Post-thaw Wash Research Articles

A three-step method for preparing cryopreserved samples of apheresis products for post-thaw analysis yields a high recovery of viable cells.

Flow cytometry protocols for counting fresh CD34+ cell samples are not ideal for cryopreserved products due to cryoprotectant cytotoxicity. For cryopreserved samples, often large volumes of hypotonic solutions, which can cause cell death, are used to remove the cryoprotectant with a post-thaw wash. We recently developed a novel multistep dilution method with subsequent flow cytometry analysis to allow for accurate and reproducible results. The previous method involved washing steps which invalidate the ability to enumerate cell recovery, and success had to be gauged solely on viability. The new method allows for assessment of total cell recovery and viable cell recovery. Apheresis products were cryopreserved in 10% DMSO at a target WBC concentration of 300 × 106/mL. Cryovials from these products were thawed at 37°C, and samples were diluted 1:2 by three additions of 1/3 sample volume using 1%-Human Albumin in Dextran 40 (10% Low Molecular Weight Dextran in 0.9% NaCl) separated by 5 min between each addition. A 1:10 dilution was performed to obtain the correct cell concentration for flow cytometric analysis resulting in a 1:20 dilution. End WBC concentrations were ~15 × 106/mL with DMSO diluted to 0.5%. Fifty-two samples were tested with this new method. Total and viable cell recoveries were calculated based on pre-cryopreservation data. Median total cell recoveries for CD34 and CD3 were >85%, while median viable cell recoveries were >75%. Cryopreserved samples can be reliably prepared for flow cytometric testing using a step-wise dilution to preserve cell integrity and robust recoveries.

Cryopreserved platelets washed with a dialysis machine for dimethyl sulphoxide removal.

Cryopreserved platelets (cPLTs) can be stored for years and are mainly used in military settings. However, the commonly used cryoprotectant dimethyl sulphoxide (DMSO) has toxic side effects when utilized in high quantities. We developed a novel method to aseptically remove DMSO from thawed cPLTs by dialysis. One unit of platelets (N = 6) was mixed with 75 mL of 27% DMSO within 4 days after collection and stored at -80°C for 1 week. The platelet counts, platelet distribution width, mean platelet volume (MPV), platelet activity, platelet release, platelet aggregation, platelet metabolism indicators and platelet ultrastructural features (determined by electron microscopy) of the samples at the pre-freeze, post-thaw wash (post-TW) and 24 h post-thaw wash (24-PTW) stages were determined and compared. The DMSO clearance rate from the post-TW platelets was 95.56 ± 1.3%, and the platelet recovery rate after washing was 74.66 ± 6.34%. The total count, activity, release factors, aggregation and thrombolytic ability of the post-TW platelets were lower, whereas the MPV and apoptosis rates were higher compared with those of the pre-freeze platelets. The lactic acid, glucose and potassium ions released from the platelets during washing were filtered away by the dialyser, which significantly reduced their concentration. However, 24-PTW platelets were metabolically active, resulting in a decrease in pH and glucose content and an increase in lactic acid content. The level of potassium ions remained low after 24 h of storage and washing. The pre-freeze platelets maintained their normal disc shape and exhibited an open canalicular system (OCS) and a dense tubular system. The cPLTs appeared irregular after washing, with protruding pseudopodia and an extensive OCS, which increased the release of their contents. We developed a novel dialysis method to effectively remove DMSO from cPLTs under aseptic conditions and maintain platelet quality. The clinical efficacy of our method remains to be determined. However, the function of the platelets declined 24 h after washing, making them unsuitable for transfusion.

Effects of pre-freeze pathogen reduction with riboflavin and UV light on red cells stored post-thaw in AS-3 additive solution.

