**5. Conclusions**

It is often the case that an assay will be generic and adapted to fit the intended application. The present communication describes an assay that has been specifically designed and validated for the purpose of measuring stem cell quality and potency for hematopoietic cellular therapeutic products derived from mobilized peripheral blood, umbilical cord blood and even bone marrow (data not shown). A similar potency and quality assay has also been developed for mesenchymal stem cells. The assays incorporate an instrument-based, biochemical marker in the form of ATP, the concentration of which is directly proportional to the proliferation ability and potential of the stem cell populations being measured. The bioluminescence signal detection system is the most sensitive, non-radioactive readout available allowing the assay to incorporate external standards and controls. The implementation of a fully compliant potency and quality assay specific for hematopoietic stem cell products should not only help standardize cell processing procedures, but also reduce the risk of graft failure and improve safety and efficacy for the patient.

#### **6. References**


platelets and erythrocytes. This is the reason why the presence and number of progenitor cell colonies counted in the CFU assay, especially GM-CFC and Mk-CFC, relates to the appearance and number of neutrophils and platelets in the circulation of the patient (Page et

Although the number of UCB transplants has increased almost exponentially since the first published UCB transplant in 1989 (Gluckman et al. 1989), approximately 20% of patients receiving an unrelated UCB transplant exhibit graft failure (Page et al. 2011a, 2011b). This has, in part, been attributed to inadequate UCB potency (Page et al. 2011a, 2011b). Previous publications have focused on the need for standardized laboratory procedures (Rich, 1997; Wagner E et al. 2006; Brand A et al. 2008). A recent publication by Spellman et al. (2011) discusses problems facing the cord blood community and the guidelines and requirements for "standardized testing methodologies" to be established. The cell-based, ATP bioluminescence assay platform described in this communication to measure both stem cell potency and quality and, in addition, help define release criteria, constitutes the next generation of assays that addresses all of the necessary requirements including, but not limited to, standardized methodology, reproducibility with limited variability between testing sites, automated testing outputs, high throughput capability and rapid turnaround

It is often the case that an assay will be generic and adapted to fit the intended application. The present communication describes an assay that has been specifically designed and validated for the purpose of measuring stem cell quality and potency for hematopoietic cellular therapeutic products derived from mobilized peripheral blood, umbilical cord blood and even bone marrow (data not shown). A similar potency and quality assay has also been developed for mesenchymal stem cells. The assays incorporate an instrument-based, biochemical marker in the form of ATP, the concentration of which is directly proportional to the proliferation ability and potential of the stem cell populations being measured. The bioluminescence signal detection system is the most sensitive, non-radioactive readout available allowing the assay to incorporate external standards and controls. The implementation of a fully compliant potency and quality assay specific for hematopoietic stem cell products should not only help standardize cell processing procedures, but also reduce the risk of graft failure and improve safety and

Botnick LE, Hannon EC, Hellman S. (1979). Nature of the hematopoietic stem cell

Brand A, Eichler H, Szczepiorkowski ZM, Hess JR, Kekomaki E, McKenna DH,

Broxmeyer HE, Douglas GW, Hangoc C, Cooper S, Bard J, English D, Arny M, Thomas L,

PamphilionD, Reems J, Sacher RA, Takahashi TA, van der Watering LM (2008). Viability does not necessarily reflect the hematopoietic cell potency of a cord blood

Boyse EA. (1989) Human umbilical cord blood as a potential source of

compartment and its proliferative potential. Blood Cells. 5:195-210.

unit: results ofan interlaboratory exercise. Transfusion. 48:546-549.

al. 2011a).

time.

**5. Conclusions** 

efficacy for the patient.

**6. References** 

transplantable hematopoietic stem/progenitor cells. Proc Natl Acad Sci USA 86:3828-3832.


RegulatoryInformation/Guidances/Blood/UCM187144.pdf.


Hematopoietic Stem Cell Potency for Cellular Therapeutic Transplantation 405

Rich IN. (2003). In vitro hematotoxicity testing in drug development: A review of past, present, and future applications. Curr Opinion Drug Disc Devel. 6:100-109. Rich IN. (2007). High-throughput in vitro hemotoxicity testing and in vitro cross-platform

Rodriguez L, Garcia J, Querol S. (2005). Predictive utility of the attached segment in

Santos GW, Sensenbrenner LL, Burke PJ, Mullins GM, Vias WB, Tutschka PJ & Slavin RE.

Solves P, Planelles D, Mirabet V, Blasco I, Carbonell-Uberos F, Soler MA, Roig RJ. (2004)

Strong M, Farrugia A & Rebulla P. (2009). Stem cell and cellular thereapy developments.

