Co-Infusion of Umbilical Cord Blood and Haploidentical CD34+ Cells Following Nonmyeloablative Conditioning as Treatment for Severe Aplastic Anemia and MDS Associated With Severe Neutropenia Refractory to Immunosuppressive Therapy
Phase: PHASE2
Conditions: Myelodysplastic Syndrome (MDS) With Refractory Anemia (RA), Severe Aplastic Anemia (SAA)
Sponsor: National Heart, Lung, and Blood Institute (NHLBI)

 
 
08-H-0046 
Richard Childs, M.D. 
May 13, 2019 (Amendment OO)  
1
CLINICAL RESEARCH PROJECT 
 
 
 
 
Protocol #08-H-0046  
 
 
 
 
 
 
 
 
IND 13,500  
 
 
Product: Miltenyi CliniMACS® CD34 Reagent System + Unrelated Cord Blood Transplant 
IND Sponsor-Investigator: Richard W. Childs, M.D. 
 
Date:     May 13, 2019 
 
Title: Co-Infusion of umbilical cord blood and haploidentical CD34+ cells following nonmyeloablative 
conditioning as treatment for severe aplastic anemia and MDS associated with severe neutropenia 
refractory to immunosuppressive therapy  
 
Other Identifying Words:  Severe Aplastic Anemia (SAA), umbilical cord blood, haploidentical, 
nonmyeloablative 
 
Principal Investigator:    
*Richard W. Childs, MD, CMTB, NHLBI (E) 
 
 
 
594-8008 
Bldg 10, CRC 3-5332 
 
Medically Responsible and Accountable Investigator:  
Richard W. Childs, MD, CMTB, NHLBI (E) 
 
 
 
594-8008 
Bldg 10, CRC 3-5332 
 
Associate Investigators:  
Neal S. Young, MD, HB, NHLBI (E)     
496-5093 
Bldg 10, CRC 3-5142 
Charles Bolan, MD, NHLBI, (E) 
 
 
 
 
   496-4506 
Bldg 10, 1C711 
Ramaprasad Srinivasan, MD, UOB, NCI (E) 
594-8007 
Bldg 10, CRC 3-5272 
Jennifer Wilder, RN, Umbilical Cord Transplant Coordinator, ETIB,NCI(C)451-3722 
Bldg 10 CRC 3-3121 
Lisa Cook, RN, NHLBI/OCD (E) 
402-5609 
Bldg 10, CRC 3-3485 
Xin Tian, PhD, Biostatistician, OBR/NHLBI (E) 
435-1298 
RKL2  Room 9208  
Nancy Geller, PhD, Director, Office of Biostatistics, NHLBI (E)   
435-0434 
RKL2, 9202 
Margaret Bevans, RN, PhD, AOCN, Clinical Nurse Scientist, CC (E) 
402-9383 
Bldg. 10, 2B13 
*Georg Aue, MD, CMTB, NHLBI (E) 
 
 
451-7141 
Bldg 10, CRC 3-3216 
Brian Wells, NHLBI/OCD (E) 
827-0035  
Bldg 10, CRC 3-5140 
Tatyana Worthy, RN, NHLBI/OCD (E) 
  
 
594-8013 
Bldg 10, CRC 3-3485 
Jeffrey Cohen, MD, NIAID (E) 
  
 
496-5265 
Bldg 50, Rm 6134 
Steven Highfill, Ph.D, CC (E)  
  
 
435-4801 
Bldg. 10 RM 3C720 
Kevin Camphausen, MD, NCI (E) 
 
 
 
 
   496-5457 
Bldg. 10 B2 3675 
Ryan Jones, MD, Walter Reed Medical Center (V)                                        402-2399       Bldg. 10 CRC 4-5140 
Rosa Nadal Rios, MD, CMTB, NHLBI (E)                                                    451-7128       Bldg. 10 CRC 3-5330 
Kristen Wood, RN, NHLBI/OCD(E)                                                              827-2977       Bldg. 10 CRC 3-3485 
 
 
 
 
    
* Investigators who may obtain informed consent 
 
Collaborators:  
Christopher Buck, Ph.D, NCI (E) 
A. Gretchen Buckler, M.D., M.P.H., CC (E) 
Meryl Waldman, M.D., NIDDK (E) 
 
 
 
 
 

 
 
08-H-0046 
Richard Childs, M.D. 
May 13, 2019 (Amendment OO)  
2
 
Subjects of Study: 
 
Number 
 
 
 
Sex 
 
Age range 
Subjects: 
  
    
Recipients: 40  
 
 
either  
Recipients: 4-55  
 
 
 
 
Donors: Up to 60 
 
 
 
 
inclusive 
 
 
 
 
 
 
 
 
 
 
 
Donors: 4-75 inclusive 
Project involves ionizing radiation?  
Yes (medically indicated) 
Off site project? 
 
 
No 
Multi-institutional project? 
 
No 
DSMB involved? 
 
 
Yes 
  
Amendment Number:  41 (OO)

 
 
08-H-0046 
Richard Childs, M.D. 
May 13, 2019 (Amendment OO)  
3
PRECIS     
 
Severe aplastic anemia (SAA) and myelodysplastic syndrome (MDS) are life-threatening bone marrow 
disorders. For SAA patients, long term survival can be achieved with immunosuppressive treatment. 
However, of those patients treated with immunosuppressive therapy, one quarter to one third will not 
respond, and about 50% of responders will relapse.  
 
Allogeneic bone marrow transplantation from either HLA-matched sibling or matched unrelated donor 
cures about 70% of patients with SAA and 30-60% of patients with MDS. Unfortunately, most patients 
with these disorders are not suitable candidates for hematopoietic stem cell transplantation (HSCT) due to 
advanced age or lack of a histocompatible donor. For such patients, transplantation using unrelated cord 
blood (UCB) has been shown to be a reasonable alternative transplant strategy. The advantage to UCB 
transplant is the ease and rapidity of availability, requirement of less than perfect HLA match, and lower 
rates of graft versus host disease compared to mismatched bone marrow or peripheral blood stem cell 
transplants.  The major disadvantage of UCB transplantation in adults is the limited number of nucleated 
cells contained within the cord unit resulting in prolonged neutropenia and failure of engraftment which 
contributes to infection and transplant related mortality (TRM). In order to harness the advantage of UCB 
availability and to overcome the disadvantage of delayed neutrophil recovery, we propose to test whether 
co-administration of unrelated umbilical cord blood and a relatively low number of highly purified 
haploidentical peripheral blood CD34+ cells from a related donor might promote rapid engraftment and 
reduce TRM secondary to prolonged neutropenia associated with conventional UCBT. 
 
This research protocol is therefore designed to evaluate the safety and effectiveness of co-infusion of 
unrelated umbilical cord blood and haploidentical CD34+ cells from a related donor following non-
myeloablative conditioning for neutropenic patients with SAA or MDS with refactory anemia (RA) that 
has proven to be refractory to medical therapy.  Subjects will receive a novel non-myeloablative 
immunosuppressive conditioning regimen of cyclophosphamide, fludarabine, horse ATG and one dose of 
total body irradiation (200cGy) followed by an infusion of the allografts. The haploidentical stem cell 
product will be T-cell depleted and enriched for CD34+ cells using the Miltenyi CliniMacs system. To 
reduce TRM secondary to prolonged neutropenia associated with conventional UCB transplantation, 
haploidentical CD34+ stem cells will be co-infused with a single UCB unit (serologically matched at t 
4/6 HLA loci).   
 
The primary endpoint is donor engraftment by day 42 (defined as an ANC of >500 from either the haplo 
donor, the cord, or both combined).  Secondary endpoints will include standard transplant outcome 
variables such as non-hematological toxicities, incidence and severity of acute and chronic GVHD, and 
relapse of disease. We will also evaluate ANC recovery (ANC > 500 cells/Pl) at day 22,  and 100 day and 
200 day treatment related mortality (TRM) of this novel transplant approach.  Health related quality of 
life will also be assessed pre-transplant, 30 and 100 days post-transplant, and every 6 months until 5 years 
post-transplant. 
 
 
 
 
 
 
 
 
 
 
 

 
 
08-H-0046 
Richard Childs, M.D. 
May 13, 2019 (Amendment OO)  
4
TABLE OF CONTENTS 
 
1.0  
Objectives 
 
 
 
 
 
 
   
 
  
2.0  
Background 
3.0 
Scientific and Clinical Justification 
4.0 
Study Design 
5.0 
Eligibility Assessment 
6.0 
Clinical Evaluation of the Recipient 
7.0 
Treatment Plan 
8.0 
Donor Evaluation, Stem Cell Collection and Processing Plan 
9.0 
Umbilical Cord Blood Processing 
10.0 
Management of Complications 
11.0 
Ancillary Laboratory Research Studies 
12.0 
Biostatistical Considerations 
13.0 
Data Safety and Monitoring  
14.0 
Human Subject Protections 
15.0 
Pharmaceuticals 
16.0 
References 
 
 
 
 
 
 
 
 
 
 
APPENDICES 
 
 
 
 
 
 
 
 
A. 
IRB Approved NHLBI Laboratory Research Studies 
B. 
Grading of GVHD 
 
 
 
 
 
 
 
 
SEE ALSO THE SUPPORTIVE CARE GUIDELINES NHLBI, 
 IRB reviewed 10/2003, 6/2006, and 1/2011

 
 
08-H-0046 
Richard Childs, M.D. 
May 13, 2019 (Amendment OO)  
5
1.0  
OBJECTIVES   
 
1.1 Primary objective:  
x 
To evaluate the potential of this modality to achieve donor engraftment in >80% of 
recipients by day 42 (defined as an ANC of >500 from either the haplo donor, the cord, 
or both combined). 
 
1.2 Secondary objectives:  
x 
To evaluate the day 22 ANC recovery rate (ANC > 500 cells/Pl)  
x 
To evaluate the safety of this novel transplant regimen (non-hematologic toxicities)  
x 
To evaluate day 100 and 200 TRM 
x 
To determine the incidence and severity of acute and chronic graft-versus-host-disease 
(cGVHD) following this transplant regimen 
x 
To evaluate the effect of a reduced conditioning transplant regimen on quality of life. 
 
2.0 BACKGROUND 
 
2.1 
Aplastic Anemia (AA) 
 
2.1.1 Pathophysiology 
 
Aplastic anemia is a disease characterized by pancytopenia and a hypocellular bone marrow.  
Although the exact etiology of aplastic anemia is not known, clinical experiences and laboratory 
data suggest that the ultimate mechanism leading to development of bone marrow failure is 
immune-mediated. [1, 2]  Suppression of hematopoiesis in aplastic anemia is likely secondary to 
specific populations of effector T-cells.  Activated cytotoxic T-cells expressing HLA-DR, the IL-2 
receptor, and IFN-J are elevated in aplastic anemia patients and localized in the bone marrow.[3-5]  
Hematopoietic progenitor and stem cells are targets for the immune attack by these cells. The 
effects exerted by cytotoxic T-lymphocytes are at least in part due to Fas ligand-induced apoptosis 
of hematopoietic progenitor cells; IFN-γ, in addition to its intrinsic inhibitory activity on 
hematopoietic progenitor and stem cells, can induce over-expression of Fas on target cells.[6, 7] 
Despite its often acute presentation, aplastic anemia is now being recognized as a chronic disease 
with frequent flares of the immune process and the need for long-term immunosuppression. High 
resolution Vβ CDR3 analysis in patients with aplastic anemia shows significantly increased 
nonrandom skewing of the Vβ-chain families of the T cell receptor, suggestive of disease specific 
clonal expansion.[8] 
 
 
2.1.2  Clinical consequences of aplastic anemia  
 
Symptoms derive from low blood counts. Anemia leads to fatigue, weakness, lassitude, 
headaches, and in older patients dyspnea and chest pain. These manifestations are most 
commonly responsible for the clinical presentation. Thrombocytopenia produces mucosal 
bleeding.  Petechiae of the skin and mucous membranes, epistaxis, and gum bleeding are frequent 
and early complaints.  Bleeding can be brisk in the presence of accompanying physical lesions, as 
in gastritis and fungal infection of the lungs.  The most feared complication of thrombocytopenia 
is intracranial hemorrhage. Bacterial and fungal infections in the setting of neutropenia are a 
major cause of morbidity and mortality, and are most often the causes of death in refractory 
cytopenias. 
 
 

 
 
08-H-0046 
Richard Childs, M.D. 
May 13, 2019 (Amendment OO)  
6
2.1.3  Treatment options for aplastic anemia 
 
x 
Horse antithymocyte globulin (ATGAM; h-ATG) is currently approved for the treatment 
of aplastic anemia by the Food and Drug Administration.  The mechanism by which h-ATG 
improves bone marrow failure in aplastic anemia is not fully known.  While it is generally 
believed that h-ATG administration leads to depletion of immune competent cells, its exact 
mechanism of action remains unclear. h-ATG preparations contain a variety of antibodies 
recognizing human T-cell epitopes, many directed against activated T-cells or activation 
antigens.[9, 10] After treatment with h-ATG, circulating levels of lymphocytes drop to 10% 
pretreatment level through a variety of mechanisms: Fc receptor complement-dependent lysis, 
opsonization and phagocytosis by macrophages; and immunomodulation leading to long-term 
depletion via antibody dependent cell-mediated cytotoxicity and activation-induced 
apoptosis.  Although the decline in circulating levels of lymphocytes is transient, the number 
of activated T -cells is decreased for more prolonged periods of time; this effect is also 
reflected in decreased IFN-γ and possibly TNF production after h-ATG.[11, 12] Tissue 
culture preparations of peripheral blood lymphocytes treated with h-ATG produce 
hematopoietic colony stimulating factors, suggesting a possible stimulatory role in vivo.  h-
ATG binds to numerous other cell types in addition to lymphocytes, including cells of the 
bone marrow.[13-15] The response to h-ATG may be mediated by circulating factors 
produced in this immunologically activated state, although response to h-ATG has not 
correlated with severity or presence of clinical serum sickness.  
 
x 
Rabbit ATG (Thymoglobulin®; r-ATG) is similar to h-ATG, except that gamma immune 
globulin is obtained by immunization of rabbits with human thymocytes.  r-ATG contains 
cytotoxic antibodies directed against antigens expressed on T lymphocytes.  r-ATG has been 
commercially available in Europe since 1985 and is currently licensed in more than 44 
countries and has been used in more than 44,000 patients worldwide. In an Italian multi-
center study, 30 aplastic anemia patients who failed h-ATG plus CsA received second line 
treatment with r-ATG and cyclosporine.[16]Overall response, defined as transfusion-
independence was achieved in 23/30 (77%) patients after a median of 95 days, and overall 
survival was 93% with a median follow up of 914 days, with no patient having relapsed at 
time of publication.  In our limited recent NIH experience, employing r-ATG as standard 
treatment in patients who had failed h-ATG, response rates have been less impressive: three 
partial responses; two late responses occurring more than six months after treatment and 
therefore of uncertain relationship to therapy; and nine patients without a response; one 
patient died of complications of pancytopenia soon after r-ATG treatment.   Initial treatment 
with r-ATG has also been evaluated. Stein et al used r-ATG in combination with prednisone 
± cyclosporine ± androgens in 57 patients with aplastic anemia, 30 being severe and 27 
moderate.[17] Response occurred in 16 cases (28%), being more frequent in females, those 
with less than 6 months duration and with severe disease. Eight patients had previously 
received r-ATG and 2 (25%) responded.  
 
x 
Cyclosporine (CsA) is a major immunosuppressive drug and probably secondary only to 
corticosteroids in world wide popularity in this role. In addition to its longstanding use in 
bone marrow and solid organ transplant recipients, CsA has been widely employed as an 
immunosuppressive drug in many autoimmune diseases. In contrast to ATG, CsA has a very 
selective inhibitory effect on T lymphocytes, suppressing early cellular response to antigenic 
and regulatory stimuli. By blocking expression of nuclear regulatory proteins, it leads to 
reduced T cell proliferation and activation with diminished release of cytokines such as 
interleukin-2 and interferon-J. The addition of CsA to ATG has improved response rates to 

 
 
08-H-0046 
Richard Childs, M.D. 
May 13, 2019 (Amendment OO)  
7
70-80% and the 5 year survival in responding patients to 80-90%.[18, 19] CsA binds to 
intracellular receptors termed immunophilins, inhibiting in turn the activity of calcineurin, 
which results in the blocking of interleukin-2 production and T cell activation and 
proliferation. In vivo, CsA inhibits the release of IL-2 from activated T-cells and 
consequently decreases T cell proliferation.[20-22] 
 
x 
Alternative immunosuppressive agents have been used with debated success. At Johns 
Hopkins, high dose cyclophosphamide without stem cell rescue produced hematologic 
responses in SAA patients similar to those seen with ATG combined with CsA, with no 
relapse or evolution to PNH or myelodysplasia being observed. [23] However, a prospective 
randomized trial conducted at the NHLBI, which compared ATG and cyclosporine to 
cyclophosphamide and cyclosporine, was terminated prematurely due to excessive toxicity, 
severe fungal infections and deaths in the group that received cyclophosphamide.[24] In 
contrast to the Hopkins experience, some of our patients relapsed or developed cytogenetic 
abnormalities. [25]  The explanation for the increased toxicity seen in the cyclophosphamide 
treated patients is the serious immunosuppression and resulting neutropenia.  The NIH is 
currently investigating the efficacy of alemtuzumab (Campath®) in subjects with MDS and 
SAA who have received no prior ATG based immunosuppressive therapy, in subjects who 
are refractory or had a suboptimal response to a course of horse ATG-based 
immunosuppression, and in subjects who have responded and relapsed following a prior 
course of ATG or are refractory to prior courses of horse and rabbit ATG. 
 
2.1.4  Transplant options for patients with Aplastic anemia 
 
x 
HLA- identical related donor transplantation for patients with SAA:  Allogeneic bone 
marrow transplantation from a histocompatible matched sibling is curative therapy in the 
majority of aplastic anemia patients who undergo the procedure.[26-29]   Survival rates with 
allogeneic hematopoietic stem cell transplantation from a histocompatible sibling have been 
reported to be as high as 90% from a single institution and approximately 70% for registry 
data, which more likely reflects the general experience.[30, 31] 
   
High dose immunosuppression both eliminates active patient immunity that is involved in the 
pathogenesis of aplastic anemia and suppresses the recipient’s immune system to prevent 
rejection of the graft. Cyclophosphamide as a single agent can be used to condition patients 
undergoing HSCT. The addition of ATG to cyclophosphamide conditioning appears to 
significantly reduce the risk of graft rejection. A report of 39 consecutive patients who 
received cyclophosphamide (total 200mg/kg in four daily doses) and ATG (total 90mg/kg in 
three doses using an HLA matched sibling donor) conditioning for a first transplant from an 
HLA matched sibling donor reported a graft rejection rate of only 5%. The 3 year survival 
rate was 92% compared to the 72% in matched historical controls receiving 
cyclophosphamide alone.[30]  A more recent series of 81 patients with SAA undergoing this 
transplant approach reported 96% of the patients had sustained engraftment with 88% of 
patients being long term survivors at a median follow up of 9.2 years. [32]  
  
Patients with SAA who are older (i.e. >40 years of age), have failed prior immunosuppressive 
therapy, or who are heavily transfused have a higher risk of graft failure after HSCT. 
Minimizing the numbers of transfusions and using irradiated, leukocyte-depleted blood 
products appears to decrease this risk. Improved transfusion practices may be responsible, in 
part, for lower graft failure rates and better transplant outcomes observed in recent years.[27] 
Several strategies have been employed to decrease graft failure. One such approach is the 
addition of radiation, either total body, total lymphoid, or thoraco-abdominal.[33, 34] 

 
 
08-H-0046 
Richard Childs, M.D. 
May 13, 2019 (Amendment OO)  
8
However, radiation is associated with a higher risk of secondary malignancies [35-38], 
pneumonitis and chronic GVHD. The introduction of fludarabine in combination with 
cyclophosphamide with or without ATG allows for the elimination of irradiation from the 
conditioning regimen and was recently shown to result in excellent engraftment rates, even in 
heavily transfused and alloimmunized patients that have received prior immunosuppressive 
therapy [39-41]  
 
x 
Matched unrelated donor transplantation for patients with SAA:  Current practice 
guidelines do not recommend transplantation from an alternative donor as first-line treatment. 
However, only about one third of otherwise eligible transplant candidates will have an HLA-
identical related donor. Alternative donor sources include HLA-mismatched related donors or 
a closely-HLA matched unrelated donors.[39] The probability of survival after unrelated 
donor transplantation is about half that observed in matched sibling transplants. Therefore, 
HSCT using unrelated donors is reserved for children identified to have a suitably matched 
unrelated donor that have failed a single course of immunosuppression or for adults who are 
refractory to multiple courses of ATG +/- alternative therapies such as androgens. Causes of 
transplant failure include a higher frequency of graft rejection, regimen-related toxicity 
(because of intensification of the conditioning regimens aimed at preventing rejection), and a 
higher incidence of acute graft-versus-host disease (GVHD) compared with HLA-identical 
sibling transplants. An analysis of 318 matched unrelated donor transplants followed over a 
median period of 5 years from 1988-1998 reported a graft failure rate of 15%, a 48% 
incidence of grade II -IV GVHD and a 5-year survival of 39%[42]. Many conditioning 
regimens for alternative donor transplants incorporate irradiation to facilitate engraftment. A 
prospective multicenter study conducted from 1994-2004 in 62 patients with aplastic anemia 
undergoing HLA matched unrelated donor HSCT determined that 200cGy (in combination 
with cyclophosphamide and ATG) was the minimal effective dose of total body irradiation 
required to achieve sustained engraftment without inducing prohibitive toxicity. Graft failure 
occurred in 2%, acute GVHD (grade II –IV) in 70%, chronic GVHD in 52%, and overall 
survival was 61%.[43]  
 
The EBMT-SAA Working Party recently reported results using a conditioning regimen 
substituting fludarabine for irradiation in 38 related and family mismatched donor transplants; 
graft rejection occurred in 18% with a 2-year survival rate of 73%.[44] 
 
x 
 Unrelated cord blood transplantation for patients with SAA:   Umbilical cord blood (UCB) 
is also increasingly being used as a source of stem cells, as this allows the transplantation of 
patients without an HLA-matched donor. A group in China treated nine young adults 
suffering from severe AA with umbilical transplants using cyclophosphamide and 
antilymphocyte globulin conditioning.[45] Seven of the nine patients had some level of 
engraftment, evidenced by the presence of stable mixed chimerism. After a median follow-up 
of 32 months, survival was nearly 80%. Successful complete chimerism was reported in a 
pediatric patient after an umbilical cord graft; stable engraftment was also achieved after 
UCB grafts in children with congenital AA in two other reports. Umbilical cord transplants 
therefore appear to be an option for patients with AA who lack a suitable HLA-matched 
donor, but more experience is needed to determine the long-term outcome of this approach. 
Transplantation using unrelated cord blood (UCB) has been shown to be a reasonable 
alternative transplant approach for patients with hematological disorders curable by 
transplantation who lack an HLA matched sibling to serve as a stem cell source.  Advantage 
of UCB transplantation include the rapidity by which UCB units can be obtained, the 
requirement for less than a perfect HLA match, and lower rates of graft versus host disease 
associated with HLA mismatching compared to bone marrow or peripheral blood stem cell 

 
 
08-H-0046 
Richard Childs, M.D. 
May 13, 2019 (Amendment OO)  
9
transplants.  The major disadvantage of UCB transplantation in adults is the limited number 
of nucleated cells contained within the cord unit resulting in prolonged neutropenia and 
failure of engraftment, which contributes, to infection and TRM.  
 
The COBLT study was a prospective study of unrelated cord blood transplantation designed 
to better define the role of this stem cell source for subjects requiring unrelated allogeneic 
transplantation; 34 adult subjects (median age 34.5 years) were enrolled. Most of these 
patients had hematological malignancies and were transplanted with a single umbilical cord 
blood unit (containing greater than 10e7 nucleated cells per Kg of recipient weight) after a 
myeloablative conditioning regimen. For the primary end point, survival at 180 days was 
dismally low at 30%. Median time to neutrophil and platelet engraftment were unacceptably 
prolonged at 31 days (range, 13 to 55 days) and 117 days respectively and the primary graft 
failure incidence was 34%. Acute and chronic GVHD were the most common cause of death 
after CBT in this study.[46] 
 
Although increasing the cell dose has been reported to improve engraftment kinetics, it is 
unclear whether higher cord blood cell doses will increase the percentage of subjects who 
will successfully engraft. Whereas doses of < 1.5 x 107 total nucleated cells (TNCs) per 
kilogram are associated with a higher incidence of graft failures[47], Rubinstein et al. [48] 
did not detect a decrease in graft failures above a dose of 2.5 x 107 TNCs per kilogram, thus 
suggesting that there may be a threshold dose for successful engraftment. This study and 
previous CBT studies [46, 49-51]indicate that despite the slow engraftment times, 
approximately 85% to 90% of subjects undergoing CBT will eventually engraft. 
 
It is now clear that the limited umbilical cord blood (UCB) cell dose is the major factor that 
compromises outcome in adult recipients of UCB transplantation. Therefore, to augment graft 
cell dose, combinations of 2 partially human leukocyte antigen (HLA) matched UCB units 
were evaluated prospectively in twenty-three patients with high-risk hematological 
malignancies.[52] Engraftment was quicker using this approach, with neutrophil recovery 
occurring at a median of 23 days (range, 15-41). Such “dual cord” transplants may shorten 
neutropenia compared to transplantation approaches using a single umbilical cord blood unit, 
but conditioning associated neutropenia of 22 days is still considerably longer than the 
median 12 days of neutropenia associated with the use of peripheral blood stem cell 
transplants.  
 
There are currently no published prospective studies using dual cord transplants as a stem cell 
source in patients with severe aplastic anemia.  
 
2.2 
Myelodysplastic Syndrome (MDS) 
2.2.1 Pathophysiology of MDS 
The myelodysplastic syndromes are a heterogeneous group of clonal hematological disorders 
characterized by bone marrow failure, multiple refractory cytopenias (anemia, neutropenia, and 
thrombocytopenia) and morphologic evidence of marrow dysplasia (proliferation of myeloblastic 
leukemia cells). As a result, patients with MDS are at variable risk of progression to acute 
leukemia which is often refractory to standard treatment. These disorders may arise de novo or 
appear years after exposure to potentially mutagenic chemotherapy. The majority of patients with 
myelodysplasia have a hypercellular or normocellular bone marrow.  In a minority of patients, 
less than 15%, a hypocellular bone marrow is found and the abnormality is referred to as 
hypoplastic myelodysplasia.[53]  Hypoplastic MDS bears morphologic similarity to aplastic 

 
 
08-H-0046 
Richard Childs, M.D. 
May 13, 2019 (Amendment OO)  
10
anemia. In some patients with aplastic anemia who initially respond to immunosuppressive 
therapy, progression to MDS may occur also suggesting that these two entities may share a 
common pathophysiology, T-cell-mediated suppression of a primitive bone marrow stem cells.    
 
