**3. Results**

The procedure for determining stem cell potency, quality and release criteria is a 3-step process. However, only the first step requires cell culture and provides all the information for the remaining steps of the procedure. The culture step involves a 3-point cell dose response for two stem cell populations (CFC-GEMM and HPP-SP) for both the RS and samples.

#### **3.1 Step 1 – Measuring stem cell potency of mobilized peripheral blood**

The first step in the procedure is illustrated in Figure 1. This shows the cell dose responses for the mature multipotential stem cell, CFC-GEMM (Figs. 1A and 1B), and the more primitive stem cell HPP-SP (Figs. 1C and 1D) from 4 different mPB samples cultured for 5 (Figs. 1A and 1C) and 7 days (Figs. 1B and 1D). The graphs demonstrate that an approximate 3-fold increase in ATP concentration occurs within 2 days when the incubation time is increased from 5 to 7 days. A 7 day incubation period allows for increased assay sensitivity as well as the ability to perform the assay to accommodate a work schedule. Since the increase is cell dose dependent, it demonstrates that the assay is directly measuring an increase in the number of cells as a result of cell proliferation. It should be noted however, that although measurements at both 5 and 7 days are on the exponential part of the growth curve for both HPP-SP and CFC-GEMM, measurement of proliferation on day 7 will exhibit slightly greater coefficients of variation (CVs) and will also include cells that have initiated differentiation. On day 5, little or no differentiation occurs (data not shown).

The ATP bioluminescence assay has been previously validated in accordance with bioanalytical method validation (FDA, 2001). For this specific application, the assay exhibited an accuracy (proportion of correct outcomes) of greater than 90%. Sensitivity (proportion of correctly identified positive samples) and specificity (proportion of correctly identified negative samples) were determined using receiver operator characteristic (ROC) statistics (DeLong et al. 1985) in which the area under the curve (AUC) was determined for background (no stimulatory cocktail) versus CFC-GEMM and background versus HPP-SP. For the former, the AUC was 0.752 (95% confidence intervals; 0.71-0.8; p < 0.0001), while for the latter the AUC was 0.73 (95% confidence intervals; 0.68-0.78, p<0.001). Since the AUC must be between 0.5 and 1, the results demonstrated that the assay could differentiate between sensitivity and specificity. Assay precision (reliability and reproducibility) was performed on background, CFC-GEMM and HPP-SP over a cell dose range from 2,500 to 10,000 cells/well and demonstrated coefficients of variation (CV) of 15% or less. This was in compliance with regulatory requirements (FDA, 2011). Robustness, in this case transferability of the assay from one laboratory to another, had been previously reported (Reems et al. 2008). The results demonstrated a correlation coefficient (R) between

Concentrations of ATP (µM) are provided as the mean ± 1 standard deviation of 8 replicate wells. The slope of the 3-point cell dose response was obtained from the linear regression using least squares analysis (Prism version 5, GraphPad Software, LaJolla, CA). For correlations, the slope of the linear regression, goodness of fit (r2) and correlation coefficient (R) are reported. Tests of significance for correlation were performed using the Pearson two-

The procedure for determining stem cell potency, quality and release criteria is a 3-step process. However, only the first step requires cell culture and provides all the information for the remaining steps of the procedure. The culture step involves a 3-point cell dose response for

The first step in the procedure is illustrated in Figure 1. This shows the cell dose responses for the mature multipotential stem cell, CFC-GEMM (Figs. 1A and 1B), and the more primitive stem cell HPP-SP (Figs. 1C and 1D) from 4 different mPB samples cultured for 5 (Figs. 1A and 1C) and 7 days (Figs. 1B and 1D). The graphs demonstrate that an approximate 3-fold increase in ATP concentration occurs within 2 days when the incubation time is increased from 5 to 7 days. A 7 day incubation period allows for increased assay sensitivity as well as the ability to perform the assay to accommodate a work schedule. Since the increase is cell dose dependent, it demonstrates that the assay is directly measuring an increase in the number of cells as a result of cell proliferation. It should be noted however, that although measurements at both 5 and 7 days are on the exponential part of the growth curve for both HPP-SP and CFC-GEMM, measurement of proliferation on day 7 will exhibit slightly greater coefficients of variation (CVs) and will also include cells that have initiated

two stem cell populations (CFC-GEMM and HPP-SP) for both the RS and samples.

