Tag Archives: quantification

October 24, 2019

Rapid Pathogen Detection for the 21st Century: A Look at PathogenDx

By Aaron G. Biros

In 1887, Julius Petri invented a couple of glass dishes, designed to grow bacteria in a reproducible, consistent environment. The Petri dish, as it came to be known, birthed the scientific practice of agar cultures, allowing scientists to study bacteria and viruses. The field of microbiology was able to flourish with this handy new tool. The Petri dish, along with advancements in our understanding of microbiology, later developed into the modern field of microbial testing, allowing scientists to understand and measure microbial colonies to detect harmful pathogens in our food and water, like E. coli and Salmonella, for example.

The global food supply chain moves much faster today than it did in the late 19^th century. According to Milan Patel, CEO of PathogenDx, this calls for something a little quicker. “Traditional microbial testing is tedious and lengthy,” says Patel. “We need 21^st century pathogen detection solutions.”

Milan Patel first joined the parent company of PathogenDx back in 2012, when they were more focused on clinical diagnostics. “The company was predominantly built on grant funding [a $12 million grant from the National Institute of Health] and focused on a niche market that was very specialized and small in terms of market size and opportunity,” says Patel. “I realized that the technology had a much greater opportunity in a larger market.”

Milan Patel, CEO of PathogenDx
Photo: Michael Chansley

He thought that other markets could benefit from that technology greatly, so the parent company licensed the technology and that is how PathogenDx was formed. Him and his team wanted to bring the product to market without having to obtain FDA regulatory approval, so they looked to the cannabis market. “What we realized was we were solving a ‘massive’ bottleneck issue where the microbial test was the ‘longest test’ out of all the tests required in that industry, taking 3-6 days,” says Patel. “We ultimately realized that this challenge was endemic in every market – food, agriculture, water, etc. – and that the world was using a 140-year-old solution in the form of petri dish testing for microbial organisms to address challenges of industries and markets demanding faster turnaround of results, better accuracy, and lower cost- and that is the technology PathogenDx has invented and developed.”

While originally a spinoff technology designed for clinical diagnostics, they deployed the technology in cannabis testing labs early on. The purpose was to simplify the process of testing in an easy approach, with an ultra-low cost and higher throughput. Their technology delivers microbial results in less than 6 hours compared to 24-36 hours for next best option.

Out of all the tests performed in a licensed cannabis testing laboratory, microbial tests are the longest, sometimes taking up to a few days. “Other tests in the laboratory can usually be done in 2-4 hours, so growers would never get their microbial testing results on time,” says Patel. “We developed this technology that gets results in 6 hours. The FDA has never seen something like this. It is a very disruptive technology.”

When it comes to microbial contamination, timing is everything. “By the time Petri dish results are in, the supply chain is already in motion and products are moving downstream to distributors and retailers,” Patel says. “With a 6-hour turnaround time, we can identify where exactly in the supply chain contaminant is occurring and spreading.”

The technology is easy to use for a lab technician, which allows for a standard process on one platform that is accurate, consistent and reproduceable. The technology can deliver results with essentially just 12 steps:

Take 1 gram of cannabis flower or non-flower sample. Or take environmental swab
Drop sample in solution. Swab should already be in solution
Vortex
Transfer 1ml of solution into 1.5ml tube
A look at how the sample is added to the microarray
Conduct two 3-minute centrifugation steps to separate leaf material, free-floating DNA and create a small pellet with live cells
Conduct cell lysis by adding digestion buffer to sample on heat blocks for 1 hour
Conduct Loci enhancement PCR of sample for 1 hour
Conduct Labelling PCR which essentially attaches a fluorescent tag on the analyte DNA for 1 hour
Pipette into the Multiplex microarray well where hybridization of sample to probes for 30 minutes
Conduct wash cycle for 15 minutes
Dry and image the slide in imager
The imager will create a TIFF file where software will analyze and deliver results and a report

Their DetectX product can test for a number of pathogens in parallel in the same sample at the same time down to 1 colony forming unit (CFU) per gram. For bacteria, the bacterial kit can detect E. coli, E. coli/Shigella spp., Salmonella enterica, Listeria and Staph aureus, Stec 1 and Stec 2 E.coli. For yeast and mold, the fungal kit can test for Aspergillus flavus, Aspergillus fumigatus, Aspergillus niger and Aspergillus terreus.

Their QuantX is the world’s first and only multiplex quantification microarray product that can quantify the microbial contamination load for key organisms such as total aerobic bacteria, total yeast & mold, bile tolerant gram negative, total coliform and total Enterobacteriaceae over a dynamic range from 100 CFU/mL up to 1,000,000 CFU/mL.

