**4. Post-thaw manipulations**

banking, because of the uniqueness of the cellular content and the importance of cell dose

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*

**Red cell reduction 1st generation MaxCell 2nd/3rd**

10% 14% 20%

20% 37% 53%

32% 28% 27% 22% Variable depends on

Sepax\* AXP\* MaxCell\*\*

vs. PrepaCyte *p* < 0.001 vs. Sepax *p* < 0.01

vs. PrepaCyte *p* < 0.001

**generation**

(n = 188)

8.89% 4.8%\*\*\*

<10%

#neutrophils in CB

in outcome of HSCT.

= 0.004).

TNC 18%

CFU 47%

Viable NCs by Trypan Blue

**3. Thawing of cryopreserved CB products**

14 Umbilical Cord Blood Banking for Clinical Application and Regenerative Medicine

Hetastarch\* Prepa

vs. PrepaCyte *p* < 0.01 vs. Sepax *p* < 0.05 14%\*\*\* vs. MaxCell *p* = 0.004

vs. PrepaCyte *p* < 0.05

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

Cyte-CB\*

Total MNC 8% 13% 7% 20% <10% CD34+ Cells 32% 24% 19% 37% 8.12% There are generally three main methods of manipulations after thaw of CB products, with many variations among different banks and transplant centers. **Table 7** summarizes whether each method reduces the total amount or just dilutes the concentration of DMSO, free hemoglobin or WBC lysates from thawing, safe thaw-to-dilution and thaw-to-infusion times, as well as summarizes the advantages and disadvantages of each method. Automated procedures using the Biosafe Sepax system can be used on all CB product types and was shown to be as effective as manual washing in terms of cell recovery and viability [46]. The Duke group found equal TNC recovery, identical viability, among 30 Sepax and 195 manual washed products with slightly better CFU and lower CD34+ recovery for the Sepax products [47].


**Table 7a.** Comparisons of the three major CB product thaw methods and the various parameters, Pros and Cons. [8, 21, 44, 48].



**Table 7b.** Pros and Cons of the three major CB product thaw methods [8, 21, 44, 48].

#### **4.1. Bedside thaw/direct infusion method**

**Pros Cons**

\*Potential for AE if used improperly not according to Manufacturer's Validated & Recommended

\*Largest infusion volume of three methods

lysate as Direct Infusion/Bedside Thaw \*Initial Dilution Step should be performed preferably within the first 10 minutes, in no case longer than the first 20 minutes for MaxCell CB and

30 minutes for RCR CB.

Protocol.

\*Same total mass of DMSO, Free Hgb, RBC & WBC

\*Potential for AE if used improperly not according to Manufacturer's Validated & Recommended

\* Compared to Direct Infusion, Dilution/Wash of MaxCell CB resulted in Worse OS, DFS, TRM, ANC 500, Platelet 20K and Platelet 50K engraftment,

\*Most significant cell loss of three methods during discard of post-centrifugation supernatant

\*Risk of cell aggregation with centrifugation \*Longest time and highest technical complexity of

\*Initial Dilution Step should be performed preferably within the first 10 minutes, in no case longer than the first 30 mins \*Potential for AE if used improperly not according to Manufacturer's Validated & Recommended Protocol.

Lower Limited cGvHD but Higher

Extensive cGvHD.

\*Risk of bag breakage

three methods

Protocol.

\*Least technically challenging—with most important element being control of thaw-toinfusion time \*Similar to other cryopreserved cellular product \*Recommended Thaw Method for MaxCell CB Products by Manufacturer for most situations.

16 Umbilical Cord Blood Banking for Clinical Application and Regenerative Medicine

\*Minimum infusion volume

\*More time from thaw to infusion \*Controlled Thaw in Cell Therapy Lab \*Hyperosmolar re-equilibration resulted in Diluted (though Equal Amount of)

Method for 1st

Dilution/Wash \*Removal of more than 80% DMSO and RBC

and WBC Lysate

colloidal agents

CB Banks

DMSO, Free Hgb, RBC & WBC lysate as Direct Infusion/Bedside Thaw \*Recommended Thaw

Gen RCR CB Products by some CB banks

\*Removal of Hetastarch, PrepaCyte or other

\*Longest Safe Thaw to Infusion Time as long as

the initial dilution is performed within the first 10 minutes \*Recommended Thaw Method for 1st Gen RCR CB Products by some

\*Recommended 1:7 Dilution-Wash Thaw Method for MaxCell CB products by Manufacturer for certain situations.

