The facilities at PeploBio meet the only global medical laboratory standard, ISO 15189. Our flow cytometry workflows are streamlined by the Clinical and Laboratory Standard Institute (CLSI), the British Pharmacopoeia, and the International Council for Harmonisation (ICH) to ensure our services are held to a high standard.
The pitfalls associated with non-standardised clinical flow cytometry assays, the requisite for standardisation, and recommended practice guidelines are covered in collaborative publications jointly authored by the International Council for Standardization of Haematology (ICSH) and the International Clinical Cytometry Society (ICCS) [1-5]. Our adherence to these standards provides the framework for PeploBio to align with global harmonisation and standardisation to reduce inter-laboratory and inter-operator variabilities in data generation and ensure product reproducibility, quality, safety, and efficacy of Advanced Therapy Medical Products (ATMPs) throughout its product lifecycle [6]. Alongside our ICO registration and BIVDA membership, we have the capability to develop robust, validated, and quality-assured assays that align with local regulatory requirements.
Further information on the flow cytometry standardisation framework we employ at PeploBio laboratories can be accessed directly through the websites:
Clinical and Laboratory Standards Institute guidelines, H62, EP06, EP17-A2 and H52-A2
British Pharmacopeia, Advanced Therapy Medicinal Products Guidance: Applications of Flow Cytometry
International Clinical Cytometry Society, Quality and Standards modules
European Society for Clinical Cell Analysis, The ESCCA 2022 Annual Conference presentation
Cell-based flow cytometry (FC) assays often fall into the criterion of Laboratory Developed Tests (LDTs) and therefore must be developed to meet Quality Assurances (QA) requirements. This section will explore the processes involved in the development of FC assays at PeploBio Ltd.
Controls are essential components of FC assay development for clinical, research, and diagnostic applications. There are many types of controls, each one serving as a reference point to ensure the accuracy, reliability, and robustness of the assay results.
Negative Control or a blank typically consists of the sample matrix in the absence of target cells or measurand. A blank is used to establish the baseline signal and background noise. This control ensures that the assay is specific to the target and not influenced by the sample matrix.
Positive Control or stained positive control consists of cells that are known to express the target antigen at high levels and are stained with specific antibodies or fluorochromes. It verifies if the assay can detect the target reliably.
Fluorescence Minus One (FMO) Control is the evaluation of a sample stained with all antibodies to target components of interest except for one. FMO controls are essential in multiparametric FC assay to gate positive and negative populations accurately. FMO controls are setup for each fluorochrome separately in multicolour FC assays. FMO controls can also be utilised to establish the Limit of Blank (LOB) for determining analytical sensitivity during FC assay validations.
Internal Controls are controls included within the assay to monitor the entire assay process. These controls can be added at various steps, such as during sample preparation or staining, to detect issues such as cell loss or staining variability.
Instrument Controls involve running microbeads or particles with known fluorescence characteristics through the flow cytometer to monitor instrument performance, reliability, calibration, and cross instrument standardisation. Instrument controls are important for Quality Control (QC). Instrument setup and controls at PeploBio are discussed in detail in the Quality Control and Companion Diagnostics (CDx) section.
Isotype controls are used to test non-specific binding of antibodies and are used for gating in flow cytometry. As discussed in the British Pharmacopoeia: Applications of flow Cytometry, these types of controls are imperfect, but can be beneficial when measuring intracellular staining.
Compensation Controls are used to correct for spectral overlap between fluorochromes in multi-colour FC assays. Compensation controls are single-stained samples or compensation beads for each fluorochrome used in the assay. Compensation controls are essential to reduce false positives and for accurate data analysis. As advised in the British Pharmacopoeia, compensation is assessed regularly at PeploBio and in situations where laser fluctuations are detected, after lasers are replaced and when using new antibody lots [6].
Staff education and training, Quality Control (QC) procedures (Internal QC [IQC] and External Quality Assessment [EQA]) are guaranteed at PeploBio to generate precise and accurate FC data [4]. PeploBio participates in EQA programmes operated by the UK NEQAS and the College of American Pathologists (CAP) to ensure we our services are held to the highest standards. Our bioanalytical team is formed of highly qualified individuals with diverse research backgrounds holding Master’s degrees, PhDs or equivalent. QC checks are performed at the start of the day and/or the start of a new batch.
