If the blood is human, the consensus is to leave the heparinized blood at room temperature overnight. No fridge, no add -ons like growth medium. Next day, PBMNC isolation with Ficoll-Paque or other available.

Hello, Philip!

You also look at folowing article and protocol ELISpot

Article Isolation And Preservation Of Peripheral Blood Mononuclear c...

Time of day for blood-drawing

Apart from the fasting/feeding state, many immune functions exhibit circadian rhythmicity [21], and thus the time of day at which blood is taken may also be an important variable. In response to tetanus toxoid, the ratio of IFN-γ/IL-10 secretion in whole blood attained its zenith during early morning (3 a.m.) and reached its nadir in the late morning and afternoon (10 a.m.–8 p.m.) [22]. The zenith corresponded with the peak of plasma melatonin, while the nadir was related with the peak of plasma cortisol. These hormones have reversed circadian rhythms and opposed immune effects, with cortisol inhibiting and melatonin enhancing proinflammatory immune responses (e.g. IFN-γ and IL-1 production). Further suggesting a cause–effect link between these hormone–cytokine associations, the IFN-γ/IL-10 ratio was reduced significantly by late evening administration of cortisone (due mainly to reduced IFN-γ levels) [22]. The circadian rhythmicity of immune function suggests that blood drawing for T cell studies may be performed at the same time of day for more accurate comparison (level of evidence: E). Further studies are needed to clarify whether this would apply to PBMCs purified from whole blood, as washing steps would greatly reduce cortisol and melatonin concentrations during T cell stimulation.

Anti-coagulants

It is often assumed that ethylenediamine tetraacetic acid (EDTA)-anti-coagulated blood is not suitable for T cell analyses, because its Ca2+-chelating properties may inhibit T cell activation. Bull et al. [24] compared EDTA, acid–citrate–dextrose (ACD) and sodium heparin as anti-coagulants for collecting blood samples. Contrary to common belief, they concluded that these anti-coagulants did not differ in terms of cell recovery, viability and function (as assessed by IFN-γ ELISPOT and intracellular staining of PBMCs stimulated with CD8+ T cell peptide epitopes). However, the performance of EDTA-stored blood tended to deteriorate more quickly with increasing storage time (24 h) before processing. This deterioration was less pronounced with heparin, as both viability and IFN-γ secretion were more stable over time (8 versus 24 h).

Similar conclusions were also obtained by comparing physical and phenotypic parameters with older technologies [25]. Light-scatter distributions were stable for ACD- or heparin-treated blood, whereas EDTA caused changes in the granulocyte distributions. Phenotypic determination of CD4+ (defined as Leu3+) and CD8+ (Leu2+) lymphocytes was also most stable for ACD or heparinized blood compared to EDTA [25].

No significant differences were recorded when cytomegalovirus (CMV)-specific responses were analysed by intracellular IFN-γ assays on PBMCs isolated from blood drawn on sodium heparin, lithium heparin or sodium citrate. However, when whole blood assays were performed in parallel, lithium heparin yielded slightly higher CMV-specific responses [26]. Hence, we recommend using heparin as anti-coagulant for T cell assays (level of evidence: C). Lithium heparin may have an additional edge over sodium heparin for blood assays (level of evidence: D).

Blood storage until processing

This issue has been analysed thoroughly in the human immunodeficiency virus (HIV) field, where it was found that time from venipuncture to cryopreservation was the most critical parameter affecting subsequent cell recovery and function [24]. In this multi-centre study, the other parameters considered were type of anti-coagulant used, method of PBMC isolation and procedure for sample shipping. Although the performance of fresh PBMCs was not considered, an increase from 8 h to 24 h before processing and cryopreservation greatly affected sample quality. When PBMCs were drawn in sodium heparin and separated on a Ficoll gradient, 24 versus 8 h reduced recovery by 30% (from 83% to 53%), viability by 4% (from ∼96% to 92%) and viral peptide-reactive T cells (IFN-γ ELISPOT) by 36–56% [24]. Similar results were obtained in an HIV vaccine trial, in which processing of blood samples within 12 h led to threefold higher IFN-γ ELISPOT responses [27].

