**2. Processing**

MaxCell CB, and patients receiving MaxCell CB showed superior engraftment, survival,

In addition, the three main post-thaw manipulation methods are reviewed. Comparison data for some of the thaw methods are presented, and matched-pair analysis was used to confirm the superiority of direct infusion thaw method over post-thaw wash

**Keywords:** cord blood banking, cord blood processing, MaxCell cord blood processing, MaxCB cord blood processing, stem cell processing, cord blood thawing, cord blood cryopreservation, cord blood transplantation, 2nd and 3rd generation cord

Today, hematopoietic stem cell transplantation (HSCT) can be performed using stem cells derived from three sources: bone marrow (BM), peripheral blood (PB), and umbilical cord blood (CB) from autologous, related allogeneic, or unrelated allogeneic donors. Autologous HSCT is not indicated for most indications, and only about one-third of patients in need of HSCT have a suitable related donor. Although there are currently tens of millions of adult volunteer donors registered worldwide, about 60% of patients will not find a suitably HLAmatched unrelated adult donor, and thus cannot access HSCT [1]. For minority patients, the probability of finding an HLA-matched unrelated adult donor is further reduced. Since the search process for an adult donor often takes several months, a significant proportion of patients will become higher risk or ineligible for HSCT or even die while waiting for the donor search [2]. In contrast, without the possibility of last minute donor refusal, CB products are available upon request and can be shipped to any transplant center in the world immediately. Importantly, cord blood transplantation (CBT) is associated with a lower incidence and severity of graft-versus-host disease (GvHD), and a partial HLA match between the donor and recipient is tolerable [3], making it an ideal donor source for minority patients without

Compared with historical controls of BM or PB HSCT, CBT has shown favorable clinical outcome despite significantly worse HLA match [3]. In the pediatric setting, CBT can be now considered established practice. For adults, CBT adoption has been slower because of cell dose limitation. Many strategies focused on overcoming this limitation are being developed, including double CBT, the utilization of two CB unit grafts [4–6], the combination of an unrelated CB graft with haplo-identical donor stem cells [7], and foregoing the post-thaw wash with direct infusion or reconstitution/dilution, which have shown promising results [8, 9]. Engraftment and survival appear to be at least equivalent to or may even be superior to historical data using post-thaw wash for CB products versus when CB was not washed. One

and transplant-related mortality, confirming pre-match observations.

4 Umbilical Cord Blood Banking for Clinical Application and Regenerative Medicine

for MaxCell CB products.

blood technologies

**1. Introduction**

large adult donor registries.

The world's first CB banks were established at the New York Blood Center, Düsseldorf and Milan in 1992 [13]. Initially, CB was processed and cryopreserved as whole cord blood, which we refer to in this chapter as zero generation process [19]; however, in 1995, Rubinstein et al. pointed out that "the first problem is that stockpiling ("banking") a sufficiently large number of cryoprotected Whole PCB (placental cord blood) units requires vast amounts of costly storage space in liquid nitrogen (LN). To establish an adequate panel, therefore, the hematopoietic cells of PCB units need to be concentrated into units of much smaller volume."[13] It was for this reason that volume reduction processing of CB units was developed, with associated reduction of the 'bulk of the erythrocytes and plasma'. It was reported that the red blood cell (RBC) reduction processing method increased the number of units that could be stored in the same freezer space by as much as 10-fold, and thus provided significant economic advantage [13].

Rubinstein et al. reported in 1998 the first comparison of engraftment rates using RBC-replete units (whole CB) and RBC-reduced (RCR) units [19]. In their **Table 2**, they indicated that the majority of patients in their series (337 of 546 patients) were transplanted with whole CB that were RBC-replete, and of the 365 patients (out of 546 patients) who had myeloid engraftment, the Kaplan-Meier engraftment rate was 80% [95% confidence interval (CI) 75–85%] among the RBC-replete CB transplant recipients, and 82% with RCR units (95% CI 76–88%). Whether the unit used for the transplants was RBC-replete or RCR was not considered among the variables associated with the engraftment- and transplantation-related events.

Many volume reduction methods of processing umbilical CB which reduce RBCs prior to the cryopreservation have been considered since then (**Table 1**); however, the most common method for processing CB units today is the hetastarch or HES RBC reduction (RCR), whether manual or automated. For the sake of convenience, we will call these first generation (1st Gen) cord blood processing techniques. It should be noted that these methods are often erroneously referred to as red cell depletion when all of these techniques retain considerable RBCs and do not deplete, but only reduce the number of RBCs.

