A review of analytical parameters in 'rapid' liquid chromatographic methods for bioanalysis: Can we do better?

Rapid bioanalysis is beneficial to many applications. However, how 'rapid' a method is, or could be, is often an unanswered question. In this statistical review, the authors have assessed multiple pre-analytical (i.e. sample preparation), and analytical method parameters specifically for liquid chromatography to assist researchers in developing and validating 'rapid' bioanalytical methods. We restricted the search to urine and plasma matrices only. Data were extracted from over 2,000 recent studies and evaluated to assess how these parameters affected the 'on-instrument' analysis time. In addition to methods using ultra-violet (UV) detection, there were a large number of mass spectrometric (MS) methods, allowing additional review of the differences between high- and low-resolution MS on analysis time. We observed that most (N = 922, 70 %) methods used 5 or 10 cm columns, and that whilst uptake of ultra-high performance (U)HPLC columns was good, the use of sub-5 cm columns and/or flow rates in excess of 1 mL/min was incredibly rare (N = 25, 3 %). The detector of choice for quantitative (U)HPLC-MS remains the triple quadrupole, although a number of groups report the use of high-resolution MS for such methods.

Predictive factors for docking site procedure in bone transport for large lower extremity segmental defects.

BACKGROUND: Segmental bone transport is a common technique for treating large segmental bone defects. However, a docking site procedure is often necessary in segmental bone transport. To date, no prognostic factors for the need of docking site procedure have been reported. Thus, the decision is often made at random, based on the surgeon's subjective judgment and experience. The aim of this study was to identify prognostic factors for the need of docking site operation. METHODS: Patients with segmental bone transport in lower extremity bone defects were included regardless of age, aetiology, and defect size. We excluded patients undergoing treatments that were not yet completed, and those who discontinued therapy by any reason. The need for docking site operation was modelled with logistical and linear regression as well as univariate analysis of variances (ANOVA). Receiver operating characteristics (ROC) curve analysis was also performed. RESULTS: Twenty-seven patients from age 12 to 74 years (mean age: 39.07 ± 18.20 years) were included. The mean defect size was 76.39 ± 41.10 mm. The duration of transport (days) showed a significant influence (p = 0.049, 95%CI: 1.00-1.02) on the need for docking site operation. No other significant influences were detected. CONCLUSION: A link between the duration of transport and the need for docking site operation was detected. Our data showed that if a threshold of about 188 days is exceeded, docking surgery should be considered.

Adverse impact of a high CD4/CD8 ratio in the allograft may be overcome by methotrexate- but not mycophenolate- or post-transplant cyclophosphamide-based graft versus host disease prophylaxis.

INTRODUCTION: A high CD4/CD8 T cell ratio in hematopoietic stem cell transplant (HSCT) allografts was observed to predict graft-versus-host disease (GVHD) and nonrelapse mortality (NRM) but has not been comparatively examined in settings of various GVHD-prophylaxis regimens. METHODS: This retrospective monocentric study included all consecutive HSCT performed with peripheral blood stem cells between January 2000 and June 2021. The impact of the graft CD4/CD8 ratio was analyzed in three cohorts with different GVHD-prophylaxis platforms. RESULTS: In the cyclosporine/mycophenolate-mofetil (CSA/MMF) cohort (n = 294, HLA-matched HSCT), a high (>75th percentile) CD4/CD8 ratio was associated with increased overall mortality (HR: 1.56; p = .01), increased NRM (HR: 1.85; p = .01) and GVHD-associated mortality (HR: 2.13; p = .005). In the post-transplant cyclophosphamide (PTCy)/tacrolimus/MMF cohort (n = 113, haploidentical-related or mismatched-unrelated HSCT), a high CD4/CD8 ratio was associated with increased overall mortality (HR 2.07; p = .04) and aGVHD3-4 (HR: 2.24; p = .02). By contrast, in the CSA/methotrexate (CSA/MTX) cohort (n = 185, HLA-matched HSCT) the CD4/CD8 ratio had no significant impact on any of the investigated endpoints. CONCLUSION: A high CD4/CD8 ratio in the allograft has an adverse impact on GVHD and survival in CSA/MMF- and PTCy-based HSCT, while MTX-based prophylaxis may largely alleviate this important risk factor.