Thomas ES, Buckner CD, Banaji M, Clift RA, Fefer A, Flournoy N, Goodell BW, Hickman

Thorpe R. Wadhwa M, Page C, Mire-Sluis A. (1999). Bioassays for the characterization and

Till JE & McCulloch EA. (1971). A direct measurement of the radiation sensitivity of normal

U.S. Food and Drug Administration (FDA) (2001). Guidance for Industry. Bioanalytical

U.S. Food and Drug Administration (FDA) (2011). Guidance for Industry. Potency tests for

with data issued by cord blood banks. Transfusion. 46: 1190-1198.

typing in umbilical cord blood banking. Clin Lab Haematol. 26:413-418. Spellman S, Hurley CK, Brady C, Phillips-Johnson L, Chow R, Laughlin M, McMannis J,

Santos GW. (1983). History of bone marrow transplantation. Clin Haematol. 12:611-639. Sohn SK, Kim JG, Seo KW, Chae YS, Jung JT, Suh JS, Lee KB. (2002). GM-CSF-based

transplantation. Bone Marrow Transplant. 30:81-86.

cord blood. Cyotherapy (March, Epub ahead of print).

mouse bone marrow cells. Radiat Res. 175:145-149.

mation/Guidances/ucm070107.pdf.

cellular and gene therapy products.

the quality control of a cord blood graft. Biol Blood Marrow Transplant. 11:247-

(1972). The use of cyclophosphamide for clinical transplantation. Transplant Proc.

mobilized effect in normal healthy donors for allogeneic peripheral blood stem cell

Utility of bag segment and cryovial samples for quality and confirmatory HLA

Reems J-A, Regan D, Rubinstein P, Kurtzberg J. (2011). Guidelines for the development and validation of new potency assays for the evaluation of umbilical

RO, Lerner KG, Neiman PE, Sale GE, Sanders JE, Singer J, Stevens M, Storb R & Weiden PI. (1977).One hundred patients with acute leukemia treated by chemotherapy, total body irradiation, and allogeneic marrow transplantation.

control of therapeutic cytokines; determination of potency. Dev Biol Stand. 97:61-

http://www.fda.gov/downloads/Drugs/GuidanceComplianceRegulatoryInfor

http://www.fda.gov/downloads/BiologicsBloodVaccines/GuidanceComplianceR egulatoryInformation/Guidances/CellularandGeneTherapy/UCM243392.pdf. Wagner E. Duval M, Dalle JH, Morin H, Bizier S, Champagne J, Champagne MA. (2006)

Assessment of cord blood unit characteristics on the day of transplant: comparison

comparative toxicity. Expert Opin. Drug Metab Toxicol. 3:295-307.

251.

4:559-564.

Biologicals. 37:103-107.

Blood. 49:511-533.

method validation.

71.

advanced Hodgkin's disease and prior radiation to the pelvic site. J Clin Oncol. 8:978-985.


Lansky D. (1999). Validation of bioassay for quality control. Dev Biol Stand. 97:157-68. Leung W, Ramirez M, Civin CI. (1999). Quantity and quality of engrafting cells in cord

8:978-985.

5:69-76.

National Marrow Donor Program (NMDP).

neic\_Tx\_Perfor/index.html#grafts.

(Aug. 2. Epub ahead of print).

index.aspx#CiteSummarySlides.

translation. Stem Cells. 28:996-1004.

42:289-97.

48:620-628.

assay. Tox Sci. 87:427-41.

Hematother. 6:191-193.

set of drug candidates. Toxicol Let 188:98-103.

Marrow Transplant. (Jan 28. Epub ahead of print).

transplantation. CIBMTR Summary Slides, 2010.

advanced Hodgkin's disease and prior radiation to the pelvic site. J Clin Oncol.

blood and autologous mobilized peripheral blood. Biol Blood Marrow Transplant.

http://www.marrow.org/PHYSICIAN/URD\_Search\_and\_Tx/Number\_of\_Alloge

concordance of a high throughput assay for bone marrow toxicity across a diverse

(2011a). Total colony-forming units are a strong, independent predictor of neutrophil and platelet engraftment after unrelated umbilical cord blood transplantation: A single-center analysis of 435 cord blood transplants. Biol Blood

Kurtzberg J. (2011b). The cord blood Apgar: a novel scoring system to optimize selection of banked cord blood grafts for transplantation. (2011b). Transfusion.

http://www.cibmtr.org/ReferenceCenter/SlidesReports/SummarySlides/pages/

unrelated transplantation: a transplant center perspective. Transfus Apher Sci.

characterizing and quantifying cell- and tissue-based therapies for clinical

evaluate the functional potential of umbilical cord blood progenitors. Transfusion.

hemotoxicity using a multifunctional bioluminescence colony-forming proliferation

Olaharski AJ, Uppal H, Cooper M, Platz S, Zabka TS, Kolaja KL. (2009). In vitro to in vivo

Page KM, Zhang L, Mendizabai A, Weasse S, Carter S, Gentry T, Balber E, Kurtzberg J.