Unfortunately, all 1st Gen RCR processing methods lose significant numbers of nucleated cells, stem cells, and progenitor cells as measured by CD34+ cells or colony forming units (CFU) enumeration, with an approximately 25% nucleated cell loss on average among various reports in the literature [10–17] and a range of 14–42% for the various methods reported by Takahashi et al. [10]. This is of concern because it is well-known that the success of CBT is critically dependent on cell dose, and insufficient cell dose is widely regarded as the most important limitation for umbilical CB transplantation, especially for adults and large children [1, 3]. **Table 1** lists some of the most common 1st Gen CB processing techniques.


\* Proprietary Technologies: SEPAX from BioSafe; AXP-Express from Thermogenesis; MaxCell 2nd Gen from StemCyte; MaxCell 3rd Gen from CyteTherapeutics, Inc.

**Table 1.** Technical comparison of some of the most popular 1st Gen CB processing techniques and the proprietary 2nd and 3rd MaxCell CB processing technologies.

To maximize CB cell dose, R. Chow has developed two proprietary alternative CB volume reduction technologies (2nd Gen and 3rd Gen) which he calls MaxCell CB processing technologies, because both share the common characteristics of (1) depleting or reducing plasma, (2) maximizing stem cell, progenitor, nucleated and mononuclear cell recovery after processing and most importantly, and (3) having identical cellular composition upon infusion for one version of the third generation technology (**Tables 1**–**3**).

Rubinstein et al. reported in 1998 the first comparison of engraftment rates using RBC-replete units (whole CB) and RBC-reduced (RCR) units [19]. In their **Table 2**, they indicated that the majority of patients in their series (337 of 546 patients) were transplanted with whole CB that were RBC-replete, and of the 365 patients (out of 546 patients) who had myeloid engraftment, the Kaplan-Meier engraftment rate was 80% [95% confidence interval (CI) 75–85%] among the RBC-replete CB transplant recipients, and 82% with RCR units (95% CI 76–88%). Whether the unit used for the transplants was RBC-replete or RCR was not considered among the variables

Many volume reduction methods of processing umbilical CB which reduce RBCs prior to the cryopreservation have been considered since then (**Table 1**); however, the most common method for processing CB units today is the hetastarch or HES RBC reduction (RCR), whether manual or automated. For the sake of convenience, we will call these first generation (1st Gen) cord blood processing techniques. It should be noted that these methods are often erroneously referred to as red cell depletion when all of these techniques retain considerable RBCs and do

Unfortunately, all 1st Gen RCR processing methods lose significant numbers of nucleated cells, stem cells, and progenitor cells as measured by CD34+ cells or colony forming units (CFU) enumeration, with an approximately 25% nucleated cell loss on average among various reports in the literature [10–17] and a range of 14–42% for the various methods reported by Takahashi et al. [10]. This is of concern because it is well-known that the success of CBT is critically dependent on cell dose, and insufficient cell dose is widely regarded as the most important limitation for umbilical CB transplantation, especially for adults and large children [1, 3]. **Table 1** lists some of the most common 1st Gen CB processing techniques.

Zero generation Whole cord blood Manual None None

First generation PrepaCyte Red cell reduced Manual Yes Yes First generation Top & bottom Optipress II Red cell reduced Manual Yes Yes First generation Ficoll Red cell reduced Manual Yes Yes

reduced

Replete

RBC Reduced + RBC

\* Proprietary Technologies: SEPAX from BioSafe; AXP-Express from Thermogenesis; MaxCell 2nd Gen from StemCyte;

**Table 1.** Technical comparison of some of the most popular 1st Gen CB processing techniques and the proprietary 2nd

First generation hetastarch Red cell reduced Manual SEPAX\* or

**Technique Manual/automated**

**processing**

AXP-Express\*

Manual SEPAX

Manual SEPAX AXP-Express **RBC reduction** 

Yes Yes

No Yes

Yes/No Yes

**Plasma depletion/ reduction**

associated with the engraftment- and transplantation-related events.

6 Umbilical Cord Blood Banking for Clinical Application and Regenerative Medicine

not deplete, but only reduce the number of RBCs.

Second generation Plasma depleted/

Third generation MaxCord

MaxCell 3rd Gen from CyteTherapeutics, Inc.

and 3rd MaxCell CB processing technologies.

MaxCell (MC) Technologies


1st generation RBC-reduced CB Products—Manual: Hetastarch most commonly used; however, also include PrepaCyte, Optipress, Ficoll Hypaque; Automated: Sepax & AXP-AutoXpress.

MaxCell: 2nd generation plasma depleted/reduced CB products and 3rd generation MaxCord CB products.

**Table 2.** A comparison of the volumes and amount of some of the various components of first (hetastarch), second and third generation CB technologies.