2.2.2 
Cytogenetic abnormalities present in MDS  
 
Cytogenetic abnormalities are present in 40-70% of de novo MDS cases[54-58] . Many times, 
patients may present with cytopenias but minimal or no evidence of marrow dysplasia and a 
diagnosis of MDS is based on abnormal cytogenetics.  Additionally, survival and the risk of 
leukemic transformation can be prognosticated based upon cytogenetic abnormalities.  Deletion 
5q, deletion 20q, or deletion Y, when occurring as the only cytogenetic abnormality are 
considered low risk MDS with the most favorable median survival (5.7 years) and lowest risk of 
progression to acute myeloid leukemia (AML).  Complex karyotypes are associated with the 
poorest median survival (0.4 years) and greatest risk of progression to AML.  Intermediate 
karyotypes such as trisomy 8 and other abnormalities that are not listed above fall in between 
with a median survival of 1.2 to 3.5 years.[58]   
 
2008 WHO Classification Criteria for Myelodysplastic Syndromes 
  Disease 
Blood Findings 
Bone Marrow Findings 
Refractory cytopenia with unilineage 
dysplasia (RCUD): Refractory anemia 
(RA) 
Refractory neutropenia (RN); Refractory  
Thrombocytopenia (RT) 
Unicytopenia or Bicytopenia  
No or rare blasts  
< 1 x 109/L monocytes 
Unilineage dysplasia only  
< 5% blasts  
< 15% ringed sideroblasts 
Refractory anemia with  
ringed sideroblasts (RARS) 
Anemia  
No blasts 
Erythroid dysplasia only  
< 10% grans or megas dysplastic  
15% ringed sideroblasts  
< 5% blasts 
Refractory cytopenia with  
multilineage dysplasia (RCMD) 
Cytopenias (bicytopenia or 
pancytopenia)  
No or rare blasts  
No Auer rods  
< 1 x 109/L monocytes 
Dysplasia in 10% of cells in two or 
more  
myeloid cell lines  
< 5% blasts in marrow  
No Auer rods  
< 15% ringed sideroblasts 
Myelodysplastic syndrome,  
unclassified (MDS-U) 
Cytopenias  
No or rare blasts  
No Auer rods 
Dysplasia in <10% of cells in one or 
more 
Lineages with an MDS-associated 
Cytogenetic abnormality  
< 5% blasts  
No Auer rods 
 
2.2.3 Clinical consequences of MDS 
 
Symptoms that derive from low blood counts include: anemia which leads to fatigue, weakness, 
lassitude, headaches, and in older patients, dyspnea and chest pain. These manifestations are most 
commonly responsible for the clinical presentation. Thrombocytopenia produces mucosal 
bleeding: petechiae of the skin and mucous membranes, epistaxis, and gum bleeding are frequent 
and early complaints.  Bleeding can be brisk in the presence of accompanying physical lesions, as 
in gastritis and fungal infection of the lungs.  The most feared complication of thrombocytopenia 
is intracranial hemorrhage. Bacterial and fungal infections in the setting of neutropenia are a 
major cause of morbidity and mortality and are often the cause of death in refractory cytopenia 

 
 
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Richard Childs, M.D. 
May 13, 2019 (Amendment OO)  
11
associated with MDS.  Patients with RA and RAS have a low risk of developing AML and their 
median survival is more than 30 months. The majority of these patients die of consequences of 
bone marrow failure or due to iron overload as a result of repeated blood transfusions. The 
median survival of patients presenting with RAEB and RAEB-t is generally shorter than 12 
months.[59] 
  
2.2.4 
Treatment options for MDS 
 
x 
Growth factors:   Until recently, most patients were treated only with transfusions and 
growth factors to improve blood counts.  While many people assume that the majority of 
patients die from leukemic transformation, progression to acute myeloid leukemia (AML) 
occurs in up to 40% of patients with MDS [60] but an equal proportion of patients die from 
bleeding or infection resulting from cytopenias. Additionally, symptoms of anemia, and 
frequent trips to the treating physician’s office for blood and/or platelet transfusions 
significantly impact quality of life.  In low risk MDS patients, erythropoietin provides a 
modest benefit for the treatment of anemia and many studies have shown a superior benefit 
when granulocyte stimulating factor (GCSF) is added to erythropoietin.[61-64] Additionally, 
GCSF alone can stimulate the production of neutrophils to prevent infectious complications 
from long-term neutropenia.[65, 66]  Unfortunately, growth factors work only for a finite 
period of time and patients eventually succumb to anemia requiring transfusion support and 
prolonged neutropenia.  The prognosis for patients with MDS/AML is very poor with high 
chemotherapy complication rates and a short duration of remission in responders. 
 
x 
Immunosuppresive therapy:  Indeed, antithymocyte globulin (ATG), the mainstay of non-
transplantation therapy for aplastic anemia, has a role in certain patients with MDS. A report 
from the National Cancer Institute suggests that patients with MDS who are young and who 
have low platelet counts may benefit from a trial of ATG, with response rates as high as 50% 
.[67, 68] Interesting recent findings have added DR15 histocompatibility type as another 
predictor for response to ATG in MDS. [68, 69]Although it was originally thought that only 
patients with hypoplastic MDS would respond, it now seems clear that bone marrow 
hypoplasia is not a requirement to respond to rabbit or horse ATG, although  patients with 
excess blasts rarely respond .[70]Investigators from Prague have shown that about 40% of 
patients with indolent-subtype MDS may respond to cyclosporine[71]. Although a large 
randomized trial testing the utility of ATG in MDS is ongoing, young patients with MDS 
who do not have excess marrow blasts can be considered for a trial of immunosuppression. 
 
x 
Decitabine and Azacitidine (AzA-CR, 5-AZC):  Azacitidine has been designated an orphan 
product for the use in the treatment of MDS but has shown only a 10-30% response rate.[72, 
73]Its use requires close monitoring as cytopenias and central nervous system toxicities 
(lethargy, somnolence, confusion, coma) are frequent complications. The drug was recently 
licensed and is widely available to those who wish to use it off study. 5-azacytidine, can 
reduce transfusion requirements, delay time to leukemic transformation, and improve quality 
of life  when compared to supportive care.[74]  This trial will not exclude patients who have 
tried and failed Azacitidine, nor does it preclude its use in patients who have failed treatment.   
 
Decitabine, another hypomethylating agent, has shown to increase survival in patients with 
average and high risk MDS. A study of 170 average and high risk MDS patients showed an 
overall response of 17% with a 9% complete response and 13% hematologic response.  In 
this population there was an increased leukemia free progression time.  While overall survival 
is improved with both drugs, some patients are limited in their ability to tolerate these 

 
 
08-H-0046 
Richard Childs, M.D. 
May 13, 2019 (Amendment OO)  
12
medications secondary to worsening cytopenias.  Additionally, all patients eventually 
progress or die of complications of their disease despite treatment.[74] 
 
Revlimid® (Lenalidomide):  Revlimid®, a drug structurally similar to Thalidomide, was 
approved by the United States Food and Drug Administration (FDA) on December 27, 2005 
for the treatment of low-or-intermediate risk MDS associated with deletion of 5q, a 
chromosomal (cytogenetic) abnormality, with or without other cytogenetic abnormalities.[75]  
 
2.2.5 
Transplant options for patients with MDS 
 
x 
Allogeneic stem cell transplantation for MDS.   It is now well established that 
myelodysplastic syndromes (MDS) can be cured with allogeneic hematopoietic stem cell 
transplantation. Disease-free survival (DFS) ranges from 29% to 40%, with corresponding 
non-relapse mortality of 37 to 50% with the rate of relapse ranging from 23% to 48% with an 
HLA-identical sibling donor.[76-79] Longer disease duration before transplantation and older 
age is associated with an increased risk of treatment-related death after transplantation, 
particularly in those with life threatening cytopenias, thereby mandating consideration of 
transplant early in the course of the disease.[80] Outcome of SCT in patients with RAEB and 
RAEBt is less favorable than the outcome in patients with RA(RS), due largely to a higher 
risk of relapse. The European Group for Blood and Marrow Transplantation (EBMT) 
reported a 5-year actuarial relapse rate of 44% and 52% in 35 RAEB patients and 28 RAEBt 
patients.[81] The Fred Hutchinson Cancer Research Center (FHCRC) reported a 49% relapse 
rate for patients with excess of blasts compared to 4% for patients without marrow blast 
elevations, with actuarial DFS of 31% versus 54%, respectively.[82] 
 
This curative effect has been ascribed to the use of myeloablative chemo-radiotherapy and the 
antileukemic effect of the transplant (the graft-versus-leukemia [GVL] effect). The 
assumption that the intensive myeloablative preparative regimen is essential for the cure of 
the malignancy went unchallenged until the demonstration by Kolb et al [83], subsequently 
confirmed by numerous investigators, that donor lymphocytes alone exert a powerful 
antileukemic effect [84] in the context of patients relapsing with myeloid leukemias after 
BMT.[85-88]  This observation has important implications: 
 
o First, it may be possible to cure some hematological malignancies with preparative 
regimens of lower intensity, designed to immunosuppress the recipient to allow 
lymphocyte and stem cell engraftment without major cytoreduction of the 
malignancy by myeloablative conditioning.[89]   
o Second, such low-intensity preparative regimens appear to have lower toxicity and 
may make transplantation appropriate in patients where procedural mortality is 
usually prohibitive, including patients with more indolent hematological diseases as 
well as patients with co-morbid diseases and older patients.[90]   
Several groups, including our own, have begun to investigate this approach to improve the 
applicability and outcome following allogeneic BMT.  Our experience at the NIH has included 
>150 subjects and has also been quite favorable, with a lower incidence of treatment-related 
complications than is typically observed with a standard “myeloablative” transplant approach. 
The application of reduced intensity conditioning (RIC) in MDS has had favorable results. 
Kröger, et al.[91] reported 37 patients with MDS, who were ineligible for transplants using 
conventional myeloablative conditioning. The reduced intensity conditioning consisted of 
fludarabine, a reduced dosage of busulphan and antithymocyte globulin. Overall TRM was 27%, 
with significantly higher mortality in those with poor-risk cytogenetics (75% versus 29%) or 

 
 
08-H-0046 
Richard Childs, M.D. 
May 13, 2019 (Amendment OO)  
13
with an HLA-matched unrelated donor (45% versus 12%). In total, 32% of patients relapsed, and 
actuarial disease free survival (DFS) at 3 years was 38% with a median follow-up of 20 months. 
A Spanish study showed a TRM of only 5% after transplantation of 37 patients with MDS and 
AML (median age: 57 years) utilizing a regimen of fludarabine and busulphan 10 mg/kg.[92] 
The 1-year progression-free survival was 66% with a corresponding frequency of disease-
progression in patients with and without graft-versus-host disease (GVHD) of 13% (95% CI, 
4%–34%) and 58% (95% CI, 36%–96%), respectively (P = 0.008). These results support the 
notion that a graft-versus-MDS/AML response is critical in reducing the risk of relapse after an 
RIC transplant. However the risk of relapse may be higher using RIC; a recent EBMT study 
reported  a 54% relapse risk for 24 patients transplanted with RIC protocols, which translated 
into an increased hazard ratio (HR) of 6.0 (p = 0.02) for relapse compared to conventional 
transplantation regimens in a multivariate Cox model.[93]  
 
In the current study, we will only include patients with refractory anemia (RA) orrefractory 
anemia with ringed sideroblasts (RARS) subtypes of MDS who have life-threatening 
neutropenia and who are at a lower risk of relapse with reduced intensity conditioning but are 
not candidates for a transplant utilizing an HLA matched related or unrelated donor.  
 
x 
Stem cell transplantation using matched unrelated donors for MDS.  Among 118 patients 
who received a stem cell transplant from an unrelated donor in the EBMT database, DFS at 2 
years, relapse risk and TRM were 28%, 35% and 58%, respectively. The TRM was 
significantly influenced by age (younger than 18 years: 40%; 18–35 years: 61%; older than 
35 years: 81%). Patients with more severe acute GVHD experienced a lower relapse risk, 
suggesting an increased graft-versus-MDS effect in these patients.[94]  
x 
Stem cell transplantation using unrelated cord blood for MDS. At present, no case series 
reporting outcome of adult patients with MDS receiving unrelated cord blood transplants has 
yet been published. However, two recent retrospective analyses have been published 
comparing transplant outcome in adult patients with a variety of hematological malignancies 
undergoing umbilical cord blood (UCB) transplants versus matched unrelated donor (MUD) 
transplants. In an EBMT analysis of 682 patients undergoing UCB (n=98) vs MUD 
transplants, recipients of UCB transplants had a significantly lower incidence of acute GVHD 
(relative risk [RR] 0.57) with a similar incidence of CGVHD and similar leukemia free 
survival and overall survival.[50]  An IBMTR analysis of 600 adult patients with leukemia 
undergoing transplantation from a matched unrelated donor (MUD) vs a single antigen 
mismatched unrelated donor versus a 1 or 2 antigen mismatched UCB transplant reported 
long-term leukemia free survival and overall survival to be slightly superior with MUD 
donors.[49] In this analysis, leukemia free survival and overall survival was similar among 
recipients of a single antigen mismatched unrelated donor transplant compared to UCB 
transplant recipients.  These and other studies have now defined a clear role of UCB 
transplantation in the treatment of adults with hematological malignancies who lack an HLA 
matched sibling donor or fully matched unrelated donor. However, graft failure, which is 
most closely associated with patient size, age, and low cord stem cell doses, remains a 
significant problem that limits the applicability of this approach in adults. A number of 
approaches are being developed to overcome the obstacle of low-cell doses in adults, 
including the use of dual cord blood transplants in which UCB grafts are combined from 
different cord donors and the ex vivo expansion of umbilical cord progenitor cells.  This 
protocol utilizes reduced intensity conditioning followed by cord blood transplantation with 
the co-infusion of haploidentical purified CD34+ cells to expedite neutrophil recovery. If 
successful, this approach could expand the application of allogeneic transplantation to a 
greater numbers of patients with MDS.   
 

 
 
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Richard Childs, M.D. 
May 13, 2019 (Amendment OO)  
14
2.3 
Haplo-identical Donor Transplantation 
 
Another viable stem cell alternative for allogeneic transplantation is to utilize relatives who are only 
partially HLA-matched. These donors have one identical HLA haplotype and variable mismatches 
for zero, one, two or all three HLA-A, -B or DR loci of the unshared haplotype. Almost all patients 
have at least one HLA-haploidentical mismatched family member (parent, child, or sibling) who is 
immediately available as a donor. Unfortunately, in full haplotype mismatched transplants, a high 
frequency of alloreactive donor T cells in unmanipulated grafts exist that recognize mismatched 
MHC antigens, resulting in an extremely high incidence of severe, acute GVHD.[95-97] Although 
extensive T cell depletion prevents GVHD, the rejection rates rise steeply because the balance 
between competing host and donor T cells shifts in favor of the unopposed host-versus-graft 
reaction.[98, 99] Furthermore, T-cell depletion is associated with a greater risk of post-transplant 
viral infections and disease relapse.  
 
2.4 
Unrelated Cord Blood Combined with Haploidentical CD34+ Cell Infusion Transplantation 
 
To harness the advantage of UCB availability and to overcome the disadvantage of delayed 
neutrophil recovery, a pilot study was conducted by Fernandez et al where 27 adult patients with 
high risk leukemia received single unit UCB transplantation co-administered with an infusion of a 
relatively low number of highly purified haploidentical peripheral blood CD34+ cells from a related 
donor.[100] Rapid engraftment of a purified CD34+ haploidentical cells was observed with the 
median time to the ANC >500/L of 10 days (range 9-36). This engraftment was much more rapid 
than is typically observed with a single UCB transplant (range 22-31 days). The time to a calculated 
cord blood ANC >500 cells/ul ranged from 13-55 days with a median of 22 days. With a median 
follow-up of 10 months (range 1-75 months), 19/27 patients survived. No major early infections, 
bacterial or fungal were observed and the need of antimicrobials during the period of neutropenia 
was lower than typically required for recipients of conventional UCB transplant. Chimerism studies 
revealed initial engraftment of the haplo-CD34+ cells, followed by engraftment of the cord unit 
resulting in dual chimerism. With time, chimerism from the haplo-CD34+ donor was observed to 
disappear with full chimerism from the UCB unit ultimately being observed. This approach appears 
to provide the benefit of rapid neutrophil engraftment that occurs as a consequence of the CD34+ 
cell infusion with the cord blood unit engrafting thereafter restoring defective cellular immunity 
that typically occurs as a consequence of the rigorous T-cell depletion associated with 
haploidentical stem cell infusion.   
 
2.5 
T Cell Depletion of G-CSF Mobilized PBSCs 
 
 
T cell depletion is a technique to select and/or enrich specific cell populations from allografts.   In 
this protocol we will use the Miltenyi CliniMacs immunomagenetic T cell depletion system that is 
widely used in Europe and available in the USA only under an IND/IDE. The CliniMACS® CD34 
Reagent System is used in vitro to select CD34+ cells from heterogeneous hematological cell 
populations prior to transplantation.  The Department of Transfusion Medicine (DTM) Cell 
Processing Section has evaluated Miltenyi CliniMacs CD34-positive selection as a method for T 
cell depletion of filgrastim (G-CSF) mobilized PBSC collections from normal donors.  Results 
(mean ± SD) are as follows (n=11 products): CD34 % recovery = 60 ± 13 % (range 41-77%) and 
CD3+ T cell depletion = 5.3 ± 0.2 log (range 5.1-5.7 log) (personal communication with EJ Read).  
The T cell depletion is approximately one log greater than the Isolex CD34 positive selection only 
process, which routinely achieves up to 4 log T cell depletion.   
 
 
 

 
 
08-H-0046 
Richard Childs, M.D. 
May 13, 2019 (Amendment OO)  
15
2.6. 
Triple Drug Regimen for GVHD Prophylaxis 
 
The prophylaxis of graft-versus-host disease (GVHD) has varied significantly among 
various studies of unrelated cord blood transplantation. Most have incorporated either tacrolimus 
or cyclosporine combined with other immunosuppressive agents such as mycophenolate mofetil, 
methotrexate, or a brief course of prednisone. At present, there is no accepted standard regimen for 
GVHD prophylaxis in this setting. This protocol will utilize tacrolimus in combination with 
mycophenolate mofetil and a brief course of prednisone as GVHD prophylaxis. 103 
 
3.0 SCIENTIFIC AND CLINICAL JUSTIFICATION 
 
Severe aplastic anemia (SAA) is a life-threatening bone marrow failure disorder characterized by 
pancytopenia and a hypocellular bone marrow.   Transplant and immunosuppressive treatment with 
ATG and CsA have dramatically changed the natural course of aplastic anemia, with 5 year 
survival of 75% in patients undergoing treatment[39]. However, of those patients treated with 
ATG/CsA, 25-33% will not respond; and about 50% of responders will relapse.[101] The 
introduction of more potent immunosuppressive medications such as alemtuzumab (Campath®) 
and r-ATG[16, 17] may have a favorable impact in response and relapse rates.  However, definite 
evidence that these novel immunosuppressive therapies can reduce morbidity, induce response and 
alleviate relapse as compared to the conventional h-ATG/CsA treatment regimen currently does not 
exist. Patients who fail immunosuppression who have an ANC’s <500 cells/Pl are at high risk for 
death from opportunistic infections, particularly invasive fungal infections. Likewise, patients with 
refractory anemia (RA) or refractory anemia with ringed sideroblasts (RARS)subtypes of MDS 
who have severe neutropenia refractory to immunosuppression are also at high risk of death from 
opportunistic bacterial and fungal infections.   
 
Allogeneic bone marrow transplantation from an HLA-matched sibling or matched unrelated donor 
offers the opportunity for cure in approximately 70-80% of patients. [30, 31] For patients lacking a 
matched related donor, a MUD transplant is the preferred therapy. However, many patients don’t 
have a matched unrelated donor identified or don’t have resources required for such a transplant. 
 
Transplantation using unrelated cord blood (UCB) has been shown to be a reasonable alternative 
transplant approach for patients with hematological disorders curable by transplantation that lack an 
HLA matched sibling to serve as a stem cell source.  The major disadvantage of UCB 
transplantation in adults is the limited number of nucleated cells contained within the cord unit 
resulting in prolonged neutropenia and failure of engraftment which contributes to infection and 
TRM. To harness the advantage of UCB availability and to overcome the disadvantage of delayed 
neutrophil recovery, a pilot study was conducted by Fernandez et al[100] in 27 patients (median age 
29 years; range 16-60) with high risk leukemia who received a single unit UCB transplantation co-
administered with an infusion of a relatively low number of highly purified haploidentical 
peripheral blood CD34+ cells from a related donor following a fully myeloablative conditioning 
regimen that included TBI. Rapid engraftment of purified CD34+ haploidentical cells was observed 
with the median time to the ANC >500 cells/ul of 10 days (range 9-36). This engraftment was much 
more rapid than is typically observed with a single UCB transplant (typically 22-31 days).  
Chimerism studies showed initial predominant chimerism in mononuclear cells and granulocytes 
being derived from the haplo-CD34+ cells followed by progressive replacement of these cells by 
cells derived from the cord blood unit. The time to a calculated cord blood ANC >500 cells/ul 
ranged from 13-55 days with a median of 22 days and a maximum cumulative incidence of the cord 
blood ANC being > 500 cells/μl of 0.93 (95% CI 0.83-1.00). Ultimately, full cord blood chimerism 
occurred in 25/27 patients at a median 55 days (range 11-96). Seventy-four percent of patients 

 
 
08-H-0046 
Richard Childs, M.D. 
May 13, 2019 (Amendment OO)  
16
developed acute GVHD although only 4 (14.8%) patients developed more severe grade III-IV 
GVHD. With a median follow-up of 10 months (range 1-75 months), 19/27 patients survived. Eight 
of 27 patients died as a consequence of non-relapse mortality.  Causes of death included infection 
(n=3; 1 toxoplasmosis, 2 cytomegalovirus), vascular occlusive disease (VOD) (n=1), conditioning 
associated multisystem organ failure (MOF) (n=1), graft failure (n=1), and GVHD (n=2).   No 
major early infections, bacterial or fungal were observed.  The need of antimicrobials during the 
period of neutropenia was lower than typically required for recipient of conventional UCB 
transplantation.[100] 
 
We therefore propose this research protocol of co-infusion of umbilical cord blood and 
haploidentical CD34+ cells following nonmyeloablative conditioning as treatment for subjects with 
SAA and RA and RARS subtypes of MDS refractory to immunosuppressive therapy.  The study is 
being done to confirm the promising results published by Fernandez et al and to further establish 
whether this transplant approach can be effectively used in patients with aplastic anemia who are at 
high risk for graft rejection. Finally, it will investigate the engraftment potential of a novel 
nonmyeloablative conditioning regimen in this setting. Should engraftment rates be good (defined 
as an ANC > 500 in 80% of patients by day 42), this protocol will serve as a platform for 
subsequent trials investigating the impact of infusing in vitro expanded NK cells from the cord 
blood unit to reduce GVHD and to facilitate engraftment.   
 
 
Justification for health related quality of life measures 
 
The Food and Drug Administration (Johnson & Temple, 1985) and the Institute of Medicine (IOM, 
2008) endorse Health Related Quality of life (HRQL) and its relevant dimensions as meaningful 
patient-reported indicators of the effects of treatment. The effects of allogeneic HSCT on HRQL 
dimensions are well established relative to standard conditioning regimens and disease populations. 
However, our understanding of the effects of reduced intensity conditioning regimens on specific 
disease populations such as aplastic anemia, bone marrow failure syndromes, PNH and 
myelodysplastic syndromes is less clear.  
 
 
Therefore, we are proposing to incorporate a systematic evaluation of HRQL in adult individuals 
(English and Spanish when available) pre-transplant and throughout the trajectory of their recovery 
and follow-up post-transplant. Specifically, we request to incorporate patient-reported outcome 
measurements initially when the transplant recipient is on-study (pre-transplant prior to inpatient 
admission for transplant), then following transplant at day 30, day 100, every at each 6-month 
interval until the 5th year post transplant.  
 
4.0 STUDY DESIGN  
 
This is a phase II clinical trial designed to evaluate a novel non-myeloablative but highly 
immunosuppressive conditioning regimen consisting of cyclophosphamide, fludarabine, ATG and 
one dose total body irradiation, followed by an infusion of a stem cell product prepared from a 
haplo-identical donor using the Miltenyi CliniMacs system for CD34 selection, and a single t 4/6 
HLA antigen matched UCB unit in subjects with severe aplastic anemia or refractory anemia (RA) 
or refractory anemia with ringed sideroblasts (RARS)subtypes of MDS who have life-threatening 
neutropenia and who are not candidates for a transplant utilizing an HLA matched sibling or 
unrelated donor.    
  

 
 
08-H-0046 
Richard Childs, M.D. 
May 13, 2019 (Amendment OO)  
17
Transplant
recipient
Fludarabine Day -5 to -1
TBI Day -1
Cyclophosphamide day -7 and -6
Haplo identical
family donor
T cell
depleted
PBSCT
Day 0
Daily
assessments
until
discharge
Follow up
to 5 years
post
transplant
Contact every
6-12
months for
quality of life
measures and
survival
stem cell
apheresis
CD34
selection for
T cell
depletion
h-ATG -5 to -2
Umbilical Cord
blood
Tracrolimus day -4 to day 180
MMF day 0 to day 100
Methylprednidone day -5 to day 19
G-CSF
mobilization
5 days prior
to stem cell
apheresis
 
5.0 ELIGIBILITY ASSESSMENT 
 
 
5.1 
Inclusion Criteria-Recipient 
 
5.1.1 
Diagnosed with severe aplastic anemia characterized by all of the following: 
  
 
a) Bone marrow cellularity <30% (excluding lymphocytes) 
 
b) Transfusion dependence for platelets and/or RBCs 
c) Neutropenia [(absolute neutrophil count < 500 cells/ PL) OR for patients receiving 
granulocyte transfusions, absolute neutrophil count < 500 cells/ PL before 
beginning granulocyte transfusions].  
 
 
 
 
 
OR 
Diagnosed with myelodysplastic syndrome characterized by refractory anemia OR 
refractory anemia with ringed sideroblasts (RARS) and at least one of the following: 
a) Neutropenia [(absolute neutrophil count < 500 cells/ μL) OR for patients receiving 
granulocyte transfusions, absolute neutrophil count < 500 cells/ μL before beginning 
granulocyte transfusions] and history of 1 or more opportunistic infections related to 
neutropenia. Or 
b) History of severe aplastic anemia transformed to MDS 
 
5.1.2 
Intolerance of or failure to respond to standard immunosuppressive therapy. 
5.1.3 
Availability of at least one HLA- haploidentical (i.e. t 5/10 and  d 8/10 HLA match) 
related donor (HLA-A, B, C, DR, and DQ loci) who is available to donate CD34+ cells (4-
75 years old).   
5.1.4 
Availability of at least one 4/6 HLA-matched (HLA-A, B, and DR loci) cord blood unit 
from the National Marrow Donor Program (NMDP). The cord blood unit must contain a 
minimum TNC (prior to thawing) of at least 1.5 x 107 cells per kilogram of recipient body 
weight with the following exception: if the minimum criterion of TNC is not met the cord 
unit must contain at least 1.7 x 105 CD34+ cells/kg (prior to thawing).  
5.1.5 
Ages 4-55 years inclusive.  
5.1.6 
Ability to comprehend the investigational nature of the study and provide informed 
consent.  The procedure will be explained to subjects aged 4-17 years with formal consent 
being obtained from parents or legal guardian. 
5.1.7 
Telomere Length Testing. 

 
 
08-H-0046 
Richard Childs, M.D. 
May 13, 2019 (Amendment OO)  
18
5.1.8 
In patients where a suspicion for a familial bone marrow failure syndrome (BMFS) exist, 
TERC and TERT mutation testing will be performed on protocol 04-H-0012 or performed 
elsewhere prior to enrolling on 04-H-0012.  
 
5.2     Exclusion Criteria – Recipient (any of the following) 
 
5.2.1 
Availability of an HLA identical or 9/10 HLA matched (HLA A, B, C, DR, and DQ loci) -
relative to serve as a stem cell donor. 
5.2.2 
The patient is deemed to be a candidate for a 6/6 HLA matched unrelated stem cell 
transplant (availability of a donor and resources required for such a transplant). 
5.2.3 
ECOG performance status of 2 or more 
5.2.4 
Major anticipated illness or organ failure incompatible with survival from transplant. 
5.2.5 
Severe psychiatric illness. Mental deficiency sufficiently severe as to make compliance 
with the transplant treatment unlikely and making informed consent impossible. 
5.2.6 
Positive pregnancy test for women of childbearing age. 
5.2.7 
HIV positive.  
5.2.8 
Diagnosis of Fanconi’s anemia (by chromosome breakage study) 
5.2.9 
Diffusion capacity of carbon monoxide (DLCO) <40% using DLCO corrected for Hgb or 
lung volumes (patients under the age of 10 may be excluded from this criterion if they have 
difficulty performing the test correctly and thus are unable to have their DLCO assessed) 
using DL Adj and DL/VA/Adj.  
5.2.10 Left ventricular ejection fraction < 40% (evaluated by ECHO)  
5.2.11 Transaminases > 5x upper limit of normal (when transaminases are elevated, the subject 
may be excluded at the discretion of the PI). 
5.2.12 Serum bilirubin >4 mg/dl. 
5.2.13 Creatinine clearance < 50 cc/min by 24 hr urine collection (adjusted for body surface area, 
i.e.50 ml/min/1.73m2). 
5.2.14 Serum creatinine > 2.5 mg/dl 
5.2.15 Failure to collect an adequate number of CD34+ cells (i.e. t 2 x 106 CD34+ cells/kg) for 
transplantation from the subject’s haploidentical relative.  
5.2.16 Presence of an active infection not adequately responding to appropriate therapy. 
5.2.17 History of a malignant disease liable to relapse or progress within 5 years. 
 
