A Phase II Study Using the CliniMACS® Device for CD34+ Cell Selection and T Cell Depletion for Graft-versus-Host Disease Prophylaxis in Alternative Donor Stem Cell Transplant Recipients
Phase: PHASE2
Conditions: Acute Lymphoblastic Leukemia, Acute Myeloid Leukemia, Chronic Myeloid Leukemia, Myelodysplastic Syndrome, Lymphomas, Bone Marrow Failure, Hemoglobinopathy, Immune Deficiency, Osteopetrosis
Sponsor: Wake Forest University Health Sciences

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A Phase II Study Using the CliniMACS® Device for CD34+ Cell Selection and T Cell 
Depletion for Graft-versus-Host Disease Prophylaxis in Alternative Donor Stem Cell 
Transplant Recipients 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Principal Investigator 
Andrew Gilman, MD 
 
 
 
 
Levine Children’s Hospital 
 
 
 
 
Carolinas Medical Center 
 
 
 
 
1000 Blythe Boulevard  
 
 
 
 
Charlotte, NC 28203 
 
 
 
 
Phone: (704) 381 – 6847 
 
 
 
 
Fax:   (704) 381 – 6841 
 
 
 
 
E-mail: Andrew.gilman@carolinashealthcare.org 
 
 
Investigational New Drug# BB-IND-14045 
 
Version 1.9 (1/30/2015) 
 
 
Previous Versions: 
 
Version 1.8 (3/11/2014) 
 
Version 1.7 (5/27/2012) 
 
Version 1.6 (3/29/2012) 
 
Version 1.5 (1/25/2012) 
 
Version 1.4 (4/7/2011) 
Version 1.3 (1/8/2010) 
 
Version 1.2 (8/31/2009) 
 
Version 1.1 (6/11/2009) 
 
Version 1.0 (3/26/2009) 
 
 
 
 
 
 
 
 

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TABLE OF CONTENTS 
STUDY COMMITTEE ............................................................................................................................................. 4 
SHORT ABSTRACT ................................................................................................................................................ 5 
SCHEMA ..................................................................................................................................................................... 6 
LIST OF ABBREVIATIONS .................................................................................................................................. 7 
1.0 
GOALS AND OBJECTIVES .................................................................................................................... 8 
1.1 
PRIMARY OBJECTIVE ............................................................................................................................... 8 
1.2 
SECONDARY OBJECTIVES ........................................................................................................................ 8 
2.0 
BACKGROUND AND RATIONALE ...................................................................................................... 8 
2.1 
STEM CELL TRANSPLANTATION .............................................................................................................. 8 
2.2 
MISMATCHED (HAPLOCOMPATIBLE) RELATED DONOR STEM CELL TRANSPLANT............................ 9 
2.3 
NK ALLOREACTIVITY ............................................................................................................................. 11 
2.4 
PRELIMINARY DATA ............................................................................................................................... 12 
2.5 
IMMUNE RECONSTITUTION .................................................................................................................... 12 
2.6 
RATIONALE FOR INCLUDING ALTERNATIVE CONDITIONING REGIMENS .......................................... 12 
3.0 
STUDY DESIGN AND ELIGIBILITY CRITERIA ........................................................................... 13 
3.1 
STUDY DESIGN ........................................................................................................................................ 13 
3.2 
RECIPIENT INCLUSION CRITERIA ......................................................................................................... 13 
3.3 
RECIPIENT EXCLUSION CRITERIA ........................................................................................................ 14 
3.4 
DONOR ELIGIBILITY CRITERIA ............................................................................................................. 15 
3.5 
DONOR SELECTION ................................................................................................................................ 15 
3.6 
PATIENT RECRUITMENT ........................................................................................................................ 15 
3.7 
PATIENT REGISTRATION ........................................................................................................................ 15 
4.0 
INVESTIGATIONAL TREATMENT PLAN ....................................................................................... 15 
4.1 
DOSE ........................................................................................................................................................ 15 
4.2 
DURATION OF THERAPY ........................................................................................................................ 16 
5.0 
DOSE MODIFICATIONS AND TOXICITIES .................................................................................. 16 
5.1 
RECIPIENT TOXICITY FROM THE CONDITIONING REGIMEN .............................................................. 16 
5.2 
RECIPIENT TOXICITY FROM TRANSPLANT ......................................................................................... 17 
5.3 
TOXICITY FROM MILTENYI BIOTEC INC. CLINIMACS® REAGENT SYSTEM PROCESSING .......... 18 
5.4 
DONOR  PBSC TOXICITY ...................................................................................................................... 18 
6.0 
DEVICE INFORMATION ....................................................................................................................... 19 
6.1 
CD34+  CELL PROCESSING .................................................................................................................... 19 
6.2 
RELEASE OF PRODUCT FOR TRANSPLANT .......................................................................................... 21 
6.3 
SHIPPING OF CD34+ SELECTED PBSC .............................................................................................. 21 
7.0 
SCHEDULE OF ASSESSMENTS AND THERAPY ........................................................................ 21 
7.1 
SCREENING ASSESSMENTS ................................................................................................................... 21 
7.2 
THERAPY ................................................................................................................................................. 23 
7.3 
MOBILIZATION AND COLLECTION OF DONOR PERIPHERAL BLOOD STEM CELLS ........................... 26 
7.4 
TREATMENT OF GRAFT FAILURE .......................................................................................................... 27 
7.5 
THERAPEUTIC DONOR LYMPHOCYTE INFUSION (DLI)....................................................................... 27 
7.6 
FOLLOW-UP ASSESSMENTS ................................................................................................................... 29 
7.7 
SCHEDULE OF IMMUNOLOGICAL AND ENGRAFTMENT STUDIES ....................................................... 29 
 

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8.0 
CRITERIA FOR TERMINATION ........................................................................................................ 30 
8.1 
CONDITIONS FOR TERMINATING THE STUDY ...................................................................................... 30 
8.2 
CONDITIONS FOR INDIVIDUAL PATIENT TERMINATION ..................................................................... 30 
9.0 
STATISTICAL CONSIDERATIONS ................................................................................................... 30 
9.1 
HYPOTHESES .......................................................................................................................................... 30 
9.2 
ACCRUAL OBJECTIVES .......................................................................................................................... 30 
9.3 
ENDPOINT DEFINITIONS ........................................................................................................................ 31 
9.4 
PLAN OF ANALYSIS ................................................................................................................................. 32 
9.5 
SAMPLE SIZE ........................................................................................................................................... 33 
9.6 
ESTIMATED DURATION OF STUDY ........................................................................................................ 34 
9.7 
REPLACEMENT POLICY .......................................................................................................................... 34 
10.0 
CRITERIA FOR EVALUATION ........................................................................................................... 34 
10.1 
MONITORED OUTCOMES ........................................................................................................................ 34 
10.2 
TOXICITY DEFINITIONS/STOPPING RULES .......................................................................................... 34 
11.0 
DATA SAFETY MONITORING PLAN ............................................................................................... 34 
11.1 
OVERSIGHT AND MONITORING PLAN ................................................................................................... 34 
11.2 
MONITORING AND REPORTING GUIDELINES ....................................................................................... 35 
11.3 
REVIEW AND OVERSIGHT REQUIREMENTS ......................................................................................... 35 
12.0 
ETHICAL ASPECTS ............................................................................................................................... 37 
12.1 
REGULATORY CONSIDERATIONS .......................................................................................................... 37 
12.2 
INDEPENDENT ETHICS COMMITTEES/INSTITUTIONAL REVIEW BOARD ........................................ 37 
13.0 
DATA FORMS AND SUBMISSION SCHEDULE ............................................................................ 38 
APPENDIX I – ALTERNATIVE CONDITIONING REGIMENS: ............................................................... 39 
APPENDIX II- ACUTE GVHD STAGING AND GRADING FOR CHILDREN** ................................... 41 
APPENDIX III - CHRONIC GVHD STAGING AND GRADING FOR CHILDREN** .......................... 42 
APPENDIX IV - DEVICE INFORMATION ..................................................................................................... 46 
APPENDIX V – RESEARCH SAMPLES .......................................................................................................... 47 
REFERENCES ........................................................................................................................................................ 51 
 

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STUDY COMMITTEE 
 
Principal Investigator 
Andrew Gilman, MD 
Levine Children’s Hospital 
Carolinas Medical Center 
1000 Blythe Boulevard  
Charlotte, NC 28203 
Phone: (704) 381 – 6847 
Fax:  
 (704) 381 – 6841 
E-mail: 
Andrew.gilman@carolinashealthcare.org 
 
Sub-Investigator 
Javier Oesterheld, MD 
Levine Children’s Hospital 
Carolinas Medical Center  
1000 Blythe Boulevard  
Charlotte, NC 28203 
Phone: (704) 381 – 9900  
Fax:  
 (704) 381 – 9901  
E-mail: 
Javier.Oesterheld@carolinashealthcare.org 
 
Michael J. Eckrich, MD, MPH 
Levine Children’s Hospital 
Carolinas Medical Center 
1000 Blythe Boulevard 
Charlotte, NC 28203 
Phone: (704) 381 – 9900  
Fax:  
 (704) 381 – 9901 
E-mail: 
Michael.Eckrich@carolinashealthcare.org 
 
 
Study Coordinator  
Mark Cannon 
Levine Children’s Hospital 
Levine Cancer Institute 
Carolinas Medical Center 
1021 Morehead Medical Drive, Suite 2200 
Charlotte, NC 28204 
Phone: (980) 442 – 2324 
Fax:     (980) 442 – 2301 
E-mail: Mark.Cannon@carolinashealthcare.org 
 
 
 
 
 
 
 
 
 
 
 
Statistician 
Anastasia Ivanova, PhD 
Associate Professor, Dept. of Biostatistics 
School of Public Health, CB# 7420 
3103-C McGavran-Greenberg Hall 
University of North Carolina at Chapel Hill 
Chapel Hill, NC 27599-7420 
Phone: (919) 843 - 8086 
Fax:  
(919) 966 - 3804  
E-mail: aivanova@bios.unc.edu 
 
Blood and Marrow Transplant Nurse 
Carrie Barnhart, RN, BSN, CHTC 
Blood and Marrow Transplant Coordinator 
Levine Children’s Hospital 
Carolinas Medical Center 
1000 Blythe Boulevard  
Charlotte, NC 28203 
Phone: (704) 355 – 0425  
Fax:  
 (704) 381 – 6841  
E-mail: 
Carrie.Barnhart@carolinashealthcare.org 
 
Blood and Marrow Transplant Pharmacist 
Stacy Epstein, Pharm.D., BCOP 
Clinical Pharmacy Specialist, Pediatric Blood 
and Marrow Transplantation  
Levine Children's Hospital 
Carolinas Medical Center 
1000 Blythe Boulevard 
Charlotte, North Carolina 28203 
Phone: 704-381-7173 
Fax: 704-381-7171 
Email: Stacy.Epstein@carolinashealthcare.org 
 
Data and Safety Monitoring Committee  
Chair: 
Paul Martin, MD 
Director, Long-Term Follow-Up program 
Fred Hutchinson Cancer Research Center 
Tel 206- 667-4415 
Fax 206- 667-5619 
Email: pmartin@fhcrc.org 
 
DSMC Coordinator 
Laura Hancock 
PBMTC Operations Center 
440 E. Huntington Drive, 4th Floor 
Arcadia, CA 91006-3776 
Ph:  (626) 241-1619 
Fax: (626) 445-4334 
email: lhancock@pbmtc.org 

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SHORT ABSTRACT 
 
A major issue in alternative donor (mismatched related and unrelated donor) transplantation is 
the development of graft-versus-host disease (GVHD).  Several clinical trials have shown that 
the use of T-cell depleted peripheral blood stem cells (PBSC) reduces GVHD in alternative 
donor transplants.  The purpose of this study is to determine the ability of CD34 positive 
selection and T cell depletion using the CliniMACS® Device as the only GVHD prophylaxis to 
prevent severe acute GVHD in recipients of an alternative donor PBSC transplant.  Mismatched 
related donors will match at least 4 of 8 HLA antigens (haplocompatible) and unrelated donors 
will match at least 6 out of 8 HLA antigens with the transplant recipient.  The patients will 
receive conditioning therapy based on their diagnosis that may include chemotherapy, anti-
thymocyte globulin (ATG), +/- total body irradiation (TBI).  The transplant recipients will be 
followed for 5 years post-transplant for the development of GVHD, engraftment, post-
transplant infections, disease relapse, and overall survival.  In addition, this study will serve as 
a platform for a companion study of therapy to accelerate immune recovery after transplant. 
 
 

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SCHEMA 
 
 
 
 
 
 
RECIPIENT 
Eligible for BMT with an 
eligible mismatched 
related donor or an 
unrelated donor  
DONOR 
Eligible for 
apheresis 
DONOR  
Apheresis 
RECIPIENT 
PBSC transplant 
RECIPIENT 
Admitted to BMT unit 
to begin conditioning 
therapy 
RECIPIENT  
Outcome measured 
by: Occurrence of 
severe (grade III/IV 
acute GVHD) by Day 
+30. 
CD34+ processing 
with CliniMACS® 
system  

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LIST OF ABBREVIATIONS 
 
AE 
 
Adverse Event 
ALL 
 
Acute Lymphocytic Leukemia 
AML 
 
Acute Myelogenous Leukemia 
ANC 
 
Absolute Neutrophil Count 
APC 
 
Antigen Presenting Cells 
ASCO  
American Society of Clinical Oncology  
BMT 
 
Bone Marrow Transplant 
BSA 
 
Body Surface Area 
CB 
 
Cord Blood 
CIBMTR 
Center for International Blood and Marrow Transplant Research 
CML 
 
Chronic Myelogenous Leukemia 
CMV 
 
Cytomegalovirus 
CR 
 
Complete Response 
CRF 
 
Case Report Form 
CTC 
 
Common Toxicity Criteria 
DFS 
 
Disease-Free Survival 
DLI 
 
Donor Lymphocyte Infusion 
DLT 
 
Dose Limiting Toxicity 
EBV 
 
Epstein Barr Virus 
EFS 
 
Event-Free Survival 
FA 
 
Fanconi Anemia 
FDA 
 
Food and Drug Administration 
FHCRC 
Fred Hutchinson Cancer Research Center 
GCP 
 
Good Clinical Practice 
G-CSF human Granulocyte-Colony Stimulating Factor 
GVHD  
Graft versus Host disease 
HLA 
 
Human Leukocyte Antigen 
HSC 
 
Hematopoietic Stem Cell 
ICH 
 
International Conference on Harmonization 
IND 
 
Investigational New Drug 
IEC 
 
Independent Ethics Committee 
IRB 
 
Institutional Review Board 
JMML  
Juvenile Myelomonocytic Leukemia 
KIR 
 
Killer cell Immunoglobulin-like Receptor 
MDS 
 
Myelodysplastic Syndrome 
MRI 
 
Magnetic Resonance Imaging 
MUD  
Matched Unrelated Donor 
PBSC  
Peripheral Blood Stem Cells 
PCR 
 
Polymerase Chain Reaction 
PI 
 
Principal Investigator 
PTLD  
Post-transplant Lymphoproliferative Disorder 
PRC 
 
Protocol Review Committee, Lineberger Comprehensive Cancer Center 
rATG  
polyclonal rabbit Anti-Thymocyte Globulin  
SAE 
 
Serious Adverse Event 
SCT 
 
Stem Cell Transplant 
TBI 
 
Total Body Irradiation 
TMP/SMX 
Trimethoprim-sulfamethoxazole 
TRM 
 
Transplant Related Mortality 
 

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1.0 
GOALS AND OBJECTIVES 
 
1.1 
Primary objective 
To determine the ability of CD34+ cell selection using the CliniMACS® device as the sole 
GVHD prophylaxis to prevent severe (grade III-IV) acute GVHD in recipients of 
alternative donor (mismatched related donor and unrelated donor) hematopoietic stem 
cell transplants. 
 
