hardly any human help. This will be illustrated by four modern analysis systems. These diagnostic tools are not only of high importance for the treatment of patients but also for the screening of blood donors and blood products.