QC material can be used to monitor performance over time and detect assay or instrument deviations from expected results. QC samples are samples of known characteristics that are used as a reference to perform cross checks against predefined expected results and other clinical samples. Stabilized QC samples are used as a full process control, to ensure constancy in staining, lysing, acquisition and analysis between batches and days [4]. There are various types of microbead designed to test FC instrument parameters [4]. At PeploBio, QC on our BD FACSLyric flow cytometers are performed using manufacturer’s BD CS&T beads to achieve standardised QC for the instrument optics, electronics, fluidics, and detector voltage adjustments. According to BD’s data, performing daily setup and checks using CS&T adjusts more than 70 parameters to stabilise the BD FACSLyric, achieving a Coefficient of Variation <0.4%. The BD CS&T fluorospheres are also used to optimise fluorescence compensation during performance QCs every 60 days.
A key aspect of the BD FACSLyric instrument relevant to the services provided by PeploBio, is adherence of the software to the main elements of the FDA’s Title 21 of the Code of Federal Regulations (21 CFR) Part 1121 CRF part 11. The 21 CRF part 11 outlines the requirements for electronic records and electronic signatures in the pharmaceutical and healthcare industries, with a focus on data security, data integrity and regulatory compliance. The BD FACSLyric system also allows for precise assay setting transfer, delivering highly reproducible results over time and between instrument to instrument.
In line with most clinical laboratories, our SOPs, QC records, Quality Assurance (QA) records and Equipment Maintenance Logs are stored in secure PeploBio servers for at least 2 years [4].
At PeploBio we use FMOs to advice our gating strategy as FMOs are more effective than isotype controls [6]. We give significant attention to gating in order to accurately distinguish the cell population or antigen of interest from other cell subsets and non-specific events which must be excluded [5, 6]. An effective gating strategy is critical to ensuring assay precision.
The BD FACSLyrics Systems we use in our laboratories can run up to a rate of 35,000 events per second without compromising on quality allows for rapid detection as well as the capability to resolve rare populations. The BD FACSLyric have no limit to the number events that can be acquired and introduce negligible sample carryover. As such, our bioanalytical team can perform FC assays to meet the individual requirements of each clinical assay, whether that is for the detection and characterisation of rare cell populations or for high-throughput screening.
The performance of a FC assay can be measured by considering several parameters such as assay precision, stability, analytical measuring range, and reference ranges.
The ISO 3534-1 definition of accuracy is the degree of agreement between the average of measured quantitative value to a define standard reference [5]. It is widely acknowledged that it is extremely difficult measure accuracy due to the lack of appropriate biological reference specimen [5]. As with most laboratories, at PeploBio we set an appropriate acceptance criterion for accuracy for each assay using commercially available reference material where possible.
Linearity measurements assess how well the assay’s measurements correlate with the concentration of the analyte over a specified range [5]. Demonstrating linearity is necessary when quantifying the target analyte in FC experiments, however this is often not feasible as most FC assays generate quasi-quantitative data (e.g., expressed as cells/µl, % cells in a population or mean fluorescence intensity [MFI] values) due to the lack of a reference standard [5]. Receptor Occupancy (RO) assays, which measures the relationship between a drug and its target receptor, can be designed to be quantitative. As such, linearity is measured using a calibration curve with linear regression analysis to measure the gradient, y-intercept, and correlation coefficient value (R2).
The reportable range refers to the range of results or values that can be precisely, accurately, and reliably reported by the assay as they have met the acceptable analytical and linearity criteria [5]. The Lower Limit of Quantitation (LLOQ) helps define the lower limits of the range. The upper limit of the range is only defined in FC if extremely bright fluorescence signals are expected or if the assay requires it. The reportable range is important for quasi-quantitative FC assays and is combined with precision and linearity validations. Qualitative assays do not always require a reportable range.
Analytical Specificity determines whether the detected signal is specific to the cell marker or target antigen [5]. Analytical specificity is shaped by gating strategy and reagent cross-reactivity e.g., antibody [5]. Where possible, our bioanalytical team uses previously validated antibodies for clinical FC tested on appropriate sample types.
Precision examines intra-assay (reproducibility) and inter-assay/intermediate (repeatability) imprecision to establish conditions at which the assay reproduces consistent results [5,6]. Parameters that an introduce assay variability include biological specimen, instrument, operator, date, laboratory, location, and reagent lot [5].