Pathogen reduction technology (PRT) may improve the safety of RBCs for transfusion. As the Czech Republic considers PRT, we asked what effects riboflavin and UV light PRT pre-freezing has on the post-thaw recovery and properties of cryopreserved RBCs (CRBCs) after deglycerolization and liquid storage. 24 Group O whole blood (WB) units were leukoreduced and then treated with riboflavin and UV light PRT (Mirasol, Terumo BCT, USA) before cryopreservation (T-CRBC); 20 similarly-collected units were untreated controls (C-CRBC). Units were processed to RBCs and then cryopreserved with 40% glycerol (wt/vol), frozen at -80°C, stored >118 days, reconstituted as deglycerolized RBC units in AS-3, and stored at 4 ± 2°C for 21 days. One treated unit sustained massive hemolysis during the post-thaw wash process and was removed from data analysis. The remaining units were assessed pre-PRT, post-PRT, and post-thaw-wash on days 0, 7, 14, and 21 for hematocrit, volume, hemoglobin per transfusion unit, pH, % hemolysis, hemoglobin in the supernatant, potassium, phosphorus, NH3 , osmolality, ATP, and 2,3-diphosphoglycerate. PRT with leukoreduction caused a 5% loss of RBC followed by a 24% freeze-thaw-wash related loss for a total 28% loss but treated units contained an average of 45 g of hemoglobin, meeting European Union guidelines for CRBC. T-CRBCs displayed higher post-wash hemolysis, potassium, and ammonia concentrations, and lower ATP at the end of storage. Cryopreserved RBCs from Riboflavin and UV light-treated WB meet the criteria for clinical use for 7 days after thawing and provide additional protection against infectious threats.

C-11: Microfluidic processing of cryopreserved blood

Cryopreservation of human red blood cells (RBCs) in the presence of 40% glycerol allows a shelf-life of 10 years, as opposed to only 6 weeks for refrigerated RBCs. Nonetheless, cryopreserved blood is rarely used in clinical therapy, in part because of the requirement for a time-consuming (∼1 h) post-thaw wash process to remove glycerol before the product can be used for transfusion. The current deglycerolization process involves a series of saline washes in an automated centrifuge, which gradually removes glycerol from the cells in order to prevent osmotic damage. We recently demonstrated that glycerol can be extracted in as little as 3 min without excessive osmotic damage if the composition of the extracellular solution is precisely controlled. Here, we explore the potential for carrying out rapid glycerol extraction using a membrane-based microfluidic device, with the ultimate goal of enabling inline washing of cryopreserved blood. To assist in experimental design and device optimization, we developed a mass transfer model that allows prediction of glycerol removal, as well as the resulting cell volume changes. Experimental measurements of solution composition and hemolysis at the device outlet are in reasonable agreement with model predictions, and our results demonstrate that it is possible to reduce the glycerol concentration by more than 50% in a single device without excessive hemolysis. Based on these promising results, we present a design for a multistage process that is predicted to safely remove glycerol from cryopreserved blood in less than 3 min.

Review of in vivo studies of dimethyl sulfoxide cryopreserved platelets.

A literature review was conducted to assess the efficacy and safety of dimethyl sulfoxide (DMSO) cryopreserved platelets for potential military use. In vivo DMSO cryopreserved platelet studies published between 1972 and June of 2013 were reviewed. Assessed were the methods of cryopreservation, posttransfusion platelet responses, prevention or control of bleeding, and adverse events. Using the Department of Defense's preferred 6% DMSO cryopreservation method with centrifugation to remove the DMSO plasma before freezing at -65°C and no postthaw wash, mean radiolabeled platelet recoveries in 32 normal subjects were 33% ± 10% (52% ± 12% of the same subject's fresh platelet recoveries), and survivals were 7.5 ± 1.2 days (89% ± 15% of fresh platelet survivals). Using a variety of methods to freeze autologous platelets from 178 normal subjects, mean radiolabeled platelet recoveries were consistently 39% ± 9%, and survivals, 7.4 ± 1.4 days. More than 3000 cryopreserved platelet transfusions were given to 1334 patients. There were 19 hematology/oncology patient studies, and, in 9, mean 1-hour corrected count increments were 11 100 ± 3600 (range, 5700-15 800) after cryopreserved autologous platelet transfusions. In 5 studies, bleeding times improved after transfusion; in 3, there was either no improvement or a variable response. In 4 studies, there was immediate cessation of bleeding after transfusion; in 3 studies, patients being supported only with cryopreserved platelets had no bleeding. In 1 cardiopulmonary bypass study, cryopreserved platelets resulted in significantly less bleeding vs standard platelets. In 3 trauma studies, cryopreserved platelets were hemostatically effective. No significant adverse events were reported in any study. In summary, cryopreserved platelets have platelet recoveries that are about half of fresh platelets, but survivals are only minimally reduced. The platelets appear hemostatically effective and have no significant adverse events.

Continuous removal of glycerol from frozen-thawed red blood cells in a microfluidic membrane device.

