Basics of flow cytometry-based sterility testing of platelet concentrates

BACKGROUND: Flow cytometry (FACS) is a common technique in blood banking. It is used, for example, for the enumeration of residual white blood cells in plasma and in cellular blood products. It was investigated whether it can also be applied for sterility testing of buffy coat-derived platelet concentrates (PCs). STUDY DESIGN AND METHODS: Plasma-reduced PCs were spiked with bacteria and stored at 20 to 24 or 37 degrees C for various times. The following 10 species were used: Bacillus cereus, Enterobacter cloacae, Escherichia coli, Klebsiella pneumoniae, Propionibacterium acnes, Pseudomonas aeruginosa, Staphylococcus aureus, Staphylococcus epidermidis, Serratia marcescens, and Yersinia enterocolitica. Bacterial DNA was stained with thiazole orange. After the platelets were lysed, bacteria were enumerated by FACS. RESULTS: All bacteria species used were detectable by FACS. The lower detection limit was approximately 100 bacteria per microL, that is, 10(5) per mL. In general, the titers measured were 1.2- to 3-fold higher than those determined by colony forming assay. In one case (K. pneumoniae) in which the dot plot of the bacteria cloud overlapped with that of bacteria debris, they were consistently lower. When PC samples were inoculated with approximately 1 colony-forming unit per mL of bacteria and kept at 37 degrees C, most species were detected within 21 hours or less. Exceptions were E. cloacae and P. acnes, which were detected after 24 to 40 and 64 hours, respectively. At 20 to 24 degrees C, the detection times were strongly prolonged. CONCLUSION: Sterility testing of PCs by FACS is a feasible approach. The present data suggest incubating PC samples for 20 to 24 hours at 37 degrees C before testing. For slow-growing bacteria, the incubation period must be prolonged by 1 to 2 days.     

Effect of vitamin D3 treatment on bone density in neurofibromatosis 1 patients: A retrospective clinical study

ObjectivesWe have previously demonstrated reduced bone density and an increased incidence of 25-hydroxy vitamin D₃ (25-OH D₃) deficiency in adults with neurofibromatosis 1 (NF1) compared to healthy controls. Vitamin D₃ is a cheap, safe, and effective supplement in the general population, but its value in NF1 patients has not been demonstrated. This study investigates the therapeutic potential of oral vitamin D₃ on bone mineral density (BMD) in NF1 patients with vitamin D₃ deficiency.MethodsWe measured serum 25-OH D₃, parathyroid hormone, calcium, and bone alkaline phosphatase concentrations, urinary deoxypyridinoline concentrations, and BMD in 35 adults with NF1. Nineteen patients received vitamin D₃ supplementation for 2 years, six patients received supplementation for 1 year and 10 patients received no supplementation. Supplementation was administered in a dose that maintained the serum 25-OH D₃ level above 30 μg/l. BMD was measured again at 1 and 2 years, and biochemical assessments of bone metabolism were measured at least every half year during therapy.ResultsTreated subjects had significantly reduced loss of BMD, as measured by T score at the hip (p = 0.011) and lumbar spine (p = 0.022). The effect on hip BMD was apparent at 1 year in comparison to baseline (p = 0.02) and was greater at 2 years in comparison to measurements at 1 year (p = 0.02).ConclusionsVitamin D₃ supplementation improves BMD in adult NF1 patients. Further studies are needed to elucidate the mechanisms responsible for reduced BMD in NF1 patients.     

Characterization of grlA, grlB, gyrA, and gyrB Mutations in 116 Unrelated Isolates of Staphylococcus aureus and Effects of Mutations on Ciprofloxacin MIC

One hundred sixteen unrelated clinical isolates of Staphylococcus aureus (70 ciprofloxacin resistant and 46 ciprofloxacin susceptible) from eight countries were studied for the presence of mutations in the grlA, grlB, gyrA, and gyrB gene loci. Two mutations within grlA (located at codons 80 and 84) and two mutations within gyrA (located at codons 84 and 88) were clearly associated with ciprofloxacin resistance, although other mutations detected within the four genes studied may also contribute to decreased susceptibility.     

