Frequently Asked Questions

Timing

Does DivergeTSL provide “rush service”?

We do not have specific “rush service” charges, but we do attempt to ensure that our clients get their data by the time they need it. The best way to ensure a faster-than-usual turnaround time for a given study is to provide us with notice so that we schedule the work to begin as soon as your samples are available for testing. Once testing is started, all the work is performed as quickly as possible while ensuring maximal quality, regardless of whether a client communicates that a certain study is a “rush”.

What are the timelines associated with developing or validating various assays?

Timelines for developing histology assays depend on the difficulty of detecting the target and the overall complexity of the assay (i.e. the number of targets multiplexed on a given slide). A typical development timeline for a double- or triple-immunofluorescence stain is 4-8 weeks.

Timelines for validating histology assays also depend on the type of validation requested. For a clinical assay-style validation consistent with the recommendations of the College of American Pathologists, validation experiments typically require 3 weeks. In cases where additional regulatory rigor is desired (for instance, if the histology assay is a primary endpoint in a clinical trial), validation experiments can be expanded to include more rounds of staining, multiple operators, etc. The timeline for these expanded validations depends on the client’s preferences.

Timelines for developing western blot assays depend on the availability of suitable antibodies and the client’s performance needs (for instance, whether there’s a need to detect and quantify protein in extremely low amounts). Typical development timelines for western blot assays are 8-12 weeks.

Timelines for validating western blot assays according to FDA guidance are typically 4-8 weeks after the approval of a western blot validation plan by the client.

Timelines for image analysis development and validation are typically 4-6 weeks after we have acquired images suitable for image analysis (which is typically done as a part of the histology assay validation work).

Staining

How does DivergeTSL validate a histology stain?

The most basic approach is the one used for the validation of non-predictive clinical immunostaining assays by the College of American Pathologists (CAP), which involves the staining and assessment of a collection of tissue samples that have expected staining outcomes. The associated criteria for validation is that a blinded evaluation of the validation slide set produces a >90% concordance with the expected results. It is also possible to perform expanded validation studies to more completely assess the assay’s performance, including accuracy, precision, and specificity. The design of an expanded validation is based on a client’s specific desires and needs, and we work with our clients to design this type of work.

Imaging and Image Analysis

How does DivergeTSL perform image analysis?

Our in-house experts work with our clients to identify the signals of interest on histological assays. We then use our robust image analysis platform, NIS-Elements, to develop the optimal strategy to characterize and quantify the signals of interest.

For multiplexed fluorescence markers, our image analysis approach emphasizes multifaceted quantification of signals of interest in anatomically/biologically appropriate areas delineated by a reference marker. This approach is best exemplified in the method that our Scientific Director, Dr. Tatyana Vetter, developed to perform dystrophin image analysis in the context of Duchenne muscular dystrophy. This general image analysis strategy allows us to assess the signals of interest with high relevance, sensitivity, and precision. Our image analysis platform enables us to visualize how the quantification is performed and where each value originated to allow efficient quality control and high transparency related to how the results correspond to the images.

Fixation and Freezing

What freezing and fixing procedures does DivergeTSL use or recommend?

The optimal tissue preparation and fixation approach depends on the main goals of the assays in question, but general guidelines are summarized as follows:

For histology studies of skeletal muscle, the optimal preparation is usually rapid freezing of fresh tissue using isopentane. We can provide resources to ensure that this is performed appropriately.

For histology studies of other organs including the heart, the optimal tissue preparation is usually formalin-fixed paraffin embedded tissue. It is important to keep the tissue in formalin for no more than 24 hours, and then to switch to 70% ethanol to avoid over-fixation. This limits the degree of autofluorescence that will be encountered in the tissue as a result of the fixation process.

For western blot studies, frozen tissue is needed. It does not matter whether this tissue was frozen using isopentane or other methods.

About DivergeTSL

Is work performed at DivergeTSL GxP compliant?

We provide GxP compliant services upon request and for an additional charge. We have the most experience running GCP-compliant studies in the clinical trial setting, but we have also performed GLP-compliant work upon request.

What can DivergeTSL supply to support setting up clinical trials?

We can provide training and operations assistance with biopsy studies in human clinical trials. The specifics of what we provide depend on client needs, but we have previously provided clinical trial freezing training, biopsy kits, freezing manuals and freezing tests.

Does DivergeTSL offer a list of “off the shelf” validated assays?

We optimize assays for specific client needs and then perform a validation study using those optimized conditions if requested by the client. In cases where a client has already identified conditions they believe to be optimal, then a technique transfer to Diverge can be performed, followed by a validation experiment. As individual assays and the validation material belong to the client for whom we developed the assay, we do not provide “off the shelf” validated assays, but our strategy allows validate assays for established procedures.

Does DivergeTSL have access to tissue for use in development activities?

We have limited access to tissue for development activities, including human surgical skeletal muscle and autopsy heart tissue. This tissue is consented for use for our own technical development and test performance but is not available for further distribution. For other tissue types (including tissues related to specific disease states), we rely on our clients to obtain and provide appropriate samples for testing. We are happy to discuss the most useful tissues for our experiments to facilitate this process.