[Interdisciplinary extremity board in the treatment of complex injuries]. / Interdisziplinäres Extremitäten-Board in der Behandlung von Komplexverletzungen.

BACKGROUND: Interdisciplinary case conferences are well-established in the field of oncology in order to provide the best possible treatment for patients with complex disease patterns which overlap several disciplines. METHODS: After studying the available literature the aims, indications, frequency, patient enrolment and documentation modalities, disciplines necessary to create the reconstruction plan and evaluation parameters of the board, were agreed in an interdisciplinary discussion among colleagues. The utilization of the extremity board and demographic features of the cases presented in the extremity board within the first 6 months were subsequently descriptively analyzed. RESULTS: The agreed primary aim of an extremity board is the timely and transparent preparation of a high-quality holistic reconstruction plan for optimized treatment of a challenging patient collective with complex injuries of the extremities. Decisive interfaces of an extremity board are the participation of interdisciplinary disciplines, established enrolment and documentation modalities and a longitudinal analysis of parameters of the acute medical treatment, the long-term function and quality of life of those affected. The patient collective so far mostly includes men under 40 years old with traumatic soft tissue defects and combined injuries. On average, reconstruction plans for 1-2 patients were approved in an interdisciplinary team per session. CONCLUSION: The extremity board serves as a platform for coordinated planning of treatment for patients with complex injuries. The limited personnel and time resources represent the greatest challenge for the successful implementation. The extremity board enables a high degree of interdisciplinary networking. The digital registration and documentation modality within the internal hospital documentation system is of utmost relevance. The preparation of the reconstruction plan is of decisive importance for the qualitative success of treatment and the restoration of function. The longitudinal analysis of appropriate parameters is imperative to measure the quality of treatment.

Training prediction models for individual risk assessment of postoperative complications after surgery for colorectal cancer.

BACKGROUND: The occurrence of postoperative complications and anastomotic leakage are major drivers of mortality in the immediate phase after colorectal cancer surgery. We trained prediction models for calculating patients' individual risk of complications based only on preoperatively available data in a multidisciplinary team setting. Knowing prior to surgery the probability of developing a complication could aid in improving informed decision-making by surgeon and patient and individualize surgical treatment trajectories. METHODS: All patients over 18 years of age undergoing any resection for colorectal cancer between January 1, 2014 and December 31, 2019 from the nationwide Danish Colorectal Cancer Group database were included. Data from the database were converted into Observational Medical Outcomes Partnership Common Data Model maintained by the Observation Health Data Science and Informatics initiative. Multiple machine learning models were trained to predict postoperative complications of Clavien-Dindo grade ≥ 3B and anastomotic leakage within 30 days after surgery. RESULTS: Between 2014 and 2019, 23,907 patients underwent resection for colorectal cancer in Denmark. A Clavien-Dindo complication grade ≥ 3B occurred in 2,958 patients (12.4%). Of 17,190 patients that received an anastomosis, 929 experienced anastomotic leakage (5.4%). Among the compared machine learning models, Lasso Logistic Regression performed best. The predictive model for complications had an area under the receiver operating characteristic curve (AUROC) of 0.704 (95%CI 0.683-0.724) and an AUROC of 0.690 (95%CI 0.655-0.724) for anastomotic leakage. CONCLUSIONS: The prediction of postoperative complications based only on preoperative variables using a national quality assurance colorectal cancer database shows promise for calculating patient's individual risk. Future work will focus on assessing the value of adding laboratory parameters and drug exposure as candidate predictors. Furthermore, we plan to assess the external validity of our proposed model.

[3D printing in the field of shoulder surgery]. / 3D-Druck im Bereich der Schulterchirurgie.