The MaxCell second generation (2nd Gen) technology or plasma depletion/reduction reduces or depletes plasma without reducing RBCs. Plasma is removed or reduced from the product prior to the cryopreservation [18]. Although the resulting volume of CB products processed by such a method is two to three times larger, 2nd Gen results in greater than 99.9% processing recovery of nucleated cells and all critical cell types prior to the testing and archival sampling. True nucleated cell loss was less than 0.01% from 2nd Gen CB processing, as evidenced by complete blood count (CBC) enumeration of the discarded plasma fraction. In practice, because of the testing and archival sampling, the actual loss will be raised to around 5–10% of the collected CB cell dose. Such minimal loss was reproducible from validation to an actual MaxCell inventory of more than 12,000 CB products that had pre- and post- processing CBC available. Even after thawing, in 188 post-thaw segment samples, a median of 90.33% *postsampling/post-thaw* TNC recovery and a median of 91.84% *post-sampling/post-thaw* CD34 cell recovery of preprocessing values were achieved for 2nd Gen CB products. The resultant volume, DMSO load, RBC, WBC and RBC lysate content of 2nd Gen CB products are similar to cryopreserved peripheral blood products (**Tables 1**–**3**).

The newest MaxCell technology, MaxCord processing technology, referred to as third generation (3rd Gen) CB processing technology in this chapter, combines the advantages of ease and convenience of thawing for the 1st Gen products and maximum stem (VSEL), progenitor (CFU and CD34+), nucleated, and mononuclear cell recovery of the 2nd Gen, without any of the associated disadvantages. While the processing methods and cryopreserved products are distinctly different, with the preferred embodiment of 3rd Gen technology, the cellular compositions of the final infused products are virtually identical between the two MaxCell technologies. **Table 2** outlines some of the component differences in volume, RBC content, and amount of additional components, such as DMSO, dextran-40, HES, and anticoagulant CPD.


Comparisons between HES 1st Gen and MaxCell 2nd and 3rd Gen are performed using parallel processing comparison using one half of the same cord blood unit for first generation hetastarch and the other half for MaxCell CB Products [18]. Cell types which exhibit significant differences are highlighted by boldfaced fonts. Second and third generation cord blood products have similar yield for all cell lineages, and only differ in the final RBC yield depending on the embodiment of 3rd Gen used. Third generation is similar to first generation in RBC reduction in RBC reduction. Sample size n = 10 for all the cell types, except for VSEL, which had a sample size n = 3. P values are calculated using the paired-sample *t*-test.

**Table 3.** Stem cell (VSEL), progenitor cell (CFU and CD34+), nucleated cell, mononuclear cell, nucleated RBC, RBC, and viability recovery of 1st (hetastarch method) MaxCell 2nd and MaxCell 3rd generation CB processing.

Third generation CB processing technology is perhaps the most flexible CB processing technology developed to date, with the additional option to reduce RBCs without losing as much stem, progenitor, nucleated, and mononuclear cells as 1st Gen RCR techniques (**Table 3**). **Table 1** highlights some of the processing technical differences among various popular 1st Gen techniques versus 2nd Gen and 3rd Gen CB processing technologies. Unlike Chow's 2nd Gen CB technology which is only amenable to SEPAX-automated processing and not accessible to AXP-Express, Chow's latest MaxCell technology can be automated using both SEPAX or AXP-Express. **Table 3** shows the differences in CB stem cell (VSEL), hematopoietic progenitor cell (CD34+ and CFUs), total nucleated cells (TNC), mononuclear cells, nucleated RBCs (nRBC), RBCs, and viability between the 1st Gen HES technique and the proprietary MaxCell 2nd/3rd Gen technologies. While the processing methods and cryopreserved products are distinctly different, the cellular composition of the final infused product is identical between the two MaxCell technologies using the preferred embodiment of 3rd Gen (**Table 3**) technology, while other embodiments can be adapted to reduce RBC, free hemoglobin, DMSO, and WBC lysate load for certain situations to better suit the needs of the individual patients (**Table 2**).

and CD34+), nucleated, and mononuclear cell recovery of the 2nd Gen, without any of the associated disadvantages. While the processing methods and cryopreserved products are distinctly different, with the preferred embodiment of 3rd Gen technology, the cellular compositions of the final infused products are virtually identical between the two MaxCell technologies. **Table 2** outlines some of the component differences in volume, RBC content, and amount of additional components, such as DMSO, dextran-40, HES, and anticoagulant CPD.

