A “No-wash” Albumin-Dextran Dilution Strategy for Cord Blood Unit Thaw: High Rate of Engraftment and a Low Incidence of Serious Infusion Reactions

Abstract

Preparation of cord blood (CB) units for infusion by albumin-dextran dilution without centrifugation may be advantageous for adult patients to minimize cell loss and, unlike a bedside thaw, is still conducted in the controlled laboratory environment. Therefore, we studied CB transplantation (CBT) using this technique in 54 consecutive CBT recipients ≥20 kg. Patients [median age 42 years (range 7–66); median weight 71 kg (range 24–109)] were transplanted for high-risk hematological malignancies with ablative (n=35) or non-ablative (n=19) conditioning and 4–6/6 human leukocyte antigen (HLA)-matched double unit grafts. One hundred and seven units were thawed with dilution whereas 1 red blood cell (RBC) replete unit was washed. A 5:1 dextran 40/ 25% albumin solution was used. RBC depleted units (n=104) were diluted ≥5.5 fold [median final volume 200 ml (range 200–500)] whereas RBC replete units (n=3) were diluted ≥4 fold [median final volume 400 ml (range 400–535)]. Total nucleated cell (TNC) recovery was 86%; the median infused TNC dose was 2.17 × 10⁷/kg/unit. While 35 patients (65%) had a total of 45 infusion reactions (6 nausea, 31 hypertension, 3 pain, 1 rigors/fever, 2 transient hypoxia, 2 renal impairment) requiring additional therapy, there were no infusion-related serious adverse events, and reactions were not related to DMSO dose/kg. Cumulative incidence of sustained donor engraftment was 94% (95%CI: 87–100) with neutrophil recovery occurring at a median of 25 days (range 13–43) in ablative and 10 days (range 7–36) in non-myeloablative recipients. CB thaw with albumin-dextran dilution reduces unit manipulation, and minimizes cell loss, speeds time to infusion, is associated with a tolerable infusion reaction profile, and a high rate of sustained engraftment in CBT recipients ≥20 kg.

Introduction

Rubinstein et al originally described the preparation of cord blood (CB) units for infusion using albumin-dextran dilution with centrifugation (“wash”) to remove dimethyl sulfoxide (DMSO) and optimize the viability of the thawed product¹. This strategy was initially used for the transplantation of small children but has been widely adopted in adult CB transplantation (CBT) as a matter of convention. While DMSO infusion can be associated with infusion reactions,² adult and larger pediatric autologous transplant recipients can tolerate small amounts of DMSO without clinically significant toxicity. Therefore, an alternative method to prepare CB for infusion originally proposed by the St. Louis Cord Blood Bank³ is to perform an albumin-dextran dilution without centrifugation. Such an approach could be advantageous for adults and larger children as cell loss is minimized⁴ and time to infusion is shortened, both desirable for a cryopreserved graft. Finally, unlike bedside thaw⁵, this technique is still conducted in the controlled environment of the Cytotherapy Laboratory where samples can be immediately taken to analyze cell recoveries and unit quality. Therefore, we conducted a prospective study of albumin-dextran dilution after CB thaw in 54 consecutive recipients of double unit CBT. Our hypothesis was that preparation of units for infusion with the “no wash” technique would be associated with tolerable infusion reactions and a high rate of sustained donor engraftment.

Materials and Methods

Patients, Conditioning, and CB Grafts

Recipients of first allograft who were ≥20 kg were eligible. Table 1 summarizes characteristics of patients, preparative regimens, and grafts. Fifty-four consecutive patients [median age 42 years (range 7–66), median weight 71 kg (range 24–109)] were transplanted for high-risk hematological malignancies with myeloablative (n=35) or non-myeloablative (n=19) conditioning according to their age, extent of prior therapy, co-morbidities, and diagnosis. All received immune suppression with cyclosporine-A (CSA) and mycophenolate mofetil (MMF). CSA was continued until at least day 100 and then tapered in the absence of graft-versus-host disease (GVHD), whereas MMF was continued until day 45 and then either stopped or tapered according to protocol. All patients received post-transplant granulocyte colony stimulating factor (G-CSF).

Table 1.

Characteristics of 54 double unit CBT recipients and grafts.