The 3D printing technology is a relatively new procedure with a high potential, especially in the field of shoulder surgery. The 3D printing procedures are increasingly being developed and also gaining new users. Principally, 3D printing procedures can be applied preoperatively in planning the surgical procedure, patient clarification and in teaching; however, the technology is increasing being used intraoperatively. In addition to intraoperative visualization of the models, 3D printing permits the use of individual and specific instruments and implants. This allows the precise transfer of the preoperative planning to the surgical procedure. Inaccuracies are mainly caused by soft tissues. The 3D printing can be beneficial in the fields of arthroplasty, shoulder instability as well as orthopedic trauma. The literature shows promising results in relation to duration of surgery, blood loss and clinical results of the procedure. On the other hand, it is still unclear which indications warrant the use of 3D printing. Other aspects that raise questions are the time of planning, the production time and the additional cost that the use of 3D printing entails. Nonetheless, 3D printing represents a meaningful enhancement of the portfolio of surgeons, which becomes highly beneficial and useful in complex situations. Furthermore, this procedure enables a certain amount of flexibility when reacting to certain circumstances.

Lesion topography of posterior cerebral artery infarcts.

This study analyzed the topography of acute ischemic stroke in the posterior cerebral artery (PCA) territory. We studied 84 patients with unilateral ischemic PCA stroke. Patients were classified according to lesion levels as cortico-subcortical (superficial), combined (cortical and mesodiencephalic) or isolated thalamic. To receive a lesion map, data from acute MR and CT imaging were normalized and labelled automatically by mapping to stereotaxic anatomical atlases. Cortical lesions accounted for 41.7%, combined for 36.9%, and isolated thalamic lesions for 21.4%. The maximum overlay of ischemia and, thus, highest occurrence of PCA ischemic stroke was found in the ventral and medial occipito-temporal cortex and adjacent white matter association tracts. Dorsal and peripheral segments of the occipito-temporo-parietal region were only rarely lesioned. This configuration was similar in both hemispheres. Consistent with this lesion pattern, visual field defects (VFD) were the most frequent signs, followed by sensorimotor signs, dizziness and sopor, cognitive and oculomotor deficits, and ataxia. The three vascular subgroups differed not only by their anatomical lesion profile and lesion load, but also by their clinical manifestation; although patients with combined and thalamic lesions were sigificantly younger, they were more disabled than participants with cortical lesions. VFD were only found in cortical and combined, and oculomotor deficits only in mesodiencephalic lesions. White matter lesions were common in the cortico-subcortical and the combined group. Basal occipito-temporal and calcarine regions, and neighbouring white matter tracts have the highest risk of ischemia in acute PCA stroke.

On the convergence of projective-simulation-based reinforcement learning in Markov decision processes.

In recent years, the interest in leveraging quantum effects for enhancing machine learning tasks has significantly increased. Many algorithms speeding up supervised and unsupervised learning were established. The first framework in which ways to exploit quantum resources specifically for the broader context of reinforcement learning were found is projective simulation. Projective simulation presents an agent-based reinforcement learning approach designed in a manner which may support quantum walk-based speedups. Although classical variants of projective simulation have been benchmarked against common reinforcement learning algorithms, very few formal theoretical analyses have been provided for its performance in standard learning scenarios. In this paper, we provide a detailed formal discussion of the properties of this model. Specifically, we prove that one version of the projective simulation model, understood as a reinforcement learning approach, converges to optimal behavior in a large class of Markov decision processes. This proof shows that a physically inspired approach to reinforcement learning can guarantee to converge.

Diagnostic value of whole-body-focused ultrasonography in high-acuity patients in the emergency department: a prospective single-center cross-sectional study.

