Clinical Investigation to Evaluate the New Health Sciences Hemanext® Oxygen Reduction System for Leukoreduced Red Blood Cells With CP2D Anticoagulant and AS-3 Additive - Pivotal Trial
Phase: PHASE2
Conditions: Whole Blood Donation and Leukoreduction
Sponsor: Hemanext

 
Study Protocol 
Protocol : 
CLIN-0001 
161 First St, Suite 1A 
Cambridge MA 02142 USA 
Clinical Investigation to Evaluate the New Health 
Sciences Hemanext® Oxygen Reduction System 
for Leukoreduced Red Blood Cells with CP2D 
Anticoagulant and AS-3 Additive - Pivotal Trial 
Date: 
28 Nov 2017 
Author:  Michael Dioguardi 
 
 
Page 1 of 65 
 
 
Sponsor: 
New Health Sciences Inc. 
 
161 First St. Suite 1A  
 
Cambridge, MA 02142 
Protocol Number:  
CLIN-0001 
Sponsor Contact: 
 
Michael Dioguardi, Director of Research and Clinical Affairs 
New Health Sciences Inc. 
 
 
161 First St. Suite 1A 
 
 
Cambridge, MA 02142 
 
Principal Investigators/sites: 
 
Site A:  
Fay Bernadette West, M.D. 
 
 
Medical Director, American Red Cross Mid-Atlantic Blood Services 
American Red Cross Mid-Atlantic Research Facility 
400 Gresham Drive, Suite 100 
Norfolk, VA  23507 
 
 
Site B:  
Waseem Anani, M.D. 
 
 
Associate Medical Director 
 
 
Blood Center of Wisconsin 
638 N. 18th Street 
Milwaukee, WI 53233 
 
 
Site H: 
Jose A. Cancelas-Perez, M.D., Ph.D. 
Deputy Director, Research Division Director and Medical Director 
for Cell Therapies 
Hoxworth Blood Center 
University of Cincinnati 
3130 Highland Avenue 
Cincinnati, OH 45267-0055 
 
 
 
 
 
 

 
Study Protocol 
Protocol : 
CLIN-0001 
161 First St, Suite 1A 
Cambridge MA 02142 USA 
Clinical Investigation to Evaluate the New Health 
Sciences Hemanext® Oxygen Reduction System 
for Leukoreduced Red Blood Cells with CP2D 
Anticoagulant and AS-3 Additive - Pivotal Trial 
Date: 
28 Nov 2017 
Author:  Michael Dioguardi 
 
 
Page 2 of 65 
 
 
Revision History 
REVISION 
DESCRIPTION 
v. 1.0 
Final Draft 
v. 1.1 
Draft with changes in response to FDA conditional IDE approval 
v. 1.2 
Changes to add FDA requested rejuvination at end of storage and clarify 
protocol for sites and CRF design.   

 
Study Protocol 
Protocol : 
CLIN-0001 
161 First St, Suite 1A 
Cambridge MA 02142 USA 
Clinical Investigation to Evaluate the New Health 
Sciences Hemanext® Oxygen Reduction System 
for Leukoreduced Red Blood Cells with CP2D 
Anticoagulant and AS-3 Additive - Pivotal Trial 
Date: 
28 Nov 2017 
Author:  Michael Dioguardi 
 
 
Page 3 of 65 
 
 
Table of Contents 
Table of Contents .......................................................................................................................................... 3 
Table of Tables ............................................................................................................................................. 5 
Table of Figures ............................................................................................................................................ 5 
2.0 
Introduction / Background ................................................................................................................. 6 
2.1 
Background ................................................................................................................................... 6 
2.2 
Product Information ....................................................................................................................... 6 
2.2.1 
Investigational Product .............................................................................................................. 6 
Figure 4: Diffusion Mechanism for O2 Removal ................................................................................... 10 
 
Figure 5: Hemanext Process ........................................................................................................... 10 
2.3 
Previous clinical experience ........................................................................................................ 11 
3.0 
Purpose / Scope .............................................................................................................................. 11 
4.0 
Safety / Regulatory Requirements .................................................................................................. 11 
4.1 
Rationale for Human Use ............................................................................................................ 11 
4.2 
Regulatory Requirements ........................................................................................................... 12 
5.0 
Study Design / Analysis .................................................................................................................. 13 
5.1 
Study Design ............................................................................................................................... 13 
5.2 
Study Primary Endpoints and Acceptance Criteria ..................................................................... 14 
5.3 
Donor/Data Criteria ..................................................................................................................... 14 
5.3.1 
Sample Size ............................................................................................................................ 14 
5.3.2 
Intended Study Enrollment ...................................................................................................... 14 
5.3.3 
Inclusion/Exclusion Criteria ..................................................................................................... 15 
5.3.4 
Donor Withdrawal Criteria ....................................................................................................... 17 
5.3.5 
Evaluable and Non-Evaluable Whole Blood Collections ........................................................ 17 
5.4 
Statistical Analysis Plan .............................................................................................................. 19 
6.0 
Test Material and Study Methodology ............................................................................................ 20 
6.1 
Test Material................................................................................................................................ 20 
6.1.1 
Product Storage, Accountability and Disposition .................................................................... 20 
6.2 
Methodology ................................................................................................................................ 20 
6.2.1 
Tare Weight Measurements .................................................................................................... 20 
6.2.2 
Donor Selection, Consent and Enrollment Rate ..................................................................... 21 
6.2.3 
Study Donors:  Assigning to Study Arm and Randomization to Study Product ...................... 21 
6.2.4 
Whole Blood Collections ......................................................................................................... 22 
6.2.5 
Leukoreduction of Whole Blood Units ..................................................................................... 22 
6.2.6 
Slow Filtrations ........................................................................................................................ 22 
6.2.7 
Whole Blood Processing and Preparation of PRBC ............................................................... 22 
6.2.8 
Blood Product Sampling .......................................................................................................... 22 
6.2.9 
Laboratory Storage and Testing Methods ............................................................................... 23 
6.2.10 
Radiolabeled Red Cell Procedures ..................................................................................... 24 
6.2.11 
Blood Product Disposition ................................................................................................... 24 
6.2.12 
Endpoint Calculations ......................................................................................................... 24 

 
Study Protocol 
Protocol : 
CLIN-0001 
161 First St, Suite 1A 
Cambridge MA 02142 USA 
Clinical Investigation to Evaluate the New Health 
Sciences Hemanext® Oxygen Reduction System 
for Leukoreduced Red Blood Cells with CP2D 
Anticoagulant and AS-3 Additive - Pivotal Trial 
Date: 
28 Nov 2017 
Author:  Michael Dioguardi 
 
 
Page 4 of 65 
 
 
6.2.13 
RBC Filtration Recovery Calculation ................................................................................... 26 
6.2.14 
Hemolysis Calculation ......................................................................................................... 26 
6.2.15 
Study Activities .................................................................................................................... 26 
7.0 
Study Risks and Safety Reporting .................................................................................................. 43 
7.1 
Analysis of Risks ......................................................................................................................... 43 
7.1.1 
Test System ............................................................................................................................ 43 
7.1.2 
Control System ........................................................................................................................ 43 
7.1.3 
Risk Assessment of Whole Blood Donation ............................................................................ 43 
7.1.4 
Anemia .................................................................................................................................... 43 
7.1.5 
Risk to Those Conducting the Research ................................................................................ 44 
7.1.6 
Radiation Exposure ................................................................................................................. 44 
7.1.7 
Receiving Allogeneic Blood ..................................................................................................... 45 
7.1.8 
Study Donor Benefits .............................................................................................................. 45 
7.2 
Adverse Events ........................................................................................................................... 45 
7.2.1 
Monitoring ................................................................................................................................ 45 
7.2.2 
Definitions ................................................................................................................................ 46 
8.0 
Study Sites ...................................................................................................................................... 49 
8.1 
Study Sites .................................................................................................................................. 49 
8.2 
Central Laboratory ...................................................................................................................... 49 
9.0 
Data Management ........................................................................................................................... 49 
9.1 
Case Report Forms ..................................................................................................................... 49 
9.2 
Monitoring .................................................................................................................................... 50 
9.3 
Protocol Deviations ..................................................................................................................... 50 
10.0 
Data Use ......................................................................................................................................... 50 
10.1 
Test Report .................................................................................................................................. 50 
10.2 
Confidentiality .............................................................................................................................. 51 
10.3 
Positive TTD Results – Result Reporting Requirements and Unit Disposition ........................... 51 
10.4 
Publication ................................................................................................................................... 51 
10.5 
Anticipated Study Schedule ........................................................................................................ 51 
11.0 
Responsibilities ............................................................................................................................... 51 
12.0 
References ...................................................................................................................................... 52 
13.0 
Appendices...................................................................................................................................... 52 
13.1 
Study Flow Chart ......................................................................................................................... 52 
13.2 
Control Product/Leukotrap System – Instructions for Use .......................................................... 52 
13.3 
Investigational Product/Hemanext RBC Processing System – Instructions for Use .................. 60 
 
 
 

 
Study Protocol 
Protocol : 
CLIN-0001 
161 First St, Suite 1A 
Cambridge MA 02142 USA 
Clinical Investigation to Evaluate the New Health 
Sciences Hemanext® Oxygen Reduction System 
for Leukoreduced Red Blood Cells with CP2D 
Anticoagulant and AS-3 Additive - Pivotal Trial 
Date: 
28 Nov 2017 
Author:  Michael Dioguardi 
 
 
Page 5 of 65 
 
 
Table of Tables 
Table 1: Prior HemanextTM Studies ............................................................................................................. 12 
Table 2:  HemanextTM Clinical Trial Design Summary .............................................................................. 13 
Table 3:  Inclusion/Exclusion criteria ........................................................................................................... 16 
Table 4:  Non-analysis-related data collection ............................................................................................ 29 
Table 5:  In vitro specimen acquisition and analyses ................................................................................. 30 
  
 
Table of Figures 
Figure 1:  Investigational Product – Hemanext Processing System Assembly ............................................ 7 
Figure 2: Investigational Product, ORB components .................................................................................... 8 
Figure 3: Investigational Device, HSB Components ..................................................................................... 9 
Figure 4:  RBC Recovery calculations (Standard and Alternate equations) ............................................... 26 
Figure 5:  Hemolysis calculation ................................................................................................................. 26 
 
 
 

 
Study Protocol 
Protocol : 
CLIN-0001 
161 First St, Suite 1A 
Cambridge MA 02142 USA 
Clinical Investigation to Evaluate the New Health 
Sciences Hemanext® Oxygen Reduction System 
for Leukoreduced Red Blood Cells with CP2D 
Anticoagulant and AS-3 Additive - Pivotal Trial 
Date: 
28 Nov 2017 
Author:  Michael Dioguardi 
 
 
Page 6 of 65 
 
 
2.0 
Introduction / Background 
2.1 
Background 
The primary goal of New Health Sciences, Inc. is to improve red cell storage through 
novel storage methods.  Based on our review of the pertinent literature, there is 
substantial evidence suggesting that prolonged exposure to oxygen during storage 
results in oxidative damage to the red blood cells leading to decreased therapeutic 
potential.  Therefore, removal of oxygen from red blood cell products prior to storage has 
potential to preserve the cells in a more physiologically relevant state.  
Currently, NHSi has focused on the design and development of a dual compartment bag 
system designated as the Hemanext Red Blood Cell Processing System.  After standard 
processing of donated whole blood units into leukoreduced packed red blood cells (LR-
RBCs) with the appropriate additive solutions, the LR-RBCs would then be placed in the 
oxygen reduction bag (ORB) which allows for the rapid diffusion of oxygen out of the 
blood, through a sterile, oxygen-permeable membrane, and into iron-based oxygen 
sorbents.  After processing, the blood is transferred again from the ORB into the 
Hemanext storage bag (HSB) which will preserve the anaerobic state of the LR-RBC 
product for the duration of cold storage.   
NHSi has conducted preliminary storage tests to ascertain the effects of anaerobic 
storage on overall blood health in various storage solutions.  The research team has 
focused primarily on percent hemolysis, which is mandated by the FDA to remain below 
1% for the duration of storage, as well as ATP and 2,3-DPG levels.   
 