1.2 
Secondary objectives  
1.2.1 To assess the ability of this approach to serve as a platform for strategies 
to accelerate post-transplant immunological recovery 
1.2.2 To evaluate the rate of engraftment in recipients of CD34+ cell selected, T 
cell-depleted transplants from alternative donors 
1.2.3 To evaluate post-transplant infections 
1.2.4 To evaluate the rate of EBV-related post-transplant lymphoproliferative 
disorder (PTLD) 
1.2.5 To evaluate post-transplant leukemia relapse 
1.2.6 To evaluate transplant-related mortality 
1.2.7 To evaluate transplant-related toxicities 
1.2.8 To evaluate overall survival 
1.2.9 To monitor device performance: 
1.2.9.1 
Purity of selected product 
1.2.9.2 
Yield of CD34+ cells 
1.2.9.3 
CD3+ cell depletion 
1.2.9.4 
Viability 
1.2.9.5 
Sterility 
1.2.10 To evaluate the correlation of NK alloreactivity with relapse, engraftment, 
transplant-related mortality, and survival 
 
 
2.0 
BACKGROUND AND RATIONALE 
 
2.1 
Stem cell transplantation 
Stem cell transplantation (SCT) can cure some children with marrow failure syndromes, 
inherited immunodeficiencies, myelodysplastic syndrome (MDS), acute lymphocytic 
leukemia (ALL), acute myelogenous leukemia (AML), chronic myelogenous leukemia 
(CML), lymphoma, white blood cell disorders (chronic granulomatous disease, 
osteopetrosis), and red blood cell disorders (sickle cell disease, thalassemia). The 
preferred donor for transplantation is a HLA identical sibling. However, less than 20% of 
patients requiring a transplant will have such a donor. Alternative stem cell sources 
include volunteer matched unrelated donors (MUD) and unrelated donor cord blood 
(CB). Approximately 90% of Caucasians and 60% of African-Americans will have a 5/6 
or 6/6-matched unrelated marrow donor. Approximately 80% of Caucasians and 40% of 
African-Americans will have a 5/6 or 6/6-matched unrelated cord blood donor. Most 
patients will have a 4/6-matched cord blood available (1). 
 
The largest experience using unrelated donors is with unrelated donor bone marrow 
transplants. Single center data for children include a report from Fred Hutchinson 
Cancer Research Center (FHCRC) that described 47% 2 yr event-free survival (EFS) for 
ALL in CR1/2 and 46% for AML in CR1/2 (2). The incidence of severe, life-threatening 
(grade III-IV) acute GVHD was 37% and 62% of HLA-matched and mismatched 
recipients, respectively. Data from the Center for International Blood and Marrow 
Transplant Research (CIBMTR) from 1996-2001 shows a 50% 2 yr EFS for ALL in CR1 
and 42% 2 yr EFS for ALL in CR2+. This compares to CIBMTR data for matched sibling 

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transplants over the same time period that shows a 70% 2 yr EFS for ALL in CR1 and 
60% 2 yr EFS for ALL in CR2+ (3).  
For unrelated donor cord blood transplants, the University of Minnesota reported a 55% 
2 yr EFS for ALL in CR1, 32% 2 yr EFS for ALL in CR2+, and 33% for AML in CR2+. The 
incidence of grade III-IV acute GVHD was 11%. The use of a 4/6-matched donor was 
associated with a relative risk of 2.4 for death compared to a 5/6 or 6/6-matched donor 
(p=0.01) (4). A larger review (562 patients) by Rubinstein et al. reported a 23% incidence 
of grade III-IV acute GVHD. The cumulative incidence of transplant-related events 
increased as the number of HLA disparities increased (5). 
 
A comparison of MUD and CB donor sources in 541 children transplanted for acute 
leukemia showed a 2 year event-free survival of 43% for unmanipulated MUD 
transplant, 37% for T cell-depleted MUD transplant, and 31% for cord blood transplant. 
The incidence of grade III-IV acute GVHD was 29%, 8%, and 21% in the respective 
groups (6). A recent retrospective review of MUD and CB donor transplant for 503 
children with leukemia transplanted in the US showed the following results (7): 
 
2 yr EFS ~ 40% after matched and mismatched unrelated donor bone marrow 
transplant, 
 
2 yr EFS ~ 40% after 4/6 matched unrelated donor cord blood, ~ 45% after 5/6, 
~ 65% after 6/6 matched (there were only 35 patients with a 6/6 matched 
donor), 
 
Severe (Grade III/IV) acute GVHD 18% and 32% after matched and mismatched 
unrelated donor bone marrow transplant, respectively, 
 
Severe (Grade III/IV) acute GVHD 27% after 4/6 matched unrelated donor cord 
blood, 20% after 5/6, 9% after 6/6 matched, 
 
Relapse 39% and 31% after matched and mismatched unrelated donor bone 
marrow transplant, respectively, 
 
Relapse 19% after 4/6 matched unrelated donor cord blood, 27% after 5/6, 31% 
after 6/6 matched. 
 
2.2 
Mismatched (haplocompatible) related donor stem cell transplant      
Another donor source that has been reported is a mismatched (haplocompatible or 
sharing only one of two haplotypes) related donor. In the past, the major challenges 
have been engraftment, graft-versus-host disease (GVHD), and an increased incidence 
of infection and relapse. Outcomes have improved markedly in recent years with the 
availability of cell selection devices that allow the administration of a large number of 
stem cells with a low dose of T cells and the development of more intensely 
immunosuppressive conditioning regimens.  In order to reduce the high risk of fatal 
GVHD associated with these mismatched donors, the stem cells need to be processed to 
significantly reduce the number of donor T cells present in the graft. CD34 is a receptor 
that is expressed on early hematopoietic stem cells (HSC). Monoclonal antibodies are 
available that efficiently bind to the CD34 antigen, and studies have shown that positive 
selection of CD34+ marrow or blood cells results in a significant (> 4 log) depletion of T 
cells from the preparation (8-10). This approach is less time-consuming and may be 
more efficient than earlier approaches because it specifically targets the HSC. A cell 
separation system for clinical use is available for evaluation in the US after extensive 
use in Europe (11,12). The Miltenyi Biotec Inc. CliniMACS® CD34+ Reagent System 
has the advantages of the best T cell depletion efficiency achievable and a very high 
efficiency of CD34+ cell recovery so that fewer apheresis are necessary. The CliniMACS® 
device uses a sterile, closed magnetic sorting system to isolate CD34+ stem cells. The 
peripheral blood stem cell product obtained from the donor is incubated with a murine 
anti-CD34 monoclonal antibody conjugated to small super-paramagnetic beads 
composed of iron dextran (commercially available to treat iron deficiency). The murine 
antibody has been used in clinical trials in humans. The dose administered to the 

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transplant after processing is 100x lower than therapeutic levels. Following incubation, 
the product passes through a strong magnet. The CD34+ cells remain at the level of the 
magnet and other cells (T, B, and NK cells, monocytes, and neutrophils) pass through 
into a waste bag. The magnet is then turned off and the CD34+ cells are released into a 
bag containing the final product.  
 
In addition, it has been well established that allogeneic peripheral blood stem cells 
(PBSC) recruited into the blood by administering G-CSF can successfully and durably 
engraft in the matched (13,14) or mismatched (15,16) relative. PBSC recruited with 
cytokines engraft earlier and contain a larger number of HSC compared to marrow HSC 
(14,15). 
 
Much of the work that has been done with this approach has been reported by a group 
in Perugia, Italy. They used a conditioning regimen of total body irradiation (TBI), 
thiotepa, fludarabine, and rabbit ATG for 101 patients. They used the CliniMACS® 
device to lower the T cell content of the donor PBSC to a median of 1 x 104/kg (range 
0.04 – 3 x 104/kg). Ninety-one percent of patients had primary engraftment. Six of 
seven patients who did not have primary engraftment were successfully engrafted after 
a second transplant, making the overall engraftment rate 99%. Grade III-IV acute GVHD 
occurred in 2% of patients. The transplant-related mortality was 37%, with the majority 
of deaths due to infection (bacterial, viral, and fungal). Relapse occurred in 16% of 66 
patients who were in remission at the time of transplant. The 2 year EFS for patients in 
remission was 48% for AML and 46% for ALL (17). 
 
The results of haplocompatible transplantation in children have been encouraging. 
Handgretinger reported a 46% 2 yr EFS in patients with ALL in CR1-3 (18) Ortin et al. 
reported 75% EFS of patients with ALL in CR2/3 with median follow-up of 18 mo (range 
6-29) (19) The international experience was reviewed at a conference in Naples in 2004. 
Lang updated the Tubingen, Germany experience when he reported a 44% 2 yr EFS for 
21 children with ALL in CR1-3 (20) Advantages of haplocompatible transplantation are 
rapid engraftment and the very low incidence of severe acute GVHD. Ortin et al. 
reported a median time to ANC > 500 and to platelet count > 50,000 of 12 and 20 days, 
respectively, with the incidence of grade III-IV acute GVHD being  5% (19). Lang 
reported a 1% incidence of grade III-IV acute GVHD (21). 
 
In a retrospective review of the experience of 3 investigators in the U.S. (Cowan, Gilman, 
Sleight) with alternative (haplocompatible) donor transplant using CD34+ cell selection 
for T cell depletion, 13/18 (72%) were surviving with follow-up of 7 mo – 7 yrs (median 
31 mo)(22). The median pt age was 8 yrs (range 1-20). Patient with malignancy (n=13) 
included: AML - CR1 (primary induction failure, failed cord blood transplant) [1], CR2 
[3]; MDS - RA/RARS [2], RAEB [2] AML (and Fanconi anemia, FA) [1]; CML - CP2 [1]; 
ALL - CR3 [2]; NHL - CR2 [1]. Patients with non-malignant (n=5) disease included severe 
aplastic anemia (1 with prior BMT 3 yrs earlier) [4] and Wiskott-Aldrich syndrome [1]. 
Fourteen donors were a 3/6 HLA match and 5 were a 4/6 match (one patient had two 
transplants using different donors). A CD34-positive selection device – Miltenyi 
CliniMACS® (15), Isolex (3) – was used to select stem cells and deplete T lymphocytes. 
The conditioning regimen was TBI 12-14 Gy in 6 fractions, thiotepa, fludarabine, and 
ATG. Fractionated TBI was replaced with single fraction TBI (2 pts) or melphalan (3 pts) 
as clinically indicated. Cyclophosphamide was used instead of thiotepa for one pt with 
FA. No post-transplant graft-versus-host disease (GVHD) prophylaxis such as 
tacrolimus or methotrexate was given. Patients received a median of 18 x 106 CD34+ 
(stem) cells/kg (range 6-28) and 3 x 104 CD3+ (T) cells/kg (range 0.3-11).  
 
Sustained primary engraftment occurred in 15/18 (83%) patients.  Primary graft failure 
occurred in one patient.  Two patients had immunological rejection following HHV-6 

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reactivation. They both engrafted after a second transplant; therefore the overall 
engraftment rate was 94%. The median time to an ANC >0.5 x 109/L was 12 days 
(range 9-21).  Platelet recovery occurred in 16/18 at a median of 17 days (range 9-22). 
Primary (occurring after SCT and prior to donor lymphocyte infusion [DLI]) grade II 
acute GVHD was seen in 4/17 patients (24%). Grade III-IV acute GVHD was seen in 1 
pt (6%) with overlap syndrome (acute + chronic GVHD) associated with HHV-6 
reactivation. Nine patients received DLI and/or stem cell boosts (boosts for graft 
rejection); 4 had grade II GVHD (3/4 had a history of acute GVHD) and none had grade 
III-IV GVHD.  After DLI and/or stem cell boost, two patients developed extensive chronic 
GVHD and one developed overlap syndrome. The Day 100 mortality and 1 year 
transplant-related mortality were 11% and 19%, respectively.  Four patients (of 13 at 
risk, 31%) have relapsed; 1 pt with cytogenetic relapse is in CR > 1 year later. The 2 yr 
predicted survival is 64% (60% for 13 patients with malignant disease and 75% for 5 
patients with non-malignant disease). 
 
Infections were common. All patients were at risk for CMV reactivation.  Seven patients 
(39%) reactivated CMV.  All cases were responsive to anti-viral therapy and/or DLI.  No 
CMV disease was seen. Seven patients had adenovirus reactivation and 6 had HHV-6 
reactivation. EBV reactivation occurred in 5/18 (28%) patients, 3 of whom manifested 
signs of post-transplant lymphoproliferative disorder. Patients received a median of 3 x 
104 CD3+ cells/kg at the time of transplant. Some patients received additional donor T 
cells (DLI) for viral reactivation. At 3 months post-transplant, only 4 of the 15 evaluable 
patients had a CD4 count > 100. By 9 months post-transplant, 10 of the 13 evaluable 
patients had a CD4 count > 200. 
 
In summary, the use of megadose CD34+ selected PBSC without post-transplant GVHD 
prophylaxis for children was associated with rapid engraftment, a low 100-day 
mortality, a very low incidence of severe GVHD, and excellent survival. The overall 
survival compares favorably with MSD and MUD HSCT. Immune reconstitution was 
slow and post-transplant infections contributed to morbidity and mortality. 
 
2.3 
NK alloreactivity 
The risk of relapse with haplocompatible transplantation may be reduced by utilizing 
both donor NK cells for a graft-versus-leukemia effect and donor lymphocyte infusions 
(DLI) to accelerate T cell reconstitution. NK alloreactivity has been shown to be present 
for myeloid leukemias and for lymphoid leukemias (23,24). Alloreactivity can be 
determined by HLA-C typing with even better results with the addition of NK cell killer 
immunoglobulin-like receptor (KIR) typing (23,24). In addition to HLA compatibility, the 
choice of the optimal donor source may depend on the age and size of the recipient, the 
type of leukemia, disease status, pre-transplant organ dysfunction, and pre-transplant 
infections. 
 
There is limited experience with unrelated donor transplants for children following 
CD34+ cell selection and T cell depletion with the CliniMACS device (25). In 30 patients 
with leukemia, primary engraftment was observed in 84% with the remainder engrafted 
after a second transplant. Grade III-IV acute GVHD occurred in 7% of patients and 
followed HHV-6 infection in both patients. The 2 year survival was 44% for patients in 
remission at the time of transplant (25). The same approach was used for unrelated 
donor transplantation for 14 children with non-malignant disease. Observed overall 
survival was 100% (follow-up 1-7 years) and no grade III-IV acute GVHD occurred (21).  
 
In a large prospective study of T cell depletion versus immunosuppressive drugs for 
GVHD prophylaxis for adults undergoing matched unrelated donor (MUD) transplant, 
there was no difference in 3 year EFS, 27 vs 34%, respectively (26). However, the T cell 
depletion used for this trial was much less intense than that achieved with the 

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CliniMACS® device, as demonstrated by an 18% incidence of grade III-IV acute GVHD 
(37% in the non-T cell-depleted arm) (26). Post-transplant infections including CMV and 
aspergillus were more problematic after T cell depletion (27). Interestingly, the outcome 
for recipients of T cell-depleted bone marrow who did not have a fungal infection was 
better than that for patients receiving T replete bone marrow. This suggests that 
successful prevention of post-transplant infection by hastening immune recovery may 
result in a superior outcome for T cell-depleted transplants. 
 
2.4 
Preliminary data 
We have collaborated with Dr. Cowan at UCSF for his prospective study of CD34+ 
selected (with the CliniMACS® device) PBSC from mismatched related donors. Patients 
received a conditioning regimen including TBI 1200 cGy, thiotepa, fludarabine, and 
rabbit ATG (3.5 mg/kg). Patients received a fixed T cell dose of 3 x 104/kg at the time of 
transplant. Seventeen evaluable patients have been enrolled with the following  
diagnoses: ALL (3), AML (6), bilineage leukemia (1), CML (1), MDS (1), aplastic anemia 
(2), congenital amegakaryocytic thrombocytopenia (1) combined immunodeficiency (1), 
and hemophagocytic lymphohistiocytosis (1).   
 
