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Optimization of Blood Handling and Peripheral Blood Mononuclear Cell Cryopreservation of Low Cell Number Samples.

Author(s): Hope CM, Huynh D, Wong YY, Oakey H, Perkins GB, Nguyen T, Binkowski S, Bui M, Choo AYL, Gibson E, Huang D, Kim KW, Ngui K, Rawlinson WD, Sadlon T, Couper JJ, Penno MAS, Barry SC, On Behalf Of The Endia Study Group

Publication: Int J Mol Sci, 2021, Vol. 22, Page

PubMed ID: 34502038 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   This paper aimed to identify acceptable durations and temperatures of a delay in peripheral blood mononuclear cell (PBMC) isolation by examining changes in total cell number, cell viability, cell functionality, and cell populations in PBMCs from healthy volunteers.  Blood from 10 healthy volunteers was collected in EDTA tubes and stored at room temperature (22-27°C) or in a refrigerator at 4°C for 0 (processed the same day as collection), 1, 2, 3, 4, 5, 6, 7, 8, or 9 days. Blood in EDTA tubes were centrifuged at 1700 x g for 10 min, and the buffy coat was removed.  Samples then underwent Ficoll gradient separation by centrifugation at 1000 x g for 20 min at room temperature, and the PBMC monolayer was collected, washed, any remaining red blood cells were lysed, and PBMCs were resuspended and counted with a Countess II cell counter with trypan blue to determine viability.  PBMCs were then centrifuged (300 x g for 10 min), resuspended in 1% Cosmic Calf Serum (CCS) at a concentration of 2 x 107 viable cells/mL), and an equal volume of pre-cooled 20% DMSO in CCS was added dropwise.  Samples were frozen in a CoolCell device (-1°C/min) overnight in a -80°C freezer and then transferred to liquid nitrogen. Frozen PBMC samples were transferred to dry ice before thawing in a 37°C water bath at a rate of 10 min/mL cryopreservation media. Pre-warmed (37°C) X-Vivo culture media with 0.04% DNase was added to tubes that were then inverted, centrifuged (500 x g for 10 min), washed, resuspended, and counted on a Countess II cell counter for post-thaw measurements of cell number, viability, and recovery (post-thaw measurement). Cells were then added to cell culture plates, incubated at 56°C for 16-18 h, washed, and recounted (post-incubation measurement). Cell functionality was assessed by ELISOPT analysis of thawed and cultured PBMC samples after treatment with either Phytohemagglutinin (PHA) or a cytomegalovirus/Eppstein-Barr virus/Flu (CEF) peptide pool. Enumeration of the following cell types was performed by staining for respective cell surface markers and quantified by flow cytometry on a BD FACS symphony machine: neutrophils (CD16-PE), monocytes (CD14-FITC), T cells (CD3-BUV737), Helper T cells (CD4-BUV496), Cytotoxic T cells (CD8-BUV395), NK cells (CD56-PerCP-Cy5.5), and B cells (CD19-BV711).

   Summary of Findings:

   Both the temperature and duration of a blood processing delay significantly affected the total number of viable cells and viability (%) in PBMCs (p<0.001). Relative to samples that were processed the same day as collection, significant reductions in the total number of viable cells were observed in PBMCs after a 1 d delay in blood processing at 4°C (1.04 versus 0.65 x 107; p<0.05) and a 2 d delay at room temperature (1.06 versus 0.42, p<0.001); viability also declined by 18.9% when processing was delayed for 1 d at 4°C (p<0.05) and by 7.3% when delayed for 2 d at room temperature (p<0.001). Notably, viability and the total number of viable cells were lower when blood experienced a delay at room temperature than at 4°C after 2 and 3 days, respectively. Post-thaw PBMC viability and recovery did not differ among samples from blood that experienced a delay at room temperature versus 4°C, although the duration of the delay did have an effect. PBMC samples from blood specimens that experienced a delay of 2 d had a 35.2% decline in post-thaw viability relative to those that were immediately processed; post-thaw recovery was not affected for this timepoint but did decline significantly after a 3 d delay relative to the 0 d control. When PBMC viability and recovery were measured post-incubation, viability was 34.7% lower in samples from blood that experienced a 2 h delay in processing and recovery was 29.7% lower in samples from blood that experienced a 1 h delay relative to immediately processed controls. 
   CEF- or PHA-stimulated IFN-γ production by cryopreserved PBMCs did not differ significantly among samples from blood that experienced a delay in processing of 0 to 4 d regardless of the temperature of the delay. 

   
   When the percentages of different cell populations were examined among PBMC samples from blood that experienced a delay of 0 to 2 d at either 4°C or room temperature, no significant changes in the percentage of T-cells or lymphocytes were observed. The percentage of monocytes declined by 8.3% when blood processing was delayed by 1 d relative to specimens that were processed immediately but did not decline further. The percentage of CD56+CD16+ NK cells declined by 25% when blood processing was delayed 1 d relative to immediately processed specimens (p<0.05), and an additional albeit nonsignificant decrease was also observed after a 2 d delay. Relative to immediately processed blood specimens, the percentage of low-density neutrophils increased from 3.7 to 22% in samples from blood that experienced a 2 d delay in processing at room temperature (p<0.001), but no change was observed when the delay occurred at 4°C. The percentage of B-cells was higher among blood specimens that experienced a 3 d processing delay at 4°C compared to those stored at room temperature (6.9 versus 2.3%, respectively). 
    

