Impact of age and gender on lymphocyte subset counts in patients with COVID-19.

In symptomatic patients with acute Coronavirus disease 2019 (COVID-19), lymphocytopenia is one of the most prominent laboratory findings. However, to date age and gender have not been considered in assessment of COVID-19-related cell count alterations. In this study, the impact of COVID-19 as well as age and gender on a large variety of lymphocyte subsets was analyzed in 33 COVID-19 patients and compared with cell counts in 50 healthy humans. We confirm that cell counts of total lymphocytes, B, NK, cytotoxic and helper T cells are reduced in patients with severe COVID-19, and this tendency was observed in patients with moderate COVID-19. Decreased cell counts were also found in all subsets of these cell types, except for CD4+ and CD8+ effector memory RA+ (EMRA) and terminal effector CD8+ cells. In multivariate analysis however, we show that in addition to COVID-19, there is an age-dependent reduction of total, central memory (CM), and early CD8+ cell subsets, as well as naïve, CM, and regulatory CD4+ cell subsets. Remarkably, reduced naïve CD8+ cell counts could be attributed to age alone, and not to COVID-19. By contrast, decreases in other subsets could be largely attributed to COVID-19, and only partly to age. In addition to COVID-19, male gender was a major factor influencing lower counts of CD3+ and CD4+ lymphocyte numbers. Our study confirms that cell counts of lymphocytes and their subsets are reduced in patients with COVID-19, but that age and gender must be considered when interpreting the altered cell counts.

Influence of Turkish origin on hematology reference intervals in the German population.

Reference intervals for laboratory test results have to be appropriate for the population in which they are used to be clinically useful. While sex and age are established partitioning criteria, patients' origin also influences laboratory test results, but is not commonly considered when creating or applying reference intervals. In the German population, stratification for ethnicity is rarely performed, and no ethnicity-specific hematology reference intervals have been reported yet. In this retrospective study, we investigated whether specific reference intervals are warranted for the numerically largest group of non-German descent, individuals originating from Turkey. To this end, we analyzed 1,314,754 test results from 167,294 patients from six German centers. Using a name-based algorithm, 1.9% of patients were identified as originating from Turkey, in line with census data and the algorithm's sensitivity. Reference intervals and their confidence intervals were calculated using an indirect data mining approach, and Turkish and non-Turkish reference limits overlapped completely or partially in nearly all analytes, regardless of age and sex, and only 5/144 (3.5%) subgroups' reference limits showed no overlap. We therefore conclude that the current practice of using common reference intervals is appropriate and allows correct clinical decision-making in patients originating from Turkey.

One Year after Mild COVID-19: The Majority of Patients Maintain Specific Immunity, But One in Four Still Suffer from Long-Term Symptoms.

After COVID-19, some patients develop long-term symptoms. Whether such symptoms correlate with immune responses, and how long immunity persists, is not yet clear. This study focused on mild COVID-19 and investigated correlations of immunity with persistent symptoms and immune longevity. Persistent complications, including headache, concentration difficulties and loss of smell/taste, were reported by 51 of 83 (61%) participants and decreased over time to 28% one year after COVID-19. Specific IgA and IgG antibodies were detectable in 78% and 66% of participants, respectively, at a 12-month follow-up. Median antibody levels decreased by approximately 50% within the first 6 months but remained stable up to 12 months. Neutralizing antibodies could be found in 50% of participants; specific INFgamma-producing T-cells were present in two thirds one year after COVID-19. Activation-induced marker assays identified specific T-helper cells and central memory T-cells in 80% of participants at a 12-month follow-up. In correlative analyses, older age and a longer duration of the acute phase of COVID-19 were associated with higher humoral and T-cell responses. A weak correlation between long-term loss of taste/smell and low IgA levels was found at early time points. These data indicate a long-lasting immunological memory against SARS-CoV-2 after mild COVID-19.

Sustained cellular immunity in adults recovered from mild COVID-19.

Transient lymphocytopenia is frequently observed in acute phase of coronavirus disease 2019 (COVID-19). It remains a concern whether impairment of cellular immunity may be retained after COVID-19. Here, we demonstrate by extensive lymphocyte profiling in 44 adults after mild COVID-19 that cellular immunity is not fundamentally altered in convalescent patients. Except for increased activated CD8+ lymphocytes, total counts of B, T, and NK cells and their subsets did not differ significantly between patients after COVID-19 and healthy controls after a median of 27 days (range 13-45) suggesting no residual cellular immune deficiency after recovery from mild COVID-19.

