Integration of ζ-deficient CARs into the CD3-zeta gene conveys potent cytotoxicity in T and NK cells.

Chimeric antigen receptor (CAR)-redirected immune cells hold significant therapeutic potential for oncology, autoimmune diseases, transplant medicine, and infections. All approved CAR-T therapies rely on personalized manufacturing using undirected viral gene transfer, which results in non-physiological regulation of CAR-signaling and limits their accessibility due to logistical challenges, high costs and biosafety requirements. Random gene transfer modalities pose a risk of malignant transformation by insertional mutagenesis. Here, we propose a novel approach utilizing CRISPR-Cas gene editing to redirect T-cells and natural killer (NK) cells with CARs. By transferring shorter, truncated CAR-transgenes lacking a main activation domain into the human CD3ζ (CD247) gene, functional CAR fusion-genes are generated that exploit the endogenous CD3ζ gene as the CAR's activation domain. Repurposing this T/NK-cell lineage gene facilitated physiological regulation of CAR-expression and redirection of various immune cell types, including conventional T-cells, TCRγ/δ T-cells, regulatory T-cells, and NK-cells. In T-cells, CD3ζ in-frame fusion eliminated TCR surface expression, reducing the risk of graft-versus-host disease in allogeneic off-the-shelf settings. CD3ζ-CD19-CAR-T-cells exhibited comparable leukemia control to T cell receptor alpha constant (TRAC)-replaced and lentivirus-transduced CAR-T-cells in vivo. Tuning of CD3ζ-CAR-expression levels significantly improved the in vivo efficacy. Notably, CD3ζ gene editing enabled redirection of NK-cells without impairing their canonical functions. Thus, CD3ζ gene editing is a promising platform for the development of allogeneic off-the-shelf cell therapies using redirected killer lymphocytes.

Integration of ζ-deficient CARs into the CD3-zeta gene conveys potent cytotoxicity in T and NK cells.

Chimeric antigen receptor (CAR)-reprogrammed immune cells hold significant therapeutic potential for oncology, autoimmune diseases, transplant medicine, and infections. All approved CAR-T therapies rely on personalized manufacturing using undirected viral gene transfer, which results in non-physiological regulation of CAR-signaling and limits their accessibility due to logistical challenges, high costs and biosafety requirements. Here, we propose a novel approach utilizing CRISPR-Cas gene editing to redirect T cells and natural killer (NK) cells with CARs. By transferring shorter, truncated CAR-transgenes lacking a main activation domain into the human CD3 ζ (CD247) gene, functional CAR fusion-genes are generated that exploit the endogenous CD3 ζ gene as the CAR's activation domain. Repurposing this T/NK-cell lineage gene facilitated physiological regulation of CAR-expression and reprogramming of various immune cell types, including conventional T cells, TCRγ/δ T cells, regulatory T cells, and NK cells. In T cells, CD3 ζ in-frame fusion eliminated TCR surface expression, reducing the risk of graft-versus-host disease in allogeneic off-the-shelf settings. CD3 ζ-CD19-CAR-T cells exhibited comparable leukemia control to T cell receptor alpha constant ( TRAC )-replaced and lentivirus-transduced CAR-T cells in vivo . Tuning of CD3 ζ-CAR-expression levels significantly improved the in vivo efficacy. Compared to TRAC -edited CAR-T cells, integration of a Her2-CAR into CD3 ζ conveyed similar in vitro tumor lysis but reduced susceptibility to activation-induced cell death and differentiation, presumably due to lower CAR-expression levels. Notably, CD3 ζ gene editing enabled reprogramming of NK cells without impairing their canonical functions. Thus, CD3 ζ gene editing is a promising platform for the development of allogeneic off-the-shelf cell therapies using redirected killer lymphocytes. Key points: Integration of ζ-deficient CARs into CD3 ζ gene allows generation of functional TCR-ablated CAR-T cells for allogeneic off-the-shelf use CD3 ζ-editing platform allows CAR reprogramming of NK cells without affecting their canonical functions.

Recognition mechanisms of hemoglobin particles by monocytes - CD163 may just be one.

