Extension of whole blood sample stability for flow cytometry … · 2017-11-17 · Sample Stability...
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Sample Stability in Various Tube Types Transfix® Effect on Cell Gating Stability of Multiple Markers in CYTO-CHEX® BCT • Whole blood sample stability can be significantly extended with the use of specialty blood collection tubes (CYTO-CHEX® and Transfix®), enabling batching of samples. • The Transfix® can be cryopreserved, offering long-term storage possibilities. • Stability of expression/detection must be confirmed for each markers, and can be influenced by different storage temperature. Some markers (ICOS, PD1 and CD45RA) show stability for ≤28 days when samples are stored at RT. Conclusions Extension of whole blood sample stability for flow cytometry analysis through the use of specialized blood collection tubes Philippe Pouliot, Scott Sugden, Josianne Chagnon-Choquet, Martin Lelaidier, Katie Young, Benoit Belanger, Dominic Gagnon, Jean-François Poulin Caprion Biosciences Inc, Montreal, Canada. Flow cytometry (FCM) is an information-rich methodology most commonly used to enumerate cells and monitor antigen expression. The use of FCM in multicenter clinical trials often requires sample shipment to specialized laboratories and hence requires prolonged sample stability. One of the most standardized FCM enumeration assays is the TBNK assay. This is an in vitro diagnostic procedure used to determine absolute counts of T, B and NK cells from whole blood samples ≤24h post- venipuncture. This small sample stability window presents a challenge for clinical trials. Introduction Objective: To identify vacutainer blood collection tubes and storage conditions that can extend sample stability. Methodology Overview: To assess the stability of cell populations and their frequencies, blood was collected from different healthy donors using EDTA tubes (K2EDTA), CYTO-CHEX® BCT or TransFix® vacutainers, and stored at different temperatures for up to 14 days prior to analysis using a validated TBNK assay. To assess marker stability, samples were also collected from healthy donors using CYTO-CHEX® BCT tubes and stored at different temperatures for ≤28 days prior to analysis. Samples were analyzed using a FCM antibody panel which includes the following types of markers: (1) gating/lineage (2) activation/naïve/memory (3) functional/monitoring. The performance of the panel was established previously in whole blood spiked with a cell line or with in-vitro stimulated PBMC, through the following steps: 1 – Identification of optimal antibody titers. 2 - FMO controls and isotype control testing for fluorescence spill over and specificity 3 – Full panel testing for Intra-assay precision on a minimum of 3 donors Objective and Methodology Figure 1: Cellular enumeration in various types of vacutainer tubes at Day 0 Whole blood was collected from 2 healthy donors into EDTA, CYTO-CHEX® or Transfix® vacutainers. A TBNK assay was performed immediately to enumerate cells at Day 0. Shown: Absolute cellular count (black columns) and cellular frequencies (grey columns) for the various cell types investigated for each donor. ➢ The 3 types of vacutainer tubes performed similarly at Day 0 and provided comparable results in terms of absolute counts and cellular frequencies. Cellular Enumeration A C E F D B Figure 2: Stability of cellular counts and frequencies in different vacutainer tubes (≤14 days). Whole blood was collected from 2 healthy donors into EDTA, CYTO-CHEX® or Transfix® vacutainers, stored for 0, (4*), 7, 10 or 14 days at Room Temperature (RT) or 4 o C and analyzed using the TBNK assay. Shown: Stability time course data for cellular counts and frequency for each tube type tested. 