Not all of the PathogenDx technology is designed for just microbial testing of cannabis or food products. Their EnviroX technology is designed to help growers, processors or producers across any industry identify areas of microbial contamination, being used as a tool for quality assurance and hazard analysis. They conducted industry-wide surveys of the pathogens that are creating problems for cultivators and came up with a list of more than 50 bacterial and fungal pathogens that the EnviroX assay can test for to help growers identify contamination hotspots in their facilities.

Using the EnviroX assay, growers can swab surfaces like vents, fans, racks, workbenches and other potential areas of contamination where plants come in contact. This helps growers identify potential areas of contamination and remediate those locations. Patel says the tool could help growers employ more efficient standard operating procedures with sanitation and sterilization, reducing the facility’s incidence of pathogens winding up on crops, as well as reduction in use of pesticides and fungicides on the product.

Deploying this technology in the cannabis industry allowed Milan Patel and the PathogenDx team to bring something new to the world of microbial testing. Their products are now in more than 90 laboratories throughout the country. The success of this technology provides another shining example of how the cannabis market produces innovative and disruptive ideas that have a major impact on the world, far beyond cannabis itself.

May 9, 2019

The Need for Standardization in Medical Cannabis Testing

By Andrew James

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There has been a move towards the legalization of cannabis for medical and/or recreational use across many countries and US states in recent years, leading to greater demand for accurate potency and safety testing. Despite this, there are currently no standardized regulations between states or countries for quality control including content, composition, adulterants, potency or levels of toxic residues. As such, in many cases where regulations are in place, testing is generally carried out at a small number of approved independent testing laboratories.

The need for self-regulation has led to the growth of portable gas chromatography (GC) being used in the field of cannabis testing.This lack of clarity makes it difficult for consumers to make informed decisions about what they are purchasing, an issue which could be damaging to the industry’s changing reputation. As it stands, producers of cannabis and cannabis-derived products can supply goods with potentially harmful contaminants such as fungi or pesticide residues, which are potentially threatening to human health. Most cannabis products sold legally in the US are required to be tested and labelled for THC, the chemical responsible for most of cannabis’s psychoactive effects. A US study found that as few as 20% of recreational cannabis products are accurately labelled with only 17% of products reviewed accurately labelled for THC content (i.e. within 10% of the labeled value). It also found 23% were under labelled, and 60% were over labelled.

If cannabis were to be categorized as a regulated pharmaceutical drug, it would be rigorously tested to comply with stringent rules and regulations regarding quality and safety of the product, as are all other drugs. However, as there is currently no centralized regulatory body that oversees this, the responsibility of quality assurance falls to the grower, manufacturer and sometimes the consumer.

The Need for Cannabis Analysis

The most common requirement when testing cannabis is positive identification and quantification of the total THC:CBD ratio. In a highly competitive marketplace, this information is important, as cannabis consumers tend to equate THC levels with price. In many instances, lower THC products are cheaper and higher THC concentrations make products more expensive. Without robust systems in place for sufficient testing, this information cannot be accurately determined, meaning the customer often cannot make an informed decision.

Pesticide use is surprisingly common in the cannabis cultivation industry

In addition to potency testing, one of the core issues facing the industry and by extension, the end consumer, is the prevalence of pesticides in cannabis products. In the Netherlands, the Ministry of Environment and Health reported that over 90% of cannabis plants tested had pesticides on them. While steps have been taken to tackle this, the lack of cohesion in testing standards combined with the onus on individual labs to carry out testing, has led to some issues within the industry.

Many individual retailers in the U.S. and internationally are self-testing for impurities such as pesticides, heavy metals and microbials. While there is a clear need for standard testing across all locations, the need for self-regulation at present has led to the growth of portable gas chromatography (GC) being used in the field of cannabis testing.

Using GC as an analytical tool

With the increased need for quality control and quality assurance in the largely unregulated cannabis industry, technology is now more accessible to smaller companies and to people with minimal laboratory experience. There are a range of cannabis testing packages available for smaller individual labs which offer more mobile testing with affordable packages. The lower entry price makes GC analysis affordable for more laboratories while still delivering reliable, high quality results.

Portable GC instruments can offer high quality potency testing, pesticide screening, terpene and flavor profiling, and residual solvents analysis. These instruments can give growers and processors an accurate result of cannabinoid percentages, a fundamental piece of information for growers and dispensaries. Systems can be configured for manual injection or a range of autosampler options can be added.

The structure of cannabidiol (CBD), one of 400 active compounds found in cannabis.

GC enables the rapid and accurate identification and quantification of the THC:CBD ratio. This is important for companies which are carrying out self-testing as it allows their customers to have assurances in the short term over the quality of their product, as well as reducing any potential risks to public health.

An example of this in practice is the use of GC by Dutch company Shamanics which carries out testing service for coffee shops in the Netherlands. The company conducts terpene analysis and potency testing to assure the quality of the products it supplies, with a portable GC, which offers the flexibility required without any established guidelines on testing in place.