Hemoglobin-based oxygen carriers (HBOCs) as blood substitutes are one of the great hopes of modern transfusion and emergency medicine. After the major safety-relevant challenges of the last decades seem to be largely overcome, current developments have in common that they are affected by degradation and excretion at an early stage in test organisms. Several possible mechanisms that may be responsible for this are discussed in the literature. One of them is CD163, the receptor of the complex of haptoglobin (Hp) and hemoglobin (Hb). The receptor has been shown in various studies to have a direct affinity for Hb in the absence of Hp. Thus, it seems reasonable that CD163 could possibly also bind Hb within HBOCs and cause phagocytosis of the particles. In this work we investigated the role of CD163 in the uptake of our hemoglobin sub-micron particles (HbMPs) in monocytes and additionally screened for alternative ways of particle recognition by monocytes. In our experiments, blockade of CD163 by specific monoclonal antibodies proved to partly inhibit HbMP uptake by monocytes. It appears, however, that several other phagocytosis pathways for HbMPs might exist, independent of CD163 and also Hb.

An Efficient and Scalable Method for the Production of Immunogenic SARS-CoV-2 Virus-like Particles (VLP) from a Mammalian Suspension Cell Line.

The rapid evolution of new SARS-CoV-2 variants poses a continuing threat to human health. Vaccination has become the primary therapeutic intervention. The goal of the current work was the construction of immunogenic virus-like particles (VLPs). Here, we describe a human cell line for cost-efficient and scalable production of immunogenic SARS-CoV-2 VLPs. The modular design of the VLP-production platform facilitates rapid adaptation to new variants. Methods: The N, M-, and E-protein genes were integrated into the genome of Expi293 cells (ExpiVLP_MEN). Subsequently, this cell line was further modified for the constitutive expression of the SARS-CoV-2 spike protein. The resulting cell line (ExpiVLP_SMEN) released SARS-CoV-2 VLP upon exposure to doxycycline. ExpiVLP_SMEN cells were readily adapted for VLP production in a 5 L bioreactor. Purified VLPs were quantified by Western blot, ELISA, and nanoparticle tracking analysis and visualized by electron microscopy. Immunogenicity was tested in mice. Results: The generated VLPs contained all four structural proteins, are within the size range of authentic SARS-CoV-2 virus particles, and reacted strongly and specifically with immunoserum from naturally infected individuals. The VLPs were stable in suspension at 4 °C for at least 10 weeks. Mice immunized with VLPs developed neutralizing antibodies against lentiviruses pseudotyped with the SARS-CoV-2 spike protein. The flexibility of the VLP-production platform was demonstrated by the rapid switch of the spike protein to a new variant of concern (BA.1/Omicron). The present study describes an efficient, scalable, and adaptable production method of immunogenic SARS-CoV-2 VLPs with therapeutic potential.

Combining different CRISPR nucleases for simultaneous knock-in and base editing prevents translocations in multiplex-edited CAR T cells.

BACKGROUND: Multiple genetic modifications may be required to develop potent off-the-shelf chimeric antigen receptor (CAR) T cell therapies. Conventional CRISPR-Cas nucleases install sequence-specific DNA double-strand breaks (DSBs), enabling gene knock-out or targeted transgene knock-in. However, simultaneous DSBs provoke a high rate of genomic rearrangements which may impede the safety of the edited cells. RESULTS: Here, we combine a non-viral CRISPR-Cas9 nuclease-assisted knock-in and Cas9-derived base editing technology for DSB free knock-outs within a single intervention. We demonstrate efficient insertion of a CAR into the T cell receptor alpha constant (TRAC) gene, along with two knock-outs that silence major histocompatibility complexes (MHC) class I and II expression. This approach reduces translocations to 1.4% of edited cells. Small insertions and deletions at the base editing target sites indicate guide RNA exchange between the editors. This is overcome by using CRISPR enzymes of distinct evolutionary origins. Combining Cas12a Ultra for CAR knock-in and a Cas9-derived base editor enables the efficient generation of triple-edited CAR T cells with a translocation frequency comparable to unedited T cells. Resulting TCR- and MHC-negative CAR T cells resist allogeneic T cell targeting in vitro. CONCLUSIONS: We outline a solution for non-viral CAR gene transfer and efficient gene silencing using different CRISPR enzymes for knock-in and base editing to prevent translocations. This single-step procedure may enable safer multiplex-edited cell products and demonstrates a path towards off-the-shelf CAR therapeutics.