Cryopreservation of human red blood cells (RBCs) in the presence of 40% glycerol allows a shelf-life of 10 years, as opposed to only 6 weeks for refrigerated RBCs. Nonetheless, cryopreserved blood is rarely used in clinical therapy, in part because of the requirement for a time-consuming (∼1 h) post-thaw wash process to remove glycerol before the product can be used for transfusion. The current deglycerolization process involves a series of saline washes in an automated centrifuge, which gradually removes glycerol from the cells in order to prevent osmotic damage. We recently demonstrated that glycerol can be extracted in as little as 3 min without excessive osmotic damage if the composition of the extracellular solution is precisely controlled. Here, we explore the potential for carrying out rapid glycerol extraction using a membrane-based microfluidic device, with the ultimate goal of enabling inline washing of cryopreserved blood. To assist in experimental design and device optimization, we developed a mass transfer model that allows prediction of glycerol removal, as well as the resulting cell volume changes. Experimental measurements of solution composition and hemolysis at the device outlet are in reasonable agreement with model predictions, and our results demonstrate that it is possible to reduce the glycerol concentration by more than 50% in a single device without excessive hemolysis. Based on these promising results, we present a design for a multistage process that is predicted to safely remove glycerol from cryopreserved blood in less than 3 min.

Analysis of 120 Pediatric Patients with Non-Malignant Disorders Transplanted Using Unrelated Plasma Depleted Cord Blood and the Role of Post-Thaw Wash

Unrelated cord blood (UCB) is an important stem cell source for unrelated hematopoietic cell transplantation of patients with non-malignant disorders. Cell dosage is a critical factor for successful UCB hematopoietic stem cell transplantation (HSCT). The red cell reduced (RCR) and post-thaw wash techniques that are widely used incur significant nucleated cell loss. Three strategies were employed to maximize cell dose and improve outcome–use of cord blood processed with plasma depletion without red blood cell reduction (PD CB), avoidance of post-thaw wash, and the use of double cords (2X) when necessary. A CIBMTR-audited analysis was performed on all 120 pediatric patients with non-malignant disorders transplanted with PD CB at 29 U.S. and 17 international centers. Transplant characteristics: median age 3.5 years (range 0.1–14); median patient weight 15 kg (range 4–61); male 58%. The majority of patients (n=58; 55%) were Asian. Twenty-two (21%) patients were Hispanic, 15 (14%) were Caucasian, 6 (6%) were African-American, and three (3%) were of Middle Eastern background. HLA ABDR matches: 6/6–26; 5/6–48; 4/6–47; 3/6 or 2/6–6; median pre-freeze nucleated cell dose 10.5×107/kg; median pre-freeze CD34+ dose 3.7×105/kg; non-myeloablative regimen 24%; 58% infused without post-thaw wash (NW). Myeloid engraftment defined as ANC≥500 and 6-month platelet engraftment defined as ≥ 20K and ≥ 50K are 89±8%, 88±8%, and 84±6% respectively. The median time to myeloid and platelet engraftment are 21 days (range 11–64), 49 days (range 13–155), and 61.5 days (range 21–205) respectively. No major adverse event was observed in either the W or the NW group. The cumulative incidence of reported grade II–IV acute GVHD was 38±5%, and 19±4% had grade III–IV acute GVHD. 36±6% developed limited chronic GVHD, and 12±4% developed extensive chronic GVHD. With a median follow-up of 329 days (range 3–1928 days), the Kaplan-Meier estimates of 1-year TRM, OS and diseasefree survival were 20±6%, 88±6% and 72±6% respectively. Foregoing post-thaw wash for PD CB transplantation improved neutrophil (RR=1.75; p=0.01) and platelet engraftment (RR=1.72; p=0.02) and reduced TRM (RR=0.38; p=0.04). This series demonstrated that unrelated PD CB transplantation can be performed safely and effectively in children with life-threatening, non-malignant disorders. Additionally, the results demonstrate possible improvement in myeloid and platelet engraftment, overall and disease-free survival when post-thaw wash is not employed.Table 1. Summary of overall resultsOutcomeAll Patients N = 120Washed CB N = 48Unwashed CB N = 71RR (Wash=Ref)P-valueANC500 Engraftment Cumulative Incidence Median # Days to Engraftment87±6% d+2186±9% d+2589±8% d+191.750.01Platelet 20K Engraftment Cumulative Incidence Median # Days to Engraftment81±6% d+4975±9% d+5288±9% d+431.720.02Autologous Recovery3±2%2±2%4±3%1.060.95Acute GvHD II–IV Acute GvHD III–IV38±5% 19±4%31±7% 17±6%45±7% 21±6%1.74 1.380.11 0.50Chronic GvHD Limited Chronic GvHD Extensive36±6% 12±4%14±6% 19±6%60±10% 6±4%5.69 0.24<0.001 0.08Transplant-Related Mortality–100 Day Transplant-Related Mortality–3 Yr10±3% 20±4%11±5% 34±8%9±4% 11±4%0.380.04Overall Survival–1 Yr Overall Survival–3 Yr79±4% 79±4%66±8% 66±8%88±4% 88±4%0.430.06Disease-Free Survival–1 Yr Disease-Free Survival–3 Yr72±5% 70±6%58±9% 51±10%84±5% 84±5%0.480.07

