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Viability and engraftment of hematopoietic progenitor cells after long-term cryopreservation: effect of diagnosis and percentage dimethyl sulfoxide concentration
Cytotherapy, 2010; 12: 764–766
SHORT COMMUNICATION
Viability and engraftment of hematopoietic progenitor cells after long-term cryopreservation: effect of diagnosis and percentage dimethyl sulfoxide concentration
MUTHU VEERAPUTHIRAN1, JOHN W. THEUS1, GINA PESEK1, BART BARLOGIE2 & MICHELE COTTLER-FOX1 1Department
of Pathology and 2Myeloma Institute for Research and Therapy, University of Arkansas for Medical Sciences, Little Rock, Arkansas, USA
Abstract Background aims. We carried out a retrospective analysis of viability by diagnosis and dimethyl sulfoxide (DMSO) concentration in patients who had undergone autologous transplants using hematopoietic progenitor cells (HPC) after long-term storage (up to 17.8 years). Methods. Viability was tested using flow cytometry for HPC that were harvested and preserved using a controlled rate freezer and 5% or 10% DMSO with human serum albumin, then stored in liquid nitrogen. Data from 262 samples were analyzed (249 myeloma patients and 13 other diagnoses): 100 consecutively thawed samples with a storage time of ⬍1 year (all 10% DMSO), 50 consecutive samples stored for 1–4.9 years (10% DMSO), 50 samples stored for 5–9 years (5% DMSO) and all samples stored and used for transplant after ⬎9 years (60 samples, 5% DMSO; two samples, 10% DMSO). Results. No statistically significant difference in viability between the 5% DMSO and 10% DMSO groups was observed (P ⫽ 0.08), so the 1–4.9 years and 5–9 years were combined and the three groups (⬍1 year, 1–9 years and ⬎9 years) were compared using an anova test. There was no difference in viability based on cryostorage period (P ⫽ 0.23) or between myeloma and other diagnoses (P ⫽ 0.45). No difference was seen in time to White blood cell (WBC) engraftment (P ⫽ 0.10) or to platelet engraftment between groups (P ⫽ 0.52). Conclusions. These data suggest that long-term storage in 5% DMSO and human serum albumin is safe. Key Words: cryostorage, engraftment, hematopoietic progenitor cell viability
Introduction Most hematopoietic progenitor cells (HPC) harvested by apheresis are cryopreserved before transplantation. The major risk in cryopreservation lies in the freezing step, where cells may be damaged by formation of intracellular ice crystals. Dimethyl sulfoxide (DMSO) in combination with human serum albumin or other proteins has been used to prevent ice crystal formation. Nevertheless, thawing after cryopreservation is associated with a variable loss of cells for transplant. Some authors have suggested that the loss may be related to the amount of DMSO used (1). It has also been suggested that HPC from myeloma patients may survive storage less well than those from patients with other diseases (2).
Our program has collected and stored HPC since November 1989, changing from 5% DMSO to 10% DMSO in August 2000. We present a retrospective analysis of viability by diagnosis and DMSO concentration in patients who had undergone autologous transplantation using HPC after long-term storage (up to 17.8 years). We also examined the time to WBC and platelet engraftment based on the percentage DMSO and length of time in storage.
Methods HPC were collected by apheresis then preserved in 5% human serum albumin and either 5% DMSO in Hank’s balance salt solution (Sigma, St Louis,
Correspondence: Muthu Veeraputhiran, MD, MPH, Fellow, Transfusion Medicine, Division of Blood Bank/Transfusion Medicine, Department of Pathology, University of Arkansas for Medical Sciences, 4301 W Markham St, Mail slot 517, Little Rock, AR 72205, USA. E-mail: [email protected] (Received 6 November 2009; accepted 1 March 2010) ISSN 1465-3249 print/ISSN 1477-2566 online © 2010 Informa Healthcare DOI: 10.3109/14653241003745896
Viability of HPC after long-term cryostorage
765
Table I. Viability and engraftment data for the cells by length of cryostorage period. Cryostorage period ⬍1 year 1–9 years ⬎ 9 years
Mean CD34 viability
Mean time to platelet engraftment
92.33 (91.48, 93.18), n ⫽ 100 91.39 (90.29, 92.49), n ⫽ 100 92.82 (90.92, 94.73), n ⫽ 62
13.48 (11.99, 14.96), n ⫽ 42 12.55 (11.10, 14.0), n ⫽ 29 12.43 (11.17, 13.69), n ⫽ 21
Mean time to WBC engraftment 12.0 (11.69,12.31), n ⫽ 51 11.44 (10.79, 12.09), n ⫽ 34 11.14 ( 10.14,11.96), n ⫽ 29
95% Confidence intervals of the mean are given within parentheses.