> **CD34+ cells (×106 )**

1.16 84.0%

1.42 99.9% **Viability (%)**

96% 98.3%

98% 99.3% **CFU-GM (×105 )**

3.5 42.1%

7.8 78.1%

118% 117% **124% 121%** 102% **225%** 128% **202% 186% 181%**

0.18 0.27 **0.002 0.003** 0.24 **0.05** 0.50 **0.001 0.01 0.007**

**CFU-E (×105 )**

3.6 48.5%

4.7 111.2% **CFU-GEMM (×105 )**

4.6 50.4%

9.3 88.4% **Total CFU (×105 )**

11.7 47.1%

21.8 88.1% **Total VSEL (×103 )**

4.434 54.6%

8.134 100.1%

**Post-processing Recovery Cell # Recovery %**

RCR Gen 1- HES

MaxCell Gen 2/3

Gen 2 and/or 3 MaxCellGen/ RCR

Recovery Ratio

P value **Gen 2**

**RBC (×1010) Total**

8 32.9%

Gen 2–24 100.2% Gen 3–8 33%

**Gen 2 312%** Gen 3 100–312%

**<0.0001** Gen 3 Variable

values are calculated using the paired-sample *t*-test.

**nRBC (×107 )**

8 Umbilical Cord Blood Banking for Clinical Application and Regenerative Medicine

2.1 78.1%

2.5 91.5% **Total MNC (×107 )**

26 80.7%

31 92.4% **TNC (×107 )**

65.77 78.7%

81.56 96.4%

Comparisons between HES 1st Gen and MaxCell 2nd and 3rd Gen are performed using parallel processing

Products [18]. Cell types which exhibit significant differences are highlighted by boldfaced fonts.

viability recovery of 1st (hetastarch method) MaxCell 2nd and MaxCell 3rd generation CB processing.

comparison using one half of the same cord blood unit for first generation hetastarch and the other half for MaxCell CB

**Table 3.** Stem cell (VSEL), progenitor cell (CFU and CD34+), nucleated cell, mononuclear cell, nucleated RBC, RBC, and

Third generation CB processing technology is perhaps the most flexible CB processing technology developed to date, with the additional option to reduce RBCs without losing as much stem, progenitor, nucleated, and mononuclear cells as 1st Gen RCR techniques (**Table 3**). **Table 1** highlights some of the processing technical differences among various popular 1st Gen techniques versus 2nd Gen and 3rd Gen CB processing technologies. Unlike Chow's 2nd Gen CB technology which is only amenable to SEPAX-automated processing and not accessible to AXP-Express, Chow's latest MaxCell technology can be

Second and third generation cord blood products have similar yield for all cell lineages, and only differ in the final RBC yield depending on the embodiment of 3rd Gen used. Third generation is similar to first generation in RBC reduction in RBC reduction. Sample size n = 10 for all the cell types, except for VSEL, which had a sample size n = 3. P Barker et al. [20] have questioned whether the MaxCell data [8] indicating significantly higher TNC doses with red cell-replete MaxCell CB products are meaningful. Indeed, it was speculated that a 'correction factor' of 0.75 should be applied to the reported TNC content of RBCreplete products, arguing that such products contain the noncritical nucleated RBC (nRBC) and neutrophils, and implying that critical stem and progenitor cells are somehow not retained to a similar extent as TNC. Unfortunately, no hard data accompanied this implication or their recommendation and the authors ignored the data on tens of thousands of CB units presented by Chow et al. [18] in **Table I**–**IV** of their paper, which showed significantly higher recovery of stem (VSEL), progenitor (CD34+ and CFU), nucleated and mononuclear cells with parallel processing of the two halves of the same CB units using MaxCell 2nd Gen technologies versus RCR techniques. More importantly, the superior outcome data for the MaxCell CB products [8] were similarly disregarded. Petz and Chow [21] pointed out that implementation of Barker's recommendation, if incorrect, would underestimate the progenitor cell content of RBC-replete CB products, resulting in the inappropriate rejection of certain optimal units by transplant physicians in favor of RCR products with an apparently higher TNC dose after application of the 'correction factor' for RBC-replete products.


This outcome assumes that large cord blood banks have normal and similar distribution of patients, disease indications, and disease stage.

**Table 4.** Published overall clinical outcome of CBT patients receiving RCR CB products from NYBC, COBLT, NMDP, and CIBMTR [19, 23–34] versus MaxCell CB products from CIBMTR audited outcome data [8].