BACKGROUND: A fast and diagnostic accurate tool to assess the unselected category of high-acuity patients, in whom the underlying pathology is not always obvious, is needed in the emergency departments (ED). We aim to describe the feasibility, validity and diagnostic yield of a routine whole-body-focused ultrasonography (wbf-us) in an unselected group of high-acuity ED patients. METHODS: In a prospective observational study, a convenience sample of ED patients (≥ 18 years) with a high-acuity score or systolic blood pressure < 100 mmHg received a routine wbf-us of the heart, lungs, abdomen and deep veins by two non-expert sonographers. Final diagnosis was established by experienced auditors. Investigators were blinded to the patients' medical history and emergency physicians and auditors were blinded to the investigators assessments. Diagnostic accuracy was assessed by comparing the investigators' ultrasonography findings to a structured double-blinded clinical audit of patient files. RESULTS: We included 171 patients, initiated a whole-body-focused ultrasonography examination (wbf-us) in 160 and completed it in 128 patients with an average time of a full examination of 28 min. We found pathology in 65/171 (38%) of the patients whose most frequent symptoms upon arrival were cardiopulmonary. Among the patients who received wbf-us, we found the majority of pathology by wbf-us of the lungs (n = 50, 31%), the heart (n = 26, 16%), few in the abdomen (n = 5, 3%) and none in the deep veins. The overall sensitivity was 50-100%, specificity 84-94%, positive predictive value 11-44% and negative predictive value 94-100%. CONCLUSION: Focused cardiopulmonary ultrasonography might be considered for routine use in high-acuity ED patients with cardiopulmonary symptoms whereas focused ultrasonography of the abdomen and deep veins performed by non-expert sonographers only seems indicated in selected patients. Trial registration Danish Data Protection Agency (ID 13/12076). Committee on Biomedical Research Ethics for the Region of Southern Denmark (ID S-20130047).

Accidental hypothermia as an independent risk factor of poor neurological outcome in older multiply injured patients with severe traumatic brain injury: a matched pair analysis.

PURPOSE: Patients with multiple injuries are particularly susceptible to accidental hypothermia which is correlated with an increased risk of post-traumatic complications and mortality; however, its impact on neurological outcome in cases where there is concomitant traumatic brain injury is underexplored. METHODS: We analyzed severely injured patients (ISS ≥ 16) including a moderate-to-severe traumatic brain injury (AISHead ≥ 3). The primary endpoint was objective neurological recovery, expressed as Glasgow Outcome Scale (GOS) score at time of discharge. Secondary endpoints were mortality, systemic inflammatory response syndrome (SIRS), sepsis, acute respiratory distress syndrome (ARDS) and multiple organ dysfunction syndrome (MODS). Statistical analysis included logistic regression (odds ratio). The significance level in all analyses was p = 0.05. RESULTS: We analyzed 278 patients (M age = 43 years, SD 19; M ISS = 32.8, SD 10.7). Mortality was 17% (n = 14). 102 patients (37%) were hypothermic on admission. Hypothermic patients were more severely injured (ISS 35.6 ± 11.1 vs. 31.2 ± 10.1, p = 0.001; APACHE II 18.1 ± 7.4 vs. 16.2 ± 7.3, p = 0.045) and had a higher transfusion requirement. Mortality rate in hypothermic patients was increased (23.5 vs. 13.1%, p = 0.03); however, hypothermia was not an independent predictor of mortality. Median GOS at discharge was 3 (IQR 3); in 47% of patients the outcome was favorable (GOS 4 or 5) and 36% it was poor (GOS 2 or 3). There were no differences in post-traumatic complications. Analysis of 73 matched pairs of hypothermic and normothermic patients could not prove hypothermia as an independent predictor of poor neurological outcome (OR 1.7, 95% CI 0.8-3.6, p = 0.1) in the total population. However, older patients (> 41 years) had a 4.2-times higher risk (95% CI 1.4-12.7; p = 0.01) of poor neurological outcome, if they were hypothermic on admission. CONCLUSIONS: Accidental hypothermia seems to have a negative impact on neurological recovery in older patients with multiple injuries including traumatic brain injury which outweighs potential benefits.

Fractures of the transverse processes of the fourth and fifth lumbar vertebrae in patients with pelvic ring injuries: indicator of biomechanical instability but not shock.

