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Abstract

A contiguous segment attached to the cord blood unit (CBU) is required for verifying HLA types, cell viability, and, possibly, potency before transplantation since such a segment is considered to be representative of the CBU. However, little is known regarding the characteristics of contiguous segments in comparison to main bag units due to the difficulty experienced in accessing a large number of cryopreserved CBUs. In this study, we used 245 nonconforming CBUs for allogeneic transplantation. After thawing the cryopreserved CBU, the number of total nucleated cells (TNCs), CD34⁺ cells, and CFUs in CB from main bags and segments, as well as cell viability and apoptosis, were examined. The comparative analysis showed that the number of TNCs was significantly higher in CB from main bags, whereas the numbers of CD34⁺ cells and CFU-GM were significantly higher in CB from segments. While the cell viability of TNCs in segments was higher, the proportion of apoptotic TNCs was also higher. In contrast, no difference was observed between the proportion of apoptotic CD34⁺ cells in main bags and segments. In the correlation analysis, the numbers of TNCs, CD34⁺ cells, and CFU-GM in main bags were highly correlated with those in segments, indicating that CB from segments is indeed representative of CB in main bags. Taken together, we conclude that segments have higher CD34⁺ cells and CFU-GM and lower TNCs than the main cryopreserved bag, although the two compartments are highly correlated with each other.

Keywords

Cord blood Cryopreservation Quality

Introduction

Cord blood units (CBUs) are cryopreserved for prolonged periods prior to use in hematopoietic stem cell transplantation (HSCT), whereas hematopoietic stem cells collected from bone marrow and cytokine-mobilized peripheral blood are used quickly after collection for transplantation. The number of total nucleated cells (TNCs) in the CBUs has been reported to be associated with the speed of engraftment and posttransplantation survival after HSCT (2,9,13,21). Therefore, among the CBUs that are human leukocyte antigen (HLA) compatible with the recipients, those with the highest TNC number before cryopreservation are selected (8). In addition, since the number of cluster of differentiation 34-positive (CD34⁺) cells has also been reported to be associated with posttransplantation survival and the manifestation of graftversus-host disease, it is considered another selection criterion for the CBUs (16,22,24). However, because the absolute numbers of TNCs and CD34⁺ cells decrease after cryopreservation (3 –6,25), a quality assessment after thawing, also known as a release test, should be performed on select CBUs (12). Specifically, before the release of CBUs to transplantation centers, the number and viability of TNCs and CD34⁺ cells, the number of colony-forming units (CFUs), HLA confirmatory typing, and other tests could be performed using CB segments (15,17).

Since CB from the main bags is used for HSCT, segments provide the only means to determine the quality of the CB main bags before transplantation. Preshipment testing using CB segments serves as not only a tool for quality validation but also the backup plan for the selection of CBUs for transplantation (18). Studies focusing on the representation of parameters of CB segments have been performed; however, these previous studies analyzed only a small number of cryopreserved CBUs (10,20).

In this study, a large number of cryopreserved CBUs were used to determine whether CB from segments is representative of CB from main bags by comparing various CB parameters measured after cryopreservation.

Materials and Methods

Sample Selection

CB collection and cryopreservation were performed as described previously (14). Briefly, 20 ml of nucleated cell concentrate was obtained after the removal of extra plasma and red blood cells from CB using a transfer/freezing bag set (Pall Medical, Covina, CA, USA). Adding 5 ml of cryoprotectant [2.5 ml of dimethyl sulfoxide (DMSO; Mylan, Rockford, IL, USA) and 2.5 ml of dextran (Daihan, Kyounggido, Korea)] to the nucleated cell concentrate resulted in the final 25 ml CBU with two segments attached. The CBU with attached segments was wrapped and frozen using a controlled-rate freezer (CryoMed, Thermo Forma, Marietta, OH, USA). The temperature was lowered to −90°C in the controlled-rate freezer (to sample temperature of −6°C with 1°C/min -> to chamber temperature of −50°C with 25°C/min -> to chamber temperature of −14°C with 10°C/min -> to chamber temperature −45°C with 1°C/min -> to chamber temperature −90°C with 10°C/min) and then the CBU was stored in the liquid phase of liquid nitrogen.