To study this issue, Chow et al. divided 10 CB units in half and processed one half by RCR using the most commonly used hetastarch method, and the other half by MaxCell in parallel [18]. As indicated in **Table 3**, the average TNC of MaxCell CB products after processing was 124% of that in RCR units (*p* = 0.002). Moreover, MaxCell caused virtually no loss of CD34 cells compared to RCR produced CB, with the mean post-processing CD34 cell count in MaxCell products being 121% of that in RCR units (*p* = 0.003). Page et al. [22] have reported on their experience with 435 CB transplants and concluded that total CFU is the best predictor of engraftment. Of major importance is that the mean recoveries of CFUs were also higher for MaxCell products [11], as shown in **Table 3**, 225% for CFU-GM (*p* = 0.05), 202% for CFU-GEMM (*p* = 0.001) and 186% for total CFU (*p* = 0.01). Taken together, this means that CFU, the most important cellular predictors of engraftment success, showed an even bigger difference than TNC between RCR and MaxCell products, in contrast to the speculation by Barker and NYBC [20].

In fact, in clinical studies of MaxCell CB products, outstanding clinical outcome in terms of engraftment and patient survival have been achieved. **Table 4** showed published engraftment and survival outcome for transplantation using 1st Gen RCR CB products from diverse CB banks (NYBC, EuroCord, COBLT CBB, London CBB, French CBB) or large outcome data registries, such as CIBMTR or NMDP [19, 23–34] versus 2nd/3rd Gen Max Cell CB products [8].

Ballen et al. [34] studied the outcome of manually processed RCR CB products, automated processed RCR CB products, and manually processed MaxCell 2nd Gen CB products. While acknowledging some of the significant design flaws in their study, the authors concluded that automated processing systems resulted in higher day-28 neutrophil recovery than manually processed plasma-reduced products; however, overall survival by day-100 was not different among the three groups. Many study decisions and design issues were not clearly explained or delineated in this study, such as patient selection or study end point selection. For patients receiving Max Cell 2nd Gen CB products and manually processed RCR CB products, there were only 133 and 279 patients, respectively, when hundreds to thousands more were available fitting the study criteria during the study period for both of these groups. For example, even in a 2008 study with just pediatric hematological malignancy patients from one transplant center (U. Arizona) and one CBB (StemCyte), Graham et al. presented data for 105 matched pairs of patients from 128 patients transplanted with PDR 2nd Gen CB and 112 patients transplanted with RCR 1st Gen CB [35]. In fact, it is obvious that of the 16 public CB banks in the study, at least four of the banks each alone had more patients fitting the exact criteria for patients transplanted with manually processed CB in the study, so the reasons why these other patients were not selected and the reasons for inclusion of the particular patients in the study were not clear. The decision to use only 100-day survival, and not 1 year, 3-year, or 5-year overall survival, was also quite unconventional. All three patient groups had approximately 80% 100-day overall survival, which should not give an erroneous impression that long-term survival is that high or that long-term survival may be the same for all three groups. Previous studies with far larger patient populations not restricted to acute leukemia/MDS from NYBC, COBLT, CIBMTR, and NMDP have confirmed that transplants with RCR CB yielded around 40–45% 1-year survival and much higher 100-day transplant-related mortality [19, 23–33]. Indeed, long-term survival data on many more patients fitting the study criteria were available from CIBMTR, NMDP, and the various banks when the study was analyzed, so it was odd that 100-day overall survival (OS) was chosen as one of the study's primary end points, when it would have been far more meaningful and just as easy to calculate and show longer term survival and transplant-related mortality data, as is typical for such CIBMTR studies. In fact, our experience of a large MaxCell CB bank

with thousands of patients transplanted with its products have shown that survival or transplant-related mortality during the first 100 days is often not predictive for long-term survival or patient mortality [8, 35–43].

of engraftment. Of major importance is that the mean recoveries of CFUs were also higher for MaxCell products [11], as shown in **Table 3**, 225% for CFU-GM (*p* = 0.05), 202% for CFU-GEMM (*p* = 0.001) and 186% for total CFU (*p* = 0.01). Taken together, this means that CFU, the most important cellular predictors of engraftment success, showed an even bigger difference than TNC between RCR and MaxCell products, in contrast to the speculation by

10 Umbilical Cord Blood Banking for Clinical Application and Regenerative Medicine

In fact, in clinical studies of MaxCell CB products, outstanding clinical outcome in terms of engraftment and patient survival have been achieved. **Table 4** showed published engraftment and survival outcome for transplantation using 1st Gen RCR CB products from diverse CB banks (NYBC, EuroCord, COBLT CBB, London CBB, French CBB) or large outcome data registries, such as CIBMTR or NMDP [19, 23–34] versus 2nd/3rd Gen Max