Three different pathways of IgM-antibody-dependent hemolysis are mainly regulated by complement.

Antibodies to red blood cells (RBCs) may hemolyze erythrocytes via Fc-mediated phagocytosis or complement-dependent. Complement activation on RBCs can be detected by C3d-direct antiglobulin test (DAT), which is the only test in immune hematology that directly targets complement. However, a positive DAT with anti-C3d cannot distinguish between C3b-mediated extravascular hemolysis, C5b-C9-mediated intravascular hemolysis and C5b-C8-mediated eryptosis. Furthermore, DAT is not suitable to estimate the strength of hemolysis. Autoimmune hemolytic anemia (AIHA) is a rare disease that is caused by autoantibodies to red blood cells that is divided in warm AIHA and in cold agglutinin disease (CAD). The causative antibodies in CAD and sometimes in warm AIHA are from the IgM class. Depending on strength of complement activation they can induce extravascular hemolysis, intravascular hemolysis and eryptosis. We studied the three types of hemolysis by use of sera from patients with CAD under various conditions. We found that additionally to the routinely applied C3d-DAT, indirect tests for complement activity (free hemoglobin and Annexin V-binding to phosphatidylserine-exposing RBCs) should be used to determine the portion of extravascular, intravascular and eryptotic hemolysis. Eryptotic hemolysis may have a significant share in clinical relevant CAD or IgM warm AIHA, which should be considered for successful treatment.

Allografts: expanding the surgeon's armamentarium.

In Germany, bone allografts are widely used and their application in clinics has increased over the years. Successful use of allografts depends on many factors such as the procurement, processing, sterilization and the surgeon's surgical experience. Tissue banks have provided safe and sterile allografts for decades ranging from hard to soft tissue. Allografts are obtained from various tissues such as bone, tendon, amniotic membrane, meniscus and skin. An advantage of allografts is their wide applicability that has never been limited by indication restrictions thus providing a huge benefit for surgeon's. The use of the correct allograft in different indications is extremely important. Thereby surgeons have access to various allograft forms such as mineralized, demineralized, freeze-dried, paste, powder, chips strips and putty. The vast options of allografts allow surgeon's to use allografts in indications they deem fit. Currently, the application of allografts is at the discretion of the expert surgeon. However, regulations are often changed locally or internationally and may impact/limit allograft use to certain indications. Here, we report the different indications where our peracetic acid (PAA) sterilised bone allografts were used as well as general literature on bone allograft use in other indications.

SARS-CoV-2 Virus-like Particles (VLPs) Specifically Detect Humoral Immune Reactions in an ELISA-Based Platform.

A key in controlling the SARS-CoV-2 pandemic is the assessment of the immune status of the population. We explored the utility of SARS-CoV-2 virus-like particles (VLPs) as antigens to detect specific humoral immune reactions in an enzyme-linked immunosorbent assay (ELISA). For this purpose, SARS-CoV-2 VLPs were produced from an engineered cell line and characterized by Western blot, ELISA, and nanoparticle tracking analysis. Subsequently, we collected 42 serum samples from before the pandemic (2014), 89 samples from healthy subjects, and 38 samples from vaccinated subjects. Seventeen samples were collected less than three weeks after infection, and forty-four samples more than three weeks after infection. All serum samples were characterized for their reactivity with VLPs and the SARS-CoV-2 N- and S-protein. Finally, we compared the performance of the VLP-based ELISA with a certified in vitro diagnostic device (IVD). In the applied set of samples, we determined a sensitivity of 95.5% and a specificity of 100% for the certified IVD. There were seven samples with an uncertain outcome. Our VLP-ELISA demonstrated a superior performance, with a sensitivity of 97.5%, a specificity of 100%, and only three uncertain outcomes. This result warrants further research to develop a certified IVD based on SARS-CoV-2 VLPs as an antigen.