5.3 
Inclusion Criteria- Related Haploidentical Donor Donating Purified CD34+ Cells  
 
5.3.1 
HLA mismatched family donor ( t 5/10   and  d 8/10 HLA match (HLA-A, B, C, DR, and 
DQ loci)) who is available to donate CD34+ cells.  
5.3.2 
Ages 4-75 inclusive 
5.3.3 
Weight > 15 kg. 
5.3.4 
For adults: Ability to comprehend the investigational nature of the study and provide 
informed consent.  For minors: Written informed consent from one parent or guardian who 
is not the recipient of the transplant and informed assent.   The process will be explained to 
the minor on a level of complexity appropriate for their age and ability to comprehend. 
5.3.5 
 If there is a suspicion of familial BMFS in the recipient, then the donor must have 
undergone genetic testing for genes associated with BMFS - performed at a CLIA-certified 
laboratory, prior to enrolling in this protocol. 
 
 
 
 
 

 
 
08-H-0046 
Richard Childs, M.D. 
May 13, 2019 (Amendment OO)  
19
5.4 
Exclusion Criteria – Related Donor (any of the following) 
 
5.4.1 
Pregnant or breastfeeding. 
5.4.2 
A suitable adult haplo identical donor is available.  
5.4.3 
Unfit to receive filgrastim (G-CSF) and undergo apheresis (history of stroke, MI, unstable 
angina, uncontrolled hypertension, severe heart disease or palpable spleen). 
5.4.4 
HIV positive (Donors who are positive for HBV, HCV or HTLV-I/II, T.cruzi [Chagas] may 
be used at the discretion of the investigator following counseling and approval from the 
recipient). 
5.4.5 
Sickling hemoglobinopathies including HbSS or HbSC. Donors with HbAS are acceptable. 
5.4.6 
Severe psychiatric illness. Mental deficiency sufficiently severe as to make compliance with 
the BMT treatment unlikely, and making informed consent impossible. 
5.4.7 
Screening test positive for Chagas disease (Trypanosoma cruzi /T. cruzi/trypanosomiasis) 
confirmed by the Center for Disease Control (CDC). 
 
 
6.0 
CLINICAL EVALUATION OF THE RECIPIENT 
Bone marrow aspirates will be read by a hematologist.  Samples will be ordered and tracked 
through the CRIS screens.  Should a CRIS screen not be available, the NIH form 2803-1 will be 
completed and will accompany the specimen and be filed in the medical record. 
 
6.1 
Pre-Study Evaluation and Procedures 
 
Lab test results and consults will be performed within 28 days of enrollment (unless otherwise 
specified); bone marrow evaluations will be performed within 3 months of enrollment; and 
pregnancy test will be performed within 72 hours of conditioning. 
 
6.1.1 
History and physical exam including ECOG assessment 
6.1.2 
Temperature, pulse, blood pressure, respiratory rate, height and weight  
6.1.3 
CBC with differential 
6.1.4 
Reticulocyte count 
6.1.5 
Acute care, mineral panel, hepatic panel, uric acid CK 
6.1.6 
Coagulation (PT, PTT)  
6.1.7 
Lipid panel 
6.1.8 
Prealbumin 
6.1.9 
C-reactive protein 
6.1.10 Iron panel (ferritin, transferrin % saturation)  
6.1.11 Folate level, B12 level  
6.1.12 Thyroid function (TSH, T4), Adrenal function (FSH, LH) 
6.1.13 Gonadal function (testosterone, estradiol), cortisol  
6.1.14 Growth hormone 
6.1.15 IgG 
6.1.16 Lymphocyte Phenotyping TBNK 
6.1.17 Isohemagglutinin titer 
6.1.18 Hemoglobin electrophoresis 
6.1.19 PNH flow cytometry of the peripheral blood for GPI anchored proteins on neutrophils 
6.1.20 growth chart, (<18 years of age) 
6.1.21 bone age (prepubertal children; within 3 months) 
6.1.22 Infectious disease screens: 
            Anti-HBs antibody  
             HBsAg 

 
 
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Richard Childs, M.D. 
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             Anti-HBc antibody 
             Anti-HCV Antibody  
             Anti –HTLV1/2 antibody 
            Anti HIV ½ antibody 
             West Nile Virus 
             HIV-1/HCV/HBV NAT 
             T.cruzi Antibody 
             Anti- HSV Type ½ Antibody,serum   
             Anti CMV  IgG,IgM 
            Anti EBV antibody panel 
            Anti- Toxoplasma IgG 
            Anti-ToxoplasmaIgM antibody (Mayo)  
            Anti-Adenovirus Antibody  
            HHV6 PCR Quantitative, Blood  
            RPR 
           Anti Varicella Zoster Virus Antibody IgG, IgM 
           PPD                                                                                                                                                             
6.1.23 High Resolution HLA-A, B, C, DR, DQ typing (once) 
6.1.24 Antibody screen for HLA alloantibodies (once) 
HLA Antibody screen-Class 1A, B, C 
HLA Antibody screen-Class 2 DRB1, DQB1 
KIR Genotype (call 301-496 8852) for approval prior to ordering 
Sequenced based HLA-C, this procedure requires advance approval by HLA 
supervisor prior to ordering (301-496-8852) 
6.1.25 Red cell ABO, Rh antibody screen, DAT (direct antiglobulin test) 
6.1.26 Type and antibody screen 
6.1.27 Direct Antiglobulin Screen 
6.1.28 Short tandem repeats (STR) profile (once) 
6.1.29 Chromosomal breakage studies to rule out Fanconi anemia (once) 
6.1.30 Telomere length testing 
6.1.31 FISH for MDS (if clinically indicated) 
6.1.32 Quantitative immunoglobulins (IGG, IGA, IGM) 
6.1.33 Dexa scan (within one year) radius, femur, spine 
6.1.34 24-hour urine collection for creatinine clearance.  
             Urinalysis (includes microscopic) 
6.1.35 For females of childbearing potential: pregnancy test (serum or urine) 
             Human Chorionic Gonadotropin, Preg 
             Pregnancy Test, Urine 
6.1.36 Bone marrow aspirate and biopsy with cytogenetic analysis (morphology, cellularity, 
percentage of blast cells, and/or chromosomal analysis by PCR) as appropriate to stage 
and classify underlying disease 
6.1.37 Chest radiograph (if patient does not have a chest CT) 
6.1.38 Pulmonary function tests (patients under the age of 10 in which it has been established 
that they have difficulty performing the tests correctly will not be required to have their 
pulmonary function assessed.) 
6.1.39 Sinus CT scan and other body CT scans as medically required 
6.1.40 Cardiac function: EKG, ECHO  
In addition, all subjects age 50 or greater or subjects age 40 or over with one of the 
following risk factors: a history of high blood pressure or increased cholesterol, family 
history of coronary disease, smoking or diabetes, will have a baseline cardiac workup, 
which will include: 

 
 
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21
 
1.  Stress nuclear perfusion imagery (sestamibi) (if clinically indicated) 
 2. Cardiac consultation (all subjects age 50 and over, within 6 months of 
conditioning) 
6.1.41 Nutritional assessment, as needed (within 6 months of conditioning) 
6.1.42 Dental consult, as needed (within 6 months) 
6.1.43 Social worker interview 
6.1.44 Ophthalmology consultation (within 6 months of conditioning) 
6.1.45 Discussion of the potential for infertility (within 6 months of conditioning) 
6.1.46 Durable power of attorney form completed (within 6 months of conditioning) 
6.1.47 Testing of genes related to inherited BMFS performed at a CLIA-certified laboratory - 
hTERC and hTERT, GATA2 mutations to be conducted in patients where a suspicion for 
familial bone marrow failure syndrome exist, on protocol 04-H-0012 or elsewhere prior 
to enrolling on 04-H-0012 to be eligible for this protocol (once) 
6.1.48 HCT Comorbidity Index 
 
6.2 
In-Patient Monitoring (day -7 to discharge [approximately day 30]) 
 
* As the subject’s condition stabilizes and they approach hospital discharge this monitoring 
frequency may decrease as clinically indicated (testing marked with an asterisk)  
 
x 
Interim clinical assessments (daily while in patient) 
x 
Temperature, pulse, blood pressure, respiratory rate, weight (*daily) 
x 
CBC with differential (*daily)  
x 
Acute care, mineral panel, hepatic panel, uric acid CK (*daily) 
x 
Reticulocytes (*twice weekly) 
x 
C-reactive protein (*weekly) if clinically indicated 
x 
Pre-albumin (*twice weekly) if clinically indicated 
x 
Lipid panel (*twice weekly) if clinically indicated 
x 
Coagulation (PT, PTT) screen (*twice weekly) if clinically indicated 
x 
Type and screen (*q4 days) only if indicated (patient still requiring transfusions) 
x 
CMV, EBV (twice weekly)  
x 
Toxoplasma gondii PCR, BLD (weekly if clinically indicated) 
x 
HHV6, adenovirus blood PCR and urine PCR surveillance (weekly) 
x 
BK blood PCR and urine PCR (*) (weekly if clinically indicated) 
x 
Repeat pulmonary function tests (if patient is stable enough to perform PFTs): (Day 30 
+/- 7 days) (patients under the age of 10 in which it has been established that they have 
difficulty performing the tests correctly will not be required to have their pulmonary 
function assessed.) 
x 
PNH flow cytometry (every 2 weeks until negative for a PNH clone x2) 
x 
Bone marrow aspirate or peripheral blood to quantitate engraftment (chimerism) in the 
lymphoid and myeloid cell lines day 15 +/- 7 days and day 30 +/- 7 days or when clinical 
conditions warrant testing. 
x 
Drug levels where appropriate (e.g., gentamicin, vancomycin, tacrolimus (daily while 
inpatient, mycophenolate) 
x 
TBNK Lymphocyte Phenotyping (Day 30 +/- 7 days) 
x 
IgG (Day 30 +/- 7 days) 
x 
Pregnancy test for females of childbearing potential (serum or urine) (within 72h of 
conditioning) 
- 
Human Chorionic Gonadotropin, Preg 

 
 
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Richard Childs, M.D. 
May 13, 2019 (Amendment OO)  
22
- 
Pregnancy Test, Urine 
 
6.3 
Follow Up Period from Hospital Discharge to Return Home (about days 30- 180) 
   
At least weekly (+/- 5 days) unless otherwise noted: 
NOTE: If patients have had extended stays past day 180, visits may decrease, some tests listed 
below may not be medically indicated at the frequency listed.  If this is the case, notes will be 
made in the charts.  
x 
Clinical assessment 
x 
Temperature, pulse, blood pressure, respiratory rate, weight 
x 
CBC with differential 
x 
Acute care, mineral panel, hepatic panel, uric acid CPK 
x 
Reticulocyte count 
x 
C-reactive protein (if clinically indicated) 
x 
Ionized magnesium (if clinically indicated) 
x 
Coagulation screen (if clinically indicated) 
x 
EBV, CMV, PCR quantitative blood (twice weekly) 
x 
HHV6 and adenovirus surveillance (weekly) 
x 
BK blood PCR and urine PCR, andnovirus urine (if clinically indicated) 
x 
DAT (direct antiglobulin test) and type and screen (as clinically indicated) 
x 
Bone marrow aspirate or peripheral blood to quantitate engraftment (chimerism) in the 
lymphoid and myeloid cell lines day 42 (+/- 3 days), 60 (+/- 3 days) and 100 (+/- 7 days) 
or when clinical conditions warrant testing (for example, patients with MDS done at 30 
+/-7 days, 100 +/- 14 days and 6 months +/- 30 days)  
x 
Pregnancy test (at least monthly +/- 1 week) (for females of childbearing age) (as 
clinically indicated) 
x 
Drug levels as appropriate (i.e.tacrolimus, MMF) 
x 
TBNK Lymphocyte Phenotyping (Day 30, 60, and 100 +/- 7days) 
x 
Vitamin D (Day 100 +/- 7 days) 
x 
Iron Panel (Day 100 +/- 7 days) 
x 
IgG (Day 30, 60, and 100 +/- 7 days) 
 
6.4 
Follow Up After Return Home  
 
Subjects and/or their referring physician will remain in contact with the NIH and are asked to 
return to the NIH or send the results of clinical assessments and/or laboratory testing at least 
every 3 months (+/- 2 weeks) for 2 years, then every 6 months (+/- 1 month (national) and +/- 3 
months for international patients) to 5 years. Additional labs/tests may be done as clinically 
indicated. 
 
x 
Clinical assessment at NIH only 
x 
CBC with differential  
x 
Acute care, mineral panel, hepatic panel, uric acid CPK 
x 
CD4/CD8 on day 180 (± 30 days) and day 360 (± 30 days) 
x 
TBNK Lymphocyte Phenotyping (For all NIH visits) 
x 
Vitamin D Day 180 (± 30 days) 
x 
Iron Panel (NIH visits only) 
x 
IgG (NIH visits only) 
x 
Lipid panel (NIH visits only) 

 
 
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Richard Childs, M.D. 
May 13, 2019 (Amendment OO)  
23
x 
Reticulocytes (NIH visits only) 
x 
Chest radiograph (when clinically indicated) 
x 
Pulmonary function tests (every 3 months x 2 years then every 6 months until 5 years 
after transplant then annually) (patients under the age of 10 in which it has been 
established that they have difficulty performing the tests correctly will not be required to 
have their pulmonary function assessed.)  (± 30 days) 
NOTE: International patients who do not have the means to travel every 3 months after 
discharge home nor the money to obtain a PFT at home will not be required to have their 
pulmonary function assessed.  Also, international patients who are stable enough to only 
need to return every 12 months may have the PFTs at PI's discretion and the PI will 
document when the patient will return. 
x 
EBV, CMV, HHV6 and adenovirus surveillance by PCR (as clinically indicated) 
x 
TBNK Lymphocyte Phenotyping (NIH visits only)  
x 
IgG (NIH visits only) 
x 
Bone marrow biopsy and aspiration with cytogenetics (for MDS patients only: 6 months, 
1 year and then yearly until 5 years, for AA at 1 year follow up and then at the discretion 
of the PI). For bone marrows +/- one month and +/- 3months for international patients 
x 
PNH flow cytometry of the peripheral blood for GPI cells (until normal x2) 
x 
Ophthalmologic exam (yearly) 
x 
Cortisol, TSH, T4, FSH, LH, and testosterone/estradiol (yearly) 
x 
Growth hormone, growth chart, bone age (prepubertal children) (yearly) 
x 
Health related quality of life measures (adults only, +/- 1 month):  PROMIS 29, PROMIS 
v 1.0 Applied Cognition-General Concerns-Short Form, FACT-BMT, and Sexual 
Functioning Questionnaire (SFQ) screening questions 1-3 and subscales (Medical Impact, 
Satisfaction and Problems), Chronic Graft vs. Host Disease measure (every year until 5 
years post-transplant). 
x 
DEXA scan (radius, femur, spine AP) (yearly) 
 
After 5 years, annual follow-up with the recipient and/or the referring physician will continue indefinitely 
to provide post-transplant standard of care and collect outcome and survival data as required by the 
National Marrow Donor Registry. 
 
7.0 
TREATMENT PLAN 
  
 
7.1 
Apheresis of Transplant Recipient for Lymphocytes (optional) 
 
One cell collection of 1010 cells by apheresis for cryopreservation for studies of donor T cell 
alloresponses and post-transplant studies will be requested prior to transplantation. 
 
7.2 
Central Venous Line Placement 
 Central Venous Catheter Placement, when indicated, will be performed by the NIH Procedures, 
Vascular Access and Conscious Sedation (PVCS) Service.  A separate consent will be obtained at 
the time of the procedure.  
 
7.3 
Infection Prophylaxis and Treatment 
 
x 
Pneumocystis pneumonia and toxoplasmosis prophylaxis:    PCP and toxoplasmosis 
prophylaxis will be given according to the NIH Supportive Care Guidelines for Allogeneic 
Hematopoietic Stem Cell Transplant Recipients. 
 

 
 
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Richard Childs, M.D. 
May 13, 2019 (Amendment OO)  
24
x 
Strongyloides prophylaxis:  Prophylaxis will be given according to the NIH Supportive Care 
Guidelines for Allogeneic Hematopoietic Stem Cell Transplant Recipients. 
 
x 
Antiviral prophylaxis:  Acyclovir or its equivalent will be given according to adult and 
pediatric doses found in the NIH Supportive Care Guidelines for Allogeneic Hematopoietic 
Stem Cell Transplant Recipients and administered for at least 1 year in recipients regardless 
of HSV serology status.   
 
x 
Antibacterial prophylaxis:  Bacterial prophylaxis to cover pseudomonas species (i.e. IV 
ceftazidime, meropenem, etc) will be given following initiation of the conditioning regimen 
in all recipients with an ANC< 200. Prophylactic antibiotic therapy will continue until the 
ANC recovers to >500.  
 
x 
EBV, CMV, HHV6 and adenovirus monitoring and treatment:  Subjects will be monitored 
for EBV, CMV, HHV6 and adenovirus PCR in the blood at baseline, then weekly until day 
180 (±30 days). Thereafter, monitoring will be performed as clinically indicated.   
o EBV reactivation will be treated with rituximab  
o CMV reactivation will be treated according to the NIH supportive Care Guidelines 
for Allogeneic Hematopoietic Stem Cell Transplant Recipients.   
o Adenovirus infection will be treated with cidofivir when clinically indicated. HHV6 
reactivation will not be treated unless there exists compelling evidence for clinical 
disease related to HHV6; such recipients may receive foscarnet upon the advice of 
NIH Infectious Disease consultants. 
 
7.4 
Fever Regimen (See NIH Supportive Care Guidelines for Allogeneic Hematopoietic Stem Cell 
Transplant Recipients) 
 
7.5 
Neutropenia Regimen 
 
x 
Filgrastim (G-CSF) will be initiated day +1 at a dose 5 mcg/kg day sq until neutrophils 
recover to ≥ 500 cells/ul.  G-CSF administration will continue for 1 to 2 days after the ANC 
is >500 cells/μL based on physician’s discretion and clinical judgment. Doses may be 
rounded +/- 10% to available vial sizes. G-CSF will be given subcutaneously as the preferred 
route and IV as the alternative route based on physician discretion.  
 
7.6 
Preparative Regimen  
 
The preparative regimen will not be initiated until the cord blood unit obtained from the 
NMDP to be used for transplantation has been transferred to the NIH Department of 
Transfusion Medicine.  
 
x 
Cyclophosphamide 60 mg/kg/d IV day -7, -6  
x 
Fludarabine 25 mg/m2/d IV day -5, -4, -3, -2, -1  
x 
Antithymocyte globulin 40mg/kg IV days -5, -4,-3,-2 for a total dose of 160 mg/kg.  
 
Subjects will receive pre-medication prior to infusion as follows:  
 
x 
IV or oral diphenhydramine 25 to 50 mg (children 4-12 yrs 12.5-25 mg) and 
x 
oral acetaminophen 650 mg (children 4-12 yrs: 325 mg)  
 

 
 
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May 13, 2019 (Amendment OO)  
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Oral prednisone at 1 mg/kg/day (or IV methylprednisolone if clinically indicated) will begin 
on day -5 prior to the first dose of h-ATG for serum sickness prophylaxis, and will be 
continued at this dose for 10 days; thereafter, prednisone will be slowly tapered over the next 
2 weeks until it is discontinued on day +19. Those recipients who develop serum sickness 
may require a longer tapering schedule and will be dosed individually as clinically indicated.   
 
Infusions reactions will be treated symptomatically (e.g., anti-emetics, IV fluid hydration, 
acetaminophen, antihistamines, inhaled bronchodilators, meperidine).  
 
In case of moderate or severe reactions hydrocortisone will be given and the infusion will be 
discontinued and restarted at a slower rate once the symptoms have subsided. The patient is 
often treated in the ICU and may require pressors. The patient may require have one or two 
days of rest prior to restarting the ATG necessitating that the conditioning regime and 
radiation may be delayed by 1 – 3 days.  If this happens, the day of the transplant will be 
delayed by the number of days that the conditioning was delayed (by up to 3 days).  If a 
recipient has a persistent severe infusion reaction that does not respond to measures to 
ameliorate the signs/symptoms associated to the infusion, the infusion will be discontinued 
and further study participation will continue as planned with the exception that no further 
ATG will be administered. 
 
x 
Total body irradiation (TBI): Day -1: All recipients should be treated with a linear 
accelerator using energies higher than 4MV. Recipients will be treated with TBI to a total 
dose of 200 cGy delivered in a single fraction over 30 minutes on Day -1 in the Department 
of Radiation Oncology.  Equally weighted opposed lateral beams will be used to encompass 
the total body with the patient positioned supine. TBI will be delivered with lateral fields 
using extended SAD values of 600cm.  Tissue compensators will be used if clinically 
appropriate.  Gonadal shielding will not be used. Occasionally, the total dose/technique of 
TBI may require modifications due to patient factors (unexpected or serious (grade 4-5) 
adverse events, serious medical illnesses not conducive to stable patient transfer, patient 
refusal, etc.) or treatment factors (linear accelerator machine offline, etc.) Modifications to 
the radiation treatment will be at the discretion of the treating radiation oncologist and will be 
discussed with the PI. 
 
7.7 
Stem Cell Transplantation Day 0   
 
7.7.1 
Cord blood unit:  
 
Minimum total nucleated cells (TNC) (prior to thawing) of 1.5 x 107 per kilogram of 
recipient body weight with the following exception: if the minimum criterion of TNC is 
not met the cord unit must contain at least 1.7 x 105 CD34+ cells/kg (prior to thawing).  
For cord blood products, there is no upper limit (maximum TNC) for the UCB unit since 
cord units are inherently limited in their number of TNCs and studies to date have not 
identified any adverse related outcomes associated with using higher cord blood cell 
doses. 
 
Confirmatory typing reveals a minimum antigen level match in 4/6 HLA loci. Shipment 
of the cord blood unit will not be requested until the DTM confirms that the G-CSF 
mobilized T-cell depleted graft collected from the haploidentical donor contains a 
suitable number of CD34+ cells (≥2 x 106/kg) and CD3+ T-cells (<1 x 104 CD3+ cells/ 
kg)  (i.e. technical failure of the depletion process has not occurred). 
 

 
 
08-H-0046 
Richard Childs, M.D. 
May 13, 2019 (Amendment OO)  
26
 
7.7.2 
Haploidentical donor:  
 
The target, minimum and maximum cell doses (given in terms of recipient body weight) 
are: 
 
CD34+ cells 
 
 
 
 
Target dose: 3 x 106/kg 
 
 
Minimum for transplant  
 
2 x 106/kg 
 
 
Maximum 
5 x 106/kg  
 
CD3+ cells 
 
 
 
 
Target dose: 5 x 103/kg 
Maximum 
 
 
 
 
1 x 104/kg  
Minimum  
 
 
 
 
None 
 
If the final CD3+ dose is greater than 1 x 104/kg the CD34+ dose will be reduced to 
achieve the targeted CD3+ dose of 5 x 103/kg 
 
If the final CD3+ dose is greater than 1 x 104/kg after reducing the CD34+ dose to < 2 x 
106/kg, this will be designated a technical failure of the depletion process. In this event, 
the subject will not be enrolled.  The recipient will then be counseled regarding further 
available treatment options at the NIH or referred back to his/her primary care physician, 
whichever is in the best interest of the subject.  
 
 
7.8 
GVHD Prophylaxis:   
 
Tacrolimus:  Tacrolimus will be initiated at a dose of 0.03 mg/kg/day by continuous intravenous 
infusion over 24 hours starting on day -4. The tacrolimus dose will be adjusted to obtain goal 
tacrolimus therapeutic drug levels of 11-15 ng/mL.  When the recipient is able to take oral 
medications, the 24-hour dose of tacrolimus will be converted to oral dosing (generally using a 
1:3 or 1:4 IV to PO conversion or upon consultation with the clinical pharmacist) and 
administered in two divided doses every 12 hours. Tacrolimus will be continued for at least 6 
months.  NOTE: there may be some circumstances when it is medically indicated to stop 
tacrolimus before the time frame indicated here. 
 
Mycophenolate mofetil (MMF):  1 gram (or approximately 15mg/kg/dose) administered orally or 
intravenously twice daily starting day 0 and continued for 100 days (possibly longer depending 
on PI’s discretion).  NOTE: there may be some circumstances when it is medically indicated to 
stop MMF before the time frame indicated here. 
 
Prednisone: Oral prednisone at 1 mg/kg/day will begin on day -5 prior to the first dose of h-ATG 
for serum sickness prophylaxis, and will be continued at this dose for 10 days; thereafter, 
prednisone will be slowly tapered over the next 2 weeks until it is discontinued on day +19. 
Prednisone may be converted to an equivalent dose of methylprednisolone for intravenous 
administration if the recipient is unable to take oral medication. 
 
7.8 
Transfusion Support:  Filtered and irradiated blood products as needed. 
 
 
7.9 
Nutrition:  Parenteral nutrition will be instituted as necessary. 
   
7.10 
Hospital Discharge:  The recipient will be in the hospital for about 4 weeks and will be 
discharged when clinically indicated to follow up as an outpatient (resolution of neutropenia in 

 
 
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Richard Childs, M.D. 
May 13, 2019 (Amendment OO)  
27
the absence of fever and uncontrolled infection or other life threatening conditions that would 
require continued hospitalization). 
 
7.11 
Discharge Home:  The recipient will remain in the vicinity of the NIH through the day 180 (±30 
days) post-transplant assessments.  Thereafter, recipients in stable condition without uncontrolled 
infection, GVHD, and/or refractory cytopenias will be eligible to be discharged home. Recipients 
may require a longer stay in the NIH vicinity if clinically indicated. 
  
7.12 
Health Related Quality of Life Measures (adults only): 
 
PROMIS 29-Profile v1.0 and PROMIS v1.0-Applied Cognition-General Concerns-Short Form  
Patient-Reported Outcomes Measurement Information System (PROMIS ®), is an initiative 
based on an NIH grant to establish and provide the public a free, reliable and validated source for 
commonly used measures of patient-reported outcomes (PROMIS, 2011). The PROMIS item 
databanks have been tested for reliability and comparability to more established measures of 
these same content areas by NIH scientists and select US academic institutions using Item-
Response Theory (Baker, 2004).  
 
The PROMIS 29 item profile instrument is a collection of profile short forms assessing seven 
primary HRQL domains (depression, anxiety, physical function, pain interference, fatigue, sleep 
disturbance, and satisfaction with participation in social roles).  The PROMIS Applied Cognition-
General Concern measure assesses problems with memory and concentration (Riley et al., 2010). 
 
Functional Assessment of Chronic Illness Therapy-BMT (FACT-BMT) 
The Functional Assessment of Cancer Therapy –BMT version 4 (FACT-BMT) measures disease 
specific QOL in transplant recipients. The FACT-BMT is an extended version of The Functional 
Assessment of Cancer Therapy –General (FACT-G) with 12 additional items that are transplant 
specific (Cella, 2004). The FACT-BMT has demonstrated validity and has evidence of internal 
consistency and reliability in the BMT population and in Spanish speaking oncology patients 
(Cella, 2004; Cella et al., 1998).  
 
Sexual Functioning Questionnaire (SFQ) [subscales: Medical Impact and Problems, gender 
specific version] 
The Sexual Functioning Questionnaire is a 30-item measure, self-report questionnaire (Syrjala, 
2000). For this study we will use screening questions 1-3, the 9-item Problems, 2-item 
Satisfaction, and the 5-item Medical Impact subscales. Respondents will be given an option to 
“opt-out” of the questions. Internal consistency and reliabilities have been reported above .80 for 
all scales and the measure has demonstrated test-retest reliability. 
 
Chronic Graft-versus-Host Disease Symptom Checklist 
The c-GHVD symptom checklist is a 30 item, 7-subscale symptom self-report scale where 
transplant recipients evaluate symptom impact of chronic Graft-versus-host disease over the last 
month. Reliability and validity testing demonstrate that this measure is more specific and 
sensitive for identifying chronic versus host disease impact when compared to other quality of life 
measures such as the SF-36 and FACT-BMT. It has demonstrated an internal consistency 
(Cronbach’s D 0.79-0.85), test-re-test reliability on all subscales (0.74-0.83) (Lee et al., 2002). 
 