Twelve of the 17 (65%) are alive and well, with follow-up ranging from 3 months to 6.5 
years (median follow-up 2.5 years). Survivors include 7/12 (58%) with malignant 
disease and 5/5 (100%) with non-malignant disease. Of note is that 9/11 patients 
treated after the last protocol amendment are alive and well, including 5/7 with 
malignant disease. Both deaths were due to late infection. One patient died at 7 months 
after transplant due to parainfluenza and Paecilomyces infections and the other died at 
23 months after transplant due to disseminated Mucor infection. There was no severe 
(grade III/IV) acute GVHD. Only 3/17 (18%) of patients achieved > 100 CD4+ T cells/uL 
by 100 days after transplant and 3/4 of these patients either had received donor T cell 
infusion for serious viral infections prior to 100 days or had GVHD (in which case the T 
cells probably represented those causing the GVHD and not providing protection 
against infection).   
 
2.5 
Immune reconstitution 
A major challenge of previous approaches to haplocompatible donor transplantation is 
the prolonged immunodeficiency that follows transplant (28). This results in viral and 
fungal infections and is the primary cause of transplant-related mortality (29). Patients 
on this study who meet the eligibility criteria will be offered the opportunity to 
participate in a companion study, which will investigate the use of a donor lymphocyte 
(T cell) infusion (DLI) to hasten immune reconstitution. The use of a subsequent 
intervention will necessitate that the success of this study will be judged by the ability 
of CD34+ cell selection with the CliniMACS® device to prevent severe, life-threatening 
acute GVHD while maintaining a satisfactory rate of engraftment. 
 
2.6 
Rationale for including alternative conditioning regimens 
Several patients have not been eligible for Dr. Cowan’s study described above because 
of the need for an individualized conditioning regimen (ie. contraindication to the total 
body irradiation (TBI), fludarabine, thiotepa, and ATG regimen) or because of 
diseases/conditions not allowed in the eligibility criteria (eg. non-Hodgkin’s lymphoma, 
poor lung function). Examples of such patients that we have transplanted include (1) 
patient with AML who failed to engraft after a cord blood transplant and had invasive 
fungal infection – alive and well 4 years after transplant, and (2) patient with rare 
immunodeficiency – NEMO syndrome – who had significant lung dysfunction and was 
on oxygen at the time of transplant – alive and well 3 years after transplant.  
 
Recent studies using the CliniMACS® device, including Dr. Cowan’s, used a CD3+ cell (T 
cell) dose of < 3 x 104/kg. Conditioning regimens include the TBI-containing regimen 

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used by the Perugia group and in Dr. Cowan’s study as well as non-TBI containing 
regimens as indicated by the patient’s disease and clinical condition. The substitution 
of Melphalan for TBI has been reported by several groups (22,30,31). Regimens have 
been developed for patients in poor clinical condition (31,32) or with DNA repair 
syndromes (33,34). Because this protocol is designed to look at the ability of the 
CliniMACS® device to prevent GVHD, the conditioning regimen used will be chosen 
based on the patient’s disease and clinical condition. The conditioning regimen will also 
include rabbit ATG with the dose based on these factors and prior in vivo therapy with 
anti-T cell antibodies. 
 
 
3.0 
STUDY DESIGN AND ELIGIBILITY CRITERIA 
 
3.1 
Study design 
Patients will be enrolled with alternative (mismatched/haplocompatible) related donors 
or unrelated donors. For patients with mismatched related donors, the majority of 
clinical experience has been with a T cell-depleted PBSC product. The CliniMACS® 
CD34+ Reagent System from Miltenyi Biotec, Inc. was approved by the FDA in January, 
2014 as a Humanitarian Use Device for the processing of PBSC to obtain a CD34-
enriched stem cell collection for adults with acute myeloid leukemia in first complete 
remission undergoing PBSC transplant from a matched related donor without the needs 
for medications to prevent GVHD.  Recent experience with the CliniMACS® device has 
produced excellent results with a 70-75% survival in children, many of whom were high 
risk patients (19,22,35).  
 
Patients that receive transplants from unrelated donors usually receive stem cells that 
are not T cell-depleted. However, this is associated with a high risk of GVHD. The 
excellent results with mismatched related donor transplants justify expanding this 
approach to unrelated donor transplant recipients. It is anticipated that the use of the 
CliniMACS® device will result in a very low risk of GVHD without the need for post-
transplant immunosuppression. The outcomes in relatively small studies for children 
receiving unrelated donor transplants using the CliniMACS® have been comparable to 
or 
better 
than 
those 
receiving 
T 
replete 
transplants 
with 
post-transplant 
immunosuppression (21,25). 
 
This protocol will allow the use of patient-specific conditioning regimens. Some patients 
have contraindications to certain components of the conditioning regimen used for Dr. 
Cowan’s study under BB-IND 8817 (UCSF 01151 protocol). An example is a patient 
with pre-existing organ dysfunction that would be better served by the use of a reduced 
intensity conditioning regimen. Another example is a patient for who total body 
irradiation is contraindicated due to very young age or prior radiation therapy. The 
target T cell dose that will be given will be < 3 x 104 /kg. The UCSF 01151 protocol uses 
a dose of 3 x 104 /kg. The T cell dose in the graft is usually < 1 x 104 /kg after 
processing and T cells need to be added to the product. There is extensive successful 
experience with this approach (17).  
 
3.2 
Recipient inclusion criteria 
3.2.1 Age < 30 years 
3.2.2 Patient must have a malignant or non-malignant disease that can benefit 
from alternative stem cell transplantation. Examples include acute and 
chronic 
leukemias, 
myelodysplastic 
syndrome, 
lymphoma, 
severe 
acquired 
and 
congenital 
cytopenias, 
white 
and 
red 
blood 
cell 
abnormalities, and immunodeficiencies. 
3.2.3 Patients with acute lymphoblastic leukemia must be in morphological 
remission (< 5% blasts) at the time of transplant. Patients with acute 

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non-lymphocytic leukemia will preferably be in morphologic remission 
but may be enrolled when aplastic after chemotherapy or with < 20% 
blasts. Patients with lymphoma must be in complete or close to complete 
remission (if residual adenopathy, PET scan must be negative or only 
have slight uptake, eg. SUV < 2). 
3.2.4 Patients must lack a healthy HLA-identical related donor of at least one 
year of age.  
3.2.5 Patient must have a mismatched related or an unrelated donor who is: 
a) Able to receive G-CSF and undergo apheresis either through 
placement of catheters in antecubital veins or a temporary central 
venous catheter, 
b) Healthy, 
c) Willing. 
d) For recipients of an unrelated donor transplant, recipient eligibility will 
be restricted as follows if in the judgment of the recipients’ transplant 
physician, the recipient cannot receive a transplant with combined 
positive and negative fractions as described in Section 6.1.3.2 or an 
unmanipulated PBSC product. The restrictions based on recipient weight 
and number of apheresis procedures for the donor for these patients will 
be: 
> If donor PBSC are collected in one day, the recipient weight must be 37 
kg or less 
> If donor PBSC are collected in two days, the recipient weight must be 
26 kg or less 
See Donor selection criteria below for additional considerations. 
3.2.6 If only one mismatched related relative is available, an acceptable 
unrelated donor must be identified as a backup.  If only one acceptable 
unrelated donor is available, a mismatched related donor must be 
identified as a backup.  Section 3.2.6 can be waived in extraordinary 
situations when the benefit of transplant outweighs the risk of graft 
failure in the judgment of the principal investigator. 
3.2.7 Patient, parent, or authorized guardian must sign informed consent for 
this study. 
 
3.3 
Recipient exclusion criteria 
3.3.1 Patient with an anticipated life expectancy of < 1 month 
3.3.2 Active infectious hepatitis or CMV infection 
3.3.3 HIV or HTLV-I/II infection 
3.3.4 Serious infection (bacterial, fungal, viral) within the last 4 weeks (the 
interval can be less than 4 weeks under extenuating circumstances) 
3.3.5 Cardiac ejection fraction < 45%; can be lower if patient is not in clinical 
cardiac failure and a reduced intensity conditioning regimen is used. 
3.3.6 Creatinine clearance <60 ml/min/1.73 m2; can be lower if a reduced 
intensity conditioning regimen is used. 
3.3.7 Pulmonary diffusion capacity (adjusted for Hgb), FEV1, or FVC <60% of 
predicted or O2 sat < 94% if unable to perform PFTs; can be lower if a 
reduced intensity conditioning regimen is used. 
3.3.8 Serum ALT > 3 x upper limit of normal (can be up to 5 x upper limit of 
normal if a reduced intensity conditioning regimen is used) or bilirubin > 
2.  The bilirubin criteria for sickle cell disease patients is direct bilirubin 
> 2 x upper limit of normal. 
3.3.9 Performance score (Lansky/Karnofsky) < 50 
3.3.10 Any condition that compromises compliance with the procedures of this 
protocol, as judged by the principal investigator. 
 

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3.4 
Donor eligibility criteria 
3.4.1 For Related donor: sibling, half-sibling, parent, cousin, aunt, uncle or 
grandparent will all be considered eligible.  
3.4.2 For Related donor: At least a 4 out of 8 HLA antigen genotypic match 
(haplocompatible). The 8 antigens are at the A, B, C, and DR loci. 
3.4.3 For unrelated donor: At least 6 out of 8 HLA antigen match (if two 
mismatches, they must be at different loci). 
3.4.4 Complete medical history, physical and screening for infectious diseases 
that are acceptable for donation.  See Section 7.1.2 for details of 
evaluation.  
3.4.5 If donor is female and of child-bearing age, negative pregnancy test. 
3.4.6 Absence of anti-HLA antibodies in recipient directed against donor 
antigens. 
3.4.7 If the recipient is CMV seropositive and has a non-malignant disease that 
is not anticipated to be fatal in the near term, the donor must be CMV 
seropositive. If the recipient has a malignant disease or potentially fatal 
non-malignant disease like HLH, the use of a CMV seronegative donor is 
discouraged but not prohibited. 
3.4.8 Donor, parent, or authorized guardian must be willing to sign informed 
consent for this study. 
 
3.5 
Donor selection 
3.5.1 Potential donors will have HLA typing. They will also be offered optional 
participation in NK cell killer immunoglobulin-like receptor (KIR) typing. 
KIR typing will not be performed on donors for recipients who express all 
3 KIR ligands. 
3.5.2 Criteria to consider when choosing among donors are: 
a). For haploidentical donors, HLA disparity i.e. 2 Ag mismatch preferred 
over 3 Ag mismatch; Dr1 match preferred over class I match 
b). KIR mismatch in GVH direction is preferred for patients with 
malignant disorders and may be helpful in other settings because it has 
been associated with improved engraftment 
c). CMV positive if recipient is CMV positive 
d). ABO compatibility 
 
3.6 
Patient recruitment 
Candidates 
are 
referred 
to 
study 
investigators 
from 
pediatric 
hematology/oncology specialists and immunologists.  Patients and parents will 
be seen in the pediatric BMT outpatient clinic or as an inpatient where consent 
will be presented.  
 
3.7 
Patient registration 
The patients will be registered by completion of eligibility forms and each patient 
will be assigned a unique subject study ID.  
 
 
4.0 
INVESTIGATIONAL TREATMENT PLAN  
4.1 
Dose 
The plan is to use the CliniMACS® device to prevent severe (grade III/IV) acute 
GVHD without the need for post-transplant immunosuppressive medications. 
The target dose of CD34+ cells is  20x106/kg for mismatched related donors and 
 10x106/kg for matched unrelated donors. The target dose of CD3+ cells is < 
3x104/kg.  The ClinicMACS® CD34+ Reagent System will be used for donor stem 
cell selection. 
 

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4.2 
Duration of therapy   
Patients will be followed for outcomes related to the device including acute and 
chronic GVHD and engraftment. Patients will also be followed long-term for 
disease-free survival. 
 
 
 
5.0 
DOSE MODIFICATIONS AND TOXICITIES 
These are part of transplant-related issues but not specific to the use of the investigational 
device (CliniMACS® device). 
 
5.1 
Recipient toxicity from the conditioning regimen 
5.1.1 Thiotepa - Risks include alopecia, mucositis, and hepatic toxicity. There 
is also cutaneous toxicity which can result in erythema and breakdown 
of the skin especially in the neck, axilla, and inguinal and perianal areas. 
This can be painful and can become secondarily infected. Significant skin 
toxicity can be prevented by frequent baths or showers when the drug is 
given. In combination with fludarabine and TBI the risk of fatal hepatic 
toxicity or fatal pulmonary toxicity is <5%. There has also been a 
multiorgan failure syndrome reported in association with thiotepa 
containing regimens. The incidence is low (<5%) and was seen when 
doses greater than 10 mg/kg were used. 
5.1.2 Fludarabine - The primary toxicity of fludarabine in the doses used has 
been severe T and B cell immunodeficiency. It has also been associated 
with the development of immune mediated hemolytic anemias, unlikely 
to occur as part of an ablative conditioning regimen for BMT. Other side 
effects include nausea, vomiting and neurotoxicity (seizures, confusion). 
Neurotoxicity is very unlikely at the doses used on this study. 
 
5.1.3 Total body irradiation (TBI) - The total TBI dose is 1200 cGy given in 6 
fractions. All fractions will use 50% shielding of the lungs. Potential 
acute toxicities associated with the use of TBI include nausea, vomiting, 
and parotitis. Subacute complications or side effects of TBI include hair 
loss, mucositis, diarrhea, veno-occlusive disease, leukoencephalopathy 
and interstitial pneumonitis. Chronic complications include cataracts in 
about 20% of cases, delayed sexual development, short stature, and 
sterility in >95% of cases, and thyroid dysfunction in 5-10% of cases. 
 
5.1.4 Rabbit anti-thymocyte globulin (rATG) - The most important risk 
associated with rATG is the development of a severe allergic reaction (i.e. 
anaphylaxis) which is rare. More common side effects of rabbit ATG 
include fever and rash. 
 
5.1.5 Melphalan – common side effects include nausea, vomiting, low blood 
counts, mucositis, sterility, and hair loss. Much less common side effects 
include liver and lung damage. 
 
5.1.6 Cyclophosphamide – common side effects include nausea, vomiting, low 
blood counts, sterility, and hair loss. Less common side effects include 
water retention, damage to the lining of the bladder leading to blood in 
the urine, and liver damage. Rare side effects include damage to the 
lungs and heart and secondary cancer (leukemia and lymphoma). 
 
5.1.7 Busulfan – common side effects include nausea, vomiting, low blood 
counts, mucositis, sterility, temporary darkening of the skin, and hair 

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loss. Less common side effects include liver damage. Rare side effects 
include lung injury and seizures (the latter are rare when prophylactic 
anti-convulsants are used). 
 
5.1.8 Rituximab – common side effects include fever, chills, nausea, 
weakness, headache, low blood pressure, itching, rash, bronchospasm, 
abdominal pain, vomiting, anemia, achy joints and muscles, dizziness, 
congestion, low blood counts, suppression of B lymphocytes resulting in 
a greater risk of infections.  Rare side effects include angioedema, severe 
reactivation of hepatitis B infection, liver failure, angioedema involving 
the skin, mouth, and GI and GU tracts, and progressive multifocal 
leukoencephalopathy.  
 
5.1.9 Infection and bleeding - In general, due to the 1-2 weeks of 
neutropenia, thrombocytopenia and mucositis, there is a significantly 
increased risk of infection and bleeding. The mortality associated with 
these complications is generally <5%. 
 
5.1.10 Mortality - The reported transplant related mortality (organ failure, 
infection, bleeding) is ~30% for patients with high-risk malignancies 
(25,30,31). In a limited number of children with non-malignant diseases 
the TRM was lower (10%) but until more patients in this category are 
evaluated, we will assume it is as high as 30%. 
 