Aims: This study aimed to analyze the correlation between transverse process (TP) fractures of the fourth (L4) and fifth (L5) lumbar vertebrae and biomechanical and haemodynamic stability in patients with a pelvic ring injury, since previous data are inconsistent. Patients and Methods: The study is a retrospective matched-pair analysis of patients with a pelvic fracture according to the modified Tile AO Müller and the Young and Burgess classification who presented to a level 1 trauma centre between January 2005 and December 2014. Results: A total of 728 patients with pelvic ring injuries were included, of whom 183 (25.1%) had a biomechanically unstable pelvic fracture. Of these patients, 84 (45.9%) had a fracture of a TP of L4 and/or L5. A total of 73 patients (13.4%) with a stable pelvic ring injury (p < 0.001) had a fracture of a TP. Patients with a fracture of a TP of L4 and/or L5 had a 5.5-fold risk (odds ratio (OR)) of having a biomechanically unstable pelvic injury. TP fractures (OR 1.6, p = 0.2) could not be confirmed as an independent predictor of haemodynamic instability. Conclusion: This is the first study that has demonstrated a positive correlation between a TP fracture of L4 and/or L5 and a biomechanically unstable pelvic ring injury. The presence of transverse process fractures of L4 and/or L5 indicates increased severity of pelvic injury and therefore can help in the planning of emergency treatment. Cite this article: Bone Joint J 2018;100-B:1214-19.

Optimization of Field and Laboratory Sample Processing for Characterization of Metallic Residues at Military Training Ranges.

Military ranges are unlike many waste sites because the contaminants, both energetics and metals, are heterogeneously distributed in soil during explosive detonation or ballistic impact and cannot be readily characterized using conventional grab sampling. The Incremental Sampling Methodology (ISM) has been successful for characterization of energetic contamination in soils, but no published ISM processing studies for soils with small arms range metals such as Pb, Cu, Sb, and Zn exists. This study evaluated several ISM sample-processing steps: (1) field splitting to reduce the sample mass shipped to the analytical laboratory, (2) necessity of milling, and (3) processing a larger subsample mass for digestion in lieu of milling. Cone-and-quartering and rotary sectorial splitting techniques yielded poor precision and positively skewed distributions. Hence, an increase in digestion mass from 2 to 10 g was evaluated with milled and unmilled samples. Unmilled samples yielded results with the largest variability regardless of aliquot mass.

Factor Xa and VIIa inhibition by tissue factor pathway inhibitor is prevented by a monoclonal antibody to its Kunitz-1 domain.

Essentials Activated FVII (FVIIa) and FX (FXa) are inhibited by tissue factor pathway inhibitor (TFPI). A monoclonal antibody, mAb2F22, was raised against the N-terminal fragment of TFPI (1-79). mAb2F22 bound exclusively to the K1 domain of TFPI (KD ∼1 nm) and not to the K2 domain. mAb2F22 interfered with inhibition of both FVIIa and FXa activities and restored clot formation. SUMMARY: Background Initiation of coagulation is induced by binding of activated factor VII (FVIIa) to tissue factor (TF) and activation of factor X (FX) in a process regulated by tissue factor pathway inhibitor (TFPI). TFPI contains three Kunitz-type protease inhibitor domains (K1-K3), of which K1 and K2 block the active sites of FVIIa and FXa, respectively. Objective To produce a monoclonal antibody (mAb) directed towards K1, to characterize the binding epitope, and to study its effect on TFPI inhibition. Methods A monoclonal antibody, mAb2F22, was raised against the N-terminal TFPI(1-79) fragment. Binding data were obtained by surface plasmon resonance analysis. The Fab-fragment of mAb2F22, Fab2F22, was expressed and the structure of its complex with TFPI(1-79) determined by X-ray crystallography. Effects of mAb2F22 on TFPI inhibition were measured in buffer- and plasma-based systems. Results mAb2F22 bound exclusively to K1 of TFPI (KD ~1 nm) and not to K2. The crystal structure of Fab2F22/TFPI (1-79) mapped an epitope on K1 including seven residues upstream of the domain. TFPI inhibition of TF/FVIIa amidolytic activity was neutralized by mAb2F22, although the binding epitope on K1 did not include the P1 residue. Binding of mAb2F22 to K1 blocked TFPI inhibition of the FXa amidolytic activity and normalized hemostasis in hemophilia human A-like plasma and whole blood. Conclusion mAb2F22 blocked TFPI inhibition of both FVIIa and FXa activities and mapped a FXa exosite for binding to K1. It reversed TFPI feedback inhibition of TF/FVIIa-induced coagulation and restored clot formation in FVIII-neutralized human plasma and blood.

Inadequacy of Conventional Grab Sampling for Remediation Decision-Making for Metal Contamination at Small-Arms Ranges.