Gender
Male	30 (56%)
Female	24 (44%)
Age (years)
Median (range)	42 (7–66)
Weight (kg)
Median (range)	71 (24–109)
Diagnosis
Acute leukemia	22
Myelodysplasia	1
Non-Hodgkins lymphoma	20
Hodgkins disease	11
Myeloablative Conditioning
Cy 120/ Flu 75/ TBI 1320–1375	18
Mel 140/ Flu 150	7
Cy 50/ Thio 10/ Flu 150/ TBI 400	8
Flu 125/ Thio 10/ TBI 1375	2
Non-myeloablative Conditioning
Cy 50/ Flu 150/ TBI 200	19
Prior Autologous Transplant	12
HLA-A,-B antigen, -DRB1 allele Match to Patient (n=108 units)
6/6	5
5/6	58
4/6	45
Infused TNC × 107/kg
Median (range) of larger unit (n=54)	2.52 (1.42–5.56)
Median (range) of smaller unit (n=54)	1.94 (0.92–4.47)

Abbreviations: Cy = Cyclophosphamide; Flu = Fludarabine; TBI = Total body irradiation; Mel = Melphalan, Thio = Thiotepa; HLA = Human leukocyte antigen; TNC = Total nucleated cell

Double unit grafts were used to augment engraftment^6–8. Units were selected primarily according to total nucleated cell (TNC) dose, human leukocyte antigen (HLA)-match, and bank of origin, and obtained from domestic (n=78) or international (n=30) banks. The majority (n=104) had undergone at least partial red blood cell (RBC) depletion prior to cryopreservation (“RBC depleted”) whereas 3 were only plasma depleted (“RBC replete”). To reduce the amount of cellular debris infused, only one RBC replete unit was permitted per double unit graft. Units were 6/6 (n=5), 5/6 (n=58), and 4/6 (n=45) HLA-A, −B antigen, −DRB1 allele matched to the recipient. Patients were transplanted between February 2006 and October 2008 and all patients or their guardians signed informed consent prior to transplantation.

Preparation of CB Units for Infusion

All units were stored in LN freezers upon arrival at −180° C. One hundred and seven units underwent albumin-dextran dilution after thaw whereas one RBC replete unit was washed prior to inclusion of such units in the study. Each unit of a double unit graft was handled separately to avoid potential mix-up. Units were placed in a zip-lock bag, thawed in a 37°C waterbath, and then transferred to a biosafety cabinet. A stock solution with a 5:1 ratio of 10% dextran 40 (molecular weight 40,000, Hospira, IL, USA) and 25% human serum albumin (CSL Behring, IL, USA) was prepared at room temperature. RBC depleted units were diluted at least 5.5 fold and RBC replete units at least 4 times their original volume with the standard dilution for units with a cryopreserved volume of 25 ml being 8 fold (final volume 200 ml). The required quantity of diluent was calculated and aliquoted into a 300 or 600 ml transfer pack for units in a single compartment bag or a cell wash infusion bag (MedSep double compartment units). A small volume of diluent was reserved to rinse bags as needed.

For single compartment RBC depleted units the cryobag was spiked with the single line of tubing and coupler of the transfer pack. For RBC replete units double spiked transfer sets were used to connect the bag with the diluent due to their increased viscosity and the risk of tubing blockage. For MedSep units the double spiked end of the cell wash infusion bag was used to spike the large and small compartments ensuring there were no leaks. For all units an amount of diluent approximately equal to the cryopreserved volume of the unit was slowly transferred to the cryobag. Once this initial 1:1 dilution was achieved the tubing was clamped. Thorough mixing was performed and the product was allowed to equilibrate for 1–2 minutes. It was then drained back into the transfer pack or cell wash infusion bag. Units frozen in two bags were combined before samples were taken.

CB Unit Assessment

A small aliquot was removed from the final cell mixture for TNC count, ABO blood group, Gram stain and culture, and assays of unit quality. Unit quality was evaluated by trypan blue exclusion, flow cytometric assay of CD34+ and CD3+ viability using 7-amino-actinomycin D, and colony-forming unit (CFU) assays. The original cryobag was sent to the Diagnostic Molecular Pathology Laboratory for DNA extraction and assessment of donor polymorphisms. TNC count per kilogram (kg) was calculated as: TNC count/ml x the volume of the final product divided by the patient’s weight. The infused viable CD34+ dose/kg was calculated as: TNC × CD34+ viability x percentage of CD34+ cells of the total sample divided by the patient’s weight. CFU assays were performed using a total of 1 × 10⁵ cells plated in duplicate and colony growth was evaluated by light microscopy at 14 days.