5.1.11 Secondary malignancy - There is also an overall reported risk of ~2-10% 
of a malignancy occurring up to 15 years post-transplant. The majority of 
these are lymphoproliferative disease related to EBV infection. Because 
these patients will not receive cyclosporine prophylaxis or post-
transplant ATG, and because the processing procedure removes almost 
all B cells from the donor graft (the source of EBV in these 
circumstances), this risk is likely to be lower.  
 
5.1.12 Decreased IQ - There is about a 30% risk of a decreased IQ (7 points) at 
1 year post transplant (36). This occurs regardless of the conditioning 
regimen and by 3 years there appears to be some recovery of IQ points. 
Of the remaining patients, 1/3 have no change in their IQ and 1/3 
increase their IQ by about 7 points. 
 
5.2 
Recipient Toxicity from Transplant 
5.2.1 Failure of engraftment - Published reports using 1-10x106 CD34+ 
cells/kg haplocompatible stem cell enriched T cell depleted PBSC with 
TBI-containing regimens have initial engraftment rates of >80% although 
with second transplants the engraftment rate has been >90%. We believe 
that the combination of thiotepa, fludarabine, ATG and TBI plus the 
large number of CD34+ cells in the graft should result in a primary 
engraftment rate of at least 80%. 
 
5.2.2 Delayed T-cell reconstitution and increased infections - The chance 
of this occurring is likely. In both children and adults who have been 
heavily pretreated it has taken as long as a year for T cell immunity (i.e. 
CD4+ cells > 200) to recover. The most commonly reported cause of 
transplant-related death with this kind of transplant is infection. 
 
5.2.3 Increased risk of leukemic relapse - The extent of this problem with 
PBSC is unknown. The studies of CD34+ PBSC haplocompatible 

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transplants that have been reported to date do not demonstrate a higher 
than expected relapse rate.  
 
5.2.4 Graft vs. Host Disease - In other studies in which <7x104 CD3+ cells/kg 
of haplocompatible PBSC are infused the incidence of grade III-IV acute 
GVHD has been <5%.  
 
5.3 
Toxicity from Miltenyi Biotec Inc. CliniMACS® reagent system processing 
5.3.1 Risk of contamination of the cell preparation with biologic or other 
foreign material. The sterility of system components that contact the cell 
sample and the detailed processing steps are designed to minimize 
potential contamination. 
 
5.3.2 Paramagnetic microspheres - Significant animal and human studies 
have been done using these super-paramagnetic beads which are small 
in size (~50 nm in diameter) and are composed of iron oxide and dextran 
conjugated to murine monoclonal antibodies. These magnetic particles 
form a stable colloidal suspension and do not precipitate or aggregate in 
magnetic fields. The concentration of the conjugate is equivalent to 22 µg 
of antibody protein per ml of reagent, 800 µg/ml of dextran and 800 
µg/ml of iron. Detailed toxicity studies have been undertaken to assess 
the safety of the antibody reagent when delivered to monkeys and rabbits 
in dosages significantly greater than the projected maximum dosage 
anticipated in clinical use (CliniMACS® Investigator brochure).  There 
have been more than 300 separations for clinical use of the CliniMACS® 
system. 
 
5.3.3 Reaction to CliniMACS® reagent - (murine monoclonal antibody 
conjugated to an iron-dextran moiety). Iron dextran is commercially 
available as a sterile solution of iron dextran complex for the treatment of 
severe iron-deficient syndromes. It contains 5% iron and 20% dextran, 
and its safety profile has been well characterized. Iron dextran solution 
contains 50 mg/ml of elemental iron, most of which is present in the 
ferric state. A total dose of iron-dextran for the average 70 kg person is 
calculated to be approximately 2 gm over several days (single dose of 100 
mg). The iron dextran exposure from a singe CliniMACS® separation is 
~0.5 mg and less than 1 mg dextran, 100x lower than a single dose and 
1000x lower than a total dose. 
 
5.3.4 CD34+ monoclonal antibody - The other reagent is the murine 
monoclonal antibody in which there is a risk of an anaphylactic reaction. 
The anti-CD34 monoclonal antibody, AC101 has been tested for safety in 
conformance with US standards. Systemic reactions appear related to 
the dose and rapidity of administration. Therapeutic levels (for cancer 
therapy or prevention of graft rejection) of mAb appear to be in the range 
of 2.5-5 mg/ml. The most commonly reported side effects have been 
myalgia, arthralgia, and flu-like symptoms. The CliniMACS® system 
results in the administration of a maximum of <15µg of antibody, 100x 
lower than therapeutic levels. Furthermore, studies have shown that the 
levels of antibody used in the CliniMACS® system do not induce 
complement activation in vitro. 
 
5.4 
Donor  PBSC toxicity 
The toxicities listed below are of concern for all donors and not specific to this protocol 
or the use of the investigational device (CliniMACS® device). 

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5.4.1 G-CSF - For the recruitment of PBSC, G-CSF is known to cause bone 
pain in most patients as well as other symptoms including headache, 
bone discomfort or ache, ankle swelling or fluid retention. These 
symptoms are dose related and generally controlled with analgesics such 
as Tylenol or ibuprofen (17-19). There is at least a theoretical risk of 
inducing a malignancy with G-CSF although the extensive experience to 
date with normal donors does not indicate that this will be a problem. 
Patients with aplastic anemia or Kostmann’s syndrome who have been 
chronically treated with G-CSF do not have any higher incidence of 
malignancy than is normally found with these disorders. 
 
5.4.2 Apheresis - This has been associated with decreased platelets and 
temporary hypotension as well as hypocalcemia and some risk of 
bleeding because of anti-coagulation. There is also pain from the 
insertion of needles into the antecubital veins and the discomfort of a 
central line if necessary. All of these are reversible and have been 
tolerated in previously reported studies. There is the small risk (<1%) 
that a healthy donor might require a platelet transfusion. There is also 
the risk that peripheral venous access might be inadequate and 
placement of a temporary central line under local anesthesia will be 
necessary (17,18). The risks of inserting a central line include bleeding, 
infection and/or pneumothorax. There are abnormalities in the 
circulating white cell populations (T cells and stem cells) that occurs 
after apheresis in normal donors. Most of these appear to resolve (i.e. 
numbers return to normal) within the first 3 months post donation, but 
there may be abnormalities or long-term side effects that at this time 
have not been identified. 
 
 
6.0 
DEVICE INFORMATION  
 
(Miltenyi Biotec, Inc. CD34+ CliniMACS® reagent system information) 
6.1 
CD34+  cell processing 
The collection will be stem cell enriched and T-cell depleted. The CD34+ cells are 
positively selected using the CliniMACS® System. 
 
The pheresis product may be stored overnight at 4oC and at a concentration < 200 x 106 
cells/ml and processed the following morning. Products may be pooled (i.e. first and 
second collection pooled and third and fourth collection pooled) for processing and cell 
selection, or the collections may be processed and CD34+cells selected on each day.  
The determination of whether to store product overnight or select daily will be 
individualized per patient and be based on patient cell counts and CD34+ column cell 
capacity.  CD34+ cell selection will be performed using the Miltenyi CliniMACS® system. 
The processing will be performed at University of California-San Francisco.   
 
6.1.1 The target cell doses after processing will be  20x106 CD34+ cells/kg for 
mismatched related donors and  10x106 CD34+ cells/kg for unrelated 
donors. A dose of > 8x106 CD34+ cells/kg will be acceptable for 
mismatched related donors and a dose of > 4 x106 CD34+ cells/kg will be 
acceptable for unrelated donors. The target T cell dose will be < 3x104 
CD3+ cells/kg.   
 
At investigator discretion, the CD34+ cell dose for PBSC transplant may 
be adjusted for overweight patients as follows:  
The dose may be reduced to a dose based on adjusted body weight if the 
patient’s actual weight is greater than 120% of the ideal body weight.  

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The ideal and adjusted body weights will be calculated according to the 
LCH Blood and Marrow Transplant Program High-dose Chemotherapy 
Orders policy.  The investigator will consider factors such as disease for 
which the transplant was performed, patient’s body frame and build, etc.  
 
6.1.2  The collection doses that correspond with the post-processing doses in 
6.1.1 will be higher than these doses because a significant portion of the 
CD34+ cells can be lost in the processing. Because of the large dose 
required, it is possible that an inadequate dose will be collected. If this 
occurs with an unrelated donor, the PI may choose to not process the 
cells or to combine the positive and negative fractions of a processed 
graft and give a T replete graft (as in Section 6.1.3.2) or to not utilize 
these cells and collect from another donor if a T cell-depleted graft is 
deemed in the patient’s best interest. In the latter case, the graft may be 
cryopreserved to be used in the future if necessary (eg. graft failure).  If 
an inadequate dose is collected from a related donor, then a backup 
donor will be used.  In the situation that a backup donor is used, it is 
likely and acceptable that the PBSC infusion from the backup donor will 
occur after the original day 0.   
 
6.1.3 In the unlikely event that the CD3+ cell count is too high in order to 
achieve the minimum acceptable CD34+ cell dose, there will be the 
following options: 
6.1.3.1 
For 
mismatched 
related 
donor 
and 
unrelated 
donor 
transplants: 
a) Patient can be given long course methotrexate. 
6.1.3.2 
For unrelated donor transplants: 
a) Positive and negative fractions can be combined to 
reconstitute the original product and then the patient can be 
started 
on 
standard 
tacrolimus 
and 
short 
course 
methotrexate. Sirolimus and/or ATG may also be used at 
physician discretion.  
 
6.1.4 The CD34+ selected stem cells will usually be cryopreserved prior to the 
transplant.  
 
6.1.5 The stem cells will be infused intravenously into the recipient. 
Prophylactic cefazolin will be given for 24 hours beginning just prior to 
the infusion unless the patient is receiving IV antibiotics with similar 
bacterial coverage. 
 
6.1.6 If >20x106 CD34+ cells/kg are available from a mismatched related 
donor, then approximately 20-22x106/kg will be infused and the 
remainder will be cryopreserved. For an unrelated donor, an aliquot of 
cells can be cryopreserved at physician discretion if >10x106 CD34+ 
cells/kg are available. For an unrelated donor, the infused dose will not 
exceed approximately 20-22x106/kg.  Depending on the clinical scenario, 
the treating physician may choose to infuse a smaller dose than above 
that still exceeds the minimum dose in section 6.1.1 to allow a backup 
graft to be cryopreserved. 
 
6.1.7 Five aliquots of the negative fraction (non CD34+ selected cells) 
containing the CD3+ cells will be made and cryopreserved for future DLI. 
Each aliquot will contain a minimum of 3x104 CD3+ cells/kg body weight 
of the recipient. 

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6.2 
Release of product for transplant 
The studies that will be done in addition to the cell immunophenotyping will be sterility 
(routine USP culture for bacteria and fungi), endotoxin testing, gram stain, and 
viability. We already have experience with all of these procedures in other protocols, 
specifically, BB-IND 8817. 
 
Viability, gram stain and endotoxin testing will be done prior to the release of the 
product for infusion. If the viability is >70%, the gram stain is negative, and the CD34+ 
cell and CD3+ cell doses meet the criteria, the product will be released for infusion. If 
any of these criteria are not met, the PI will need to assess the risk of using the product 
and to decide if it will be released. 
 
If the culture becomes positive, appropriate antibiotics will be given to the recipient. The 
donor will undergo a clinical evaluation for infection and the reagents (including the 
aliquoted reagents) and procedures will be tested and reviewed to try to identify the 
source. If the gram stain is positive, the cells will be cryopreserved until the culture 
results are back, and the donor may undergo another leukapheresis providing he or she 
has no clinical evidence of infection. 
 
6.3 
Shipping of CD34+ selected PBSC 
USCF will ship the CD34+ selected PBSC to arrive on Day 0 of transplant. A portion of 
the graft will be retained at UCSF and/or a backup donor will be assigned in case a rare 
catastrophe (eg. plane crash) leads to loss of the cells.  
 
 
7.0 
SCHEDULE OF ASSESSMENTS AND THERAPY 
 
Timing of protocol therapy administration and response assessment studies are based 
on schedules derived from the experimental design or on established standards of care. 
Minor unavoidable deviations (up to 72 hours) from protocol directed therapy and/or 
disease evaluations for valid clinical, patient or family logistical, or facility or procedure 
and/or anesthesia scheduling issues are acceptable. The infusion details are intended 
as guidelines and may be modified as clinically necessary.  Necessary minor deviations 
for common logistical or clinical reasons will not be considered violations at audit. 
Minor delays as described above will not be construed as prospectively planned or 
instituted on a routine basis so advance IRB approval will not be required. 
 
 
7.1 
Screening assessments 
7.1.1 Recipient  pre-transplant evaluation (within 30 days of admission 
unless specified below): 
7.1.1.1 
Pulmonary function tests (PFTs) if patient age allows (usually 
> 5 years of age); if not, the patient will have pulse oximetry. 
PFTs do not need to be repeated if the patient has had 
acceptable PFTs in the 3 months prior to admission and has 
not had chemotherapy with pulmonary toxicity or pneumonia 
since then. 
7.1.1.2 
Echocardiogram and ECG. These tests do not need to be 
repeated if they have been acceptable in the 3 months prior to 
admission and the patient has not had chemotherapy with 
cardiac toxicity since then. 
7.1.1.3 
CXR or chest CT scan 
7.1.1.4 
Creatinine clearance by Schwartz formula, urine collection, or 
GFR scan 

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7.1.1.5 
Testing for syphilis, CMV, HIV, EBV, HSV, VZV, Hepatitis B & 
C, HTLV I/II, and toxoplasmosis. Testing does not need to be 
repeated if it has been previously positive (ie. CMV positive). 
7.1.1.6 
ABO typing and antibody screen. The ABO typing does not 
need to be repeated within 30 days if the patient’s ABO typing 
has been performed. The antibody screen does need to be 
done within 30 days. 
7.1.1.7 
Liver function tests, electrolytes, BUN, creatinine, CBC, 
differential. 
7.1.1.8 
Bone marrow within 2 weeks of admission for patients with 
MDS/leukemia. Lumbar Puncture (LP) at this time as 
indicated. 
7.1.1.9 
HLA typing. This does not need to be repeated within 30 days 
if the patient has already had original and verification HLA 
typing. 
7.1.1.10 
Complete history and physical exam 
7.1.1.11 
IgG  level 
7.1.1.12 
Evaluation for anti-HLA antibodies against donor antigens 
prior to stem cell collection and also within 30 days of 
admission if collection occurs before this. 
7.1.1.13 
Pregnancy test if female and of childbearing age. Also, must 
be performed within 7 days of admission.  
 
7.1.2 Donor pre-apheresis evaluation (must be done within 30 days of 
donation except as noted below; the following is for related donors, 
unrelated donors will be evaluated according to the guidelines of the 
National Marrow Donor Program): 
7.1.2.1 
Complete history will include: 
7.1.2.1.1 Surgical history 
7.1.2.1.2 Review of systems 
7.1.2.1.3 History of inherited conditions and chronic illness 
7.1.2.1.4 History of hematological problems and immunological 
disorders 
7.1.2.1.5 History of cancer 
7.1.2.1.6 Donor 
health 
questionnaire 
which 
includes 
transfusion, vaccination and travel history 
7.1.2.1.7 List of current medications and allergies. 
7.1.2.2 
Screen for evidence of syphilis, CMV, HIV, EBV, Hepatitis B & 
C, 
HTLVI/II, 
West 
Nile 
virus, 
Chagas’ 
disease, 
and 
toxoplasmosis. Testing does not need to be repeated if it has 
been previously positive (ie. CMV positive). 
7.1.2.3 
ABO typing and antibody screen. The ABO typing does not 
need to be repeated within 30 days if the patient’s ABO typing 
has been performed. The antibody screen does need to be 
done within 30 days. 
7.1.2.4 
Liver function tests, electrolytes, BUN, creatinine, CBC, diff, 
PT, PTT 
7.1.2.5 
HLA typing. This does not need to be repeated within 30 days 
if the donor has already had original and verification HLA 
typing. 
7.1.2.6 
Optional NK cell KIR typing. This does not need to be done 
within 30 days of donation. KIR typing will not be performed 
on donors for recipients who express all 3 KIR ligands. 
Potential donors will be offered 
participation 
in KIR 
phenotyping by flow cytometry and KIR genotyping by PCR in 

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the laboratory of Dr. Wing Leung at St. Jude Children’s 
Research Hospital. See section 7.8.1 and Appendix V.  
7.1.2.7 
ECG. The ECG does not need to be repeated if it was 
acceptable in the 3 months prior to admission and the donor 
has been well since then. 
7.1.2.8 
Chest x-ray. The CXR does not need to be repeated if it was 
acceptable in the 3 months prior to admission and the donor 
has been well since then.   
7.1.2.9 
Pregnancy test if female and of child-bearing age. 
Donor eligibility will be determined in accordance with 21 CFR 1271.45-
.90 and for related donors the LCH BMT Program Hematopoietic 
Progenitor Cell Donor and Recipient Evaluation Guidelines. 
 