0 5 10 15 7 14 0.6 0.8 1.0 1.2 Days Normalized Raw Cell Counts (cells/mL) Absolute Cell Counts Stability Cytochex 4 o C 0 5 10 15 7 14 0.6 0.8 1.0 1.2 Days Relative Frequency Stability Cytochex RT Normalized Frequency of Cells 0 5 10 15 7 14 0.6 0.8 1.0 1.2 Days Relative Frequency Stability Cytochex 4 o C Normalized Frequency of Cells CYTO-CHEX® 0 5 10 15 7 14 0.6 0.8 1.0 1.2 Days Normalized Raw Cell Counts (cells/mL) Absolute Cell Counts Stability TransFix RT 0 5 10 15 7 14 0.6 0.8 1.0 1.2 Days Normalized Raw Cell Counts (cells/mL) Absolute Cell Counts Stability TransFix 4 o C 0 5 10 15 7 14 0.6 0.8 1.0 1.2 Days Normalized Raw Cell Counts (cells/mL) Absolute Cell Counts Stability TransFix -80 o C 0 5 10 15 7 14 0.6 0.8 1.0 1.2 Days Relative Frequency Stability TransFix RT Normalized Frequency of Cells 0 5 10 15 7 14 0.6 0.8 1.0 1.2 Days Relative Frequency Stability TransFix 4 o C Normalized Frequency of Cells 0 5 10 15 7 14 0.6 0.8 1.0 1.2 Days Relative Frequency Stability TransFix -80 o C Normalized Frequency of Cells Transfix® RT 4C -80C 0 5 10 15 7 14 0.6 0.8 1.0 1.2 Days Relative Frequency Stability K2-EDTA RT Normalized Frequency of Cells 0 5 10 15 7 14 0.6 0.8 1.0 1.2 Days Relative Frequency Stability K2-EDTA 4oC Normalized Frequency of Cells 0 5 10 15 7 14 0.6 0.8 1.0 1.2 Days Normalized Raw Cell Counts (cells/mL) Absolute cell counts stability K2-EDTA RT CD3+ CD4+ CD8+ B CELL NK CELL CD45+ 0 5 10 15 7 14 0.6 0.8 1.0 1.2 Days Normalized Raw Cell Counts (cells/mL) Absolute cell counts stability K2-EDTA 4oC EDTA Count Frequency Count Frequency Count Frequency The EDTA vacutainers demonstrate a rapid reduction of the absolute count, for all cell types already by 4 days. In addition, storage at 4 o C accelerates cell loss. Observation of the frequency shows that although all populations are affected, B cells are more dramatically reduced, an effect enhanced by storage at 4 o C. The stability assessment was stopped past 7 days due to the low quality of the sample. The CYTO-CHEX® BCT vacutainers demonstrate a stability for both cell count and frequency. This stability is observed until the last time point assayed, being 14 days. Both RT and 4 o C storage provide similar performance. The Transfix® vacutainers demonstrate a stability for both cell count and frequency. This stability is observed until the last time point assayed, being 14 days. Interestingly, the Transfix® vacutainers can be cryopreserved at -80 o C, so this condition was also assayed. The results demonstrate that storage at 4 o C or at -80 o C both enhance the stability of both cell counts and cell frequencies. ➢ The EDTA tubes have a limited stability, while both CYTO-CHEX® BCT and Transfix® vacutainers can prolong sample stability to 14 days, with Transfix® tubes being compatible with -80C storage. Figure 3: Effect of the sample stabilization in Transfix® vacutainers The Transfix® vacutainers contain a proprietary fixative that stabilizes blood cells. In FCM analysis, the use of such reagents often results in a variation of physical properties (FSC/SSC plot) and may alter epitope recognition, preventing antibody binding. Shown: TBNK assay data from samples collected in Transfix® tubes (A) Physical properties (FSC for general cell size and SSC for cell granularity) as well as CD45 expression (B) Selection of the different cell populations: CD3+ Lymphocytes (T cells), CD4 T cells, CD8 T cells, B cells and NK cells. The effect of the Transfix® fixative on SSC characteristics is visible in the form of a spreading of the SSC values. On the other hand, the fluorescence characteristics are mostly stable in both time and storage conditions. ➢ These results demonstrate that although stabilization affects the physical properties of the cells (FSC/SSC), the different