Adding a piece to the puzzle of Latin American blood donors and the potential risk of Trypanosoma cruzi transmission in Germany.

Introduction: Chagas disease (CD) is caused by the Trypanosoma cruzi (T. cruzi) infection and has become a global health concern due to population mobility, as well as non-vectorial transmission routes. Several countries outside Latin America (LA) have reported transfusion-associated transmission, but equivalent studies in Germany are lacking. This study aims to collect first data on the risk of transfusion associated transmission as well as LA blood donors originating from CD endemic countries in Germany. Materials and methods: A total of 305 blood donors who were assumed to be at risk for T. cruzi infection were retrospectively (267) as well as prospectively (38) selected at German blood donation sites in Bavaria and Berlin, and all retrospectively as well as 27 prospectively selected were serologically screened. Prospective study subjects additionally filled out a questionnaire. Results: All samples tested seronegative for T. cuzi specific antibodies. Prospectively enrolled study subjects all had high socio-economic status including good education. Knowledge regarding CD was limited but willingness to donate frequently was high. Blood donation rates from donors born in LA countries seem to increase from 2015. Discussion: Although no transfusion associated T. cruzi infection has been documented in Germany, it has likely already happened unnoticed, or will do in the near future. Performing risk-adapted serology-based blood donor screenings in Germany could avoid transfusion-associated transmission events as well as contribute to active case detection. Moreover, larger, and ongoing studies are needed to increase the evidence base as well as end the neglect of CD in Germany.

Pharmacological interventions enhance virus-free generation of TRAC-replaced CAR T cells.

Chimeric antigen receptor (CAR) redirected T cells are potent therapeutic options against hematological malignancies. The current dominant manufacturing approach for CAR T cells depends on retroviral transduction. With the advent of gene editing, insertion of a CD19-CAR into the T cell receptor (TCR) alpha constant (TRAC) locus using adeno-associated viruses for gene transfer was demonstrated, and these CD19-CAR T cells showed improved functionality over their retrovirally transduced counterparts. However, clinical-grade production of viruses is complex and associated with extensive costs. Here, we optimized a virus-free genome-editing method for efficient CAR insertion into the TRAC locus of primary human T cells via nuclease-assisted homology-directed repair (HDR) using CRISPR-Cas and double-stranded template DNA (dsDNA). We evaluated DNA-sensor inhibition and HDR enhancement as two pharmacological interventions to improve cell viability and relative CAR knockin rates, respectively. While the toxicity of transfected dsDNA was not fully prevented, the combination of both interventions significantly increased CAR knockin rates and CAR T cell yield. Resulting TRAC-replaced CD19-CAR T cells showed antigen-specific cytotoxicity and cytokine production in vitro and slowed leukemia progression in a xenograft mouse model. Amplicon sequencing did not reveal significant indel formation at potential off-target sites with or without exposure to DNA-repair-modulating small molecules. With TRAC-integrated CAR+ T cell frequencies exceeding 50%, this study opens new perspectives to exploit pharmacological interventions to improve non-viral gene editing in T cells.

SARS-CoV-2 T Cell Response in Severe and Fatal COVID-19 in Primary Antibody Deficiency Patients Without Specific Humoral Immunity.