7.2 
Therapy 
7.2.1 Recipient cytoreductive regimen - this will be patient-specific. 
The standard regimen will be: 
Total Body Irradiation (TBI) followed by chemotherapy (see table below): 
 
 
 
 
 
 
 
 
 
 
 
 
 
Drug doses may be reduced to a dose based on adjusted body weight if the 
patient’s actual weight is greater than 120% of the ideal body weight. The ideal 
and adjusted body weights will be calculated according to the LCH Blood and 
Marrow Transplant Program High-dose Chemotherapy Orders policy. The 
investigator will consider factors such as disease for which the transplant was 
performed, patient’s body frame and build, etc. 
 
Example of other conditioning regimens include (see Appendix I for 
details): 
Substitution of melphalan 140 mg/m2 for TBI 
Substitution of busulfan and melphalan for TBI and thiotepa 
Substitution of cyclophosphamide for thiotepa in patients with Fanconi anemia 
These modifications will be based upon published conditioning regimens (22,30-
34) but minor adjustments can be made to adjust for the clinical scenario. 
 
Patients with severe combined immunodeficiency and lack of NK cells or 
function and no GVHD due to maternal engraftment may be transplanted 
without conditioning. 
 
Total dose of the TBI (1200 cGy) will be delivered in 200 cGy fractions separated 
by at least 6 hours. The patient will be treated with AP and PA fields and lungs 
will be partially blocked such that the dose to the lungs will be 600cGy (50% 
clinical shielding of lungs for ALL 6 doses). Alternatively, 100% shielding of 
the lungs can be used for 3 doses and no shielding for the remainder if 
necessary for the clinical circumstances. Also, for patients with ALL, an 
Day – 9 
200 cGy TBI x 2 
Day – 8 
200 cGy TBI x 2 
Day – 7 
200 cGy TBI x 2 
Day – 6 
Fludarabine 40 mg/m2 (1.33 mg/kg if <12 kg body weight) 
Thiotepa 10 mg/kg/day divided into 2 doses 12 hours apart 
Day – 5 
Fludarabine 40 mg/m2; Rabbit ATG 1.5 mg/kg 
Day – 4 
Fludarabine 40 mg/m2;; Rabbit ATG 1.5 mg/kg 
Day – 3 
Fludarabine 40 mg/m2; Rabbit ATG 1.5 mg/kg 
Day – 2 
Fludarabine 40 mg/m2; Rabbit ATG 1.5 mg/kg 
Day – 1 
Rest 
Day 0 
Transplant 

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additional dose boost to the testes of 400 cGY in a single fraction may be 
administered.   
 
Thiotepa will be given IV on day –6 at a dose of 5 mg/kg every 12 hrs x 2. Each 
infusion will be given over 3-4 hrs. Very careful attention to skin care during 
thiotepa treatment is necessary. Supportive skin care measures will be followed 
according to LCH Blood and Marrow Transplant Program Guidelines of Use of 
Thiotepa to prevent skin toxicity.   
 
Fludarabine will be administered IV each day, from–6 to–2, at a dose of 40 
mg/m2 as a 30 minute infusion.  The fludarabine dose will be reduced for 
patients with creatinine clearance less than or equal to 70 ml/min/1.73m2. A 
reduced dose of fludarabine (i.e. 30 mg/m2/day for 5 days) will be administered 
to participants with a history of significant neurologic disease, CNS-directed 
therapy or toxicity such as CNS irradiation, grade III-IV chemotherapy-induced 
neurotoxicity, and/or those receiving a CNS radiation boost with this transplant.  
 
Rabbit ATG will be infused each day, from –5 to –2 as six hour infusion. The 
dose will be 1.5 mg/kg/day. 
 
Patients with a history of EBV infection who will not receive TBI will be given 
rituximab IV on day -1. The dose will be 375 mg/m2/dose. 
 
 
7.2.2 GVHD prophylaxis and therapy 
7.2.2.1 
There will be no post-transplant GVHD prophylaxis given 
unless the CD3+ cell dose is too high (see Section 6.1.2). 
7.2.2.2 
Initial treatment of GVHD will usually be prednisone or 
methylprednisolone 1-2 mg/kg/d. A rapid taper can be used if 
there is a quick and complete response. Additional therapy 
may be added if needed. Alternative therapy can be used 
depending on the clinical scenario (eg. It may be prudent to 
avoid steroids in patients with fungal infections). 
 
7.2.3 Infection prophylaxis and pre-emptive therapy 
The infection prophylaxis and therapy listed below can be modified if 
clinically necessary (this includes changing drugs and time period as 
clinically relevant).    
7.2.3.1 
Prophylaxis against Herpes viruses: 
a) Acyclovir 500 mg/m2/dose IV q8h started on day -1 and 
continued until day +21 or discharge. The acyclovir dose may 
be adjusted in patients > 120% of IBW. This will be the first 
choice for prophylaxis. 
b) Foscarnet 60 mg/kg/dose q12h started on day +1 and 
continued until day +21 or discharge may be given to CMV 
seropositive transplant recipients. 
c) After completing acyclovir IV or foscarnet, valganciclovir 15 
mg/kg PO daily (max 900 mg) through day +42 after 
transplant depending on blood counts. This is particularly 
important for CMV seropositive transplant recipients.  
d) After completing valganciclovir or in place of if counts do 
not permit, acyclovir approximately 20 mg/kg/dose (max 800 
mg) PO TID until the CD4>200 and for a minimum of 6 
months. Valacyclovir (Valtrex) 500 mg PO TID may be used as 
an alternative if patient weight permits. 

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7.2.3.2 
CMV: Patients will have CMV testing by PCR 2x per week (or 
weekly if donor and recipient are CMV negative) starting on 
approximately day +10 until day +100 after transplant and 
then q2 weeks until CD4>200/ul. Monitoring should be more 
frequent in patients who have prolonged CMV reactivation 
after transplant. If the PCR test becomes positive, ganciclovir 
will be started at an induction dose of 5 mg/kg IV q12 x 7-14 
days, then a maintenance dose of 5 mg/kg/day 5-7 days per 
week until PCR is negative x 2 weeks (assuming the patient 
remains asymptomatic). Alternatively, patients may be treated 
with foscarnet induction and maintenance. Valganciclovir can 
be used as prophylaxis following ganciclovir or foscarnet 
therapy if CMV reactivation occurred when the patient was on 
acyclovir prophylaxis. Valganciclovir can also be used for 
therapy at physician discretion. 
7.2.3.3 
EBV: EBV PCR will be obtained weekly starting on 
approximately day +10 post SCT and continuing until day 
+100 after SCT. Monitoring should be more frequent in 
patients 
who 
have 
EBV 
reactivation 
after 
transplant. 
Monitoring will be every 1-4 weeks from day +100 to day +180 
depending on the clinical scenario. Patients who have no T 
cell recovery during this time are at greatest risk.  If the EBV 
viral load is positive, rituximab 375 mg/m2 IV weekly x 4 will 
be given. CT scan of head, neck, chest, abdomen, and pelvis 
or PET/CT scan should be considered to evaluate for 
adenopathy. A therapeutic donor lymphocyte infusion can 
also be considered. 
7.2.3.4 
HHV-6: HHV-6 PCR will be obtained 2x/week starting on 
approximately day +10 until day +60 after transplant and 
then 1-2x/wk until day +100. HHV-6 may be followed after 
day +100 in patients who have had reactivation. If the PCR 
test becomes positive, ganciclovir will be started at an 
induction dose of 5 mg/kg IV q12 x 7-14 days, then a 
maintenance dose of 5 mg/kg/day 5-7 days per week until 
PCR is negative x 2 weeks (assuming the patient remains 
asymptomatic). Alternatively, patients may be treated with 
foscarnet induction and maintenance. Valganciclovir can be 
used as prophylaxis following ganciclovir or foscarnet therapy 
if HHV-6 reactivation occurred when the patient was on 
acyclovir prophylaxis. Valganciclovir can also be used for 
therapy at physician discretion. Reactivation after the initial 
occurrence will be treated according to the LCH BMT HHV-6 
policy and the clinical scenario. 
7.2.3.5 
Adenovirus: Stool will be monitored weekly for adenovirus 
until discharge. If the stool test is positive, then a PCR will be 
done on blood, stool, and urine. Adenovirus PCR on blood will 
be obtained weekly starting on approximately day +10 post 
SCT and continuing until day +100 after SCT. For adenovirus 
in the stool and/or blood by PCR, cidofovir will be given.  
Treatment of isolated stool positivity may be based on the 
clinical situation (eg. if patient has renal dysfunction). This 
will be given as 5 mg/kg IV weekly or 1 mg/kg IV three days 
per week until the PCR is negative. Testing for adenovirus 
should be considered in patients with fever, URI symptoms, or 
diarrhea. 

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7.2.3.6 
Toxoplasma: Toxoplasma PCR will be obtained weekly starting 
on approximately day +10 post SCT and continuing until day 
+100 after SCT for patients who are seropositive or have a 
seropositive donor. Monitoring will be every 1-4 weeks from 
day +100 to day +180 depending on the clinical scenario. 
Prophylaxis with trimethoprim/sulfamethoxazole per PCP 
guidelines is preferred for patients at risk. PCR testing will be 
continued beyond day +180 for toxo seropositive recipients 
who have CD4 < 200. 
7.2.3.7 
Pneumocystis 
carinii 
prophylaxis 
will 
be 
trimethoprim/sulfamethoxazole 5 mg/kg/day PO of TMP 
component divided BID for 3 consecutive days per week until 
the CD4 > 200 and for a minimum of 6 months after 
transplant and after discontinuation of immunosuppression. 
Therapy can be reduced to 2 days per week if needed for low 
blood counts. If TMP/SMX is not tolerated, alternative 
prophylaxis will be given per institutional policy. 
7.2.3.8 
Fungal prophylaxis: Voriconazole 4 mg/kg (max 200 mg) PO 
(or IV if PO not tolerated) BID or posaconazole 4 mg/kg (max 
200 mg) PO TID will be started on Day -1 and continued until 
CD4 > 200.  
7.2.3.9 
Bacterial prophylaxis: Ciprofloxacin 10 mg/kg PO BID or 7.5 
mg/kg IV starting on day -1 and continuing until ANC > 500 
or initiation of antibiotics for fever and neutropenia. 
7.2.3.10 
IVIG: Gammaglobulin will be administered at a dose of 200 
mg/kg IV every 2 weeks while hospitalized and then 400-500 
mg/kg IV every 4 weeks as an outpatient.  The dose may be 
rounded to the nearest vial size. IVIG will be given for at least 
6 months after transplant. After that time, IVIG may be 
discontinued when the IgM level is within the normal range 
within a few days prior to when IVIG is due. An 
isohemagglutinin (IgM) titer of >1:8 and a normal IgA level can 
be used as additional indications for discontinuation of IVIG 
at physician discretion. Isohemagglutinin titers are not 
relevant if the donor’s blood type is AB. 
 
7.2.4 Supportive care 
7.2.4.1 
Post-transplant G-CSF may be started on day +14 if the ANC 
is <500; G-CSF may be started as early as Day +6 in patients 
with active fungal infections at the discretion of the treating 
physician or for other serious infections with PI approval. 
7.2.4.2 
All transfusions (except the stem cell product) will be 
irradiated and leukodepleted. In addition, CMV negative PRBC 
and platelets will be used, when available, for CMV negative 
recipients. 
 
7.3 
Mobilization and collection of donor peripheral blood stem cells 
The donor will receive at least 4 days of granulocyte colony stimulating factor (G-
CSF, Neupogen®) administered subcutaneously (8 mcg/kg/dose) twice a day. In 
donors over 120% of IBW, an adjusted G-CSF dose may be considered. The total 
daily dose may be reduced to 10 mcg/kg and the dose may be given once a day 
for an unrelated donor if the recommended dosing is not possible. The donor will 
undergo an outpatient apheresis using antecubital veins for venous access if 
possible. If the peripheral venous access is inadequate, a temporary central line 
will be placed as an outpatient. The morning dose of G-CSF will be held on the 

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day of collection if apheresis will take place in the morning. If the dose is held in 
the morning, it should be given soon after the completion of the apheresis 
procedure if another apheresis is planned. G-CSF will be continued until the 
completion of apheresis. If the donor’s white blood count determined on the 4th 
day and after apheresis on subsequent days of G-CSF (for patients receiving 8 
mcg/kg/dose q12) is greater than 60 x 109 cells/L, the G-CSF dose should be 
decreased to 8 ug/kg daily. If the donor’s blood count is greater than 80 x 109 
cells/L, the G-CSF dose should be held. 
 
The preferred apheresis procedure for related donors will process 5 blood 
volumes (max 25 L). Pheresis performed at Carolinas Medical Center will be 
performed in accordance with the LCH BMT Program Pediatric PBSC 
Mobilization and Collection Guidelines. The related donor may require 1-4 
apheresis procedures to achieve an adequate cell dose. Apheresis performed on 
an unrelated donor will be performed according to the guidelines of the donor 
center and National Marrow Donor Program (NMDP). Unrelated donors will only 
have 1 large volume (24L in one day) or 2 standard volume (12L/day x 2 days) 
apheresis procedure(s) according to applicable NMDP protocols and procedures. 
 
The donor PBSC may be collected before the recipient has met all eligibility 
criteria if the treating physician believes that it is likely that the recipient will 
meet the criteria. The recipient, parent, or guardian should sign consent for the 
transplant prior to the donor PBSC collection. 
 
7.4 
Treatment of graft failure 
If at 4 weeks post-transplant there is no evidence of engraftment based upon 
blood counts, bone marrow examination and chimerism assays, infusion of a 
second PBSC transplant will be performed using the same or another donor (the 
other parent, sibling, or unrelated donor). Additional chemotherapy and 
immunosuppression will be given prior to the second transplant as needed.  
 
7.4.1 If pancytopenia occurs at a later time point, the same approach may be 
used.  There may be other situations like persistent cytopenias or 
significantly decreased donor chimerism for which the physician may 
choose to use this approach. 
 
 
 
7.5 
Therapeutic donor lymphocyte infusion (DLI) 
7.5.1 The following are common indications for DLI. DLI may be given for these 
indications at physician discretion based on the clinical scenario. For 
example, patients with malignant and non-malignant diseases may 
require different approaches. Patients may also receive viral-specific (eg. 
CMV and EBV-specific) T cells as an alternative. 
7.5.2 Indication: 
7.5.2.1 
Mixed Chimerism (< 80% donor) or increasing recipient 
chimerism 
7.5.2.2 
Relapse 
7.5.2.3 
EBV-related PTLD 
7.5.2.4 
Viral infections that progress despite treatment with anti-
virals 
7.5.3 Collection of therapeutic donor lymphocytes: 
7.5.3.1 
DLI will usually be obtained from the cryopreserved negative 
fraction (T cell-containing) of the CD34+ cell selected G-CSF-
mobilized PBSC.  