cell populations can be adequately identified. GATING MARKERS CD15- Clone MC-480 CD16- Clone 3G8 CD33- Clone WM53 CD56- Clone NCAM1 ACTIVATION/MEMORY MARKERS CD27- Clone M-T271 CD45RA- Clone HI100 CD62L- Clone DREG56 CD69- Clone FN50 HLA-DR- Clone G46-6 FUNCTIONAL MARKERS ICOS- Clone DX29 PD-1- Clone EH12.2H7 TIM-3- Clone 344823 PD-L1- Clone MIH1 Frequency MdFI Day 0 Day 3 Day 7 Day 14 Day 21 Day 28 -100 -50 0 50 100 % Difference to Day 0 Gating Markers Stability - Frequency CD15 RT CD16 RT CD33 RT CD56 RT CD15 4oC CD16 4oC CD33 4oC CD56 4oC Day 0 Day 3 Day 7 Day 14 Day 21 Day 28 -100 -50 0 50 100 % Difference to Day 0 Gating Markers Stability - MdFI CD15 RT CD16 RT CD33 RT CD56 RT CD15 4oC CD16 4oC CD33 4oC CD56 4oC Day 0 Day 3 Day 7 Day 14 Day 21 Day 28 -100 -50 0 50 100 100 150 200 250 300 % Difference to Day 0 Activation/Memory Markers Stability- Frequency CD27 RT CD45RA RT CD62L RT CD69 RT HLA-DR RT CD27 4oC CD45RA 4oC CD62L 4oC CD69 4oC HLA-DR 4oC Day 0 Day 3 Day 7 Day 14 Day 21 Day 28 -100 -50 0 50 100 % Difference to Day 0 Activation/Memory Markers Stability - MdFI CD27 RT CD45RA RT CD62L RT CD69 RT HLA-DR RT CD27 4oC CD45RA 4oC CD62L 4oC CD69 4oC HLA-DR 4oC Day 0 Day 3 Day 7 Day 14 Day 21 Day 28 -100 -50 0 50 100 100 150 200 250 300 % Difference to Day 0 Markers of Interest Stability- Frequency ICOS RT PD-1 RT TIM-3 RT PD-L1 RT ICOS 4oC PD-1 4oC TIM-3 4oC PD-L1 4oC Day 0 Day 3 Day 7 Day 14 Day 21 Day 28 -100 -50 0 50 100 % Difference to Day 0 Markers of Interest Stability - MdFI ICOS RT PD-1 RT TIM-3 RT PD-L1 RT ICOS 4oC PD-1 4oC TIM-3 4oC PD-L1 4oC Figure 4: Stability of various cell markers in CYTO-CHEX® BCT (≤28 days) Whole blood from 3 healthy donors was collected into CYTO-CHEX® BCT, and stimulated peripheral blood mononuclear cells (PBMC) were spiked into the blood samples to provide positive signal for weakly expressed markers. Samples were then stored for 0, 3, 7, 14, 21 or 28 days at RT or 4 o C and assayed in triplicate using a FCM antibody panel which included various markers (gating/lineage, activation/naïve/memory, functional/monitoring). Shown: Stability time course data normalized to Day 0 and averaged across the 3 donors for each marker. ➢ The stability of the various markers vary greatly and should be investigated on an individual basis. FSC SSC CD45 SSC Day 0 Day 14 RT 4C -80C A RT, Day 0 4C, Day 14 -80C, Day 14 RT, Day 14 B *EDTA was expected to be less stable, thus a Day 4 timepoint was included for this condition.
Transcript of Extension of whole blood sample stability for flow cytometry … · 2017-11-17 · Sample Stability...
Sample Stability in Various Tube Types
Transfix® Effect on Cell Gating
Stability of Multiple Markers in CYTO-CHEX® BCT
• Whole blood sample stability can be significantly extended with the use of specialtyblood collection tubes (CYTO-CHEX® and Transfix®), enabling batching of samples.
• The Transfix® can be cryopreserved, offering long-term storage possibilities.
• Stability of expression/detection must be confirmed for each markers, and can beinfluenced by different storage temperature. Some markers (ICOS, PD1 and CD45RA)show stability for ≤28 days when samples are stored at RT.
Conclusions
Extension of whole blood sample stability for flow cytometry analysis through the use of specialized blood collection tubes
Philippe Pouliot, Scott Sugden, Josianne Chagnon-Choquet, Martin Lelaidier, Katie Young, Benoit Belanger, Dominic Gagnon, Jean-François Poulin
Caprion Biosciences Inc, Montreal, Canada.
Flow cytometry (FCM) is an information-rich methodology most commonly used to enumerate cells andmonitor antigen expression. The use of FCM in multicenter clinical trials often requires sample shipment tospecialized laboratories and hence requires prolonged sample stability.
