Using molecular biology and biochemistry to detect blood doping

Posted on June 19, 2020June 30, 2020 by Michele Arduengo

Monitoring the use of performance-enhancing substances among athletes is complex and the requirements for tests and assays that detect use of such substances have changed significantly over the last few decades.

The haematological (blood) module of Athlete Biological Passport was adopted December 1, 2009 (ABP) by the World Anti-Doping Agency. The module sets out standard protocols to monitor doping of professional athletes by looking at changes in biological parameters, without relying on the detection of illegal compounds in body fluids. Such biological methods eliminate the need to develop and validate a test to detect every new compound that can be used for doping. The current version of the ABP, adopted in 2014, also adds monitoring of certain steroid use indicators from urine samples.

Blood doping which aims at increasing red blood cells so that more oxygen can be transported to muscles to increase stamina or performance is particularly difficult to detect. There are typically three ways that it is accomplished: use of erythropoietin (EPO) or synthetic oxygen carriers and blood transfusions. While transfusions of large volumes of blood or use of EPO can be detected, microdosing EPO or transfusing smaller volumes of packed red blood cells is much harder to detect.

Nicolas Leuenberger and colleagues at the Swiss Laboratory for Doping Analysis have developed a method to detect blood doping. In addition to addressing the detection of blood doping, his laboratory is also concerned about easing the transport and storage requirements for samples and ensuring that sample collection does not adversely affect athlete performance.

Improving Collection and Storage of Blood Samples

Because sample collection and storage are so critical to accurate test results, any new assays developed to detect blood doping benefit from ease of collection and storage. The Leuenberger laboratory investigated the use of the TAP™ Push Button collection device, which is billed as a simple method for blood collection that is easy to use and eliminates the need for painful needle sticks or finger pricks that can affect athlete performance. After TAP collection, 20µl of blood from the device was placed on to filter paper and dried (dried blood samples; DBS), which are much easier to store and transport from collection site to laboratory.

An RNA Biomarker for Blood Doping

Blood withdrawal and autologous transfusion or recombinant human EPO injection stimulate erythropoiesis and immature red blood cells can be distinguished based on their gene expression profiles. One of the genes that is expressed by immature red blood cells is aminoleuvulinate synthase 2, a gene that encodes an enzyme ALAS2 involved in the synthesis of heme, a pathway active during RBC maturation. RNA transcripts are unstable and tend to degrade rapidly, so isolating linear RNA transcripts from a collected sample can be difficult. However circular RNAs (circRNAs) are a class of RNA molecule produced by the backsplicing of pre-mRNAs that are high in abundance, quite stable and maintain cell-type specific expression. The Leuenberger laboratory developed a method for measuring the linear and circular forms of ALAS2 RNA in DBS to monitor erythropoiesis.

One of the greatest challenges in developing this protocol was achieving efficient RNA extraction from only 20ul of dried blood. Leuenberger and his colleagues adopted a two-step purification; beginning with a phenol:chloroform extraction on the DBS followed by a further purification on the Maxwell® RSC automated instrument, using the Maxwell RSC miRNA Serum and Plasma kit. Switching from a manual to an automated method for the second step was crucial. It reduced chances of contamination as well reduced pipetting errors, without compromising good quality and yield of RNA therefore contributing to assay reproducibility. To normalize volumes within the blood spot, the protocol uses RNA produced by housekeeping genes. The work to automate the assay has been published in Bioanalysis.

What’s Next

This protocol is being tested to see if microdosing of EPO or small transfusions can also be detected by monitoring ALAS2 RNA expression in DBS. The Swiss laboratory of Doping Analysis is also in the process of developing a method to detect gene doping by isolating plasmid DNA from whole blood samples, using the Maxwell RSC.

Additionally, the collection and storage methods used have implications for the clinic, especially for patients that need routine blood monitoring. The ability to isolate circular RNAs shows promise in forensic applications to identify body fluids.

RNA Extraction for Clinical Testing—Do Not Try this with Home-brew

Posted on March 24, 2020April 22, 2020 by Michele Arduengo

This blog was written with much guidance from Jennifer Romanin, Senior Director IVD Operations and Global Service and Support, and Ron Wheeler, Senior Director, Quality Assurance and Regulatory Affairs at Promega.