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7.5.3.2 
T cells from the G-CSF-mobilized PBSC may be less 
alloreactive than T cells collected from blood when G-CSF is 
not present. G-CSF increases Th2 cells that decrease IL-12 
production and delays immune recovery. At the discretion of 
the treating physician, a new collection can be performed to 
provide DLI depending on the clinical situation (such as 
leukemic relapse).  For larger recipients, the stem cell donor 
will undergo lymphocytopheresis to collect several CD3+ cell 
doses, for example, in aliquots of 5 X 104/kg, 1 X 105/kg, 5 
X105/kg, and 1 X 106/kg. For patients requiring small doses, 
donors may give whole blood if ABO compatibility permits the 
use of whole blood as a source of DLI. 
7.5.4 Dose: 
1 Persistent mixed chimerism requiring treatment will be donor % < 80%. 
2 Increasing mixed chimerism will be one 20 point drop in the percentage of donor cells or two 
values of greater than 10% over at least 2 weeks. A lower dose (than for persistent mixed 
chimerism) can be used for increasing recipient chimerism depending on the rate of increase 
and the clinical scenario. 
3 Disease pace: Slow = Chronic Leukemia and MDS without increased blasts; Rapid = Acute 
leukemia, lymphoma, and rapidly-progressive EBV PTLD. For diseases not falling into these 
categories, the choice of using the slow or rapid dose is at physician discretion. 
4 The doses for unrelated donors usually apply to matched unrelated donors. Donors with 
mismatch at HLA-C that is predicted to not be clinically relevant may also be treated at this 
dose at physician discretion. Mismatched unrelated donors will be treated at the Haploidentical 
Donor doses. 
5 The DLI dose for patients with 7/8-matched related donors (mismatch not at DR) can be 
treated with dose for matched unrelated donors at physician discretion. 
7.5.4.1 
If < 6 months post-SCT (high risk of GVHD because APCs are 
still recipient): At physician discretion, a dose that is 50% of 
that in the table can be given to decrease the risk of GVHD. 
The CD3+ cell dose can be repeated every1-2 months. 
7.5.4.2 
If > 6 months post-SCT (APC’s are donor so risk of GVHD 
much less): CD3+ cell dose starts at dose in table above, but 
may be increased with each dose eg. 1 X 105/kg, then 5 X 
105/kg, then 1 x 106/kg, etc.; sequential doses given every1-2 
months. 
7.5.4.3 
Prior 
to 
starting 
DLI 
in 
patients 
that 
are 
on 
immunosuppression for GVHD but without active GVHD at 
the time, immunosuppression will be discontinued based on 
indication as follows: 
a) Stopped for morphological relapse, 
b) Weaned over 1 week for rapidly increasing mixed chimerism 
and/or cytogenetic/molecular relapse, 
c) Weaned over 4 weeks for persistent mixed chimerism or 
slowly increasing chimerism; for patients with persistent 
Indication for DLI 
Persistent mixed 
chimerism1 or EBV PTLD 
or viral infection 
Increasing recipient 
chimerism2 and/or 
cytogenetic/molecular relapse 
Morphological relapse  
Donor type 
Slow3 
Rapid3 
Slow3 
Rapid3 
 
Matched unrelated 
Donor4 
1 x 105/kg 
5 x 105/kg 
5 x 105/kg 
1 x 106/kg 
Chemo 1 x 106/kg 
Haploidentical 
Donor5 
1 x 104/kg 
5 x 104/kg 
5 x 104/kg 
1 x 105/kg 
Chemo  1 x 105/kg 

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mixed 
chimerism, 
patients 
will 
be 
re-assessed 
when 
immunosuppression is stopped prior to giving DLI. 
7.5.4.4 
For patients with active GVHD, they will remain on their 
GVHD therapy and DLI will be given as soon as possible. 
 
 
 
7.6 
Follow-up assessments 
7.6.1 Follow-up history and physical evaluations which are standard for all 
transplant patients occur monthly for 6-12 months, then every 3 months 
x 1 year then every 6 months x 1 year then yearly until 5 years post-
transplant. 
7.6.2 Follow-up studies will be performed according to BMT guidelines and 
include assessment of engraftment, marrow function, immune status, 
liver 
and 
kidney 
function, 
pulmonary 
function 
tests, 
and 
echocardiogram. 
7.7 
Schedule of immunological and engraftment studies  
 
 
 
 
 
 
 
 
 
 
 
 
 
*T cell function studies (mitogens) are optional after they become normal.  
% The 15 and 21 month lymphocyte phenotyping are optional. 
©If the patient has progression of primary disease, then the above tests are optional at 
the discretion of the physician.   
 
Assessments can be +/- 7 days for evaluations through Day 120. Assessments can be 
within one month of target after Day 120 and within three months of target after 24 
months. 
 
These are the assessments for study and clinical purposes. In addition, these tests may 
be performed more often for clinical reasons as well. For example, engraftment studies 
will usually be performed to monitor for relapse in leukemia patients according to the 
LCH BMT Post-Transplant Evaluation Guidelines. Patients with non-malignant disease 
will usually be monitored every 1-2 weeks after engraftment until day +100 depending 
on the clinical scenario. 
 
7.8 
Research samples 
7.8.1 NK cell receptor typing 
7.8.1.1 
NK cell KIR typing will be performed on potential donors in 
the laboratory of Dr. Wing Leung at St. Jude Children’s 
Research Hospital. The results will be communicated to Dr. 
Gilman. The results may be used for donor selection for 
patients with leukemia at Dr. Gilman’s discretion. 
7.8.1.2 
NK cell typing will be optional and dependent on the donor’s 
willingness to participate. 
 
Weeks© 
 
Months© 
 
Years© 
4 
8 
12 
 
4 
5 6 9 12 
15 
18 
21 
24 
 
3 4 5 
Lymphocyte 
Phenotyping 
X 
X X 
 
X 
X X X X 
X% 
X 
X% 
X 
 
X X X 
T cell 
function* 
 
 
 
 
 
 
X X X 
 
X 
 
X 
 
X X X 
Engraftment 
X 
X X 
 
X 
X X X X 
 
X 
 
X 
 
X X X 
Research 
immune 
recovery 
testing 
 
 
 
 
Day 
120 
 
X  
X 
 
 
 
 
 
 
 
 

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7.8.2 Immune recovery testing at St. Jude 
Testing will include analysis of T cell receptor excision circles (TREC) and 
T cell receptor repertoire (V beta typing). This will be performed at 
approximately Day 120, 6 months, and 1 year after transplant (see 
section 7.7). Blood will be sent to Dr. Wing Leung at St. Jude Children’s 
Research Hospital. See Appendix V for details. 
7.8.3 Immune recovery testing at Emory University 
Testing will include analysis of T and B subsets and T cell activation by 
flow cytometry. Testing will also include evaluation of CMV and EBV 
specific T cells by tetramer analysis and cytokine production. This will be 
performed at approximately Day 120, 6 months, and 1 year after 
transplant (see section 7.7). Blood will be sent to Dr. Leslie Kean at 
Emory University. See Appendix V for details. 
 
 
8.0 
CRITERIA FOR TERMINATION  
8.1 
Conditions for terminating the study 
8.1.1 The Principal Investigator may terminate the study for any of the 
following reasons:   
8.1.1.1 
Significant toxicities 
8.1.1.2 
If it becomes clear that the study treatment is less effective 
than other available treatments. 
 
8.2 
Conditions for individual patient termination 
8.2.1 The Principal Investigator may terminate the participation of an 
individual patient for any of the following reasons: 
8.2.1.1 
Disease progression 
8.2.1.2 
Need for exclusionary concurrent treatment 
8.2.1.3 
Withdrawal of informed consent 
8.2.1.4 
Protocol non-compliance 
8.2.1.5 
Lost to follow-up 
 
 
9.0 
STATISTICAL CONSIDERATIONS   
9.1 
Hypotheses 
Transplantation of stem cells that have been CD34+ selected and T cell-depleted 
with the CliniMACS® device will prevent severe (grade III/IV) acute GVHD 
without the use of prophylactic post-transplant immunosuppression. The 
incidence of grade III/IV acute GVHD is predicted to be <10%. This has been our 
experience in 18 reported patients  (22) and currently 25 treated recipients of 
haplocompatible donor transplants. 
 
9.2 
Accrual objectives 
Originally, the plan was to enroll thirty patients into each of two cohorts 
(haplocompatible related donor and unrelated donor). The study objective is to 
determine the ability of CD34+ selection using the CliniMACS® device as the sole 
GVHD prophylaxis to prevent severe (grade III-IV) acute GVHD by Day +30 after 
transplant in recipients of alternative donor stem cell transplants. Thirty 
patients will allow estimating the rate of severe acute GVHD with the 95% 
confidence interval of the maximum width of 0.25 if the observed rate of severe 
acute GVHD does not exceed 10%. A very low incidence of severe GVHD is 
predicted based on a reported incidence of 2% in a large study of adults (17) and 
a 0% incidence in a large study in children (21) and our experience (22). For 
example, if 3 severe acute GVHD events are observed in 30 patients, the exact 
95% confidence interval is (0.02, 0.27). The accrual goal to the haplocompatible 

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cohort will be increased to 60. This is necessary for several reasons. First, this 
study serves to provide patients for the companion study which is evaluating an 
approach to hasten immune recovery after transplant. The companion study 
may require another 16-30 patients to complete its objective. Also, adding 
additional patients will allow us to better address the primary and secondary 
objectives in subpopulations like those with malignant disease, sickle cell 
disease, and immunodeficiency. Finally, the accrual of additional patients will 
allow the determination of acute GVHD with tighter confidence intervals. 
Because the accrual to the unrelated donor cohort may be lower, the statistical 
objective for this group can be changed to estimation of severe GVHD incidence 
as a pilot trial with less tight confidence interval goals than above. Patients who 
have primary graft failure (never engraft) will not be evaluable. They will be 
replaced by the enrollment of additional patients. Based on the literature and 
our experience, graft rejection is expected to occur in < 10% of patients. Patients 
that are enrolled but who do not receive a transplant will be replaced because 
they will not be evaluable for study objectives. 
 
It is anticipated that study accrual will fluctuate significantly from quarter to 
quarter because marked variations in numbers of transplants in common. Also, 
the accrual to the unrelated donor cohort may be less than expected. If this is 
the case, then the accrual goal for this cohort will be 10 patients and the 
statistical objective will be to estimate severe GVHD incidence as a pilot trial for 
this approach. 
 
9.3 
Endpoint definitions 
9.3.1 GVHD: Grading of GVHD will be according to Appendix II and III 
9.3.2 Engraftment: 
9.3.2.1 
Primary graft failure (lack of engraftment) will be the lack of 
recovery of ANC to > 500 by Day +28 after transplant in the 
absence of immunological graft rejection. This endpoint is 
intended to monitor graft quality after manipulation. 
9.3.2.2 
Late (secondary) graft failure 
a) Initial evidence for marrow recovery and engraftment with 
subsequent pancytopenia without another cause (ie. 
Infection and/or drug therapy) and decrease in donor 
chimerism by greater than 50% from highest level 
achieved 
b) Decrease in donor chimerism to less than 10%  
9.3.3 Immune recovery: Immune recovery will be assessed by the time to 
CD4>100 and CD4>200. 
9.3.4 Severe toxicity: Severe toxicity will include any unexpected Grade 3 (for 
transplant recipient) toxicity or any non-hematological grade 4-5 toxicity 
through 1 year post-transplant. 
9.3.5 Post-transplant infections: Infections that occur following the initiation of 
the conditioning regimen. 
9.3.6 CMV infection and disease: CMV infection will be reactivation detected by 
CMV PCR on plasma and CMV disease will be evidence of organ 
involvement (eg. CMV pneumonia, enteritis). 
9.3.7 Post-transplant lymphoproliferative disease (PTLD): PTLD will include 
patients that have clinical PTLD (for example, detectable EBV viral load 
and fever and/or adenopathy). 
9.3.8 Transplant-related mortality (TRM): TRM will include death due to 
regimen-related toxicity or GVHD (usually all causes other than disease 
relapse). 

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9.3.9 Disease free survival (DFS) and overall survival (OS):  Disease-free 
survival 
will 
be 
survival 
without 
relapse, 
including 
molecular, 
cytogenetic, and morphological relapse. DFS and OS at one, two, and 5 
years after transplant will be determined from the day of transplant. 
9.3.10 Chimerism at Day 100: Chimerism (percentage of donor cells) will be 
assessed at Day 100 post-transplant in evaluable (without disease 
progression) patients. 
9.3.11 Device (CliniMACS®) performance parameters: The parameters will 
include the purity of the CD34+ selected stem cell product, yield of CD34+ 
cells after selection, degree of CD3+ cell depletion after selection, and 
viability and sterility of the stem cell product after selection. 
 
9.4 
Plan of analysis 
The primary endpoint of the study is the incidence of severe (grade III/IV) acute 
GVHD occurring by Day +30 after transplant. In our experience, almost all 
patients who develop acute GVHD after this type of transplant do so by Day +30. 
Acute GVHD attributed to the primary graft will not be able to be assessed after 
Day +30 in patients who are enrolled on the companion prophylactic donor 
lymphocyte infusion (DLI) protocol. In this situation, there will be no way to 
know if the GVHD is due to the primary graft or the DLI. It will be more likely to 
be due to the DLI because a higher dose of T cells will be given with the DLI and 
the level of circulating ATG will be less after DLI. We anticipate that a few 
patients will not go on the companion prophylactic study and these patients can 
be assessed for acute GVHD until Day +100. Although this will not be used for 
the primary endpoint, it will corroborate the fact that patients do not develop 
acute GVHD between Day 30 and Day +100. GVHD will be graded per standard 
criteria as discussed above. 
 
The following variables will be assessed as secondary endpoints:  
a. Engraftment: The incidence of primary graft failure and late (secondary) graft 
failure will be determined. 
b. Immune recovery: The time to CD4 count >100 and >200 will be calculated.  
c. Severe toxicities: The incidence of severe toxicities will be determined. This 
will include any unexpected grade 3 (for transplant recipient) toxicity or any 
non-hematological grade 4-5 toxicity through 1 year post-transplant. 
d. Post-transplant infections: Post-transplant infections will be described by 
incidence and type. 
e. CMV infection and disease: The incidence of each will be calculated. 
f. 
Post-transplant lymphoproliferative disease (PTLD): The incidence of PTLD 
will be calculated. 
g. Transplant-related mortality (TRM): The incidence of TRM will be calculated 
at Day 100 after transplant and long term. 
h. Disease-free survival: The method of Craddock et al. (37) will be used to 
estimate DFS. The definition of DFS will parallel that of leukemia-free 
survival proposed by Craddock.  
i. 
Overall survival: Overall survival will be estimated using the Kaplan-Meier 
method (38). 
j. 
Chimerism at Day 100: The percentage of donor cells will be reported for all 
evaluable (without disease progression) patients.  
k. Device (CliniMACS®) performance parameters: The parameters will be 
summarized for all products that are processed and median and ranges will 
be determined. 
 
The endpoints will be monitored at least annually. The frequency will depend on 
the number of patients enrolled. Patients that receive chemotherapy for relapse 

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of underlying disease will only be followed for overall survival from that the time 
of chemotherapy. 
 
Additional results that will be monitored include the research studies for 
immune recovery (TREC, T cell receptor repertoire (V beta typing) T and B 
subsets and T cell activation by flow cytometry, and CMV and EBV specific T 
cells by tetramer analysis and cytokine production). This will be performed at 
approximately Day 120, 6 months, and 1 year after transplant. The analysis of 
these results will be descriptive.  
 
Stopping rules  
The study includes stopping rules based on the incidence of graft failure and on 
transplant-related mortality (TRM) by Day 100 after transplant.  
 
Stopping rule for primary graft failure: The trial is stopped if there are  bk graft 
failures out of n resolved patients. Only points where stopping is possible are 
listed. 
 