Completion of all measures is expected to take approximately 15-20 minutes.  The questionnaires 
will be sent to recipients for completion and return to NIH if the subject is unable or not 
scheduled to come to the Clinical Center for in-person follow-up. 
 

 
 
08-H-0046 
Richard Childs, M.D. 
May 13, 2019 (Amendment OO)  
28
8.0 
DONOR EVALUATION, STEM CELL COLLECTION AND PROCESSING PLAN 
 
 
8.1 
Pre-Study Consult and Evaluation (The following tests may be done under the 97-H-0041 
protocol) 
 
x 
History and physical examination 
x 
Chest X-ray in donors with underlying pulmonary disease or history of smoking 
x 
HLA typing class I (A, B, Cw) and Class II (DR,DQ) with high resolution DR, DQ High 
Resolution HLA, -A, -B, -C, -DR, -DQ typing  
x 
Low resolution class I A, B, C and class II DR,DQ and High resolution class II DR/DQ 
x 
Antibody screen Class I and Class II (A, B, C; DRB1; DQB1); HLA sequence base 
typing 
x 
Confirmatory typing when patient arrived at NIH, high resolution DR/DQ and sequence 
base A, B, C 
x 
Confirm HLA compatibility with recipient (>3/6 and < 5/6 HLA match) 
x 
Rule out sickling hemoglobinopathies including HbSS, HbSC by history and peripheral 
blood smear--donors with HbAS are acceptable 
x 
HbsAg, anti-HBc, anti-HCV, anti-HIV, anti-HTLV, anti-CMV, RPR, West Nile virus 
NAT (nucleic acid test), T.cruzi (Chagas), VZV serology--this screening for infectious 
disease markers will be repeated if more than 30 days have elapsed since baseline 
screening   
x 
Red blood cell ABO group, Rh type, antibody screen 
x 
CBC with differential,  
x 
Coagulation screen,  
x 
Acute, mineral, hepatic, uric acid, LDH, protein, cpk  
x 
pregnancy test (females of childbearing potential) 
x 
Profile STR 
x 
Fit to donate: Orientation - visit to Department of Transfusion Medicine - inspection of 
veins to determine the need for a central line for apheresis 
x 
Consent to undergo filgrastim (G-CSF) mobilization and donate leukocytes 
x 
KIR genotyping 
x 
If there is a suspicion of familial BMFS in the recipient, then the haplo donor must have 
undergone genetic testing for genes associated with BMFS – performed at a CLIA-
certified laboratory, prior to enrolling in this protocol (cohort 1 subjects only). Testing of 
genes related to inherited BMFS - telomere length, TERC and TERT mutations to be 
conducted on protocol 04-H-0012 or elsewhere prior to enrolling on 04-H-0012 to be 
eligible for this protocol 
x 
Research blood for laboratory studies 
 
8.2  
Pre-Consent Evaluation and Concurrent Care of Minor Donors (donors less than age 18  
 
only)  
 
 
For donors less than age 18, a social worker and mental health specialist (psychologist or 
psychiatrist) will meet with the minor prior to the assent process to confirm willingness to 
participate. 
 
 
For donors less than age 18, a pediatric provider (pediatrician, pediatric nurse practitioner or 
pediatric physician’s assistant) will be provided who is separate from the transplant team and is 
charged to consider the health and welfare of the minor donor. This practitioner will serve as the 
donor’s health care provider and advocate during the minor’s participation on the clinical trial. 

 
 
08-H-0046 
Richard Childs, M.D. 
May 13, 2019 (Amendment OO)  
29
 
8.3 
Apheresis of Haplo Donor for Lymphocytes (optional, ages 18 and older only) 
Collection of lymphocytes for haplo donor T cells (or lymphocytes) will be collected from the 
allograft, after CD 34 selection, for cryopreservation for studies of donor T cell alloresponses and 
post-transplant studies.  
 
8.4 
Donor Mobilization with G-CSF 
 
After medical evaluation and clearance for suitability as an allogeneic donor by the BMT service 
in consultation with DTM, the donor will undergo stem cell mobilization with filgrastim (G-CSF, 
Neupogen®, Amgen®) as an outpatient. Filgrastim (G-CSF) will be administered based on body 
weight (see below) for 5-7 days, subcutaneously. The doses for days 1-4 may be given at any 
time of day, but the doses for days 5, and 6, and if necessary day 7 must be given very early in the 
morning, prior to apheresis.  Predictable side effects of filgrastim (G-CSF), including headache, 
bone pain, and myalgia, will be treated with acetaminophen or ibuprofen. Prophylactic treatment 
of these side effects with the same medications may be elected.  Other side effects will be 
evaluated and treated accordingly. 
 
8.4.1 
Minor donor mobilization with G-CSF (≤ 18 years old) 
 
Filgrastim (G-CSF) will be administered according to a vial based algorithm to reduce wastage, 
improve donor compliance, and optimize CD34 yields. 
 
Donor Wt           Total filgrastim (G-CSF) Dose (range) 
15-18.9 kg 
   10 mcg/kg/dose 
19-30.9 kg 
 300 mcg (10.0 to 15.8 mcg/kg) 
31-37.9 kg 
 480 mcg (10.0 to 13.0 mcg/kg)                                                                           
 
 
38 – 48.9 kg           600 mcg (12.5 to 15.8 mcg/kg) 
49 – 56.9 kg           780 mcg (13.9 to 15.9 mcg/kg) 
57 – 60.9 kg           900 mcg (15.0 to 15.8 mcg/kg) 
61 – 67.9 kg           960 mcg (14.3 to 15.7 mcg/kg) 
68 – 108.9 kg         1080 mcg (10.0 to 15.9 mcg/kg) 
> 109 kg             1200 mcg (11.0 or less) 
 
 
8.4.2 Adult donor mobilization with G-CSF (> 18 years old) – per Supportive Care Guidelines  
 
Dose algorithms  
 
Two dosing algorithms are recommended for use in the Clinical Center. The standard dose and 
higher dose algorithm.  The higher dose algorithm is intended for use in donors whose 
components will undergo further processing.  As this study involves grafts that will undergo cell 
processing, the higher dose algorithm will be used for all donors enrolled on this protocol.  The 
total dose is capped at 1200 mcg/kg/day.  
 
I. Higher-Dose Filgrastim Algorithm  
 
Donor Weight   
 
Total Daily Filgrastim  
Dose (range)  
38 – 48.9 kg 
 
 
600 mcg  
 
 
(12.5 to 15.8 mcg/kg) 
49 – 56.9 kg 
 
 
780 mcg 
 
 
(13.9 to 15.9 mcg/kg) 

 
 
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Richard Childs, M.D. 
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57 – 60.9 kg 
 
 
900 mcg 
 
 
(15.0 to 15.8 mcg/kg) 
61 – 67.9 kg 
 
 
960 mcg 
 
 
(14.3 to 15.7 mcg/kg) 
68 – 108.9 kg  
 
1080 mcg 
 
 
(10.0 to 15.9 mcg/kg) 
 > 109 kg 
 
 
1200 mcg 
 
 
(11.0 or less) 
 
8.5 
Peripheral Blood Stem Cell Collection & Processing    
 
PBSC apheresis will be done on day 5, and if necessary on day 6 and day 7 of filgrastim (G-
CSF), i.e., after the 5th, and possibly the 6th, and if necessary the 7th day doses of filgrastim (G-
CSF) (which are given in the early morning).  All apheresis procedures will be done using a 2-
armed approach or by temporary central venous catheter in the femoral position, using the Baxter 
CS3000Plus or the Cobe Spectra.   The volume processed per apheresis procedure will be 
determined by DTM medical staff on the day of apheresis, based on peak CD34 cell mobilization 
response to G-CSF and the CD34 cell dose needed, based on kilogram weight of recipient.  This 
will range from 5 to 35 liters processed per day for 1 to 3 days, not to exceed a total of 75 liters 
over 3 days.  In pediatric subjects, defined as less than 40 kg, a maximum of 8 blood volumes will 
be processed per day, for up to 1 to 3 days. The second and third day apheresis collection will not 
be required if the CD34 dose is 3 x106/kg or more after the first day of collection.  
 
Anticoagulation will be accomplished with acid citrate dextrose (ACD-A).  If the donor is small 
or intolerant to ACD-A and the adverse citrate effects cannot be controlled by usual means 
(slowing flow rate, oral or IV calcium), consideration will be given to using heparin 
anticoagulation.  Donors will receive divalent cation prophylaxis to prevent citrate toxicity during 
apheresis, in accordance with standard DTM policies.    
 
If the minimum CD34+ cell dose is not achieved, a third apheresis may be performed (after an 
additional [7th] dose of filgrastim [G-CSF]), if it is predicted that the minimum CD34 cell target 
can be achieved in this way.     
 
If the minimum CD34+ cell dose from the haploidentical donor has not been achieved after one 
cycle of mobilization, a 2nd mobilization with up to two full apheresis collections to achieve the 
minimum CD34+ cell dose may be performed after an interval of at least 10 days or an alternative 
related haploidentical donor may be selected to undergo mobilization.  
 
Stem cell products will be cryopreserved after each collection and stored until transfusion into the 
recipient (see NIH Clinical Center, Department of Transfusion Medicine, Cell Processing 
Section’s SOPs).  From experience with over 100 stem cell processing procedures, we anticipate 
that in most cases both the CD34+ cell target and the CD3+ T cell target will be achieved.   
 
8.6 
Ex Vivo Processing of Peripheral Blood Stem Cells (PBSC) and Lymphocytes 
 
In general, the PBSC processing goals are to maximize CD34+ cell content and deplete CD3+ 
lymphocytes using the CD34-selection system from Miltenyi (see section 15).  From experience 
with over 100 stem cell processing procedures, we anticipate that in most cases both the CD34+ 
cell target and the CD3+ T cell target will be achieved.  In the majority of donors, peripheral 
blood stem cell processing with the Miltenyi system will result in doses of CD3+ cells well below 
1 x 104/kg. 
 
The erythrocyte content of the apheresis product is approximately 1.0 ml of packed RBCs per 
liter processed.  Although the typical RBC content of a 25-liter PBPC product would be about 25 
ml, processing by positive selection will typically result in a product with less than 1.0 ml of 

 
 
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RBCs.  Donor plasma will also be eliminated by the processing procedures. Therefore, even if 
there is ABO or other RBC incompatibility between donor and recipient, no specific processing 
or precautions will need to be taken with the PBSC product.  
 
8.7       Sterility and Viability Testing 
 
Sterility testing is done prior to cryopreservation. For the PBSC product, the results should be 
available before the final product is given to the recipient but the infusion date is variable and 
depends on the recipient's transplantation schedule. If the final result (no growth at 14 days) is not 
available before the final product is given to the recipient, the principal investigator (PI) is 
notified. If a culture is positive at any time during the incubation period, the microbiology testing 
laboratory performs a gram stain on the liquid media and notifies the cell therapy lab of the 
preliminary morphologic finding. The cell therapy lab will immediately notify the PI.  
Concurrently, the testing lab will plate the liquid media onto solid media for organism 
identification and sensitivity testing. These results are usually available within 48-72 hours. The 
results are immediately reported to the PI by the cell therapy lab personnel.  
 
Products released for infusion into the recipient are in full compliance with 21 CFR subpart C 
(1271.45-1271.90) which sets out the requirements for determining donor eligibility. 
 
Viability testing is performed with 7-AAD via flow cytometry.  
 
8.8 
Cryopreservation, Thawing and Quality Systems for the Stem Cell Product 
 
Stem cell products will be cryopreserved after each collection and stored until transfusion into the 
recipient.   
 
Post CD34 enrichment, PBSC products are usually cryopreserved in vials; rarely in 25ml bags. 
The container used at cryopreservation depends on the cell content of the product. Below is a 
table modified from DTM cryopreservation and thaw SOPs (DTM-SOP-5101 and 5102, 
respectively) which details the volumes and range of cell concentrations at cryopreservation and 
at infusion. Products cryopreserved in vials are transferred to another container for infusion. 
Transfers are performed in a biologic safety cabinet. Since the final product for infusion is 
transferred from the vial used to cryopreserve the product to another container, another sterility 
sample will be obtained. The sample for testing is obtained from rinsing the original 
cryopreservation container with the same diluent as used to dilute the final product.  50 ml bags 
will not be used for this protocol, but are included here for completeness.  
 
 
Cryo 
container 
Minimum 
concentration 
at cryo 
Maximum 
concentration 
at cryo 
Maximum 
vol at 
cryo 
Max 
TNC/ 
container 
Volume at 
infusion 
Maximum 
concentration 
of final 
product 
5 ml vial 
2.5e6/ml 
100e6/ml 
4.5ml 
450e6 
30 ml 
1.5e7/ml  
Bag, 25ml 
20e6/ml 
300e6/ml 
25 ml 
7.5e9 
31 ml 
2.4e8/ml 
Bag, 50ml 
20e6/ml 
300e6/ml 
50 ml 
1.5e10 
62 ml 
2.4e8/ml 
TNC=total nucleated cell count 
 
All products will be prepared for infusion by standard operating procedures of the DTM Cell 
Processing Section. Detailed information is provided by the Drug Master File BB MF 11054 
“Facility, Operational and Quality Systems for Manufacture of HCT/Ps” of the DTM Cell 
Processing section (FDA registration #1174694). This includes a detailed description of the 

 
 
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DTM’s quality assurance plan, facilities, environmental control and monitoring, operational 
control systems and aseptic processing, equipments, supplies and eligibility, manufacturing 
systems, process development, process validation, process control and change control, product 
evaluation and lot release, storage, product labeling, label controls and tracking, product receipt 
and distribution, final product preparation, issue and administration, environmental impact and 
good laboratory practice statements.  
 
8.9 
Donor Monitoring and Follow-up Plan 
 
Donors will be contacted by our clinical staff within 48 hours after their cell collection. Donors will 
remain on study for one year following enrollment. In the event that a recipient needs a stem cell 
boost following transplant, the donor may re-sign onto the protocol if the one-year time frame has 
elapsed.   
 
9.0 
UMBILICAL CORD BLOOD PROCESSING 
 
Pre-cryopreservation: Only licensed cord blood units will be used or if a licensed cord blood unit 
is not available, umbilical cord blood (UCB) units will be obtained through the NMDP multicenter 
access and distribution protocol for unlicensed cyropreserved cord blood units.  Cord blood units 
will be collected and cryopreserved according to the Cord Blood Bank Participation Criteria, 
including Policies and Procedures stated therein, and the NMDP requirements for product release. 
Donor eligibility determination is performed according to NMDP standards and/or FACT-Netcord 
guidelines in the case of cord blood units. The NMDP standards are set by a board of NMDP 
network representatives including cord blood banks, transplant centers, donor and recruitment 
centers, and follow the most recent FDA eligibility guidelines.    
 
The table below details the infectious disease marker testing and acceptance criteria (suitability 
criteria for cord blood units): 
Test 
NMDP action 
HBsAg 
Accept non-reactive 
Anti-HBc 
Accept non-reactive 
Anti-HCV 
Accept non-reactive 
Anti-HIV1/2 
Accept non-reactive 
HIV p24Ag 
Performed if HIV-1 NAT testing is not performed 
Accept non-reactive 
HIV-1 and HCV 
Accept non-reactive 
Anti-HTLV I/II 
Accept non-reactive 
Anti-CMV IgG or total 
Accept non-reactive 
Accept reactive 
Serologic syphilis 
Accept non-reactive 
 
Ordering procedures: Jennifer Wilder, the NIH Certified Hematopoietic Umbilical Cord 
Transplant Coordinator will conduct the search and arrange for procurement of unrelated umbilical 
cord product for this protocol. Once a suitable cord blood product is identified, shipment of the 
cord blood unit will not be requested until the DTM confirms that the G-CSF mobilized T-cell 
depleted graft collected from the haploidentical donor contains a suitable number of CD34+ cells 
(≥2 x 106/kg) and CD3+ T-cells (<1 x 104 CD3+ cells/ kg) (i.e. technical failure of the depletion 
process has not occurred). Once CD34+ cell content is confirmed, the cord blood unit will be 
shipped to the Department of Transfusion Medicine Cell Processing Section (CPS) per NMDP 
procedures, in dry shippers.  
 

 
 
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Storage of cord blood units: Upon arrival, the UCB units will be transferred to LN2 storage in 
CPS until date of infusion. Prior to infusion, the PI, in consultation with CPS, will evaluate the 
RBC content, if ABO incompatible, to determine whether washing the UCB to remove excess 
RBC is warranted. Whether the UCB units are thawed and released for infusion with or without 
washing, UCB units will be stored, processed and released for infusion per DTM CPS procedures 
(see NIH DTM SOP 5107 [product receipt] and NIH DTM SOP 5102 [thaw and infusion]). 
 
Post-cryopreservation: Each product is thawed and prepared for immediate infusion. Sterility and 
viability testing is done post thaw as is a progenitor cell assay (CFU) and a complete cell count.  
The cord units will be infused with a goal viability of >= 70%. Cord units with lower than this 
viability will still be infused and we will track the outcome of these poorer viability units and 
report them in our annual report. Viability is performed with 7-AAD via flow cytometry. The 
sample is obtained post thaw, post dilution. 
 
Laboratory staff coordinates closely with the nursing staff administering the product such that the 
product is thawed and delivered to the recipient’s bedside for immediate infusion.  Product is not 
stored post thaw.  
 
 
10.0 
MANAGEMENT OF COMPLICATIONS 
 
The major complications are acute and chronic GVHD, reactivation of cytomegalovirus (CMV), 
and graft failure. Subjects with these complications will be treated as follows: 
 
10.1 
Acute GVHD (See PBSCT Supportive Care Guidelines) 
 
10.2 
Chronic GVHD (See also PBSCT Supportive Care Guidelines) 
 
x 
Tacrolimus and/or cyclosporine at standard dose. 
x 
Prednisone dosed according to severity. 
x 
Change to alternate day steroid and Tacrolimus/cyclosporine therapy when response is 
established. 
x 
Non-responding subjects may be treated with other standard of care therapies including 
but not limited to PUVA, mycophenolate, azathioprine, photopheresis, daclizumab, 
infliximab, rituximab, or thalidomide at the discretion of the attending physician.   
 
10.3 CMV Reactivation (see section 7.3 Infection Prophylaxis and Treatment) 
 
10.4 
EBV reactivation (see section 7.3 Infection Prophylaxis and Treatment) 
 
10.5 
Graft Failure/Relapse of Marrow Aplasia 
 
Recipients who fail to achieve a neutrophil count of ≥500 cells/μl by day 42 post transplant will 
be deemed to have primary graft failure. Subjects with primary graft failure will be treated with 
supportive care (transfusions, growth factors, etc) when clinically indicated.  If the subject is 
found to have one or more additional HLA compatible (i.e. > 4/6) cord blood units, and remains 
neutropenic, they will be offered co-enrollment onto the Branch’s standard of care protocol where 
they may be treated with a subsequent single or double cord unit transplant using a conventional 
transplant approach. Subjects who are not a candidate for a salvage cord blood transplant will be 
treated with supportive care and may be referred back to their referring physician if it is in the 
best interest of the subject. Data collection to include outcome and survival will continue, 

 
 
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however complications following standard of care therapies will be reported per the standard of 
care protocol.   
 
Transplant recipients with secondary graft failure may be treated with a stem cell boost.  
Secondary graft failure is defined as the development of sustained cytopenia and/ or continued 
requirement for platelet or red blood cell transfusions after initial successful primary engraftment.  
In this setting, a boost of hematopoietic stem cells from the original stem cell donor may be 
subsequently administered to the recipient. This process will involve the infusion of CD34+ cells 
that are collected from the original haploidentical stem cell donor following conventional GCSF 
mobilization that are CD34 selected using the Miltenyi CliniMACS system. In the event the 
transplant recipient is unable to return to the Clinical Center for this infusion (i.e., due to 
hospitalization related to illness) these cells may be shipped by the Department of Transfusion 
Medicine to a hospital with a transfusion service for infusion consistent with their Human 
Subjects Protection Policies. 
 
10.6 
Pulmonary Engraftment Syndrome   
 
Subjects who develop pulmonary engraftment syndrome (most likely 10-25 days post transplant) 
will be treated with steroids.  
 
11.0 
ANCILLARY LABORATORY RESEARCH STUDIES 
   
During the course of study participation, specimens will be collected for correlative studies. 
 
11.1 
Collection, Storage and Disposition of Samples 
Intended use: These specimens will not be read by a pathologist or used for diagnostic purposes.   
These studies will not be used in assessing the primary endpoint but are undertaken for 
descriptive or exploratory ancillary research, and have been approved by the NIH Intramural IRB 
and are listed in the Appendix of the protocol. 
Hemorrhagic cystitis study:  We will share the following de-identified research samples with Dr 
Christopher Buck’s laboratory at NCI: blood, urine, serum, plasma, CSF, Lung lavage fluid.  His 
lab will perform deep sequencing on DNA extracted from BKV viral particles using the Illumina 
Nextera/MiSeq platform.  BKV neutralization serology will also be performed. 
Hepatitis E research study: We will share de-identified plasma samples with Dr A. Gretchen 
Buckler’s laboratory at DTM/Clinical Center.  Her lab will test them for hepatitis E RNA and, if 
they find positives, will work towards setting up a prospective trial to determine if future patients 
show evidence of contracting hepatitis E through transfusion using linked donor-recipient pairs.  
Etiology of post-transplant syndromes: We will share de-identified blood plasma and/or serum 
with Dr. Waldman at NIDDK.  Dr. Waldman will use blood plasma and or serum to conduct 
studies on the etiology of post-transplant syndromes that cause renal failure including nephrotic 
syndrome, TMA, and glomerulonephritis. 
Tracking:  Samples will be ordered and tracked through the CRIS research screens.  Should a 
CRIS screen not be available, the NIH form 2803-1 will be completed and will accompany the 
specimen and be filed in the medical record.  Samples will not be sent outside NIH without IRB 

 
 
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notification and an executed MTA.  In accordance with the NHLBI Biospecimen Inventory 
system (BSI) policy, the specimens are inventoried in BSI.   
Storage:  Research samples will be stored with identifiers in the secure laboratory of the 
principal investigator.  
End of study procedures:  Samples from consenting subjects will be stored until they are no 
longer of scientific value or if a subject withdraws consent for their continued use, at which time 
they will be destroyed.  
Loss or destruction of samples:  Should we become aware that a major breech in our plan for 
tracking and storage of samples has occurred, the IRB will be notified. 
11.2 
Research Sample Collection 
 
11.2.1   Recipient peripheral blood samples:  60 ml peripheral blood will be collected at days 15, 
30, 42, 60, 100 (+/- 7 days for all) and then at scheduled annual follow-up visits for 
chimerism analysis lymphocyte subset analysis.   
 
Subjects will be offered co-enrollment on protocol 07-I-0183: The collection of research 
samples and data for repository from unrelated hematopoietic stem cell transplantation 
recipients for the National Marrow Donor Program.  If consented to participate on this 
ancillary study, one ACD (acid citrate dextrose) anti-coagulated blood-20 ml drawn in a 
yellow top tube - will be sent to the NMDP research sample repository.  A MTA for these 
samples has been executed.  In pediatric recipients, the amount drawn will be 1-20 ml, 
not to exceed 3ml/kg (the NMDP has no minimum blood sample size from a pediatric 
subject). 
 
11.2.2 Recipient apheresis: One apheresis collection of approximately 1010 leukocytes in a 
volume of 200 ml, pre-transplant.  
 
11.2.3 Donor blood sample: Cells obtained during the apheresis procedures (stem cells and 
lymphocytes) additional to those required for the transplant recipient will be used for 
research studies.    
 
11.2.4 Recipient  bone marrow samples: An extra volume (up to 25 ml) of bone marrow aspirate 
will be collected for research studies at the pre transplant evaluation and when collecting 
for monitoring disease. The cells will be used to investigate lymphocyte interactions with 
bone marrow progenitor cell proliferation. No marrow aspirates solely for research 
purposes are planned. 
 
11.2.5     Pulmonary function tests 
 
Pulmonary status at baseline, day 30 and after day 100 will be analyzed to see if reduced 
intensity conditioning (RIC) effects lung function before day 100 or if the decline in lung 
function is only seen later, consistent with bronchiolitis obliterans syndrome (BOS).   
Pulmonary status will be assessed in all subjects except for those under the age of 10 
where prior attempts have failed because of the subject’s inability to perform the 
pulmonary function test. 
 
12.0   BIOSTATISTICAL CONSIDERATIONS  

 
 
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12.1   Primary Endpoint 
 
The primary endpoint is donor engraftment by day 42 +/- 3 days (defined as an ANC of >500) 
from either the haplo donor, the cord, or both combined.  
 
12.2      Secondary Endpoints 
 
Secondary endpoints will include: 
x 
ANC recovery rate (ANC ≥ 500 cells/Pl) at day 22, 100 day (+/- 3 days) and 6 months 
(+/- 2 weeks)  
x 
treatment related mortality (TRM) 
x 
standard transplant outcome variables such as non-hematologic toxicity, incidence and 
severity of acute and chronic GVHD, and relapse of disease.  
 
Health related quality of life will also be assessed as secondary outcome measures.  The 
following parameters will be recorded: 
 
x 
CD34 + cell dose, CD3 + cell dose 
x 
Degrees of cord blood, related haplo-CD34+ cell donor and recipient chimerism in T-
lymphocyte, NK cell, and myeloid lineages assessed by PCR of short tandem repeats 
(STRs) 
x 
Neutrophil recovery (days to neutrophil count of 0.5 x 109/l) 
x 
Days to an UCB neutrophil count of 0.5 x 109/l  
x 
Platelet recovery (days to platelet count of 20 x 109, days to transfusion independence) 
x 
Red cell recovery (days to transfusion independence)  
x 
Incidence and severity of acute and chronic GVHD (the DSMB will monitor for evidence 
of excessive GVHD)  
x 
Graft failure (loss of the graft as measured by chimerism) 
x 
Cumulative incidence of late graft failure 
x 
Time to transfusion independence 
x 
Non-hematological effects attributable to the preparative regimen 
x 
Disease progression or relapse 
x 
Transplant related mortality before and after day 100 and day 200 
x 
Disease-free survival and overall survival. 
x 
Health related quality of life measurements post-transplant 
 
12.3 
Sample Size 
 
We require engraftment by day 42 in at least 80% of recipients and wish to insure that 
engraftment is not as low as 60%. Let p denote the probability of engraftment by day 42. We will 
test the null hypothesis that p is less than or equal to 0.6 versus the alternative hypothesis that p is 
at least 0.8. We choose a two-stage minimax design [102] which allows early stopping if too few 
recipients engraft and minimizes the maximum sample size subject to alpha = 0.05 and power = 
0.80. 
 
In the first stage we will enter (up to) 13 recipients. If 8 or fewer have engraftment by day 42, we 
stop early because the procedure is not successful enough. This implies that we must have at least 
9 of the first 13 recipients with engraftment by day 42. If at least 9 recipients have engraftment by 
day 42, we will enter an additional 22 recipients. If 25 or fewer of the total of 35 recipients have 
engraftment by day 42, we consider the procedure unsuccessful (rejecting the alternative 

 
 
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hypothesis), but if at least 26 of the 35 recipients have engraftment by day 42, then we consider 
the procedure successful. 
 
To account for the possibility of non-treatment related deaths before day 42, an additional 5 
subjects may be enrolled to replace those subjects that are non-evaluable for the study’s primary 
endpoint.  
 
12.4     Methods of Statistical Analysis  
 
Although the primary endpoint for this protocol is persistent engraftment by day 42, subjects will 
be followed up for 60 months on protocol and then contacted indefinitely to monitor the 
incidence of chronic GVHD, long-term disease-free and overall survival. 
 
At the completion of follow-up for all subjects, time-to-event distributions (disease-free survival, 
non-relapse mortality and overall survival) will be estimated using the Kaplan- Meier method or 
competing risk methods, as appropriate, and 95% confidence intervals for the median time to 
events will be calculated. 
 
12.5     Interim Analysis and Stopping Rules  
  
Interim analysis for efficacy.   
An interim subgroup analysis is planned when at least 50% of recipients are evaluable for 
primary endpoint.  A modified Haybittle-Peto procedure with Bonferroni adjustment will be 
used:  we would require a conservative p-value <0.001 to declare significance and reject the null 
hypothesis of primary endpoint at the interim analysis.   We would not stop the trial based on 
early evidence of clinical benefit at the interim subgroup analysis, and we would continue 
enrollment to the original planned sample size.  At the final analysis we would require p<0.049 to 
declare significant improvement in the primary endpoint.  
 