Morbidity and mortality of COVID-19 is increased in patients with inborn errors of immunity (IEI). Age and comorbidities and also impaired type I interferon immunity were identified as relevant risk factors. In patients with primary antibody deficiency (PAD) and lack of specific humoral immune response to SARS-CoV-2, clinical disease outcome is very heterogeneous. Despite extensive clinical reports, underlying immunological mechanisms are poorly characterized and levels of T cellular and innate immunity in severe cases remain to be determined. In the present study, we report clinical and immunological findings of 5 PAD patients with severe and fatal COVID-19 and undetectable specific humoral immune response to SARS-CoV-2. Reactive T cells to SARS-CoV-2 spike (S) and nucleocapsid (NCAP) peptide pools were analyzed comparatively by flow cytometry in PAD patients, convalescents and naïve healthy individuals. All examined PAD patients developed a robust T cell response. The presence of polyfunctional cytokine producing activated CD4+ T cells indicates a memory-like phenotype. An analysis of innate immune response revealed elevated CD169 (SIGLEC1) expression on monocytes, a surrogate marker for type I interferon response, and presence of type I interferon autoantibodies was excluded. SARS-CoV-2 RNA was detectable in peripheral blood in three severe COVID-19 patients with PAD. Viral clearance in blood was observed after treatment with COVID-19 convalescent plasma/monoclonal antibody administration. However, prolonged mucosal viral shedding was observed in all patients (median 67 days) with maximum duration of 127 days. PAD patients without specific humoral SARS-CoV-2 immunity may suffer from severe or fatal COVID-19 despite robust T cell and normal innate immune response. Intensified monitoring for long persistence of SARS-CoV-2 viral shedding and (prophylactic) convalescent plasma/specific IgG as beneficial treatment option in severe cases with RNAemia should be considered in seronegative PAD patients.

Age of Red Cells for Transfusion and Outcomes in Patients with ARDS.

Packed red blood cells (PRBCs), stored for prolonged intervals, might contribute to adverse clinical outcomes in critically ill patients. In this study, short-term outcome after transfusion of PRBCs of two storage duration periods was analyzed in patients with Acute Respiratory Distress Syndrome (ARDS). Patients who received transfusions of PRBCs were identified from a cohort of 1044 ARDS patients. Patients were grouped according to the mean storage age of all transfused units. Patients transfused with PRBCs of a mean storage age ≤ 28 days were compared to patients transfused with PRBCs of a mean storage age > 28 days. The primary endpoint was 28-day mortality. Secondary endpoints included failure-free days composites. Two hundred and eighty-three patients were eligible for analysis. Patients in the short-term storage group had similar baseline characteristics and received a similar amount of PRBC units compared with patients in the long-term storage group (five units (IQR, 3-10) vs. four units (2-8), p = 0.14). The mean storage age in the short-term storage group was 20 (±5.4) days compared with 32 (±3.1) days in the long-term storage group (mean difference 12 days (95%-CI, 11-13)). There was no difference in 28-day mortality between the short-term storage group compared with the long-term storage group (hazard ratio, 1.36 (95%-CI, 0.84-2.21), p = 0.21). While there were no differences in ventilator-free, sedation-free, and vasopressor-free days composites, patients in the long-term storage group compared with patients in the short-term storage group had a 75% lower chance for successful weaning from renal replacement therapy (RRT) within 28 days after ARDS onset (subdistribution hazard ratio, 0.24 (95%-CI, 0.1-0.55), p < 0.001). Further analysis indicated that even a single PRBC unit stored for more than 28 days decreased the chance for successful weaning from RRT. Prolonged storage of PRBCs was not associated with a higher mortality in adults with ARDS. However, transfusion of long-term stored PRBCs was associated with prolonged dependence of RRT in critically ill patients with an ARDS.

Intraoperative blood loss as indicated by haemoglobin trend is a predictor for the development of postoperative spinal implant infection-a matched-pair analysis.

BACKGROUND: With a reported rate of 0.7-20%, postoperative spinal implant infection (PSII) is one of the most common complications after spine surgery. While in arthroplasty both haematoma formation and perioperative blood loss have been identified as risk factors for developing periprosthetic joint infections and preoperative anaemia has been associated with increased complication rates, literature on the aetiology of PSII remains limited. METHODS: We performed a matched-pair analysis of perioperative haemoglobin (Hb) and haematocrit (Hct) levels in aseptic and septic spine revision surgeries. 317 patients were included, 94 of which were classified as septic according to previously defined criteria. Patients were matched according to age, body mass index, diabetes, American Society of Anesthesiologists score and smoking habits. Descriptive summaries for septic and aseptic groups were analysed using Pearson chi-squared for categorical or Student t test for continuous variables. RESULTS: Fifty patients were matched and did not differ significantly in their reason for revision, mean length of hospital stay, blood transfusion, operating time, or number of levels operated on. While there was no significant difference in preoperative Hb or Hct levels, the mean difference between pre- and postoperative Hb was higher in the septic group (3.45 ± 1.25 vs. 2.82 ± 1.48 g/dL, p = 0.034). CONCLUSIONS: We therefore show that the intraoperative Hb-trend is a predictor for the development of PSII independent of the amount of blood transfusions, operation time, number of spinal levels operated on and hospital length of stay, which is why strategies to reduce intraoperative blood loss in spine surgery need to be further studied.