**Table 7b.** Pros and Cons of the three major CB product thaw methods [8, 21, 44, 48].

Dilution/ Reconstitution The CB product is thawed at bedside using the above thaw technique and immediately administered to the patient. Thaw to completion of infusion is typically completed within 10 min, with a maximum of 20 min, to avoid DMSO-induced toxicity and lysis of cells. Cell loss and technical complexity are minimal for bedside thaw/direct infusion of the three main thaw methods, especially if the infusion bag is flushed with saline or another approved infusion fluid to rinse out and inject the residual cells. Delay in infusion after thaw will lead to DMSO toxicity and cell lysis, resulting in loss of cell viability and release of potentially harmful cytokines, chemokines and cell debris that may potentially cause adverse events if released in sufficient amount. For all of the SAE reported to NMDP for CB infusion in 2008–09, prolonged thaw to dilution or infusion (essentially *in vivo* dilution) times were the common element among all the cases (NMDP, unpublished data). Further clinical data from the St. Louis bank's transplanted CB from the same publication [48] showed that the no-wash direct infusion provided the best post-thaw TNC recovery (median 99.0%, mean 85.6%, *p* < 0.01) and viability (median 95.0%, mean 89.3%, *p* < 0.01) over no-wash dilution (TNC recovery median 78.0%, mean 78.4%, viability median 88.0%, mean 84.8%) and post-thaw wash (TNC recovery median 78.6%, mean 77.4%, viability median 73.0%, mean 74.0%) [48].

There has been some concern that the presence of residual RBC in cryopreserved MaxCell CB may adversely affect the safety of HSCT; however, lysed RBC ghosts and free hemoglobin do not usually give rise to severe problems [49] unless a patient has compromised renal function or is on nephrotoxic drugs. The rare occurrences of acute renal failures with HSCT are frequently self-limiting or resolved with dialysis. While MaxCell manufacturers have always advocated direct infusion without post-thaw washing or dilution for most patients receiving MaxCell CB products, they also caution that for children, small patients, patients with compromised renal function (pre-existing or iatrogenic), patients with known sensitivity to DMSO, RBC or WBC lysates, chemokines and cytokines, and, lastly, for transplant centers that cannot directly infuse MaxCell CB products within 10–20 min of thawing, post-thaw washing is indicated. If post-thaw reconstitution or washing is to be performed, then it is of utmost importance to dilute the MaxCell CB product adequately (serial 1:1 dilutions three times to 1:7 final minimal dilution) within 10 min of thawing and to complete infusion of the washed product within 1–2 h, respectively (**Table 7**). The lack of SAEs when MaxCell CB products are thawed using strictly either the manufacturer's direct infusion method or post-thaw washing procedures or following proper validated good thawing practices has been documented [8].

Chow et al. [50–55] showed that for MaxCell CB products, direct infusion resulted in superior 1-year and 3-year overall survival, disease-free survival, and transplant-related mortality, as well as neutrophil ANC500 and platelet 20K and 50K engraftment, with higher limited cGvHD but lower extensive cGvHD over post-thaw wash [50–54]. **Table 8** shows the results of bedside thaw/direct infusion compared to post-thaw wash methods for MaxCell CB products in a matched-pair analysis of 258 patients forming 129 pairs of non-washed versus washed patients (95 malignancy pairs and 34 nonmalignant indication pairs), which confirmed the pre-match observations that direct infusion of MaxCell CB resulted in improved neutrophil ANC500 and platelet 20K, and 50K engraftment, as well as higher 1-year and 3-year overall survival, diseasefree survival, and lower transplant-related mortality, over transplants where MaxCell CB was post-thaw washed. Lastly, higher limited cGvHD with lower extensive cGvHD of direct infusion will maximize graft-versus-leukemia effect (GvL) without the increased mortality associated with severe cGvHD [55–59].


Paired Prentice-Wilcoxon test and log-rank tests were used to analyze 258 patients forming 129 pairs; (95 pairs malignancies and 34 pairs nonmalignant indications; relapse calculations are only for the 95 malignancy pairs). Paired Prentice-Wilcoxon test uses matched pairs of patients infused with post-thaw washed versus unwashed MaxCell CB products. Log-rank tests used univariate analysis of previously matched patients.

