Cord blood units with low CD34+ cell viability have a low probability of engraftment after double unit transplantation

Abstract

Double unit cord blood (CB) transplantation (CBT) appears to augment engraftment despite only one unit engrafting in most patients. We hypothesized that superior unit quality, as measured by a higher percentage of viable cells postthaw, would determine the engrafting unit. Therefore, we prospectively analyzed 46 double-unit transplants postthaw using flow cytometry with modified gating that included all dead cells. Using a 75% threshold (mean viability minus 2 SD), 20% of units had low CD34+ cell viability, with viability varying according to the bank of origin. Further, in the 44 patients with single unit engraftment, CD34+ cell viability was higher in engrafting units (P=.0016). Although either unit engrafted if both had high CD34+ viability, units with <75% viability were very unlikely to engraft: in 16 patients who received one high and one low CD34+ viability unit, only 1 of 16 units with viability <75% engrafted (P=.0006). Further, in the single patient without engraftment of either unit, both had CD34+ viability <75%. Finally, poor CD34+ viability correlated with lower colony forming units (CFUs) (P=.02). Our data suggests one mechanism by which double unit CBT can improve engraftment is by increasing the probability of transplanting at least one unit with adequate viability and the potential to engraft.

Conflict of interest statement

Conflict of interest disclosure: The authors declare no competing financial interests.

Copyright (c) 2010 American Society for Blood and Marrow Transplantation. Published by Elsevier Inc. All rights reserved.

Figures

Figure 1. Flow cytometric evaluation of CB units post-thaw

Live cells are shown in green, dead cells are shown in red, and debris is shown in black. A. Modified gating strategy. Plot 1: The lower FSC primary threshold excludes most debris but no dead cells. Plot 2: Viability is assessed in the FSC versus 7AAD dot plot. Debris is shown in R6 and is gated out in all subsequent plots. Dead cells (7AAD-positive) are in R7 whereas live cells (7AAD-negative) are in R5. Plot 3: For comparison traditional gating cannot adequately distinguish debris from viable cells in the 7AAD versus SSC dot plot. Live and dead cells are shown for CD45+ (Plot 4), CD34+ (Plot 5), and CD3+ (Plot 6) cell populations. Viability was calculated from the total debris-free sample for CD45+ cells (Plot 7), CD34+ cells (Plot 8), and CD3+ cells (Plot 9). An average of 300 events (range: 100–1000) were acquired per sample for the CD34+ cell analysis. B. Traditional gating strategy. The same plots as in 1A are shown with a high FSC threshold that excludes most dead cells, as frequently used in traditional gating. In CB units with a high percentage of dead cells the traditional methodology over-estimates cell viability compared with our modified gating. C. Comparison of CD34+ cell viability using modified and traditional gating in CB units with a small percentage of dead cells. The difference between the two gating strategies is small.

Figure 1. Flow cytometric evaluation of CB units post-thaw

Live cells are shown in green, dead cells are shown in red, and debris is shown in black. A. Modified gating strategy. Plot 1: The lower FSC primary threshold excludes most debris but no dead cells. Plot 2: Viability is assessed in the FSC versus 7AAD dot plot. Debris is shown in R6 and is gated out in all subsequent plots. Dead cells (7AAD-positive) are in R7 whereas live cells (7AAD-negative) are in R5. Plot 3: For comparison traditional gating cannot adequately distinguish debris from viable cells in the 7AAD versus SSC dot plot. Live and dead cells are shown for CD45+ (Plot 4), CD34+ (Plot 5), and CD3+ (Plot 6) cell populations. Viability was calculated from the total debris-free sample for CD45+ cells (Plot 7), CD34+ cells (Plot 8), and CD3+ cells (Plot 9). An average of 300 events (range: 100–1000) were acquired per sample for the CD34+ cell analysis. B. Traditional gating strategy. The same plots as in 1A are shown with a high FSC threshold that excludes most dead cells, as frequently used in traditional gating. In CB units with a high percentage of dead cells the traditional methodology over-estimates cell viability compared with our modified gating. C. Comparison of CD34+ cell viability using modified and traditional gating in CB units with a small percentage of dead cells. The difference between the two gating strategies is small.

Figure 1. Flow cytometric evaluation of CB units post-thaw

Live cells are shown in green, dead cells are shown in red, and debris is shown in black. A. Modified gating strategy. Plot 1: The lower FSC primary threshold excludes most debris but no dead cells. Plot 2: Viability is assessed in the FSC versus 7AAD dot plot. Debris is shown in R6 and is gated out in all subsequent plots. Dead cells (7AAD-positive) are in R7 whereas live cells (7AAD-negative) are in R5. Plot 3: For comparison traditional gating cannot adequately distinguish debris from viable cells in the 7AAD versus SSC dot plot. Live and dead cells are shown for CD45+ (Plot 4), CD34+ (Plot 5), and CD3+ (Plot 6) cell populations. Viability was calculated from the total debris-free sample for CD45+ cells (Plot 7), CD34+ cells (Plot 8), and CD3+ cells (Plot 9). An average of 300 events (range: 100–1000) were acquired per sample for the CD34+ cell analysis. B. Traditional gating strategy. The same plots as in 1A are shown with a high FSC threshold that excludes most dead cells, as frequently used in traditional gating. In CB units with a high percentage of dead cells the traditional methodology over-estimates cell viability compared with our modified gating. C. Comparison of CD34+ cell viability using modified and traditional gating in CB units with a small percentage of dead cells. The difference between the two gating strategies is small.

Figure 2. Post-thaw CD34+, CD3+ and CD45+ cell viability and unit engraftment in 44 double unit CB grafts

The distribution of post-thaw viabilities of the CD34+, CD3+, and CD45+ cells are shown for the engrafting (in closed symbols) and non engrafting units (open symbols) for the 44 patients with single unit engraftment.

Figure 3. Post-thaw CD34+ cell viability and CB bank of origin (N=92 units)

CB units were obtained from domestic (N=71), and international banks (INT, N=21). Each group depicts the distribution of viability for the respective units from each bank (for the domestic) or each country (for the international ones).

Figure 4. Schema depicting the difference between the dose of viable CD34+ cells/kg versus the percentage of viable CD34+ cells in two CB units

The infused viable CD34+ cells per kg of the recipient weight (CD34+ cell dose) and the percent CD34+ cell viability (7AAD-positive CD34+ cells divided by the total CD34+ cells in the unit expressed as a percentage) represent two different graft characteristics. In this example the two CB units have the same total number of viable CD34+ cells (and would have an identical infused CD34+ cell dose). However, the percentage of viable CD34+ cells is markedly different. Based on the results of this study unit #1 is of poor quality and we would predict that unit #2 would engraft in the double unit setting.