2.2 
Product Information  
2.2.1 Investigational Product 
The Hemanext Red Blood Cell Processing System is a blood storage device that 
reduces oxygen and carbon dioxide from leukocyte reduced red blood cells in 
additive solution.  The Hemanext System is an assembly of two disposables, the 
Oxygen Reduction Bag (ORB) and the Hemanext Storage Bag (HSB). See 
Figure 1 below for the concept configuration. 
 
 
 
 
 
 
 
 
 
 
 
 

 
Study Protocol 
Protocol : 
CLIN-0001 
161 First St, Suite 1A 
Cambridge MA 02142 USA 
Clinical Investigation to Evaluate the New Health 
Sciences Hemanext® Oxygen Reduction System 
for Leukoreduced Red Blood Cells with CP2D 
Anticoagulant and AS-3 Additive - Pivotal Trial 
Date: 
28 Nov 2017 
Author:  Michael Dioguardi 
 
 
Page 7 of 65 
 
 
Figure 1:  Investigational Product – Hemanext Processing System Assembly 
 
 
 
The ORB consists of the following elements: (a) an inner bag; (b) barrier bag; (c) 
spacer and (d) quilt of 9 sorbent sachets.  The inner bag holds the AS-3 LR RBC 
and is housed in an outer barrier bag.  The barrier bag ensures that there is no 
ingress of oxygen into the ORB during or post O2/CO2 reduction.  The inner bag is 
physically separated from the barrier bag by polyester fabric elements embedded 
into the outer surface of the inner bag.  Materials of construction of the inner bag 
ensure that oxygen released from the LR RBC is optimally transferred to the 
surrounding space. The sorbent quilt ensures that oxygen released from the LR 
RBC is absorbed.  The spacer (perforated patterned polyethylene stabilizer) is 
strategically placed in the space between the inner bag and the sorbent quilt to 
ensure that all of the surface area of the inner bag is clear and able to transfer 
oxygen. The oxygen indicator is designed to change color if exposed to oxygen. 
The ORB has tubing to allow sterile docking of the LR RBC bag.  The ORB is 
connected to the ASB bag via integral tubing.  The connections facilitate the 
transfer of the oxygen reduced LR RBC blood.  See Figure 2 below for the concept 
configuration. 
 
 
 
 

 
Study Protocol 
Protocol : 
CLIN-0001 
161 First St, Suite 1A 
Cambridge MA 02142 USA 
Clinical Investigation to Evaluate the New Health 
Sciences Hemanext® Oxygen Reduction System 
for Leukoreduced Red Blood Cells with CP2D 
Anticoagulant and AS-3 Additive - Pivotal Trial 
Date: 
28 Nov 2017 
Author:  Michael Dioguardi 
 
 
Page 8 of 65 
 
 
 
Figure 2: Investigational Product, ORB components 
 
 
 
The HSB consists of the following elements: (a) inner bag; (b) barrier bag; (c) 
spacer and (d) single sorbent sachet.  The materials of construction of the HSB 
ensure that it is capable of maintaining the appropriate low oxygen environment 
for additive stored LR RBC in refrigerated environments for 42 days. The inner bag 
holds the blood and is housed inside the oxygen impermeable barrier bag.  A 
sorbent is strategically placed in the space, between the inner and outer bags, to 
ensure that low oxygen levels are maintained post reduction by the HSB. See 
Figure 3 below for the concept configuration. 
 
 
 
 
 
 
 
 
 
 
 
 
O2 Indicator 
Barrier Bag 
Inner Bag 
Spacer 
Sorbent Quilt 

 
Study Protocol 
Protocol : 
CLIN-0001 
161 First St, Suite 1A 
Cambridge MA 02142 USA 
Clinical Investigation to Evaluate the New Health 
Sciences Hemanext® Oxygen Reduction System 
for Leukoreduced Red Blood Cells with CP2D 
Anticoagulant and AS-3 Additive - Pivotal Trial 
Date: 
28 Nov 2017 
Author:  Michael Dioguardi 
 
 
Page 9 of 65 
 
 
Figure 3: Investigational Device, HSB Components 
 
 
 
The LR RBC unit bag is connected using an FDA cleared, sterile docking device 
to the ORB. The sorbent quilt between the inner bag and the barrier bag creates 
an O2/CO2 starved environment. LR RBC are then transferred by gravity to the 
ORB inner bag. Once the full unit volume is in the ORB inner bag; the Hemanext 
system is transferred to a tray on an agitator at room temperature (22+2°C). Its 
contents are evenly distributed, creating a large thin layer of LR RBC contacting 
the inner surface of the ORB inner bag. The agitator moves in a side to side motion 
causing the LR RBC to travel within the inner bag in a wave-like motion at the 
same rate as the shaker, maintaining a constant agitation of RBC and additive 
solution. Solution in direct contact with the inner bag surface releases oxygen 
through the material and into the O2/CO2-starved environment between the inner 
bag and barrier bag of the ORB. Maintaining this agitation of red blood cells and 
residual plasma\additive solution ensures that oxygen saturated RBCs that are 
furthest from the walls of the bag exchange oxygen molecules with low oxygenated 
solution closest to the bag surface, which in turn lose the gained oxygen by 
constant exposure to the oxygen-starved environment on the outer side of the ORB 
inner bag. The O2/CO2 absorption rate is characteristic of the oxygen reduction 
container material, exchange surface area, volume of RBC and agitation. At the 
end of the exposure time of 3 hours, the resulting deoxygenated LR RBC is 
transferred to the HSB in the kit. The HSB utilizes the same principles as the ORB 
to preserve a low oxygen and CO2 environment within its contents. It utilizes a 
sorbent to maintain this environment and preserve the blood at low O2 and CO2 
O2 Indicator 
Barrier Bag 
Sorbent Sachet 
Spacer 
Inner Bag 

 
Study Protocol 
Protocol : 
CLIN-0001 
161 First St, Suite 1A 
Cambridge MA 02142 USA 
Clinical Investigation to Evaluate the New Health 
Sciences Hemanext® Oxygen Reduction System 
for Leukoreduced Red Blood Cells with CP2D 
Anticoagulant and AS-3 Additive - Pivotal Trial 
Date: 
28 Nov 2017 
Author:  Michael Dioguardi 
 
 
Page 10 of 65 
 
 
levels for its storage duration of up to 42 days. The HSB is also a transfusion ready 
device. 
 
 
Figure 4: Diffusion Mechanism for O2 Removal 
 
 
 
 
Figure 5: Hemanext Process 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Agitator 
LR RBC 
Oxygen 
Reduction 
Bag (ORB) 
Oxygen 
Reduction 
Bag (ORB) 
Hemanext 
Storage Bag 
(HSB) 
STEP 1 – Sterile 
Transfer LR RBC to 
ORB 
STEP 2 – Agitate in 
commercial shaker 
STEP 3 – Transfer to 
HSB and store for 
use 
3 HOURS 
Permeable Membrane  

 
Study Protocol 
Protocol : 
CLIN-0001 
161 First St, Suite 1A 
Cambridge MA 02142 USA 
Clinical Investigation to Evaluate the New Health 
Sciences Hemanext® Oxygen Reduction System 
for Leukoreduced Red Blood Cells with CP2D 
Anticoagulant and AS-3 Additive - Pivotal Trial 
Date: 
28 Nov 2017 
Author:  Michael Dioguardi 
 
 
Page 11 of 65 
 
 
2.3 
Previous clinical experience 
Two research studies were conducted to confirm the readiness of the Hemanext RBC 
processing system for clinical trials in the United States (see table 1 below).   
An initial pool and split study was performed with the partnership of Rhode Island Blood 
Center for donor collection.  The storage data was measured internally in NHSI’s Blood 
Lab.  Key blood quality metrics (such as ATP and 2,3 DPG) compared favorably for the 
experimental product to the control product.   
After a final product design was reached, a study was conducted independently at the 
American Red Cross Norfolk lab.  The study increased the hold time to 12 hours and 
was designed to be a pilot of the experimental arm of this protocol.  All acceptance 
criteria were met, and results similar to internal testing were collected.  In addition, RBC 
Morphology was equivalent, it was confirmed that Hemanext may be ATP/DPG 
rejuvenated at 42 days, and oxygenation dissociation curves matched the DPG profile of 
Hemanext. 
 
3.0 
Purpose / Scope 
This trial will serve to generate data sufficient to evaluate the in vitro and in vivo 
performance of the Hemanext product using Leukoreduced Red Blood Cells generated 
under conditions of 12-hour room temperature hold. 
 
The objective of this clinical study is to evaluate CP2D/AS-3 leukocyte-reduced RBC 
stored anaerobically for 42 days after deoxygenation with the Hemanext Storage 
System.  In addition to 24-hour recovery and hemolysis, a list of standard blood quality 
parameters will be measured.   
It is expected, based on these data, that Hemanext processing will maintain 
physiological levels of these blood quality parameters (such as 2,3 DPG). 
 
4.0 
Safety / Regulatory Requirements   
4.1 
Rationale for Human Use 
The human use portions of this study are whole blood collection, and the measurement 
of autologous, in vivo, radiolabeled 24-hour red cell recovery and survival of red blood 
cells stored in AS-3® additive.  All other study procedures involve in vitro bench testing 
only.   
While in vitro testing provides an estimate of the in vivo red blood cell recovery and 
survival, the FDA requires in vivo studies to establish efficacy of a blood storage system; 
therefore autologous, radiolabeled, in vivo red cell recoveries will be performed in this 
study.   
 
See section 7.1 for additional information regarding study-associated risks. 
 