**3.1 Step 1 – Measuring stem cell potency of mobilized peripheral blood** 

differentiation. On day 5, little or no differentiation occurs (data not shown).

**2.6 Assay validation and statistics** 

laboratories of 0.94 (p<0.001).

tailed test with an alpha of 0.05.

**3. Results** 

Fig. 1A and 1B. Measurement of Mobilized Peripheral Blood CFC-GEMM Stem Cell Potency on 5 and 7 Days of Culture.

Hematopoietic Stem Cell Potency for Cellular Therapeutic Transplantation 391

In addition to the samples, a mPB RS was also included and allows the potency ratio to be

Potency Ratio = Slope of the sample linear regression / Slope of the RS linear regression. The potency ratio therefore provides information on the dose required to obtain the same response as the RS. Since the potency of the RS is always 1, samples with a potency ratio less than 1 will require larger cell doses to produce the same response, while potency ratios

The results in Fig. 1 illustrate two fundamental concepts that are necessary for measuring stem cell potency and quality. The first concept is that the slope of the cell dose response should be greater for the more primitive stem cells (HPP-SP) than for the mature hematopoietic stem cells (CFC-GEMM), since the former have greater proliferation potential than the latter. The slope of the cell dose response therefore provides a direct measurement of stem cell proliferation potential. The greater the proliferation potential, the greater the potency. Thus, the slope of the cell dose response is also a direct measurement of potency. The second concept, also illustrated in Fig. 1, shows that as the slope increases at a specific cell dose, there

The result of combining these two concepts is shown in Figures 2A and 2B for CFC-GEMM and HPP-SP stem cell populations, respectively. The figures show that when the ATP concentration at a specific cell dose (in this case 5,000 cells/well) is plotted against the slope of the cell dose response linear regression for both stem cell populations cultured for either 5 or 7 days, there is a direct correlation between stem cell potency and quality. As a result, both stem cell potency and quality have to be taken into account to determine if the stem cell product conforms to specific, but arbitrary, acceptance values and can therefore be released for use.

Figure 3A and 3B shows the ability of CFC-GEMM and HPP-SP to proliferate at 5,000 cells/well after 5 and 7 days of culture, respectively. It had previously been found that, after 5 days in culture, an ATP concentration below 0.04µM indicated that cells could not sustain proliferation. At 7 days, this threshold was increased to 0.12µM. After 5 days of culture, the ATP concentration of samples 1 and 2 demonstrated minimal proliferation, but greater than the 0.04µM threshold. After 7 days of culture, proliferation of both samples had increased, together with samples 3 and 4. If release criteria were based solely on stem cell quality or proliferation ability, it would be assumed that all 4 samples might be acceptable for release. However, Fig. 2 demonstrates that both stem cell quality and potency have to be considered

Figure 3C shows the cumulative potency ratios of both CFC-GEMM and HPP-SP after 5 days and 7 days (Fig. 3D) in culture. Since the potency of the CFC-GEMM and HPP-SP reference standards is always 1, samples 1 and 2 exhibited potency ratios significantly less than the reference standard. In contrast, samples 3 and 4 exhibited both high stem cell quality and potency after 5 and 7 days of culture. Based on these results, mPB samples 1 and 2 would be sub-optimal or rejected, while samples 3 and 4 would be acceptable for use.

greater than 1 will require fewer cells to produce the same response as the RS.

is a concomitant increase in ATP concentration. This is a measure of stem cell quality.

**3.3 Step 3 – Using stem cell potency and quality to determine release criteria** 

**3.2 Step 2 – The relationship between stem cell potency and quality** 

calculated as follows:

as part of the release criteria.

Fig. 1C and 1D. Measurement of Mobilized Peripheral Blood HPP-SP Stem Cell Potency on 5 and 7 Days of Culture.

Fig. 1C and 1D. Measurement of Mobilized Peripheral Blood HPP-SP Stem Cell Potency on

5 and 7 Days of Culture.