Management of CB Infusion and Infusion-Related Adverse Reactions

All patients received pre-medication with oral acetaminophen and intravenous diphenhydramine (or hydroxyzine); most also received lorazepam. In addition, patients received high volume hydration 4–6 hours pre-transplant and 12–24 hours post-transplant. CB units were infused consecutively with infusion of the first unit starting within two hours of thaw. The median interval between infusion of the first and second unit was 10 minutes (range 0–100 minutes). Experienced transplant nurses were present during infusion of both units and patients were closely monitored: vital signs were recorded before each unit infusion, every 15 minutes during infusion, immediately after each unit, and then every 15 minutes x 2, every 30 minutes × 2, and every 60 minutes × 4. Emergency medications including intravenous lorazepam and hydralazine were at the bedside for immediate administration in the event of nausea, hypertension, or other reactions.

A serious infusion reaction was defined as any life-threatening event related to CB infusion such as: anaphylaxis; cardiac, pulmonary, or acute renal failure; seizure; transfer to intensive care unit; or death within 48 hours of CB infusion. Hypoxia was defined as oxygen saturation of <94%. Hypertension was defined as a systolic pressure of ≥160 and/or a diastolic pressure of ≥90 mm/Hg, and bradycardia was a heart rate of <50 beats/minute. Renal insufficiency was defined as a 1.5 fold increase in serum creatinine over the day zero baseline and greater than the upper limit of normal for age.

The amount of DMSO per unit was estimated based on the cryopreservation volume of the unit and the concentration of DMSO reported by the CB bank. When no concentration was reported, the banking standard of 10% DMSO (v/v) in the final volume of the cryopreserved product was used. The amount of DMSO infused was calculated as the total amount of DMSO contained in the two units of each patient’s graft, divided by the patient’s body weight.

Assessment of Donor Engraftment and GVHD

Time to neutrophil recovery was defined as the first of 3 consecutive days with an absolute neutrophil count (ANC) ≥0.5 × 10⁹/l. Time to platelet recovery was defined as the first of 3 consecutive days at ≥50 × 10⁹/l and at least 7 days without platelet transfusion support. Sustained donor engraftment was defined as sustained donor-derived count recovery with donor chimerism of at least 90% (both units combined). Donor chimerism was determined serially on bone marrow and/ or peripheral blood at days 21, 28, 60, 100, 180, and 360 after transplantation by quantitative polymerase chain reaction of informative polymorphic short tandem repeat (STR) regions of DNA from each donor units and the recipient using a multiplex kit (GenePrint Fluorescent STR Marker Kit, Promega, WI, USA)⁷. The engrafting unit contributed >50% of the total donor chimerism in serial testing. Patients were evaluated weekly for development and extent of GVHD. Overall staging of acute GVHD was based on IBMTR criteria⁹.

Statistical Analysis

The probability of neutrophil and platelet engraftment, acute GVHD, and TRM were computed using the cumulative incidence function. For neutrophil engraftment early death was the competing risk, whereas death or graft failure were the competing risks for acute GVHD. Relapse was the competing event for TRM. Overall and disease-free survival (DFS) were calculated using Kaplan-Meier methodology.

Results

Cell Recoveries

RBC depleted units (n=104) had a median cryopreserved volume of 25 ml (range 21–65) and were diluted ≥5.5 fold [median final volume 200 ml (range 200–500)]. The RBC replete units (n=3) had a larger median cryopreserved volume of 90 ml (range 89–95) and were diluted ≥4 fold [median final volume 400 ml (range 400–535)]. Cell doses and recoveries are summarized in Table 2.

Table 2.

Post-thaw recoveries of CB units thawed with dilution (n=107^*).

	Median or % (Range)
Pre-thaw TNC × 107/kg	2.66 (1.06–6.87)
Post-thaw TNC × 107/kg	2.27 (0.92–5.85)
% TNC Recovery	86% (50–121%)
% CD34+ Viability	90% (34–98%)
Post-thaw CD34+ × 105/kg/unit**	0.90 (0.13–3.69)
Post-thaw CFU-GM × 104/kg/unit	1.25 (0.00–5.79)

^*The single washed unit is excluded.

^**The infused dose of viable CD34+ cells.

Infusion Reactions

There were no serious adverse events related to infusion. Nineteen patients (35%) had no reactions. Thirty-five patients (65%) had a total of 45 reactions requiring additional therapy. Six patients had nausea treated with additional intravenous lorazepam. Thirty-one patients had hypertension treated with intravenous hydralazine and/or furosemide. Three patients had chest or abdominal pain treated with a small dose of intravenous narcotic, and one had rigors/ fever treated with intravenous meperidine. Two patients had transient hypoxia treated with supplemental oxygen by nasal cannula. Two patients had transient renal insufficiency: one recipient of two RBC depleted units had a serum creatinine peak of 1.6 mg/dL which returned to baseline by day 7 post-transplant, and one recipient of a RBC depleted and RBC replete unit had a serum creatinine peak of 2.7 mg/dL which returned to baseline by day 22 post-transplant. These two patients were managed by extending intravenous hydration and other supportive measures. Finally, 3 patients had transient asymptomatic bradycardia not requiring therapy. In one patient the infusion was stopped and the bradycardia spontaneously resolved after 19 minutes; the infusion was then completed uneventfully. In the other 2 patients the infusion was continued and the bradycardia resolved within 25 and 60 minutes of completion of the second unit, respectively.