Ballen et al. [34] studied the outcome of manually processed RCR CB products, automated processed RCR CB products, and manually processed MaxCell 2nd Gen CB products. While acknowledging some of the significant design flaws in their study, the authors concluded that automated processing systems resulted in higher day-28 neutrophil recovery than manually processed plasma-reduced products; however, overall survival by day-100 was not different among the three groups. Many study decisions and design issues were not clearly explained or delineated in this study, such as patient selection or study end point selection. For patients receiving Max Cell 2nd Gen CB products and manually processed RCR CB products, there were only 133 and 279 patients, respectively, when hundreds to thousands more were available fitting the study criteria during the study period for both of these groups. For example, even in a 2008 study with just pediatric hematological malignancy patients from one transplant center (U. Arizona) and one CBB (StemCyte), Graham et al. presented data for 105 matched pairs of patients from 128 patients transplanted with PDR 2nd Gen CB and 112 patients transplanted with RCR 1st Gen CB [35]. In fact, it is obvious that of the 16 public CB banks in the study, at least four of the banks each alone had more patients fitting the exact criteria for patients transplanted with manually processed CB in the study, so the reasons why these other patients were not selected and the reasons for inclusion of the particular patients in the study were not clear. The decision to use only 100-day survival, and not 1 year, 3-year, or 5-year overall survival, was also quite unconventional. All three patient groups had approximately 80% 100-day overall survival, which should not give an erroneous impression that long-term survival is that high or that long-term survival may be the same for all three groups. Previous studies with far larger patient populations not restricted to acute leukemia/MDS from NYBC, COBLT, CIBMTR, and NMDP have confirmed that transplants with RCR CB yielded around 40–45% 1-year survival and much higher 100-day transplant-related mortality [19, 23–33]. Indeed, long-term survival data on many more patients fitting the study criteria were available from CIBMTR, NMDP, and the various banks when the study was analyzed, so it was odd that 100-day overall survival (OS) was chosen as one of the study's primary end points, when it would have been far more meaningful and just as easy to calculate and show longer term survival and transplant-related mortality data, as is typical for such CIBMTR studies. In fact, our experience of a large MaxCell CB bank

Barker and NYBC [20].

Cell CB products [8].

The most rigorous method to address clinical outcome differences between different types of CB processing methods would be to conduct prospective double-blind placebo-controlled clinical trials; however, in HSCT, this is largely not feasible. Instead, matched-pair analyses allow for comparisons that are reasonably free from extraneous factors. To investigate rigorously whether clinical outcome differences exist between RCR and MaxCell CB products, Chow and collaborators performed matched-pair analysis using retrospective outcome data. Moreover, they secured an independent third party's assistance from CIBMTR to audit the outcome data with patient chart review on site at transplant centers using an audit design proposed by CIBMTR and finding the MaxCell CB outcome data to be 97.3% accurate, with no errors in survival, mortality, or engraftment. For both studies, all thalassemia and pediatric hematological malignancy patients were included and outcome were similar before and after matched-pair comparisons. A logistic regression model was used to find patients with similar characteristics to form pairs. For both studies, cumulative incidence was used for ANC500 neutrophil and platelet 20K and 50K engraftment. Kaplan–Meier was used for overall survival, disease-free survival, transplanted-related mortality, and relapse or autologous recovery. Cox regression analysis, log-rank test, univariate comparisons and the paired Prentice-Wilcoxon method were performed to compare the two matched-pair groups. Using the above methodologies, two rigorous retrospective matched-pair analysis of patients using RCR versus MaxCell CB products were conducted for thalassemia as well as for pediatric hematological malignancy patients [35–38].


Relative Risk calculated by Cox Regression for Univariate Analysis, with Red Cell Reduced CB products as reference. PPW = Paired Prentice-Wilcoxon test used in comparing matched-pair patients [**35–38**]. Absolute Neutrophil Count (ANC 500) is the first day of 3 consecutive days of an absolute neutrophil count equal to or greater than 0.5 × 109 /L prior to day 60 after transplantation (ANC 500)) engraftment. Platelet 20K engraftment is the first day of 7 consecutive days of a platelet count equal to or greater than 20 × 109 /L prior to the day 180 after transplantation.

**Table 5a.** Matched-Pair Comparison of Clinical Outcome of Patients transplanted with First Generation Red Cell Reduced CB Products versus MaxCell 2nd/3rd Generation CB products—30 pairs of thalassemia patients [36, 38] (Jaing et al. 2008) and 105 pairs of pediatric malignancy patients [35, 37, 38] (Jaing et al. 2008).