Navigation system for minimally invasive esophagectomy: experimental study in a porcine model

Background

Navigation systems potentially facilitate minimally invasive esophagectomy and improve patient outcome by improving intraoperative orientation, position estimation of instruments, and identification of lymph nodes and resection margins. The authors’ self-developed navigation system is highly accurate in static environments. This study aimed to test the overall accuracy of the navigation system in a realistic operating room scenario and to identify the different sources of error altering accuracy.

Methods

To simulate a realistic environment, a porcine model (n = 5) was used with endoscopic clips in the esophagus as navigation targets. Computed tomography imaging was followed by image segmentation and target definition with the medical imaging interaction toolkit software. Optical tracking was used for registration and localization of animals and navigation instruments. Intraoperatively, the instrument was displayed relative to segmented organs in real time. The target registration error (TRE) of the navigation system was defined as the distance between the target and the navigation instrument tip. The TRE was measured on skin targets with the animal in the 0° supine and 25° anti-Trendelenburg position and on the esophagus during laparoscopic transhiatal preparation.

Results

On skin targets, the TRE was significantly higher in the 25° position, at 14.6 ± 2.7 mm, compared with the 0° position, at 3.2 ± 1.3 mm. The TRE on the esophagus was 11.2 ± 2.4 mm. The main source of error was soft tissue deformation caused by intraoperative positioning, pneumoperitoneum, surgical manipulation, and tissue dissection.

Conclusion

The navigation system obtained acceptable accuracy with a minimally invasive transhiatal approach to the esophagus in a realistic experimental model. Thus the system has the potential to improve intraoperative orientation, identification of lymph nodes and adequate resection margins, and visualization of risk structures. Compensation methods for soft tissue deformation may lead to an even more accurate navigation system in the future.     

In vivo analysis of cervical kinematics after implantation of a minimally constrained cervical artificial disc replacement

Introduction

To better understand cervical kinematics following cervical disc replacement (CDR), the in vivo behavior of a minimally constrained CDR was assessed.

Methods

Radiographic analysis of 19 patients undergoing a 1-level CDR from C4–5 to C6–7 (DISCOVER, Depuy-Spine, USA) was performed. Neutral–lateral and flexion–extension radiographs obtained at preop, postop and late follow-up were analyzed for segmental angle and global angle (GA C2–7). Flexion–extension range of motion was analyzed using validated quantitative motion analysis software (QMA®, Medical Metrics, USA). The FSU motion parameters measured at the index and adjacent levels were angular range of motion (ROM), translation and center of rotation (COR). Translation and COR were normalized to the AP dimension of the inferior endplate of the caudal vertebra. All motion parameters, including COR, were compared with normative reference data.

Results

The average patient age was 43.5 ± 7.3 years. The mean follow-up was 15.3 ± 7.2 months. C2–7 ROM was 35.9° ± 15.7° at preop and 45.4° ± 13.6° at follow-up (?p < .01). Based on the QMA at follow-up, angular ROM at the CDR level measured 9.8° ± 5.9° and translation was 10.1 ± 7.8 %. Individuals with higher ROM at the CDR level had increased translation at that level (p < .001, r = 0.97), increased translation and ROM at the supra-adjacent level (p < .001, r = .8; p = .005, r = .6). There was a strong interrelation between angular ROM and translation at the supra-adjacent level (p < .001, r = .9) and caudal-adjacent level (p < .001, r = .9). The location of the COR at the CDR- and supra-adjacent levels was significantly different for the COR-X (p < .001). Notably, the COR-Y at the CDR level was significantly correlated with the extent of CDR-level translation (p = .02, r = .6). Shell angle, which may be influenced by implant size and positioning had no impact on angular ROM but was correlated with COR-X (p = .05, r = ?.6) and COR-Y (p = .04, r = ?.5).