**Table 8.** Matched-pair analysis results comparing 129 pairs of 258 CBT patients receiving unwashed versus washed MaxCell CB products [56–59].

#### **4.2. Dilution and wash thaw method**

In 1995, Rubinstein et al. [13] described a thaw method, which consisted of slow reconstitution of the thawed unit three times with an equal volume of isotonic solution (5% [wt/vol] dextran-40/2.5% [wt/vol] human serum albumin) to an eventual ratio of 1:7 product:diluent (1:1 → 1:3 → 1:7), followed by centrifugation at 4°C at 400 × g for 10 min. The supernatant, containing DMSO, hetastarch (if applicable), cell lysates, hemolysate (including free hemoglobin), and any chemokines and cytokines released up to that point, are removed and cellular sediment is resuspended in one volume of fresh isotonic infusion solution equal to or greater than the original product volume. For all products, this method achieves post-thaw stability in cases of prolonged thaw-to-infusion time and reduces the potential for infusion reactions, by significantly reducing the amount of DMSO, hetastarch (if applicable), cell lysates, hemolysate (including free hemoglobin), and any chemokines and cytokines. Moreover, according to Rubinstein et al. [13], this method averts post-thaw osmotic damage and stabilizes cell viability if the product is not infused immediately, and reportedly provided near total recovery of CFU progenitors. A number of recent studies have failed to confirm the latter observation as reviewed by Akel et al. [44]. The COBLT study reported no infusion-related toxicity without addressing the recovery controversy [32]. Laroche et al. [60] found 18% TNC loss with the dilution-wash method, with 11% loss due to the washing step alone. Other reports also show significant loss of 10–25% for TNC, CD34+ and CFU [44–48]. Importantly, Regan et al. [48] showed that no-wash direct infusion provided significantly improved post-thaw TNC recovery (median 99.0%, mean 85.6%, *p* < 0.01) and viability (median 95.0%, mean 89.3%, *p* < 0.01) over post-thaw wash (TNC recovery median 78.6%, mean 77.4%, viability median 73.0%, mean 74.0%). This represents a significant 20% median TNC loss and 22% median viability reduction for post-thaw wash. Recently, even the NYBC in their HEMACORD product insert recommended a second centrifugation step for the supernatant to harvest some of the lost cells, which is a revision of the original NYBC wash method [13]. Both COBLT and NYBC recommends a final wash dilution ratio at 1:7.

**Table 8** summarizes a comparison study comparing MaxCell CB products thawed and infused with bedside thaw versus post-thaw wash and showed improvement in 1-year overall survival, disease-free survival, transplant-related mortality, neutrophil, and platelet engraftment. Interestingly, limited cGvHD was higher while extensive cGvHD was lower for patients infused with unwashed MaxCell CB.

#### **4.3. Reconstitution/dilution and no-wash method**

free survival, and lower transplant-related mortality, over transplants where MaxCell CB was post-thaw washed. Lastly, higher limited cGvHD with lower extensive cGvHD of direct infusion will maximize graft-versus-leukemia effect (GvL) without the increased mortality

Paired Prentice-Wilcoxon test and log-rank tests were used to analyze 258 patients forming 129 pairs; (95 pairs malignancies and 34 pairs nonmalignant indications; relapse calculations are only for the 95 malignancy pairs). Paired Prentice-Wilcoxon test uses matched pairs of patients infused with post-thaw washed versus unwashed

**Table 8.** Matched-pair analysis results comparing 129 pairs of 258 CBT patients receiving unwashed versus washed

In 1995, Rubinstein et al. [13] described a thaw method, which consisted of slow reconstitution of the thawed unit three times with an equal volume of isotonic solution (5% [wt/vol] dextran-40/2.5% [wt/vol] human serum albumin) to an eventual ratio of 1:7 product:diluent (1:1 → 1:3 → 1:7), followed by centrifugation at 4°C at 400 × g for 10 min. The supernatant, containing DMSO, hetastarch (if applicable), cell lysates, hemolysate (including free hemoglobin), and any chemokines and cytokines released up to that point, are removed and cellular sediment is resuspended in one volume of fresh isotonic infusion solution equal to or greater than the original product volume. For all products, this method achieves post-thaw stability in cases of prolonged thaw-to-infusion time and reduces the potential for infusion reactions, by significantly reducing the amount of DMSO, hetastarch (if applicable), cell lysates, hemolysate (including free hemoglobin), and any chemokines and cytokines. Moreover, according to Rubinstein et al. [13], this method averts post-thaw osmotic damage and stabilizes cell viability if the product is not infused immediately, and reportedly provided near total recovery of CFU progenitors. A number of recent studies have failed to confirm the latter observation as reviewed by Akel et al. [44]. The COBLT study reported no infusion-related toxicity without addressing the recovery controversy [32]. Laroche et al. [60] found 18% TNC loss with the dilution-wash method, with 11% loss due to the washing step alone. Other reports also show

