After a period of using glycerol for the cryopreservation of hematopoietic progenitor cells (HPCs) [1], dimethyl sulfoxide (DMSO) has become the most commonly used cryoprotectant for this purpose because of its high cryoprotective efficiency and rapid penetration across cell membranes [2–4]. The great advantage of its use is the possibility of infusing the thawed cell product without removing the cryoprotectant, which was not possible with glycerol. However, the potential toxicity of DMSO has given rise to debates on the safety of its use. Cases of adverse reactions such as increased heart or respiratory rate, facial flushing, increased or decreased blood pressure, dyspnea, nausea, and vomiting have been described after infusion of HPC concentrates containing DMSO [3–9]. DMSO toxicity is dose-dependent. Therefore, strategies are being developed to neutralize the toxicity, to reduce the concentration of the cryoprotectant, or to wash it off before clinical administration. Infusion of an HPC product containing DMSO is generally accepted as safe in concentrations below 10 % (v/v) [10] under the condition that the maximal daily dose of DMSO does not exceed 1 g per kg patient body weight [11,12]. While the above-mentioned symptoms usually cause only transient discomfort for the patient, serious adverse reactions have been described in patients with preexisting cardiovascular, respiratory, renal or central nervous system diseases, sometimes with fatal outcomes [6–8,13]. In the practice of University Hospital Hradec Králové, the most frequent medical indications for DMSO reduction are chronic renal failure caused by secondary amyloidosis in multiple myeloma and primary or secondary amyloidosis of the heart.

The most widely used technique in DMSO washing is gradual dilution of the cell suspension with its subsequent centrifugation and the addition of cryoprotectant-free solution [14,15]. The degree of dilution and the composition of the washing solution are usually adjusted to minimize osmotic changes. The components of the washing medium should be acceptable from a clinical point of view, i.e., it should not contain components of animal origin and should contain registered drugs, and use CE-certified medical devices, or products approved by the national competent authority (State Institute for Drug Control in the Czech Republic) [15]. In clinical practice, certified saline solutions/electrolytes, such as 0.9 % NaCl, Normosol-R® (Hospira, Inc., USA), Plasma-Lyte 148® (Baxter, USA), Ringer's solution (B. Braun, Germany) with dextran-40 (5–10 %), human serum albumin (1–5 %), hydroxyethyl starch (HES - 3–6 %), or acid citrate dextrose anticoagulant are acceptable. These media are often supplemented with dextran-40, HES, or human serum albumin at various concentrations [16–19].

Commercially available closed automatic systems developed for hematopoietic cell grafts, which are usually characterized by large-transplanted volumes, can also be used to wash out cryoprotectants. Examples of such systems are devices based on the principle of dilution and subsequent centrifugation, e.g., the COBE® 2991 Cell Processor (Terumo BCT, Inc.), Sepax S-100® (Sepax 2 S-100), and Biosafe SA® (GE HealthCare) [13,15,17,19,20], or on the principle of dilution and subsequent filtration, e.g., the Haemonetics ACP215 Automated Cell Processor® (Haemonetics Corp), CytoMate® (Baxter/Nexell), or Lovo® (Fresenius Kabi) [13,15,21,22].

Our clinical center has long experience with autologous HPC transplantation in multiple myeloma [23], and the infused DMSO dose is far below the limit [24] in most cases. A controlled study performed by Horacek et al. [25] did not report any differences in monitored vital functions between infusions of autologous and allogeneic HPC concentrates. Nevertheless, in a minority of cases, it was necessary to split the HPC dose over several days [24,26].

This retrospective study reviewed cases of primary or secondary amyloidosis as a complication of multiple myeloma treated by HPC autologous transplantation requiring DMSO reduction. Data regarding the influence of the freezing/thawing and DMSO reduction processes on the content of nucleated cells (NC), mononuclear cells (MNC), and CD34+ cells were analyzed. This analysis included pre- and post-process viability, the recovery of viable cells, and repopulation potency, as measured by the colony-forming unit-granulocyte macrophage (CFU-GM) assay, for samples contained within one 100 mL cryobag.