 
Study Protocol 
Protocol : 
CLIN-0001 
161 First St, Suite 1A 
Cambridge MA 02142 USA 
Clinical Investigation to Evaluate the New Health 
Sciences Hemanext® Oxygen Reduction System 
for Leukoreduced Red Blood Cells with CP2D 
Anticoagulant and AS-3 Additive - Pivotal Trial 
Date: 
28 Nov 2017 
Author:  Michael Dioguardi 
 
 
Page 12 of 65 
 
 
 
 
Table 1: Prior HemanextTM Studies  
Study ID 
(Year 
Initiated) 
Study Name 
Purpose 
Number of 
Subjects 
Outcome 
RESPR27 
(2016) 
Hemanext Processing 
System with 42-day 
Storage and in-vitro 
Characterization of 
Resultant Oxygen 
Reduced, 
Leukoreduced, Red 
Blood Cells in AS-3 
vs. Control.   
The purpose of this 
pilot study is to 
generate data of the 
current design of the 
Hemanext Processing 
System (consisting of 
an oxygen reduction 
bag “ORB” and an 
anaerobic storage bag 
“HSB”, 60” tubing, y-
connector and 2 
ratchet clamps) and 
Hemanext process.   
20 donors.  10 
pools of two 
donors.   
Hemanext 
O2/CO2 reduced 
RBC meet all in 
vitro acceptance 
criteria.   
RESPR34 
(2017)  
12-hour Processing 
with The Hemanext 
Red Blood Cell 
Processing System 
with 42-day Storage 
and in-vitro 
Characterization of 
Resultant Oxygen 
Reduced, 
Leukoreduced, Red 
Blood Cells in 
CP2D/AS-3 
Confirm the feasibility 
of the current design of 
the HEMANEXT RBC 
PROCESSING 
SYSTEM and 
Hemanext process for 
preparing RCC in 12 
hours from blood 
donation.   
Obtain P50 values that 
correspond with DPG 
levels and rejuvenation 
data on the post Day-42 
stored products to show 
that Hemanext process 
does not compromise 
the ability of Rejuvesol 
to increase ATP and 
DPG 
levels 
in 
anticipation 
of 
FDA 
pivotal study. 
 
10 donors 
Hemanext met 
acceptance 
criteria, P50 
tracked 2,3 DPG, 
and Hemanext 
demonstrated 
ability to be 
ATP/DPG 
rejuvenated. 
 
4.2 
Regulatory Requirements 
The clinical investigation shall be conducted according to FDA regulations (21 CFR), as 
applicable.  
 

 
Study Protocol 
Protocol : 
CLIN-0001 
161 First St, Suite 1A 
Cambridge MA 02142 USA 
Clinical Investigation to Evaluate the New Health 
Sciences Hemanext® Oxygen Reduction System 
for Leukoreduced Red Blood Cells with CP2D 
Anticoagulant and AS-3 Additive - Pivotal Trial 
Date: 
28 Nov 2017 
Author:  Michael Dioguardi 
 
 
Page 13 of 65 
 
 
This study will be conducted under IRB (Institutional Review Board) approvals. All 
potentially eligible donors will read, have the opportunity to ask questions on, and sign 
Informed Consent Forms approved by the respective IRB prior to any study related 
procedures. 
 
5.0 
Study Design / Analysis 
5.1 
Study Design 
In vitro and in vivo performance of O2/CO2 reduced red blood cells produced with the 
Hemanext System will be used to demonstrate the acceptability the final product for 
clearance.  To accomplish this, the study will require a total of 100 study donors.  The 
study entails a randomized, paired, 2-x-2 crossover design where every study donor (n = 
100) who completes the study will donate a total of two whole blood units with individual 
units being donated at least 56 days (8 weeks) apart.  One unit will be used as the test 
and the other unit will be used for the control.  The order in which the IP and the CP will 
be used to collect, filter and store the whole blood and appropriate blood products (within 
the context of the crossover design) will be randomized. 
 
The full clinical investigation is summarized in  
Table 2.  See the study’s flow 
diagram in Section 13.1 for an additional reference. 
 
 
Table 2:  HemanextTM Clinical Trial Design Summary 
Hemanext Pivotal Clinical Trial Summary 
 
‘n’ (evaluable) * 
93 
Pre-filtration storage & filtration conditions 
20-24°C 
Randomized, 
Paired,  
Crossover 
Investigational unit filtration 
<8 hours + up to 4 
hours processing 
Control unit filtration  
< 8 hours 
In vivo subgroup study  (‘n’) 
√ (24-28)  
Post-filtration assessments 
√ 
Baseline assessments 
√ 
Post-storage assessments 
 
√ 
 
 
 
 
 

 
Study Protocol 
Protocol : 
CLIN-0001 
161 First St, Suite 1A 
Cambridge MA 02142 USA 
Clinical Investigation to Evaluate the New Health 
Sciences Hemanext® Oxygen Reduction System 
for Leukoreduced Red Blood Cells with CP2D 
Anticoagulant and AS-3 Additive - Pivotal Trial 
Date: 
28 Nov 2017 
Author:  Michael Dioguardi 
 
 
Page 14 of 65 
 
 
5.2 
Study Primary Endpoints and Acceptance Criteria 
The primary endpoints that will be evaluated in this clinical trial are RBC mass recovery, 
hemolysis and 24-hour in vivo red cell recovery.  The endpoint-associated study 
acceptance criteria are listed below:  
• 
A one-sided 95% lower confidence limit for the true proportion of packed RBC 
units with hemolysis at day 42 of < 1% is > 95%. 
• 
The mean 24-hour, post-transfusion, in vivo red cell recovery of at least 75% with 
a standard deviation of ≤ 9%, and the lower limit of a one-sided 95% confidence 
interval for the population proportion of successes is ≥ 70%. 
• 
Red Blood cell recovery > 85%; with 95% confidence that at least 95% of units 
are > 85%.    
 
5.3 
Donor/Data Criteria 
5.3.1 Sample Size 
The sample sizes described within the protocol were selected to demonstrate 
meeting study primary endpoints that conform to the FDA’s 95-95 rule (95% 
confidence and 95% reliability).  For successful endpoint outcomes, the study 
requires meeting the acceptance criteria in 92 of 93 test units per test condition. 
Therefore, 93 evaluable study donors will complete the study.  In the event of two 
(2) donors/units with unmet acceptance criterion occurring out of ninety-three 
(93) evaluable procedures (i.e., within one or more study arm(s) for one or more 
acceptance criteria/primary endpoints), the study will be discontinued. 
 
5.3.2 Intended Study Enrollment 
Definitions: 
• 
Study Donor: an individual who has signed the informed consent form 
and is enrolled in the study. 
• 
Evaluable Study Donor: an individual who is enrolled in the study, meets 
the inclusion criteria, and completes two Day 42 test periods.  
This clinical trial requires enrolling a total of 93 evaluable study donors.  
However, due to the study design which incorporates a 2x2 crossover and the in 
vivo RBC recovery and survival substudy, the study will purposely enroll up to 
100 study donors to account for study withdrawal, loss to follow-up and/or study 
donors who meet study criteria for the first donation of the crossover, but fail to 
meet study criteria for the second donation.   
The study sites will make efforts to balance gender during screening. 
Once enrolled, if a study donor’s status should change from evaluable to non-
evaluable per protocol definitions (see section 5.3.5), or if the study donor should 
discontinue follow-up (e.g., either voluntarily withdraw from the trial or become 

 
Study Protocol 
Protocol : 
CLIN-0001 
161 First St, Suite 1A 
Cambridge MA 02142 USA 
Clinical Investigation to Evaluate the New Health 
Sciences Hemanext® Oxygen Reduction System 
for Leukoreduced Red Blood Cells with CP2D 
Anticoagulant and AS-3 Additive - Pivotal Trial 
Date: 
28 Nov 2017 
Author:  Michael Dioguardi 
 
 
Page 15 of 65 
 
 
lost to follow-up), then that study donor may be replaced in order to maintain 
intended study enrollment goals.  Such replacement would increase the total 
number of enrolled study donors, but maintain the final number of evaluable 
study donors. 
In instances where up to 100 study donors have been enrolled and all have 
undergone their first donation, consecutive donors will return for their second 
donation until a complete set of 93 matched data pairs have been obtained for 
both in vitro and, when applicable, in vivo parameters.  As a result, it is possible 
that a study donor who donates an evaluable unit of whole blood will not be 
asked to return to provide a second unit of whole blood in instances where a 
complete data set has already been ascertained within the relative study arm. 
Site A is not participating in enrolling donors for the in vivo cohort of the study.  
Site A will target enrollment of 40 evaluable donors for in vitro testing only.  Site 
H will target enrollment 46 evaluable donors for in vitro testing of which 14 will 
also be enrolled for the cohort of in vivo testing.  Site B target enrollment 14 
evaluable donors for both in vivo and in vitro testing.   
 
 
 
Table 3: Target Enrollment by site 
Site 
In vitro 
In 
vivo 
Site A: American Red Cross, 
Norfolk 
40 
0 
Site B: Blood Center of Wisconsin 
14 
14 
Site H: Hoxworth Blood Center 
46 
14 
Total 
100 
28 
 
 
5.3.3 Inclusion/Exclusion Criteria 
The study donor must conform to the criteria listed in Table  at the time of study 
donor selection and must meet study eligibility in order to participate in the study.  
Any deferral criteria will be based on local procedures and SOPs. Note, further, 
that later evaluability determinations (see section 5.3.5) may impact study-eligible 
individuals’ eligibility/evaluability and/or full participation within the study. 
 
 
 
 
 
 

 
Study Protocol 
Protocol : 
CLIN-0001 
161 First St, Suite 1A 
Cambridge MA 02142 USA 
Clinical Investigation to Evaluate the New Health 
Sciences Hemanext® Oxygen Reduction System 
for Leukoreduced Red Blood Cells with CP2D 
Anticoagulant and AS-3 Additive - Pivotal Trial 
Date: 
28 Nov 2017 
Author:  Michael Dioguardi 
 
 
Page 16 of 65 
 
 
 
Table 4:  Inclusion/Exclusion criteria 
Inclusion  
Criteria 
Criteria to qualify for Inclusion 
Reference 
Age 
Study donor must be ≥ 18 years of age. 
Age of legal consent used 
for inclusion criteria. 
Weight 
Study donor must be ≥ 110 pounds. 
AABB Standards for Blood 
Banks and Transfusion 
Services 28th Edition 
Temperature Study donor’s body temperature must be ≤ 37.5°C / 
99.5°F (oral). 
Hemoglobin 
Study donor’s hemoglobin must be ≥12.5 g/dL if female 
and ≥13.0 g/dL if male. 
FDA Standard 21CFR 
640.3(b)(3). Study 
participants must meet 
EITHER hemoglobin or 
hematocrit criteria. 
Hematocrit 
Study donor’s hematocrit must be ≥ 38% if female and 
≥39% if male. 
Donor  
Eligibility 
Study donor must meet all criteria per respective site’s 
Research Blood Donation Record (BDR). 
Site documentation 
Prior 
Donation 
Study donor’s most recent single RBC unit donation 
must have been ≥56 days prior to study donation. 
Study donor’s most recent double RBC unit donation 
must have been ≥ 112 days prior to study donation. 
Only donors who participate in the in vivo portion of 
the study: 
An additional 2-4 weeks between donations will be 
required to ensure washout from prior in vivo 
survival procedures. 
AABB Standards for Blood 
Banks and Transfusion 
Services 28th Edition 
Informed  
Consent 
Study donor must have consented to study participation 
by reviewing and having expressed understanding the 
site-respective IRB–approved informed consent form 
prior to undergoing any study related procedures. 
ISO 14155:2011 
21 CFR 50 
Blood-borne 
Pathogens 
Study donor’s testing results from collected blood does 
not indicate a risk of transfusion-transmitted disease 
(TTD)*. 
N/A 
Adverse  
Events 
Study donors must agree to report adverse events from 
the time of signing the informed consent to twenty-four 
hours following the end of their active study 
involvement. 
N/A 
Pregnancy 
Female study donors must not be pregnant or expected 
to be pregnant.  For donors participating in the in vitro 
portion, this is an interview question only.   
N/A 

