Microparticles are 0.1-1.0 μm vesicles derived from cell membranes upon cell activation, injury, or apoptosis, and are believed to aid coagulation. Due to their clinical significance, a calibration method to quantify microparticles on an upgraded flow cytometer was developed in this study. Optimal settings were previously sest on the Apogee A40 flow cytometer. However, this flow cytometer has recently been internally upgraded to a newer model, Apogee A50, making re-calibration necessary for accurate microparticle measurements. The same beads used for calibration appeared to scatter even more light on the A50 using the same settings and no longer corresponded to the desired upper limit. As a result, steps were taken to re-calibrate the optimal settings for microparticle detection. These were achieved by using polystyrene and silica beads of known sizes, antibodies with fluorescent molecules attached, and normal patient platelet rich and platelet poor plasma. Instead of using the forward scatter curve as previously done on the A40, experiments on the A50 utilized the side scatter curve due to the histogram’s appearance. After adjusting the voltages and threshold, the 1 μm microparticle cutoff corresponded with 0.4 μm polystyrene and 0.7 μm silica beads. The compensation settings (subtraction values to correct for overlap of fluorescent light signals) also differed significantly on the A50 compared to the A40. Based on these results, the Apogee A50 needs to have these newly established settings applied in order to effectively quantitate and measure microparticles.

We validated the LIATEST® Antithrombin III (ATIII) antigen assay for use on the Stago® STA compact analyzer. The LIATEST® is an antibody agglutination assay that measures the amount of ATIII antigen in patients who are on cardiopulmonary support systems. The LIATEST® was chosen because is more stable than the STACHROM (chromogenic) gold standard.

Matrix-Associated Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS) is a high-throughput instrument with the ability to provide rapid and accurate identification of organisms in the microbial clinical setting. This powerful instrument has greatly impacted patient care in the recent decade. It has provided physicians the identification of organisms within a shorter time frame enabling more appropriate antibiotic treatments. With a newly implemented MALDI-TOF MS system at Virginia Mason Medical Center, it was necessary to validate the claim from the Bruker MALDI Biotyper CA System® against FDA cleared library list of organisms was indeed accurate. Fifteen anaerobic and twenty-three yeast isolates were selected in order to validate the instrument’s ability to identify organisms in the claim. These organisms were plated on media best suited for their growth and were incubated at 24 hour, 48 hour, and 24-hour sub culture to validate colony age, viability, and reproducibility. The purpose of this study was to validate the performance of the Bruker MALDI Biotyper CA System® on previously sequenced strains of yeasts and anaerobes, and to reliably perform testing on these frequently isolated organisms. Of the 15 different anaerobic isolates, MALDI-TOF was able to yield an accuracy of 86.7% using direct transfer methods. On the other hand, of the 23 different yeast isolates, the instrument yielded an accuracy of 87.0% with the misidentification of only one yeast isolate, which was possibly due to the resemblance to its closely related family. The validation of yeasts and anaerobes was proven to provide accurate identification to the species level with a vast number of isolates. MALDI-TOF MS has now been proven as a powerful method to be utilized in the clinical setting in order to accurately identify pathogenic organisms from patient isolates with this high-throughput instrument.

Iohexol and Iothalamate are nonradioactive contrast reagents, and are used as a marker to determine glomerular filtration rate (GFR). GFR is commonly used to evaluate the overall function of the kidneys as it reflects the rate at which toxins are filtered. It is considered the best index of kidney function. In order to switch from iothalamate to iohexol, a method validation is required to switch protocols. The method being evaluated uses iohexol that is given by bolus IV (single injection), and quantitates the concentration by high performance liquid chromatography (HPLC). HPLC uses high pressure and the affinity of molecules to elute of a column at specific times, called retention times, allowing the the quantitation of iohexol in a sample. Iohexol methodology provides many advantages including using less invasive clinical techniques, needing fewer sampling, and being less expensive than most markers used to determine GFR. The methodology is validated on two Waters® HPLC setups with analysis using EZ Chrom® and EP Evaluator®. EZ Chrom® is the program that stores and analyzes the the information, such as retention times, in order to determine the amount of iohexol. EP Evaluator®, is a program which analyzes data by calculating statistics to determine the precision and accuracy of the results. Assays performed included, inter-assays, intra-assays, analytical sensitivities, linearity, carryover, interferences, analytical comparison and cost analysis. The data showed that within limits, iohexol testing is precise, accurate, and overall valid to implement at Seattle Children’s Hospital in order to determine GFR. This makes it possible for Seattle Children's Hospital to continue to improve patient care by providing a less invasive method to determine GFR.
 

Staphylococcus aureus is a global human pathogen and a major cause of wound infections, food poisoning, and invasive disease. Its success as a pathogen requires virulence factors used to evade the human immune system and subvert host defenses. Protein A is a multifunctional immune evasion factor that inhibits bacterial uptake by immune cells, modulates inflammatory responses, and disrupts development of adaptive immunity. We found that protein A expression decreased when cultures of S. aureus were exposed to the antimicrobial molecule nitric oxide (NO). NO is a reactive radical produced at high levels by macrophages and other host cells during infection. Reverse transcriptase quantitative PCR (RT-qPCR) showed that transcription of the spa gene, encoding protein A, was significantly decreased in the presence of NO. Dose response curves were generated comparing the sensitivity of spa and other genes to NO as well as to hydrogen peroxide (H₂O₂), another host-produced antimicrobial compound. Western blots confirmed that NO inhibition of spa transcription resulted in reduced protein A levels. Through allelic exchange mutagenesis, we mutated multiple spa transcription factors of S. aureus and identified the regulator protein XdrA as a likely NO target. Given that NO modifies cysteine thiols of proteins, we hypothesized that XdrA is S-nitrosylated by NO, leading to inhibition of spa transcription and concurring decrease in protein A. Single cysteine mutants of XdrA (C54S, C73S) have shown partial resistance to NO inhibition. A double cysteine XdrA mutant will be tested to assess the cumulative effect that multiple NO modifications have on spa transcription. This will allow us to understand the molecular mechanism of NO dependent protein A inhibition. Additional experiments demonstrating that NO produced by macrophages inhibits production of an important S. aureus virulence factor would characterize one mechanism by which innate immunity controls bacterial infections.