Stopping rule for early death: If there are 5 treatment related deaths (TRM) prior to day 42, we 
will stop the trial early.  Early deaths determined to be related to treatment will be included in the 
stopping rules for safety. If we are convinced that a death before day 42 is unrelated to treatment, 
we will ask the IRB to review the data and permit continuation if deemed appropriate.  
 
Stopping rule for futility: In the first stage, if 5 recipients fail to have engraftment by day 42, we 
will stop the trial early (even before all 13 recipients are enrolled). If at least 9 recipients have 
engraftment by day 42, we will start the second stage. Then if there are a total of 10 or more 
recipients who fail to have engraftment by day 42 in the first and second stages, we stop the trial 
early (even before additional 22 recipients are enrolled in the second stage). 
 
Stopping rules for safety:   We wish to assure that this procedure has less than 50% day 200 
TRM. We will use a Bayesian stopping rule. The stopping boundary is reached if the Bayesian 
posterior probability that the true probability of 200 day TRM exceeds 0.45 is at least 90%. We 
take our prior distribution to be a beta distribution so that our prior clinical opinion is worth 20% 
of the weight we will place on the new data. This gives prior parameters alpha = 4.05, beta = 
4.95.We will begin monitoring 200 day TRM when 5 recipients are evaluable for 200 day TRM. 
 
The following table summarizes the threshold numbers for the boundary, which would lead to a 
recommendation to stop the study due to excess day 200 TRM. 
 

 
 
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Number of subjects 
Stop if the number who have developed 200 
day TRM reaches 
 Up to 5 
5 
6-7 
6 
8-9 
7 
10-11 
8 
12-13 
9 
14-15 
10 
16-17 
11 
18-19 
12 
20-21 
13 
22-23 
14 
24-25 
15 
26-27 
16 
28-29 
17 
30-31 
18 
32-33 
19 
34-35 
20 
 
We conducted a simulation to evaluate this stopping rule. We generated a study with 35 independent 
Bernoulli trials, with probability p for 200 day TRM and determined whether the study was stopped. We 
repeated the simulations 100,000 times. When the probability of 200 day TRM was 0.4, the probability of 
stopping the study was 0.10.  When the probability of 200 day TRM was 0.5, the probability of stopping 
the study was 0.40.  When the probability of 200 day TRM was 0.6, the probability of stopping the study 
was 0.78. When the probability of 200 day TRM was 0.7, the probability of stopping the study was 0.98.   
Hence we believe that this stopping rule has satisfactory statistical properties. 
 
In addition, the DSMB will evaluate all treatment related serious adverse events (TRSAEs).  The DSMB 
may recommend early study termination of the trial if other unforeseen adverse events necessitate this 
decision.    
 
12.6   Off Study Criteria 
 
 
12.6.1 Withdrawal per subject choice 
  
Recipients and their donors will be given ample opportunity to withdraw from the study 
prior to admission for conditioning and transplantation. Should they wish to withdraw 
prior to transplant they will be replaced in order to maintain appropriate statistical power 
to evaluate primary endpoints. After the transplant, the risks of withdrawal will be 
carefully explained. If the recipient chooses to withdraw before the primary endpoint of 
the study is reached, the recipient will be followed in the safety analysis. 
 
Without compromising their participation in the treatment protocol recipient and donor 
may at any time withdraw from participation in the ancillary laboratory research aspects 
of the protocol. 
 
12.6.2 Withdrawal by principal investigator decision  
 
Transplant recipients who  
x 
experience disease progression or relapse  
x 
fail to achieve hematological recovery (graft failure) or  

 
 
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x 
experience a significant decline in performance status which negates further 
treatment on protocol  
 
will be offered co-enrollment onto the standard of care protocol, evaluated for 
participation on another primary treatment protocol or referred back to their home 
physician depending on what is in the best interest of the recipient. Data collection to 
include outcome and survival will continue, however complications following alternative 
therapies will not be reported on this protocol. 
  
12.6.3 Completion of the study 
 
Following the completion of 5 years of follow up care the recipient will go off study and be 
referred back to their primary care physician.  Yearly communication with the recipient will be 
continued since outcome data for the recipient’s life span is required by the NMDP.  Donors will 
remain on study for one year following enrollment. In the event that a recipient needs a stem cell 
boost following transplant, the donor may re-sign onto the protocol if the one year time frame has 
elapsed.   
 
13.0 
DATA AND SAFETY MONITORING 
 
13.1 
Safety Monitoring 
 
Principal Investigator: Accrual and safety data will be monitored by the PI. The protocol will be 
continuously evaluated for any unusual or unpredicted complications with the aim of detecting 
and preventing unacceptable increase in morbidity and mortality over and above that anticipated 
from unmanipulated bone marrow transplants (see stopping rules in section 12.5). 
 
 NIH Intramural IRB. Accrual and safety data will be monitored reviewed annually by the 
Institutional Review Board (IRB). Prior to implementation of this study, the protocol and the 
proposed subject consent and assent forms will be reviewed and approved by the properly 
constituted Institutional Review Board (IRB) operating according to the 45 CFR 46 Code of 
Federal Regulations.  This committee must approve all amendments to the protocol or informed 
consent, and conduct continuing annual review so long as the protocol is open to accrual or 
follow up of subjects.  
 
DSMB: The NHLBI Data Safety and Monitoring Board will review the protocol at six or twelve 
month intervals.  A progress report will be forwarded to the DSMB at these times. The DSMB 
may recommend early termination of the study for considerations of safety and efficacy.  
 
Monitoring:  As per ICH-GCP 5.18 and FDA 21 CFR 312.50, clinical protocols are required to 
be adequately monitored by the study sponsor.   The monitoring of this study will be conducted 
by Clinical  Research Associates (CRAs)/Monitors employed by an independent contract 
organization working under an agreement with NHLBI to monitor aspects of the study in 
accordance with the appropriate regulations and the approved protocol. The objectives of a 
monitoring visit will be: 1) to verify the existence of signed informed consent form (ICF) and 
documentation of the ICF process for each monitored subject; 2) to verify the prompt and 
accurate recording of all monitored data points, and prompt reporting of all SAEs; 3) to compare 
abstracted information with individual subjects’ records and source  documents (subject’s charts, 
laboratory analyses and test results, physicians’ progress notes, nurses’ notes, and any other 
relevant original subject information); and 4) to help ensure investigators are in compliance with 
the protocol. The monitors also will inspect the clinical site regulatory files to ensure that 

 
 
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regulatory requirements (Office for Human Research Protections-OHRP and FDA) and 
applicable guidelines (ICH-GCP) are being followed. During the monitoring visits, the 
investigator (and/or designee) and other study personnel will be available to discuss the study 
progress and monitoring visit. 
 
The investigator (and/or designee) will make study documents (e.g., consent forms and pertinent 
hospital or clinical records readily available for inspection by the local IRB, FDA, the site 
monitors, and the NHLBI staff for confirmation of the study data. 
 
FDA (IND 13500):  An annual progress report, amendments to the protocol, and changes in the 
status of the protocol will be forwarded to FDA to:   
 
 
 
 
 
 
Candace Jarvis 
 
 
Center for Biologics Evaluation and Research (CBER) 
 
 
Office of Cellular and Gene Therapy 
 
 
Document Control Center, HFM-99, Suite 200N  
 
 
 
1401 Rockville Pike 
 
 
Rockville, MD 20852-1448 
 
 
Phone: 301-827-5357 
 
Miltenyi Biotec, Inc.: An annual progress report, amendments to the protocol, and change in the 
status of the protocol will be forwarded to Miltenyi Biotec, Inc to 
 
 
 
Tara Clark, General Manager US Clinical Operations 
  
 
and Norman Pilon, PhD  
 
 
Miltenyi Biotec, Inc. 
 
 
85 Hamilton Street,Cambridge, MA 02139. 
 
 
Phone (617) 218-0060 
 
13.2 
Definitions 
 
Adverse Event (AE):  Any untoward medical occurrence in a human subject, including any abnormal sign 
(e.g., abnormal physical exam or laboratory finding), symptom, or disease, temporally associated with the 
subject’s participation in the research, whether or not considered related to the subject’s participation in 
the research. 
An abnormal laboratory value will be considered an AE if the laboratory abnormality is characterized by 
any of the following: 
 
x 
Results in discontinuation from the study 
x 
Is associated with clinical signs or symptoms 
x 
Requires treatment or any other therapeutic intervention 
x 
Is associated with death or another serious adverse event, including hospitalization 
x 
Is judged by the Investigator to be of significant clinical impact 
x 
If any abnormal laboratory result is considered clinically significant, the investigator will provide 
details about the action taken with respect to the test drug and about the patient’s outcome. 
 
Serious Adverse Event (SAE):  A serious adverse event that: 
x 
results in death; 
x 
is life-threatening (places the subject at immediate risk of death from the event as it occurred); 
x 
results in in-patient hospitalization or prolongation of existing hospitalization; 

 
 
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x 
results in a persistent or significant incapacity; 
x 
results in a congenital anomaly/birth defect; or 
x 
based upon appropriate medical judgment, may jeopardize the subject’s health and may require 
medical or surgical intervention to prevent one of the other outcomes listed in this definition. 
 
Suspected adverse reaction: Suspected adverse reaction means any adverse event for which there is a 
reasonable possibility that the drug caused the adverse event. For the purposes of IND safety reporting, 
‘reasonable possibility’ means there is evidence to suggest a causal relationship between the drug and the 
adverse event. A suspected adverse reaction implies a lesser degree of certainty about causality than 
adverse reaction, which means any adverse event caused by a drug. 
 
Serious event: An event is serious if it meets the definition of a serious adverse event (above) or if it 
requires immediate corrective action by a PI and/or IRB to protect the safety, welfare or rights of subjects. 
 
Unexpected adverse reaction: An adverse event or suspected adverse reaction is considered “unexpected” 
if it is not listed in the investigator brochure or is not listed at the specificity or severity that has been 
observed; or, if an investigator brochure is not required or available, is not consistent with the risk 
information described in the general investigational plan or elsewhere in the current application. 
"Unexpected”, also refers to adverse events or suspected adverse reactions that are mentioned in the 
investigator brochure as occurring with a class of drugs or as anticipated from the pharmacological 
properties of the drug, but are not specifically mentioned as occurring with the particular drug under 
investigation. 
 
Unanticipated Problem (UP):  Any incident, experience, or outcome that meets all of the following 
criteria: 
1. unexpected in terms of nature, severity, or frequency in relation to 
a. the research risks that are described in the IRB-approved research protocol and informed 
consent document; Investigator’s Brochure or other study documents; and 
b. the characteristics of the subject population being studied; and 
2. related or possibly related to participation in the research; and 
3. places subjects or others at a greater risk of harm (including physical, psychological, economic, or 
social harm) than was previously known or recognized. 
 
Unanticipated Problem that is not an Adverse Event:  An unanticipated problem that does not fit the 
definition of an adverse event, but which may, in the opinion of the investigator, involves risk to the 
subject, affect others in the research study, or significantly impact the integrity of research data.  For 
example, report occurrences of breaches of confidentiality, accidental destruction of study records, or 
unaccounted-for study drug. 
 
Protocol Deviation (PD):  Any change, divergence, or departure from the IRB approved research 
protocol. 
 
Non-compliance: The failure to comply with applicable NIH HRPP policies, IRB requirements, or 
regulatory requirements for the protection of human research. Noncompliance may be further 
characterized as: 
 
1. Serious non-compliance: Non-compliance that: 
a. Increases risks, or causes harm, to participants. 
b. Decreases potential benefits to participants. 
c. Compromises the integrity of the NIH HRPP. 
d. Invalidates the study data. 

 
 
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2. Continuing non-compliance: Non-compliance that is recurring. An example may be a pattern of 
non-compliance that suggests a likelihood that, absent an intervention, non-compliance will 
continue. Continuing noncompliance could also include a failure to respond to IRB requests to 
resolve previous allegations of non-compliance. 
 
3. Minor (non-serious) non-compliance: Non-compliance that, is neither serious nor continuing. 
 
13.2.1 Event Characterization and Reporting to the IRB and Clinical Director (CD)  
Approved by HSRAC on September 30, 2013 
Date effective: October 28, 2013 
 
All adverse events occurring during the study, including those observed by or reported to the research 
team, will be recorded.  Serious unanticipated problems, and serious protocol deviations, will be reported 
to the IRB and Clinical Director as soon as possible but not more than 7 days after the PI first learns of 
the event.  Not serious unanticipated problems will be reported to the IRB and Clinical Director as soon 
as possible but not more than 14 days after the PI first learns of the event.  Not serious protocol deviations 
will be reported to the IRB as soon as possible but not more than 14 days after the PI first learns of the 
event.  SAEs that do not meet the criteria of Unanticipated Problem (UP) must be reported to the Clinical 
Director within 14 days of learning of the event. 
 
Deaths will be reported to the Clinical Director within 7 days after the PI first learns of the event. 
 
 
13.3 
Adverse Events –Transplant Recipients  
 
13.3.1 Adverse Event Reporting and Collection 
 
Adverse events used to evaluate the safety of this protocol regimen will be collected to include 
any unfavorable and unintended signs (including abnormal laboratory findings), symptoms and/or 
diseases (i.e. incidence of GVHD, graft failure, regimen related toxicities, or infectious 
complications) which either occur during the study, having been absent at baseline or if present at 
baseline, appear to worsen with the exceptions listed below.  The AE reporting period for this 
study begins when the patient starts the preparative regimen, except for AEs at least possibly 
related to a study procedure performed prior to the start of the preparative regimen (i.e., 
apheresis).  AEs will be collected until completion of the 1 year follow up.  After that time, only 
SAEs that are at least possibly related to the transplant will be recorded and reported to the IRB.  
The AEs will be attributed (unrelated, unlikely, possibly, probably or definitely) to study 
medication and graded by severity utilizing NCI Common Terminology for Adverse Events 
(CTCAE) version 3.0.  A copy of the criteria can be down-loaded from the CTEP home page at   
http://ctep.cancer.gov/reporting/ctc.html.   
 
Events not included in the CTCAE will be graded as follows: 
 
Grade 1: Mild: discomfort present with no disruption of daily activity, no treatment 
required beyond prophylaxis 
 
Grade 2: Moderate: discomfort present with some disruption of daily activity, require 
treatment 
 
Grade 3: Severe: discomfort that interrupts normal daily activity, not responding to first 
line treatment 
 
Grade 4: Life-threatening: discomfort that represents immediate risk of death 
 

 
 
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The following are expected outcomes for the transplant recipient and will be documented in the 
subject's medical record, but will not be collected or reported to IRB unless they meet the criteria 
for an SAE and/or UP: 
 
x 
Renal insufficiency 
x 
Hepatic insufficiency 
x 
Transient cardiac arrhythmias 
x 
Transient cardiac insufficiency 
x 
Pulmonary insufficiency 
x 
Neutropenia and its complications 
x 
Thrombocytopenia and its complications 
x 
Anemia and its complications 
x 
Transfusion reactions 
x 
Treatable infections from bacteria, viruses, protozoa and fungi 
x 
Late effects of transplant regimens including: cataracts, infertility, growth  
  
impairment, hypothyroidism, and dental caries 
x 
Headache, insomnia, psychosis, mood changes, disorientation, seizures from metabolic 
imbalance 
x 
Nausea, vomiting, diarrhea, mucositis, weight loss, dry mouth, hiccoughs, constipation 
x 
Well-characterized drug reactions - allergic manifestations, "red man" syndrome 
x 
Well-characterized drug side effects from drugs used routinely in transplant recipients 
(e.g.; preparative regimen chemotherapy, immunosuppressive drugs, antimicrobials)  
x 
Common side effects of antiemetics, analgesics, anti-inflammatory agent and known 
complications of steroid therapy 
x 
Complications from intravenous catheters, thrombotic occlusion, infection, local 
reactions, cardiac arrhythmia  
 
The following are expected outcomes related to cord blood infusion and will be documented in 
the subject's medical record, but will not be collected or reported to the IRB unless they meet the 
criteria for an SAE and/or UP: 
 
x 
Changes in heart rate or rhythm  
x 
Changes in blood pressure 
x 
Fever, chills, sweats 
x 
Nausea, vomiting, diarrhea, abdominal cramping, fluid overload 
x 
Headache 
x 
Dyspnea 
x 
Hemoglobinuria 
x 
Allergic reaction 
 
 
The following are expected outcomes that will be collected and reported in summary form 
at the time of continuing review but will not be reported to IRB at each occurrence unless 
they meet the criteria of an SAE and/or UP: 
 
x Acute graft-versus-host disease 
x Chronic graft-versus-host disease 
x Graft failure/graft rejection 
x Veno-occlusive disease 

 
 
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x Hemorrhagic cystitis 
x Nephrotic syndrome 
x Cytomegalovirus reactivation or disease 
x EBV reactivation or disease 
x Invasive fungal infections 
x Disease relapse or progression 
x Secondary hematologic and/or solid tumor malignancy  
x Pre-malignant conditions such as myelodysplastic syndrome 
x Hemophagocytic Lymphohistiocytosis (HLH) 
 
13.4 Adverse Events - Donors 
 
The following are expected outcomes for the donor that will not be collected or reported to 
the IRB unless they meet the criteria of an UP: 
 
• Common side effects of filgrastim (G-CSF) administration (bone pain, fatigue, 
arthralgias, headache, insomnia, fever, worsening of pre-existing skin rashes, increases of 
alkaline phosphatase, lactate dehydrogenase and/or uric acid levels, elevated blood 
leukocyte count, or thrombocytopenia 
• Hypotension during apheresis 
 
The following are expected outcomes for the donor that will be collected and reported in 
summary form at the time of continuing review but will not be reported to IRB at each 
occurrence unless they meet the criteria of an UP:  
 
  
• 
Ischemic chest pain during filgrastim (G-CSF) administration 
• 
Splenic enlargement 
• 
Cutaneous vasculitis 
• 
Bone pain, muscle aches or headaches no controlled with non-narcotic analgesics 
 
13.5 
Reporting of SAEs and UPs 
 
All serious adverse events will be reported to  
Richard Childs, M.D.; Principal Investigator 
Bldg 10, CRC, Room CRC 3-5332 
Phone: 301-594-8008 
 
 
FDA (IND 13,500) SAEs that are unexpected and suspected (original and 2 copies) will also be 
forwarded as soon as possible and no later than seven calendar days in the case of death or 
life-threatening serious adverse events or within fifteen calendar days after the occurrence 
of all other forms of serious adverse events to the FDA. A summary of all SAEs will be 
included with the annual progress report to the FDA.  
 
 
 
 
 
Candace Jarvis 
 
 
Center for Biologics Evaluation and Research (CBER) 
 
 
Office of Cellular and Gene Therapy 
 
 
Document Control Center, HFM-99, Suite 200N 
 
 
1401 Rockville Pike 
 
 
Rockville, MD 20852-1448 

 
 
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Phone: (301)827-5357 
 
DSMB: Reports of serious adverse events that are unexpected and suspected will also be 
forwarded as soon as possible and no later than seven (7) days in the case of death or life-
threatening serious adverse events or within fifteen (15) days after the occurrence of all other 
forms of serious adverse events to the Data and Safety Monitoring Board (DSMB).  A summary 
of all SAEs will be included for review semiannually by the DSMB.  
 
 If the serious adverse event is thought to be due to the experimental component of the protocol, 
accession to the protocol will be stopped until a full discussion with the IRB has been held.   
 
Miltenyi Biotec, Inc.: Reports of serious adverse events will be forwarded to Miltenyi Biotec, 
Inc. to 
 
 
 
Tara Clark, General Manager US Clinical Operations 
  
 
and Norman Pilon, PhD  
 
 
Miltenyi Biotec, Inc. 
 
 
85 Hamilton Street, Cambridge, MA 02139. 
 
 
Phone (617) 218-0060 
 
13.6       Data Management 
 
The PI will be responsible for overseeing entry of data into an in-house password protected 
electronic system and ensuring data accuracy, consistency and timeliness. The principal 
investigator, associate investigators, NHLBI fellows, research nurses and/or a contracted data 
manager will assist with the data management efforts.   Data will be abstracted from Clinical Center 
progress notes as well as from progress notes forwarded from home physician. Laboratory data 
from NIH will be imported electronically from CRIS into an in-house clinical trial 
database.  Laboratory values from referring home physicians will be entered into the system.  
 
Research data will be prospectively collected by authorized Investigator personnel and entered 
into the NHLBI Medidata Rave Database. Medidata Rave is a web-based, 21 CFR 11 compliant, 
database which will consist of the study specific set of electronic CRFs (e-CRFs) used for 
capturing, managing and reporting clinical research data.  
 
The database will maintain complete data records on each research subject. Subjective and 
objective patient experiences during the duration of the study will be documented in the patient 
medical record notes. These protocol notes will serve as the primary source material from which 
data will be collected and research analyses will be performed. Any pertinent supplementary 
information obtained from outside laboratories, outside hospitals, radiology reports, laboratory 
reports, or other patient records will be used as additional source for data collection 
 
CIBMTR: Subjects will be offered co-enrollment on protocol 07-I-0183: The collection of research 
samples and data for repository from unrelated hematopoietic stem cell transplantation recipients 
for the national marrow donor program.  If consented to participate on this ancillary study the 
National Marrow Donor program (NMDP) will be forwarded transplant outcome data.    Data 
reporting requirements for the NMDP Coordinating Center include:  Baseline confirmatory data, 
pre-conditioning, 100 day, 6 month, 1 year and annually post-transplant outcome data for the 
recipient's life span.  Data reporting requirements for the NMDP Donor Center include: 30 day, 6 
months and 1 year post transplant updates. 
 

 
 
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For the purposes of quality assurance (i.e. accreditation of the NHLBI Transplant program), de-
identified, anonymous data will be released to the Center for International Blood and Marrow 
Transplant Research (CIBMTR) according to federally mandated policies and procedures. 
 
End of study procedures:  Data will be stored in locked cabinets and in a password protected 
database until it is no longer of scientific value.  
 
Loss or destruction of data:  Should we become aware that a major breech in the plan to protect 
subject confidentiality and trial data has occurred, the IRB will be notified. 
 
Publication policy: Given the research mandate of the NIH, subject data including the results of 
testing and responses to treatment will be entered into an NIH-authorized and controlled research 
database. Any future research use will occur only after appropriate human subject protection 
institutional approval such as prospective NIH IRB review and approval or an exemption from 
the NIH Office of Human Subjects Research (OHSR). 
 
14.0 
HUMAN SUBJECT PROTECTIONS  
 
14.1 
Rational for Subject Selection 
  
Aplastic anemia (AA):   The study will be open to all AA subjects who satisfy the inclusion 
criteria and provide an informed consent to the protocol. No subjects will be excluded from 
participation based on gender, race or ethnicity.  Epidemiologic studies suggest that the gender will 
be approximately evenly split between male and females, and that 90% of the subjects will be 
Caucasian.   
 
Myelodysplastic syndrome (MDS):  The  NHLBI has a robust source of subjects with bone 
marrow failure and we do not anticipate recruitment to be problematic. Since 1997, we have 
screened over 400 subjects with MDS (about 30/year).  
  
Recruitment efforts:  The study will be listed on ClinicalTrials.gov, the Clinical Center research 
studies website, The Aplastic Anemia Foundation, and the National Heart, Lung and Blood 
Institute subject recruitment website.  If recruitment goals are not met, a recruitment plan will be 
developed by the Clinical Center Office of Patient Recruitment.  
 
Competition between Branch protocols:  There are no competing branch protocols for this 
subject population.    
 
Reimbursement for protocol participation, travel, food, and lodging will be consistent with 
NIH guidelines.  In determining reimbursement, the following factors are considered applicable 
to this protocol:   the patients are diagnosed with a rare disease; the patient population is sick; the 
protocol offers the potential for direct benefit; the protocol regimen is demanding; and in order to 
complete accrual in a reasonable timeframe a geographically dispersed participant population is 
required.  
 
Payment for participation:  $0 
 
14.2 
Participation of Children  
   
14.2.1  As recipients:  

 
 
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Although the time to engraftment is quicker in younger children receiving cord blood transplants 
compared to adults, neutrophil recovery is still delayed compared to the use of a peripheral blood 
stem cell transplant. For patients with aplastic anemia who have had prior opportunistic infections 
or who have an active infection at transplant, expediting neutrophil recovery by co-transplanting 
haploidentical stem cells could potentially reduce the risk of infectious mortality associated with 
conventional cord transplantation. Furthermore, graft rejection occurs frequently in children with 
aplastic anemia undergoing conventional cord blood transplantation. The use of haploidentical 
stem cells may also serve as a backup stem cell source should the graft be rejected, which 
potentially could prevent rejection related mortality.    
 
14.2.2  As donors: 
 
Allogeneic bone marrow (and peripheral blood) transplantation (BMT) provides an option for 
cure for aplastic anemia and is therefore is considered an accepted standard clinical intervention 
for this disease.   The donor would be donating stem cells to his/her family member regardless of 
the objectives of this research protocol.    We are however excluding from participation as donors, 
children who weigh <18 kg and are < 6 years of age. The risks of the apheresis procedure are 
related to the weight of the child, more precisely his/her extracorporeal volume, which is weight-
dependent.  The risks have to do with (1) need for a central line, (2) need for an allogeneic red 
cell prime, (3) need for systemic heparinization because subject is too small to get citrate: 
 
> 25 kg:  the procedure and associated risk is the same as that in an adult, however a central 
line is almost always needed.    
 
19 to 25 kg:  A central line is required.  Donors may or may not need a central line (at the 
discretion of the apheresis department). With concurrent magnesium and calcium infusion, 
children may be safely anti-coagulated with citrate. 
 
On sister protocol 99-H-0050, 5 of 10 (50%) of minor donors (4/15/2009) required femoral 
lines, three of which (ages 6, 7, and 10) required conscious sedation. 
 
14.2.3  As participants in research studies:   
 
Pediatric donors may participate only in those laboratory studies that the IRB finds involves no 
greater than minimal risk to children provided that adequate provisions are made for soliciting the 
assent of the children and the permission of their parents or guardians.  
 
14.3  
Hazards and Discomforts 
 
 
14.3.1  The recipient 
 
 
 
Related to venous access placement: Intravenous line placement carries a small risk of bleeding, 
bruising or pain and a very low risk of accidental injury to the adjacent artery and nerve. Some 
participants may experience a vasovagal reaction (lightheadedness, or, rarely fainting due to 
temporary lowering of blood pressure).  
 
Related to bone marrow aspiration: No major risks are involved with bone marrow aspirate and 
biopsy.  However, a small risk of infections, pain, bleeding, and hematoma formation at the site 
of the aspiration exists with the procedure. 
 

 
 
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Related to chest x-ray: The risks associated with x-rays are very small.  For example, one chest 
x-ray has the same increased risk of death (due to cancer) as smoking 1.4 cigarettes or drinking ½ 
liter of wine.  On average, all of us receive an equivalent dose of x-rays every 30 days from 
nature.  However, the more radiation received during a lifetime, the greater the risk of developing 
cancerous tumors or of inducing changes in genes.  The changes in genes could cause 
abnormalities or disease in future offspring.  We do not expect the radiation in this study to 
greatly increase these risks, but we do not know the exact increase in such risks.   
 
Related to pulmonary function test: Risks are minimal, but some subjects may become 
lightheaded or faint during the test.  The test could also trigger an asthma attack in persons with 
asthma. 
 
Related to CT scans: Oral and/or intravenous contrast agents will be used and are usually well 
tolerated.  However, some subjects will experience allergic reactions to intravenous contrast. To 
lower the risk of allergic reactions, low allergenic contrast agents are administered at NIH 
Clinical Center.  In addition, subjects will be advised that approximately 2-7% of patients who 
receive contrast agents will experience a temporary reduction in kidney function lasting up to 2 
weeks following infusion and that in rare instances, permanent renal damage can result from the 
use of the IV contrasting agent.  Therefore, in subjects with impaired kidney function, we will not 
use intravenous contrast.  The amount of radiation from a head CT is comparable to 8 months of 
natural background radiation. 
 
Related to EKG (age > 50 or > 40 with risk factor): There is no pain or risk associated with 
having an electrocardiogram. 
 
Related to echocardiogram (age > 50 or > 40 with risk factor): This test has no known side 
effects. 
 
Related to MUGA scan (age > 50 or > 40 with risk factor):  There are no known adverse events 
associated with a MUGA scan.  The radiotracer used during the scan has a radiation dose similar 
to that of a CT scan (8 months of natural background radiation). 
 