 
Study Protocol 
Protocol : 
CLIN-0001 
161 First St, Suite 1A 
Cambridge MA 02142 USA 
Clinical Investigation to Evaluate the New Health 
Sciences Hemanext® Oxygen Reduction System 
for Leukoreduced Red Blood Cells with CP2D 
Anticoagulant and AS-3 Additive - Pivotal Trial 
Date: 
28 Nov 2017 
Author:  Michael Dioguardi 
 
 
Page 17 of 65 
 
 
 
Only female donors who participate in the in vivo portion 
of the study: 
Women of child-bearing age must not be pregnant, as 
determined by a negative pregnancy test prior to each 
re-infusion, or be breastfeeding.  If acceptable by local 
procedures, post-menopausal or surgically sterile 
women may be exempt from the pregnancy testing 
requirement. 
*Study donors’ TTD test results may not be available until several days following enrollment and donation of a 
whole blood unit.  See section 5.3.5 for additional details.  
 
For the purpose of this trial, not meeting any of the inclusion criteria constitutes 
exclusion from the trial (i.e., meeting exclusion criteria). 
 
5.3.4 Donor Withdrawal Criteria 
Any study donor that enrolled in the study can withdraw from the study, 
effectively withdrawing consent, at any time, for any reason, without prejudice or 
consequence.  Study donors are to be informed that they are free to leave the 
study at any time for any reason without prejudice or consequence in the 
informed consent document.  A study donor may withdraw from the study at any 
time following blood donation and thus withdraw permission to evaluate their 
blood product.   
If a study donor withdraws from the study, whether prior to completion of the 
whole blood donation or after completion of the whole blood donation, the data 
collected up through the time of withdrawal (including testing results) will be 
included within the study database.  Withdrawn study donors will be replaced in 
all instances where incomplete data required to calculate acceptance criteria 
result from their withdrawal.  If a study donor withdraws at a point when both the 
whole blood collection and all study analyses are complete, then their withdrawal 
does not impact the data’s entry into the database, their data will be analyzed 
within the full data set and the study donor will not be replaced. 
 
5.3.5 Evaluable and Non-Evaluable Whole Blood Collections 
Collected whole blood’s evaluability status per protocol definition (see section 
5.3.5.1) will determine whether these data are included within the data set that 
will be analyzed towards study endpoints.  Blood donations deemed unevaluable 
per protocol definition will necessitate whole blood sample replacement obtained 
from alternate evaluable study donors such that intended population targets are 
maintained.  
 
5.3.5.1 
Definition of Non-Evaluable Whole Blood Collection 

 
Study Protocol 
Protocol : 
CLIN-0001 
161 First St, Suite 1A 
Cambridge MA 02142 USA 
Clinical Investigation to Evaluate the New Health 
Sciences Hemanext® Oxygen Reduction System 
for Leukoreduced Red Blood Cells with CP2D 
Anticoagulant and AS-3 Additive - Pivotal Trial 
Date: 
28 Nov 2017 
Author:  Michael Dioguardi 
 
 
Page 18 of 65 
 
 
Each of the following issues or occurrences defines a non-evaluable 
whole blood collection. Partial data sets from donors who provide data 
and/or blood donations will be entered into the database and reported 
within descriptive statistics as outlined within the study’s statistical 
analysis plan (SAP). 
• 
TTD:  Data/analyses resulting from blood acquired from donors 
who are subsequently determined to test positive for TTD will 
result in a determination of non-evaluability and will not be 
included within study endpoint analyses.  Upon TTD positive 
determination, further analyses of the blood will cease, the study 
donor will be notified via site-respective procedures, and any 
collected data will be reported as descriptive statistics.  
• 
Slow Collection:  Any whole blood collection taking longer than 
15 minutes to complete will be considered a slow collection and 
will be defined by the point that the cannula on the respective 
system’s Donor Line is broken to initiate blood flow into the Donor 
Bag until the Donor Line is clamped to cease blood flow to the 
Donor Bag at the end of collection.  All whole blood units resulting 
from a slow collection will be non-evaluable.  Information/data 
from study donors from whom the collection is slow per this 
definition will only be included within the demographics section of 
the descriptive statistics. 
• 
Incomplete Collection:  Upon collection of the full unit and prior 
to any sampling, filtration or processing, any collection that is 
stopped prior to reaching the 500 ± 50 mL acceptable volume 
range will be considered unevaluable.  With the exception of 
descriptive statistics, data resulting from incomplete collection 
donations will not be included in study-related analyses. 
• 
Protocol Deviation: Any procedural protocol deviation that the PI 
and/or sponsor determine(s) has the potential to impact (both 
positively and negatively) any acceptance criteria, study endpoint 
and/or analytical study-critical outcomes will result in a 
determination of non-evaluability.  Procedural protocol deviations 
must be documented within the eCRFs and all deviations that 
compromise data validity (i.e., one that impacts evaluability) must 
be reported to the sponsor within 24 hours (see section 9.3).  
Examples of protocol deviation that have the potential to impact 
study outcomes include but are not limited to the following: (1) 
storage deviation (e.g., electrical outage and unknown storage 
conditions, unit left out of refrigeration for >30 minutes, sample 
frozen vs. refrigerated, etc.), (2) incorrect mixing/agitation (e.g., 
unit dropped from height, unit inappropriately mixed prior to 
sampling, excess sample drawn into syringe and quickly re-
injected back into unit, etc.), (3) other deviation (e.g., incorrect 

 
Study Protocol 
Protocol : 
CLIN-0001 
161 First St, Suite 1A 
Cambridge MA 02142 USA 
Clinical Investigation to Evaluate the New Health 
Sciences Hemanext® Oxygen Reduction System 
for Leukoreduced Red Blood Cells with CP2D 
Anticoagulant and AS-3 Additive - Pivotal Trial 
Date: 
28 Nov 2017 
Author:  Michael Dioguardi 
 
 
Page 19 of 65 
 
 
centrifuge speed/duration, other)).  Deviations must further be 
communicated to the respective IRB per local regulations. 
• 
Sickle Cell Trait:  Any whole blood unit in the study from a sickle 
cell trait (SCT) positive study donor will be excluded. 
• 
Grossly Lipemic Specimens:  Any whole blood unit grossly 
evaluated (per visual inspection) as lipemic will be non-evaluable 
relative to only the RBC hemolysis endpoint at Day 42.  Any units 
in question will be reviewed by the PI or designee and if 
considered non-evaluable, the unit will be removed from the study, 
and the study donor may be replaced.   
• 
Positive Bacterial Culture:  This evaluability determination 
pertains only to study donors who participate in the in vivo portion 
of the study.  Should the outcome of the pre-infusion bacterial 
culture (see section 6.2.16.11) indicate that the stored LRRBCs 
are ‘+’ for bacteria, then the unit will be discarded.  As a result, the 
study donor and his/her unit of blood will be non-evaluable and the 
study donor may be replaced. 
 
5.4 
Statistical Analysis Plan 
The data from this study will be analyzed to determine whether the primary endpoints 
and study acceptance criteria (as recommended by FDA and as listed in section 5.2) 
were met.   
In order to assure that 95% confidence in 95% reliability of the of the lower confidence 
level of the units meet the acceptance criteria for the primary endpoints, the sample size 
shall be: 
• 
Ninety-three (93) evaluable procedures in which no more than one (1) failure 
occurs against the acceptance criteria for the primary endpoints. 
 
Secondary analyses, such as differences in populations by site and comparisons 
between arms, will be described in more detail within the statistical analysis plan.  This 
formal SAP will be developed in conjunction but separately from this protocol, and filed 
within the trial master file. 
 
The following, at a minimum, will be presented using descriptive statistics: 
• 
Donor Disposition:  All data on all study donors enrolled in trial including data 
identified as evaluable and non-evaluable donations will be presented.  
• 
Demographics:  Age, gender, weight, height, ethnicity and race will be provided 
for all study donors enrolled in the trial for both evaluable and non-evaluable 
donations.  A comparison to census data will be performed in the final report. 
• 
Collection procedure-related data:  Collection duration and ambient 
temperatures will be provided for both evaluable and non-evaluable donations. 

 
Study Protocol 
Protocol : 
CLIN-0001 
161 First St, Suite 1A 
Cambridge MA 02142 USA 
Clinical Investigation to Evaluate the New Health 
Sciences Hemanext® Oxygen Reduction System 
for Leukoreduced Red Blood Cells with CP2D 
Anticoagulant and AS-3 Additive - Pivotal Trial 
Date: 
28 Nov 2017 
Author:  Michael Dioguardi 
 
 
Page 20 of 65 
 
 
• 
Filtration & processing procedure-related data:  Temperature, head height, 
filtration time, filter time, filter weight, and final collection volumes will be provided 
for evaluable and non-evaluable donations. 
• 
Laboratory data:  Laboratory data will be provided for evaluable and non-
evaluable donations (if applicable). Other than the endpoints detailed in the 
acceptance criteria, the laboratory data will be presented for information only. 
• 
Adverse events:  Expected, unexpected and serious adverse events will be 
reported for all enrolled study donors. 
 
6.0 
Test Material and Study Methodology 
6.1 
Test Material 
 
Disposables directly related to the IP will be labeled as an investigational product. 
 
6.1.1 Product Storage, Accountability and Disposition 
Study product will be supplied by NHSi after the study contract has been 
executed.  Study product may not be used in any capacity until proof of IRB 
approval and IRB-approved informed consent(s) for the study have been 
received by the Sponsor.  Site documentation of 100% accountability of the IP is 
required beginning with receipt of the product until all product has been 
dispensed, destroyed and/or returned to the study sponsor.  A “Study Product 
Receipt and Disposition Log” will be supplied by NHSi in the Clinical Trial Binder 
for documenting each receipt and disposition (i.e., used, discarded or returned) 
of the study product.  The IP are to be stored at room temperature in a secure 
location upon receipt.  Unused product will be returned to the sponsor at the end 
of the trial unless other documented instructions are provided in advance to the 
respective clinical site. 
 
