open access

Vol 5, No 4 (2012)
Research paper
Published online: 2012-12-17
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Transient warming events and cryogenic

Zbigniew R. Mrowiec, Adla Angelina, Julio Laluf
Journal of Transfusion Medicine 2012;5(4):153-158.

open access

Vol 5, No 4 (2012)
ORIGINAL PAPERS
Published online: 2012-12-17

Abstract

Background: The term “Transient Warming Events” (TWE) has been used to describe the effects of brief periods of exposure of cryopreserved cells to ambient temperature in the course of retrieving and returning outgoing samples from the same cane. This study was designed to evaluate the effect of multiple TWEs on cryogenically preserved umbilical cord blood units (UCB) at the New Jersey Community Blood Centre using established assays of cell recovery, viability, and clonogenicity. Materials and methods: The UCBs were collected and processed according to the laboratory SOPs from consenting donors. Each UCB was split into two 25 mL samples: an experimental and a control sample. Both samples were frozen using a Controlled Rate Freezer and then stored below –180°C in the vapour phase of a liquid nitrogen (LN2) freezer. The experimental samples were exposed 10 times to room temperature until the sample reached a target temperature. We tested target temperatures of –110°C and –80°C. Finally, both experiment and control samples were simultaneously thawed in a 37°C dry bath. Each sample was tested for Total Nucleated Cell (TNC) count, CD34+ cell count, cell viability, and ability to generate Colony Forming Units (CFU) in a standardized assay. Results: When UCB units were each exposed to 10 TWE to a target temperature of –110°C, analysis of cell counts showed a 1.5% loss of TNC, 3.5% loss of CD34+ cells, and 2% drop in CFU, with no loss of viability. None of the values between experimental and control was statistically significant. When UCB units were exposed to 10 TWE to a target temperature of –80°C, the differences between experimental and control samples in the TNC, CD34+ cell count and viability were also not statistically significant. The numbers of CFUs in experimental samples was decreased by 8% compared to control, and reached statistical significance. In the New Jersey Community Blood Centre, removal of a UCB from cryogenic storage takes less than a minute to execute, typical of other UCB storage centres with well planned and executed sample retrieval protocols. It would take three times as long for a 25 ml UCB to reach a target temperature of –110°C, and five times as long to reach a target temperature of –80°C. We found no significant degradation of cell function after repeated TWE to target temperatures as high as –110°C.Conclusions: The results of this study demonstrate that the TWE that UCB units experience in day-to-day sample retrieval activities are not detrimental to the samples that are returned repeatedly to cryogenic storage at the New Jersey Community Blood Centre. This should encourage other centres to conduct their own TWE studies to evaluate the effects of TWEs with each type of cryopreserved tissue and associated methods of storage and retrieval, before considering investing in highly expensive cryostorage units that are designed to minimize TWEs.

Abstract

Background: The term “Transient Warming Events” (TWE) has been used to describe the effects of brief periods of exposure of cryopreserved cells to ambient temperature in the course of retrieving and returning outgoing samples from the same cane. This study was designed to evaluate the effect of multiple TWEs on cryogenically preserved umbilical cord blood units (UCB) at the New Jersey Community Blood Centre using established assays of cell recovery, viability, and clonogenicity. Materials and methods: The UCBs were collected and processed according to the laboratory SOPs from consenting donors. Each UCB was split into two 25 mL samples: an experimental and a control sample. Both samples were frozen using a Controlled Rate Freezer and then stored below –180°C in the vapour phase of a liquid nitrogen (LN2) freezer. The experimental samples were exposed 10 times to room temperature until the sample reached a target temperature. We tested target temperatures of –110°C and –80°C. Finally, both experiment and control samples were simultaneously thawed in a 37°C dry bath. Each sample was tested for Total Nucleated Cell (TNC) count, CD34+ cell count, cell viability, and ability to generate Colony Forming Units (CFU) in a standardized assay. Results: When UCB units were each exposed to 10 TWE to a target temperature of –110°C, analysis of cell counts showed a 1.5% loss of TNC, 3.5% loss of CD34+ cells, and 2% drop in CFU, with no loss of viability. None of the values between experimental and control was statistically significant. When UCB units were exposed to 10 TWE to a target temperature of –80°C, the differences between experimental and control samples in the TNC, CD34+ cell count and viability were also not statistically significant. The numbers of CFUs in experimental samples was decreased by 8% compared to control, and reached statistical significance. In the New Jersey Community Blood Centre, removal of a UCB from cryogenic storage takes less than a minute to execute, typical of other UCB storage centres with well planned and executed sample retrieval protocols. It would take three times as long for a 25 ml UCB to reach a target temperature of –110°C, and five times as long to reach a target temperature of –80°C. We found no significant degradation of cell function after repeated TWE to target temperatures as high as –110°C.Conclusions: The results of this study demonstrate that the TWE that UCB units experience in day-to-day sample retrieval activities are not detrimental to the samples that are returned repeatedly to cryogenic storage at the New Jersey Community Blood Centre. This should encourage other centres to conduct their own TWE studies to evaluate the effects of TWEs with each type of cryopreserved tissue and associated methods of storage and retrieval, before considering investing in highly expensive cryostorage units that are designed to minimize TWEs.
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Keywords

cord blood; cryopreservation; transient warming events

About this article
Title

Transient warming events and cryogenic

Journal

Journal of Transfusion Medicine

Issue

Vol 5, No 4 (2012)

Article type

Research paper

Pages

153-158

Published online

2012-12-17

Bibliographic record

Journal of Transfusion Medicine 2012;5(4):153-158.

Keywords

cord blood
cryopreservation
transient warming events

Authors

Zbigniew R. Mrowiec
Adla Angelina
Julio Laluf

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