Intra-assay imprecision measured by assaying at least 5 samples in triplicate within a single analytical run. At PeploBio, we often measure intra-assay imprecision by calculating the coefficient of variation (% CV = [Standard Deviation ÷ Mean] × 100%). CV normalizes variations at low levels of event detection and therefore preferred over standard deviation (SD) [5].
Inter-assay imprecision is measured by testing 3-5 independent analytical runs containing least 3 samples or commercially available QC grade whole blood for FC [5]. The instruments are powered down and recalibrated between each run. The CV is calculated.
At PeploBio, we endeavour to use homogenous and authentic samples for the measurement of assay precision. In the unlikely event this may not be achievable, artificially prepared samples or sample solution may be used as alternatives as stated in the ICH’s Validation of Analytical Procedures: Text and Methodology (2006). Our FC assays must achieve less than 20% deviation between measurements to meet our acceptability criteria for precision which ensures reliability and consistency in our results.
Sensity of FC assays is measured using Limit of Blank (LoB), Limit of Detection (LoD) and Lower Limit of Quantification (LLoQ) and is determined by the lowest observable result of a specific measurement.
Limit of Blank (LoB) is highest measured signal by the FC in samples without measurand. LOB = mean of at least 5 blanks + 1.645 SD 95% of the negative samples fall below this value. LOB can also be measured by a gating or using Florescence Minus One (FMO) control tube [5].
Limit of Detection (LoD) is the lowest level of measurand that can be reliably distinguished from the background noise (LOB) but cannot be quantified by the FC. LOD = mean of low positive samples + 1.645 SD 95% of low concentrations of measurand are detected above the value of the blank (background)
Lower Limit of Quantitation (LLoQ) is the lowest concentration of the measurand that can be quantified (LOD with bias and imprecision errors) with predefined levels of precision and accuracy.
Sample stability is defined by the latest timepoint at which the sample integrity reaches 20% deviation from the baseline measurement [5]. Stability is evaluated for every sample type under the conditions it will be transported, stored, and tested.
Specimen stability is measured by testing ≥5 samples within 2 hours of harvest, and subsequently tested at 24-, 48- and 72-hour timepoints to accommodate to transport time. The specimen stability result must not deviate more than 20% from the baseline reading to meet our acceptance criteria. PeploBio supports temperature-controlled transport of samples to our facilities to ensure minimal biological degradation and distortion of results.
Processed sample stability is evaluated to determine the time latest timepoint at which a processed sample (stained, lysed, fixed) can be acquired on the flow cytometer. Processed sample stability is measured by testing ≥5 specimens within 1 hour of processing to establish the baseline result and compare against results at appropriate subsequent timepoints. The stability of the processed sample must achieve less than 20% deviation from the baseline results to meet our acceptance criteria.
Sample carryover refers to residual specimen transfer from one sample to another on an automated FC. The BD FACSLyric flow cytometry systems we use in our laboratories have been validated by the manufacturer to ensure minimal sample carry over. BD’s brochure on the BD FACSLyric system reported that sample carryover of ≤0.1% is observed with the default sample injection tube (SIT) flush and as low as 0.05% up to 6 SIT flushes. In line with ICSH and ICCS guidelines, FC assay carryover at PeploBio is detected (to levels <1%) using beads of different fluorescent intensities. Carryover assays are performed by running negative and positive specimen in alternate and assessing the events in the final negative samples [5].
In FC, reference ranges define the minimum and maximum limits of measurand in a sample for the purpose of result interpretation. This is primarily applicable for quantitative assays but could be relevant for qualitative assays in certain circumstances e.g., in a TBNK assay.
PeploBio is a full-service clinical contract research organization (CRO) with UKAS accreditation and with facilities meeting the only global standard medical laboratory standard, ISO15189. At PeploBio, our services operate to the US and UK regulatory standards. We are ICO registered to prioritise data protection and ensure GDPR compliance and as a BIVDA member we have access to up-to-date regulatory information. Our regulatory strategies have awarded our laboratories the position as the Top Clinical Laboratory Services Company in the UK in 2022 by Life Sciences Review.
We serve the medical device manufacturing, biotechnology, and pharmaceutical industries. Our mission is to work alongside, support and bridge the scientific and medical communities to advance the global development of safe and effective medical therapeutics and devices to fight against disease such as oncology, cardiology, endocrinology and infectious disease.
