Sample Processing in the Laboratory - BAL, CSF, PK & More

Collecting samples is just the beginning of the analysis process, with a myriad of techniques and technologies used to extract meaningful data once a specimen enters the laboratory. Below, we explore some of the most widely used sample processing in laboratory methods, including BAL, CSF, PK and more.

BAL

Bronchoalveolar lavage (BAL) samples are used to analyse fluid from the lungs. Specimens are usually collected during a bronchoscopy, a procedure that sees a saline solution injected through a bronchoscope to cleanse the airways and collect a sample in the process. As samples profile the lower respiratory tract, BAL is often used to diagnose and evaluate lung diseases.

Once in the laboratory, BAL samples can be used to carry out more specific diagnostic tests, including cytopathologic stains, polymerase chain reaction (PCR) molecular assays and immunologic tests. These sample processing in laboratory tests add context to BAL specimens and allow doctors to identify specific microbial infections.

Over the past few years, next-generation diagnostic techniques like matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF) have allowed scientists to rapidly extract microbiologic data from BAL samples. PCR coupled to electrospray ionisation mass spectrometry (PCR/ESI-MS) is another diagnostic technique gaining traction in the laboratory analysis sector.

“More recently, whole-genome sequencing, including real-time metagenomic sequencing, of BAL fluid has been used to diagnose and manage viral, bacterial, and fungal pneumonias in critically ill patients with and without immunosuppression”, reads an article published in the Journal of Thoracic Disease. “In addition, shotgun sequencing of BAL fluid has been used to characterise the metagenomics and microbiome of the respiratory tract of lung transplant recipients and patients with chronic lung diseases. As whole-genome sequencing becomes more readily available in clinical laboratories, its role in the analysis of BAL fluid will likely increase.”

CSF

Cerebrospinal fluid (CSF) samples are used to detect and measure chemicals, proteins and other substances in cerebrospinal fluid. Present around the spinal cord and brain, the clear fluid acts as a protective cushion, as well as delivers nutrients and removes waste from the brain.

Specimens for CSF sample processing are collected during a spinal tap procedure, which sees a thin, hollow needle inserted into the lower spine. CSF is extracted through the needle and used to diagnose conditions that affect the brain and spine, including meningitis and demyelinating diseases such as multiple sclerosis, neuromyelitis optica and acute disseminated encephalomyelitis.

Prompt transport and storage of CSF is essential for accurate sample processing in laboratory, with the CDC recommending “Cerebrospinal fluid (CSF) should be processed in a microbiology laboratory within 1 hour after collection or inoculated into Trans-Isolate (T-I) medium for transport to the laboratory if processing within 1 hour is not feasible.”

Pharmacokinetic (PK)

PK sample processing is fundamental to the drug development industry. The process describes the study of how a drug is absorbed, distributed, metabolised and excreted within a living organism. The term is derived from the ancient Greek words “pharmakon” meaning "drug" and “kinetikos” meaning to “motion”. Basically, PK samples are used to track how a living organism affects a drug, from the moment the substance is ingested into the system to final excretion. In the drug development industry, PK data is used to determine the effectiveness of a drug and if it’s suitable for mass production. PK sample processing data is are also used to track how a drug is performing for a patient and optimise dosages.

Pharmacokinetic data is often used in conjunction with pharmacodynamic data (PD), which profiles how a drug affects a living organism, as opposed to how a living organism affects a drug. Understanding the relationship between pharmacokinetics and pharmacodynamics, a modelling technique known as PK/PD, is a key step in the drug development and approval process.

Mass spectrometry (MS) is one of the most common techniques used to study PK samples, which are usually complex. Liquid chromatography mass spectrometry (LC-MS) is a popular analytical technique and is generally carried out with a triple quadrupole mass spectrometer. LC-MS is often used alongside tandem mass spectrometry to add context to data and improve the specificity of results.

The highly sensitive nature of mass spectrometry has accelerated the concept of microdosing studies, which aim to replace animal testing with extremely low dose human trails. “The wider use of human microdosing would minimise all these animal tests by providing human-specific ADME data and, thus, identifying early those compounds destined to fail later owing to suboptimal pharmacokinetics or metabolism,” writes Gill Langley, author for correspondence at the Dr Hadwen Trust for Humane Research in the UK.