Relative Risk calculated by Cox Regression for Univariate Analysis, with Red Cell Reduced CB products as reference. PPW = Paired Prentice-Wilcoxon Test used in comparing matched-pair patients [**35–38**]. Absolute Neutrophil Count (ANC 500) is the first day of 3 consecutive days of an absolute neutrophil count equal to or greater than 0.5 × 109 /L prior to day 60 after transplantation (ANC 500)) engraftment. Platelet 20K engraftment is the first day of 7 consecutive days of a platelet count equal to or greater than 20 × 109 /L prior to the day 180 after transplantation.

**Table 5b.** Matched-Pair Comparison of Clinical Outcome of Patients transplanted with First Generation Red Cell Reduced CB Products versus MaxCell 2nd/3rd Generation CB products—105 pairs of pediatric malignancy patients [35, 37, 38].

For the thalassemia matched-pair study, 48 patients and 10 patients transplanted with MaxCell CB and RCR CB, respectively, and 3 MaxCell CBT patients were matched to each RCR CBT patients to form 30 pairs [36, 38]. Outcome comparisons of the two patient groups pre-match showed superiority in overall survival, disease-free survival, and transplant-related mortality for patients transplanted with MaxCell CB. Factors matched between the two groups were age, weight, #HLA matches, TNC dose, and transplant center experience. As the patients are mostly pediatric, there were no differences in median TNC between the two patient groups before (MaxCell 9.1 versus RCR 8.9 × 107 /kg) or after the match (MaxCell 9.1 versus RCR 8.9 × 107 /kg). **Table 5** showed significant improvement in 1- to 3-year overall survival and disease-free survival and 1-year and 3-year transplant-related mortality with the use of MaxCell CB for thalassemia patients [36, 38]. Interestingly, neutrophil engraftment, and short-term (100-day) survival or transplant-related mortality were not significantly different between MaxCell and RCR CB products.

**RCR 1st Generation CB MaxCell 2nd/3rd Generation CB**

89 ± 6%

PPW p=NS

**69 ± 6 %**

**65 ± 5 %**

**61 ± 4 %**

54 ± 4 %

**23 ± 4 %**

/L prior to the day 180 after transplantation.

Univ. RR = 0.81, *P* = 0.06 Log-Rank Test *p* = 0.12 Matched Pair 91 ± 7%

Univ. RR = 1.60; *p* = 0.007 Log-Rank test *p* = 0.006 **Matched Pair 71 ± 7 %** Matched-Pair PPW *p* = 0.001

Univ. RR = 1.41; *p* = 0.06 Log-Rank Test *p* = 0.05 **Matched Pair 68 ± 7 %** Matched-Pair PPW *p* = 0.007

Univ. RR (death) = 0.74; *p* = 0.01

Univ. RR (death) = 0.86; *p* = 0.11

Log-Rank Test *p* = 0.11 **Matched Pair 68 ± 5%** Matched-Pair PPW *p* = 0.005

Log-Rank Test *p* = 0.41 Matched Pair 61 ± 6% Matched Pair PPW *p* = 0.07

Univ. RR = 0.49; *p* < 0.001 Log-Rank Test *p* = 0.002 **Matched Pair 17 ± 4%** Matched-Pair PPW *p* = 0.0001

/L

ANC500 Neutrophil Engraftment Pediatric Leukemia Matched Pair

Platelet 20K Engraftment Pediatric Leukemia Matched Pair

Platelet 50K Engraftment Pediatric Leukemia Matched Pair

1-Yr Overall Survival

Pediatric Leukemia Matched Pair

1-Yr Disease-Free Survival Pediatric Leukemia Matched Pair

1-yr Transplant-Related Mortality Pediatric Leukemia Matched Pair

37, 38].

days of a platelet count equal to or greater than 20 × 109

85 ± 6 %

12 Umbilical Cord Blood Banking for Clinical Application and Regenerative Medicine

PPW p=NS

**55 ± 6 %**

**51 ± 6 %**

**48 ± 5 %**

46 ± 5 %

**45 ± 5 %**

Univ. RR = 0.81; *p* = 0.06 Log-Rank Test *p* = 0.12 Matched Pair 87 ± 6 %

Univ. RR = 1.60; *p* = 0.007 Log-Rank Test *p* = 0.006 **Matched Pair 56 ± 6 %** Matched Pair PPW *p* = 0.001

Univ. RR = 1.41; *p* = 0.06 Log-Rank Test *p* = 0.05 **Matched Pair 52 ± 6%** Matched-Pair PPW *p* = 0.007

