Factors influencing cord blood viability assessment before cryopreservation.
BACKGROUND
Cord blood (CB) viability determines product quality and varies with time and temperature of exposure before cryopreservation. Global viability assessment may not reflect viability of white blood cell (WBC) subsets, CD34+ cell viability, or hematopoietic stem/progenitor cells function.


METHODS
We compared trypan blue (TB) and acridine orange/propidium iodide (AO/PI) staining with flow-cytometric (7-aminoactinomycin D [7-AAD]) viability in total WBCs (Tot-AAD), granulocytes, monocytes, lymphocytes, and CD34+ cells and total nucleated cell, CD34+, and colony-forming cell (CFC) recovery as a function of time and temperature (4, 24, and 37 degrees C) before cryopreservation.


RESULTS
TB, AO/PI, and Tot-AAD viability was concordant up to 72 hours (4 degrees C) and 48 hours (24 degrees C) postcollection; however, CD34+ viability was significantly higher due to loss of viable granulocytes. In contrast, at "physiologic" temperature (37 degrees C), the decline in TB, AO/PI, and Tot-AAD viability was significantly lower than the rate of viable CD34+ and CFC loss. At all times and temperatures, CFC recovery correlated best with CD34+ viability and recovery.


CONCLUSIONS
CB cell populations exhibit differential time- and temperature-dependent susceptibility to in vitro cell death; consequently, global viability measurements using TB, AO/PI, or 7-AAD (Tot-AAD) significantly underestimate (4-24 degrees C) or overestimate (24-37 degrees C) CD34+ viability and CFC recovery. Our results demonstrate the limitations of global viability assessment with TB, AO/PI, and total AAD; endorse the routine use of CD34+ cell viability measurements; emphasize the importance of temperature control during shipment; and have implications with regard to establishing acceptable "cutoff" values for viability measurements and CB collection through processing time.
Solomon
Michael
M
Department of Pathology and Pediatrics, St Louis University School of Medicine, 1402 South Grand Blvd, St Louis, MO 63104, USA.
Wofford
Jonathan
J
Johnson
Cory
C
Regan
Donna
D
Creer
Michael H
MH
D000925:Anticoagulants / Q000494:pharmacology; D015703:Antigens, CD / Q000032:analysis; D018952:Antigens, CD34 / Q000032:analysis; D002470:Cell Survival / Q000502:physiology; D003114:Colony-Forming Units Assay / Q000379:methods; D004396:Coloring Agents; D015925:Cryopreservation / Q000379:methods*; D005260:Female; D005312:Fetal Blood / Q000166:cytology*; D005434:Flow Cytometry / Q000379:methods; D006801:Humans; D007231:Infant, Newborn; D007962:Leukocytes / Q000166:cytology; D008214:Lymphocytes / Q000166:cytology; D010920:Placenta / Q000166:cytology / Q000502:physiology; D011247:Pregnancy; D014471:Umbilical Veins
D000925:Anticoagulants; D015703:Antigens, CD; D018952:Antigens, CD34; D004396:Coloring Agents