Study inclusion criteria were met by 13 patients (nine men and four women) with an average age of 58 years (range: 44–70 years) and weight of 80 kg (range: 52–103 kg). The retrospective study period was 2013–2020. The whole process of HPC collection, transport, processing, and administration is presented in Figure 1. Twelve patients had a diagnosis of primary amyloidosis or secondary amyloidosis as a complication of multiple myeloma, and one patient (No. 3) had an allergic adverse reaction to DMSO at the beginning of HPC infusion (Table 1).

Post-thaw recovery of key hematopoietic progenitor cells parameters

Figures 2, 3, and 4 show HPC recoveries estimated from the thawed cryobags. Figure 2 shows that after cryopreservation and thawing, the number of all NC was reduced, and CD34+ cell potency was also reduced.

Figure 2.

Recovery (%) of key hematopoietic progenitor cell parameters after the freezing/thawing process, comparing data from collection and after freezing/thawing. Blue, orange, and yellow points denote outliers. TNC: total number of viable nucleated cells; TMNC: total number of viable mononuclear cells; CFU-GM: colony forming unit-granulocyte macrophage.

Figure 3.

Recovery (%) of key hematopoietic progenitor cell parameters after the dimethyl sulfoxide removal process, comparing data after freezing/thawing process and after dimethyl sulfoxide removal.

TNC: total number of viable nucleated cells; TMNC: total number of viable mononuclear cells; CFU-GM: colony forming unit-granulocyte macrophage

Figure 4.

Recovery (%) of key hematopoietic progenitor cell parameters after dimethyl sulfoxide removal, comparing data at collection and after dimethyl sulfoxide removal.

TNC: total number of viable nucleated cells; TMNC: total number of viable mononuclear cells; CFU-GM: colony forming unit-granulocyte macrophage

Figure 3 shows greater inter-individual differences in cell recovery for TNC, TMNC, and CD34+. Recoveries expressed as medians (Q1; Q3) were: TNC 120.85 % (61.16 %; 154.16 %), TMNC 104.53 % (38.98 %;139.27 %), CD34+ 51.49 % (40.35 %; 91.82 %) and CFU-GM 93.37 % (90.86 %; 97.59 %) on comparing values ​​obtained after freezing/thawing and after DMSO removal.

Figure 4 shows that DMSO removal decreased TNC, HPC potency and CD34+ content. Recoveries, expressed as medians (Q1; Q3), were: TNC 83.98 % (42.62 %; 95.26 %), TMNC 98.71 % (57.00 %; 166.40 %), CD34+ 50.69 % (35.90 %; 95.96 %), and CFU-GM 74.80 % (57.92 %; 85.07 %), on comparing values ​​obtained after leukapheresis and after DMSO removal.

Results of correlation analysis between CFU-GM and CD34+ cells

Table 4 shows considerable decreases in CFU-GM and CD34+per kg after DMSO removal. Using Evans Handbook [29], the correlation between post-thaw values of viable CD34+ content and CFU-GM content was found to be significant (r = 0.751; p-value = 0.003) (Figure 5). A comparable result was found after DMSO removal (r = 0.814; p-value = 0.001) (Figure 6).

Figure 5.

Correlation between post-thaw values of CD34+ cell content and colony forming unit-granulocyte macrophage (CFU-GM) content (r = 0.751; p-value = 0.003).

Figure 6.

Correlation between the CD34+ cell content and colony forming unit-granulocyte macrophage (CFU-GM) content after dimethyl sulfoxide removal (r = 0.814; p-value = 0.001).

Glycerol was used as a cryoprotectant in the first autologous hematopoietic cell transplantations performed before the start of the regular hematological transplantation program at University Hospital Hradec Králové, [31,32]. Later, the use of DMSO was introduced, and washing was performed as standard. Since the beginning of 1994, routine washing of DMSO was stopped, and the rule applied abroad was followed, namely that the daily dose of DMSO per kg of the recipient's weight should not exceed 1 g. Nevertheless, certain doubts persisted in the Czech Republic about the clinical use of DMSO, especially with regard to the quality of the product [12]. These doubts were definitively resolved in 1996 when the State Institute for Drug Control fully accepted DMSO for use. This acceptance, however, mandated laboratory testing of DMSO products at individual transplantation centers until a clinically applicable CE-marked product became available in the Czech Republic. If the DMSO daily dose exceeded the recommended limit, the infusions were spread over several days [12].