Related to cardiac stress test (age > 50 or > 40 with risk factor): The possible risks of this 
procedure are: an allergic reaction to the radioactive dye or a flushed feeling; lightheadedness or 
fainting from the exertion; an abnormal heart rhythm brought on by the exertion.  Although very 
rare, it is possible that a nuclear stress test could cause a heart attack. 
 
Related to the quality of life instruments:  Subjects may feel uncomfortable answering the 
questions.   
 
Related to the transplant procedure:   Stem cell transplantation is a major procedure, which 
entails serious discomforts and hazards for the recipient.  The major limitation of UCB 
transplantation in adults is the limited number of nucleated cells contained within the cord unit 
resulting in prolonged neutropenia and failure of engraftment, which contributes to infection and 
TRM.  Full haplotype mismatched transplants have been associated with a risk of transplant-
related mortality as high as 50%. The high frequency of alloreactive donor T cells in 
unmanipulated grafts that recognize mismatched MHC antigens results in an extremely high 
incidence of severe, acute GVHD. Although extensive T cell depletion prevents GVHD, the 
rejection rates rise steeply because the balance between competing host and donor T cells shifts in 
favor of the unopposed host-versus-graft reaction. Furthermore, T-cell depletion is associated 
with a greater risk of post-transplant viral infections, secondary Hemophagocytic 

 
 
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Lymphohistiocytosis (HLH), and disease relapse. Toxicities potentially associated with the 
infusion of the CBU include dimethyl sulfoxide (DMSO) toxicity and side effects from intact and 
hemolyzed red cells and may include changes in heart rate or rhythm, changes in blood pressure, 
fever, chills, sweats, nausea/vomiting, diarrhea, abdominal cramping, fluid overload, headache, 
dyspnea, hemoglobinuria, allergic reaction, acute renal failure, and in rare cases infusion reaction 
resulting in death. 
 
The mortality from a cord blood or full haplotype mismatched transplants may be as high as 40%. 
It is therefore only appropriate to carry out the procedure with its experimental component in the 
context of a life-threatening condition and with full informed consent from the recipient and 
donor and immediate family. The major hazards (graft failure/rejection, GVHD, infection, and 
disease relapse) have already been mentioned. Inevitably after transplantation, blood counts will 
decrease with an associated requirement for red blood cell and platelet transfusions, as well as a 
need for empiric antimicrobial use to prevent infectious complications.  At the time of count 
recovery, there is also a small risk of pulmonary engraftment syndrome, manifested by non-
infectious fever, pulmonary infiltrates, and dyspnea, which can be treated effectively with prompt 
administration of corticosteroids.  The major discomforts during the post-transplant period are 
those of nausea, mucositis, anorexia, diarrhea, fever, malaise, and intolerance of the isolation 
period.  Possible organ toxicities related to the high-dose preparative regimen include hepatic 
veno-occlusive disease and interstitial pneumonitis, which can be fatal. 
 
Transplant recipients have been reported to have an increased risk of solid cancers but most studies 
are small and have limited ability to evaluate the interaction of host, disease, and treatment-related 
factors. In the largest study to date to evaluate risk factors for solid cancers, a multi-institutional 
cohort of 28874 allogeneic transplant recipients developed 189 solid malignancies. Overall, patients 
developed new solid cancers at twice the rate expected based on general population rates (observed-
to-expected ratio 2.1; 95% confidence interval 1.8-2.5), with the risk increasing over time (P trend < 
0.001); the risk reached 3-fold among patients followed for 15 years or more after transplantation. 
These findings showed that the risk of developing a non-squamous cell carcinoma (non-SCC) 
following conditioning radiation was highly dependent on age at exposure. Among patients irradiated 
at ages under 30 years, the relative risk of non-SCC was 9 times that of non-irradiated patients, while 
the comparable risk for older patients was 1.1 (P interaction <.01). Chronic graft-versus-host disease 
and male sex were the main determinants for risk of SCC. These data indicate that allogeneic 
transplant survivors, particularly those irradiated at young ages, face increased risks of solid cancers, 
supporting strategies to promote lifelong surveillance among these patients. (Blood. 2009; 113:1175-
1183). 
 
Secondary MDS/AML of donor origin is also a known but rare complication following allo-transplant 
[112-115]. This phenomenon is increasingly being realized given our ability to do chimerism assays 
to distinguish donor from patient cells.  
 
 
Because of the nonmyeloablative nature of this regimen, is anticipated that recipients will be less 
likely to become infertile with this transplant approach compared to a conventional myeloablative 
transplant. Because it is possible that infertility may occur, we will discuss the possibility of sperm 
banking or egg retrieval with the subject before undergoing the transplant.   
 
Related to the apheresis procedure:   The apheresis procedures will be performed in accordance 
with standard apheresis donation policies and procedures operative in the Dept. of Transfusion 
Medicine and will be in compliance with the Blood Donor Standards of the American Association 

 
 
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of Blood Banks and the rules and regulations of the Food and Drug Administration. Adverse 
reactions to apheresis procedures are rare, but include: 
 
x 
Pain and hematoma at the needle placement site (if done via peripheral access) 
x 
Vasovagal episodes, characterized by transient hypotension, dizziness, nausea and rarely, 
syncope are seen in less than 2% of the procedures. Hypotension secondary to volume 
depletion is known for the rare potential for a cerebrovascular or cardiovascular event.  
x 
Cutaneous or circumoral parasthesias, chills, nausea, heartburn and rarely muscle spasms 
may result from the use of citrate anticoagulant used to prevent clotting in the 
extracorporeal circuit. Citrate reactions are usually relieved by slowing the rate of the 
anticoagulant infusion and by administering oral calcium carbonate tablets or with 
intravenous calcium gluconate. 
 
Related to the CliniMACS® CD34 Reagent System:  Theoretical risks to the recipient could 
include system failure, user error, or recipient reaction to selected product components (i.e. residual 
amounts of unbound CliniMACs CD34 Reagent, iron dextran, or human anti-mouse antibodies).  
For full text see Investigator’s Brochure, Version 7, dated 7/31/2011, section 6, Summary and 
Guidance for the Investigator. 
 
Prior to each apheresis, the potential risks associated with the procedure will be explained to the 
subject and a separate informed consent obtained. 
 
Related to the common transplant related drugs and radiotherapy. 
Antithymocyte globulin: Febrile reactions, allergic symptoms including rash, wheezing, 
anaphylaxis, cytopenias, serum sickness, nausea, vomiting, diarrhea, arthralgias, hypotension, 
tachycardia, bradycardia, fever, chills, myelosuppression. Several cases of a severe lung injury 
related to Atgam treatment have been reported. Although this side effect appears extremely rare, 
it is serious and can be fatal. There is no information about the mechanism or specific treatment 
for this condition. A few patients recovered after intensive medical support including use of a 
breathing machine.  
 
Cyclophosphamide: Immediate-- tingling and metallic taste, nausea and vomiting, ADH-like 
effect, cardiotoxic at high doses (>70 mg/kg). Rare -- pulmonary toxicity, urticaria and flushing, 
mucositis Delayed--marrow suppression, nausea, mucositis, rash, hemorrhagic cystitis, 
myocardial damage, alopecia, infertility. 
 
Fludarabine: Myelosuppression, fever and chills, nausea and vomiting, malaise, fatigue, anorexia, 
weakness, and rarely hemolysis and pulmonary toxicity, hemolytic anemia and interstitial 
pneumonitis. Serious opportunistic infections have occurred in CLL patients treated with fludarabine.   
  
Mycophenolate mofetil: Serious side effects include increased susceptibility to infection and 
possible development of lymphoma, and increased risk of pregnancy loss and congenital 
malformations (external ear and facial abnormalities including cleft lip and palate, and anomalies 
of the distal limbs, heart, esophagus, and kidney). Nausea, diarrhea, leukopenia, headache, 
weakness, urinary frequency, leg cramps or pain, liver function test abnormalities, infection 
related to immunosuppression, and skin rash. Hemorrhagic gastritis and pancreatitis have been 
reported rarely. Malignant neoplasms have been reported in psoriasis patients treated with oral 
mycophenolic acid, although a definite cause-effect relationship has not been established.    
Multifocal leukoencephalopathy (PML) caused by reactivated JC virus has been reported 
following MMF (10 confirmed and 7 possible cases).  The latent virus is present in about 80 
percent of adults. In response, in June 2008, a safety alert was issued and physicians were advised 

 
 
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that patients who present with or develop new neurologic signs or symptoms should be evaluated 
for PML. Neurological warning signs include: major changes in vision, unusual eye movements, 
loss of balance or coordination and/or disorientation or confusion. There are no known effective 
treatments for PML. 
 
Prednisone:   Sodium retention, hypertension, congestive heart failure in susceptible patients, 
hyperglycemia, increased requirements for insulin or oral hypoglycemic agents in diabetics, 
increased appetite, psychic disturbances, peptic ulcers, and increased risk for opportunistic 
infections.  Long term administration of glucocorticoids may be associated with myopathy, 
cataracts, osteoporosis, osteonecrosis, and growth retardation in children. 
 
 
Tacrolimus: Acute tacrolimus nephrotoxicity is usually manifested by a moderate decline in renal 
excretory function, which is readily reversible by a decrease in drug dosage.  Although some 
degree of transient renal dysfunction may occur in patients with therapeutic levels of tacrolimus, 
significant renal toxicity is associated with elevated trough or steady state levels.  In addition to 
an increase in BUN and creatinine, hyperkalemic hyperchloremic acidosis, low fractional 
excretion of sodium and the onset of hypertension with hypomagnesemia are seen with 
tacrolimus nephrotoxicity.  Hypertension occurs in up to 60% of patients.  Hypomagnesemia can 
be associated with neurologic symptoms, including seizures, cerebellar ataxia and depression.  
Dose-related hepatotoxicity, manifested by elevation of serum transaminases and bilirubin, has 
been reported. 
 
 
Related to antimicrobials in general: Allergic reactions, renal impairment (gentamicin, 
vancomycin, amphotericin, acyclovir), "red man" syndrome (vancomycin), hepatic damage 
(acyclovir, rifampin) , nausea, anorexia, abdominal discomfort, pancytopenia, neutropenia, 
peripheral neuropathy, renal damage (Ganciclovir--reversible on stopping drug). 
 
 
Related to filgrastim (G-CSF): (See section 14.3.2 below) 
 
Related to the total body radiation: The side effects of radiation have been well described. The most 
common include nausea and mucositis. There also exists a risk of hypothyroidism, cataracts, 
interstitial pneumonitis, nephropathy, and an unspecified long term risk of developing secondary 
malignancies. Importantly, the majority of the nonneoplastic effects were sub clinical and/or 
reversible.  
 
Related to fertility: Although the conditioning regimen uses low dose radiation and chemotherapy 
that usually does not compromise fertility, no data is currently available on the chances that 
subjects will maintain fertility after this type of transplant. Even those who become infertile after 
the transplant may regain fertility with time.  
 
14.3.2 The donor 
 
Related to filgrastim (G-CSF): The hazard to the donor is low. The discomforts from G-CSF 
mobilization and leukapheresis for collection of blood stem cells are probably lower than those 
associated with marrow harvesting. G-CSF has been given to large numbers of normal donors 
without major side effects or long-term consequences. The immediate side effects of G-CSF are 
bone pain, fatigue, insomnia, myalgia and headache. These are usually mild and are self-limiting. 
Reversible thrombocytopenia, with platelet counts falling to the region of 100,000 /cu mm is 
frequent. Although regarded as safe and well tolerated, G-CSF administration could result in 
splenic rupture, a potentially life-threatening complication. Five cases of splenic rupture have 
been reported in healthy donors (Becker et al. 1997, Falzetti et al. 1999, Balaguer et al. 2004, 

 
 
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Dincer et al. 2004, Nuamah et al. 2006). Donors will be asked to avoid vigorous activities and to 
report any left upper abdominal and/or shoulder pain to the research team or the on-call physician 
for the NIH Department of Transfusion Medicine at 301-496-1211.  
 
Patients with ongoing ischemic heart disease have been reported to have angina seemingly 
temporally-related to G-CSF administration and apheresis(Vij).  In addition, a rare occurrence of 
pulmonary hemorrhage has been reported in a healthy donor who was a cigarette smoker and had 
underlying pulmonary disease.104 
 
Related to central line placement: It is estimated that about 50% of the donors less than age 18 will 
require intravenous line placement (femoral vein); and about 50% of those minors will require 
conscious sedation for the line placement procedure to successfully complete apheresis. Intravenous 
line placement in the femoral vein using a temporary Quentin catheter carries a small risk of 
bleeding, bruising or pain, infection, and a very low risk of accidental injury to the adjacent artery 
and nerve. Some subjects may experience a vasovagal reaction (lightheadedness, or, rarely, fainting 
due to temporary lowering of blood pressure). Using only trained experienced staff for the 
procedure minimizes these risks.  
 
In exceptional instances the donor is required to donate PBPC a third time. There is a minimal 
additional risk that the donation of PBPC on three successive days can increase the possibility of 
thrombocytopenia. Thrombocytopenia is transient and unlikely to cause clinical sequelae.  
 
Related to Apheresis: see section 14.3.1 
 
14.4 
Risks in Relation to Benefits  
 
As of April 18, 2019, this study is now closed to new subject accrual and continues in Follow-up 
only. The level of risk is now minimal. 
 
  
14.4.1 For adult transplant subjects 
Clinically the approach is ethically acceptable because we are offering adult subjects with 
debilitating hematologic disorders who have failed or are not eligible for conventional treatments 
an alternative therapy which may be curative.   The protocol aims to decrease the risk of 
transplant-related mortality associated with UCB transplantation, thus making more patients 
candidates for potentially curative therapy. The risk of death due to complication from the 
allogeneic BMT procedure is justified by the anticipated benefit of potentially eradicating the 
patient’s SAA or MDS.  
 
Therefore, for adult transplant recipients on this protocol, the research involves greater than 
minimal risk to subjects with the prospect of direct benefit (45 CFR 46.102). 
 
14.4.2 For pediatric transplant subjects   
 
The inclusion of children satisfies the criteria set forth in 45 Code of Federal Regulations 46, 
Subpart D: 46.405 as follows: 
 
 (a) the risk is justified by the anticipated benefit to the subjects: We are offering pediatric 
subjects with debilitating hematologic disorders who have failed or are not eligible for 
conventional treatments an alternative therapy which may be curative    
 

 
 
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(b) the relation of the anticipated benefit to the risk is at least as favorable to the subjects as that 
presented by available alternative approaches. Clinically the approach is ethically acceptable 
because the protocol aims to decrease the risk of transplant-related mortality associated with UCB 
transplantation, thus making patients candidates for potentially curative therapy 
 
 (c) adequate provisions are made for soliciting the assent of the children and permission of their 
parents or guardians, as set forth in 46.408. 
 
Therefore for pediatric transplant recipients on this protocol, the research involves greater than 
minimal risk but presents the prospect of direct benefit to the individual subjects (45 CFR 
46.405). 
 
 
14.4.3 For adult donors: 
 
An HLA partially matched family member will be co-enrolled into this study as a stem cell 
donor. The stem cell collection aspect of this protocol is not investigational. Despite the risks 
associated with this procedure, potential benefit does exist for family donors. The donor derives 
psychosocial benefit from donating stem cells both at the time of donation and possibly into the 
future, especially in view of the reduced life expectancy due to this disease in a family member.  
Other potential benefits include detection of illnesses, determination of blood cell counts, and 
evaluation of kidney and liver function in the potential donor at the time of screening.  
 
 
Therefore, participation of adults as a stem cell donors on this protocol involves greater than 
minimal risk but presents the prospect for direct benefit (45 CFR 46.102).   
 
14.4.4 For pediatric donors: 
 
An HLA partially matched pediatric family member may be co-enrolled on this study as a stem 
cell donor. The stem cell collection aspect of this protocol is not investigational. Despite the risks 
associated with this procedure, potential benefit does exist for the pediatric donors. The donor 
derives psychosocial benefit from donating stem cells both at the time of donation and possibly 
into the future, especially in view of the reduced life expectancy due to this disease in a family 
member.  Other potential benefits include detection of illnesses and evaluation of overall health 
status of the pediatric participant at the time of screening.  
 
Therefore, participation as a stem cell donor on this protocol involves greater than minimal risk 
but presenting the prospect of direct benefit to individual subjects (45 CRF 46.405). 
 
14.4.5 For pediatric donors – healthy volunteers- involved in laboratory research studies.   
 
The inclusion of children satisfies the criteria set forth in 45 Code of Federal Regulations 46, 
Subpart D: 46.404 as follows: 
 
(1) The research does not involve greater than minimal risk.  Blood and bone marrow specimens 
for research are obtained concurrently with clinically indicated sampling. Immune cell 
collection will not be permitted in pediatric donors. Therefore, there is no risk associated with 
sample collection for research because research will only be performed on material obtained 
during standard research interventions. 
 
Research specimens will be stored in the PI’s laboratory. Samples will be assigned a unique 
code known only to the principal investigator, which will serve as a link to the child’s name 

 
 
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and clinical information collected as part of this research protocol.  No samples will be 
provided to investigators outside the branch without permission of the IRB, therefore 
confidentiality will be protected. 
 
Only those laboratory tests approved by the IRB and involving not greater than minimal risk 
will be conducted. Research will not include genetic testing. Therefore, there is no genetic 
testing associated risk.  
     
(2) adequate provisions are made for soliciting the assent of the children and permission of their 
parents or guardians, as set forth in 46.408.   
 
Therefore, participation of pediatric donors in laboratory research on this protocol involves not 
greater than minimal risk (45 CFR 46.404). 
 
14.5 
Informed Consent Processes and Procedures 
 
Adults:  The investigational nature and research objectives of this trial, the procedure and its 
attendant risks and discomforts will be carefully explained to the subject and a signed informed 
consent document will be obtained prior to entry onto this study.    
 
Minor subjects:   If the subject is a minor, minor assent will be sought.    Where deemed 
appropriate by the clinician, and the child's parent or guardian, the child will also be included in 
all discussions about the trial and a minor's assent will be obtained.  The parent who signs the 
consent for the minor must be a legally recognized parent or guardian.  The parent or guardian 
will sign on the designated line on the informed consent attesting to the fact that the child had 
given assent. When the assent is not age appropriate, the study will be explained to the child and 
the assent will be obtained verbally from the child.   
 
Re-Consent for Minors when they reach the age of majority:  When a pediatric subject 
reaches age 18, continued participation will require re-consenting of the now adult with the 
standard protocol consent document to ensure legally effective informed consent has been 
obtained. Should sample or data analysis continue following completion of active participation 
and the subject has reached 18 years of age, we will attempt to contact the subject using the last 
known contact information to obtain consent for continued use of data or samples collected 
during their prior visit. Given the length of time that may have transpired for some of the subjects 
since their last visit for this study, we request waiver of informed consent for those individuals 
who after good faith efforts to contact them, we are unable to contact. 
 
Requirements for Waiver of Consent consistent with 45 CFR 46.116 (d), each of which must be 
addressed in relation to the protocol: 
 
(1) The research involves no more than minimal risk to the subjects. 
a. Analysis of samples and data from this study involves no additional risks to subjects. 
(2) The waiver or alteration will not adversely affect the rights and welfare of the subjects. 
a. This is an FDA-regulated study and as such, we are mandated to retain all samples, 
once collected, regardless of the age of the subject at the time of collection.  
Retention of these samples or data does not affect the welfare of subjects.  
(3) The research could not practicably be carried out without the waiver or alteration. 
a. Considering the length of time between a minor’s enrollment and their age of 
majority, it is possible that more than a few subjects may be lost to follow up.  A 

 
 
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significant reduction in the number of samples analyzed could impact the quality of 
the research.  
(4) Whenever appropriate, the subjects will be provided with additional pertinent information 
after participation. 
a. We only plan to request a waiver of reconsent for those subjects who have been lost 
to follow-up.   
 
Consenting to pregnancy testing in minors of childbearing age:  The subject will be informed 
during the assent process that for safety, we need to do a pregnancy test.  The subject will also be 
told that if it is positive, we will counsel her and help her tell her parents.  If the minor does not 
want to proceed, she will be advised not to sign the assent and her enrollment on this screening 
protocol will end. 
 
Minor donors:  Assent will not be sought until an evaluation by a social worker and mental 
health specialist (psychologist or psychiatrist) is completed to determine the minor donor’s 
willingness to participate.    
 
Decisionally impaired:  The IRB will be consulted prior to enrolling any decisionally impaired 
subject (donor or recipient). 
 
Non-English speaking research participants:  We anticipate enrolling non-English speaking 
research participants into this study.  The IRB approved full consent document will be translated 
into the subject's native language in accordance with the Clinical MAS Policy M77-2.  If there is 
an unexpected enrollment of a research participant for which there is no translated extant IRB 
approved consent document, the principal investigator and or those authorized to obtain informed 
consent will use a short form oral consent process as described in MAS Policy M77-2, 
45CFR46.117(b)(2) and 21CFR50.27(b)(a).  The summary that will be used is the English 
version of the extant IRB approved consent document. 
 
 
At any time during participation in the protocol that new information becomes available relating 
to risks, adverse events, or toxicities, this information will be provided orally or in writing to all 
enrolled or prospective subject participants.  Documentation will be provided to the IRB and if 
necessary, the informed consent amended to reflect relevant information. 
 
14.6      Conflict of Interest 
 
The Principal Investigator assured that each associate investigator listed on the protocol title page 
received a copy of the NIH’s guide to preventing conflict of interest.  Investigators added 
subsequent to the initial circulation will be provided a copy of the document when they are added. 
Copies of the initial Conflict of Interest Statements were forwarded to the Clinical Director.  No 
initial members of the research team reported a potential conflict of interest.  
 
This protocol may have investigators who are not NIH employees. Non- NIH investigators are 
expected to adhere to the Principles of the Protocol Review Guide but are not required to report 
their personal financial holdings to the NIH. 
 
This protocol has no associated patents or CRADAs.  A CTA between Miltenyi Biotec 
Incorporated and the National Heart, Lung and Blood Institute of the use of the CliniMACS 
CD34 and CD3 reagent cell selection systems for clinical research purposes was executed on 
9/10/2007. 
  

 
 
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An MTA between the NMDP research sample repository and the NHLBI has been executed for 
the transfer of the pre transplant sample of peripheral blood.  
 
A data transfer agreement between the CIBMTR and NHLBI for the transfer of transplant 
outcome data was executed in 2009. 
 
15.0   PHARMACEUTICALS 
 
 
15.1     CliniMACS® CD34 Reagent System  
 
15.1.1   Investigational product:  The CliniMACS® CD34 Reagent System is a medical device 
that is used in vitro to select and enrich specific cell populations. When using the CD34 Reagent, 
the system selects CD34+ cells from heterogeneous hematological cell populations for 
transplantation in cases where this is clinically indicated. The CliniMACS® CD34 Reagent System 
is comprised of four primary components:  
• CliniMACS CD34 Reagent: a sterile monoclonal antibody reagent specific for CD34+ cells  
• CliniMACS Plus Instrument: a software controlled instrument that processes the blood 
sample (cell product)  
• CliniMACS Tubing Sets: single-use, sterile, disposable tubing sets with two proprietary cell 
selection columns (CliniMACS Tubing Set and CliniMACS Tubing Set LS)  
• CliniMACS PBS/EDTA Buffer: a sterile, isotonic phosphate-buffered, 1 mM EDTA, saline 
solution, used as external wash and transport fluid for the in vitro preparation of blood cells  
 
15.1.2 Physical, chemical and toxicological information  
x 
CD34 Reagent description:   The CliniMACS CD34 Reagent is a dark amber, 
nonviscous, colloidal solution containing the antibody conjugate in buffer. The conjugate 
consists of a monoclonal antibody towards the class II epitope of the human CD34 
antigen. The murine monoclonal IgG1 antibody is covalently linked to dextran beads that 
have an iron oxide/hydroxide core and are superparamagnetic.  
x 
Safety testing of the CD34 monoclonal antibody:  Cell banking, cell culture, as well as 
subsequent purification of the antibody, follow the applicable current international 
guidelines. The testing of the CD34 Master Cell Bank, the End of Production Cells, the 
CD34 mAb pooled cell culture harvest (unprocessed bulk) and the purified CD34 
monoclonal antibody (mAb) have been completed and the purified CD34 mAb has been 
released for manufacturing of the CD34 Reagent. Additionally, the viral 
inactivation/removal steps used in the purification of the CD34 monoclonal antibody 
have been validated.  
x 
Safety testing of the CD34 Reagent: Detailed toxicity studies have been undertaken to 
assess the safety of the CD34 Reagent when delivered in dosages significantly greater 
than the projected maximum dosage anticipated in clinical use. The testing was 
performed in accordance with 21 CFR §58, Good Laboratory Practices for Nonclinical 
Laboratory Studies. A summary of this testing is provided in the following table.  
 
Table 1: Summary of Toxicology of the CliniMACS CD34 Reagent  
Test  
Results  
Human Cryosection Cross 
Reactivity Study  
CD34 Reagent specifically reacted with cell types known to 
posses the CD34 antigen. Not considered toxicologically 
significant.  

 
 
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Interspecies Cross 
Reactivity Study  
CD34 antibody does not cross react with non–human primate 
hematopoietic cells expressing the CD34 antigen. These species 
could be used for safety testing.  
Subchronic Toxicity  
No toxicity  
Cardiovascular Safety 
Study in Rhesus Monkeys  
No drug-related effects on mean arterial pressure, mean right 
ventricle pressure, cardiac output, ECGs, respiration rate, heart 
rate or cageside observations were noted when escalating doses of 
CD34 Reagent  
Irritation  
No irritation  
Hemocompatibility  
Compatible with human blood  
Sterility assay of the final 
container  
Reproducibly sterile product  
 
15.1.3 Safety testing of CliniMACS® System components (Instrument, Tubing Sets and PBS/EDTA 
Buffer) Biocompatibility testing of the CliniMACS System components (Tubing Sets and 
PBS/EDTA Buffer) was performed according to ISO 10993. The requirements of ISO 10993 
were fulfilled for the CliniMACS CD34 Reagent System.   The CliniMACS plus Instrument has 
been tested for electrical safety and the potential for fire, shock, explosion, or mechanical 
damage. Potential safety issues have been reduced by using a design to meet European standards 
EN 60601-1. It is UL and CSA listed and approved.  
 
15.1.4 Overall safety of the CliniMACS® CD34 Reagent System:  The results summarized in the 
Investigator’s Brochure support that CliniMACS® CD34 Reagent System is sufficiently safe for 
clinical use with human subjects. The potential application of the CliniMACS CD34 Reagent is 
broad. Infusion of purified CD34+ cells is indicated in a number of clinical applications after 
myeloablative or lymphoablative therapy including reduction of tumor cells in the transplant and 
depletion of T cells for autologous (autoimmune diseases) and allogeneic transplantations.  
Individual risk analysis on the therapeutically used target cells isolated in conjunction with 
CliniMACS® CD34 Reagent System should be addressed by each site using these cells.  
 
The BMT CTN #0303 Study entitled “A Single Arm, Multicenter Phase II Trial of Transplants of 
HLA-Matched, CD34+ Enriched, T-cell Depleted Peripheral Blood Stem Cells Isolated by the 
CliniMACS System in the Treatment of Patients with AML in First or Second Complete 
Remission” was performed in conjunction with the Blood and Marrow Transplant Clinical Trials 
Network (BMT CTN). This multi-center, phase II study was performed to support the probable 
benefit of the CliniMACS® CD34 Reagent System for processing hematopoietic progenitor cells-
apheresis (HPC-A) to obtain a CD34 positive cell enriched population intended to provide for 
hematopoietic reconstitution following a myeloablative preparative regimen without the need for 
additional graft-vs-host disease (GVHD) prophylaxis in patients with Acute Myeloid Leukemia 
(AML) in first or second morphologic complete remission. 
 
The BMT CTN #0303 Study was designed to provide extensive T-cell Depletion (TCD) using the 
CliniMACS® CD34 Reagent System, to eliminate the requirement for post-transplant 
pharmacological GVHD prophylaxis in adult patients with AML in first or second CR 
undergoing a myeloablative transplant from a matched related donor. The goal was to 
demonstrate that the side effects of T-cell depleted allogeneic hematopoietic stem cell 
transplantation (HCT) would be reduced if combined with a conditioning regimen that was highly 
immunosuppressive and anti-leukemic. The primary objective was to achieve a disease-free 
survival (DFS) rate at six months post-transplant that exceeded 75%. Secondary objectives 
included assessments of engraftment, overall survival, EBV reactivation, transplant-related 

 
 
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mortality (TRM), acute and chronic GVHD, relapse and performance of the CliniMACS® CD34 
Reagent System. 
 
