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Peripheral whole blood is the most frequent specimen type for clinical flow cytometry. But this is not always convenient for clinical trials. The selection of the sample matrix is highly nuanced and should be experimentally determined before the start of any clinical flow cytometry program. This is because distinct cell types, surface biomarkers, and activation markers are all impacted differently by sample collection, processing, and preservation techniques. Therefore, understanding which of your markers represent critical endpoints, and how these may be affected post sample collection is central to the quality of the analysis. There are logistical challenges to sample collection and processing at clinical sites, and consistency in sample quality across clinical sites can be challenging to maintain.

FlowMetric offers a range of solutions based on the clinical protocol requirements and the limitations in capabilities at the clinical sites. We also offer qualified in-house PBMCs preparation, cryofreezing, and tissue processing services, to ensure the highest quality of analysis for your clinical study.

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Selection of Sample Matrix

Tissue, Whole Blood, PBMCs, Anticoagulant, Fixative, and Fresh versus Frozen.

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Shipping Conditions

Ambient, Cool-packs or Frozen/Dry Ice. Transit Time, Acceptance Criteria. Communication with Shipping Companies, and Couriers/Tracking

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Sample Processing

Standard Procedures for Optimal Thawing, PBMCs Isolation, RBC lysis, Tissue Processing, Viability Acceptance Criteria

Fresh Whole Blood Samples

Fresh whole blood samples are almost always the preferred sample matrix for clinical trials. However, their use requires careful coordination in sample collection, shipping, and receipt at the testing laboratory. Fresh samples can be highly labile and must be received and processed within pre-determined stability windows, or otherwise, be discarded from the study.

Studies involving Receptor Occupancy Assays almost invariably involve fresh blood samples, that are collected and transported under carefully optimized conditions in order to preserve bound drug and receptor levels on the target cell population.

An optimal anticoagulant section is another key factor for fresh samples. Over time, all anticoagulants have some impact on sample quality, biomarker stability, and cell profiles. Ideally, the anticoagulant should be selected based on the cell population and markers of interest.

EDTA is typically used for complete blood counts and white blood cell differentials but should be avoided for functional assay systems, due to its effect on certain cell proliferation-, cytotoxicity, and cytokine profiles.

Whole blood collected into either sodium heparin or acid citrate dextrose supports the longest reported sample stability window of up to 72 hours, however, since ACD is a liquid it is not conducive to absolute cell counts and quantitative flow cytometry applications. Heparin can induce white blood cell clumping, so this should be addressed before the acquisition of the sample.

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Recommended Anticoagulants and Storage Times for Commonly Performed Assays

Assay Type Commonly Employed Anticoagulant Recommended Stability
Lymphocyte immunophenotyping Sodium heparin or EDTA Store ≤72 hr
Myeloid immunophenotyping EDTA Use immediately/ within 24 hours
Neutrophil function Sodium heparin or EDTA Use immediately/ within 24 hours
Platelet activation EDTA Use immediately/ within 24 hours
Platelet markers EDTA Use immediately/ within 24 hours
Reticulocyte enumeration EDTA Store ≤72 hr at 4°C
DNA analysis Sodium heparin or EDTA Use immediate for cell-cycle analysis; store for no longer than 72 hr for ploidy analysis

Lymphocyte immunophenotyping

Commonly Employed Anticoagulant

Sodium heparin or EDTA

Recommended Stability

Store ≤72 hr

Myeloid immunophenotyping

Commonly Employed Anticoagulant

EDTA

Recommended Stability

Use immediately/ within 24 hours

Neutrophil function

Commonly Employed Anticoagulant

Sodium heparin or EDTA

Recommended Stability

Use immediately/ within 24 hours

Platelet activation

Commonly Employed Anticoagulant

EDTA

Recommended Stability

Use immediately/ within 24 hours

Platelet markers

Commonly Employed Anticoagulant

EDTA

Recommended Stability

Reticulocyte enumeration

Commonly Employed Anticoagulant

EDTA

Recommended Stability

Store ≤72 hr at 4°C

DNA analysis

Commonly Employed Anticoagulant

Sodium heparin or EDTA

Recommended Stability

Use immediate for cell-cycle analysis; store for no longer than 72 hr for ploidy analysis

Preparation of PBMCs and Freezing Down

In some instances, the clinical sites are equipped to prepare Peripheral Blood Mononuclear Cells (PBMCs) from the fresh blood samples, and these can be frozen down and shipped to clinical testing labs for analysis. The interval between blood collection and processing is a critical parameter for many functional immunological assays. Granulocytes can become activated, and this results in oxidative stress in lymphocytes that can impact their functionality. In addition, increased storage time before processing can result in the contamination of the PBMC fraction with granulocytes which can impact downstream analysis.

Cryopreservation is the optimal method for long-term PBMCs storage. The cryopreservation mechanism requires freezing the cells without instigating cryogenic damage, through slow, controlled rates of cooling that create intracellular ice crystals but discourage the formation of extracellular ice crystals. The use of cryoprotectants such as DMSO or Glycerol that help prevents water-deprivation-related cellular damage, coupled with very low storage temperatures (-80°C and LN2 vapor-phase) work to maintain cellular viability throughout storage and thawing. However, since DMSO is toxic to cells, it is typically used at 10-15% within freezing media and rapidly removed post-thaw to prevent loss in cell viability.

Sample Preservation- Fixation Solutions

Depending on the type of assay and clinical endpoints, it might be possible to fix the cells before staining and acquisition. One commonly employed protocol involves fixing the cells in 4% formaldehyde for 30 minutes, then washing and storing the samples at 2-8° C in an appropriate buffer prior to acquisition.

In some instances, it may be preferable to stain the cells first and then fix and store them in the dark before acquisition. In both scenarios, long-term exposure to formaldehyde is avoided and we test to ensure that antibody conjugates still bind their targets in fixed samples.

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There are several commercially available sample preservation solutions for flow cytometry analysis that the FlowMetric team has experience working with:

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Cyto-Chex™

Cyto-Chex™ (Streck Laboratories) vacuum tubes preserve fresh whole blood for up to 7 days for routine WBC enumeration, using a cross-linking, formalin-free method with K3-EDTA anticoagulant. For some clinical applications, this method has been demonstrated to preserve sample integrity for up to 30 days in samples from patient cohorts with hematological disorders including leukemia, lymphomas, and HIV.

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The SmartTube system

Blood drawn into vacutainers containing sodium heparin can be preserved over an extended period using the SmartTube system. This utilizes a base station in which the blood is stimulated and stabilized with partial fixation before cooling to 4°C for transportation. Samples are reportedly stable for up to 11 days.

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TransFix Reagent

TransFix Reagent is simply to use requiring the only addition of the TransFix reagent into the blood collection tube followed by gentle mixing and stored between 2-25°C for as long as 10 days. For optimal preservation, blood samples should be treated with TransFix (1:5 ratio of reagent to blood) as soon as possible after collection, but no more than 6 hours. There are some nuances to its use, including that the blood sample should not be kept on ice or in the refrigerator before treatment with TransFix.

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The FlowMetric Advantage

For every flow cytometry assay, the lab should conduct its assessment of optimal sample matrix selection and processing. Working with a CRO who provides expertise in flow cytometry assay development through standardized sample collection, transportation, and processing, ensures the quality of the analysis for every sample in your clinical trial.

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