One possible reason for blood stored for prolonged periods at room temperature performing less well in T cell assays is an increase in granulocyte contamination of the PBMC preparation. Granulocytes become activated upon prolonged storage, which affects their buoyancy profile resulting in less efficient separation by density gradient procedures [28,29]. In a recent study addressing this point [29], it was found that PBMC contamination by activated (CD11b+CD15+) granulocytes was observed within 8 h after venipuncture and room temperature storage (2·3–fold increase), and increased to 11·3-fold by 24 h, in comparison to PBMCs from fresh blood (< 3 h after venipuncture). Granulocyte contamination not only reduced the relative number of T cells present in PBMCs, but also inhibited T cell proliferation following PHA stimulation in ∼75% of samples [29] and IFN-γ ELISPOT responses to CD8+ T cell viral epitope peptides [23]. Both granulocyte contamination and inhibition of T cell responses were effectively limited when granulocyte activation was reduced by diluting blood in phosphate-buffered saline (PBS) or RPMI-1640 (1:1) prior to storage [23,29]. Similar observations were made in a macaque study, in which granulocyte contamination affected the quality and quantity of spots in IFN-γ ELISPOT assays [30]. It should be noted, however, that granulocyte contamination is not the only factor altering T cell responsiveness upon prolonged blood storage. Indeed, blood samples kept under conditions minimizing granulocyte contamination (i.e. gentle agitation) also displayed decreased CD8+ T cell responsiveness against viral epitopes compared to short-stored blood and functional recovery upon preliminary dilution [23]. Interestingly, a trend towards increased counts of viral epitope-loaded HLA tetramer-positive CD8+ T cells was observed instead upon prolonged blood storage. Also in this case, blood dilution prior to storage corrected this phenomenon. The mechanism underlying this observation may be the same one at play for reduced IFN-γ ELISPOT responses. T cells from stored blood may be less responsive to antigen-specific stimulation, as elicited by peptide-loaded antigen-presenting cells in ELISPOT assays and by peptide-loaded HLA molecules in HLA multimer assays. While this translates into lower T cell responses in the case of ELISPOT, it may lead to increased multimer binding when tetramer assays are performed, as the T cell receptor may be less down-regulated upon peptide-HLA interaction. This mechanism is similar to that proposed for the facilitating effect of protein kinase inhibitors on HLA multimer staining [31].

Comparison of samples kept under gentle agitation on a rocking platform versus tubes stored horizontally without any agitation showed similar recoveries, but non-agitated samples were highly contaminated with granulocytes after prolonged (18 h) storage [23]. Also in this case, the granulocyte contamination was associated with a significant decrease in IFN-γ ELISPOT CD8+ T cell responses against viral peptide epitopes. Blood storage at room temperature rather than at 4°C is also most suitable for PBMC preservation and to avoid non-specific T cell stimulation. In line with this widespread laboratory tradition, light-scatter distributions as well as CD4+ and CD8+ T cell frequencies were found to be more stable upon storage at room temperature versus 4°C, due mainly to changes in granulocyte distribution [25,32]. Granulocyte contamination also increases substantially upon storage at 4°C [29].

Based upon these data, blood should be processed as soon as possible after drawing, ideally within 8–12 h (level of evidence: C). While waiting to be processed, blood should be agitated gently at room temperature (18–22°C) (level of evidence: D). If blood is not processed immediately (i.e. waiting time > 8 h), it should be stored diluted in RPMI-1640 (1:1) under gentle agitation, in order to decrease granulocyte contamination and to preserve detectable T cell responses (level of evidence: C). HLA multimer-based assays may instead perform better with undiluted blood (level of evidence: D).

PBMC PREPARATION

Article A whole blood monokine-based reporter assay provides a sensi...

I think you're right. It's plausible that the granus would degranulate and damage the T-cells. Catalase is not a half-bad idea. But even in theory, it can mitigate only a fraction of the granule arsenal. I'd believe that it could buy you some time, but I fear that it would be unreliable, and dependent on subtle differences in conditions. You'll never know if an effect reflects an interesting difference between two batches, or a chaotic response to the preservation treatment.

My low-tech suggestion would be to deplete the granulocytes immediately using gradient centrifugation. Alternatively, and probably more effectively, immunomagnetic purification of the T-cells. With some luck that might even salvage the frozen samples. Or might not.

Much thanks Nicole. I am currently storing my whole blood overnight by first diluting it 1:1 with DPBS then placing it on a gentle rocker overnight at room temperature. The rocker is per the literature's recommendations for overnight blood storage, as was nicely cited by Alexandr. The literature seems to say that diluting the blood corrects a lot of issues with overnight storage

I am completely agree with Nicole.

I isolate PBMC from human blood sample many a times after 24-48 hrs. I hardly find granulocytes contamination ( I keep my sample at RT only).

Keeping sample at 4 Degree will cause cold shock to cells as well as shaking blood will cause mechanical harm.

Leave your sample at RT upto 48 hrs and isolate PBMCs with lymphoprep or histopaque.

I am starting to work with human donor derived PBMCS and I am seeing some structures in the cells which I am not sure about. I would really appreciate if any of you could help in understanding what I am seeing. I have posted a question regarding it at the link https://www.researchgate.net/post/Please_advise_on_what_are_the_black_filamentous_structures_we_are_seeing_in_our_human_PBMCs_cell_culture

I believe it is some sort of contamination but not sure if these could be cell debris too.