A total of 245 nonconforming CBUs (male/female = 129:116) for allogeneic transplantation were obtained from the Seoul Metropolitan Government Public Cord Blood Bank (Allcord, Seoul, Korea). All CBUs were processed and cryopreserved from August 2006 to December 2011. This study was reviewed and approved by the Institutional Review Board of Boramae Hospital (06–2012–126).

Thawing of CBUs and Measurements of Postthaw CB Parameters

After the selected CBUs were thawed in a 37°C water bath, the numbers and viability of TNCs and CD34⁺ cells, cell apoptosis, and the number of CFUs in CB from both main bags and attached segments were examined as given below.

Number of TNCs and CD34⁺ Cells

The number of TNCs (including neutrophils, lymphocytes, and monocytes) was measured by using the automated cell counter XE-2100 (Sysmex, Kobe, Japan). CD34⁺ cell enumeration was conducted as previously described using Stem-kit (BD Bioscience, San Jose, CA, USA, #IM3630) (20), based on the single-platform International Society of Hematotherapy and Graft Engineering method.

Cell Viability Test

After thawing, the viability of TNCs was either tested by 0.4% trypan blue (#23606; Gibco, Grand Island, NY, USA) staining or measured using 7-aminoactinomycin D (7-AAD) by excluding nonviable cells in the process of CD34⁺ cell enumeration. The viability of CD34⁺ cells was tested using the 7-AAD Stem-kit (#IM3630; BD Bioscience).

Analysis of Apoptotic TNCs Using Caspase 3

Briefly, 100 μl of cell suspensions (adjusted to 1 × 10⁴ cells/μl using phosphate-buffered saline) was placed in test tubes. Cells were stained with 20 μl of fluorescein isothiocyanate (FITC)-conjugated anti-CD45 (BD Biosciences) for 15 min at room temperature in the dark. Erythrocytes were then lysed by adding 2 ml of an ammonium chloride-based buffered solution (BD Bioscience). After washes, cells were adjusted to a density of 1 × 10⁶ cells in 500 μl of Cytofix/Cytoperm solution (BD Biosciences) and incubated for 20 min on ice. After centrifugation, cell pellets were washed twice with perm/wash buffer solution (500 μl; BD Biosciences). Cells resuspended in 100 μl of perm/wash buffer solution were stained with 20 μl of phycoerythrin (PE)-conjugated caspase 3 (#550914; BD Pharmingen) for 20 min at room temperature in the dark. After washes and addition of 500 μl perm/wash buffer solution, cells were analyzed by flow cytometry (FC-500; Beckman Coulter, Inc., Fullerton, CA, USA). For each sample, 50,000 events were acquired and analyzed.

Analysis of Apoptotic CD34⁺ Cells Using Annexin V

As described above, 100 μl of cell suspensions (10 × 10³ cells/μl) was placed into test tubes. Cells were stained with 10 μl of PE-conjugated anti-CD34 (#A07776; Beckman Coulter) and 5 μl of PE cyanine 7 (PC7)-conjugated anti-CD45 (#IM3546; Beckman Coulter) for 15 min at room temperature in the dark. For annexin V/7-AAD staining, we used a kit (#IM3614; Beckman Coulter) and followed manufacturer's instruction. Briefly, erythrocytes were then lysed by adding an ammonium chloride-based buffered solution. After washes, cells were adjusted to a density of 1 × 10⁶ cells in 100 μl of annexin V-binding buffer and stained with 10 μl of FITC-conjugated annexin V for 15 min on ice in the dark. After addition of 10 μl peridinin–chlorophyll–protein complex-conjugated 7-AAD and 380 μl of annexin V-binding buffer, the cells were analyzed by flow cytometry (FC-500). For each sample, 50,000 events among live cells (negative for 7-AAD) were acquired and analyzed.