Unrelated Cord Blood Transplantation (UCBT) for Transfusion- Dependent Thalassemia a CIBMTR Audited Retrospective Analysis of 30 Consecutive Patients from a Single Center

UCBT offers a cure for thalassemia and has advantages as a stem cell source because the requirement for an HLA match between donor and recipient is less stringent. Previous reports of UCBT for thalassemia have yielded transplant related mortality (TRM) that was quite high and it is known that pre-freeze total nucleated cell (TNC) dose is critical for UCBT. With strategies that maximize TNC dose – using non-red cell reduced but plasma depleted (PD) CB, no post-thaw wash (NW), and double cord transplantation (DCT) when necessary – we have achieved promising results with UCBT in young patients with transfusion-dependent β-thalassemia. Between 10/2003 and 5/2008, 39 CB products were infused after Bu/Cy/ATG myeloablation into 30 pediatric thalassemia major patients (9 DCT) using NW PD CB exclusively. Patient status: 21 Pesaro class−1, 8 Pesaro class−2, and 2 unknown. Median age was 5 years (range 1–14 years) with a median weight of 18 kg (range 11–37 kg). The data was audited by the transplant center (TC) and on-site by CIBMTR (98.5% accuracy for Chang Gung). Median pre-freeze TNC cell dose was 10.9×107/kg, and median pre-freeze CD34+ cell dose was 4.0×105/kg. The number of CB products HLA ABDR matched were 4, 11, and 24 for 6/6, 5/6, and 4/6 or fewer matches. No significant adverse events were observed despite major ABO incompatibility in some cases and forgoing post-thaw wash. Cumulative incidences of donor-derived (determined by chimerism studies) neutrophil (ANC500), platelet 20K and 50K (plt 20K & 50K) engraftment were 96±11%, 92±11% and 92±12%, and median times to ANC500, plt 20K and 50K engraftment were 17.5 days (range 11–26), 49.5 days (range 28–135) and 63.5 days (range 35–203), respectively. Grade III/IV acute GvHD occurred in 40±9%, and extensive chronic GvHD occurred in 4±4% of the patients. Transplant related mortality were 10±5% at 100 days and 13±6% at 1 year. Overall survival at 1 and 3 years were 87±6% and 82±8%, while disease-free survival at 1 and 3 years were 85±7% and 78±9%. Five patients expired including 1 death prior to day +10 due to traumatic head injury. Mean and median follow up times were 24 and 16 months respectively (range 0.3–58 months) as of August 2008. To our knowledge, this is the largest single-institutional UCBT series for thalassemia, and shows the favorable clinical results that are attainable when TNC dose is optimized with PD CBU, no post-thaw wash and DCT when necessary.

Avoidance of Post-Thaw Wash Prior to Transplantation of Plasma Depleted Cord Blood (PD CB) Is Associated with Improved Engraftment & Decreased Severity of Chronic GvHD (cGvHD) without Increased Relapse.