Research shows grab sampling is inadequate for evaluating military ranges contaminated with energetics because of their highly heterogeneous distribution. Similar studies assessing the heterogeneous distribution of metals at small-arms ranges (SAR) are lacking. To address this we evaluated whether grab sampling provides appropriate data for performing risk analysis at metal-contaminated SARs characterized with 30-48 grab samples. We evaluated the extractable metal content of Cu, Pb, Sb, and Zn of the field data using a Monte Carlo random resampling with replacement (bootstrapping) simulation approach. Results indicate the 95% confidence interval of the mean for Pb (432 mg/kg) at one site was 200-700 mg/kg with a data range of 5-4500 mg/kg. Considering the U.S. Environmental Protection Agency screening level for lead is 400 mg/kg, the necessity of cleanup at this site is unclear. Resampling based on populations of 7 and 15 samples, a sample size more realistic for the area yielded high false negative rates.

Applying Incremental Sampling Methodology to Soils Containing Heterogeneously Distributed Metallic Residues to Improve Risk Analysis.

This study compares conventional grab sampling to incremental sampling methodology (ISM) to characterize metal contamination at a military small-arms-range. Grab sample results had large variances, positively skewed non-normal distributions, extreme outliers, and poor agreement between duplicate samples even when samples were co-located within tens of centimeters of each other. The extreme outliers strongly influenced the grab sample means for the primary contaminants lead (Pb) and antinomy (Sb). In contrast, median and mean metal concentrations were similar for the ISM samples. ISM significantly reduced measurement uncertainty of estimates of the mean, increasing data quality (e.g., for environmental risk assessments) with fewer samples (e.g., decreasing total project costs). Based on Monte Carlo resampling simulations, grab sampling resulted in highly variable means and upper confidence limits of the mean relative to ISM.

Taxonomy of Tricholoma in northern Europe based on ITS sequence data and morphological characters.

Based on molecular and morphological data we investigated the taxonomy and phylogeny of the ectomycorrhizal genus Tricholoma in northern Europe. Our phylogenetic tree confirmed the presence of at least 72 well circumscribed species within the region. Of these, three species, viz. T. boreosulphurescens, T. bryogenum and T. ilkkae are described as new to science, based on morphological, distributional, ecological and molecular data. Several other terminal branches represent putative cryptic taxa nested within classical species or species groups. Molecular type studies and/or designation of sequenced neotypes are needed in these groups, before the taxonomy can be settled. In general our phylogenetic analysis supported previous suprageneric classification systems, but with some substantial changes. Most notably, T. virgatum and allies were found to belong to sect. Tricholoma rather than sect. Atrosquamosa, while T. focale was found to be clearly nested in sect. Genuina rather than in sect. Caligata. In total, ten sections are accepted, with five species remaining unassigned. The combination of morphological and molecular data showed pileus colour, pileipellis structure, presence of clamp connections and spore size to be rather conservative characters within accepted sections, while the presence of a distinct ring, and especially host selection were highly variable within these.

[Complex joint reconstruction and joint transplantation with the FLOCSAT concept-planning and surgical implementation]. / Komplexe Gelenkrekonstruktion und Gelenktransplantation mit dem FLOCSAT-Konzept ­ Planung und chirurgische Umsetzung.

Cartilage defects in adult patients are so far incurable. Fresh osteochondral allograft (OCA) transplantation is based on the insertion of mature, living, mechanically sound hyaline cartilage into an osteochondral defect where it undergoes osseointegration. Intact hyaline cartilage of OCA does not cause immune reactions in the recipient. Many reports show that small OCA have good osseointegration and show good long-term results. These observations have been incorporated into the development of the fresh large (> 10 cm2) osteochondral shell allograft transplantation (FLOCSAT) concept, which is based on the following principles: 1) the thickness of the osseous layer should be kept as thin as possible (target < 6-8 mm) so that the transplant remains stable and fixable. This results in reduced segments of vascularization, simplified ossification and reduced immunogenic bone volume. 2) The bone surface is processed and enlarged (oscillating saw: pie crust technique, drill holes) and areas of sclerosis are simultaneously broken off. 3) Cell reduction and washing out of the bony layer with a pulsatile jet lavage. 4) Prevention of impaction and dessication: cartilage with its living chondrocytes are very sensitive to mechanical contusion and dessication. When introducing the transplant, the tissue must therefore be continually moistened and the pressure acting on the cartilage must be controlled. 5) Stable fixation: extensive uniplanar osteochondral transplants cannot be inserted by the press-fit method; therefore, fixation is carried out with small implants. In this publication we demonstrate how severe and complex posttraumatic or degenerative delayed problems can be solved using FLOCSAT.

