Ex vivo hemolysis: Three cases demonstrating mechanically-induced hemolysis from the extracorporeal circuit during hemodialysis.

Sporadic mechanically-induced hemolysis associated with kinks in extracorporeal blood circuits during hemodialysis is a rare but potentially serious complication that exhibits laboratory features consistent with both in vivo and in vitro hemolysis. Misclassification of clinically significant hemolysis as in vitro can lead to inappropriate test cancellation and delayed medical interventions. Here, we report three cases of hemolysis attributed to kinked hemodialysis blood lines, which we have defined as "ex vivo" hemolysis. All three cases demonstrated an initial mixed picture of laboratory features consistent with both classifications of hemolysis. Specifically, absent features of in vivo hemolysis on blood film smear despite normal potassium led to the misclassification of these samples as in vitro hemolysis and their cancellation. A proposed mechanism for these overlapping laboratory features is the recirculation of damaged red blood cells from the kinked or pinched hemodialysis line back into the patient circulation producing an "ex vivo" hemolysis presentation. In two of the three cases, the patients developed acute pancreatitis as a result of hemolysis and required urgent medical follow up. We developed a decision pathway to help laboratories in identifying and handling these samples by recognizing that in vitro and in vivo hemolysis have overlapping laboratory features. These cases highlight the need for laboratorians and the clinical care team to be vigilant about mechanically-induced hemolysis from the extracorporeal circuit during hemodialysis. Communication is critical to identify the cause of hemolysis in these patients and prevent unnecessary delays in result reporting.

CRISPR technology incorporating amplification strategies: molecular assays for nucleic acids, proteins, and small molecules.

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR-associated (Cas) protein systems have transformed the field of genome editing and transcriptional modulation. Progress in CRISPR-Cas technology has also advanced molecular detection of diverse targets, ranging from nucleic acids to proteins. Incorporating CRISPR-Cas systems with various nucleic acid amplification strategies enables the generation of amplified detection signals, enrichment of low-abundance molecular targets, improvements in analytical specificity and sensitivity, and development of point-of-care (POC) diagnostic techniques. These systems take advantage of various Cas proteins for their particular features, including RNA-guided endonuclease activity, sequence-specific recognition, multiple turnover trans-cleavage activity of Cas12 and Cas13, and unwinding and nicking ability of Cas9. Integrating a CRISPR-Cas system after nucleic acid amplification improves detection specificity due to RNA-guided recognition of specific sequences of amplicons. Incorporating CRISPR-Cas before nucleic acid amplification enables enrichment of rare and low-abundance nucleic acid targets and depletion of unwanted abundant nucleic acids. Unwinding of dsDNA to ssDNA using CRISPR-Cas9 at a moderate temperature facilitates techniques for achieving isothermal exponential amplification of nucleic acids. A combination of CRISPR-Cas systems with functional nucleic acids (FNAs) and molecular translators enables the detection of non-nucleic acid targets, such as proteins, metal ions, and small molecules. Successful integrations of CRISPR technology with nucleic acid amplification techniques result in highly sensitive and rapid detection of SARS-CoV-2, the virus that causes the COVID-19 pandemic.

Molecular Diagnosis of COVID-19: Challenges and Research Needs.

Molecular diagnosis of COVID-19 primarily relies on the detection of RNA of the SARS-CoV-2 virus, the causative infectious agent of the pandemic. Reverse transcription polymerase chain reaction (RT-PCR) enables sensitive detection of specific sequences of genes that encode the RNA dependent RNA polymerase (RdRP), nucleocapsid (N), envelope (E), and spike (S) proteins of the virus. Although RT-PCR tests have been widely used and many alternative assays have been developed, the current testing capacity and availability cannot meet the unprecedented global demands for rapid, reliable, and widely accessible molecular diagnosis. Challenges remain throughout the entire analytical process, from the collection and treatment of specimens to the amplification and detection of viral RNA and the validation of clinical sensitivity and specificity. We highlight the main issues surrounding molecular diagnosis of COVID-19, including false negatives from the detection of viral RNA, temporal variations of viral loads, selection and treatment of specimens, and limiting factors in detecting viral proteins. We discuss critical research needs, such as improvements in RT-PCR, development of alternative nucleic acid amplification techniques, incorporating CRISPR technology for point-of-care (POC) applications, validation of POC tests, and sequencing of viral RNA and its mutations. Improved assays are also needed for environmental surveillance or wastewater-based epidemiology, which gauges infection on the community level through analyses of viral components in the community's wastewater. Public health surveillance benefits from large-scale analyses of antibodies in serum, although the current serological tests do not quantify neutralizing antibodies. Further advances in analytical technology and research through multidisciplinary collaboration will contribute to the development of mitigation strategies, therapeutics, and vaccines. Lessons learned from molecular diagnosis of COVID-19 are valuable for better preparedness in response to other infectious diseases.

Beacon-mediated exponential amplification reaction (BEAR) using a single enzyme and primer.

Beacon-mediated Exponential Amplification Reaction (BEAR) enables isothermal, exponential signal amplification. BEAR uses only a single enzyme and a single primer. Detection of 0.2 amol of a mitochondrial DNA with a point mutation in less than an hour demonstrates an application of the BEAR technique for nucleic acid research.

The Effects of SELEX Conditions on the Resultant Aptamer Pools in the Selection of Aptamers Binding to Bacterial Cells.

Aptamers of high affinity and specificity have a wide range of analytic and clinical applications. Selection of DNA or RNA aptamer molecules usually involves systematic evolution of ligands via exponential enrichment (SELEX), in which a random DNA or RNA library is incubated with a target molecule, and the oligonucleotides that bind the target are then separated from the nonbinders, PCR amplified, and used as refined libraries in the next round of selection. Conventional SELEX methodologies require the use of purified target molecules and their immobilization onto a solid support. However, purified targets from cells are not always available, and fixing the target to a support may alter its conformation. To overcome these problems, we have developed a SELEX technique using live bacterial cells in suspension as targets, for selecting DNA aptamers specific to cell-surface molecules. Through the selection of aptamers binding to Lactobacillus acidophilus and Streptococcus pyogenes, we report here optimization of this technique and show how varying selection conditions impact the characteristics of resultant aptamer pools, including the binding affinity, selectivity, and the secondary structures. We found that the use of larger starting library sequence diversity, gel purification of the subsequent pools, and the introduction of counter-selection resulted in a more efficient SELEX process and more selective aptamers. A SELEX protocol with lower starting sequence diversity, the use of heat denaturation, and the absence of counter-selection still resulted in high-affinity aptamer sequences specific to the target cell types; however, the SELEX process was inefficient, requiring 20 rounds, and the aptamers were not specific to the strain of the bacterial cells. Strikingly, two different SELEX methodologies yielded the same sequence that bound strongly to the target S. pyogenes cells, suggesting the robustness of the bacterial cell-SELEX technique.