In addition to the samples, a mPB RS was also included and allows the potency ratio to be calculated as follows:

Potency Ratio = Slope of the sample linear regression / Slope of the RS linear regression.

The potency ratio therefore provides information on the dose required to obtain the same response as the RS. Since the potency of the RS is always 1, samples with a potency ratio less than 1 will require larger cell doses to produce the same response, while potency ratios greater than 1 will require fewer cells to produce the same response as the RS.

#### **3.2 Step 2 – The relationship between stem cell potency and quality**

The results in Fig. 1 illustrate two fundamental concepts that are necessary for measuring stem cell potency and quality. The first concept is that the slope of the cell dose response should be greater for the more primitive stem cells (HPP-SP) than for the mature hematopoietic stem cells (CFC-GEMM), since the former have greater proliferation potential than the latter. The slope of the cell dose response therefore provides a direct measurement of stem cell proliferation potential. The greater the proliferation potential, the greater the potency. Thus, the slope of the cell dose response is also a direct measurement of potency. The second concept, also illustrated in Fig. 1, shows that as the slope increases at a specific cell dose, there is a concomitant increase in ATP concentration. This is a measure of stem cell quality.

The result of combining these two concepts is shown in Figures 2A and 2B for CFC-GEMM and HPP-SP stem cell populations, respectively. The figures show that when the ATP concentration at a specific cell dose (in this case 5,000 cells/well) is plotted against the slope of the cell dose response linear regression for both stem cell populations cultured for either 5 or 7 days, there is a direct correlation between stem cell potency and quality. As a result, both stem cell potency and quality have to be taken into account to determine if the stem cell product conforms to specific, but arbitrary, acceptance values and can therefore be released for use.

### **3.3 Step 3 – Using stem cell potency and quality to determine release criteria**

Figure 3A and 3B shows the ability of CFC-GEMM and HPP-SP to proliferate at 5,000 cells/well after 5 and 7 days of culture, respectively. It had previously been found that, after 5 days in culture, an ATP concentration below 0.04µM indicated that cells could not sustain proliferation. At 7 days, this threshold was increased to 0.12µM. After 5 days of culture, the ATP concentration of samples 1 and 2 demonstrated minimal proliferation, but greater than the 0.04µM threshold. After 7 days of culture, proliferation of both samples had increased, together with samples 3 and 4. If release criteria were based solely on stem cell quality or proliferation ability, it would be assumed that all 4 samples might be acceptable for release. However, Fig. 2 demonstrates that both stem cell quality and potency have to be considered as part of the release criteria.

Figure 3C shows the cumulative potency ratios of both CFC-GEMM and HPP-SP after 5 days and 7 days (Fig. 3D) in culture. Since the potency of the CFC-GEMM and HPP-SP reference standards is always 1, samples 1 and 2 exhibited potency ratios significantly less than the reference standard. In contrast, samples 3 and 4 exhibited both high stem cell quality and potency after 5 and 7 days of culture. Based on these results, mPB samples 1 and 2 would be sub-optimal or rejected, while samples 3 and 4 would be acceptable for use.

Hematopoietic Stem Cell Potency for Cellular Therapeutic Transplantation 393

Fig. 3. Combining Mobilized Peripheral Blood Stem Cell Potency and Quality to Provide

**3.4 Umbilical cord blood stem cell potency, quality and release and the relationship to** 

A total of 28 UCB samples were analyzed for potency and quality using the same procedure described for mPB above, except that all assays were terminated after 5 days in culture, rather than performing both 5 and 7 day cultures. A 3-point cell dose response was performed for both the CFC-GEMM and HPP-SP stem cell populations and the slope of the linear regression was calculated for each cell dose response. The respective potency ratio for each CFC-GEMM and HPP-SP sample was then calculated using a UCB reference standard that was prepared from cord blood unit cells that did not meet the necessary criteria for storage. The slopes and potency ratios for each stem cell population are shown in Table 1. Also shown are the reported times to neutrophil and platelet engraftment. For one sample (sample 10), insufficient cells were obtained to perform a cell dose response for both stem cell populations. In two other samples (samples 18 and 25), insufficient cells were obtained after thawing to perform a HPP-SP stem cell dose

Release Criteria.

response.