CFU Assay

The CFU assay was performed as described previously (20) using commercially available methylcellulose medium (MethoCult H4435 Enriched; Stemcell Technologies, Vancouver, BC, Canada).

Statistical Analysis

Paired t tests were performed to compare various parameters of CB from main bags and segments, whereas the simple linear regression analysis was carried out to determine any associations between them. Differences were considered to be statistically significant when p < 0.05. All statistical calculations were performed using the Statistical Package for Society Sciences v. 12.0 Systems (IBM Corp., Armonk, NY, USA).

Results

Number of TNCs and CD34⁺ Cells and Cell Viability Changes After Thawing Cryopreserved CB

Table 1 shows the number of TNCs and CD34⁺ cells, as well as cell viability, before and after thawing. While no significant difference was observed in the number of TNCs (p = 0.500), the number of CD34⁺ cells and cell viability were decreased significantly after cryopreservation (p < 0.001). The recovery rates of TNCs and CD34⁺ cells after cryopreservation were 95.8% ± 2.9% (range, 87.9–100%) and 68.2% ± 12.3% (range, 42.3–98.4%), respectively.

Table 1.

Number of TNCs and CD34⁺ Cells and Cell Viability Before and After Cryopreservation

	Before Freezing	After Thawing	p
TNCs (×10⁸/unit)	7.7 ± 3.3	8.0 ± 3.4	0.500
	(3.9–25.5)	(3.8–26.5)
CD34+ cells (×10⁶/unit)	2.8 ± 1.5	1.9 ± 1.0	<0.001
	(0.5–9.3)	(0.4–6.3)
Cell viability^* (%)	89.3 ± 2.4	86.1 ± 2.7	<0.001
	(80.0–94.0)	(74.0–92.0)

Results have been presented as mean ± SD (range). TNCs, total nucleated cells; CD34⁺, cluster of differentiation 34 positive.

Viability of TNCs, as measured using trypan blue.

Comparison Between Various Parameters of CB From Main Bags and Segments

Table 2 shows that the number of TNCs in CB from main bags was significantly higher than that in CB from segments (p < 0.001); in contrast, the number of CD34⁺ cells was significantly higher in CB from segments (p < 0.001). In addition, the viability of TNCs (determined by trypan blue and 7-AAD staining) or CD34⁺ cells (determined by 7-AAD) was also significantly higher in CB from segments (p < 0.001, p < 0.001, and p = 0.005, respectively). While the proportion of apoptotic TNCs was significantly higher in CB from segments (p < 0.001), no difference was observed in the proportion of apoptotic CD34⁺ cells (p = 0.105). Despite no significant difference in the number of CFU-granulocyte/erythrocyte/macrophage/megakaryocyte (GEMM) between CB from main bags and segments (p = 0.990), the numbers of both total CFUs and CFU-granulocyte/macrophage (GM) were significantly higher in CB from segments (p = 0.005).

Table 2.

Comparison Between Parameters of Cord Blood From Main Bags and Segments

	Main Bag	Segment	p
TNCs (×10³/μl)	32.0 ± 13.5	31.4 ± 13.5	<0.001
CD34⁺ cells (/μl)	67.6 ± 49.7	74.8 ± 52.6	<0.001
Viability
TNCs
Trypan blue (%)	86.1 ± 2.7	86.7 ± 2.6	<0.001
7-AAD (%)	54.5 ± 8.1	59.7 ± 9.4	<0.001
CD34⁺ cells
7-AAD (%)	95.5 ± 4.0	96.6 ± 4.6	0.005
Apoptosis
TNCs
Caspase 3 (%)	4.9 ± 2.2	5.3 ± 2.5	<0.001
CD34⁺ cells
Annexin V (%)	4.9 ± 4.0	4.5 ± 4.0	0.105
Total CFUs (×10⁵/unit)	16.53 ± 12.27	17.87 ± 11.66	0.017
CFU-GEMM (×10⁵/unit)	0.15 ± 0.52	0.15 ± 0.49	0.990
CFU-GM (×10⁵/unit)	1.99 ± 2.73	2.53 ± 3.19	0.005

Results have been presented as mean ± SD. TNCs, total nucleated cells; 7-AAD, 7-aminoactinomycin D; CFU, colony-forming unit; CFU-GEMM, CFU-granulocyte/erythrocyte/macrophage/megakaryocyte; CFU-GM, CFU-granulocyte/macrophage (GM).