MO, USA) or 10% DMSO in Plasmalyte A (Baxter, Deerfield, IL, USA), frozen using a controlled rate freezer and stored in liquid nitrogen. Viability was assessed by flow cytometry using unwashed samples and propidium iodide or 7-AAD uptake within the CD34⫹ population identified using the International Society of Hematotherapy and Graft Engineering (ISHAGE) method (3). The change from Hank’s solution to Plasmalyte A and from propidium iodide to 7-AAD was validated appropriately. Data from 263 samples were analyzed (249 myeloma patients and 13 other diagnoses): 100 consecutively thawed samples with a storage time of ⬍1 year (all 10% DMSO), 50 consecutive samples stored for 1–4.9 years (all 10% DMSO), 50 consecutive samples stored for 5–9 years (all 5% DMSO) and all samples used for transplantation after ⬎9 years (60 samples, 5% DMSO; two samples, 10% DMSO). To compare the effect of DMSO concentration they were also regrouped based on the concentration of DMSO used (101 samples, 5% DMSO; 150 samples, 10% DMSO). An unpaired t-test was used to compare any statistical difference between the groups. As there was no difference between 5% and 10% DMSO, the 1–4.9year and 5–9-year groups, they were combined and compared with ⬍1-year and ⬎9-year groups using an anova test. We also looked at two patients who had HPC harvested at two different periods and compared viability for varying periods of cryostorage.
prior to two consecutive CBC with platelets ⬎20 000/mm3, we are unable to give a specific date for engraftment. HPC collections remained viable after 17.8 years, the longest time reported for use after cryostorage to date. Although a significant decrease in nucleated cell recovery and viability in patients with myeloma has been reported previously (2), no significant difference in CD34 viability was observed here, in agreement with another study (4). Storage time did not affect engraftment. One patient had three cell infusions from the same collection (two after storage ⬍1 year and one after ⬎9 years), with viabilities of 91.5% (⬍1 year) and 83% (⬎9 years), respectively. Another patient had four infusions of cells from the same collection (three after 5–9 years and one after ⬎9 years), with viabilities of 93.6% (5–9 years) and 92% (⬎9 years), respectively. The infusion of DMSO has been related to adverse events such as nausea, vomiting, flushing, fever, dyspnea, transient hypertension, cardiac events and anaphylaxis, for which reasons it has been suggested that the least amount of DMSO possible should be administered to a patient at any given time (5–7). Until now there has been no data to show that long-term storage in 5% DMSO yields a clinically viable product. We now provide these data, as there was no statistically significant difference in viability and engraftment between the 5% and 10% DMSO groups after storage for up to 18 years.
Results and discussion The overall results are summarized in Table I and Figure I. The mean viabilities for the ⬍1-year, 1–9year and ⬎9-year groups were 92.33%, 91.39% and 92.82% respectively, with no statistically significant difference in viability between them (P ⫽ 0.23). Using a unpaired t-test, no difference was seen in viability based on diagnosis (P ⫽ 0.45) or between 5% and 10% DMSO (P ⫽ 0.08). The means for 5% DMSO and 10% DMSO were 92.85% and 91.6%, respectively. No difference was seen in the time to White blood cell before (WBC) engraftment, as defined by Absolute neutrophil count (ANC) ⬎ 500 on two consecutive Complete blood count (CBC) (P ⫽ 0.10) or platelet engraftment to 20 000, between the groups (P ⫽ 0.52). All patients had platelet engraftment, but for those patients discharged home
Figure 1. Whisker box-plot comparing the minimum, maximum, median first and third quartile viabilities for the three groups (<1year, 1-9-year, >9-year) based on cryostorage period.