Validation of Microbiological Testing of Tissue Preparations with Different Incubation Temperatures.

INTRODUCTION: The European Pharmacopoeia (Ph. Eur.) provides principles for microbiological testing of tissue preparations. According to the Ph. Eur., tests should be performed at different temperatures for detection of aerobic bacteria and fungi (20-25°C) vs. anaerobic bacteria (30-35°C). Semiautomated systems using blood culture bottles are already widely used and they are adequate for growth detection. Resin-containing bottles and the addition of penicillinase permit testing of culture media containing antibiotics. MATERIALS AND METHODS: At 3 temperatures (21, 30, and 35°C) cornea culture media with and without dextran (CM II and CM I) and thermal disinfected femoral head medium (FH) were spiked with the 6 reference strains recommended by the Ph. Eur. (additionally: Enterococcus faecalis, Staphylococcus epidermidis, and Cutibacterium acnes). Microbial growth was monitored with the BACTECTM FX unit or visually at 21°C. RESULTS: Growth for all strains was detected with each medium at all 3 temperatures, except for C. acnes at 21°C (all media) and 30°C with FH. C. acnes had the highest times to detection, requiring test durations of 14 days. Microbial growth was faster at 30 and 35°C compared to 21°C. CONCLUSION: The requirements according to the Ph. Eur. for a successful method suitability test could be fulfilled for the semiautomated blood culture bottle system with the BACTECTM FX unit for the media and microorganisms used. In the presented validation study 35°C was shown to be the incubation temperature with the fastest growth, of the majority of the test strains used, and complete detection within 14 days.

Algorithms for the Testing of Tissue Donors for Human Immunodeficiency Virus, Hepatitis B Virus, and Hepatitis C Virus.

BACKGROUND: Although transmission of pathogenic viruses through human tissue grafts is rare, it is still one of the most serious dreaded risks of transplantation. Therefore, in addition to the detailed medical and social history, a comprehensive serologic and molecular screening of the tissue donors for relevant viral markers for human immunodeficiency virus (HIV), hepatitis B virus (HBV), and hepatitis C virus (HCV) is necessary. In the case of reactive results in particular, clear decisions regarding follow-up testing and the criteria for tissue release must be made. METHODS: Based on the clinical relevance of the specific virus markers, the sensitivity of the serological and molecular biological methods used and the application of inactivation methods, algorithms for tissue release are suggested. RESULTS: Compliance with the preanalytical requirements and assessment of a possible hemodilution are mandatory requirements before testing the blood samples. While HIV testing follows defined algorithms, the procedures for HBV and HCV diagnostics are under discussion. Screening and decisions for HBV are often not as simple, e.g., due to cases of occult HBV infection, false-positive anti-HBc results, or early window period positive HBV NAT results. In the case of HCV diagnostics, modern therapies with direct-acting antivirals, which are often associated with successful treatment of the infection, should be included in the decision. CONCLUSION: In HBV and HCV testing, a high-sensitivity virus genome test should play a central role in diagnostics, especially in the case of equivocal serology, and it should be the basis for the decision to release the tissue. The proposed test algorithms and decisions are also based on current European recommendations and standards for safety and quality assurance in tissue and cell banking.

Comparison of Total Immunoglobulin G in Ante- and Postmortem Blood Samples from Tissue Donors.