Correlation Between Parameters of CB From Main Bags and Segments

Table 3 shows the Pearson's correlation coefficient (r) between various parameters of CB from main bags and segments. The results show that the number of TNCs, CD34⁺ cells, total CFUs, CFU-GEMM, and CFU-GM in CB from segments all showed significant correlations with the respective parameters of CB from main bags. While a correlation was observed between the viability of TNCs in the two parts of the CBUs, no relationship was detected between the viability of CD34⁺ cells. In addition, the data also indicate a correlation between the proportion of apoptotic TNCs or CD34⁺ cells in CB from main bags and segments.

Table 3.

Correlation Between Parameters of Cord Blood From Main Bags and Segments

	r ^*	p ^†
TNCs (×10³/μl)	0.991	<0.001
CD34⁺ cells (/μl)	0.947	<0.001
Viability
TNCs
Trypan blue (%)	0.838	<0.001
7-AAD (%)	0.629	<0.001
CD34⁺ cells
7-AAD (%)	0.104	0.107
Apoptosis
TNCs
Caspase 3 (%)	0.893	<0.001
CD34⁺ cells
Annexin V (%)	0.510	<0.001
Total CFU (×10⁵/unit)	0.793	<0.001
CFU-GEMM (×10⁵/unit)	0.362	<0.001
CFU-GM (×10⁵/unit)	0.609	<0.001

r, Pearson's correlation coefficient. Abbreviations: see Table 2.

†

Significance of Pearson's correlation coefficients.

Discussion

In this study, to determine whether CB from segments is representative of that from main bags, we compared various CB parameters after thawing cryopreserved CBUs.

CB from segments and CB from main bags are thought to be exposed to the same processing, including cryopreservation and the thawing process; therefore, CB banks have been using CB from segments as a source for the quality assessment of CBUs before releasing them to transplantation centers (10). For release tests, the American Association of Blood Banks recommends that the CBUs have at least two segments integrally attached to the main bag (1).

Previous studies have compared the parameters of CB from segments with those of CB from main bags (10,20). The numbers of TNCs and CD34⁺ cells in CB from segments were found to be similar to those in CB from main bags (20). The number of total CFUs in CB was also similar between the two (10,20). However, interestingly, our present study shows different results from those of previous studies. We found that while the number of TNCs was lower in CB from segments than in CB from main bags, the number of CD34⁺ cells was higher in CB from segments. In addition, the numbers of total CFUs and CFU-GM were also higher in CB from segments, compared to CB from main bags.

We observed that the viability of TNCs, which was determined by trypan blue staining, was significantly higher in CB from segments than that from main bags. These results are consistent with a previous study (10), in which the use of 7-AAD was suggested for the evaluation of cell viability, due to the discrepancy between the data determined by trypan blue and 7-AAD (10). Therefore, in this study, the viability of TNCs and CD34⁺ cells was also examined using 7-AAD and was found to be consistent with the results of trypan blue staining. The higher viability of TNCs and CD34⁺ cells in segments might be attributed to the even thickness of the tube diameter, which may provide favorable conditions for maintaining the cell viability.

In this study, we introduced apoptosis as another parameter. Since annexin V is attached to phosphatidylserine and is translocated from the inner to the outer cell membrane during early apoptosis, it stains apoptotic cells. On the other hand, trypan blue and 7-AAD stain only dead cells because they only penetrate nonintact cell membranes (7,19). A previous study reported that apoptotic CD34⁺ cells may influence the outcome of transplantation (23); therefore, we employed the apoptosis assay as a parameter for the quality assessment of cryopreserved CBUs. Our results show that the percentage of apoptotic TNCs was higher in CB from segments, whereas no significant difference was observed in the percentage of apoptotic CD34⁺ cells between CB from segments and main bags.