One of the most standardized FCM enumeration assays is the TBNK assay. This is an in vitro diagnosticprocedure used to determine absolute counts of T, B and NK cells from whole blood samples ≤24h post-venipuncture. This small sample stability window presents a challenge for clinical trials.
Introduction
Objective:To identify vacutainer blood collection tubes and storage conditions that can extend sample stability.
Methodology Overview:To assess the stability of cell populations and their frequencies, blood was collected from different healthydonors using EDTA tubes (K2EDTA), CYTO-CHEX® BCT or TransFix® vacutainers, and stored at differenttemperatures for up to 14 days prior to analysis using a validated TBNK assay.
To assess marker stability, samples were also collected from healthy donors using CYTO-CHEX® BCT tubesand stored at different temperatures for ≤28 days prior to analysis. Samples were analyzed using a FCMantibody panel which includes the following types of markers: (1) gating/lineage (2)activation/naïve/memory (3) functional/monitoring. The performance of the panel was establishedpreviously in whole blood spiked with a cell line or with in-vitro stimulated PBMC, through the followingsteps:
1 – Identification of optimal antibody titers.2 - FMO controls and isotype control testing for fluorescence spill over and specificity3 – Full panel testing for Intra-assay precision on a minimum of 3 donors
Objective and Methodology
Figure 1: Cellular enumeration in various types of vacutainer tubes at Day 0Whole blood was collected from 2 healthy donors into EDTA, CYTO-CHEX® or Transfix® vacutainers. ATBNK assay was performed immediately to enumerate cells at Day 0. Shown: Absolute cellular count(black columns) and cellular frequencies (grey columns) for the various cell types investigated for eachdonor.
➢ The 3 types of vacutainer tubes performed similarly at Day 0 and provided comparable results in termsof absolute counts and cellular frequencies.
Cellular Enumeration
A
C
E F
D
B
Figure 2: Stability of cellular counts and frequencies in differentvacutainer tubes (≤14 days).Whole blood was collected from 2 healthy donors into EDTA,CYTO-CHEX® or Transfix® vacutainers, stored for 0, (4*), 7, 10 or14 days at Room Temperature (RT) or 4oC and analyzed using theTBNK assay. Shown: Stability time course data for cellular countsand frequency for each tube type tested.
0 5 10 157 14
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rmalized
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Cell C
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Absolute Cell Counts Stability Cytochex 4oC
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Relative Frequency Stability Cytochex RT
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Relative Frequency Stability Cytochex 4oC
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Absolute Cell Counts Stability TransFix RT
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Absolute Cell Counts Stability TransFix -80oC
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Relative Frequency Stability TransFix RT
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Relative Frequency Stability TransFix 4oC
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Relative Frequency Stability K2-EDTA RT
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Relative Frequency Stability K2-EDTA 4oC
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Absolute cell counts stability K2-EDTA RT
CD3+
CD4+
CD8+
B CELL
NK CELL
CD45+
0 5 10 157 14
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Absolute cell counts stability K2-EDTA 4oC
EDTA
Count Frequency Count Frequency
Count Frequency
The EDTA vacutainers demonstrate a rapid reduction of the absolute count, for all cell types already by 4days. In addition, storage at 4oC accelerates cell loss. Observation of the frequency shows that although allpopulations are affected, B cells are more dramatically reduced, an effect enhanced by storage at 4oC. Thestability assessment was stopped past 7 days due to the low quality of the sample.
The CYTO-CHEX® BCT vacutainers demonstrate a stability for both cell count and frequency. This stability isobserved until the last time point assayed, being 14 days. Both RT and 4oC storage provide similarperformance.
The Transfix® vacutainers demonstrate a stability for both cell count and frequency. This stability isobserved until the last time point assayed, being 14 days. Interestingly, the Transfix® vacutainers can becryopreserved at -80oC, so this condition was also assayed. The results demonstrate that storage at 4oC orat -80oC both enhance the stability of both cell counts and cell frequencies.
➢ The EDTA tubes have a limited stability, while both CYTO-CHEX® BCT and Transfix® vacutainers canprolong sample stability to 14 days, with Transfix® tubes being compatible with -80C storage.