766
M. Veeraputhiran et al.
Declaration of interest: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.
3.
4.
References 1.
2.
Abrahamsen J, Bakken A, Bruserud O. Cryopreserving human peripheral blood progenitor cells with 5-percent rather than 10-percent DMSO results in less apoptosis and necrosis in CD 34⫹ cells. Transfusion. 2002;42: 1573–80. Fois E, Desmartin M, Benhamida S, Xavier F, Vanneaux V, Rea D, et al. Recovery, viability and clinical toxicity of thawed and washed haematopoetic progenitor cells: analysis of 952 autologous peripheral blood stem cell transplantations. Bone Marrow Transplant. 2007;40:831–35.
5.
6.
7.
Sutherland DR, Anderson L, Keeney M, Nayar R, Chin-Yee I. The ISHAGE guidelines for CD34⫹ cell determination by flow cytometry. International Society of Hematotherapy and Graft Engineering. J Hematother. 1996;5:213–26. Sartor M, Antonenas V, Garvin F, Webb M, Bradstock KF. Recovery of viable CD 34⫹ cells from cryopreserved hemopoietic progenitor cell products. Bone Marrow Transplant. 2005;36:199–204. Galmes A, Besalduch J, Bargay J, Novo A, Morey M, Jose M et al. Long term storage at –80°C of hematopoietic progenitor cells with 5-percent dimethysulfoxide as the sole cryoprotectant. Transfusion. 1999;39:70–3. Davis JM, Rowley SD, Braine HG, Piantadosi S, Santos GW et al. Clinical toxicity of cryopreserved bone marrow graft infusion. Blood. 1990;75:78-1-86. Stroncek DF, Fautsch SK, Lasky LC, Hurd DD, Ramsay Nk, McCullough J et al. Adverse reactions in patients transfused with cryopreserved marrow. Transfusion. 1991;31:521–26.
SHORT COMMUNICATION
Viability and engraftment of hematopoietic progenitor cells after long-term cryopreservation: effect of diagnosis and percentage dimethyl sulfoxide concentration
MUTHU VEERAPUTHIRAN1, JOHN W. THEUS1, GINA PESEK1, BART BARLOGIE2 & MICHELE COTTLER-FOX1 1Department
of Pathology and 2Myeloma Institute for Research and Therapy, University of Arkansas for Medical Sciences, Little Rock, Arkansas, USA
Abstract Background aims. We carried out a retrospective analysis of viability by diagnosis and dimethyl sulfoxide (DMSO) concentration in patients who had undergone autologous transplants using hematopoietic progenitor cells (HPC) after long-term storage (up to 17.8 years). Methods. Viability was tested using flow cytometry for HPC that were harvested and preserved using a controlled rate freezer and 5% or 10% DMSO with human serum albumin, then stored in liquid nitrogen. Data from 262 samples were analyzed (249 myeloma patients and 13 other diagnoses): 100 consecutively thawed samples with a storage time of ⬍1 year (all 10% DMSO), 50 consecutive samples stored for 1–4.9 years (10% DMSO), 50 samples stored for 5–9 years (5% DMSO) and all samples stored and used for transplant after ⬎9 years (60 samples, 5% DMSO; two samples, 10% DMSO). Results. No statistically significant difference in viability between the 5% DMSO and 10% DMSO groups was observed (P ⫽ 0.08), so the 1–4.9 years and 5–9 years were combined and the three groups (⬍1 year, 1–9 years and ⬎9 years) were compared using an anova test. There was no difference in viability based on cryostorage period (P ⫽ 0.23) or between myeloma and other diagnoses (P ⫽ 0.45). No difference was seen in time to White blood cell (WBC) engraftment (P ⫽ 0.10) or to platelet engraftment between groups (P ⫽ 0.52). Conclusions. These data suggest that long-term storage in 5% DMSO and human serum albumin is safe. Key Words: cryostorage, engraftment, hematopoietic progenitor cell viability
Introduction Most hematopoietic progenitor cells (HPC) harvested by apheresis are cryopreserved before transplantation. The major risk in cryopreservation lies in the freezing step, where cells may be damaged by formation of intracellular ice crystals. Dimethyl sulfoxide (DMSO) in combination with human serum albumin or other proteins has been used to prevent ice crystal formation. Nevertheless, thawing after cryopreservation is associated with a variable loss of cells for transplant. Some authors have suggested that the loss may be related to the amount of DMSO used (1). It has also been suggested that HPC from myeloma patients may survive storage less well than those from patients with other diseases (2).