**engraftment potential** 

Fig. 2. Relationship Between Stem Cell Potency and Quality for Mobilized Peripheral Blood CFC-GEMM and HPP-SP Detected on Day 5 or 7 of Culture.

Fig. 2. Relationship Between Stem Cell Potency and Quality for Mobilized Peripheral Blood

CFC-GEMM and HPP-SP Detected on Day 5 or 7 of Culture.

Fig. 3. Combining Mobilized Peripheral Blood Stem Cell Potency and Quality to Provide Release Criteria.

#### **3.4 Umbilical cord blood stem cell potency, quality and release and the relationship to engraftment potential**

A total of 28 UCB samples were analyzed for potency and quality using the same procedure described for mPB above, except that all assays were terminated after 5 days in culture, rather than performing both 5 and 7 day cultures. A 3-point cell dose response was performed for both the CFC-GEMM and HPP-SP stem cell populations and the slope of the linear regression was calculated for each cell dose response. The respective potency ratio for each CFC-GEMM and HPP-SP sample was then calculated using a UCB reference standard that was prepared from cord blood unit cells that did not meet the necessary criteria for storage. The slopes and potency ratios for each stem cell population are shown in Table 1. Also shown are the reported times to neutrophil and platelet engraftment. For one sample (sample 10), insufficient cells were obtained to perform a cell dose response for both stem cell populations. In two other samples (samples 18 and 25), insufficient cells were obtained after thawing to perform a HPP-SP stem cell dose response.

Hematopoietic Stem Cell Potency for Cellular Therapeutic Transplantation 395

Fig. 4. Correlation Between Proliferation Potential (Potency) and Proliferation Ability (Quality) for CFC-GEMM and HPP-SP Stem Cells from Umbilical Cord Blood.


IE = Insufficient cells to perform cell dose response. NE = No engraftment.

Table 1. Stem Cell Proliferation Potential / Potency Characteristics and Time to Engraftment of 28 Umbilical Cord Blood Samples.

1 2.94E-05 2.91 2.23E-05 0.70 28 237 2 3.14E-05 3.12 2.19E-05 0.69 14 2 3 1.41E-05 1.40 1.01E-05 0.31 6 45 4 2.09E-05 2.07 3.04E-05 0.95 30 49 5 2.19E-05 2.18 2.59E-05 0.81 17 39 6 9.28E-06 0.92 1.79E-05 0.56 12 9 7 2.25E-05 2.23 1.67E-05 0.52 17 45 8 2.50E-05 2.48 1.48E-05 0.46 22 39 9 1.52E-05 1.50 9.70E-06 0.30 56 13 10 IE - IE - 43 103 11 1.77E-05 1.75 9.39E-06 0.29 34 7 12 2.83E-05 2.81 1.99E-05 0.62 20 26 13 1.09E-05 1.08 7.43E-06 0.23 NE NE 14 8.12E-06 0.81 3.55E-06 0.11 19 183 15 6.69E-06 0.66 4.77E-06 0.15 31 122 16 1.26E-05 1.25 1.02E-05 0.32 13 39 17 1.81E-05 1.80 1.90E-05 0.60 5 40 18 3.07E-05 3.05 IE - NE NE 19 2.01E-05 1.99 4.45E-05 1.39 27 38 20 2.52E-05 2.50 3.18E-05 1.00 22 62 21 1.54E-05 1.53 3.30E-05 1.03 29 55 22 2.31E-05 2.29 3.30E-05 1.03 18 23 23 1.20E-05 1.19 1.70E-05 0.53 28 70 24 1.63E-05 1.62 2.22E-05 0.70 15 46 25 1.46E-05 1.45 IE - 26 39 26 2.12E-05 2.10 2.73E-05 0.85 28 61 27 1.37E-05 1.35 1.74E-05 0.54 37 126 28 1.50E-05 1.49 2.16E-05 0.68 114 113

Potency Ratio for HPP-SP

Days to Neutrophil Engraftment (>500/ul)

Days to Platelet Engraftment (>50k/ul)

Slope for HPP-SP

Sample Number Slope for CFC-GEMM

Potency Ratio for CFC-GEMM

IE = Insufficient cells to perform cell dose response. NE = No engraftment.

of 28 Umbilical Cord Blood Samples.