6.2 
Methodology 
6.2.1 Tare Weight Measurements 
During the study initiation visit and prior to enrolling the first study donor, tare 
weight measurements must be determined for the IP.  Tare weight instructions 
and source documents will be supplied within the study’s Regulatory Binder, and 
tare weights will be obtained and confirmed at the individual study sites.  Tare 
weight data must be entered onto the sponsor-provided worksheet(s) and into 
the appropriate eCRF within the study’s EDC system. 
All blood and blood component weight measurements will be gross weight 
measurements.  Each site participating in the clinical trial will either be provided 
with a prepared Tare Kit or will develop a Tare Kit during the SIV.  The Tare Kits 
are to be retained until the completion of the study.   

 
Study Protocol 
Protocol : 
CLIN-0001 
161 First St, Suite 1A 
Cambridge MA 02142 USA 
Clinical Investigation to Evaluate the New Health 
Sciences Hemanext® Oxygen Reduction System 
for Leukoreduced Red Blood Cells with CP2D 
Anticoagulant and AS-3 Additive - Pivotal Trial 
Date: 
28 Nov 2017 
Author:  Michael Dioguardi 
 
 
Page 21 of 65 
 
 
NOTE: The ORB component of the Hemanext RBC Processing system must be 
weighed individually and cannot be a part of a standard Tare Kit.   
 
6.2.2 Donor Selection, Consent and Enrollment Rate 
This study will enroll up to 93 evaluable study donors (see sections 5.3.2, 5.3.3 
and 5.3.5 for details regarding intended enrollment, inclusion/exclusion criteria 
and donor evaluability determinations).  Study donors will be selected from site-
managed databases of known donors as well as new donors who meet study 
eligibility criteria.  Clinical sites may choose to employ IRB-approved 
advertisements as a means to assist recruitment. 
Individuals who express interest in study participation must undergo site-specific 
informed consent processes per local requirements.  Study donors will be 
consented by the respective sites’ PI or appropriate PI delegate(s) prior to 
undergoing any study-related activities or assessments using the respective 
sites’ IRB-approved Informed Consent Form, and will follow consenting 
procedures per local SOP and 21CFR 50.27.   
For the purpose of this study, any study donor who has signed the study’s 
informed consent documentation is considered to be enrolled in the study 
regardless of whether the enrolled individual engages in any study activity 
beyond ICF signing, donates blood, or is evaluable.   
Each study sites’ rate of enrollment will be dependent on limitations resulting 
from both individual study donor’s evaluability and sites’ ability to process the 
required number of evaluable study donors per study arm.  As a result, the 
distribution of evaluable study donors may vary per study arm and per site.   
 
6.2.3 Study Donors:  Assigning to Study Arm and Randomization to Study 
Product 
Study donors must be randomized to either IP or CP prior to their first donation.  
A donor randomized to IP will donate their first whole blood unit to be stored with 
the Investigational Product, and will donate their second whole blood unit to be 
stored with the Control Product.  A study donor randomized to CP will donate 
their first whole blood unit with the Control Product, and will donate their second 
whole blood unit with the Investigational Product.   
The randomization process will be a manual process that will be recorded within 
the Subject Enrollment and Screening Log.  The randomization procedure will be 
based on a form generated wholly with SAS that will provide sequentially listed 
randomized product.    Further details regarding the randomization process and 
associated documentation will be captured within the study’s Operation Manual.  
Documentation relative to randomization will be stored within the study’s 
Regulatory Binder. 
 
 

 
Study Protocol 
Protocol : 
CLIN-0001 
161 First St, Suite 1A 
Cambridge MA 02142 USA 
Clinical Investigation to Evaluate the New Health 
Sciences Hemanext® Oxygen Reduction System 
for Leukoreduced Red Blood Cells with CP2D 
Anticoagulant and AS-3 Additive - Pivotal Trial 
Date: 
28 Nov 2017 
Author:  Michael Dioguardi 
 
 
Page 22 of 65 
 
 
6.2.4 Whole Blood Collections 
After study donors are consented, initial screen procedures have been 
completed, and individual donors have been assigned to their study arm and 
randomized, study donors will donate 500mL ± 10% of whole blood with each 
whole blood donation.  Approximately 20 - 40mL of blood (included within the 
500mL ± 10%) will be collected during phlebotomy for CBC, ABO typing, TTD 
and SCT testing.  The total volume of blood removed from any donor at collection 
will not exceed 550mL. 
Study donors who also participate in the in vivo cohort will have whole blood 
samples of various volumes drawn as part of the cohort.  The in vivo study does 
not require an additional whole blood unit donation.  However, as part of the in 
vivo study, whole blood samples will be drawn on Day 42 (50-65 mL total) and 
additional whole blood samples will be drawn on Day 43 (10 mL) and 5ml for 
each subsequent time point for in vivo survival.  See section 6.2.11 for details. 
 
6.2.5 Leukoreduction of Whole Blood Units 
Each collected whole blood unit will be leukoreduced warm using the 
leukoreduction filter per the IFU for the Haemonetics WB Leukotrap System 
(Appendix 13.2).  The collection set with leukoreduction filter will be used 
according to the instructions for use provided at study initiation with the head 
height specifications included. 
 
6.2.6 Slow Filtrations 
Any filtration with a Bag Empty Time (emptying of the Donor Bag) that is greater 
than one hour will be considered a slow filtration.  Slow filtrations will be 
documented, the Sponsor will be notified immediately and testing will continue to 
completion if the product is stored in < 12 hours for the IP or < 8 hours for the CP 
groups.   
 
6.2.7 Whole Blood Processing and Preparation of PRBC  
As dictated, each leukoreduced whole blood unit will be processed into PRBCs 
and platelet-poor plasma (PPP) using a representative hard-spin centrifugation 
and subsequent aspiration (PRBC/PPP separation).  
The PRBC unit will be mixed with the AS-3 additive per the product’s instructions 
for use.  PPP will not be analyzed, and this plasma will be discarded per Section 
6.2.12. 
 
6.2.8 Blood Product Sampling 
Blood product containers will be thoroughly mixed prior to sampling to ensure a 
representative sample is obtained from a homogeneous blood product.  

 
Study Protocol 
Protocol : 
CLIN-0001 
161 First St, Suite 1A 
Cambridge MA 02142 USA 
Clinical Investigation to Evaluate the New Health 
Sciences Hemanext® Oxygen Reduction System 
for Leukoreduced Red Blood Cells with CP2D 
Anticoagulant and AS-3 Additive - Pivotal Trial 
Date: 
28 Nov 2017 
Author:  Michael Dioguardi 
 
 
Page 23 of 65 
 
 
Thorough mixing of each whole blood unit prior to taking pre-filtration and post-
filtration samples is of critical importance for the calculation of filtration red cell 
recovery.   
All sample aliquots will be taken without damaging the integrity of the test system 
or blood unit.  Evaluation samples from whole blood and RBC units will be taken 
via a sterile connection between a sterile transfer bag to the blood product 
container using an FDA approved sterile connection device and then transferring 
an aliquot to the transfer bag. 
When sampling, care must be taken to ensure that air is not transferred to the 
blood component container from the sample transfer bag.    If a red cell unit is 
removed from cold storage for sampling purposes it must be returned to cold 
storage (1-6°C) within 30 minutes of the time of removal. 
 
6.2.9 Microvascular Analysis 
This study incorporates an evaluation of deformability using an experimental 
assay to characterize red cell deformability called the Microvascular Analyzer 
(MVA).  The MVA measured perfusion of an artificial microvascular network 
under physiological conditions.   Additional details on the procedure for this 
assay are documented in the study operations manual.  This testing will only be 
performed on donors participating in the in vivo portion of the study.   
For the purposes of this study, the assay will be considered optional and inability 
to complete it will not be documented as a protocol deviation.  The Sponsor and 
sites may mutually agree to discontinue the testing at any time should difficulties 
with the equipment be encountered. 
 
6.2.10 Laboratory Storage and Testing Methods 
All study blood products will be stored under standard blood banking conditions.  
All study blood products will be segregated from normal blood product inventory.  
PRBC units are stored for 42 days at 1-6°C.   
All testing will be conducted using laboratory equipment and instruments that are 
in current calibration, according to site standard procedures or using a validated 
scientific method.  Site research laboratory standard procedures for testing 
performed in the study are to be available for inspection by the Sponsor.  When 
study testing is performed by a certified clinical laboratory, copies of current CLIA 
and/or CAP certification must be provided to the Sponsor. 
Refrigerators used to store blood products will be equipped with a temperature 
recorder or linked to a temperature recording system that will alarm and initiate 
contact with site staff when out of the protocol-indicated temperature ranges. 
 
 
 

 
Study Protocol 
Protocol : 
CLIN-0001 
161 First St, Suite 1A 
Cambridge MA 02142 USA 
Clinical Investigation to Evaluate the New Health 
Sciences Hemanext® Oxygen Reduction System 
for Leukoreduced Red Blood Cells with CP2D 
Anticoagulant and AS-3 Additive - Pivotal Trial 
Date: 
28 Nov 2017 
Author:  Michael Dioguardi 
 
 
Page 24 of 65 
 
 
6.2.11 Radiolabeled Red Cell Procedures 
This study incorporates an in vivo cohort to evaluate Day 42 RBC recovery and 
subsequent survival.  Measures to ensure the study donor receives his or her 
own (autologous) radiolabeled red cells begin at donation when the study donor’s 
name, birth date and unique identifying number will be recorded on the donor 
registration form.  Clinical sites must have a standard labeling and handling 
process intended to eliminate errors in linking packed RBC units with the correct 
study donor. 
On the first day of each in vivo cohort (42-days following each donation for in vivo 
cohort participants), female study donors must not be pregnant as determined by 
a negative pregnancy test (whether urine and/or serum pregnancy tests are used 
will be dictated by local procedures; women who are post-menopausal or 
surgically sterile may be exempt from the pregnancy test requirement if allowed 
by local procedures).  The outcome of the pregnancy test will be recorded in both 
the source documentation and EDC. 
RBC survival testing will be performed at 24h (+1h), 48h (+4h), 72h (+4h), 7 days 
(+1day), 14 days (+1day), 21 days (+1day), and 28 days (+1day). 
 
Radiolabeling RBC procedures are derived from the literature (Davey, 1987; 
Heaton, 1992).  However, individual clinical sites will follow specific local labeling, 
infusion, sampling and recovery/mass determination procedures and SOPs.   
 
6.2.12 Blood Product Disposition 
Each whole blood unit collected in the study will generate one unit of RBC in 
additive solution and one unit of PPP. All additive RBC units are to be discarded 
per site procedure after completion of the study following receipt of Sponsor’s 
written consent. 51Cr and 99mTc-labeled RBC samples will be discarded 
following receipt of written permission from the sponsor and will be discarded per 
study site-specific SOP relative to the destruction of radiolabeled biological 
materials.   Written consent will not be required for any unit with a confirmed 
positive infectious disease result.     
 