Currently, there are new trends, the goal of which is either to find other equally effective cryoprotective solutions or to reduce the risk of adverse reactions [15]. In general, the rule of "three Rs" – Replace, Reduce, Remove [33] – is applied. This involves the search for suitable combinations with well-known, but individually less effective, cryoprotectants such as ethylene glycol, hydroxycellulose, sucrose, maltose, trehalose, and also some macromolecules (dextran, polyvinylpyrrolidone, etc.) [13]. Automatic washing systems working in a closed system are available. Compared with classic manual DMSO washing, their advantage is high viability of HPCs and minimal risk of microbial contamination. The disadvantage is the high price of the device [13,17,19,20].

Previous studies at this center with multiple myeloma patients who underwent autologous transplantation demonstrated that DMSO doses per kg administered at transplantation were, in the majority of cases, well below the maximum allowable daily dose [24]. The problem is posed by occasional poorly mobilized patients, with whom it was necessary to split the transplant dose into two after several days.

In accordance with Yang et al. [34], this study demonstrated a correlation between the level of CD34+ cells and CFU-GM content. This correlation does not mean, however, that in individual cases such a correlation may not exist as demonstrated by Watts et al. [35] and Morgestern et al. [36]. The results of this study for the CD34+ cell freeze/thaw recovery process were higher that the results of Yang et al. [34], who established a median recovery of viable CD34+ cells in the freeze/thaw process of 66.4 % (versus 90.18 % in the present study), and CFU-GM of 63.0 % (versus 80.6 % in the present study). This difference can be explained by more efficient cryoprotection based on a combination of DMSO and HES. Mean CD34+ cell recovery after manual DMSO removal, as determined by Chen et al. [37] using Trypan blue solution, was 85.4 % which was higher than the present results determined by flow cytometry.

The results of this study were achieved in a relatively small group, as DMSO reduction after thawing was performed only for patients with a known higher risk of arrhythmia (amyloidosis of the heart) or who were at risk of impaired elimination of the DMSO (renal failure caused by amyloidosis of the kidney). In only one case, the removal of DMSO was not planned but was performed in an emergency situation, namely a severe adverse reaction after initiation of an infusion of thawed concentrate.

It was confirmed that the removal of DMSO by washing the cells leads to a significant decrease in the viability of MNCs and the dose of CD34+ cells per kg of recipient weight (Table 2) and that the results of the washing process and of the entire process show large individual differences. Decreased viability may be a manifestation of cryopreservation-induced delayed cell death [38]. Nevertheless, in all these patients, a sufficient dose was administered, and delayed engraftments of neutrophils or platelets were not reported. In our practice, we routinely estimate the dose of CD34+ cells and CFU-GM from thawed control samples, always comparing the resulting values with the doses determined before cryopreservation.

The results of this study confirm that DMSO washing should be limited to indicated cases only, which is in line with the European Directorate for the Quality of Medicines & HealthCare (EDQM) 2022 recommendation [11]. Another problem is that the result of determining the washed product sterility is known only after administration to the recipient. However, this risk is minimal if the thawed product is handled in purity Grade A clean rooms with Class B background.

We still regard the use of DMSO as safe if the daily dose of 1 g per kg is not exceeded [11].

DMSO removal should only be performed in indicated cases, as it leads to significant loss of progenitor cells. Despite the fact that data from only 13 patients were analyzed and that the resulting CD34+ dose was suboptimal, engraftments were achieved in all cases. The minimal CD34+ dose should be 1 × 106 per kg of patient body weight with optimum being 2 × 106 per kg of patient body weight as recommended by EBMT standards. Determination of the CD34+ level should be performed simultaneously with determination of CFU-GM to minimize the risk of prolonged engraftment or non-engraftment.

This study was supported by MH CZ-DRO (UHHK, 00179906).

M.J., M.L., and P.M.: study design, writing, editing; M.J. and J.G.: practical performance of the DMSO reduction and data collection; A.B.: statistical analysis of data; D.C. and J.R.: review and editing.