A Trip Down Memory Lane

Back in the day when we all walked two miles uphill in the snow to get to our laboratories, RNA and DNA extraction was a home-brew experience. You made your own buffers, prepped your own columns and spent hours lysing cells, centrifuging samples, and collecting that fluorescing, ethidium bromide-stained band of RNA in the dark room from a tube suspended over a UV box. Just like master beer brewers tweak their protocols to produce better brews, you could tweak your methodology and become a “master isolater” of RNA. You might get mostly consistent results, but there was no guarantee that your protocol would work as well in the hands of a novice.

Enter the biotechnology companies with RNA and DNA isolation kits—kits and columns manufactured under highly controlled conditions delivering higher quality and reproducibility than your home-brew method. These systems have enabled us to design ever more sensitive downstream assays–assays that rely on high-quality input DNA and RNA, like RT-qPCR assays that can detect the presence of a specific RNA molecule on a swab containing only a few hundred cells. With these assays, contaminants from a home-brew isolation could result in false positives or false negatives or simply confused results. Reagents manufactured with pre-approved standard protocols in a highly controlled environment are critical for ultra sensitive tests and assays like the ones used to detect SARS-CoV-2 (the virus that causes COVID-19).

The Science of Manufacturing Tools for Scientists

There are several criteria that must be met if you are producing systems that will be sent to different laboratories, used by different people with variable skill sets, yet yield results that can be compared from lab to lab.

Building a Collaborative Research Network to Address a Rare Disease

Posted on February 29, 2020March 1, 2020 by Michele Arduengo

Early June 2016

Chris had extreme leg pain off and on for about a month. Pain that came and went, creeping in slowly but sometimes with extreme intensity. Based on x-rays an orthopedist diagnosed a torn hamstring that was on the mend. We were sent home to rest and ice his muscles.

One Sunday Chris played in the pool for 5 hours straight and didn’t wince once. The following week he was fine so he went to soccer practice on Wednesday and swim team practice the next day. At 11:30 that night he woke up screaming in pain. Same leg. Same spot. Back again.

Late June 2016

We were on vacation in Greece. The pain started again, severe and intense and scary, so bad he couldn’t sleep lying down in a bed. Desperate, we ended up in a Greek hospital… the local pediatrician was wonderful and recommended we fly home and see an orthopedic doctor as soon as possible…a terrifying flight home: No answers and a pit in our stomachs. Chris was in a wheelchair.

July 2016

We finally got the orthopedist to order the MRI. The MRI results were what every parent fears: “leukemia or lymphoma” and a referral to an oncologist. After many invasive tests, the oncologist said it was probably not cancer. We felt such relief, but we were left with no answers for all his pain. We moved on to infectious disease.

August 2016

The infectious disease specialist said they could not culture anything so they didn’t believe that Chris had an infection. Again, incomplete answers. We were then passed off to rheumatology. The frustration of not having any answers and our child still in pain was heart-breaking, isolating, and terrifying.

Based on the bone biopsy and MRIs the rheumatologists finally gave Chris a diagnosis: Chronic Recurrent Multifocal Osteomyelitis (CRMO often pronounced “chromo” for short).

The good news: it was not cancer; the bad news: very little is known about CRMO because it is a rare disease.

Rapid Test to Detect SARS-2-CoV Developed in Brazil

Posted on February 22, 2020February 22, 2020 by Michele Arduengo

The Virology lab at the Universidade Federal da Bahia (UFBA), led by Dr. Gúbio Soares, has developed a fast and specific real-time PCR assay using GoTaq 1-Step RT-qPCR for detection of SARS-2-CoV (the coronavirus previously named 2019-nCoV), which causes the respiratory disease COVID-19. The Maxwell RSC instrument is used for automated extraction of RNA from oral-pharyngeal secretion collected by swab or bronchial wash prior to the assay. This coronavirus-specific assay can shorten the time to identify SARS-2-CoV from 48 hours to 3 hours (1), providing critical information to public health officials in a timely fashion.

“Promega has been providing all our reagents for standard and real-time PCR and also for nucleic acid extraction. It’s a company I can rely on the relationship; they are our partners and have provided excellent support both technically and financially. Promega is the base of all our assays.” Dr. Gúbio Soares.