Approximation of emicizumab plasma levels in emergency situations. A practical approach.

INTRODUCTION: A dedicated emicizumab assay based on the modified one-stage factor VIII (FVIII) assay (mOSA) is mainly available in haemophilia treatment centres (HTC). A method to estimate emicizumab plasma levels based on a widely available assay would be desirable, especially for emergency situations. AIM: A method for emicizumab plasma level approximation (ELA) using a routine FVIII activity measurement with standard one-stage assay (sOSA) was developed and evaluated. METHOD: Within this pilot study, 59 samples from patients with severe haemophilia A with (n = 8) and without (n = 8) inhibitors under emicizumab treatment were analysed using sOSA following a manual 1:8 sample pre-test dilution with saline. The sOSA was determined in two different laboratories, using two different analyser platforms each. RESULTS: The results demonstrated an excellent correlation of approximated emicizumab plasma levels (ELA) with the emicizumab plasma concentration determined with mOSA (r > .9; p < .05). The ELA showed a sensitivity of 93.3% and a specificity of 89.6% to predict a pre-defined cut-off-value of ≤30 µg/ml for the discrimination between subtherapeutic and therapeutic emicizumab plasma levels. CONCLUSION: Approximation of emicizumab levels by standard one-stage FVIII assay discriminates between subtherapeutic and therapeutic emicizumab levels and might facilitate clinical decision-making in emergency situations, such as bleeding, trauma or urgent surgery in case that dedicated emicizumab assays are not available.

Next-generation reference intervals for pediatric hematology.

Background Interpreting hematology analytes in children is challenging due to the extensive changes in hematopoiesis that accompany physiological development and lead to pronounced sex- and age-specific dynamics. Continuous percentile charts from birth to adulthood allow accurate consideration of these dynamics. However, the ethical and practical challenges unique to pediatric reference intervals have restricted the creation of such percentile charts, and limitations in current approaches to laboratory test result displays restrict their use when guiding clinical decisions. Methods We employed an improved data-driven approach to create percentile charts from laboratory data collected during patient care in 10 German centers (9,576,910 samples from 358,292 patients, 412,905-1,278,987 samples per analyte). We demonstrate visualization of hematology test results using percentile charts and z-scores (www.pedref.org/hematology) and assess the potential of percentiles and z-scores to support diagnosis of different hematological diseases. Results We created percentile charts for hemoglobin, hematocrit, red cell indices, red cell count, red cell distribution width, white cell count and platelet count in girls and boys from birth to 18 years of age. Comparison of pediatricians evaluating complex clinical scenarios using percentile charts versus conventional/tabular representations shows that percentile charts can enhance physician assessment in selected example cases. Age-specific percentiles and z-scores, compared with absolute test results, improve the identification of children with blood count abnormalities and the discrimination between different hematological diseases. Conclusions The provided reference intervals enable precise assessment of pediatric hematology test results. Representation of test results using percentiles and z-scores facilitates their interpretation and demonstrates the potential of digital approaches to improve clinical decision-making.

Development of the competency-based medical curriculum for the new Augsburg University Medical School.

Aim: With the resolution from April 28, 2014, the Bavarian state government in Germany decided to found a new medical school at Augsburg University, thereby requiring the development of a competency-based medical curriculum. Methods: Two interdisciplinary groups developed a spiral curriculum (following Harden) employing the model of Thumser-Dauth & Öchsner. The curriculum focuses on specifically defined competencies: medical expertise, independent scientific reasoning, argumentation and scholarship, as well as communication skills. Results: The spiral curriculum was developed as a hybrid curriculum. Its modular structure incorporates the mandatory subjects required by the German regulations for medical licensure (Approbationsordnung) into organ- and system-centered blocks which are integrated both horizontally and vertically. Basic preclinical sciences are covered in the blocks "Movement," "Balance" and "Contact." The clinical sciences are organized according to six pillars (conservative medicine, surgical medicine, men's-women's-children's medicine, the senses, the nervous system and the mind, and general medicine) which students revisit three times each over the course of the program. A longitudinal clinical course incorporates interdisciplinary education. A particular focus is on scientific education encompassing a longitudinal course in the sciences (including interdisciplinary classes with other university departments), block practicums, and two scientific projects. Conclusion: It is not only the degree of integration und intensity of the Augsburg University undergraduate medical degree program, but also its targeted advancement of academic, social and communication skills that have not yet been realized to such an extent elsewhere in Germany. On July 8, 2016, the German Council of Science and Humanities unanimously gave this concept a positive evaluation. Future research will examine and evaluate the Augsburg medical curriculum and the impact of the new medical school on the hospital and university in Augsburg.