When testing for potency using the GC, they look for total THC and CBD by converting the acidified versions of the cannabinoids into neutral forms within the heat of the GC injector. The process has flexibility which means that if they need to see both the acidified and neutral versions, they can do this by derivatizing the sample. The accuracy of this process is crucial to Shamanics and similar companies within the industry so that they can accurately judge the quality of a product, and relay this information to retailers and consumers.

The future of GC in standardized testing

While the growing availability of portable GC instruments and the increasing sophistication of individual labs means more companies are able to self-test products, there is still a significant hurdle to overcome in terms of standardising and regulating both the medical and recreational cannabis markets. Where regulation is brought in it should be consistent across states and countries and most importantly, it should be monitored and enforced. In the meantime, responsible producers are using the technology available to them to provide consumers with guarantees that their cannabis products are safe and of a high quality.

February 7, 2018

Managing Cannabis Testing Lab Workflows using LIMS

By Dr. Susan Audino

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With the state led legalization of both adult recreational and medical cannabis, there is a need for comprehensive and reliable analytical testing to ensure consumer safety and drug potency. Cannabis-testing laboratories receive high volumes of test requests from cannabis cultivators for testing quantitative and qualitative aspects of the plant. The testing market is growing as more states bring in stricter enforcement policies on testing. As the number of testing labs grow, it is anticipated that the laboratories that are now servicing other markets, including high throughput contract labs, will cross into cannabis testing as regulations free up. As the volume of tests each lab performs increases, the need for laboratories to make effective use of time and resource management, such as ensuring accurate and quick results, reports, regulatory compliance, quality assurance and many other aspects of data management becomes vital in staying competitive.

Cannabis Testing Workflows

To be commercially competitive, testing labs offer a comprehensive range of testing services. These services are available for both the medical and recreational cannabis markets, including:

Detection and quantification of both acid and neutral forms of cannabinoids
Screening for pesticide levels
Monitoring water activity to indicate the possibility of microbiological contamination
Moisture content measurements
Terpene profiling
Residual solvents and heavy metal testing
Fungi, molds, mycotoxin testing and many more

Although the testing workflows differ for each test, here is a basic overview of the operations carried out in a cannabis-testing lab:

Cannabis samples are received.
The samples are processed using techniques such as grinding and homogenization. This may be followed by extraction, filtration and evaporation.
A few samples will be isolated and concentrated by dissolving in solvents, while others may be derivatized using HPLC or GC reagents
The processed samples are then subjected to chromatographic separation using techniques such as HPLC, UHPLC, GC and GC-MS.
The separated components are then analyzed and identified for qualitative and quantitative analysis based on specialized standards and certified reference materials.
The quantified analytical data will be exported from the instruments and compiled with the corresponding sample data.
The test results are organized and reviewed by the lab personnel.
The finalized test results are reported in a compliant format and released to the client.

In order to ensure that cannabis testing laboratories function reliably, they are obliged to follow and execute certain organizational and regulatory protocols throughout the testing process. These involve critical factors that determine the accuracy of testing services of a laboratory.

Factors Critical to a Cannabis Testing Laboratory

Accreditations & Regulatory Compliance: Cannabis testing laboratories are subject to regulatory compliance requirements, accreditation standards, laboratory practices and policies at the state level. A standard that most cannabis testing labs comply to is ISO 17025, which sets the requirements of quality standards in testing laboratories. Accreditation to this standard represents the determination of competence by an independent third party referred to as the “Accreditation Body”. Accreditation ensures that laboratories are adhering to their methods. These testing facilities have mandatory participation in proficiency tests regularly in order to maintain accreditation.
Quality Assurance, Standards & Proficiency Testing: Quality assurance is in part achieved by implementing standard test methods that have been thoroughly validated. When standard methods are not available, the laboratory must validate their own methods. In addition to using valid and appropriate methods, accredited laboratories are also required to participate in appropriate and commercially available Proficiency Test Program or Inter-Laboratory Comparison Study. Both PT and ILC Programs provide laboratories with some measure of their analytic performance and compare that performance with other participating laboratories.
CloudLIMS Cannabis Testing LIMS: Multi-analyte Configuration for Cannabis Testing Services
Real-time Collaboration: Testing facilities generate metadata such as data derived from cannabis samples and infused products. The testing status and test results are best served for compliance and accessibility when integrated and stored on a centralized platform. This helps in timely data sharing and facilitates informed decision making, effective cooperation and relationships between cannabis testing facilities and growers. This platform is imperative for laboratories that have grown to high volume throughput where opportunities for errors exist. By matching test results to samples, this platform ensures consistent sample tracking and traceability. Finally, the platform is designed to provide immediate, real-time reporting to individual state or other regulatory bodies.
Personnel Management: Skilled scientific staff in cannabis-testing laboratories are required to oversee testing activities. Staff should have experience in analytical chromatography instruments such as HPLC and GC-MS. Since samples are often used for multi-analytes such as terpenes, cannabinoids, pesticides etc., the process often involves transferring samples and tests from one person to another within the testing facility. A chain of custody (CoC) is required to ensure traceability and ‘ownership’ for each person involved in the workflow.