Post-thaw washing of frozen CB prior to transplantation is widely practiced to remove DMSO, red cell debris and free hemoglobin, however, washing a red cell containing product may result in cell loss. We analyzed outcomes of engraftment, survival, relapse and GvHD in 299 patients transplanted with plasma depleted but not red cell reduced (PD) CB where 154 were washed (W) and 145 were directly infused - (non-washed or NW). 207 (69%) of 299 patients had malignant diagnosis. The two groups were comparable for patient, disease and graft characteristics except for a higher % of Asian patients in the NW group in contrast to a higher % of Caucasian/Hispanic patients, higher % of US transplant centers (TC) and higher number of malignant diagnoses for the W group. Nucleated cell recovery after thawing, as reported by TC, was higher for NW (median 85% vs. 79%, p=0.003). The NW group had higher cumulative incidences of and faster neutrophil (ANC500) and platelet engraftment (Plt 20K and 50K) than the W group. No difference was seen for acute GvHD (aGvHD); however, the NW group had significantly lower rates of extensive (Ext) cGvHD and higher rates of limited (Ltd) cGvHD (p=0.0005 and <0.0001). Relapse rates were similar between W and NW. Multivariate analysis and matched pair analysis between W versus NW confirm these observations. In univariate analysis, overall survival (OS) and disease-free survival (DFS) were significantly higher for the NW group. This retrospective analysis represents the largest series comparing different thaw methods for PD CB and is the first to demonstrate a potential association of no post-thaw wash with significant decrease in extensive cGvHD rate without concomitant increase in relapse rate. These improvements may contribute to the apparently better survival for the NW group. Prospective trials which may include infusion of wash supernatants into patients are necessary to confirm and better understand these observations.ANC 500 & Plt 20K100 Day TRM1 & 3 Yr OS1 & 3 Yr DFS1 Yr RelapseII-IV/III-IV aGvHDLtd/Ext cGvHDOverall88±4/69±417±261±3 56±351±3 39±523±335/1420/11W87±5/64±517±354±4 48±443±5 30±627±536/159/17NW91±6/76±616±369±4 67±460±5 57±520±436/1436/1P value0.002/0.0010.050.020.050.290.93/0.80<0.0001/0.0005ANC 500 & Plt 20KTRMOSDFSRelapseII-IV/III-IV aGvHDLtd/Ext cGvHDMV adjusted Cox RR & P value1.51 0.002/1.70 0.0010.67 0.110.71 0.090.74 0.110.76 0.34N/A3.88/<0.001Matched Pairs P value0.008/0.00020.530.320.220.190.98/0.440.02/0.001All results in % except for p-values & where indicated. Engraftment presented as cumulative incidence, GvHD as proportions; all others estimated by Kaplan-Meier.

166: Unrelated cord blood transplantation (UCBT) for transfusion-dependent thalassemia – a retrospective audited analysis of 41 consecutive patients

UCBT offers a cure for thalassemia and CB has advantages as a stem cell source for minorities, where thalassemia is prevalent. Previously mortality risks outweigh benefits and cell dose is critical for UCBT. With strategies that maximize cell dose - using non-red cell reduced plasma depleted (PD) CB, no post-thaw wash (NW), and double cord transplantation (DCT), promising results may be achieved with UCBT in selected thalassemics. Between 7/01 and 9/06, 47 UCBT were performed after myeloablative therapy in 41 pediatric patients with thalassemia major (7 DCT & 1 re-transplant) at 14 transplant centers (TC) using 81% PD CB with 91% NW.

Unrelated Cord Blood Transplantation (CBT) for Thalassemia.