Table 1. Stem Cell Proliferation Potential / Potency Characteristics and Time to Engraftment

Fig. 4. Correlation Between Proliferation Potential (Potency) and Proliferation Ability (Quality) for CFC-GEMM and HPP-SP Stem Cells from Umbilical Cord Blood.

Hematopoietic Stem Cell Potency for Cellular Therapeutic Transplantation 397

Figure 5A shows the stem cell quality (proliferation ability at a specific cell dose) and Fig. 5B, the cumulative stem cell potency ratio (proliferation potential measured as the slope of the linear regression of the cell dose response and compared to that of the reference standard) for each of the stem cell populations. Samples 18 and 25 only show the potency for the CFC-GEMM populations since insufficient cells were available to measure the potency of the more primitive HPP-SP population. For 21 of the samples, stem cell quality of both populations was greater than the arbitrary ATP concentration cutoff level of 0.04µM, below which cells cannot sustain proliferation. The same 21 samples also exhibited a cumulative potency above the RS potency of 1. Sample 6 exhibited a CFC-GEMM potency below the RS, while sample 13 demonstrated a CFC-GEMM potency slightly greater than the RS. However, the additional potency provided by the HPP-SP stem cell populations

It is now possible to consider the interpretation of the results. Samples 14 and 15 pose an interesting anomaly. The CFC-GEMM quality is below the ATP concentration cutoff point for both samples and slightly greater than the cutoff point for HPP-SP. However, both samples exhibit a cumulative potency below the RS. These results would indicate that both sample 14 and 15 would exhibit limited or no engraftment potential. From Table 1, the time to neutrophil engraftment for sample 14 was only 19 days while that for sample 15 was 31 days. Platelet engraftment was 183 and 122 days for sample 14 and 15, respectively. Therefore, these two samples did not agree with the reported clinical outcome. Table 1 also shows that samples 13 and 18 did not engraft. As described above, sample 13 exhibited a CFC-GEMM potency that was in a questionable range and may not have provided the necessary short-term engraftment and reconstitution. In contrast, sample 18, appeared to exhibit sufficient CFC-GEMM quality and potency, although insufficient cells did not allow information to be obtained for the primitive HPP-SP stem cell population. Despite the four sample outliers, the assay exhibits an accuracy of greater than 85%. Nevertheless, further studies that correlate *in vitro* data with more detailed clinical outcome for both engraftment and reconstitution would be prudent to ascertain a range for both stem cell quality and

**3.5 Correlation between the ATP concentration and TNC, MNC, viability, CD34** 

There was no correlation between the ATP concentration for both cord blood stem cell populations with either dye exclusion viability or CD34+ counts. This was to be expected since neither viability nor CD34 membrane expression are cell functionality or proliferation markers. However, ATP concentration did correlate with both the TNC and MNC, but only when calculated on a per kilogram patient body weight basis. These results are shown in Fig. 6A for TNC and 6B for MNC. In both cases, the ATP concentration was calculated based on the patient body weight of the number of cells transplanted. The results in Fig. 6A demonstrate that when TNC is used, a strong correlation is obtained for the CFC-GEMM, but although still statistically significant, the primitive HPP-SP stem cell population exhibited a lower correlation coefficient. In contrast, Fig. 6B shows that the correlation between the ATP concentration and the MNC, both based on kilogram body weight, for CFC-GEMM and HPP-SP is highly significant with lower variation compared to the TNC values. The results clearly demonstrate that the greater the number of cells transplanted, the greater the number of stem cells transplanted that can exhibit proliferation ability. However,

increased the cumulative potency above that of the RS.

potency that would improve the accuracy of the assay.