LIMS for Laboratory Automation

Gathering, organizing and controlling laboratory-testing data can be time-consuming, labor-intensive and challenging for cannabis testing laboratories. Using spreadsheets and paper methods for this purpose is error-prone, makes data retrieval difficult and does not allow laboratories to easily adhere to regulatory guidelines. Manual systems are cumbersome, costly and lack efficiency. One way to meet this challenge is to switch to automated solutions that eliminate many of the mundane tasks that utilize valuable human resources.^. Laboratory automation transforms the data management processes and as a result, improves the quality of services and provides faster turnaround time with significant cost savings. Automating the data management protocol will improve the quality of accountability, improve technical efficiency, and improve fiscal resources.

Real Time Test Status in CloudLIMS

A Laboratory Information Management System (LIMS) is a software tool for testing labs that aids efficient data management. A LIMS organizes, manages and communicates all laboratory test data and related information, such as sample and associated metadata, tests, Standard Operating Procedures (SOPs), test reports, and invoices. It also enables fully automated data exchange between instruments such as HPLCs, GC-FIDs, etc. to one consolidated location, thereby reducing transcription errors.

How LIMS Helps Cannabis Testing Labs

LIMS are much more capable than spreadsheets and paper-based tools for streamlining the analytical and operational lab activities and enhances the productivity and quality by eliminating manual data entry. Cloud-enabled LIMS systems such as CloudLIMS are often low in the total cost of acquisition, do not require IT staff and are scalable to help meet the ever changing business and regulatory compliance needs. Some of the key benefits of LIMS for automating a cannabis-testing laboratory are illustrated below [Table 1]:

Key Functionality	Benefit
Barcode label designing and printing	Enables proper labelling of samples and inventory Follows GLP guidelines
Instant data capture by scanning barcodes	Facilitates quick client registration and sample access
360⁰ data traceability	Saves time and resources for locating samples and other records
Inventory and order management	Supports proactive planning/budgeting and real time accuracy
Custodian management	Promotes overall laboratory organization by assigning custodians for samples and tests Maintains the Chain-of-custody (CoC)
Test management	Accommodates pre-loaded test protocols to quickly assign tests for incoming samples
Accounting for sample and inventory quantity	Automatically deducts sample and inventory quantities when consumed in tests
Package & shipment management	Manages incoming samples and samples that have been subcontracted to other laboratories
Electronic data import	Electronically imports test results and metadata from integrated instruments Eliminates manual typographical errors
Report management	Generates accurate, customizable, meaningful and test reports for clients Allows user to include signatures and additional sections for professional use
21 CFR Part 11 compliant	Authenticates laboratory activities with electronic signatures
ISO 17025 accreditation	Provides traceable documentary evidence required to achieve ISO 17025 accreditation
Audit trail capabilities	Adheres to regulatory standards by recording comprehensive audit logs for laboratory activities along with the date and time stamp
Centralized data management	Stores all the data in a single, secure database facilitating quick data retrieval
Workflow management	Promotes better data management and resource allocation
High-configurability	Enables modification of screens using graphical configuration tools to mirror testing workflows
State compliance systems	Integrates with state-required compliance reporting systems and communicates using API
Adheres to regulatory compliance	Creates Certificates of Analysis (CoA) to prove regulatory compliance for each batch as well as batch-by-batch variance analysis and other reports as needed.
Data security & confidentiality	Masks sensitive data from unauthorized user access Cloud-based LIMS encrypts data at rest and in-transit while transmission between the client and the server
Global accessibility	Cloud-based LIMS provides real-time access to laboratory data from anytime anywhere
Real-time collaboration	Cloud-based LIMS enhances real-time communication within a laboratory, between a laboratory and its clients, and across a global organization with multiple sites

Table 1. Key functionality and benefits of LIMS for cannabis testing laboratories

Upon mapping the present day challenges faced by cannabis testing laboratories, adopting laboratory automation solutions becomes imperative. Cloud-based LIMS becomes a valuable tool for laboratory data management in cannabis testing laboratories. In addition to reducing manual workloads, and efficient resource management, it helps labs focus on productive lab operations while achieving compliance and regulatory goals with ease.

For more information on this, check out a webinar here: Webinar: How to Meet Cannabis Testing Standards and Regulatory Requirements with LIMS by Stephen Goldman, laboratory director at the State of Colorado certified Cannabis testing facility, PhytaTech.