CBT is a curative therapy for thalassemia, with cell dosage is a critical factor for CBT. We previously showed that by combining strategies that maximize cell dose-using non-red cell reduced plasma depleted cord blood (PD CB), forego post-thaw wash, and double cord transplantation - promising results may be achieved with unrelated CBT in selected patients. Between 7/91 and 4/06, 45 unrelated CBT were performed after myeloablative therapy (Bu/Cy/ATG in all 33 patients with known regimen) in 36 pediatric patients with transfusion-dependent thalassemia and 2 SCD patients at 14 transplant centers (TC) using 80% PD CB (6 double cords & 1 re-transplant). For the thalassemics, 19 were Lucarelli class 1, 4 class 2, 1 class 3, and 14 status unknown. Median age was 6 yo (range 0.3–20 yo) and median weight was 19 kg (range 8–76 kg). Data was audited. No significant adverse events were observed despite major ABO incompatibility in 9 cases. Unadjusted ANC500, platelet 20K and 50K engraftment with donor chimerism were achieved in 81±7%, 79±8% and 76±8% of the cases, and median times to ANC500 engraftment, platelet 20K and 50K engraftment were at +16.5 (range 11–33), +37 (range 16–133) and +55 (range 26–144 days) days, respectively. 8 patients died including 2 early deaths prior to day +20 and one accidental trauma caused cerebral hemorrhage. All remaining 30 patients are alive, with 25 disease-free and 5 autologous recovery for an 1-Yr OS of 77±7% and DFS at 65±8%. With a mean and median follow up time of 392 and 257 days respectively (range 7–1,760 days), the median day to hospital discharge was day +58 (range 22–137 days). The two experienced TC (>5 cases of CBT for hemoglobinopathy) were compared with less experienced centers, and CBU that were post-thaw washed (14%) were compared with CB that were not washed (86%). Results show potential benefits with TC experience and no post-thaw wash; however, since both experienced centers do not practice post-thaw wash, these two factors cannot be distinguished. Unadjusted ANC500, platelet 20K and 50K engraftment were 87±7%, 86±8% & 86±8% and 1-Yr OS of 87±7% and DFS at 77±9% were achieved for experienced centers not performing post-thaw wash. The role of plasma depletion vs. RBC depletion was also studied by matching age, weight, TNC dose, #HLA matches, conditioning regimen, thalassemia diagnosis, and post-thaw wash between the groups at the 2 experienced TC. Outcome trended towards improvements in engraftment and thalassemia-free survival for PD CB; however, larger numbers and rigorous matched pair analysis are needed for definitive conclusions. Overall, this is the largest known study of unrelated cord blood transplantation for thalassemia and preliminary results show that when cell dose (PD CBU, double cord, and no wash) and other conditions (TC experience and judicious patient selection) are optimal, CBT may be a promising approach for the curative therapy of thalassemia major with unadjusted ANC500, platelet 20K and 50K engraftment rates of 93±7%, 91±8% & 82±8% respectively and 1-Yr OS of 86±9% and DFS at 86±9%.

Hematopoietic Stem Cell Transplantation (HSCT) for Benign Indications Using Plasma Depleted Umbilical Cord Blood Units (UCB) That Were Not Depleted of Red Blood Cells.

UCB is an attractive source for unrelated HSCT of benign indications; however, cell dosage is a critical factor for UCB HSCT. The red cell depletion (RD) and post-thaw wash techniques that are widely used incur significant nucleated cell loss; therefore, two strategies to minimize cell loss are to deplete plasma, but not the red blood cells (PD) during processing and forego post-thaw wash. A retrospective audited analysis was performed on 61 patients with benign disorders who were transplanted with 68 PD UCB units (8 double cords) with 29 thalassemias, 8 AA, 5 WAS, 5 SCID, 2 osteoporosis, 2 sickle cell disease, 2 Hemophagocytic Lymphohistiocytosis, 2 Hurler Syndrome, 1 CGD, 1 Fanconi's Anemia, 1 Leroy I-Cell Disease, 1 Lymphohistiocytosis, 1 OIMD, and 1 Alpha Mannosidosis. Transplant characteristics: patient median age 4.25 years old (range 0.3–39); median weight 17 kg (range 5–76); male 56%; median # HLA ABDR matches of 5.0 (12–6/6; 19–5/6; 23–4/6; 5–3/6, 1–2/6); median pre-freeze TNC dose 8.1 x 107/kg; median post-thaw TNC dose as reported by TC 7.7 x 107/kg; median pre-freeze CD34 dose 3.1 x 105/kg; transplants outside of U.S.− 32 (52%); non-myeloablative − 9; 44% post-thaw washed (W), 56% infused without post-thaw wash (NW). The Kaplan-Meier estimates of 3-month ANC500 and 6-month platelet 20K and 50K engraftment are 87±5%, 83±6%, and 84±6% respectively. The median time to engraftment for ANC 500, platelet 20K, and 50K are 20 days (range 11–64), 46 days (range 13–153), and 61 days (range 21–171) respectively. No major adverse event was observed in either the W or the NW group, and the median time to engraftment for ANC 500, platelet 20K and 50K for W vs. NW were 21 vs. 19 days, 55 vs. 44.5 days, and 76 vs. 59 days respectively. The incidence of reported grade II–IV acute GVHD was 29%, and 10% had grade III–IV acute GVHD. 33% developed limited chronic GVHD, and 15% developed extensive chronic GVHD. With a median follow-up of 219 days (range 4–1402 days), the Kaplan-Meier estimates of 1-year TRM, OS and disease-free survival were 20±6%, 78±6% and 72±6% respectively. These results demonstrate that HSCT using unrelated PD UCB can be performed safely with outstanding results in patients with benign disorders, and post-thaw washing may delay engraftment of HSCT using PD UCB.