While the absolute numbers of TNCs, CD34⁺ cells, total CFUs, and CFU-GM in CB from segments and main bags were significantly different, strong linear correlations were observed between these parameters. Especially, a strong correlation (r > 0.9) was found between the number of TNCs or CD34⁺ cells in segments and main bags. Therefore, we conclude that CB from segments may be representative of the quality of CB from main bags that are used for HSCT.

In patients receiving CB for HSCT, the number of TNCs has been used as the primary selection criterion for CBUs (11,16). However, since the number of CD34⁺ cells was previously reported as a more reasonable selection criterion for CBUs (26), the lowest dose of CD34⁺ cells has been adopted for the selection of appropriate CBUs at some transplantation centers (24). Generally, the CB banks measure the numbers of CD34⁺ cells and TNCs using CB from segments before releasing the CBUs that are HLA compatible with the recipients. At transplantation centers, the numbers of these cells are measured using CB from main bags prior to transplantation. Because there is a difference between the number of CD34⁺ cells in CB from segments and main bags, the CB banks and transplantation centers would report different numbers of CD34⁺ cells. The fact that the number of CD34⁺ cells is higher in CB from segments and the number of TNCs is higher in that from main bags could cause problems in communicating accurate cell counts between the CB banks and transplantation centers. To provide more accurate CD34⁺ cell counts to transplantation centers, a linear regression equation relating the number of CD34⁺ cells in CB from segments to that in CB from main bags (Fig. 1) may be used to predict the number of CD34⁺ cells in CB from main bags, although the equations established and provided by different CB banks might vary.

Figure 1.

Regression equation relating the number of CD34⁺ cells in CB from segments to that in CB from main bags. This equation may be used to predict the number of CD34⁺ cells in CB from main bags (Y) by measuring the number of CD34⁺ cells in CB from segments (X). Regression equation is Y = 0.864X + 0.943, r = 0.947.

Since determination of shelf life of cryopreserved CBUs is still an ongoing process (5,6), the study design and the results of our report may add some answers to those related shelf-life decisions.

In summary, our study indicates that CB from segments and CB from main bags are highly correlated with regard to the number of cells reflecting the hematopoietic activity of CBUs, despite significant differences in the absolute numbers of those cells. We expect that the results of this present study are useful not only for better interpreting the release tests of CBUs but also for assisting the selection of high-quality CBUs suitable for HSCT.

Footnotes

Acknowledgments

This study was supported by a grant of the Research Project, Seoul Metropolitan Public Cord Blood Bank (2012–005). We thank all of the staff members of Allcord, especially Jee Young Jang and Jung Ja Hong, for their technical assistance, and all the Korean mothers who donated cord blood to the public cord blood bank. The authors declare no conflicts of interest.

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Attached Segment has Higher CD34 + Cells and CFU-GM than the Main Bag after Thawing

Abstract

Keywords

Introduction

Materials and Methods

Sample Selection

Thawing of CBUs and Measurements of Postthaw CB Parameters

Number of TNCs and CD34+ Cells

Cell Viability Test

Analysis of Apoptotic TNCs Using Caspase 3

Analysis of Apoptotic CD34+ Cells Using Annexin V

CFU Assay

Statistical Analysis

Results

Number of TNCs and CD34+ Cells and Cell Viability Changes After Thawing Cryopreserved CB

Comparison Between Various Parameters of CB From Main Bags and Segments

Correlation Between Parameters of CB From Main Bags and Segments

Discussion

Footnotes

Acknowledgments

References

Number of TNCs and CD34⁺ Cells

Analysis of Apoptotic CD34⁺ Cells Using Annexin V

Number of TNCs and CD34⁺ Cells and Cell Viability Changes After Thawing Cryopreserved CB