Page KM, Zhang L, Mendizabai A, Weasse S, Carter S, Shoulars K, Gentry T, Balber E,

Pasquini MC & Wang Z. (2010). Current use and outcome of hematopoietic stem cell

Picardi A, Arcese W. (2010). Quality assessment of cord blood units selected for

Querol S, Gomez SG, Pagliuca A, Torrabadella M, Madrigal JA. (2010). Quality rather than quantity: the cord blood bank dilemma. Bone Marrow Transplant. 45:970-8. Rayment EA & Williams DJ. (2010). Concise rewiew: Mind the gap: challenges in

Reems J-A, Hall KM, Gebru LH, Taber G, Rich IN. (2008). Development of a novel assay to

Rich IN & Hall KM. (2005). Validation and development of a predictive paradigm for

Rich IN, Kubanek B. (1982). The effect of reduced oxygen tension on colony formation of

Rich IN. (1997). Standardization of the CFU-GM assay using hematopoietic growth factors. J.

erythropoietic cells in vitro. Brit J Haematol. 52:579-88.


http://www.fda.gov/downloads/Drugs/GuidanceComplianceRegulatoryInfor mation/Guidances/ucm070107.pdf.


**18** 

*Spain* 

**Detection of CMV Infection in Allogeneic SCT** 

Pilar Blanco-Lobo, Omar J. BenMarzouk-Hidalgo and Pilar Pérez-Romero

Cytomegalovirus (CMV) end-organ disease is a serious complication after stem cell transplantation (SCT) (Boeckh M, 2003). Within the first one hundred days after SCT, 50% of recipients develop CMV infection determined by positive antigenemia and 65 to 86.5% when viral replication is determined by real-time PCR (RT-PCR) (Ljungman et al., 2006; Solano et al., 2001). Described risk factors for CMV infection concern donor type, graft source, positive CMV serostatus of donor and recipient, CD34+ graft selection, preconditioning regimen, GvHD prophylaxis regimen, incidence of acute and chronic GvHD and prophylaxis and treatment for GvHD (Ljungman et al., 2002; Ozdemir et al., 2007). Pre-emptive therapy is currently based on viral replication determined by either antigenemia or RT-PCR (Drew, 2007). Although antigenemia has been extensively used (Drew, 2007), RT-PCR has been shown to be more sensitive (Hakki et al., 2003; Solano et al.,

The use of techniques based on nucleic acid amplification for the detection of CMV in clinical samples are in expansion and in many hospitals have replaced the use of other assays such as viral cultures or pp65 antigenemia. Several studies have assessed the performance between the different CMV viral load assays available. However, no many studies have compared the differences between the DNA extraction methods used (Fahle & Fischer, 2000; Caliendo et al., 2007; Kalpoe et al., 2004; Leruez-Ville et al., 2003; Avetisyan et al., 2006; Boeckh et al., 2009; Gerna et al., 2008; Gimeno et al., 2008). Although during the DNA extraction the majority of the methods use internal controls as a measurement of the DNA loss during the extraction procedure, in the downstream amplification not many of the assays use DNA standards that will facilitate the comparison among the different kits and standardization of the results between hospitals. In fact the availability of an optimal and efficient DNA extraction procedure that can use a broad type of samples with minimum modifications in the procedure may be practical and affordable for use in the clinical

One of the differences between results using the methods available is type of clinical specimen used to perform the CMV DNA extraction. Samples collected vary from plasma,

**1. Introduction** 

2001).

practice (Fahle & Fischer, 2000).

**Recipients: The Multiple Assays** 

*University Hospital Virgen del Rocio, Sevilla,* 

*Unit of Infectious Disease, Microbiology and Preventive Medicine, Instituto de Biomedicina de Sevilla (IBiS)/CSIC/Universidad de Sevilla,* 

Zubair AC, Kao G, Daley H, Schott D, Freedman A, Ritz J. (2006). CD34(+) CD38(-) and CD34(+) HLA-DR(-) cells in BM stem cell grafts correlate with short-term engraftment but have no influence on long-term hematopoietic reconstitution after autologous transplantation. Cytotherapy. 8:399-407.