Hematopoietic Stem Cell Transplantation (HSCT) for Malignancies Using Plasma Depleted Umbilical Cord Blood (UCB) That Were Not Red Blood Cell Depleted.

Cell dosage is a limiting factor for UCB HSCT, especially for adult patients. Most UCB banks practice red cell depletion (RCD) techniques to save storage space, which incur significant nucleated cell loss after processing. One method to minimize cell loss and still reduce volume after processing is to deplete plasma (PD), but not the red blood cells. Not washing UCB (NW) after thawing also minimizes cell loss. A large racially diverse PD UCB inventory of over 25,000 units is now available on stem cell registries. There were 70 ALL, 54 AML, 19 CML, and 45 others transplanted for malignant indications. A retrospective audited analysis was performed on 170 patients with engraftment and/or survival information. Of the ALL/AML/CML cases with available information, there were 32 1CR/CP, 33 2CR, and 13 3CR/CP. The median age of patients was 13 years old (range 05–59); median weight 50 kg (range 5–137); male 45%. Transplant characteristics indicated a median # HLA ABDR matches of 4.0; median pre-freeze TNC dose 4.1 × 107/kg; median post-thaw TNC dose as reported by TC 3.7 × 107/kg; median pre-freeze CD34 dose 1.4 × 105/kg; transplants outside of U.S. 34%; double unit transplant 30%; non-myeloablative 16%. Sixty-one percent of the transplanted UCB were washed post-thaw (W), 39% were infused without post-thaw wash (NW), with 20% of the units without available post-thaw data. Kaplan-Meier estimates of 3-month ANC500 and 6-month platelet 20K and 50K engraftment are 89±3%, 81±5%, and 73±5% respectively. Median time to engraftment for ANC 500, platelet 20K, and 50K were 22 days (range 7–69 days), 52.5 days (range 12–181 days), and 64 days (range 25–374 days) respectively. Median time to engraftment for post-thaw washed units (W) versus PD CBU not washed and infused directly after thawing (NW) were 24 vs. 22 days for ANC500, and 57 vs. 48 days for platelet 20K respectively. The incidence of reported grade II-IV and III-IV acute GVHD were 32% and 14% respectively. Twelve percent developed limited chronic GVHD and 14% developed extensive chronic GVHD. With a median follow-up of 170 days (range 4–1402 days), the Kaplan-Meier estimates of 1-year TRM, OS and relapse-free survival were 31±4%, 51±4% and 47±4% respectively. These results demonstrate that HSCT using unrelated PD UCB can be performed safely and effectively in patients with malignancies, and post-thaw wash may not be necessary.

Post-That Washing Prior to Transplantation of Umbilical Cord Blood (UCB) That Were Depleted of Plasma but Not of Red Blood Cells.

Washing after thawing of frozen UCB used for hematopoietic stem cell transplantation (HSCT) is widely practiced for the purpose of removal of the cryoprotectant DMSO and free hemoglobin from lysed red cells. An updated retrospective audited analysis was performed on the outcomes of 208 PD UCB used in 186 patients with known thaw conditions that have engraftment and/or survival data −106 washed (W) and 80 non-washed (NW). When the DMSO dose was kept under the recommended 1 g per kg of recipient weight, the only severe adverse reaction was a seizure and encephalopathy following infusion of a NW PD UCB that resolved without any sequelae. Total nucleated cell recovery after thawing as reported by transplant centers was higher for NW (median 89% vs. 75%). The Kaplan-Meier estimate of 3-month neutrophil (ANC500) engraftment were 91±4% for NW versus 88±4% for W with median time to neutrophil of 21 versus 24 days (p = 0.02). The K-M estimate for 6-month platelet 20K engraftment were 86±6% for NW and 78±5% for W with median time to engraftment at 46 days for NW and 55 days for W (p = 0.002). Acute grade III–IV GvHD were similar at 12% (NW) and 13% (W), but extensive chronic GvHD were 4% (NW) and 19% (W). 1-year TRM were 25±5% for NW and 34±5% for W (p=0.75), with relapse rate at 16±5% for NW and 28±5% for W (p=0.09). K-M estimates of 1-year OS were 63±6% versus 53±5% (p = 0.54), and for 1-year DFS were 62±6% versus 48±5% (p = 0.23) for NW and W respectively. To our knowledge, this was the largest transplant outcome study comparing no post-thaw wash versus wash for UCB, though the conclusions are confined to PD CBU. No clear benefits of post-thaw washing were seen for PD UCB. HSCT with NW PD UCB was at least as efficacious as that using W PD UCB with respect to GvHD, TRM, relapse rate, 1-year OS and DFS. Moreover, washing may have a negative impact on neutrophil and platelet engraftment of PD UCB.

