With the rapid progress of immune-monitoring drug development, flow cytometry has found itself increasingly at the forefront of clinical trial assessment of safety and efficacy. This is not without challenges since flow cytometry analysis can be complicated and expensive, too often employs idiosyncratic experimental and analytical methods. So how can a platform without standardized methods and processes, be successfully applied to evaluate clinical endpoints?

Several novel approaches to instrument calibration and experimental design are now helping to establish harmonization of flow cytometry across multiple clinical labs. In this blog we explore the first step in this workflow and the importance of sample collection and shipment processes for clinical trial samples.

Sample Collection

With most clinical trial now performed using centralized networks, the standardization of efficient and reproducible sample collection and processing is a critical consideration. Sample integrity begins as soon as the blood sample is collected, even the timing of the collection can result in circadian variation in cellular profiles [1, 2, 3]. The anticoagulant used can also influence the functional phenotype of cells, as well as the stability of different cell types and biomarkers. Typically, the expression of surface markers and population distribution are more stable in Na-Heparin blood than EDTA. However, Na-Heparin use is associated with a more rapid decrease in leukocyte counts over time.

Fixation of Samples

Fixation can help preserve biomarkers and provide disinfection for infectious samples, but the fixation process, and timing should be optimized and standardized for clinical applications. The most widely used fixative agents for flow cytometry applications includes basic paraformaldehyde (PFA) treatment (1-4%) as well as specialized reagents such as TransFix™, Cyto-Chex™. In our hands, we find absolute count preservation is typically better in Cyto-Chex fixed peripheral blood samples. PFA has several drawbacks including the fixation of red blood cells that precludes their later lysis and should be avoided for panels examining granulocytes since it represses their responsiveness to stimulation and can damage several surface epitopes including CD13, CD32, and CD62L.  It should be noted that the distribution of the main leukocyte subsets changes over type with most fixation approaches, with a gradual loss of granulocytes after ~24 hours.  However, since the sample quality for flow cytometry analysis relies on both cell count and biomarker expression, each of these should be assessed for the specific goals of the clinical program.

Cryopreservation of Samples

Cryopreservation can overcome many of the issues of sample stability and preservation but requires more processing at the clinical sites which in itself can introduce sample variability. Ficoll preparation of PBMCs for freezing requires significant technical processing and may induce biased distribution of specific immune cell types such as CD8+ T-cells [7]. Simple methods for rapid whole blood freezing have been developed, for example using CryoStor™ CS10 [8, 9, 10]. A key advantage to these simple methods is that they minimize the volume of blood required, which is especially critical for pediatric studies.

Cooling and thawing rates both can impact the quality of the cryopreserved sample. A target cooling rate of -1˚C per minute or slower is generally preferred since it generates smaller, less damaging ice crystals. Warming rates are less impactful, but generally, rapid warming to enable rapid removal of DMSO is recommended [11].

The Importance of Shipping Conditions

Shipping conditions may also impact the quality of the samples. The key factors being the duration of the shipping process and the temperature. Most carriers offer next day morning delivery and providing a tracking number to the recipient lab is essential if missed and lost shipments are to be minimized. Prolonged shipping of fresh blood can result in the selective loss of cell populations such as eosinophils and neutrophils [4]. Furthermore, biochemical changes including changes in blood gases, decreased pH, and changes in amino acids, carbohydrate moieties, lipids and cofactors can all influence cell viability, in a temperature dependent manner [5]. Whereas monocytes and natural killer cell subsets are relatively stable at 4˚C, lymphocyte stability is affected by lower temperatures, in fact optimal temperatures for T-cells is described as 14-16˚C [6].

Each sample should be labelled with two unique patient identifiers, along with the initials of the phlebotomist, date and time of sample collection. Samples should be packaged within secondary packaging materials to protect the samples and adequate absorbent material to absorb the entire contents from any damaged receptacles. The outer package should consist of a polystyrene foam insulated, corrugated fiberboard shipper. According to 49 CFR 173.199(b), packages containing specimens transported by air, must be capable of withstanding, without leaking, an internal pressure producing a pressure differential of not less than 95 kPa (0.95 bar, 14 psi). So, it is important to verify compliance with the manufacture of the blood tube or secondary packaging material before use. The polystyrene foam-insulated, corrugated fiberboard shipper should be lined with absorbent material and a single layer of refrigerator packs laid on the bottom of the container. The packaged specimens should you plated directly on top of the packs with cushioning material in place to minimize shifting while in transit. A second layer of refrigerator packs can then be applied to help maintain a temperature of 1-10˚C. The shipping manifest should be placed into a sealable plastic bag for protection and the package well sealed, before placing of the UN 3373 label and inclusion of the words “Biological Substance, Category B”

Final Thoughts

Central to high quality flow cytometry analysis is sample quality, and for clinical trials this can represent an area of significant variability. Since most clinical trials now involve multiple collection site, it is important that protocols for sample collection, processing and shipping are established and adhered to according to the program needs.

Our team at FlowMetric have experience working with all different types of clinical specimens, for a wide range of flow cytometry applications. We can provide you with guidance on the initial sample collection and best practical approaches for the most favorable sample preservation, to ensure optimal determination of your clinical end points.

References

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  2. Bollinger T, et al. 2009 Clin Exp Immunol. 155:231–238. [PubMed: 19040608]
  3. Dimitrov, S. et. al. 2004. Brain Behav Immun. 18:341–348. [PubMed: 15157951]
  4. Park, Y. M. and Bocher, B. S. 2010 Eosinophils survival and apoptosis in health and disease. Allergy, Asthma Immunol. Res. 2 (2), 87-101.
  5. Ng. et. al., 2012. Optimal cellular preservation for high dimensional flow cytometric analysis of multicenter trials. J. Immunol. Methods. 385 (1), 79-89.
  6. Ekong, et. al. 1992 The effect of the temperature and duration of sample storage on the measurement of lymphocyte subpopulations from HIV-1 positive and control subjects. J. Immunol. Methods 151 (1-2) 217-225.
  7. Appay V. et. al. 2006 Immuno-monitoring of CD8+ T cells in whole blood versus PBMC samples. J Immunol Methods. 309(1–2):192–9.
  8. Langenskiöld C. et. al. 2018 Determination of blood cell subtype concentrations from frozen whole blood samples using TruCount beads. Cytometry B Clin Cytom. 94(4):660–6.
  9. Verschoor C. P. et. al. 2018 A comprehensive assessment of immunophenotyping performed in cryopreserved peripheral whole blood. Cytometry B Clin Cytom. 94(5):662–70
  10. Braudeau, C. et. al. 2020 An easy and reliable whole blood freezing method for flow cytometry immunophenotyping and functional analyses. Cytometry Part B. Clinical Cytom. Doi: 10.1002/cyto.b.21994
  11. Baboo, J. et. al. 2019 The impact of varying cooling and thawing rates on the quality of cryopreserved human peripheral blood T-cells. Scientific Reports 9 article number 3417.