Figure 3: Effect of the sample stabilization in Transfix® vacutainersThe Transfix® vacutainers contain a proprietary fixative that stabilizes blood cells. In FCM analysis, the useof such reagents often results in a variation of physical properties (FSC/SSC plot) and may alter epitoperecognition, preventing antibody binding. Shown: TBNK assay data from samples collected in Transfix®tubes (A) Physical properties (FSC for general cell size and SSC for cell granularity) as well as CD45expression (B) Selection of the different cell populations: CD3+ Lymphocytes (T cells), CD4 T cells, CD8 Tcells, B cells and NK cells.
The effect of the Transfix® fixative on SSC characteristics is visible in the form of a spreading of the SSCvalues. On the other hand, the fluorescence characteristics are mostly stable in both time and storageconditions.
➢ These results demonstrate that although stabilization affects the physical properties of the cells(FSC/SSC), the different cell populations can be adequately identified.
GATING MARKERSCD15- Clone MC-480
CD16- Clone 3G8CD33- Clone WM53CD56- Clone NCAM1
ACTIVATION/MEMORY MARKERS
CD27- Clone M-T271CD45RA- Clone HI100CD62L- Clone DREG56
CD69- Clone FN50HLA-DR- Clone G46-6
FUNCTIONALMARKERS
ICOS- Clone DX29PD-1- Clone EH12.2H7TIM-3- Clone 344823PD-L1- Clone MIH1
Frequency MdFI
Day 0 Day 3 Day 7 Day 14 Day 21 Day 28-100
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Gating Markers Stability - Frequency
CD15 RT
CD16 RT
CD33 RT
CD56 RT
CD15 4oC
CD16 4oC
CD33 4oC
CD56 4oC
Day 0 Day 3 Day 7 Day 14 Day 21 Day 28-100
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Gating Markers Stability - MdFI
CD15 RT
CD16 RT
CD33 RT
CD56 RT
CD15 4oC
CD16 4oC
CD33 4oC
CD56 4oC
Day 0 Day 3 Day 7 Day 14 Day 21 Day 28-100
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Activation/Memory Markers Stability- Frequency
CD27 RT
CD45RA RT
CD62L RT
CD69 RT
HLA-DR RT
CD27 4oC
CD45RA 4oC
CD62L 4oC
CD69 4oC
HLA-DR 4oC
Day 0 Day 3 Day 7 Day 14 Day 21 Day 28-100
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Activation/Memory Markers Stability - MdFI
CD27 RT
CD45RA RT
CD62L RT
CD69 RT
HLA-DR RT
CD27 4oC
CD45RA 4oC
CD62L 4oC
CD69 4oC
HLA-DR 4oC
Day 0 Day 3 Day 7 Day 14 Day 21 Day 28-100
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Markers of Interest Stability- Frequency
ICOS RT
PD-1 RT
TIM-3 RT
PD-L1 RT
ICOS 4oC
PD-1 4oC
TIM-3 4oC
PD-L1 4oC
Day 0 Day 3 Day 7 Day 14 Day 21 Day 28-100
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Markers of Interest Stability - MdFI
ICOS RT
PD-1 RT
TIM-3 RT
PD-L1 RT
ICOS 4oC
PD-1 4oC
TIM-3 4oC
PD-L1 4oC
Figure 4: Stability of various cell markers in CYTO-CHEX® BCT (≤28 days)Whole blood from 3 healthy donors was collected into CYTO-CHEX® BCT, and stimulated peripheral bloodmononuclear cells (PBMC) were spiked into the blood samples to provide positive signal for weaklyexpressed markers. Samples were then stored for 0, 3, 7, 14, 21 or 28 days at RT or 4oC and assayed intriplicate using a FCM antibody panel which included various markers (gating/lineage,activation/naïve/memory, functional/monitoring). Shown: Stability time course data normalized to Day 0and averaged across the 3 donors for each marker.
➢ The stability of the various markers vary greatly and should be investigated on an individual basis.
FSC
SSC
CD45
SSC
Day
0D
ay 1
4
RT 4C -80C
A
RT, Day 0 4C, Day 14
-80C, Day 14RT, Day 14
B
*EDTA was expected to be less stable, thus a Day 4 timepoint was included for this condition.