Outcome Comparisons between Two Cord Blood Banks on Different Continents Using Identical Procedures.

Theoretically, cord blood (CB) banked using identical procedures by different banks should yield similar outcome if standard operating procedures (SOP) and quality programs are followed rigorously. To compare and validate the quality of the CB products, we studied whether all CB with available engraftment and/or survival transplant data from two banks, one in the USA and one in Taiwan, banked using virtually identical procedures, yielded similar transplantation outcome. Unadjusted engraftment of ANC500 were 91±3% for US (n=144) and 79±6% for Taiwan (n=49), while 1-year overall survival (OS) were 56±4% for US (n=153) vs. 68±7% for Taiwan (n=50). A number of obvious differences in transplants for the two banks were identified, for example, CB Transplants from the Taiwan CB bank (CBB) had more patients with benign diseases versus US (54% vs. 22%), which also influenced the proportion of pediatric patients and cell dose. Moreover, the Taiwan CBB had most units transplanted outside of the USA, whereas the USA CBB shipped most of its products within the USA. Another difference was that a higher proportion of CB were washed post-thaw for the USA CBB because USA transplant centers were more likely to practice post-thaw washing. We also analyzed the benign and malignant groups separately. For malignant patients, unadjusted engraftment of ANC500 were 92±3% for USA (n=114) and 73±10% for Taiwan (n=22), while 1-year OS were 50±5% for USA (n=120) vs. 40±11% for Taiwan (n=23). For benign indications, unadjusted engraftment of ANC500 were 89±6% for USA (n=30) and 85±7% for Taiwan (n=27), while 1-year OS were 66±9% for USA (n=33) vs. 92±5% for Taiwan (n=27). To adjust for some of these differences, a multivariate analysis was performed adjusting for malignancy, TNC dose, CD34+ dose, #HLA matches, recipient age, recipient sex, and washed status of CBU. Collectively, these data suggest that most of the apparent differences in outcome between the two banks may be explained by factors such as % malignant indications, and use of post-thaw wash. Overall, after adjustment for the differences between transplants using CB from the two banks, outcomes were quite similar, suggesting that if SOPs and quality programs were followed rigorously, results can be reproducible for CBB at disparate locations.

Comparison of Cord Blood Transplant Outcome between U.S. Versus Non-U.S. Transplant Centers for Cord Blood from a Single Source.

Background: Cord blood transplantations (CBT) are performed in transplant centers (TC) worldwide; however, conditions for TC located around the world may be quite different, in particular, few studies have focused on CBT outcome in Asian TC. Factors such as patient populations, experiences, disease indications, available resources, and protocols may affect outcome as some have suggested.Methods: In an effort to compare CBT performed by U.S. (n=129) and non-U.S. TC (n=63), a retrospective audited analysis was performed for all 192 patients with available engraftment and/or survival data using cord blood units (CBU) from a single source to minimize potential differences in quality.Results: Kaplan-Meier estimates of engraftment for ANC500, platelet 20K and 50K were 91±3%, 80±5% and 74±5% for U.S. respectively, and 81±5%, 85±6%, and 81±7% for non-U.S. TC respectively. The relapse rate for the U.S. and non-U.S. TC were 26±5% and 17±5% respectively. TRM were 31±4% for U.S. TC and 22±5% for non-U.S. TC. The 3-year OS and DFS were 51±5% and 41±7% for U.S. and 68±6% and 61±7% for non-U.S. centers respectively. Factors including recipient age, CD34+ cell dose, #HLA matches, malignancy and wash status of the CBU were taken into consideration in a multivariate analysis which will be presented. In this study, non-U.S. TC were more likely to forego post-thaw wash of CB and perform CBT for benign indications, which appeared to affect outcome. Upon closer inspection of the individual TC, it appeared that there may be more heterogeneity in the TC results overseas, and that centers in the U.S. produced more consistent outcome. While it is possible that, due to many complex reasons, outcome at some non-U.S. TC may be somewhat uneven, many including several in Asia, produced outstanding results that rival the best TC anywhere.Conclusion: It appears that many factors, including available resources, post-thaw wash, and CBT experience affect outcome, and outstanding outcome can be achieved, regardless of TC location.