Figures 4A and 4B show the correlation of ATP concentrations at 5,000 cells/well with the slope of the UCB dose response for both CFC-GEMM and HPP-SP stem cell populations. The correlation coefficient (R) for HPP-SP was greater than that for CFC-GEMM, but the correlation for both stem cell populations was statistically significant (p < 0.001). The relationship between stem cell potency and quality is an indication that both parameters have to be taken into consideration when defining release criteria. Although stem cell quality could be ascertained for sample 10, insufficient cells were available to measure stem cell potency. Insufficient cells for samples 18 and 26 were also the reason why potency could not be determined for the HPP-SP stem cell population.

Fig. 5. Combining Umbilical Cord Blood Stem Cell Potency and Quality to Provide Release Criteria.

Figures 4A and 4B show the correlation of ATP concentrations at 5,000 cells/well with the slope of the UCB dose response for both CFC-GEMM and HPP-SP stem cell populations. The correlation coefficient (R) for HPP-SP was greater than that for CFC-GEMM, but the correlation for both stem cell populations was statistically significant (p < 0.001). The relationship between stem cell potency and quality is an indication that both parameters have to be taken into consideration when defining release criteria. Although stem cell quality could be ascertained for sample 10, insufficient cells were available to measure stem cell potency. Insufficient cells for samples 18 and 26 were also the reason why potency could

Fig. 5. Combining Umbilical Cord Blood Stem Cell Potency and Quality to Provide Release

Criteria.

not be determined for the HPP-SP stem cell population.

Figure 5A shows the stem cell quality (proliferation ability at a specific cell dose) and Fig. 5B, the cumulative stem cell potency ratio (proliferation potential measured as the slope of the linear regression of the cell dose response and compared to that of the reference standard) for each of the stem cell populations. Samples 18 and 25 only show the potency for the CFC-GEMM populations since insufficient cells were available to measure the potency of the more primitive HPP-SP population. For 21 of the samples, stem cell quality of both populations was greater than the arbitrary ATP concentration cutoff level of 0.04µM, below which cells cannot sustain proliferation. The same 21 samples also exhibited a cumulative potency above the RS potency of 1. Sample 6 exhibited a CFC-GEMM potency below the RS, while sample 13 demonstrated a CFC-GEMM potency slightly greater than the RS. However, the additional potency provided by the HPP-SP stem cell populations increased the cumulative potency above that of the RS.

It is now possible to consider the interpretation of the results. Samples 14 and 15 pose an interesting anomaly. The CFC-GEMM quality is below the ATP concentration cutoff point for both samples and slightly greater than the cutoff point for HPP-SP. However, both samples exhibit a cumulative potency below the RS. These results would indicate that both sample 14 and 15 would exhibit limited or no engraftment potential. From Table 1, the time to neutrophil engraftment for sample 14 was only 19 days while that for sample 15 was 31 days. Platelet engraftment was 183 and 122 days for sample 14 and 15, respectively. Therefore, these two samples did not agree with the reported clinical outcome. Table 1 also shows that samples 13 and 18 did not engraft. As described above, sample 13 exhibited a CFC-GEMM potency that was in a questionable range and may not have provided the necessary short-term engraftment and reconstitution. In contrast, sample 18, appeared to exhibit sufficient CFC-GEMM quality and potency, although insufficient cells did not allow information to be obtained for the primitive HPP-SP stem cell population. Despite the four sample outliers, the assay exhibits an accuracy of greater than 85%. Nevertheless, further studies that correlate *in vitro* data with more detailed clinical outcome for both engraftment and reconstitution would be prudent to ascertain a range for both stem cell quality and potency that would improve the accuracy of the assay.

#### **3.5 Correlation between the ATP concentration and TNC, MNC, viability, CD34**

There was no correlation between the ATP concentration for both cord blood stem cell populations with either dye exclusion viability or CD34+ counts. This was to be expected since neither viability nor CD34 membrane expression are cell functionality or proliferation markers. However, ATP concentration did correlate with both the TNC and MNC, but only when calculated on a per kilogram patient body weight basis. These results are shown in Fig. 6A for TNC and 6B for MNC. In both cases, the ATP concentration was calculated based on the patient body weight of the number of cells transplanted. The results in Fig. 6A demonstrate that when TNC is used, a strong correlation is obtained for the CFC-GEMM, but although still statistically significant, the primitive HPP-SP stem cell population exhibited a lower correlation coefficient. In contrast, Fig. 6B shows that the correlation between the ATP concentration and the MNC, both based on kilogram body weight, for CFC-GEMM and HPP-SP is highly significant with lower variation compared to the TNC values. The results clearly demonstrate that the greater the number of cells transplanted, the greater the number of stem cells transplanted that can exhibit proliferation ability. However,