Our program has collected and stored HPC since November 1989, changing from 5% DMSO to 10% DMSO in August 2000. We present a retrospective analysis of viability by diagnosis and DMSO concentration in patients who had undergone autologous transplantation using HPC after long-term storage (up to 17.8 years). We also examined the time to WBC and platelet engraftment based on the percentage DMSO and length of time in storage.
Methods HPC were collected by apheresis then preserved in 5% human serum albumin and either 5% DMSO in Hank’s balance salt solution (Sigma, St Louis,
Correspondence: Muthu Veeraputhiran, MD, MPH, Fellow, Transfusion Medicine, Division of Blood Bank/Transfusion Medicine, Department of Pathology, University of Arkansas for Medical Sciences, 4301 W Markham St, Mail slot 517, Little Rock, AR 72205, USA. E-mail: [email protected] (Received 6 November 2009; accepted 1 March 2010) ISSN 1465-3249 print/ISSN 1477-2566 online © 2010 Informa Healthcare DOI: 10.3109/14653241003745896
Viability of HPC after long-term cryostorage
765
Table I. Viability and engraftment data for the cells by length of cryostorage period. Cryostorage period ⬍1 year 1–9 years ⬎ 9 years
Mean CD34 viability
Mean time to platelet engraftment
92.33 (91.48, 93.18), n ⫽ 100 91.39 (90.29, 92.49), n ⫽ 100 92.82 (90.92, 94.73), n ⫽ 62
13.48 (11.99, 14.96), n ⫽ 42 12.55 (11.10, 14.0), n ⫽ 29 12.43 (11.17, 13.69), n ⫽ 21
Mean time to WBC engraftment 12.0 (11.69,12.31), n ⫽ 51 11.44 (10.79, 12.09), n ⫽ 34 11.14 ( 10.14,11.96), n ⫽ 29
95% Confidence intervals of the mean are given within parentheses.
MO, USA) or 10% DMSO in Plasmalyte A (Baxter, Deerfield, IL, USA), frozen using a controlled rate freezer and stored in liquid nitrogen. Viability was assessed by flow cytometry using unwashed samples and propidium iodide or 7-AAD uptake within the CD34⫹ population identified using the International Society of Hematotherapy and Graft Engineering (ISHAGE) method (3). The change from Hank’s solution to Plasmalyte A and from propidium iodide to 7-AAD was validated appropriately. Data from 263 samples were analyzed (249 myeloma patients and 13 other diagnoses): 100 consecutively thawed samples with a storage time of ⬍1 year (all 10% DMSO), 50 consecutive samples stored for 1–4.9 years (all 10% DMSO), 50 consecutive samples stored for 5–9 years (all 5% DMSO) and all samples used for transplantation after ⬎9 years (60 samples, 5% DMSO; two samples, 10% DMSO). To compare the effect of DMSO concentration they were also regrouped based on the concentration of DMSO used (101 samples, 5% DMSO; 150 samples, 10% DMSO). An unpaired t-test was used to compare any statistical difference between the groups. As there was no difference between 5% and 10% DMSO, the 1–4.9year and 5–9-year groups, they were combined and compared with ⬍1-year and ⬎9-year groups using an anova test. We also looked at two patients who had HPC harvested at two different periods and compared viability for varying periods of cryostorage.