Forty-seven patients were enrolled and 44 transplanted at eight different centers in the BMT CTN 
network. The mean age was 46.3 years with 28 female and 16 male patients. Of the 37 AML 
CR1patients on protocol, 27% had an unfavorable cytogenetic or molecular risk profile. The 
conditioning regimen consisted of hyperfractionated total body irradiation (1375cGy in eleven 
fractions) with partial lung shielding, thiotepa (10 mg/kg), cyclophosphamide (120 mg/kg), and 
rabbit antithymocyte globulin (2.5 mg/kg). The donors, all HLA-identical siblings, were given 
GCSF for mobilization and scheduled to undergo at least 2 leukapheresis procedures to ensure a 
graft with a high CD34+ cell content. All allografts were CD34 enriched and were targeted to 
contain > 5.0 x106 CD34+ cells/kg and < 1.0 x 105 CD3+ cells/kg. No pharmacological GVHD 
prophylaxis was given post-transplant. 
 
The primary endpoint of the trial was met, in that the probability of disease-free survival (DFS) at 
six months post transplantation was 81.8% for all transplanted patients. Additionally, the data 
from the study showed that the performance of the CliniMACS® CD34 Reagent System was safe 
and consistent in this multicentric trial because of the reproducible, passive depletion of CD3+ 
cells (median 4.8 log10 depletion), the enrichment of CD34+ cells, through the sterility of the 
transplanted grafts and the absence of infusional toxicity and attributable adverse events and low 
rates of acute and chronic GVHD without the need for additional post-transplant GVHD 
prophylaxis. 
 
A European safety study, ACS 950101, for the CliniMACS System was published in Bone 
Marrow Transplantation 25; 243-49, February 20001. The study was designed to meet European 
Essential Requirements 3 and 14 (MDD 93/42/EEC) and was conducted per EN540 to support the 
CE Marking of the device (received December 1997). 
 
The initial clinical study with the CliniMACS System was conducted in subjects undergoing high-
dose chemotherapy for breast cancer. The purpose of the European Safety Study was to show:  
 
x 
Suitability of the CD34 Reagent and other CliniMACS components for selection 
ofCD34+cells with high, yield, purity viability and safety  
x 
CD34+ cells can safely be administered to subjects after myeloablative chemotherapy  
x 
Selected CD34+ cells are effective in reconstituting the hematopoietic system after 
myeloablative chemotherapy  
x 
Rate of device failures  
 
Cells were isolated from leukapheresis products from sixty-five subjects enrolled in the study. 
Fifty-four subjects received selected CD34+ cells and fifty-two were evaluable for engraftment as 
summarized in Table 2 (one subject died 5 days post-transplant and prior to engraftment and one 
subject did not recover platelet counts even after back-up cells were infused). All subjects receiving 
selected cells completed 60 and 100-day follow-up after infusion, during which time their 
hematological and immune status were monitored, as was human murine antibodies (HAMA) 
production. A summary of the results of the European clinical trial is provided below.  
 
Table 2: Time to Hematological Engraftment After Infusion of CliniMACS Selected CD34+ Cells 
Criteria for Engraftment  
 
Time to Engraftment (Days)  
Platelets (≥ 20 x 109/L)  
Neutrophils (≥ 500/PL) 

 
 
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Median (Kaplan –Meier) 
11.6 
9.1 
Standard Deviation 
6.05 
5.81 
Quartile Range (Kaplan-Meier) 
10.0 – 12.0 
8.0 – 10.0 
Range 
8 – 29 
8 - 11 
Number 
52 
52 
 
The following conclusions were made regarding this clinical study:  
 
• The CliniMACS® CD34 Reagent System selects CD34+ cells from heterogeneous 
hematological cell populations. The resulting CD34+ product is of high purity (median of 
96.1 %, range 27.4 – 99.4 %); the median recovery of CD34+ cells was 52.3 % (range 15.2 – 
146.3%). The reported performance results are similar to those seen in pre-clinical studies.  
• Infusion of selected, autologous CD34+ cells after high dose chemotherapy resulted in rapid 
engraftment (see Table 2). These data are comparable to previously reported results, using 
bone marrow or peripheral blood cells as stem cell source. Following cyclophosphamide, 
thiotepa and carboplatin (CTCb), Weaver et al. reported median times to platelet and 
neutrophil engraftment of 9 days (range 0-53 days) and 9 days (range 5-26 days), 
respectively. Also after CTCb, Elias et al., reported median times to platelet and neutrophil 
recovery of 12 days (range 8-134 days) and 14 days (range 10-57 days), respectively (Elias et 
al., 1992; Weaver et al., 1995).  
• The selection process has no discernable effect on cell viability or sterility.  
• There were no adverse events or device malfunctions reported as related to the infusion of the 
cells or use of the CliniMACS® CD34 Reagent System. None of the subjects were reactive for 
HAMA post infusion. There were no reports of late engraftment failure or evidence of delayed 
immune reconstitution.  
 
15.2 
Anti-Thymocyte Globulin (equine) Sterile Solution (ATGAM®) 
Other: Antithymocyte gammaglobulin, Antithymocyte globulin, ATGAM, Antithymocyte 
immunoglobulin, lymphocyte immune globulin and h-ATG. 
Supply/availability: commercially available (Pharmacia & Upjohn Company). 
Product description: Anti-thymocyte globulin (equine) sterile solution (ATGAM®) is available in 
5 ml ampoules containing 50 mg of horse gamma globulin/mL (250 mg per ampoule). 
Preparation: The calculated dose of anti-thymocyte globulin should be diluted in 0.9% sodium 
chloride injection to a concentration not to exceed 4 mg/mL.  
Storage/stability: Anti-thymocyte globulin (equine) ampoules should be stored in a refrigerator at 
2o to 8o C. Once diluted, anti-thymocyte globulin (equine) is physically and chemically stable for 
up to 24 hours at concentrations of up to 4 mg/mL in the recommended diluents. It is 
recommended that diluted anti-thymocyte globulin (equine) be stored in a refrigerator if it is 
prepared prior to the time of infusion.  
Administration: Anti-thymocyte globulin (equine) should be administered into a high-flow 
central vein through an in-line filter with a pore size of 0.2 to 1 micron. The dose should be 
infused over no less than 4 hours. Infusion times may be extended to up to 24 hours for 
intolerance. Patients should be closely monitored for infusion / allergic reactions.  
Compound: Principally monomeric IgG, prepared from plasma or serum of healthy horses 
hyperimmunized with human thymus lymphocytes. 
Action: Immunosuppressive agent. Exact mechanism of immunosuppression of ATgam has not 
been fully elucidated but may involve elimination of antigen-reactive T-cells in peripheral blood 
and/or alteration of T-cell function. 
 

 
 
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15.3 
Cyclophosphamide (Cytoxan®, Neosar®) 
Supply: Commercially available. 
Product description: Cyclophosphamide is available as a lyophilized powder for injection in 
multiple vial sizes.  
Preparation: Cyclophosphamide powder for injection should be reconstituted with sterile water 
for injection to yield a concentration of 20 mg/mL as described in the product labeling. Once 
reconstituted, the prescribed dose will be further diluted in 250 mL of 0.9% sodium chloride 
injection or 5% dextrose in water for intravenous administration over 60 minutes. 
Storage and stability: Vials of cyclophosphamide are stored at room temperature. Once 
reconstituted as directed, solutions of cyclophosphamide are stable for 24 hours at room 
temperature, or 6 days when refrigerated at 2-8o C.  
Route of administration: The prescribed dose of cyclophosphamide will be diluted in an 
additional 250 mL of 0.9% sodium chloride injection or 5% dextrose in water for intravenous 
administration over 60 minutes. 
 
 
15.4 
Fludarabine Phosphate (Fludara®) 
Supply:  commercially available. 
Product description: Fludarabine phosphate is commercially available as both a lyophilized 
powder for injection in vials containing 50 mg of fludarabine phosphate with mannitol 50mg and 
sodium hydroxide for pH adjustment and a solution for injection in 2 mL vials containing 50 mg 
of fludarabine phosphate (25 mg/mL of fludarabine) with 25 mg/mL mannitol and sodium 
hydroxide for pH adjustment. 
Preparation: Fludarabine lyophilized powder for injection should be reconstituted with 2 mL of 
sterile water for injection, USP to a concentration of 25 mg/mL. The prescribed dose of 
fludarabine should be diluted in 100 mL of either 0.9% sodium chloride or 5% dextrose in water 
for intravenous administration over 30 minutes. 
Storage and stability: Fludarabine vials should be stored under refrigeration between 2o-8o Celcius 
(36o- 46o F).  Reconstituted fludarabine phosphate is chemically and physically stable for 24 
hours at room temperature or for 48 hours if refrigerated. The manufacturer recommends use of 
either the reconstituted powder for injection or the solution for injection (once diluted for 
administration) within 8 hours because neither product contains an antimicrobial preservative. 
Administration: The prescribed dose of fludarabine should be diluted in 100 mL of either 0.9% 
sodium chloride or 5% dextrose in water for intravenous administration over 30 minutes. 
 
15.6 
Filgrastim (G-CSF, Neupogen®) 
 
Supply: Commercially available. 
 
Product description: Filgrastim injection is available in a concentration of 300mcg/ml in 1ml 
(300mcg) and 1.6ml (480mcg) vials. 
 
Preparation: For subcutaneous administration, the appropriate prescribed dose is drawn up from 
the vial with no further dilution prior to administration. For intravenous administration, the 
commercial solution for injection should be diluted prior to administration. It is recommended 
that the prescribed dose be diluted with dextrose 5% in water (DO NOT DILUTE WITH 
NORMAL SALINE) to a concentration greater than 5mcg/ml. Filgrastim diluted to 
concentrations between 5 and 15mcg/ml should be protected from absorption to plastic materials 
by the addition of albumin (human) to a final concentration of 2mg/ml. When diluted in 5% 
dextrose or 5% dextrose plus albumin (human), filgrastim is compatible with glass bottles, PVC 
and polyolefin IV bags, and polypropylene syringes.   
 
Storage and stability:  Filgrastim for injection should be stored in the refrigerator at 2o to 8oC (36o 
to 46oF). Avoid shaking. 
 
Route of administration: Subcutaneous injection or intravenous infusion over 15-30 minutes. 

 
 
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15.7 
Mycophenolate Mofetil (MMF) (MMF, CellCept®) 
 
Supply: Commercially available. 
Product description: Oral dose formulations of mycophenolate mofetil include a 250 mg capsule, 
a 500 mg tablet, and an oral powder for suspension (200 mg/mL when reconstituted). The 
parenteral formulation of mycophenolate mofetil is available as a 500 mg vial containing a 
lyophilized powder that requires reconstitution. 
Preparation: The parenteral formulation of mycophenolate mofetil requires both reconstitution 
and dilution with 5% dextrose injection (5% dextrose in water or D5W) in a two step process. 
Each 500 mg vial should first be reconstituted with 14 mL of 5% dextrose injection followed by 
gentle shaking of the vial to dissolve the drug. Once reconstituted, the prescribed dose should be 
further diluted with 5% dextrose injection to a final concentration of 6 mg/mL. Inspect the 
infusion solution for particulate matter or discoloration. Discard if particulate matter or 
discoloration is observed. 
Storage and stability: Oral tablets, capsules and oral suspension should be stored at controlled 
room temperature 15-30oC (59-86oF). Once the oral suspension is reconstituted, the suspension 
may be stored at room temperature for up to 60 days. Refrigeration of the reconstituted oral 
suspension at 2-8oC (36 to 46oF) is also acceptable.  Parenteral vials should also be stored at 
controlled room temperature 15-30oC (59-86oF). Parenteral doses following dilution with 5% 
dextrose injection are stable for 7 days under refrigeration (4oC) or room temperature. NIH 
Clinical Center guidelines utilize a 48 hour expiration of prepared doses. 
Route of administration: Oral or intravenous administration. For intravenous administration, each 
dose should be infused over a period of no less than 2 hours by either peripheral or central vein. 
 
15.8     Prednisone 
Supply: Commercially available. 
Product description:  Prednisone tablets are available in the following tablet sizes: 1 mg, 2.5 mg, 
5 mg, 20 mg, and 50 mg. Prednisone is also available in an oral solution (5 mg per 5 ml). 
Storage and stability:  Store at controlled room temperature 15-30oC (59-86oF). 
Route of administration: Oral.  
 
  
15.9 
Tacrolimus (FK506, Prograf®) 
 
Supply: Tacrolimus will be obtained by the NIH Clinical Center Pharmacy Department from 
commercial sources and is available in capsules (0.5 mg, 1mg, and 5mg), and as a parenteral 
concentrate for injection (5 mg/ml, 1 ml ampules). 
Preparation: For parenteral doses, tacrolimus injection concentrate (5 mg/ml) should be diluted to 
a final concentration of 0.004 to 0.02 mg/ml in dextrose 5% in water or sodium chloride 0.9%.  
Parenteral doses of tacrolimus will be prepared in non-PVC containers and infused with non-PVC 
administration sets/tubing 
Storage and Stability: Capsules and ampules of parenteral concentrate bear expiration dates and 
are stored at room temperature.  Tacrolimus conentrate for injection that has been diluted to a 
final concentration of 0.004 to 0.02 mg/ml is stable for 24 hours in 5% dextrose or 0.9% sodium 
chloride injection in glass, PVC or non-PVC plastic containers.  To minimize the potential for 
sorption to PVC plastic bags and tubing as well the leaching of phthalate plasticizer (DEHP) into 
the solution, only non-PVC plastic bags and intravenous administration sets should be utilized. 
Administration: Tacrolimus may be given intravenously over 24 hours or orally. 
 
 

 
 
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16.  REFERENCES 
 
1. 
Young, N.S. and A.J. Barrett, The treatment of severe acquired aplastic anemia. Blood, 1995. 
85(12): p. 3367-77. 
2. 
Young, N.S. and J. Maciejewski, The pathophysiology of acquired aplastic anemia. N Engl J 
Med, 1997. 336(19): p. 1365-72. 
3. 
Zoumbos, N.C., et al., Circulating activated suppressor T lymphocytes in aplastic anemia. N 
Engl J Med, 1985. 312(5): p. 257-65. 
4. 
Platanias, L., et al., Lymphocyte phenotype and lymphokines following anti-thymocyte globulin 
therapy in patients with aplastic anaemia. Br J Haematol, 1987. 66(4): p. 437-43. 
5. 
Maciejewski, J.P., et al., Bone marrow and peripheral blood lymphocyte phenotype in patients 
with bone marrow failure. Exp Hematol, 1994. 22(11): p. 1102-10. 
6. 
Maciejewski, J.P., et al., Increased expression of Fas antigen on bone marrow CD34+ cells of 
patients with aplastic anaemia. Br J Haematol, 1995. 91(1): p. 245-52. 
7. 
Maciejewski, J., et al., Fas antigen expression on CD34+ human marrow cells is induced by 
interferon gamma and tumor necrosis factor alpha and potentiates cytokine-mediated 
hematopoietic suppression in vitro. Blood, 1995. 85(11): p. 3183-90. 
8. 
Risitano, A.M., et al., Oligoclonal and polyclonal CD4 and CD8 lymphocytes in aplastic anemia 
and paroxysmal nocturnal hemoglobinuria measured by V beta CDR3 spectratyping and flow 
cytometry. Blood, 2002. 100(1): p. 178-83. 
9. 
Bourdage, J.S. and D.M. Hamlin, Comparative polyclonal antithymocyte globulin and 
antilymphocyte/antilymphoblast globulin anti-CD antigen analysis by flow cytometry. 
Transplantation, 1995. 59(8): p. 1194-200. 
10. 
Raefsky, E.L., et al., Biological and immunological characterization of ATG and ALG. Blood, 
1986. 68(3): p. 712-9. 
11. 
Sloand, E., et al., Intracellular interferon-gamma in circulating and marrow T cells detected by 
flow cytometry and the response to immunosuppressive therapy in patients with aplastic anemia. 
Blood, 2002. 100(4): p. 1185-91. 
12. 
Zeng, W., et al., Characterization of T-cell repertoire of the bone marrow in immune-mediated 
aplastic anemia: evidence for the involvement of antigen-driven T-cell response in cyclosporine-
dependent aplastic anemia. Blood, 1999. 93(9): p. 3008-16. 
13. 
Gascon, P., et al., Lymphokine abnormalities in aplastic anemia: implications for the mechanism 
of action of antithymocyte globulin. Blood, 1985. 65(2): p. 407-13. 
14. 
Greco, B., et al., Antithymocyte globulin reacts with many normal human cell types. Blood, 1983. 
62(5): p. 1047-54. 
15. 
Barrett, A.J., et al., Crossreaction of antilymphocyte globulin with human granulocyte colony-
forming cells. J Clin Pathol, 1978. 31(2): p. 129-35. 
16. 
Di Bona, E., et al., Rabbit antithymocyte globulin (r-ATG) plus cyclosporine and granulocyte 
colony stimulating factor is an effective treatment for aplastic anaemia patients unresponsive to a 
first course of intensive immunosuppressive therapy. Gruppo Italiano Trapianto di Midollo Osseo 
(GITMO). Br J Haematol, 1999. 107(2): p. 330-4. 
17. 
Stein, R.S., et al., Treatment of aplastic anemia with an investigational antilymphocyte serum 
prepared in rabbits. Am J Med Sci, 1994. 308(6): p. 338-43. 
18. 
Bacigalupo, A., et al., Antilymphocyte globulin, cyclosporin, and granulocyte colony-stimulating 
factor in patients with acquired severe aplastic anemia (SAA): a pilot study of the EBMT SAA 
Working Party. Blood, 1995. 85(5): p. 1348-53. 
19. 
Rosenfeld, S.J., et al., Intensive immunosuppression with antithymocyte globulin and 
cyclosporine as treatment for severe acquired aplastic anemia. Blood, 1995. 85(11): p. 3058-65. 
20. 
Shevach, E.M., The effects of cyclosporin A on the immune system. Annu Rev Immunol, 1985. 3: 
p. 397-423. 

 
 
08-H-0046 
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21. 
Orosz, C.G., et al., Analysis of cloned T cell function. II. Differential blockade of various cloned T 
cell functions by cyclosporine. Transplantation, 1983. 36(6): p. 706-11. 
22. 
Nussenblatt, R.B. and A.G. Palestine, Cyclosporine: immunology, pharmacology and therapeutic 
uses. Surv Ophthalmol, 1986. 31(3): p. 159-69. 
23. 
Brodsky, R.A., et al., Durable treatment-free remission after high-dose cyclophosphamide 
therapy for previously untreated severe aplastic anemia. Ann Intern Med, 2001. 135(7): p. 477-
83. 
24. 
Tisdale, J.F., et al., High-dose cyclophosphamide in severe aplastic anaemia: a randomised trial. 
Lancet, 2000. 356(9241): p. 1554-9. 
25. 
Tisdale, J.F., et al., Late complications following treatment for severe aplastic anemia (SAA) with 
high-dose cyclophosphamide (Cy): follow-up of a randomized trial. Blood, 2002. 100(13): p. 
4668-70. 
26. 
Storb, R., et al., Changing trends in marrow transplantation for aplastic anemia. Bone Marrow 
Transplant, 1992. 10 Suppl 1: p. 45-52. 
27. 
Horowitz, M.M., Current status of allogeneic bone marrow transplantation in acquired aplastic 
anemia. Semin Hematol, 2000. 37(1): p. 30-42. 
28. 
Tsai, T.W. and C.O. Freytes, Allogenic bone marrow transplantation for leukemias and aplastic 
anemia. Adv Intern Med, 1997. 42: p. 423-51. 
29. 
Margolis, D.A. and B.M. Cammita, Hematopoietic stem cell transplantation for severe aplastic 
anemia. Curr Opin Hematol, 1998. 5(6): p. 441-4. 
30. 
Storb, R., et al., Cyclophosphamide combined with antithymocyte globulin in preparation for 
allogeneic marrow transplants in patients with aplastic anemia. Blood, 1994. 84(3): p. 941-9. 
31. 
Deeg, H.J., et al., Long-term outcome after marrow transplantation for severe aplastic anemia. 
Blood, 1998. 91(10): p. 3637-45. 
32. 
Kahl, C., et al., Cyclophosphamide and antithymocyte globulin as a conditioning regimen for 
allogeneic marrow transplantation in patients with aplastic anaemia: a long-term follow-up. Br J 
Haematol, 2005. 130(5): p. 747-51. 
33. 
Gluckman, E., et al., Bone marrow transplantation for severe aplastic anemia: influence of 
conditioning and graft-versus-host disease prophylaxis regimens on outcome. Blood, 1992. 79(1): 
p. 269-75. 
34. 
Gluckman, E., et al., Bone marrow transplantation in 107 patients with severe aplastic anemia 
using cyclophosphamide and thoraco-abdominal irradiation for conditioning: long-term follow-
up. Blood, 1991. 78(9): p. 2451-5. 
35. 
Deeg, H.J., et al., Malignancies after marrow transplantation for aplastic anemia and fanconi 
anemia: a joint Seattle and Paris analysis of results in 700 patients. Blood, 1996. 87(1): p. 386-
92. 
36. 
Pierga, J.Y., et al., Secondary solid malignant tumors occurring after bone marrow 
transplantation for severe aplastic anemia given thoraco-abdominal irradiation. Radiother 
Oncol, 1994. 30(1): p. 55-8. 
37. 
Socie, G., et al., Malignant tumors occurring after treatment of aplastic anemia. European Bone 
Marrow Transplantation-Severe Aplastic Anaemia Working Party. N Engl J Med, 1993. 329(16): 
p. 1152-7. 
38. 
Socie, G., et al., Increased incidence of solid malignant tumors after bone marrow 
transplantation for severe aplastic anemia. Blood, 1991. 78(2): p. 277-9. 
39. 
Young, N.S., R.T. Calado, and P. Scheinberg, Current concepts in the pathophysiology and 
treatment of aplastic anemia. Blood, 2006. 108(8): p. 2509-19. 
40. 
Srinivasan, R., et al., Overcoming graft rejection in heavily transfused and allo-immunised 
patients with bone marrow failure syndromes using fludarabine-based haematopoietic cell 
transplantation. Br J Haematol, 2006. 133(3): p. 305-14. 
41. 
Kumar, R., et al., Fludarabine, cyclophosphamide and horse antithymocyte globulin conditioning 
regimen for allogeneic peripheral blood stem cell transplantation performed in non-HEPA filter 

 
 
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rooms for multiply transfused patients with severe aplastic anemia. Bone Marrow Transplant, 
2006. 37(8): p. 745-9. 
42. 
Passweg, J.R., et al., Bone marrow transplants from mismatched related and unrelated donors for 
severe aplastic anemia. Bone Marrow Transplant, 2006. 37(7): p. 641-9. 
43. 
Deeg, H.J., et al., Optimization of conditioning for marrow transplantation from unrelated donors 
for patients with aplastic anemia after failure of immunosuppressive therapy. Blood, 2006. 
108(5): p. 1485-91. 
44. 
Bacigalupo, A., et al., Fludarabine, cyclophosphamide and anti-thymocyte globulin for 
alternative donor transplants in acquired severe aplastic anemia: a report from the EBMT-SAA 
Working Party. Bone Marrow Transplant, 2005. 36(11): p. 947-50. 
45. 
Mao, P., et al., Sustained and stable hematopoietic donor-recipient mixed chimerism after 
unrelated cord blood transplantation for adult patients with severe aplastic anemia. Eur J 
Haematol, 2005. 75(5): p. 430-5. 
46. 
Cornetta, K., et al., Umbilical cord blood transplantation in adults: results of the prospective 
Cord Blood Transplantation (COBLT). Biol Blood Marrow Transplant, 2005. 11(2): p. 149-60. 
47. 
Laughlin, M.J., et al., Hematopoietic engraftment and survival in adult recipients of umbilical-
cord blood from unrelated donors. N Engl J Med, 2001. 344(24): p. 1815-22. 
48. 
Rubinstein, P., et al., Outcomes among 562 recipients of placental-blood transplants from 
unrelated donors. N Engl J Med, 1998. 339(22): p. 1565-77. 
49. 
Laughlin, M.J., et al., Outcomes after transplantation of cord blood or bone marrow from 
unrelated donors in adults with leukemia. N Engl J Med, 2004. 351(22): p. 2265-75. 
50. 
Rocha, V., et al., Transplants of umbilical-cord blood or bone marrow from unrelated donors in 
adults with acute leukemia. N Engl J Med, 2004. 351(22): p. 2276-85. 
51. 
Takahashi, S., et al., Single-institute comparative analysis of unrelated bone marrow 
transplantation and cord blood transplantation for adult patients with hematologic malignancies. 
Blood, 2004. 104(12): p. 3813-20. 
52. 
Barker, J.N., et al., Transplantation of 2 partially HLA-matched umbilical cord blood units to 
enhance engraftment in adults with hematologic malignancy. Blood, 2005. 105(3): p. 1343-7. 
53. 
Nand, S. and J.E. Godwin, Hypoplastic myelodysplastic syndrome. Cancer, 1988. 62(5): p. 958-
64. 
54. 
Jacobs, R.H., et al., Prognostic implications of morphology and karyotype in primary 
myelodysplastic syndromes. Blood, 1986. 67(6): p. 1765-72. 
55. 
Yunis, J.J., et al., Refined chromosome study helps define prognostic subgroups in most patients 
with primary myelodysplastic syndrome and acute myelogenous leukaemia. Br J Haematol, 1988. 
68(2): p. 189-94. 
56. 
Morel, P., et al., Cytogenetic analysis has strong independent prognostic value in de novo 
myelodysplastic syndromes and can be incorporated in a new scoring system: a report on 408 
cases. Leukemia, 1993. 7(9): p. 1315-23. 
57. 
Toyama, K., et al., Clinical implications of chromosomal abnormalities in 401 patients with 
myelodysplastic syndromes: a multicentric study in Japan. Leukemia, 1993. 7(4): p. 499-508. 
58. 
Greenberg, P., et al., International scoring system for evaluating prognosis in myelodysplastic 
syndromes. Blood, 1997. 89(6): p. 2079-88. 
59. 
Greenberg, P., Myelodysplastic Syndrome. 3rd ed. Hematology: Basic Principles and Practice, ed. 
R. Hoffman, Benz, EJ, Shattil, SJ. 2000, New York: Churchill Livingstone. 1106. 
60. 
Heaney, M.L. and D.W. Golde, Myelodysplasia. N Engl J Med, 1999. 340(21): p. 1649-60. 
61. 
Mantovani, L., et al., Treatment of anaemia in myelodysplastic syndromes with prolonged 
administration of recombinant human granulocyte colony-stimulating factor and erythropoietin. 
Br J Haematol, 2000. 109(2): p. 367-75. 
62. 
Hellstrom-Lindberg, E., et al., Treatment of anemia in myelodysplastic syndromes with 
granulocyte colony-stimulating factor plus erythropoietin: results from a randomized phase II 
study and long-term follow-up of 71 patients. Blood, 1998. 92(1): p. 68-75. 