Overall, of the 35 patients with infusion reactions, 31 received RBC depleted units, 3 received one RBC depleted and one RBC replete unit, and 1 received a RBC depleted and a washed unit. Further, 21 (60%) patients reacted during the first unit, and 14 (40%) reacted during the second unit. Nausea, hypertension, pain, or hypoxia occurred during either the first or second unit, whereas all 3 episodes of bradycardia occurred during the second unit. There was no relationship between the timing of administration of pre-medications and the occurrence of infusion reactions (data not shown). The mean total dose of DMSO administered with the two units was 0.117 g/kg (range: 0.05–0.28), and there was no relationship between the DMSO dose and infusion reactions except at the highest dose level where all 4 patients had a reaction (Figure 1).

Figure 1. Distribution of infused DMSO doses/kg with double unit CB grafts prepared with albumin dilution and incidence of infusion reactions in 53 patients.

To most accurately represent the DMSO dose with two unwashed units the single patient who received one washed unit is excluded from this analysis.

Transplant Outcomes

Three patients (2 recipients of myeloablative and 1 of non-myeloablative conditioning) had graft failure. Thus, the cumulative incidence (CI) of sustained donor engraftment was 94% by day 50 (95%CI: 87–100) with neutrophil recovery to ≥0.5 × 10⁹/l occurring at a median of 25 days (range 13–43) in myeloablative and 10 days (range 7–36) in non-myeloablative CBT recipients (Figure 2A). The CI of platelet recovery to ≥50 × 10⁹/l was 80% at day 180 (95%CI: 68–92) overall, and occurred at a median of 50 days (range 35–182) in recipients of ablative and 38 days (range 24–59) in non-myeloablative conditioning (Figure 2B). Of the 51 patients with sustained donor derived hematopoiesis, 50 engrafted with a single unit [median infused TNC 2.21 × 10⁷/kg (range 1.32–5.56) and median infused CD34+ dose 0.90 × 10⁵/kg (range 0.13–3.69)], whereas a single patient had sustained engraftment of both units.

Figure 2. Cumulative incidence of sustained neutrophil engraftment (2A) and platelet engraftment (2B) after CBT with double unit grafts prepared with albumin-dextran dilution.

Recipients of ablative and non-myeloablative conditioning are shown separately. While early recovery in recipients of non-myeloablative conditioning was due to transient autologous recovery all engrafting patients shown converted to sustained donor-derived hematopoiesis.

The CI of day 100 grade II–IV acute GVHD was 44% (95%CI: 30–58). Nine patients had grade II, 13 had grade III, and one had grade IV aGVHD. Eleven of 43 (26%) of evaluable patients have either late acute or chronic GVHD to date. With a median follow-up of 11 months, 14 patients have died of transplant-related causes and 10 patients have relapsed. Thus, the one year overall survival is 65% (95%CI: 51–79) and the one year disease-free survival is 56% (95%CI: 42–70) (Figure 3).

Figure 3. Kaplan-Meier probabilities of overall survival and disease-free survival after CBT with double unit grafts prepared with albumin-dextran dilution.

Discussion

The methodology of preparation of cryopreserved CB units for infusion was originally described by Rubinstein and colleagues in 1995¹. Notably, while the authors recommended thaw with removal of the cryoprotectant by centrifugation (“wash”), they emphasized that (p10119): “Progenitor cell viability is measurably decreased when thawed cells, still suspended in hypertonic cryopreservative solutions, are rapidly mixed with large volumes of isotonic solutions or plasma. The osmotic damage inflicted by the severe solute concentration gradient, however, can be averted by a simple 2-fold dilution after thawing, providing almost total recovery of viable hematopoietic progenitor cells.”¹ This suggests that the most important event in the thaw of cryopreserved CB may be the initial dilution when the first osmotic shifts occur. We hypothesized that for larger CBT recipients the centrifugation step may therefore not be necessary. Dilution alone is an appealing simplification of the thaw technique given that centrifugation can result in cell loss especially if the interface is not clear, and can lead to increased unit manipulation especially if clumping necessitates filtration of the product. While there may be concern that DMSO could be toxic to the hematopoietic stem cells (HSC)¹⁰ we speculated that this could be mitigated by adequate product dilution as well as prompt patient infusion. We also reasoned that clinical DMSO toxicity would be tolerable since similar amounts of DMSO are routinely administered to patients undergoing autologous transplantation². Therefore, we investigated a dilution only strategy for the preparation of CB units for infusion. A lower limit of 20 kg patient weight was chosen for inclusion in this study given the risk of DMSO infusion may be higher in smaller patients, and these patients may not tolerate the large volumes of the diluted products. Furthermore, young children usually have higher cell dose products and the potential cell loss that may occur with a wash may be acceptable.