[Partial and complete joint transplantation with fresh osteochondral allografts-the FLOCSAT concept]. / Partielle und komplette Gelenktransplantation mit frischen osteochondralen Allografts ­ das FLOCSAT-Konzept.

Cartilage defects in adult patients do not heal well. Fresh osteochondral allograft (OCA) transplantation is based on mature, living, mechanically sound hyaline cartilage attached to a bone interface, which is brought into an osteochondral defect, where it becomes osseointegrated. According to current knowledge, intact hyaline cartilage tissue is immune privileged and does not, in contrast to bone, meniscus or ligaments, cause an immune reaction. The technique has the unique advantage of transplanting viable, mature and mechanically stable hyaline cartilage into the affected area. An OCA is the only biological surgical technique for chondral and osteochondral lesions after failed cell-based techniques or autologous osteochondral transplantation.Fresh osteochondral allografts with mainly small cylindrical transplants show survival of 20 years and more. Based on this experience the FLOCSAT (fresh large [> 10 cm2] osteochondral shell allograft transplantation) concept with the thinnest possible (< 6-8 mm) bone thickness has been developed. Cells survive in special media at 4 °C for 4 weeks or more and are transplanted with a minimum of 70% living cells in a live/dead assay and cell density > 200 cells/mm2.FLOCSATs can replace parts or entire joints as uniplanar or multiplanar(n-planar)-FLOCSAT, as unipolar, bipolar or tripolar(n-polar)-FLOCSAT, and in combination with meniscus and/or ligaments (nMnL)-FLOCSAT (n number of structures).The FLOCSAT concept was applied successfully to knee, ankle and elbow joints. All transplants showed sound osseointegration. Cartilage failure was the reason for unsuccessful outcome. Challenges remain regarding graft availability, precise size matching, complex logistics, demanding surgical technique in complex geometries, and open questions in immunology and chimerism.

Two-stage late reconstruction with a fresh large osteochondral shell allograft transplantation (FLOCSAT) for a large ostechondral defect in a non-union after a lateral tibia plateau fracture 2-year follow up.

This is the description of a 58-year-old female patient presenting 8 months after a horse riding accident with significant pain and inability to walk independently. Imaging revealed a large osseous defect of the lateral tibia plateau which was not united posteriorly. The patient refused knee replacement and we developed a patient specific two-step procedure for her. Step 1: Filling of the defect with a large cortico-cancellous autograft from the posterior iliac crest; step 2: Transplantation of a fresh large osteochondral shell allograft (FLOCSAT). The postoperative protocol included continuous passive motion (CPM), partial weight bearing for three months, and physiotherapy. Based on the concept of immuno-privileged cartilage tissue, the patient did not get any immuno-suppressive therapy. Pain-, activity of daily living, Lysholm and Tegner scores were evaluated before defect filling surgery with autograft, before allograft transplantation, and at 12 and 24 months after allograft transplantation. There were no complications. Radiographic analyses with plain films and CT scans revealed solid osseous integration within 3 month. The patient regained excellent functionality in both, activities of daily living and sports (back to horse riding, trampolin jumping). Knee arthroscopy after 1year showed excellent condition of the lateral meniscus and the cartilage of the lateral tibia plateau. Chimerism/DNA analysis of a cartilage biopsy showed, that at 1year 32% of the donor cells have been already replaced by the patient's own cells. To our knowledge, this is the first case of a patient who sustained such a large defect during a tibia plateau fracture, and got successfully treated with a fresh large osteochondral shell allograft transplantation in a two-step procedure.