Sputum

Sputum samples are used to detect and diagnose bacterial infections in the lungs and other parts of the lower respiratory tract. While they’re usually coughed up by the patient, sputum samples can also be extracted manually by a doctor. Deep coughs are essential for the processing of sputum samples, as the specimen must contain phlegm and mucus from the lungs, not simply saliva from the mouth. Ideally sputum samples should be collected in the morning and over the course of three consecutive days.

Once obtained, sputum samples are stored in a sterile cup and transported for sample processing in laboratory. If they will be in transit for more than one hour refrigeration is necessary. Once at the laboratory, a microscope slide is used for analysis and processing of sputum samples. A staining dye and acid wash solution is used to detect the presence of mycobacterium and diagnose conditions like bronchitis and bacterial pneumonia.

Stool samples

While patients often find stool samples uncomfortable to collect, they can reveal valuable information regarding the health of the gastrointestinal system. Stool sample processing usually involves microscopic examination, as well as microbiologic, immunologic and chemical tests. Stool microscopy is a useful diagnostic tool for detecting parasitic organisms such as helminths and protozoa, as well as the presence of white blood cells, a condition known as faecal leukocytes.

Liquid chromatography is a common technique used for stool sample processing, though it can be slow and time consuming. Over the past few years, a new high-throughput technique called rapid evaporative ionisation mass spectrometry (REIMS) has revolutionised stool sample processing and allowed scientists to unlock faster and more reliable results.

Next-generation Macromolecular Crystallography (MX)

Over the past decade, Macromolecular Crystallography (MX) has set a new benchmark for research. Used by both academic researchers and global pharmaceutical companies, the technique is used to analyse the shape and structure of biological molecules. This gives scientists an in-depth overview of their function and mechanics.

The Diamond Light Source national synchrotron science facility in the UK is a global leader in Macromolecular Crystallography, with seven dedicated beamlines used to carry out MX analysis. Samples are generally in protein crystal form and transported to the facility in in liquid nitrogen to preserve integrity. The samples sent to Diamond are diverse, ranging from ancient dinosaur bones to dangerous disease proteins.

The importance of sample provenance and transparency

Sample provenance, i.e. the ability to track and trace the original source of a sample, is fundamental to the life sciences sector. Without transparency, researchers put the reliability of their data at risk. This not only wastes valuable resources but also delays the development of potentially lifesaving drugs and therapies.

Industry experts around the world are advocating for better sample provenance and transparency, particularly when it comes to the use of biospecimens to develop drugs, diagnostic techniques and vaccines. Without detailed knowledge of the origins of the biospecimen, data cannot be considered reliable and accurate. This includes valuable specimens used for blood sample processing.

Not-for-profit company Biosample Hub is working hard to address the issue, with a purpose-built online platform used to connect companies with reliable biobanks. Instead of being discarded as medical waste, Biosample Hub aims to redirect leftover clinical samples like blood and tissue to biotech and pharmaceutical research companies. With a recent State of the Discovery Nation Report of the BioIndustry Association and Medicines Discovery Catapult revealing 80% of British companies find it difficult to access patient samples, the platform is a gamechanger for the life sciences industry.

“The current system is broken" says Biosample Hub founder Robert Hewitt. "It’s not working well for biobanks or for the companies looking for samples. Therefore, I’ve made it my mission to create a solution - Biosample Hub is that solution.”

The challenges of biosample access

Despite access to a wide range of methods and instruments, collecting, accessing and analysing biosamples remains a challenge for small biotech companies looking to spearhead new developments. Unlike big pharma companies, small-scale biotech companies are often open to risk exposure and willing to explore new opportunities. This makes them central to the development of new therapies, diagnostic techniques and vaccines.

Ideas and concepts pioneered by small biotech companies are often passed on to well-funded pharmaceutical companies, where they can be transformed on a commercial scale. Unfortunately, access to the high-quality clinical samples needed to accelerate research is vastly difficult for small biotech companies. Writing on behalf of Biosample Hub, author Robert Hewitt explores the issue further in ‘The challenges of biosample access and what needs to change’