We found that the amount of DMSO estimated to have been infused with the double unit grafts (range 0.05–0.28 g/kg of recipient weight) compared favorably to that infused with autologous products². Further, infusion of unwashed CB was relatively well tolerated even with the use of double unit grafts. It has previously been reported that 14 of 23 (61%) recipients of washed double unit grafts had no symptoms associated with infusion⁷. While this incidence appears lower than that of infusion reactions we observed with the “no wash” dilution, a larger study comparing the two techniques with uniform criteria for defining a reaction would be required to truly reflect the differences between these strategies. Further, we found that the infusion reactions were safely managed with the appropriate supportive care. Importantly, units were infused with close nursing and medical supervision which is mandatory in the care of patients receiving these products. Pre-medication and prompt administration of additional medications to treat nausea, hypertension, or other reactions during the infusion is needed to ensure the safety of this approach as well as patient comfort. In addition, although only two patients in the group of 54 had renal insufficiency after the infusion, clinicians should be aware of the potential for nephrotoxicity of the unwashed grafts which is presumably due to free hemoglobin and cellular debris. We recommend hydration with crystalloids at a minimum of 2 ml/kg/hour six hours pre-CBT and a minimum of 24 hours after completion of the infusion. If a rise in creatinine is observed, aggressive hydration should be continued.

Whether our “no wash” technique is an acceptable alternative for units that are not RBC depleted is difficult to determine based on only 3 units. It is notable that all 3 recipients of RBC replete units reacted to infusion including one patient with a significant rise in creatinine to 2.7 mg/dL. Dilution of these units may be more appealing than a bedside thaw given that it is performed in the controlled laboratory environment. Moreover, avoiding centrifugation may also be an advantage, since washing of RBC replete units is associated with inferior engraftment¹¹. However, as the “no wash” method does not remove RBC debris and free hemoglobin, hydration and close monitoring with infusion of these units is extremely important, and the study of a larger number of patients is needed before it could be concluded that the “no wash” method of preparation for RBC replete units is safe.

In addition to the tolerable infusion reaction profile a high rate of sustained donor engraftment was observed in this study. This is likely contributed to by all patients having hematologic malignancies (reducing the risk of rejection as compared to the transplantation of non-malignant disorders), the use of highly immunosuppressive preparative regimens, and the use of double unit grafts. However, the finding of a 94% incidence of sustained donor engraftment suggests that DMSO does not have substantial toxicity to hematopoietic progenitors, at least as practiced with our methodology. Therefore, hematopoietic DMSO toxicity cannot be used as an argument to substantiate the practice of CB thaw with wash.

A further observation of this study was a lower rate of acute GVHD than that previously reported with double unit CBT in similar studies using washed products. For example, incidences of 65% ⁷ and 59%⁸ grade II–IV acute GVHD have been reported in similar studies of ablative and non-myeloablative CBT grafts, respectively. While this observation would require a controlled trial to confirm, one could postulate that the infusion of cells or soluble factors usually removed in the washed product could mediate an immunosuppressive effect and thus possibly account for a modest reduction in the incidence of acute GVHD.

Finally, as compared to the traditional wash, albumin-dextran dilution offers a number of additional advantages. The reduction in technician time may translate to cost savings for the Cytotherapy Laboratory. This is especially relevant given the additional time required for the preparation of double unit grafts for infusion. Further, this technique may assist in a more accurate evaluation of unit quality. We perform a flow cytometric assay to determine the viability of the CD34+ cells in the thawed product using 7-amino-actinomycin D staining¹². By evaluating the unit without wash no dead cells are removed. Therefore, on the day of transplant we can ascertain the true percentage of live, viable cells in the thawed units of the those originally present, and therefore ensure that a graft containing at least one unit with a high percentage of viable cells and thus adequate quality has been transplanted¹².

In summary, the albumin-dextran dilution of CB units is associated with a tolerable infusion reaction profile, a high rate of engraftment, and minimal manipulation of the unit in the Laboratory. It may therefore be the method of choice for CBT in adults and larger children ≥ 20kg.