Adjuvant therapeutic vaccination in patients with non-small cell lung cancer made lymphopenic and reconstituted with autologous PBMC: first clinical experience and evidence of an immune response.

BACKGROUND: Given the considerable toxicity and modest benefit of adjuvant chemotherapy for non-small cell lung cancer (NSCLC), there is clearly a need for new treatment modalities in the adjuvant setting. Active specific immunotherapy may represent such an option. However, clinical responses have been rare so far. Manipulating the host by inducing lymphopenia before vaccination resulted in a magnification of the immune response in the preclinical setting. To evaluate feasibility and safety of an irradiated, autologous tumor cell vaccine given following induction of lymphopenia by chemotherapy and reinfusion of autologous peripheral blood mononuclear cells (PBMC), we are currently conducting a pilot-phase I clinical trial in patients with NSCLC following surgical resection. This paper reports on the first clinical experience and evidence of an immune response in patients suffering from NSCLC. METHODS: NSCLC patients stages I-IIIA are recruited. Vaccines are generated from their resected lung specimens. Patients undergo leukapheresis to harvest their PBMC prior to or following the surgical procedure. Furthermore, patients receive preparative chemotherapy (cyclophosphamide 350 mg/m2 and fludarabine 20 mg/m2 on 3 consecutive days) for induction of lymphopenia followed by reconstitution with their autologous PBMC. Vaccines are administered intradermally on day 1 following reconstitution and every two weeks for a total of up to five vaccinations. Granulocyte-macrophage-colony-stimulating-factor (GM-CSF) is given continuously (at a rate of 50 microg/24 h) at the site of vaccination via minipump for six consecutive days after each vaccination. RESULTS: To date, vaccines were successfully manufactured for 4 of 4 patients. The most common toxicities were local injection-site reactions and mild constitutional symptoms. Immune responses to chemotherapy, reconstitution and vaccination are measured by vaccine site and delayed type hypersensitivity (DTH) skin reactions. One patient developed positive DTH skin tests so far. Immunohistochemical assessment of punch biopsies taken at the local vaccine site reaction revealed a dense lymphocyte infiltrate. Further immunohistochemical differentiation showed that CD1a+ cells had been attracted to the vaccine site as well as predominantly CD4+ lymphocytes. The 3-day combination chemotherapy consisting of cyclophosphamide and fludarabine induced a profound lymphopenia in all patients. Sequential FACS analysis revealed that different T cell subsets (CD4, CD8, CD4CD25) as well as granulocytes, B cells and NK cells were significantly reduced. Here, we report on clinical safety and feasibility of this vaccination approach during lymphoid recovery and demonstrate a patient example. CONCLUSION: Thus far, all vaccines were well tolerated. The overall trial design seems safe and feasible. Vaccine site reactions associated with infusion of GM-CSF via mini-pump are consistent with the postulated mechanism of action. More detailed immune-monitoring is required to evaluate a potential systemic immune response. Further studies to exploit homeostasis-driven T cell proliferation for the induction of a specific anti-tumor immune response in this clinical setting are warranted.

Immunotherapy of lung cancer: an update.

In Germany lung cancer is the leading cause of cancer-associated death in men. Surgery, chemotherapy and radiation may enhance survival of patients suffering from lung cancer but the enhancement is typically transient and mostly absent with advanced disease; eventually more than 90% of lung cancer patients will die of disease. New approaches to the treatment of lung cancer are urgently needed. Immunotherapy may represent one new approach with low toxicity and high specificity but implementation has been a challenge because of the poor antigenic characterization of these tumors and their ability to escape immune responses. Several different immunotherapeutic treatment strategies have been developed. This review examines the current state of development and recent advances with respect to non-specific immune stimulation, cellular immunotherapy (specific and non-specific), therapeutic cancer vaccines and gene therapy for lung cancer. The focus is primarily placed on immunotherapeutic cancer treatments that are already in clinical trial or well progressed in preclinical studies. Although there seems to be a promising future for immunotherapy in lung cancer, presently there is not standard immunotherapy available for clinical routine.