Univ. RR (death) = 0.74; *p* = 0.01

Univ. RR (death) = 0.86; *p* = 0.11

Log-Rank Test *p* = 0.11 **Matched Pair 50 ± 5%** Matched-Pair PPW *p* = 0.005

Log-Rank Test *p* = 0.41 Matched Pair 47 ± 5% Matched Pair PPW *p* = 0.07

Univ. RR = 0.49; *p* < 0.001 Log-Rank Test *p* = 0.002 **Matched Pair 44 ± 5%** Matched-Pair PPW *p* = 0.0001

Relative Risk calculated by Cox Regression for Univariate Analysis, with Red Cell Reduced CB products as reference. PPW = Paired Prentice-Wilcoxon Test used in comparing matched-pair patients [**35–38**]. Absolute Neutrophil Count (ANC 500) is the first day of 3 consecutive days of an absolute neutrophil count equal to or greater than 0.5 × 109

prior to day 60 after transplantation (ANC 500)) engraftment. Platelet 20K engraftment is the first day of 7 consecutive

**Table 5b.** Matched-Pair Comparison of Clinical Outcome of Patients transplanted with First Generation Red Cell Reduced CB Products versus MaxCell 2nd/3rd Generation CB products—105 pairs of pediatric malignancy patients [35,

92 pairs

For the pediatric hematological malignancy matched-pair study, factors matched between the two groups were age, weight, #HLA matches, TNC dose, disease, and disease status [35]. Combining audited outcome data from one CBB (StemCyte) and data from one transplant center (U. Arizona), Graham et al. presented data for 105 matched pair of patients (paired from 128 patients transplanted with MaxCell 2nd Gen CB and 112 patients transplanted with RCR 1st Gen CB). **Table 5** shows that for the 105 pairs of pediatric hematological malignancy patients, 1- and 3-year overall survival, 100-day, 1-year and 3-year transplant-related mortality, and platelet (20K and 50K) engraftment were significantly improved with the use of MaxCell CB, while disease-free survival trended towards improvement [35, 37, 38]. Superior outcome of the MaxCell CB patient group in pre-match comparisons were confirmed by the results seen in the matched-pair analysis. Again, neutrophil engraftment and short-term (100-day) survival or transplant-related mortality were not significantly different between MaxCell and RCR CB products despite significant advantages in platelet engraftment, overall survival and transplant-related mortality.

Using data supplied by the NMDP for its large CB inventory of its CBB network (as of June 30, 2006) derived from almost 50,000 units (10,912 MaxCell and 38,819 RCR), Chow et al. [8] showed that a 24% superior nucleated cell recovery amplified into a 200% increase for MaxCell over RCR for the proportion of the inventories with products that had TNC counts higher than 150 × 107 (20% versus 10% of the inventory; test for difference in proportions, *p* < 0.0001) and a threefold difference for the proportion of products that had TNC counts higher than 200 × 107 (6% versus 2% of the inventory; test for difference in proportions, *p* < 0.0001). Therefore, the effectiveness of MaxCell CB processing is supported by data derived from the NMDP CB inventories of almost 50,000 units, which proves that the MaxCell CB inventories have significantly higher proportions of products with high TNC doses than the RCR CB inventories. Indeed, the MaxCell CB inventories had two to three times the proportion of high-cell dose products with TNC number of 150 × 107 or above and 200 × 107 or above (*p* < 0.0001) than the inventories of RCR units. Thus, MaxCell CB processing provides more efficient utilization of this valuable resource. This would seem to be particularly significant for those patients who participate in directed CB donation and private banking, because of the uniqueness of the cellular content and the importance of cell dose in outcome of HSCT.

#### **3. Thawing of cryopreserved CB products**

Unlike cryopreservation, thawing should be performed as quickly as possible by immersing the body (but not the ports) of cord blood bag in a 37°C water bath to an icy slush mixture. After thawing, there will be invariably some cell lysis, including 5–20% of the RBCs [44], a certain amount of WBC, principally neutrophils which do not survive freezing and thawing well, and occasional cell clumping and viscosity due to the release of chromosomal DNA. **Table 6** shows the expected cell loss for the various 1st Gen methods [44] as well as for the MaxCell 2nd/3rd Gen technologies [8]. The published cell loss and death associated with the CB processing method is listed below in **Table 6**, which showed the least TNC, CD34, and CFU loss for MaxCell CB products, followed by PrepaCyte, and with AXP-Express coming in last of the four techniques tested by Akel et al. [44]. Screnci et al. [45] independently confirmed that 42 un-manipulated RBC-replete CB products had significantly better post-thaw and wash recovery of TNC than 36 RCR CB, 95.2 ± 14.7% versus 85 ± 15.4% (*p* = 0.004).


**Table 6.** Post-thaw cell loss vs. pre-freeze cell count [11\*\*, 44\*, 45\*\*\*].