Dr. Soares’ laboratory has experience developing assays to identify and detect emerging viral pathogens. Their laboratory first identified the Zika virus as the etiologic agent in the large outbreaks of acute exanthematous illness (AEI) in northeast Brazil in April 2015 (2). Zika was eventually declared a public health emergency of international importance by the World Health Organization in February 2016, after increased incidence of microcephaly was detected in the infants of women infected during pregnancy. Many of the lessons learned in the management of the Zika crisis are informing how scientists are addressing SARS-2-CoV. The Zika response was characterized by a collaborative spirit to share data, samples and resources among scientific labs across the globe.

Below is a video link from Brazil (audio in Portuguese) describing Dr. Soares’ group work on SARS-2-CoV. https://globoplay.globo.com/v/8302334/

Sources Cited

TV Bahia (2020) Test developed at UFBA can identify coronavirus in 3 hours, says researcher. [Internet: https://g1.globo.com/ba/bahia/noticia/2020/02/07/teste-desenvolvido-na-ufba-pode-identificar-coronavirus-em-3h-diz-pesquisador.ghtml Accessed: February 19, 2020]
Sandler, N. (2016) Zika: Personal Perspectives, Global Responses Promega Corporation. [Internet: https://www.promega.com/Resources/PubHub/Inspiration/Zika%20Perspectives%20Responses/ Accessed : February 19, 2020]

The Race to Develop New Therapeutics Against Coronaviruses

Posted on February 11, 2020February 12, 2020 by Michele Arduengo

Once the purview of virology researchers, the word “coronavirus” is now part of the vernacular in the mainstream media as reports of quarantined cruise ships (1) and makeshift hospitals (2) fill our online news feeds. While there is currently no approved anti-viral treatment for coronavirus infection (3), a team led by researchers from Vanderbilt University recently published work characterizing the anti-CoV activity of a compound, which they now plan to test against 2019-nCoV (4).

Developing New Therapeutics Against Coronaviruses

Coronaviruses (CoVs) are enveloped, single-stranded RNA viruses that exhibit cross-species transmission—the ability to spread quickly from one host (e.g., civet) to another (e.g., human). Scientists classify CoVs into four groups based on the nature of the spikes on their surface: alpha (α), beta (ß), gamma (γ) and delta (δ, 1). Only the alpha- and beta-CoVs can infect humans. Four coronaviruses commonly circulate within human populations: Human CoV 229E (HCoV229E), HCoVNL63, HCoVOC43, and HCoVHKU1. Three other CoVs have emerged as infectious agents, jumping from their normal animal host species to humans: SARS-CoV, MERS-CoV and most recently, 2019-nCoV (5).

TE micrograph of a single MERS-CoV — Digitally colorized transmission electron micrograph reveals ultrastructural details of a single Middle East respiratory syndrome coronavirus (MERS-CoV) virion. Image credit: National Institute of Allergy and Infectious Diseases

The need for an effective, broad spectrum treatment against HCoVs, has been brought into sharp focus by the recent outbreak of the 2019 Novel Coronavirus (2019-nCoV; 6).

Popular Papers from Promega Authors

Posted on January 21, 2020January 21, 2020 by Michele Arduengo

Promega is a chemistry and instrument supplier to scientists in diverse industries and research labs around the world. True. But we are more than just a supply company; we are scientists dedicated to supporting the work of other scientists. We want the science behind the technologies we develop to be both vetted and valued by the scientific community at large, which is one reason our scientists take the time to prepare and submit manuscripts to peer-reviewed journals. Here we call out some of our published research papers that were highly read in 2019. In the journal ACS Chemical Biology alone, five Promega-authored papers were among the top 10 most read papers in 2019. Here’s a quick review of the highlights from these ACS papers.

MSI Testing of Tumor Cells for Better Tailored Treatment

Posted on November 7, 2019November 13, 2019 by Michele Arduengo

There are as many different cancers as there are people with cancer. Unlike infectious diseases, which are caused by pathogens that are foreign to our bodies (bacteria, viruses, parasites), cancer cells arise from our body—our own cells gone rogue. Because cancer is a dysfunction of a person’s normal cells, every cancer reflects the genetic differences that mark us as individuals. Add to that environmental influences like diet, tobacco use, the microbiome and even occupation, and the likelihood of finding a “single” pharmaceutical cure for cancer becomes virtually impossible.