MaxCell CB products. Log-rank tests used univariate analysis of previously matched patients.

associated with severe cGvHD [55–59].

18 Umbilical Cord Blood Banking for Clinical Application and Regenerative Medicine

MaxCell CB products [56–59].

**4.2. Dilution and wash thaw method**

The St. Louis CB Bank described a basic dilution and no-wash strategy [48]. Reconstitution with 1:1 ratio and ≥1:2 ratio of dextran-human serum albumin stabilized the hetastarchprocessed RCR CB and PrepaCyte-CB, respectively, decreased viability loss with prolonged thaw-to-infusion time (for up to 8 h for HES-RCR CB), limited wash-related cell loss, and reduced preparation time and complexity. Barker et al. used a 1:4 ratio dilution and no-wash procedure for 104 RCR and 3 MaxCell products without severe AE [9]. However, the St. Louis group's own data [48] showed that no-wash direct infusion provided significantly better postthaw TNC recovery (median 99.0%, mean 85.6%, *p* < 0.01) and viability (median 95.0%, mean 89.3%, *p* < 0.01) over no-wash dilution (TNC recovery median 78.0%, mean 78.4%, viability median 88.0%, mean 84.8%). This represents a substantial 21% median TNC loss and 7% median viability reduction for reconstitution compared to direct infusion.

Most CB banks now agree on the reconstitution, dilution, and washing solution composition of one volume of 25% human serum albumin (HSA) mixed with five volumes of 10% lowmolecular-weight dextran 40), though the degree of CB product dilution varies between 2- and 16-fold. It should be noted that post-thaw washing or reconstitution can further reduce cell dose and viability as shown above [48], and is recommended for RCR CB by some CB banks, but not recommended by the manufacturer for MaxCell CB except in special circumstances [18, 21]. Regan et al. [48] showed a greater reduction of viability, TNC, and CD34 for unwashed and undiluted CB after 2 h *in vitro*. However, in clinical practice, direct infusion performed with bedside thaw should always be performed within 10–20 min to avoid DMSO toxicity (**Table 6**) and is almost never performed after such a prolonged post-thaw interval, making such *in vitro* comparisons clinically irrelevant except in cases where CB product manufacturer's recommendation is not followed. Importantly, the manufacturer of MaxCell CB products recommends completion of infusion of MaxCell CB products using the bedside thaw method within 10 min of initiation of thawing for adults at 5–10 mL/min for a 75-mL product. Several other groups have demonstrated that direct infusion without washing produces excellent results for CB thawing [44, 47, 48, 60–68]. Chow et al. [18, 21, 50–59] was the first group to report superior clinical outcome with bedside direct infusion over post-thaw wash, with improved neutrophil and platelet engraftment, reduced transplant-related mortality, decreased extensive cGvHD, increased limited cGvHD, and enhanced overall and disease-free survival.

To address the question of whether the presence of residual RBC in the cryopreserved MaxCell products may adversely affect the outcome of HSCT, we have previously reported on the outcomes of 118 MaxCell CB transplants for patients with both hematological malignancies and nonmalignant indications [8]. Our experience indicates a 90.3% cumulative incidence for neutrophil (ANC500) engraftment, 75.5% for platelet 20K engraftment, 16.3% for 100-day transplant-related mortality, 65.5% for 1-year overall survival, and 51.6% for 1-year diseasefree survival. This was followed by another series with 120 patients with nonmalignant indications [39] with similar outcome. At this point, after thousands of MaxCell CB products have been transplanted at around 300 transplant centers, that favorable experience reported previously has been maintained [[18, 35–43, 50–59], Chow et al. unpublished observations].