6.2.13 Endpoint Calculations 
Study sites will perform real-time calculations (RBC mass) and batch testing 
(hemolysis) to determine whether study endpoints meet study criteria and 
specifications.  Clinical sites will employ the formulas that follow to perform real-
time calculations for the purpose of informing the study sponsor quickly when it 
appears that data relative to the study acceptance criteria appear to be out of the 
acceptable range.  However, for the purpose of the database and resulting data, 
the raw values will be entered into the EDC database, and the reported values 
will be calculated separately. 

 
Study Protocol 
Protocol : 
CLIN-0001 
161 First St, Suite 1A 
Cambridge MA 02142 USA 
Clinical Investigation to Evaluate the New Health 
Sciences Hemanext® Oxygen Reduction System 
for Leukoreduced Red Blood Cells with CP2D 
Anticoagulant and AS-3 Additive - Pivotal Trial 
Date: 
28 Nov 2017 
Author:  Michael Dioguardi 
 
 
Page 25 of 65 
 
 
24-hour in vivo RBC recovery and survival will occur per the procedures 
summarized in section 6.2.11, and clinical sites will use the raw data to perform 
their own 24-hour % RBC recovery and survival calculations.  Only % Recovery 
and % Survival values will be entered into the study’s EDC system.  Formulas 
used and the means to perform calculations relative to RBC recovery are site-
specific.  Calculated values will be confirmed per data captured within the eCRF 
relative to confirmation of calculations during monitoring visits. 
Upon performing calculations for recovery, should a calculated value fall outside 
of the protocol-specified endpoint criteria, then the sponsor will be notified as 
soon as possible and within 24 hours. 

 
Study Protocol 
Protocol : 
CLIN-0001 
161 First St, Suite 1A 
Cambridge MA 02142 USA 
Clinical Investigation to Evaluate the New Health 
Sciences Hemanext® Oxygen Reduction System 
for Leukoreduced Red Blood Cells with CP2D 
Anticoagulant and AS-3 Additive - Pivotal Trial 
Date: 
28 Nov 2017 
Author:  Michael Dioguardi 
 
 
Page 26 of 65 
 
 
6.2.14  RBC Filtration Recovery Calculation 
RBC filtration recovery will be calculated via both a standard and the alternative 
method (using post filtration and pre-filtration weights).  These calculations will be 
performed during the data analysis phase of the study based on hematocrit, 
volume and pre-/post filtration weights provided by sites.  To better ensure 
accurate data, it is recommended that unit volumes be calculated from the weight 
of the full product.  The results from both calculations will be reported. 
RBC filtration recovery will be determined according to Figure 4.  The acceptance 
criteria will be determined by the “standard equation” below. 
 
Figure 4:  RBC Recovery calculations (Standard and Alternate equations) 
Standard equation: 
RBC Recovery = (A x Post-filtration unit volume) / (B x Pre-filtration unit volume) x 100 
 
Where: 
‘A’ = Post-Hemanext HCT or RBC count  
 
 
‘B’ = Pre-filtration HCT or RBC count 
 
 
Unit volume (mL) = unit weight (g) / 1.056 (g/mL) 
 
 
1.056 = relative density of whole blood 
Alternate equation: 
RBC Recovery = (Post-filtration net unit weight) / (Pre-filtration net unit weight) x 100                               
 
6.2.15 Hemolysis Calculation 
Supernatant hemoglobin will be tested and % hemolysis calculated.   
% Hemolysis will be determined according to the equation in Figure 5 and using 
Spun Hematocrit only. 
 
Figure 5:  Hemolysis calculation  
% Hemolysis = (Supernatant Hgb (mg/dL) x 0.001) x ((1-(Hct x 0.01)) x 100) 
 
 
                                Total Hgb (g/dL) 
 
 
6.2.16 Study Activities  
Study donors who are eligible for study participation will be assigned to either the 
in vitro portion only or in vivo and in vitro groups.  Study donors will be enrolled 
until the intended total of evaluable units has been collected.  Note that cited 
days reference the number of days post collection.   
 
See Tables 5-7for summary of analyses and data collection. 

 
Study Protocol 
Protocol : 
CLIN-0001 
161 First St, Suite 1A 
Cambridge MA 02142 USA 
Clinical Investigation to Evaluate the New Health 
Sciences Hemanext® Oxygen Reduction System 
for Leukoreduced Red Blood Cells with CP2D 
Anticoagulant and AS-3 Additive - Pivotal Trial 
Date: 
28 Nov 2017 
Author:  Michael Dioguardi 
 
 
Page 27 of 65 
 
 
 
a) After signing the respective consent documentation, collect and 
record data relative to inclusion/exclusion criteria, demographics, 
concomitant medications and medical history. 
b) Collect and record the collection set’s identification numbers. 
c) Collect a whole blood unit using the product per the IFU.  Draw a 
sample of the study donor’s native whole blood from the diversion bag 
and place aliquots in tubes for the indicated analyses. 
d) As soon as reasonably possible after collection, place anticoagulated 
whole blood units into room temperature storage until filtration.  
e) Immediately prior to filtration, weigh, sample and re-weigh each whole 
blood unit.   
f) Initiate room temperature filtration for each unit (5-7 hours post-
collection).  Record filtration-associated data.   
g) Weigh, sample and then re-weigh filtered whole blood units.   
h) As soon as practical following the completion of filtration, the LRWB 
units will be centrifuged and processed at room temperature to 
separate the unit into PRBCs and PPP.  Record centrifugation-
associated data. 
i) 
AS-3 will be mixed with the PRBCs as soon as practical following 
separation from the plasma.   
j) 
Following processing, the additive RBC unit will be weighed. 
k) Plasma will be discarded.  
l) 
The unit will be sampled and weighed. 
m) If the unit is in the CP arm then at this point it will be placed in 
refrigerated storage (1-6℃; within 8 hours of donation). 
n) If the unit is in the IP arm then the AS-3 LR-RBC unit will be 
transferred to the Hemanext ORB (within 8 hours of donation) at 
which point the unit will be weighed to ensure that the volume of the 
unit is less than 350 mL. 
o) The ORB will be placed on a Helmer Platelet Shaker for 3 hours + 15 
minutes.   
p) The O2 reduced LR-RBC will be transferred to the HSB at a head 
height of up to 72 inches at which point the unit will be sampled and 
weighed again prior to refrigerated storage (1-6ºC; within 12 hours of 
donation) or on Day 1 until subsequent testing on Day 21 (+/- 1 day) 
and Day 42 (+ 1 day). 
q) For study participants participating in the in vivo portion of the study, 
refer to the instructions in section 6.2.11.  Dual radiolabeled RBCs will 

 
Study Protocol 
Protocol : 
CLIN-0001 
161 First St, Suite 1A 
Cambridge MA 02142 USA 
Clinical Investigation to Evaluate the New Health 
Sciences Hemanext® Oxygen Reduction System 
for Leukoreduced Red Blood Cells with CP2D 
Anticoagulant and AS-3 Additive - Pivotal Trial 
Date: 
28 Nov 2017 
Author:  Michael Dioguardi 
 
 
Page 28 of 65 
 
 
be infused on Day 42 and post-infusion blood samples will be drawn 
per local procedure.  Study Participants will return to the site 
periodically on Days 43-70, and blood samples will be drawn per local 
procedure such that they are drawn within the time specified in 
Section 6.2.10. 
r) Study donors will return at ≥ 56 days following their first donation. If 
the study donor continues to meet the inclusion/exclusion criteria, 
then they will donate their second unit of whole blood using product 
alternate to their randomization.   
s) Follow steps b) through m) if second unit is in the CP arm and follow 
steps b) through p) if second unit is in the IP arm.. 
 

 
Study Protocol 
Protocol : 
CLIN-0001 
161 First St, Suite 1A 
Cambridge MA 02142 USA 
Clinical Investigation to Evaluate the New Health 
Sciences Hemanext® Oxygen Reduction System 
for Leukoreduced Red Blood Cells with CP2D 
Anticoagulant and AS-3 Additive - Pivotal Trial 
Date: 
28 Nov 2017 
Author:  Michael Dioguardi 
 
 
Page 29 of 65 
 
 
Table 5:  Procedure related data collection 
 
 
Donor 
Collection 
Leukoreduction 
Separation 
Post-Separation 
Donor, Donation & Sample 
Handling  
Data Collection 
Selection 
(Pre)Collection  
Pre-filtration 
Filtration 
Post-filtration 
Centrifugation 
Baseline 
Day 0,21 42 
Storage 
Units /  
Blood Product 
WB  
WB  
WB  
WB  
LRWB  LRWB  
RBC +  
AS 
 
RBC +  
AS 
 
+ anticoagulant 
 
Date(s) 
dd-MMM-yyyy 
X 
 
 
 
 
 
X 
X 
Time(s) 
hh:mm 
X 
 
X 
 
X 
X 
X 
X 
Temperature 
°C / °F 
 
X 
X 
 
X 
X 
X 
 
IFU confirmation 
Yes/No 
 
X 
 
X 
 
 
X 
 
Study Donor 
Informed consent 
Y/N, date, time 
X 
 
 
 
 
 
 
 
Eligibility criteria 
See protocol 
X 
 
 
 
 
 
 
 
Demographics 
See protocol 
X 
 
 
 
 
 
 
 
Concomitant Medications 
Donor-reported 
X 
 
 
 
 
 
 
 
Medical History 
Donor-reported 
X 
 
 
 
 
 
 
 
Donation History 
Donor-reported 
X 
 
 
 
 
 
 
 
Temperature 
°C / °F 
X 
 
 
 
 
 
 
 
Blood pressure 
mmHg 
X 
 
 
 
 
 
 
 
Pulse 
BPM 
X 
 
 
 
 
 
 
 
Donation 
System Manufacturer 
See product 
 
X 
 
 
 
 
 
 
Part #/ Product code/ Lot # 
See product 
 
X 
 
 
 
 
 
 
Collection date 
dd-MMM-yyyy 
 
X 
 
 
 
 
 
 
Needle in 
hh:mm:ss 
 
X 
 
 
 
 
 
 
Needle out 
hh:mm:ss 
 
X 
 
 
 
 
 
 
Adverse Events 
Donor-reported 
 
X 
 
 
 
 
 
 
Unit Handling 
Pre-sample Gross Weight 
g 
 
 
X 
 
X 
 
X 
X 
Post-sample Gross Weight 
g 
 
 
X 
 
X 
 
X 
X 
Filtration start/end time 
hh:mm:ss 
 
 
 
X 
 
 
 
 
Filtration temperature 
°C 
 
 
 
X 
 
 
 
 
Filter weight 
g 
 
 
 
X 
 
 
 
 
Centrifuge details 
See protocol 
 
 
 
 
 
X 
 
 
Centrifuge settings 
See protocol 
 
 
 
 
 
X 
 
 
Hemanext Gross Weight 
g 
 
 
 
 
 
 
X 
 
Hemanext Processing Start 
hh:mm 
 
 
 
 
 
 
X 
 
Hemanext Processing End 
hh:mm 
 
 
 
 
 
 
X 
 
HSB Gross Weight 
g 
 
 
 
 
 
 
 