prior to two consecutive CBC with platelets ⬎20 000/mm3, we are unable to give a specific date for engraftment. HPC collections remained viable after 17.8 years, the longest time reported for use after cryostorage to date. Although a significant decrease in nucleated cell recovery and viability in patients with myeloma has been reported previously (2), no significant difference in CD34 viability was observed here, in agreement with another study (4). Storage time did not affect engraftment. One patient had three cell infusions from the same collection (two after storage ⬍1 year and one after ⬎9 years), with viabilities of 91.5% (⬍1 year) and 83% (⬎9 years), respectively. Another patient had four infusions of cells from the same collection (three after 5–9 years and one after ⬎9 years), with viabilities of 93.6% (5–9 years) and 92% (⬎9 years), respectively. The infusion of DMSO has been related to adverse events such as nausea, vomiting, flushing, fever, dyspnea, transient hypertension, cardiac events and anaphylaxis, for which reasons it has been suggested that the least amount of DMSO possible should be administered to a patient at any given time (5–7). Until now there has been no data to show that long-term storage in 5% DMSO yields a clinically viable product. We now provide these data, as there was no statistically significant difference in viability and engraftment between the 5% and 10% DMSO groups after storage for up to 18 years.
Results and discussion The overall results are summarized in Table I and Figure I. The mean viabilities for the ⬍1-year, 1–9year and ⬎9-year groups were 92.33%, 91.39% and 92.82% respectively, with no statistically significant difference in viability between them (P ⫽ 0.23). Using a unpaired t-test, no difference was seen in viability based on diagnosis (P ⫽ 0.45) or between 5% and 10% DMSO (P ⫽ 0.08). The means for 5% DMSO and 10% DMSO were 92.85% and 91.6%, respectively. No difference was seen in the time to White blood cell before (WBC) engraftment, as defined by Absolute neutrophil count (ANC) ⬎ 500 on two consecutive Complete blood count (CBC) (P ⫽ 0.10) or platelet engraftment to 20 000, between the groups (P ⫽ 0.52). All patients had platelet engraftment, but for those patients discharged home
Figure 1. Whisker box-plot comparing the minimum, maximum, median first and third quartile viabilities for the three groups (<1year, 1-9-year, >9-year) based on cryostorage period.
766
M. Veeraputhiran et al.
Declaration of interest: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.
3.
4.
References 1.
2.
Abrahamsen J, Bakken A, Bruserud O. Cryopreserving human peripheral blood progenitor cells with 5-percent rather than 10-percent DMSO results in less apoptosis and necrosis in CD 34⫹ cells. Transfusion. 2002;42: 1573–80. Fois E, Desmartin M, Benhamida S, Xavier F, Vanneaux V, Rea D, et al. Recovery, viability and clinical toxicity of thawed and washed haematopoetic progenitor cells: analysis of 952 autologous peripheral blood stem cell transplantations. Bone Marrow Transplant. 2007;40:831–35.
5.
6.
7.
Sutherland DR, Anderson L, Keeney M, Nayar R, Chin-Yee I. The ISHAGE guidelines for CD34⫹ cell determination by flow cytometry. International Society of Hematotherapy and Graft Engineering. J Hematother. 1996;5:213–26. Sartor M, Antonenas V, Garvin F, Webb M, Bradstock KF. Recovery of viable CD 34⫹ cells from cryopreserved hemopoietic progenitor cell products. Bone Marrow Transplant. 2005;36:199–204. Galmes A, Besalduch J, Bargay J, Novo A, Morey M, Jose M et al. Long term storage at –80°C of hematopoietic progenitor cells with 5-percent dimethysulfoxide as the sole cryoprotectant. Transfusion. 1999;39:70–3. Davis JM, Rowley SD, Braine HG, Piantadosi S, Santos GW et al. Clinical toxicity of cryopreserved bone marrow graft infusion. Blood. 1990;75:78-1-86. Stroncek DF, Fautsch SK, Lasky LC, Hurd DD, Ramsay Nk, McCullough J et al. Adverse reactions in patients transfused with cryopreserved marrow. Transfusion. 1991;31:521–26.