But, while looking for a single cure for all cancers may not be a fruitful activity, defining a best practice for understanding the genetic and protein biomarkers of individual tumors is proving worthwhile.

qPCR: The Very Basics

Posted on August 2, 2019January 2, 2020 by Michele Arduengo

Real-Time (or quantitative, qPCR) monitors PCR amplification as it happens and allows you to measure starting material in your reaction. Data are presented graphically rather than as bands on a gel.

For those of us well versed in traditional, end-point PCR, wrapping our minds and methods around real-time or quantitative (qPCR) can be challenging. Here at Promega Connections, we are beginning a series of blogs designed to explain how qPCR works, things to consider when setting up and performing qPCR experiments, and what to look for in your results.

First, to get our bearings, let’s contrast traditional end-point PCR with qPCR.

End-Point PCR	qPCR
Visualizes by agarose gel the amplified product AFTER it is produced (the end-point)	Visualizes amplification as it happens (in real time) via a detection instrument
Does not precisely measure the starting DNA or RNA	Measures how many copies of DNA or RNA you started with (quantitative = qPCR)
Less expensive; no special instruments required	More expensive; requires special instrumentation
Basic molecular biology technique	Requires slightly more technical prowess

Quantitative PCR (qPCR) can be used to answer the same experimental questions as traditional end-point PCR: Detecting polymorphisms in DNA, amplifying low-abundance sequences for cloning or analysis, pathogen detection and others. However, the ability to observe amplification in real time and detect the number of copies in the starting material can quantitate gene expression, measure DNA damage, and quantitate viral load in a sample and other applications.

Anytime that you are preforming a reaction where something is copied over and over in an exponential fashion, contaminants are just as likely to be copied as the desired input. Quantitative PCR is subject to the same contamination concerns as end-point PCR, but those concerns are magnified because the technique is so sensitive. Avoiding contamination is paramount for generating qPCR results that you can trust.

Use aerosol-resistant pipette tips, and have designated pipettors and tips for pre- and post-amplification steps.
Wear gloves and change them frequently.
Have designated areas for pre- and post-amplification work.
Use reaction “master mixes” to minimize variability. A master mix is a ready-to-use mixture of your reaction components (excluding primers and sample) that you create for multiple reactions. Because you are pipetting larger volumes to make the reaction master mix, and all of your reactions are getting their components from the same master mix, you are reducing variability from reaction to reaction.
Dispense your primers into aliquots to minimize freeze-thaw cycles and the opportunity to introduce contaminants into a primer stock.

These are very basic tips that are common to both end-point and qPCR, but if you get these right, you are off to a good start no matter what your experimental goals are.

If you are looking for more information regarding qPCR, watch this supplementary video below.

Meet the Mighty Masked Masters of Measurement

Posted on July 3, 2019July 3, 2019 by Michele Arduengo

Scientific investigation is an iterative process, for which reproducibility is key. Reproducibility, in turn, requires accuracy and precision—particularly in measurement. The unsung superheroes of accuracy and precision in the research lab are the members of your local Metrology Department. According to Promega Senior Metrologist, Keela Sniadach, it’s good when the metrology department remains unsung and behind the scenes because that means everything is working properly.

Holy Pipettes, Scientists! We have a metrology department?! Wait…what’s metrology again?

Metrology (the scientific study of measurement) got its start in France, when it was proposed that an international length standard be based on a natural source. It was from this start that the International System of Units (SI), the modern metric system of measurement, was born.

Metrology even has its own day: May 20, which is the anniversary of the day that the International Bureau of Weights and Measures (BIPM) was created by the Meter Convention in Paris in 1875. The job of BIPM is to ensure worldwide standards of measurement.

For life scientists, metrology centers around making sure the equipment used everyday—from pipettes to heating blocks to centrifuges—is calibrated and measuring correctly. Continue reading “Meet the Mighty Masked Masters of Measurement”

Promega Connections

Thoughts, tech tips and news about science from Promega Corporation

Author: Michele Arduengo

Using molecular biology and biochemistry to detect blood doping