X 
Refrigerator 
Date/Time/Temp  
standard 
 
 
 
 
 
 
X 
X 
Bacterial culture notification 
(if needed) 
dd-MMM-yyyy 
 
 
 
 
 
 
 
X 
 
 

 
Study Protocol 
Protocol : 
CLIN-0001 
161 First St, Suite 1A 
Cambridge MA 02142 USA 
Clinical Investigation to Evaluate the New Health 
Sciences Hemanext® Oxygen Reduction System 
for Leukoreduced Red Blood Cells with CP2D 
Anticoagulant and AS-3 Additive - Pivotal Trial 
Date: 
28 Nov 2017 
Author:  Michael Dioguardi 
 
 
Page 30 of 65 
 
 
Table 6: Specimen acquisition and analyses for IP arm  
Specimen Acquisition, Analysis & Data 
Collection 
Study Donor 
Selection-  
Pre-collection 
Pre-filtration 
Post-filtration 
Baseline 
(Day 0) 
Post-Hemanext 
(RBC Day0-1) 
During Storage 
(RBC 20-22d) 
Post-Storage 
(RBC 42-43d) 
 
Units / Blood 
Product 
WB  
WB  
LR 
WB  
 
LR-
RBC 
LR RBC + 
AS-3 
 
LR RBC 
+ AS-3 
LR RBC + 
AS-3 
+ Anticoagulant 
 
 
Hct/Hgb – finger stick 
% / g/dL 
x 
 
 
 
 
 
 
RBC Count 
cells x 106/µL 
 
x 
x 
x 
x 
x 
x 
WBC Count 
cells x 103/µL 
 
x 
x 
x 
x 
x 
x 
PLT Count 
cells x 103/µL 
 
x 
x 
x 
x 
x 
x 
Hemoglobin 
g/dL 
 
x 
x 
x 
x 
x 
x 
Hematocrit 
% 
 
x 
x 
x 
x 
x 
x 
MCV 
fL 
 
x 
x 
x 
x 
x 
x 
MCH 
pg 
 
x 
x 
x 
x 
x 
x 
MCHC 
g/dL 
 
x 
x 
x 
x 
x 
x 
RDW 
% 
 
x 
x 
x 
x 
x 
x 
Spun Hematocrit 
% 
 
x 
x 
x 
x 
x 
x 
Sickle Cell Trait 
+ / - 
x 
 
 
 
 
 
 
Viral markers 
+ / - 
x 
 
 
 
 
 
 
ABO w/Rh factor 
A/B/AB/O    +/- 
x 
 
 
 
 
 
 
Supernatant Hgb 
mg/dL 
 
x 
x 
x 
x 
x 
x 
ATP  
µmol/L 
 
 
 
x 
 
x 
x 
2,3-DPG 
µmol/L 
 
 
 
x 
 
x 
x 
ATP Rejuvenation 
µmol/L 
 
 
 
 
 
 
x 
2,3-DPG Rejuvenation 
µmol/L 
 
 
 
 
 
 
x 
rWBC Count 
cells/µL 
 
 
x 
 
 
 
 
SO2 
% 
 
 
 
x 
 
x 
x 
pO2 
mmHg 
 
 
 
x 
 
x 
x 
pCO2 
mmHg 
 
 
 
x 
 
x 
x 
HCO3 
mmHg 
 
 
 
x 
 
x 
x 
pH 
at 37ºC 
 
 
 
x 
 
x 
x 
Potassium 
mmol/L or 
mEq/L 
 
 
 
x 
 
x 
x 
Sodium 
mmol/L or 
mEq/L 
 
 
 
x 
 
x 
x 
Lactate 
mmol/L or 
mg/dL 
 
 
 
x 
 
x 
x 
Glucose 
mmol/L or 
mg/dL 
 
 
 
x 
 
x 
x 
RBC Morphology 
0-100 
 
 
 
x 
 
x 
x 
RBC Deformability 
MVA 
 
 
 
x 
 
x 
x 
RBC Microparticles 
#MV (1000 
beads-1) 
 
 
 
x 
 
 
x 
x 
RBC Metabolomic Panel 
 
 
 
 
x 
 
x 
x 
Day 35-37 Bacterial culture 
+/-  
(& notification) 
 
 
 
 
 
 
x 
Day 42 Pregnancy test  
+/- (♀only) 
 
 
 
 
 
 
x 
Day 42 In vivo recovery 
% 
 
 
 
 
 
 
x 
 In vivo Survival 
% 
 
 
 
 
 
 
x* 
 
 
*Survival Calculated at 48h, 72h, 7d, 14d, 21d, 28d 

 
Study Protocol 
Protocol : 
CLIN-0001 
161 First St, Suite 1A 
Cambridge MA 02142 USA 
Clinical Investigation to Evaluate the New Health 
Sciences Hemanext® Oxygen Reduction System 
for Leukoreduced Red Blood Cells with CP2D 
Anticoagulant and AS-3 Additive - Pivotal Trial 
Date: 
28 Nov 2017 
Author:  Michael Dioguardi 
 
 
Page 31 of 65 
 
 
Table 7: Specimen acquisition and analyses for CP arm  
 
Specimen Acquisition, Analysis & Data 
Collection 
Study Donor 
Selection-  
Pre-collection 
Pre-filtration 
Post-filtration 
Baseline 
(Day 0) 
During Storage 
(RBC 20-22d) 
Post-Storage 
(RBC 42-43d) 
 
Units / Blood 
Product 
WB  
WB  
LR 
WB  
 
LR-
RBC 
 
LR 
RBC + 
AS-3 
LR RBC + 
AS-3 
+ Anticoagulant 
 
 
Hct/Hgb – finger stick 
% / g/dL 
x 
 
 
 
 
 
RBC Count 
cells x 106/µL 
 
x 
x 
x 
x 
x 
WBC Count 
cells x 103/µL 
 
x 
x 
x 
x 
x 
PLT Count 
cells x 103/µL 
 
x 
x 
x 
x 
x 
Hemoglobin 
g/dL 
 
x 
x 
x 
x 
x 
Hematocrit 
% 
 
x 
x 
x 
x 
x 
MCV 
fL 
 
x 
x 
x 
x 
x 
MCH 
pg 
 
x 
x 
x 
x 
x 
MCHC 
g/dL 
 
x 
x 
x 
x 
x 
RDW 
% 
 
x 
x 
x 
x 
x 
Spun Hematocrit 
% 
 
x 
x 
x 
x 
x 
Sickle Cell Trait 
+ / - 
x 
 
 
 
 
 
Viral markers 
+ / - 
x 
 
 
 
 
 
ABO w/Rh factor 
A/B/AB/O    +/- 
x 
 
 
 
 
 
Supernatant Hgb 
mg/dL 
 
x 
x 
x 
x 
x 
ATP  
µmol/L 
 
 
 
x 
x 
x 
2,3-DPG 
µmol/L 
 
 
 
x 
x 
x 
ATP Rejuvenation 
µmol/L 
 
 
 
 
 
x 
2,3-DPG Rejuvenation 
µmol/L 
 
 
 
 
 
x 
rWBC Count 
cells/µL 
 
 
x 
 
 
 
SO2 
% 
 
 
 
x 
x 
x 
pO2 
mmHg 
 
 
 
x 
x 
x 
pCO2 
mmHg 
 
 
 
x 
x 
x 
HCO3 
mmHg 
 
 
 
x 
x 
x 
pH 
at 37ºC 
 
 
 
x 
x 
x 
Potassium 
mmol/L or 
mEq/L 
 
 
 
x 
x 
x 
Sodium 
mmol/L or 
mEq/L 
 
 
 
x 
x 
x 
Lactate 
mmol/L or 
mg/dL 
 
 
 
x 
x 
x 
Glucose 
mmol/L or 
mg/dL 
 
 
 
x 
x 
x 
RBC Morphology 
0-100 
 
 
 
x 
x 
x 
RBC Deformability 
MVA 
 
 
 
x 
x 
x 
RBC Microparticles 
#MV (1000 
beads-1) 
 
 
 
x 
x 
x 
RBC Metabolomic Panel 
 
 
 
 
x 
x 
x 
Day 35-37 Bacterial culture 
+/-  
(& notification) 
 
 
 
 
 
x 
Day 42 Pregnancy test  
+/- (♀only) 
 
 
 
 
 
x 
Day 42 In vivo recovery 
% 
 
 
 
 
 
x 
 In vivo Survival 
% 
 
 
 
 
 
x* 
 
 
*Survival Calculated at 48h, 72h, 7d, 14d, 21d, 28d 

 
Study Protocol 
Protocol : 
CLIN-0001 
161 First St, Suite 1A 
Cambridge MA 02142 USA 
Clinical Investigation to Evaluate the New Health 
Sciences Hemanext® Oxygen Reduction System 
for Leukoreduced Red Blood Cells with CP2D 
Anticoagulant and AS-3 Additive - Pivotal Trial 
Date: 
28 Nov 2017 
Author:  Michael Dioguardi 
 
 
Page 32 of 65 
 
 
6.2.16.1 Detailed Data Collection and Study Analyses 
 
Each data point and analysis is listed here in order to evaluate unmet 
acceptance criteria endpoints.  Refer to the study-specific Operations 
Manual (separate from this protocol) for a concise listing of data collection 
and analyses per the various study scenarios. 
Data in bold, black font will only be collected once in this study, and 
will not be re-collected when the study donor returns for their 
second donation. 
 
6.2.16.2  Study Donor Selection: Consent and Demographics 
 
Consent will be obtained from all study donors prior to the collection of 
demographic information and any study-related screening, tests or blood 
donation.   
Data collected and samples processed during study donor selection are 
as follows: 
• 
Consent obtained (Yes/No)       
 
 
 
• 
Informed Consent date and time        
 
      
• 
Inclusion/Exclusion criteria        
 
 
 
• 
Date of birth       
 
 
 
 
 
• 
Gender       
 
 
 
 
 
 
• 
Race       
 
 
 
 
 
 
• 
Ethnicity        
 
 
 
 
 
• 
Height        
 
 
 
 
 
 
• 
Weight       
 
 
 
 
 
 
• 
Concomitant Medications        
 
 
 
• 
Medical History       
 
 
 
 
 
• 
Donation History        
 
 
 
 
• 
Body temperature        
 
 
 
 
• 
Blood Pressure       
 
 
 
 
 
• 
Pulse       
 
 
 
 
 
 
• 
Hct/Hgb (finger stick)       
• 
Randomization  
 
 
 
 
 

 
Study Protocol 
Protocol : 
CLIN-0001 
161 First St, Suite 1A 
Cambridge MA 02142 USA 
Clinical Investigation to Evaluate the New Health 
Sciences Hemanext® Oxygen Reduction System 
for Leukoreduced Red Blood Cells with CP2D 
Anticoagulant and AS-3 Additive - Pivotal Trial 
Date: 
28 Nov 2017 
Author:  Michael Dioguardi 
 
 
Page 33 of 65 
 
 
For the purpose of this study: 
• 
Concomitant medications will include all study donor self-reported 
medications (e.g., prescription medication and/or over the counter 
medications and nutritional supplements) that the study donor 
takes or has taken within the last 14 days. 
• 
Medical history will include all study donor self-reported chronic 
diseases for which the study donor is currently being treated and 
any surgeries the study donor experienced within the previous 6 
months. 
• 
Donation history will include whether the study donor is a first-time 
donor and, if not, when the study donor’s last donation occurred.  
This data will be considered in the presence of a vaso-vagal-
related adverse event. 
• 
Sites will have the option to perform the hematocrit and/or the 
hemoglobin analysis via whole blood obtained by finger stick.  
Only the analysis performed will be recorded. 
 