Conclusion

The COR is an important parameter for assessing the ability of non-constrained CDRs to replicate the normal kinematics of a FSU. CDR size and location, both of which can impact shell angle, may influence the amount of translation by affecting the location of the COR. Future research is needed to show how much translation is beneficial concerning clinical outcomes and facet loading.     

Comparison of revision strategies for failed C2-posterior cervical pedicle screws: a biomechanical study

Study purpose

With increasing usage within challenging biomechanical constructs, failures of C2 posterior cervical pedicle screws (C2-pCPSs) will occur. The purpose of the study was therefore to investigate the biomechanical characteristics of two revision techniques after the failure of C2-pCPSs.

Materials and methods

Twelve human C2 vertebrae were tested in vitro in a biomechanical study to compare two strategies for revision screws after failure of C2-pCPSs. C2 pedicles were instrumented using unicortical 3.5-mm CPS bilaterally (Synapse/Synthes, Switzerland). Insertion accuracy was verified by fluoroscopy. C2 vertebrae were potted and fixed in an electromechanical testing machine with the screw axis coaxial to the pullout direction. Pullout testing was conducted with load and displacement data taken continuously. The peak load to failure was measured in newtons (N) and is reported as the pullout resistance (POR). After pullout, two revision strategies were tested in each vertebra. In Group-1, revision was performed with 4.0-mm C2-pCPSs. In Group-2, revision was performed with C2-pedicle bone-plastic combined with the use of a 4-mm C2-pCPSs. For the statistical analysis, the POR between screws was compared using absolute values (N) and the POR of the revision techniques normalized to that of the primary procedures (%).

Results

The POR of primary 3.5-mm CPSs was 1,140.5 ± 539.6 N for Group-1 and 1,007.7 ± 362.5 N for Group-2; the difference was not significant. In the revision setting, the POR in Group-1 was 705.8 ± 449.1 N, representing a reduction of 38.1 ± 32.9 % compared with that of primary screw fixation. For Group-2, the POR was 875.3 ± 367.9 N, representing a reduction of 13.1 ± 23.4 %. A statistical analysis showed a significantly higher POR for Group-2 compared with Group-1 (p = 0.02). Although the statistics showed a significantly reduced POR for both revision strategies compared with primary fixation (p < 0.001/p = 0.001), the loss of POR (in %) in Group-1 was significantly higher compared with the loss in Group-2 (p = 0.04).

Conclusions

Using a larger-diameter screw combined with the application of a pedicle bone-plastic, the POR can be significantly increased compared with the use of only an increased screw diameter.     

Intra-operative adjustment of standard planes in C-arm CT image data

Purpose

With the help of an intra-operative mobile C-arm CT, medical interventions can be verified and corrected, avoiding the need for a post-operative CT and a second intervention. An exact adjustment of standard plane positions is necessary for the best possible assessment of the anatomical regions of interest but the mobility of the C-arm causes the need for a time-consuming manual adjustment. In this article, we present an automatic plane adjustment at the example of calcaneal fractures.

Methods

We developed two feature detection methods (2D and pseudo-3D) based on SURF key points and also transferred the SURF approach to 3D. Combined with an atlas-based registration, our algorithm adjusts the standard planes of the calcaneal C-arm images automatically. The robustness of the algorithms is evaluated using a clinical data set. Additionally, we tested the algorithm’s performance for two registration approaches, two resolutions of C-arm images and two methods for metal artifact reduction.

Results

For the feature extraction, the novel 3D-SURF approach performs best. As expected, a higher resolution (\(512^3\) voxel) leads also to more robust feature points and is therefore slightly better than the \(256^3\) voxel images (standard setting of device). Our comparison of two different artifact reduction methods and the complete removal of metal in the images shows that our approach is highly robust against artifacts and the number and position of metal implants.

Conclusions

By introducing our fast algorithmic processing pipeline, we developed the first steps for a fully automatic assistance system for the assessment of C-arm CT images.