 
 
08-H-0046 
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63. 
Negrin, R.S., et al., Maintenance treatment of the anemia of myelodysplastic syndromes with 
recombinant human granulocyte colony-stimulating factor and erythropoietin: evidence for in 
vivo synergy. Blood, 1996. 87(10): p. 4076-81. 
64. 
Negrin, R.S., et al., Treatment of the anemia of myelodysplastic syndromes using recombinant 
human granulocyte colony-stimulating factor in combination with erythropoietin. Blood, 1993. 
82(3): p. 737-43. 
65. 
Negrin, R.S., et al., Treatment of myelodysplastic syndromes with recombinant human 
granulocyte colony-stimulating factor. A phase I-II trial. Ann Intern Med, 1989. 110(12): p. 976-
84. 
66. 
Negrin, R.S., et al., Maintenance treatment of patients with myelodysplastic syndromes using 
recombinant human granulocyte colony-stimulating factor. Blood, 1990. 76(1): p. 36-43. 
67. 
Molldrem, J.J., et al., Antithymocyte globulin for treatment of the bone marrow failure associated 
with myelodysplastic syndromes. Ann Intern Med, 2002. 137(3): p. 156-63. 
68. 
Saunthararajah, Y., et al., HLA-DR15 (DR2) is overrepresented in myelodysplastic syndrome and 
aplastic anemia and predicts a response to immunosuppression in myelodysplastic syndrome. 
Blood, 2002. 100(5): p. 1570-4. 
69. 
Saunthararajah, Y., et al., A simple method to predict response to immunosuppressive therapy in 
patients with myelodysplastic syndrome. Blood, 2003. 102(8): p. 3025-7. 
70. 
Steensma, D.P., et al., Antithymocyte globulin has limited efficacy and substantial toxicity in 
unselected anemic patients with myelodysplastic syndrome. Blood, 2003. 101(6): p. 2156-8. 
71. 
Jonasova, A., et al., Cyclosporin A therapy in hypoplastic MDS patients and certain refractory 
anaemias without hypoplastic bone marrow. Br J Haematol, 1998. 100(2): p. 304-9. 
72. 
Silverman, L.R., et al., Randomized controlled trial of azacitidine in patients with the 
myelodysplastic syndrome: a study of the cancer and leukemia group B. J Clin Oncol, 2002. 
20(10): p. 2429-40. 
73. 
Kornblith, A.B., et al., Impact of azacytidine on the quality of life of patients with myelodysplastic 
syndrome treated in a randomized phase III trial: a Cancer and Leukemia Group B study. J Clin 
Oncol, 2002. 20(10): p. 2441-52. 
74. 
Kantarjian, H.M., Treatment of myelodysplastic syndrome: questions raised by the azacitidine 
experience. J Clin Oncol, 2002. 20(10): p. 2415-6. 
75. 
List, A., et al., Lenalidomide in the myelodysplastic syndrome with chromosome 5q deletion. N 
Engl J Med, 2006. 355(14): p. 1456-65. 
76. 
Anderson, J.E., et al., Allogeneic bone marrow transplantation for 93 patients with 
myelodysplastic syndrome. Blood, 1993. 82(2): p. 677-81. 
77. 
De Witte, T., et al., Allogeneic bone marrow transplantation for secondary leukaemia and 
myelodysplastic syndrome: a survey by the Leukaemia Working Party of the European Bone 
Marrow Transplantation Group (EBMTG). Br J Haematol, 1990. 74(2): p. 151-5. 
78. 
Sutton, L., et al., Factors influencing outcome in de novo myelodysplastic syndromes treated by 
allogeneic bone marrow transplantation: a long-term study of 71 patients Societe Francaise de 
Greffe de Moelle. Blood, 1996. 88(1): p. 358-65. 
79. 
Nevill, T.J., et al., Treatment of myelodysplastic syndrome with busulfan-cyclophosphamide 
conditioning followed by allogeneic BMT. Bone Marrow Transplant, 1992. 10(5): p. 445-50. 
80. 
Anderson, J.E., et al., Allogeneic marrow transplantation for refractory anemia: a comparison of 
two preparative regimens and analysis of prognostic factors. Blood, 1996. 87(1): p. 51-8. 
81. 
Runde, V., et al., Bone marrow transplantation from HLA-identical siblings as first-line 
treatment in patients with myelodysplastic syndromes: early transplantation is associated with 
improved outcome. Chronic Leukemia Working Party of the European Group for Blood and 
Marrow Transplantation. Bone Marrow Transplant, 1998. 21(3): p. 255-61. 
82. 
Anderson, J.E., F.R. Appelbaum, and R. Storb, An update on allogeneic marrow transplantation 
for myelodysplastic syndrome. Leuk Lymphoma, 1995. 17(1-2): p. 95-9. 

 
 
08-H-0046 
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83. 
Kolb, H.J., et al., Graft-versus-leukemia reactions in allogeneic chimeras. Blood, 2004. 103(3): p. 
767-76. 
84. 
Woods, W.G., et al., A comparison of allogeneic bone marrow transplantation, autologous bone 
marrow transplantation, and aggressive chemotherapy in children with acute myeloid leukemia 
in remission. Blood, 2001. 97(1): p. 56-62. 
85. 
Horowitz, M.M., et al., Graft-versus-leukemia reactions after bone marrow transplantation. 
Blood, 1990. 75(3): p. 555-62. 
86. 
Weiden, P.L., et al., Antileukemic effect of graft-versus-host disease in human recipients of 
allogeneic-marrow grafts. N Engl J Med, 1979. 300(19): p. 1068-73. 
87. 
Gorin, N.C., et al., Retrospective evaluation of autologous bone marrow transplantation vs 
allogeneic bone marrow transplantation from an HLA identical related donor in acute myelocytic 
leukemia. A study of the European Cooperative Group for Blood and Marrow Transplantation 
(EBMT). Bone Marrow Transplant, 1996. 18(1): p. 111-7. 
88. 
Zittoun, R.A., et al., Autologous or allogeneic bone marrow transplantation compared with 
intensive chemotherapy in acute myelogenous leukemia. European Organization for Research 
and Treatment of Cancer (EORTC) and the Gruppo Italiano Malattie Ematologiche Maligne 
dell'Adulto (GIMEMA) Leukemia Cooperative Groups. N Engl J Med, 1995. 332(4): p. 217-23. 
89. 
Feinstein, L., et al., Nonmyeloablative hematopoietic cell transplantation. Replacing high-dose 
cytotoxic therapy by the graft-versus-tumor effect. Ann N Y Acad Sci, 2001. 938: p. 328-37; 
discussion 337-9. 
90. 
Slavin, S., et al., Nonmyeloablative stem cell transplantation and cell therapy as an alternative to 
conventional bone marrow transplantation with lethal cytoreduction for the treatment of 
malignant and nonmalignant hematologic diseases. Blood, 1998. 91(3): p. 756-63. 
91. 
Kroger, N., et al., Allogeneic stem cell transplantation after a fludarabine/busulfan-based 
reduced-intensity conditioning in patients with myelodysplastic syndrome or secondary acute 
myeloid leukemia. Ann Hematol, 2003. 82(6): p. 336-42. 
92. 
Martino, R., et al., Evidence for a graft-versus-leukemia effect after allogeneic peripheral blood 
stem cell transplantation with reduced-intensity conditioning in acute myelogenous leukemia and 
myelodysplastic syndromes. Blood, 2002. 100(6): p. 2243-5. 
93. 
T. De Witte, R.B.a.A.V.B.e.a., Allogeneic stem cell transplantation with matched related and 
unrelated donors for patients with refractory anemia: T-cell depletion and reduced intensity 
regimens are associated with increased relapse risk. Blood, 2003. 102: p. 422a. 
94. 
Arnold, R., et al., Unrelated bone marrow transplantation in patients with myelodysplastic 
syndromes and secondary acute myeloid leukemia: an EBMT survey. European Blood and 
Marrow Transplantation Group. Bone Marrow Transplant, 1998. 21(12): p. 1213-6. 
95. 
Powles, R.L., et al., Mismatched family donors for bone-marrow transplantation as treatment for 
acute leukaemia. Lancet, 1983. 1(8325): p. 612-5. 
96. 
Beatty, P.G., et al., Marrow transplantation from related donors other than HLA-identical 
siblings. N Engl J Med, 1985. 313(13): p. 765-71. 
97. 
Szydlo, R., et al., Results of allogeneic bone marrow transplants for leukemia using donors other 
than HLA-identical siblings. J Clin Oncol, 1997. 15(5): p. 1767-77. 
98. 
Aversa, F., et al., Full haplotype-mismatched hematopoietic stem-cell transplantation: a phase II 
study in patients with acute leukemia at high risk of relapse. J Clin Oncol, 2005. 23(15): p. 3447-
54. 
99. 
Kim, H.J., et al., Haplotype mismatched transplantation using high doses of peripheral blood 
CD34+ cells together with stratified conditioning regimens for high-risk adult acute myeloid 
leukemia patients: a pilot study in a single Korean institution. Bone Marrow Transplant, 2005. 
35(10): p. 959-64. 
100. 
Magro, E., et al., Early hematopoietic recovery after single unit unrelated cord blood 
transplantation in adults supported by co-infusion of mobilized stem cells from a third party 
donor. Haematologica, 2006. 91(5): p. 640-8. 

 
 
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Richard Childs, M.D. 
May 13, 2019 (Amendment OO)  
68
101. 
Rosenfeld, S., et al., Antithymocyte globulin and cyclosporine for severe aplastic anemia: 
association between hematologic response and long-term outcome. Jama, 2003. 289(9): p. 1130-
5. 
102. 
Simon, R., Optimal two-stage designs for phase II clinical trials. Control Clin Trials, 1989. 
10(1): p. 1-10. 
 
104   Kopp HG, Horger M, Faul C, Hartmann JT, Kanz L, Lang P, Vogel W,  Granulocyte colony-
stimulating factor induced pulmonary hemorrhage in a healthy stem cell donor. Granulocyte 
colony-stimulating factor induced pulmonary hemorrhage in a healthy stem cell donor. J Clin 
Oncol. 2007 Jul 20;25(21):3174-5. 
104. 
Baker, F., & Kim, S. The Basics of Item Response Theory (2nd ed.). 2010; New York: Marcel 
Dekker. 
 
Johnson J, Temple R. Food and Drug Administration requirement for approval of new anti-cancer 
drugs. Cancer Treat Rep. 1985;69:1155-1157. 
105. 
PROMIS. (2011). Patient-Reported Outcomes Measurement Information System: Dynamic Tools 
to Measure Health Outcomes From the Patient Perspective 
  
Retrieved January 31, 2011, from http://www.nihpromis.org/default.aspx 
106. 
Riley, W. T., Rothrock, N., Bruce, B., Christodolou, C., Cook, K., Hahn, E. A., et al. Patient-
reported outcomes measurement information system (PROMIS) domain names and definitions 
revisions: Further evaluation of content validity in IRT-derived item banks. Quality of Life 
Research. 2010 19(9), 1311-1321. 
107. 
Institute of Medicine. Cancer Care for the Whole Patient: Meeting Psychosocial Health Needs. 
Washington, DC: IOM; 2008. 
108. 
Lee S, Francis Cook E, Soiffer R, Anttin JH. Development and Validation of a Scale to Measure 
Symptoms of Chronic Graft-versus-Host Disease. Biology of Blood and Marrow Transplantation. 
2002, 8:444-452. 
109. 
Cella D: Manual of the Functional Assessment of Chronic Illness Therapy (FACIT Scales) 
(Version 4.1) 2004. 
110. 
Cella D, Hernandez L, Bonomi AE, et al: Spanish language translation and initial validation of 
the functional assessment of cancer therapy quality-of-life instrument. Med Care. 1998; 36:1407-
18. 
111. 
Syrjala KL, Schroeder TC, Abrams JR, Atkins TZ, Sanders JE, Brown W, Heiman JR.Sexual 
function measurement and outcomes in cancer survivors and matched controls. Journal of Sex 
Research. 2000; 37(3) 213-225. 
112. 
Geller NL, Follmann D, Leifer ES, Carter SL, Design of Early Trials in Stem Cell 
Transplantation: A Hybrid Frequentist-Bayesian Approach.  In Advances in Clinical Trial 
Biostatistics (ed. Geller NL), 2004. New York: Marcel Dekker, Inc. 
113. 
Lechowicz et al. Allogeneic hematopoietic cell transplantation for mycosis fungoides and Sezary 
syndrome. Bone Marrow Transplantation (2014) 49, 1360–1365. 
114. 
Duarte RF et al. Allogeneic Hematopoietic Cell Transplantation for Patients With Mycosis 
Fungoides and Se´zary Syndrome: A Retrospective Analysis of the Lymphoma Working Party of 
the European Group for Blood and Marrow Transplantation. J Clin Oncol 2010. 
115. 
Duarte RF et al. Long-Term Outcome of Allogeneic Hematopoietic Cell Transplantation for 
Patients With Mycosis Fungoides and Se´zary Syndrome: A European Society for Blood and 
Marrow Transplantation Lymphoma Working Party Extended Analysis. J Clin Oncol 2014. 
 
 
 
 

 
 
08-H-0046 
Richard Childs, M.D. 
May 13, 2019 (Amendment OO)  
69
APPENDIX A:  IRB APPROVED NHLBI LABORATORY RESEARCH STUDIES  
 
v. 2/5/2013 
 
 
DESCRIPTION OF LABORATORY STUDY BY BRANCH SECTION 
Does this test 
pose a greater 
than minimal 
risk to 
pediatric 
subjects per 45 
CFR 46.404? 
Does this test 
pose a 
greater than 
minimal risk 
to healthy 
pediatric 
donors per 45 
CFR 46.404? 
 
 
 
 
A 
Stem Cell Allotransplantation Section (Dr. A. John Barrett) 
 
 
A.1 
Measurement of lymphocyte function and immune responses directed toward 
allogeneic tissues, malignant cells, and infectious agents.  Assay of a variety of 
antigens, including standard proliferation, cytotoxicity, and intracellular cytokine  
detection including GVHD predictive markers. Measurement of antigen-specific 
responses including employment of tetramers, ELISPOT technique, gene 
amplification-based assays, and flow cytometry.  Selection of cells using 
immunomagnetic beads or flow cytometry.  Culture, expansion, and selection of 
cells. Surface marker analysis of PB MC using flow cytometry. Cytokine/chemokine 
analysis of plasma/serum samples using ELISA and/or Luminex techniques. 
 
No 
No 
A.2 
Generation of cell lines for the study of immune cell interactions with other cells.  
Transformation of B-lymphocytes using Epstein-Barr virus.  Derivation of malignant 
cell lines from patient leukemic or solid tumor samples. 
No 
No 
A.3 
Infection of cells and cell lines with recombinant genes to ascertain the effects of 
expressed molecules on immune responses and on growth and development. 
Transfection of cell lines with specific molecules to study antigen-specific responses. 
No 
No 
A.4 
Assays of peripheral blood and bone marrow progenitor cells including primitive and 
late erythroid progenitor-derived colonies, myelomonocytic colonies, and primitive 
multi- potential progenitor-derived colonies. 
No 
No 
A.5 
Injection of human cells into experimental animals to study the immune system and 
the growth of normal and malignant cells under varying conditions. 
No 
No 
A.6 
Testing of selection methods, cell isolation, and cell expansion leading to the 
development of new cell-based therapies requiring scale-up for clinical application. 
No 
No 
A.7 
Identification of individual T cell clones by their T cell receptor sequence. 
No 
No 
A.8 
Measurement of tumor and tissue specific antigens in cells of subjects and donors by 
mRNA,protein, or peptide expression in cells or fluids. 
No 
No 
A.9 
Laser capture micro dissection of cells from biopsies for GVHD to determine 
clonotypes. 
No 
No 
A.10 
DNA and RNA typing of genes that control immune responses in lymphocytes. 
 
No 
No 
A.11 
Microassay studies utilizing cellular DNA, cDNA, and RNA for neoplasia and host-
tumor interactions. 
No 
No 
 
 
 
 
B 
Molecular Hematopoiesis  Section  (Dr. Cynthia Dunbar)  
 
 
B.1 
Flow cytometric analysis of cell surface and cytoplasmic proteins, including cell 
adhesion molecules, putative retroviral receptors, and markers of differentiation, 
using bone marrow and mobilized peripheral blood cells. 
No 
No 

 
 
08-H-0046 
Richard Childs, M.D. 
May 13, 2019 (Amendment OO)  
70
B.2 
Hematopoietic progenitor-derived colony ascertainment in vitro (as described above), 
and engraftment of immunodeficient mice for detection of human stem cell number 
and function. 
No 
No 
B.3 
Testing ability of hematopoietic progenitor cells to be transduced with retroviral, 
lentiviral, and novel gene transfer vectors in vitro. 
No 
No 
B.4 
Reprogramming of adult mature cells, including skin fibroblasts and blood cells, into 
induced pluripotent stem cells in vitro. 
No 
No 
 
 
 
 
C 
Cell Biology Section (Dr. Neal Young)  
 
 
C.1 
Studies of blood and bone marrow hematopoietic progenitor numbers, including 
early and late erythroid progenitors, myelomonocytic progenitors, and multi-potential 
progenitor cells.  In addition, bone marrow may be placed in long-term bone marrow 
culture to assess the function of stroma and stem cells and to assay more primitive 
progenitors, as well as organelle culture.  Whole or selected bone marrow 
populations are cultured short-term for CD34 cell expansion. 
No 
No 
C.2 
Assays of apoptosis in hematopoietic cells and their progeny, using flow cytometric 
methods such as annexin and caspase-3 staining, propidium iodide uptake, and 
mitochondrial permeability tests. 
No 
No 
C.3 
Separation and functional study of cell populations characteristic of paroxysmal 
nocturnal hemoglobinuria, identified by absence of glycosylphosphatidylinositol 
anchored proteins. 
No 
No 
C.4 
Studies of mutation rates in hematopoietic cells and in buccal mucosa cells, using 
conventional hypoxanthine phosphoribosyltransferase activity functional assays, 
sequencing of mitochondrial DNA after specific gene amplification, and 
measurement of GPI-anchored deficient cells in blood and bone marrow. 
No 
No 
C.5 
Assays of immune function of T-cells, including intracellular cytokine staining, 
ELISPOT, semiquantitative gene amplification for gamma-interferon, tumor necrosis 
factor, interleukin-2, and other cytokines, and functional assessment in co-culture 
using specific neutralizing monoclonal antibodies.  In addition, peripheral blood 
lymphocytes are subjected to spectratyping for CDR3 size distribution as well as 
nucleotide sequence of CDR3 peaks obtained. 
No 
No 
C.6 
Studies of engraftment of human normal and diseased bone marrow and peripheral 
blood in immunodeficient mice in order to determine the presence of hematopoietic 
repopulating stem cells as well as functional differences among selected populations. 
No 
No 
C.7 
Flow cytometric analysis of blood and bone marrow for lymphocyte phenotype, 
especially for evidence of activation of lymphocytes, for markers of apoptosis, and 
for antigens associated with primitive and mature hematopoietic cell populations. 
No 
No 
C.8 
Flow cytometric analysis of blood and bone marrow for hematopoietic stem cell 
progenitors and CD34 positive cells. 
No 
No 
C.9 
Studies of chromosomal instability in myelopdysplastic syndromes including BM 
cell and CD34 cell response to PAS crosslinking and examination of the cytotoxic 
effect of lymphocytes to the abnormal clone of cells. 
No 
No 
C.10 
Surface Enhanced Laser/Desorption Ionization (SELDI)  time-of-flight mass 
spectrometry (Ciphergen)  (proteomics methodology). 
No 
No 
C.11 
Mitochondrial DNA (mtDNA) sequence heterogeneity. 
No 
No 
C.12 
Measurement of EBV viral load. 
No 
No 
C.13 
Measurement of EBV LMP-1 via RT-PCR for LMP-1 RNA or flow cytometry for 
LMP-1. 
No 
No 
C.14 
Outgrowth assay of EBV transformed B cells. 
No 
No 
C.15 
Quantification of serumchemokines and cytokines (e.g. SDF-1, IL-10, IL-6, CXCR4, 
CXCL12). 
No 
No 
C.16 
Quantification of EBV cytotoxic T cells (tetramerstaining). 
No 
No 

 
 
08-H-0046 
Richard Childs, M.D. 
May 13, 2019 (Amendment OO)  
71
C.17 
Telomere length measurement by Southern blot, Q-PCR, flow-fish, in situ 
hybridization and STELA 
No 
No 
C.18 
Telomere repair complex gene mutations by nucleotide sequencing of some or all of 
the following:  DKC1 ,TERC, TERT, SBDS, NOp10 , NHP2. 
No 
No 
C.19 
Analysis of  inflammatory markers and/or bacterial, viral, fungal or protozoal 
elements in plasma or serum using molecular, colorimetric, enzymatic, flow 
cytometric or other assays in subjects receiving immunosuppressive therapy, 
chemotherapy and/or bone marrow transplantation. 
No 
No 
C.20 
Confocal microscopic imaging of bone marrow. 
No 
No 
C.21 
Characterization of intracellular signaling proteins by cell permeabilization and flow 
cytometry, and quantitative immunoblots. 
No 
No 
C.22 
Assays for chromosomal aneuploidy by florescence in situ hybridization (FISH) and 
other molecular techniques. 
No 
No 
C.23 
Conversion of human dermal fibroblasts into hematopoietic progenitors using Oct4 
transfection. 
No 
No 
C.24 
Quantification of gene expression with RNA-seq 
No 
No 
C.25 
Characterization of chromatin and promoter/enhancer landscapes with ATAC-seq 
No 
No 
C.26 
Measurement of protein markers with SomaLogic's SOMAscan assay 
No 
No 
 
 
 
 
D 
Virus Discovery Section (Dr. Neal Young) THESE ASSAYS WILL NOT BE 
PERFORMED ON SAMPLES FROM HEALTHY PEDIATRIC DONORS 
 
 
D.1 
Assays of serum, blood cells, and bone marrow cells for B19 parvovirus and possible 
B19  variants using gene amplification, cell culture, and hematopoietic colony 
inhibition assays. 
No 
N/A 
D.2 
Assays of blood, bone marrow, liver, and other tissues for potentially novel viruses, 
using a variety of techniques including RNA and DNA assays, differential display, 
gene amplification with conserved and random primers, cell culture assays, 
immunohistochemical methods, and inocculation of mice, rabbits, and monkeys, as 
well as antibody measurements. 
No 
N/A 
D.3 
Assays of blood, bone marrow, and liver for known viruses, including herpesviruses 
such as cytomegalovirus, human herpesviruses 6, 7, and 8, enteric viruses such as A-
6, circiviruses, and parvoviruses, using assays as in (2). 
No 
N/A 
D.4 
Spectra-typing of blood cells to determine response to known or putative viral 
infections. 
No 
N/A 
D.5 
HLA typing or subtyping to determine risk factors/determinants for hepatitis-AA 
studies. 
No 
N/A 
D.6 
Cytotoxic lymphocyte assays with intracellular cytokine measurement for 
determining anti-viral response and lymphocyte cloning to obtain clones with 
specific antiviral activity. 
No 
N/A 
 
 
 
 
E 
Solid Tumor Section (Dr. Richard Childs) 
 
 
E.1 
Cr51 cytotoxicity assay to evaluating killing of patient tumor cells by patient NK cell 
clones and T-cells. 
No 
No 
E.2 
ELISA for IL-12  maturity of DC's made from subjects monocytes. 
No 
No 
E.3 
ELISA for IFN ã to evaluate specificity of CTL clones. 
No 
No 
E.4 
H thymidine uptake to evaluate proliferation potential of antigen specific T-cells. 
No 
No 
E.5 
PCR of STR to assess chimerism status of cellular subsets grown in-vitro or retrieved 
from subjects post-transplant. 
No 
No 
E.6 
Flow sorting of PBL and/or tissue samples to evaluate chimerism of different subsets. 
No 
No 
E.7 
Surface marker analysis of peripheral blood mononuclear cells using flow cytometry. 
No 
No 
E.8 
cDNA expression arrays to evaluate T-cells expression/gene patterns in subjects with 
GVHD and a GVT effect. 
No 
No 

 
 
08-H-0046 
Richard Childs, M.D. 
May 13, 2019 (Amendment OO)  
72
 
 
E.9 
Geno typing of tumor or tissue samples by high density cDNA arrays. 
No 
No 
E.10 
VHL mutation analysis on kidney cancer tissue. 
No 
No 
E.11 
Transduction of dendritic and tissue cells with tumor antigens using plasmids, viral 
vectors and hybrid fusions. 
No 
No 
E.12 
Lasar capture microdisection of cells from tumor biopsies and tissue samples  to 
determine origin (donor vs patient). 
No 
No 
E.13 
Quantification of polyoma virus BK exposure by serology and PCR in stem cell 
transplant donors and recipients from blood and urine samples. 
No 
No 
E.14 
Quantification of polyoma virus BK specific T cells in stem cell transplant donors 
and recipients from peripheral blood samples. 
No 
No 
E.15 
Determination of origin of neovasculature endothelial cells in tumor and tissue 
samples obtained from subjects post transplant. 
No 
No 
E.16 
Quantification of lymphocyte subsets CD34 progenitors and endovasculator 
progenitors in G-CSF mobilized peripheral cell allografts. 
No 
No 
E.17 
Testing for polyoma virus BK latency in CD34 progenitors, B cells and T cells in the 
G-CSF mobilized peripheral cell allografts. 
No 
No 
E.18 
Determination of etiology of membraneous nephropathy using serum from subjects. 
No 
No 
E.19 
Serum Proteomic patterns analysis to diagnose complications related to allogeneic 
transplantation. 
No 
No 
E.20 
Determine cell origin (donor vs patient) of tissue samples using IHC, IF, sorting, and 
FISH. 
No 
No 
 
 
 
 
F 
Lymphoid Malignancies Section (Dr. Adrian Wiestner) 
 
 
F.1 
Culture of cells from research subjects to investigate molecular disease mechanisms, 
model host tumor interactions, and to test effect of drugs on cell survival and cellular 
functions. 
No 
No 
F.2 
Generation of stable cell lines for the study of hematologic malignancies. 
No 
No 
F.3 
Modifications of cells using standard expression systems or biologic molecules, e.g. 
interfering RNA, to investigate the effects of candidate genes on cellular functions. 
 
 
F.4 
Identification and monitoring of B or T cell populations as identified by flow 
cytometry and by their B cell or T cell receptor expression. 
No 
No 
F.5 
Measurement of gene expression in cells or tissues. Techniques frequently used 
include gene expression profiling on microarrays, quantitative RT-PCR, Western 
blotting, flow cytometry and ELISA assays. 
No 
No 
F.6 
Analysis of chromosomal abnormalities or mutations in malignant cells and non-
malignant cells including FISH technology and DNA sequencing. 
No 
No 
F.7 
Assays of immune function of B-cells and T-cells, including intracellular cytokine 
staining, ELISPOT, quantitative RT-PCR for cytokines or other immune regulatory 
genes. 
No 
No 
F.8 
Analysis of antibody specificities in serum and antigen specificity of the B-cell 
receptor on cells. Techniques may include expression of antibodies in phage display 
systems, generation of antibodies in cell culture systems and use of such antibodies to 
screen for cognate antigens. 
No 
No 
F.9 
Transplantation of human cells into mice (xenograft model) to study disease biology 
and to investigate the effect of experimental therapy. 
No 
No 
F.10 
Measurements of drug concentrations, biologic molecules and disease markers in 
blood, serum, and plasma. 
No 
No 

 
 
08-H-0046 
Richard Childs, M.D. 
May 13, 2019 (Amendment OO)  
73
APPENDIX B Grading of GVHD 
 
GVHD is staged based on degree of severity of skin, liver and gastrointestinal (GI) tract involvement ranging 
from mild to severe (table 1).  Acute GVHD is then graded for prognostic and treatment purposes based on a 
four-grade scale, with grade I considered mild, grade II as moderate, grade III as severe, and grade IV being a 
life-threatening (Table 2).  The likelihood of severe and life-threatening acute GVHD increases with the 
degree of mismatch between the major histocompatibility complex (MHC) antigens of the donor and those of 
the recipient as well as factors such as donor and recipient age, cytomegalovirus (CMV) donor and recipient 
status, underlying disease, conditioning regimen and type of GVHD prophylaxis.  For the purposes of 
secondary endpoint assessment the following grading schemas will be used:  
 
Table 1:   CLINICAL GRADING.   Organ systems are staged for severity as follows:  
 
SKIN 
 
 
 
 
  
 
Stage   
 
1 
 
2 
 
 3 
 
4 
 
 
Area affected 
 
<25 
  
25-50  
>50%    
exfoliation 
GASTROINTESTINAL TRACT 
Stage  
 
1 
 
2 
 
3 
 
4 
Diarrhea (l/day)  
<0.5 
 
<1.0 
 
<1.5 
 
ileus, blood loss 
LIVER 
Stage  
 
1 
 
2 
 
3 
 
4 
Bilirubin mg/dl  
3 
 
3-6 
 
6-15 
 
>15 
 
Table 2: OVERALL GRADING is made from the individual organ stages as follows:  
 
 
Skin            Gut /Liver     GRADE      
 
0  
 
0 
 
0 
 
 
 
1 
 
0 
 
I 
not clinically significant 
 
2 
 
0 
 
II 
clinically significant 
 
0-2 
 
1-2 
 
 
 
3 
 
0-2 
 
III 
 
 
 
0-3 
 
3 
 
Any 
 
4 
 
IV