Hematopoietic Stem Cell Potency for Cellular Therapeutic Transplantation 399

For biopharmaceutical products, potency is measured by comparing the dose response to that of an established RS (Thorpe et al. 1999; Lansky, 1999; FDA, 2011). For these materials, a parallel dose response to that of the RS should be obtained (Thorpe et al, 1999; Gottscalk & Dunn, 2005; Jonkman & Sidik, 2009). Lack of parallelism indicates either contamination or a different material to that of the RS. When the dose response curves are parallel to the RS, the horizontal displacement to the left or right indicates a greater or lower potency, respectively. The dose of the compound can be compared and, if necessary, adjusted to that of the reference standard. In this way, the same dose can always be used with compound batches

Cells, in particular, stem cells, pose significant differences and challenges to this paradigm. First, unlike biopharmaceutical products, where relatively large quantities of the material would be available to establish several batches of reference standards, it might be extremely difficult to establish cell reference standards (Strong et al. 2009; Rayment & Williams, 2010). There are several alternatives to establishing reference standards for cell therapeutics that have already been discussed above. However, a prerequisite for establishing reference standards and to compare results within and between laboratories is the use of a standardized and validated assay that is sufficiently robust so that it can be transferred and established in different laboratories. Lack of such an assay has been the reason why laboratories have not been able to directly compare processing, cryopreservation and thawing procedures for hematopoietic and other stem cell therapeutic products prior to use. It might be argued that the present study should have been performed in parallel with the CFU assay. This has been the functional assay used previously in hematopoietic stem cell processing laboratories and is still used by the cord blood community today. More recently, the CFU assay has been suggested as a potency assay (Page et al. 2011a, 2011b), in addition to other parameters normally measured. These include total nucleated cell count (TNC), viability and CD34 membrane expression (FDA, 2009). There were three reasons for not performing parallel studies using the CFU assay. First, the ATP bioluminescence assay was originally derived from the methylcellulose CFU assay. Unlike the CFU assay, however, the ATP assay has undergone several major technical advances culminating in the assay used for the present study. It was also previously shown that even the methylcellulose-free format used in this study is not only equivalent to the original CFU assay, but is clearly a more reliable, reproducible and robust assay (Rich, 2007, Reems et al, 2008). Furthermore, lower sensitivity and precision (high variations) of the CFU assay, coupled with the lack of standardization (see below), would have resulted in inconclusive results. Second, unlike the CFU assay, the ATP assay can and has been validated in compliance with bioanalytical regulatory requirements (FDA, 2001). Furthermore, for an assay to be a potency assay, the regulatory agencies require demonstration of specific assay characteristics (FDA, 2011). Assay validation is just one of these characteristics, but to validate an assay, standards and controls are required. The CFU assay lacks standards and controls and cannot be validated according to regulatory requirements. Finally, it is often assumed that the CFU assay measures proliferation, whereas the CFU assay is actually a clonogenic differentiation assay. Proliferation is certainly involved in the formation of hematopoietic colonies. However, the colonies produced in methylcellulose are identified and counted by the ability of the cells producing the colonies to differentiate and mature. Therefore, the CFU assay detects

**4. Discussion** 

of different potency.

the results also demonstrates that using the mononuclear cell count rather than total nucleated cell count produces a better estimate for the stem cell response. However, cell counts alone cannot be used as a potency assay and cannot replace the information and value provided by a standardized cell functionality assay.

Fig. 6. Correlation between ATP Concentration as a Measure of Stem Cell Proliferation and the Number of Cord Blood Cells Transplanted Expressed as either Total Nucleated Cell Counts (TNC) or Mononuclear Cell Counts (MNC)/Kilogram Body Weight.