Number of Patients, n
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
Boundary, b n
-
2
3
3
3
4
4
4
4
5
5
5
5
5
6
6
6
6
6
6
Number of Patients, n
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
Boundary, b n
7
7
7
7
7
7
8
8
8
8
8
8
9
9
9
9
9
9
10
10
Number of Patients, n
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Boundary, b n
10
10
10
10
10
11
11
11
11
11
11
11
12
12
12
12
12
12
13
13  
 
The stopping rule for graft failure yields the probability of stopping the trial of 
0.05 if the rate of graft failure is 0.1. The probability of stopping the trial is 0.61 
if the graft failure rate is 0.2 and 0.97 if the graft failure rate is 0.3.  These 
probabilities were calculated based on the binomial distribution. The stopping 
rule was generated as described by Ivanova et al. (39).     
 
Stopping rule for Day 100 TRM: The trial is stopped if there are  bn patients 
with TRM out of n resolved patients.  
 
Number of Patients, n
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
Boundary, b n
-
-
3
4
4
4
5
5
5
5
6
6
6
6
7
7
7
7
8
8
Number of Patients, n
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
Boundary, b n
8
8
9
9
9
9
10
10
10
10
11
11
11
11
11
12
12
12
12
12
Number of Patients, n
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Boundary, b n
13
13
13
13
14
14
14
14
14
15
15
15
15
15
16
16
16
16
16
17  
 
The stopping rule for TRM yields the probability of stopping the trial of 0.05 if 
the TRM rate is 0.15. The probability of stopping the trial is 0.21 if the TRM rate 
is 0.2, and 0.80 if the TRM rate is 0.3. These probabilities were calculated based 
on the binomial distribution. The stopping rule was generated as described by 
Ivanova et al. (39).     
   
If the study reaches a stopping boundary, the study will be suspended.  At this 
point it may be terminated by the PI or submitted to the DSMC with a 
description of the failures to date and a rationale for why the study should be 
continued. Proper use of the stopping rule table will be ensured by the Study 
Investigator. 
 
9.5 
Sample size 

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Patients will be recruited by the PI and his co-investigator. 30 patients will be 
enrolled into the unrelated donor cohort and 60 patients into the alternative 
related donor cohort.  
 
9.6 
Estimated duration of study   
 
The study will require 8 years for accrual and up to 5 years for follow-up 
studies. 
 
9.7 
Replacement policy 
 
If a patient becomes unevaluable for engraftment at 4 weeks post-BMT,  then 
an additional patient will be enrolled. 
 
 
10.0 
CRITERIA FOR EVALUATION 
10.1 
Monitored outcomes   
10.1.1 The primary outcome will be severe (grade III/IV) graft-versus-host 
disease.   
10.1.2 Secondary outcomes will be engraftment (ANC>500 and >80% donor cells 
in blood), survival, disease-free survival, infection, transplant-related 
toxicity and mortality, grade 3/4 stem cell product infusion-related 
toxicity, and relapse. 
 
10.2 
Toxicity definitions/stopping rules 
10.2.1 If any patient develops grade IV acute GVHD by 4 weeks post BMT, the 
protocol will be halted and the processing re-evaluated before proceeding. 
10.2.2 Stopping rules will be used for graft failure. 
 
 
11.0 
DATA SAFETY MONITORING PLAN  
11.1 
Oversight and monitoring plan 
The study will utilize the Data and Safety Monitoring Committee (DSMC) of the 
Pediatric Blood and Marrow Transplant Consortium (PBMTC). The DSMC will be 
responsible for safeguarding the interests of participants in this trial.  This 
responsibility will be exercised by providing recommendations for continuation 
or early termination of the trial, based on assessment of safety.  The DSMC may 
also formulate recommendations related to the selection, recruitment or 
retention of participants, their management and adherence to protocol-specified 
regimens, and the procedures for data management and quality control. 
 
The DSMC will be advisory to Andrew Gilman, MD who will serve as sponsor and 
principal investigator and to his co-investigators.  Dr. Gilman and his co-
investigators will be responsible for promptly reviewing any recommendations 
and deciding how to respond.       
 
  
 
The DSMC will:  
 
Review the current protocol   
 
Review progress of the trial on a semi-annual basis  
 
Review of all serious adverse events including expected and unexpected 
events according to PBMTC DSMC guidelines. 
 
If at any time the Investigator stops enrollment or stops the study due to safety 
issues, the DSMC and IRB will be notified within 24 business hours via e-mail. 
A formal letter will be submitted within 10 business days and the FDA will be 
notified.  
 

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11.2 
Monitoring and reporting guidelines 
The PI will conduct continuous review of data and patient safety at weekly Blood 
and Marrow Transplant meeting where the results of each patient’s treatment 
are discussed.  The discussion will be documented by the BMT research team.  
The discussion may include the number of patients, significant toxicities as 
described in the protocol, dose adjustments, and observed responses.  Semi-
annual reports will be submitted to the PBMTC DSMC for review according to 
PBMTC guidelines.  Grade 3 (unexpected for transplant recipient) adverse 
events, non-hematologic grade 4 adverse events, all grade 5 adverse events and 
all serious adverse events related to study participation will be submitted to the 
FDA, IRB and DSMC in accordance with their reporting requirements.   
 
11.3 
Review and oversight requirements 
11.3.1 Oversight of cell processing 
The results of the cell processing at UCSF will be reviewed by the PI.  The 
outcomes and AE's will also be reviewed. 
 
11.3.2 Adverse event definition 
An adverse experience is any untoward medical occurrence in a patient or 
clinical investigation subject administered a pharmaceutical product and which 
does not necessarily have a causal relationship with this treatment.  An adverse 
experience or event (AE) can therefore be any unfavorable and unintended sign 
(including an abnormal laboratory finding), symptom, or disease temporally 
associated with the use of a medicinal (investigational) product, whether or not 
related to the medicinal (investigational) product. 
 
11.3.3 Adverse event recording   
Adverse events will be graded using the NCI Common Terminology Criteria for 
Adverse Events (CTCAE) version 3.0 found on the following website: 
http://ctep.cancer.gov/reporting/ctc.html. All non-hematologic grade 3 – 4 
adverse events and all grade 5 adverse events will be recorded until 1 year post-
transplant. All deaths and other endpoints in Section 9.3 will continue to be 
recorded through the duration of follow-up.    Grading of acute and chronic 
GVHD will be according to Appendix II and III.  Causality will be rated as 
definitely, probably, possibly, or unlikely related, or unrelated to the CD34+ 
CliniMACS® collection.  The investigator is responsible for making an 
assessment of whether or not it is reasonable to suspect a causal 
relationship between the adverse event and the study treatment. 
 
 
 
 
11.3.4 Serious adverse events definition 
An unexpected adverse event is any adverse drug experience where the 
specificity or severity of which is not consistent with the current investigator 
brochure; or, if an investigator brochure is not required or available, the 
specificity or severity of which is not consistent with the risk information 
described in the general investigational plan or elsewhere in the current 
application. 
 
A serious adverse experience (SAE) or serious adverse drug reaction (ADR) is any 
adverse drug experience occurring at any dose that results in any of the 
following outcomes: 
11.3.4.1 
Death 

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11.3.4.2 
Life-threatening (places the subject, in the view of the 
investigator, at immediate risk of death from the reaction as it 
occurred) 
11.3.4.3 
Inpatient hospitalization or prolongs existing hospitalization 
11.3.4.4 
Persistent or significant disability/incapacity (a substantial 
disruption of a person’s ability to conduct normal life 
functions) 
11.3.4.5 
Birth defect/congenital anomaly 
11.3.4.6 
Any important medical event that may not result in prior 
listed outcomes but, based upon appropriate medical 
judgment, may jeopardize the subject, and may require medic 
al and surgical intervention to prevent one of the prior listed 
outcomes. 
 
11.3.5 Targeted AE reporting guidelines for this protocol 
11.3.5.1 
Any evidence of grade III/IV acute or extensive chronic GVHD 
using protocol appendices II and III 
11.3.5.2 
Grade 
3 
(unexpected 
for 
transplant 
recipient), 
non-
hematological grade 4, and all grade 5 adverse events until 1 
year post-transplant. See Appendix VI for expected adverse 
events. 
11.3.5.3 
Hospitalization 
11.3.5.4 
Relapse 
11.3.5.5 
Any problems with CliniMACS device during cell separation or 
inability to achieve CD34+ cell and CD3+ cell target doses. 
11.3.5.6 
Failure to engraft or late graft failure. 
 
11.3.6 Adverse event reporting procedures 
Reporting will be in accordance with 21 Code of Federal Regulation (CFR) 
Part 312.32. 
 
Grade 3 (unexpected for transplant recipient) adverse events, non-
hematologic grade 4 adverse events, and all grade 5 adverse events will 
be recorded in the Case Report Forms until 1 year post-transplant.  This 
will include the severity or toxicity grade, the relationship to the study 
drug, treatment and the outcome of the event.   
 
FDA website for guidance in reporting serious adverse events 
http://www.accessdata.fda.gov/scripts/cdrh/cfdocs/cfcfr/CFRSearch.cf
m?fr+312.32 
 
MedWatch forms and information: 
http://www.fda/gov/medwatch/getforms.html 
 
CliniMACS® CD34+ Reagent System: 
Fax MedWatch form directly to the Safety Officer, Miltenyi Biotec Inc. 
(781-782-1920) 
 
Serious Adverse events will be reported on the MedWatch form when it 
meets the definition of expedited FDA reporting.  
 
Serious Adverse events will be reported to PBMTC DSMC within 10 days 
of event.    
 

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If the SAE is death and determined to be possibly, probably, or definitely 
related to the investigational drug or any research related procedure, the 
event must be reported to the DSMC within 24 business hours. The 
reporting procedure is by personal communication via phone with 
written documentation of the 1:1 communication or via e-mail with a 
copy of the e-mail to be placed in the regulatory binder.   
 
11.3.7 Review of adverse event rates 
If the study has an increased incidence of grade 3 or grade 4 adverse 
events associated with the infusion of the CD34+ selected stem cell 
product above the rate reported in the protocol or investigational 
brochure, this will be reported to the DSMC at the time of identifying the 
increased rate. 
 
11.3.8 Semi-annual review of study progress 
Principal Investigator is required to submit semi-annual study progress 
reports to the PBMTC DSMC.  The progress reports will describe toxicity 
and include the rate of grade 3 (unexpected for transplant recipient) 
adverse events, all non-hematological grade 4 adverse events, all grade 5 
adverse events and all serious adverse events through 1 year post-
transplant. In addition, the report will include an updated accrual 
summary, all deaths reported on study, and a brief synopsis of study 
status.  
 
These semi-annual reports are reviewed by the PBMTC DSMC. These 
reports are scheduled for May and November as per DSMC procedures. 
The cut-off date for data will be 2 weeks prior to this date. The reports 
will be submitted within at least 1 month from the scheduled date.  
 
 
12.0 
ETHICAL ASPECTS  
12.1 
Regulatory considerations 
This study will be reviewed by the Carolinas Healthcare System (CHS) 
Institutional Review Board. In addition, an Investigational New Drug application 
will be filed with the FDA.  As per FDA regulations (21 CFR 312.33), annual 
reports will be submitted to the FDA within 60 days of the anniversary date that 
the IND went into effect. 
 
12.2 
Independent Ethics Committees/Institutional Review Board 
This protocol and the informed consent will be approved by the CHS IRB.  The 
Principal Investigator is responsible for keeping the IRB advised of the progress 
of the study and of any changes made in the protocol prior to implementation.  
The Principal Investigator will also keep the IRB informed of any significant 
adverse reactions, and any protocol exceptions or deviations.  Records of all 
study review and approval documents must be kept on file by the Principal 
Investigator and are subject to FDA inspection during or after completion of the 
study.  The IRB will receive notification of the termination of the study. 
 
 
 
 
 
 
 
 

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13.0 
DATA FORMS AND SUBMISSION SCHEDULE  
Forms will be completed and documented for each patient. The Completion Schedule 
below is intended as a reasonable guideline only.  The exception is the Recipient/Donor 
eligibility form which should be completed before the recipient receives study treatment.  
A research chart with completed paper case report forms and supporting 
documentation of reportable  AEs and SAEs for all enrolled patients will be maintained 
in the BMT Research office.  Patient enrollment information and toxicity/reporting 
information will also be entered by the coordinator into the BMT database. If the patient 
is enrolled on LCH BMT 09-02 protocol (09-02), then completion of acute and chronic 
GVHD forms for LCH BMT 09-01 are not required until completion of follow up ends at 
2 years for 09-02.    
 
 
 
 
 
FORMS 
Submission Schedule 
Enrollment Forms: 
 
Recipient Eligibility 
 
Donor  Eligibility 
 
Recipient Baseline Information 
 
Complete recipient eligibility form and 
baseline form after recipient, parent, or 
guardian has signed consent and prior to 
treatment of the recipient.  Complete 
donor eligibility form after donor, parent, 
or guardian has signed consent and prior 
to treatment of the donor.  Complete 
baseline information. 
Treatment Forms: 
 
Transplant Data Form 
 
CD34+ Cell Processing 
 
Complete within one month of last dose of 
conditioning therapy.     
 
Follow Up Forms: 
 
Engraftment Assessment & 
Lymphocyte Phenotyping 
 
Acute GvHD 
 
Chronic GvHD 
 
Adverse Events Reporting 
Complete within one month of time points 
listed in table in Section 7.7). 
Toxicity Reporting: 
 
SAE Forms 
 
CHS IRB Form 
 
FDA MedWatch  
Complete as required based on reporting 
criteria for each institution. See sections 
11.2 to 11.3.6 for further information. 

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APPENDICES 
APPENDIX I – Alternative conditioning regimens: 
1. Chemotherapy Alone Regimen (for patients who have a contraindication to TBI or non-
malignant disease (22). 
1.1. On day –7, administer melphalan 140mg/m2 IV over 20-30 minutes x 1 dose.   
1.2. On day –6, thiotepa 5mg/kg/dose will be administered intravenously in two doses 
(each dose over 3-4 hours) for a total dose of 10mg/kg/day. 
1.3. On days –6 through –2, fludarabine 40 mg/m2/d (infuse over 30 minutes) will be 
administered in the early morning hours on each of the five days (there will be at least 
24 hrs between the last dose of fludarabine and  the stem cell infusion).  Fludarabine 
dose will be reduced by 20% for creatinine clearance of < 70 ml/min/1.73m2. 
1.4. Rabbit ATG (thymoglobulin) 1.5-2.5 mg/kg on days –5 to day –2 infused over 6 hours. 
The dose will be at PI discretion. Examples are that patients with non-malignant 
disease will usually receive the higher dose and patients with solid tumors who have 
received intensive chemotherapy prior to transplant will usually receive the lower dose. 
 
2. Reduced Intensity Regimen (for patients with decreased organ function) (30,31) 
2.1. On day -7 through day -3, fludarabine 30 mg/m2/d (infused over 30 minutes) will be 
administered in the early morning hours on each of the five days.  Fludarabine dose 
will be reduced by 20% for creatinine clearance of 30-70 ml/min/1.73m2. 
2.2. On day –3, thiotepa 5mg/kg/dose will be administered intravenously in two doses 
(each dose over 3-4 hours) for a total dose of 10mg/kg/day. 
2.3. On days –2 and –1, melphalan 60 mg/m2/d (infuse over 20-30 minutes) will be 
administered in the morning (there will be at least 24 hrs between the last dose of 
melphalan and  the stem cell infusion). 
2.4. Rabbit ATG (thymoglobulin) 0.5 mg/kg on day -7 infused over 4 hrs and then 2.5 
mg/kg for four daily doses from day –6 to day –3 infused over 6 hours. 
 