BACKGROUND: A serology testing for infectious diseases (HIV, hepatitis B and C, syphilis) is mandatory in tissue donors. In many donors postmortem blood is the only sample available. Even though serological tests and nucleic acid amplification tests (NAT) used are validated for postmortem blood, a characterization of those blood samples is not yet established. We therefore investigated the total immunoglobulin G (IgG) content in postmortem blood of tissue donors and compared it to a corresponding antemortem blood sample. METHODS: Ante- and postmortem blood samples were obtained from 100 consecutive tissue donors. The total IgG of all samples was measured using an immune-turbidometric test on the AU 480 Chemistry Analyzer (Beckman Coulter). RESULTS: The mean total IgG concentration of antemortem blood samples from all 100 donors was 8.9 g/L ± 3.7 g/L (median 8.9 g/L, range 1.5 to 23.8 g/L). In 80 donors the IgG concentration in the antemortem blood sample was within the normal range with values ≥6 g/L (mean 10.0 g/L ± 3.3 g/L, median 9.3 g/L,). The total IgG concentration of the postmortem blood samples was lower with 7.2 g/L ± 3.2 g/L (median 6.7 g/L, range 0.6 to 18.2 g/L). The difference between the values of the antemortem and postmortem blood samples was 1.7 g/L ± 2.6 g/L (16.3%) (median 1.6 g/L, range -7.7 to 10.1 g/L). In 36 donors this difference was less than 10%, in 23 it was between 10 and 25%, in 33 between 26 and 50%, and in 8 over 50%. In 57 donors the total IgG in the postmortem blood sample was within the normal range with ≥6 g/L, in 53 of them also the value of the antemortem blood sample was within the normal range. No correlation for total IgG was found regarding the donor characteristics (age, sex, disease) and the sample characteristics (hemolysis, postmortem time). CONCLUSION: Total IgG values in antemortem samples were below the lower limit of 6 g/L in 20% of the cases. Total IgG was significantly lower in the postmortem samples compared to the antemortem samples, while 57% were still above the lower limit. No correlation with the postmortem time could be found. This lowered IgG levels should be payed attention to when using postmortem blood for infectious serology testing. Additional NAT testing should be considered.

[A clinical radiological score for femoral head grafts : Establishment of the Tabea FK score to ensure the quality of human femoral head grafts]. / Ein klinisch-radiologischer Score für Femurkopftransplantate : Etablierung des Tabea-FK-Scores zur Sicherung der Qualität humaner Femurkopftransplantate.

INTRODUCTION: Transplantation of cancellous tissue from human femoral heads (FK) is an established method in the reconstruction of bony defects in orthopedic and trauma surgery. Standardized rating systems with respect to the morphological quality of this tissue are not available. MATERIALS AND METHODS: In 91/105 patients who had been a regular, clinically-indicated surgery (arthroplasty of the hip joint) the respective femoral head (FK) was taken under standardized conditions. Using a checklist defined clinical and radiological criteria of FK are judged in terms of their quality (cysts, necrosis, calcification, deformities, osteoporosis) and divided by the Tabea FK score into three classes (best/middle/poor quality). This was followed by a blinded repeated scoring, now as macroscopic assessment of three sawed layers from the same femoral head. The femoral heads are examined by peripheral quantitative computed tomography (pQCT) and a standardized histological examination of the bony tissue. We evaluated the accordance of the Tabea FK score with complementary assessments by calculation of sensitivity and specificity. RESULTS: Femoral heads from 91/105 patients (ages: 68.4 ± 9.9 , n = 60 women, n = 31 men) were explanted and included in the study. The correlation between the primary radiologic clinical score (Tabea FK score) and the macroscopic second review of the sawn FK with respect to middle/best and poor/middle quality was classified as good (sensitivity 77% and 81%, respectively; specificity 76% and 84%, respectively). The correlation of histology and macroscopic second review was worse and in relation to discrimination of middle/best and poor/middle quality had a sensitivity of 85% and 54%, respectively, and a specificity of 66% and 97%, respectively. The pQCT showed a sensitivity of 82% only in discrimination of middle/best, while sensitivity in discrimination of poor/middle and poor/middle + best, respectively, was <10%. DISCUSSION: The corresponding correlation between the primary and the second clinical score was evaluated as good. This emphasizes the long-standing skills of operationally active orthopedic surgeons to classify the quality of cancellous bone correctly already on the basis of X­ray images and intraoperative findings. In this respect, the introduction of the Tabea FK score as a quality assurance tool in the routines of bone banks can be recommended.