   Biospecimens

   • Cell - White Cells
   • Bodily Fluid - Blood

   Preservative Types

   • Other Preservative
   • Frozen

   Diagnoses:

   • Normal

   Platform:

   Analyte	Technology Platform
   Cell count/volume	Flow cytometry
   Cell count/volume	Hematology/ auto analyzer
   Cell count/volume	ELISpot

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Storage	Storage temperature	Room temperature 4°C
   Storage	Storage duration	0 d 1 d 2 d 3 d 4 d 5 d 6 d 7 d 8 d 9 d
   Biospecimen Aliquots and Components	Centrifugation	Centrifugation delays investigated

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2. Study Purpose

   This paper explored the smallest volumes of cryopreservative in an aliquot that could be used without compromising PBMC recovery or viability. PBMCs from three healthy volunteers were obtained from a secondary source and were verified to have a viability ≥80%.  Samples were aliquoted such that they contained different volumes of cryopreservative (20% DMSO/CCS; 25, 50, 100, 150, 200, 300, 500 µl) but the same final PBMC concentration of 10 × 106 cells/mL. Aliquots were cryopreserved and stored in liquid nitrogen for an average of 4.4 months. Frozen PBMC samples were transferred to dry ice before thawing in 37°C water bath at a rate of 10 min/mL cryopreservation media (thaw).  Pre-warmed (37°C) X-Vivo culture media with 0.04% DNase was added to tubes that were then inverted, centrifuged (500 x g for 10 min), washed, resuspended, and counted on a Countess II cell counter for post-thaw measurements of cell number, viability, and recovery (post-thaw measurement). Cells were then added to cell culture plates, incubated at 56°C for 16-18 h, washed, and recounted (post-incubation measurement). PBMCs were counted with a Countess II cell counter with trypan blue to determine viability.

   Summary of Findings:

   While all of the cryopreservative volumes evaluated remained within the acceptable criteria specified by the authors (≥80% post-thaw viability and ≥55% recovery), the volume of cryopreservative did significantly affect post-thaw viability (p<0.001). Post-hoc analysis revealed that the post-thaw viability of samples containing 25 and 50 µl of cryoprotectant was approximately half that of samples that contained ≥200 µl of cryoprotectant. The volume of cryopreservative in PBMC samples also significantly affected the post-incubation viability and recovery (p<0.001), as viability was lower among samples with 25 or 50 µl compared to those with ≥150 µl and recovery was lower in samples with 100 µl cryoprotectant than ≥200 µl (p<0.05).  

   Biospecimens

   • Cell - White Cells

   Preservative Types

   • Other Preservative
   • Frozen

   Diagnoses:

   • Normal

   Platform:

   Analyte	Technology Platform
   Cell count/volume	Hematology/ auto analyzer

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Biospecimen Preservation	Volume/ concentration of preservative	25 µl 50 µl 100 µl 150 µl 200 µl 300 µl 500 µl

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3. Study Purpose

   This paper explored the lowest concentration of PBMCs that could be cryopreserved without compromising PBMC recovery or viability. PBMCs from four healthy volunteers were obtained from a secondary source and were verified to have a viability ≥80%.  Samples were aliquoted such that they contained different concentrations of PBMCs (10 × 10⁶, 6.67 × 10⁶, 3.33 × 106, 1.67 × 10⁶ cells/mL) but the same final aliquot volume (150 µl). Samples were cryopreserved and stored in liquid nitrogen for an average of 1.7 months. Frozen PBMC samples were transferred to dry ice before thawing in 37°C water bath at a rate of 10 min/mL cryopreservation media (thaw).  Pre-warmed (37°C) X-Vivo culture media with 0.04% DNase was added to tubes that were then inverted, centrifuged (500 x g for 10 min), washed, resuspended, and counted on a Countess II cell counter for post-thaw measurements of cell number, viability, and recovery. Cells were then added to cell culture plates, incubated at 56°C for 16-18 h, washed, and recounted (post-incubation measurement). PBMCs were counted with a Countess II cell counter with trypan blue to determine viability.

   Summary of Findings:

   Of the total cell concentrations that were evaluated, only the two highest concentrations (total cell concentration of 6.67 × 10⁶ and 10 × 10⁶ cells/mL) met the acceptable criteria of ≥80% post-thaw viability and ≥55% recovery, with lower recovery observed among samples of a lower PBMC concentration. Significant differences in post-incubation viability among PBMC concentrations were limited to when the lowest concentration (1.67 ×10⁶ cells/ml) was compared to the two highest concentrations (6.67 × 10⁶ and 10 × 10⁶ cells/mL) (p-value not reported).

   Biospecimens

   • Cell - White Cells

   Preservative Types

   • Other Preservative
   • Frozen

   Diagnoses:

   • Normal

   Platform:

   Analyte	Technology Platform
   Cell count/volume	Hematology/ auto analyzer

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Biospecimen Aliquots and Components	Aliquot size/volume	10 × 10^6 cells/mL 6.67 × 10^6 cells/mL 3.33 × 10^6 cells/mL 1.67 × 10^6 cells/mL

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