3. Alternative Reduced Intensity Regimen (without thiotepa) (32) 
3.1. On day –8 and -7, administer busulfan 0.8 mg/kg (1 mg/kg if > 10 kg and < 4 yrs of 
age) q 6 hrs IV over 2 hrs. 
3.2. On days –7 through –3, fludarabine 40 mg/m2/d (infuse over 30 minutes) will be 
administered in the morning on each of the five days.  Fludarabine dose will be 
reduced by 20% for creatinine clearance of 30-70 ml/min/1.73m2. 
3.3. On day –2, melphalan 140mg/m2 IV over 20-30 minutes x 1 dose.  Prepare melphalan 
immediately prior to administration to maintain stability. 
3.4. Rabbit ATG (thymoglobulin) 0.5 mg/kg on day -6 infused over 4 hrs and then 2.5 
mg/kg for four daily doses from day –5 to day –1 infused over 6 hours. 
 
4. Regimen for chromosome breakage disorders like Fanconi anemia with aplasia (33) 
4.1. On day -6 through day -3, cyclophosphamide 10mg/kg/dose will be administered 
intravenously (each dose to run over 1 hour) on each of the four days. 
4.2. On day -6 through day -3, fludarabine 40 mg/m2/d (infused over 30 minutes) will be 
administered in the morning on each of the four days.  Fludarabine dose will be 
reduced by 20% for creatinine clearance of < 70 ml/min/1.73m2. 
4.3. Rabbit ATG (thymoglobulin) 0.5 mg/kg on day -6 infused over 4 hrs and then 3.75 
mg/kg for four daily doses from day –5 to day –2 infused over 6 hours. 
 
5. Regimen for chromosome breakage disorders like Fanconi anemia with MDS/AML (34) 
5.1. On day -7, a 450 cGy single dose of total body irradiation is delivered. 
5.2. On day -5 through day -2, cyclophosphamide 10mg/kg/dose will be administered 
intravenously (each dose to run over 1 hour) on each of the four days. 
5.3. On day -6 through day -2, fludarabine 30 mg/m2/d  (infused over 30 minutes) will be 
administered in the early morning hours on each of the five days (there will be at least 

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24 hrs between the last dose of fludarabine and  the stem cell infusion).  Fludarabine 
dose will be reduced by 20% for creatinine clearance of < 70 ml/min/1.73m2. 
5.4. Rabbit ATG (thymoglobulin) 0.5 mg/kg on day -6 infused over 4 hrs and then 2.5 
mg/kg for four daily doses from day –5 to day –2 infused over 6 hours. 
 
6. Alternative Regimen (for patients with immunological disorders with immune activation and 
those with high risk of autologous marrow recovery including beta-thalassemia major) (40) 
6.1. On day –10 and -9, administer cyclophosphamide 50 – 60 mg/kg/dose will be 
administered intravenously (each dose to run over 1 hour) on each day with 
adjustments per referenced article.  
6.2. On day –8 through -6, administer busulfan daily IV over 2 hrs on days -8 and -7 and 
q12 on day -6 with adjustments per referenced article. 
6.3. On day –5, thiotepa 5mg/kg/dose will be administered intravenously in two doses 
(each dose over 3-4 hours) for a total dose of 10mg/kg/day. 
6.4. On days –8 through –4, fludarabine 40 mg/m2/d (infuse over 30 minutes) will be 
administered in the morning on each of the five days.  Fludarabine dose will be 
reduced by 20% for creatinine clearance of 30-70 ml/min/1.73m2. 
6.5. Rabbit ATG (thymoglobulin) 2.5 mg/kg for four daily doses from day –4 to day –1 
infused over 6 hours. 
6.6. Dose adjustments will be made based on ferritin, hepatomegaly, and age for patients 
with thalassemia and also for other patients (at physician discretion) per referenced 
article. Busulfan Css levels will be targeted at 500 – 600 mcg/L. 
6.7. Azathioprine and hydroxyurea will be used prior to transplant (as described in the 
reference) for patients with beta-thalassemia major 

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       APPENDIX II- Acute GVHD staging and grading for children** 
 
ORGAN 
STAGE 
DESCRIPTION 
SKIN 
1 
Maculopapular rash < 25% of 
BSA 
 
2 
25 – 50% of BSA 
 
3 
Generalized erythroderma 
 
4 
Desquamation and bullae 
 
 
 
LIVER 
1 
Bilirubin 2 - 3 mg/dL 
 
2 
Bilirubin 3.1 - 6 mg/dL 
 
3 
Bilirubin 6.1 - 15 mg/dL 
 
4 
Bilirubin > 15 mg/dL 
 
 
 
GUT 
1 
Diarrhea > 500 – 1000 ml/day 
(> 10 mL/kg - 20 mL/kg/day) 
OR persistent UGI symptoms 
 
2 
Diarrhea > 1000 – 1500 ml/day 
(> 20mL/kg – 30 mL/kg/day) 
 
3 
Diarrhea >1500 ml/day 
(>30 mL/kg/day) 
 
4 
Severe abdominal pain or ileus 
 
 
 
GRADE 
Skin 
    Liver 
 
Gut 
I 
1-2 
       0  
 
0 
II 
3 and/or 
       1      and/or 
1 
III 
-- 
     2-3      and/or 
2-3 
IV 
4 and/or 
       4      and/or 
4 
 
 
 
 
** Adapted from Glucksberg and Jacobsohn articles (41,42). 

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APPENDIX III - Chronic GVHD staging and grading for children** 
Chronic GVHD grading will be performed with both a limited/extensive grading system 
and with the NIH Consensus scoring system. 
The limited/extensive grading will use the revised Seattle classification (43) shown 
below. 
 
 
. 
 
 
 
 
 
  

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Grading of Chronic GVHD severity by NIH Consensus Guidelines using Organ Scoring Table (44) 
 
Severity 
 
 
Definition 
 
 
Mild 
 
Involves on 1 or 2 organs or sites (except the lung; see below), with no clinically significant 
functional impairment (maximum of score 1 in all affected organs or sites) 
 
 
Moderate 
 
(1) At least one organ or site with clinically significant but no major disability (maximum score of 2 
in any affected organ or site OR (2) 3 or more organs or sites with no clinically significant 
functional impairment (maximum score of 1 in all affected organs or sites) OR a lung score of 1. 
 
 
Severe 
 
 
(1) Major disability caused by chronic GVHD (score of 3 in any affected organ) or site OR a lung 
score of > 2. 
 
 

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NIH Consensus Chronic GVHD Organ Scoring 
 

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Organ scoring of chronic GVHD. *AP may be elevated in growing children, and not reflective of liver dysfunction. †Pulmonary 
scoring should be performed using both the symptom and pulmonary function testing (PFT) scale whenever possible. When 
discrepancy exists between pulmonary symptom or PFT scores the higher value should be used for final scoring. Scoring using the 
Lung Function Score (LFS) is preferred, but if DLCO is not available, grading using FEV1 should be used. The LFS is a global 
assessment of lung 
function after the diagnosis of bronchiolitis obliterans has already been established [29]. The percent predicted FEV1 and DLCO 
(adjusted for hematocrit but not alveolar volume) should be converted to a numeric score as follows: > 80% = 1; 70-79% = 2; 60-
69% = 3; 50-59% = 4; 40-49% = 5; < 40% = 6. The LFS = FEV1 score + DLCO score, with a possible range of 2-12. GVHD 
indicates graft versus host disease; ECOG, Eastern Cooperative Oncology Group; KPS, Karnofsky Performance Status; LPS, 
Lansky Performance Status; BSA, body surface area; ADL, activities of daily living; LFTs, liver function tests; AP, alkaline 
phosphatase; ALT, alanine aminotransferase; AST, aspartate aminotransferase; ULN, upper limit of normal. 

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APPENDIX IV - Device information  
 
Brand: CliniMACS® CD34 Reagent System Information 
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 
 
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  
 
 
University of California, San Francisco CliniMACS® cell sorter Device information: 
1) Serial number: 000288 
2) Catalogue number: 15101 
3) Part number:  44085 
 
 
Distributor:                          
 
Manufacturer: 
Corporate Headquarters 
  
 
Mitenyi Biotec GmbH, Clinical Products 
Miltenyi Biotec Inc.  
 
 
Friedrich-Ebert Strasse 
12740 Earhart Avenue 
 
 
Technologiepark H-13 
Auburn, CA 95602 
 
 
 
D51429 
 
 
 
 
 
 
Bergisch Gladbach, Germany 
 
 
Sponsor Contact: 
Miltenyi Biotec Inc., Suite 305 
120 Presidential Way 
Woburn, MA  01801 
Phone: (781) 782-1910 
Fax: (781) 782-1920 
 

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APPENDIX V – RESEARCH SAMPLES 
 
Sample for KIR/NK Cell Biology Studies 
 
Blood samples will be obtained for potential donors in order to perform KIR typing. Donor 
samples for KIR typing will usually be obtained at the time of confirmatory HLA typing of 
potential donors and after HLA typing results are available for related donors. KIR typing will 
only be performed upon optional donor consent. 
 
Sample Collection 
 
Two 10 mL ACD yellow-top tubes of peripheral blood will be collected from potential donor and 
sent to the address below for KIR typing. 
 
Shipping 
 
All samples will be shipped at room temperature by overnight mail to Dr. Wing Leung at 
SJCRH. No refrigeration is necessary. Blood samples must be protected in shipping containers 
inside Styrofoam boxes to minimize temperature change because the cells must be tested in a 
viable condition. Samples sent for KIR typing must be sent via FedEx and must be sent with a 
specimen transmittal form. Label sample with Patient ID, Protocol ID, and time of sample 
collection. 
 
Research personnel at LCH will notify Dr. Leung that a donor sample will be sent for KIR 
typing. For related donors, research personnel will provide the study Donor ID and for 
unrelated donors, research personnel will provide the study Donor ID, DID, RID and estimated 
collection date. 
 
All samples for KIR typing should be sent to the address below: 
 
Barbara Rooney  
Laboratory of Dr. Wing Leung 
St. Jude Children’s Research Hospital 
262 Danny Thomas Place, Room D5032 
Memphis, TN 38105 
Phone: (901) 595-4155 
Fax: (901) 595-4023 
E-mail: wing.leung@stjude.org or Barbara.rooney@stjude.org 
Saturday deliveries are permissible; Sunday deliveries are not permissible. 
Note: If a Saturday delivery is planned, please notify Dr Leung at the number listed above and 
clearly mark the package “For Saturday Delivery.” 
 
Ship each specimen individually by overnight air freight on the day of its collection. Maximum 
time from sample collection to shipment should be no greater than 24 hours. Exception: 
Samples collected on weekends or holidays should be shipped the first working day following 
collection. Store and ship samples at room temperature. Send fresh. 
 
 
 
 
 
 
 
 
 

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Samples for Immune Recovery Studies 
 
The St. Jude and Emory samples can be drawn on different days to allow the target 
amount to be obtained for each lab. The samples for immune recovery studies at St. Jude 
and Emory University can be sent within 1 week of each time point if the amount of 
blood taken is limited by patient size. The limit for blood taken in each blood draw is 2 
mL/kg (max 60 mL).  
 
Sample for Immune Recovery Studies at St. Jude 
 
Blood samples will be obtained at approximately Day 120, 6 months, and 1 year after 
transplant as described in Sections 7.7 and 7.8.2.   
 
Sample Collection 
 
Two 10 mL ACD yellow-top tubes of peripheral blood will be collected. 
 
Shipping 
 
All samples will be shipped at room temperature by overnight mail to Dr. Wing Leung at 
SJCRH. No refrigeration is necessary. Blood samples must be protected in shipping containers 
inside Styrofoam boxes to minimize temperature change, because the cells must be tested in a 
viable condition. Samples will be sent via FedEx and must be sent with a specimen transmittal 
form. Label sample with Patient ID, Protocol ID, and time of sample collection. 
 
All samples for immune recovery studies should be sent to the address below: 
Barbara Rooney 
Laboratory of Dr Wing Leung 
St. Jude Children’s Research Hospital 
262 Danny Thomas Place, Room D5032 
Memphis, TN 38105 
Phone: (901) 595-4155 
Fax: (901) 595-4023 
E-mail: wing.leung@stjude.org or Barbara.rooney@stjude.org 
Saturday deliveries are permissible; Sunday deliveries are not permissible. 
Note: If a Saturday delivery is planned, please notify Dr Leung at the number listed above and 
clearly mark the package “For Saturday Delivery.” 
 
Ship each specimen individually by overnight air freight on the day of its collection. Maximum 
time from sample collection to shipment should be no greater than 24 hours. Store and ship 
samples at room temperature. Send fresh. 
 
Sample for Immune Recovery Studies at Emory University 
 
Blood samples will be obtained at approximately Day 120, 6 months, and 1 year after 
transplant as described in Sections 7.7 and 7.8.3.   
 
Sample Collection 
 
Peripheral blood will be collected as follows: 
 
Samples will consist of the following: 
1. 8 mL into cytochex tubes (can do 4 cc on smaller children if needed). 

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a. Cyto-Chex BCT is a blood collection tube for the preservation of whole blood 
samples for immunophenotyping by flow cytometry. Cell morphology and 
surface markers are maintained in these tubes for up to 7 days allowing for 
blood to be sent to a central location for processing. 
b. Fill a Cyto-Chex tube with 4 ml whole blood 
c. Immediately mix the collected tube by gentle inversion 8-10 times. 
d. Collected blood should be shipped by priority overnight express to Dr. Kean’s 
laboratory on the same day as collection at ambient temperature. 
 
2. 16-32 mL into CPT tubes for viral specific assays as feasible based on patient weight.  
a. Cell Processing Tubes (CPT) are tubes used for the collection of blood for 
cryopreservation of peripheral blood mononuclear cells (PBMCs) for functional T 
cell Assays and plasma preservation. 
b. Collected blood should be shipped by priority overnight express to Dr. Kean’s 
laboratory on the same day as collection at ambient temperature. 
 
Shipping 
 
All samples will be shipped at room temperature by overnight mail to Dr. Leslie Kean’s 
laboratory at Emory University. No refrigeration is necessary. Blood samples must be protected 
in shipping containers inside Styrofoam boxes to minimize temperature change, because the 
cells must be tested in a viable condition. Samples will be sent via FedEx and be sent with a 
specimen transmittal form. Label sample with Patient ID, Protocol ID, and time of sample 
collection. 
 
Prior to shipping, send an email to Aneesah Garrett:  aneesah.d.polnett@emory.edu notifying 
her of an impending shipment.  Include the Fed Ex tracking number in the email. Aneesah’s 
phone #: 404-727-4738 
 
Copy Jennifer Cheeseman  (jcheese@emory.edu) on the email to Aneesah.   
 
Samples are to be shipped to the Dr. Leslie Kean’s Laboratory at: 
 
 Emory Transplant Center Biorepository 
                                   101 Woodruff Circle, #5014-WMB 
                                   Emory University 
                                   Atlanta, GA 30322 
 
Ship each specimen individually by overnight air freight on the day of its collection. Maximum 
time from sample collection to shipment should be no greater than 24 hours. Store and ship 
samples at room temperature. Send fresh. 
 
 
 
 
 
 
 
 
 
 
 
 
 

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Appendix VI –Grade 3 Adverse Events Expected for Transplant Recipient 
 
 
Cardiac 
Infection 
Hypertension 
Colitis, infectious 
Constitutional Symptoms 
Febrile Neutropenia 
Fatigue 
Infection 
Weight gain 
Opportunistic infection 
Gastrointestinal 
Metabolic/ Laboratory 
Anorexia 
ALT 
Colitis 
AST 
Diarrhea 
GGT 
Dysphagia 
Hypomagnesemia 
Enteritis 
Hypophosphatemia 
Esophagitis 
Hypokalemia 
Gastritis 
Hyponatremia 
Ileus, GI 
Musculoskeletal/ Soft Tissue 
Mucositis/ stomatitis (clinical exam) 
Muscle weakness 
Mucositis/ stomatitis (functional) 
Osteonecrosis (avascular necrosis) 
Nausea 
Neurology 
Typhlitis 
Mood Alteration (agitation, anxiety, depression) 
Vomiting 
Pain 
Growth and Development 
Sexual/ Reproductive function 
Growth velocity 
Irregular menses 
Hemorrhage/ Bleeding 
Petechiae/ purpura 
 
 

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