6.2.16.3  Collection:  Pre-Collection, Collection and Unit Transfer 
 
Data regarding whole blood donation and study donor sample collection 
and sample processing are listed below.  The collection set’s identifier 
numbers will be recorded.  Routine phlebotomy will be conducted for 
venous access and blood donation per the IFU.  A sample of the study 
donor’s native (non-anti-coagulated) whole blood will be drawn from the 
diversion bag and aliquots will be placed in tubes for the indicated 
analyses.  
Data in bold, black font will only be collected once in this study, and 
will not be re-collected when the study donor returns for their 
second donation. 
 
Items followed by an “*” are analyses performed on the pre-collection 
study donor sample. 
• 
System manufacturer       
 
 
 
 
• 
Part number/Product code       
 
 
 
• 
Lot #       
 
 
 
 
 
 
• 
Filter ID       
 
 
 
 
 
 
• 
Collection date       
 
 
 
 
 
• 
Donation start time (“Needle in”)        
 
 
• 
Donation end time (“Needle out”)        
 
 

 
Study Protocol 
Protocol : 
CLIN-0001 
161 First St, Suite 1A 
Cambridge MA 02142 USA 
Clinical Investigation to Evaluate the New Health 
Sciences Hemanext® Oxygen Reduction System 
for Leukoreduced Red Blood Cells with CP2D 
Anticoagulant and AS-3 Additive - Pivotal Trial 
Date: 
28 Nov 2017 
Author:  Michael Dioguardi 
 
 
Page 34 of 65 
 
 
• 
Collection room temperature       
 
 
 
• 
Procedural IFU confirmation (Yes/No)       
 
 
• 
Adverse event documentation       
 
 
 
• 
Sickle Cell Trait *       
 
 
 
 
• 
Viral Markers *       
 
 
 
 
 
• 
Blood typing (ABO) with Rh factor *       
 
 
 
For the purpose of this study: 
• 
For the procedural IFU confirmation, a response indicative of a 
deviation from the IFU will result in a prompt to record a protocol 
deviation in a separate Comments eCRF or other log. 
• 
The study donor’s evaluability confirmation will reflect the criteria 
listed in section 5.3.5.1 which defines each item that could impact 
evaluability. 
• 
For the adverse event documentation, a response indicative of the 
occurrence of an adverse event will result in a prompt to record 
adverse event details in a separate Adverse Event eCRF. 
 
6.2.16.4  Pre-filtration Storage and Sampling  
Following transfer to the medical laboratory, whole blood mixed with 
anticoagulant will be stored prior to filtration.  Samples for analysis will be 
obtained immediately prior to filtration.  Two (2) plasma samples will be 
processed from the pre-filtration unit just prior to filtration and frozen for 
future analysis in instances of confounding data in subsequent analyses. 
Pre-filtration data/specimen collection and sample processing are as 
follows: 
• 
Unit placed in pre-filtration storage: time and temperature       
• 
Time pre-filtration samples obtained       
 
• 
WB NLR Unit Pre-sample Gross Weight        
 
• 
WB NLR Unit Post-sample Gross Weight       
 
• 
Spun Hematocrit        
 
 
 
 
• 
CBC       
 
 
 
 
 
 
o WBC Count         
 
 
 
 
o RBC Count        
 
 
 
 
 
o MCV        
  
 
 
 
 
o MCH         
 
 
 
 
 

 
Study Protocol 
Protocol : 
CLIN-0001 
161 First St, Suite 1A 
Cambridge MA 02142 USA 
Clinical Investigation to Evaluate the New Health 
Sciences Hemanext® Oxygen Reduction System 
for Leukoreduced Red Blood Cells with CP2D 
Anticoagulant and AS-3 Additive - Pivotal Trial 
Date: 
28 Nov 2017 
Author:  Michael Dioguardi 
 
 
Page 35 of 65 
 
 
o MCHC        
 
 
 
 
 
o RDW 
o PLT Count       
 
 
 
 
 
o Total Hemoglobin        
 
 
 
 
o Hematocrit        
 
 
 
 
 
• 
Supernatant Hemoglobin        
 
 
 
• 
Two (2) reference plasma aliquots 
 
6.2.16.5  Filtration (Leukoreduction) 
The whole blood unit will be filtered for leukoreduction per the study IFU 
and under Study Arm-specific conditions.  
Filtration data collection is as follows: 
• 
Filtration Start: time and temperature        
  
• 
Head Height        
 
 
 
 
 
• 
Prime Time        
 
 
 
 
 
• 
Filtration End:  date and time        
 
 
 
• 
Drain Time       
 
 
 
 
 
• 
Filter Weight (following filtration completion)       
 
• 
Procedural IFU confirmation (Yes/No)       
 
 
 
For the purpose of this study: 
• 
For the procedural IFU confirmation, a response indicative of a 
deviation from the IFU will result in a prompt to record a protocol 
deviation in a separate Comments eCRF or other log. 
 
6.2.16.6  Post-filtration  
The unit will be weighed, sampled, and re-weighed.  
• 
Sampling time and temperature        
  
• 
Filtered WB Unit Pre-sample Gross Weight        
  
• 
Filtered WB Unit Post-sample Gross Weight        
 
• 
CBC       
 
 
 
 
 
 
o WBC Count  
o RBC Count  

 
Study Protocol 
Protocol : 
CLIN-0001 
161 First St, Suite 1A 
Cambridge MA 02142 USA 
Clinical Investigation to Evaluate the New Health 
Sciences Hemanext® Oxygen Reduction System 
for Leukoreduced Red Blood Cells with CP2D 
Anticoagulant and AS-3 Additive - Pivotal Trial 
Date: 
28 Nov 2017 
Author:  Michael Dioguardi 
 
 
Page 36 of 65 
 
 
o MCV  
o MCH  
o MCHC 
o RDW 
o PLT Count  
o Total Hemoglobin  
o Hematocrit 
• 
Supernatant Hgb 
• 
Spun Hematocrit 
• 
rWBC Count       
 
 
 
 
 
 
6.2.16.7  Filtered Whole Blood Separation 
Following the completion of filtration, the filtered whole blood units are to 
be processed into additive PRBCs and PPP.  The first step of this 
process involves centrifugation of the filtered whole blood unit using a 
hard spin method followed by expressing the PPP from the RBCs (no 
data is collected relative to plasma expressing).  
Separation data collection is as follows: 
• 
Centrifugation time and temperature       
 
• 
Centrifuge Make         
 
 
 
 
• 
Centrifuge Model         
 
 
 
 
• 
Centrifuge Serial #        
 
 
 
 
• 
Centrifuge Rotor #        
 
 
 
 
• 
Centrifuge Cup Type        
 
 
 
 
• 
Centrifuge Duration        
 
 
 
 
• 
Centrifuge RCF         
 
 
 
 
• 
Centrifuge Brake        
 
 
 
  
• 
Centrifuge Temperature         
 
 
 
 
For the purpose of this study: 
• 
Standard information regarding each centrifuge used will be 
captured prior to the initiation of study activities.  Only deviations 
from the standard equipment will be entered into the eCRF. 
 

 
Study Protocol 
Protocol : 
CLIN-0001 
161 First St, Suite 1A 
Cambridge MA 02142 USA 
Clinical Investigation to Evaluate the New Health 
Sciences Hemanext® Oxygen Reduction System 
for Leukoreduced Red Blood Cells with CP2D 
Anticoagulant and AS-3 Additive - Pivotal Trial 
Date: 
28 Nov 2017 
Author:  Michael Dioguardi 
 
 
Page 37 of 65 
 
 
6.2.16.8  Baseline Sampling 
The AS-3 is mixed with the RBCs per the IFU.  The RBC units will be 
sampled for Baseline tests.  If the LR-RBC is in the CP arm it willthen be 
placed in 1-6ºC storage for 42 days post-collection.   
 
Baseline data collection and sample processing are as follows:   
• 
Time AS-3 added to RBC unit     
• 
IFU confirmation         
 
 
 
 
• 
Sampling time       
 
 
 
 
• 
Sampling room temperature       
 
 
 
• 
Pre-sample Gross Weight       
 
 
 
• 
Post-sample gross weight       
 
 
 
• 
CBC      
 
 
 
 
 
  
o WBC Count  
o RBC Count  
o MCV  
o MCH  
o MCHC  
o RDW  
o PLT Count  
o Total Hemoglobin  
o Hematocrit  
• 
Spun Hematocrit   
 
 
 
 
 
 
• 
RBC morphology 
• 
Supernatant Hemoglobin  
• 
ATP  
• 
2,3-DPG  
• 
SO2 
• 
pCO2 
• 
HCO3   
• 
pH 
• 
Potassium  

 
Study Protocol 
Protocol : 
CLIN-0001 
161 First St, Suite 1A 
Cambridge MA 02142 USA 
Clinical Investigation to Evaluate the New Health 
Sciences Hemanext® Oxygen Reduction System 
for Leukoreduced Red Blood Cells with CP2D 
Anticoagulant and AS-3 Additive - Pivotal Trial 
Date: 
28 Nov 2017 
Author:  Michael Dioguardi 
 
 
Page 38 of 65 
 
 
• 
Sodium 
• 
Glucose  
• 
Lactate  
• 
RBC Morphology 
• 
Time RBC placed in refrigerated storage  
 
• 
Storage temperature     
 
 
 
  
 
For the purpose of this study: 
• 
For the procedural IFU confirmation, a response indicative of a 
deviation from the IFU will result in a prompt to record a protocol 
deviation in a separate Comments eCRF or other log. 
 
6.2.16.9 Hemanext Processing (IP only) 
If the unit is in the IP arm it will be transferred to the ORB and will 
undergo the Hemanext process per the Hemanext IFU. 
 
Hemanext Processing data collection are as follows:   
• 
Lot # 
• 
ORB Gross Weight (net weight should be < 370g) 
• 
Time ORB placed on agitator 
• 
Time ORB removed from agitator 
• 
IFU confirmation 
 
 
 
 
For the purpose of this study: 
• 
For the procedural IFU confirmation, a response indicative of a 
deviation from the IFU will result in a prompt to record a protocol 
deviation in a separate Comments eCRF or other log. 
 
 
 
 
 
  
 
6.2.16.10 Post –Hemanext Processing (IP only) 
After undergoing the Hemanext process the unit will be transferred to the 
HSB and placed in 1-6ºC storage for 42 days post-collection.   
 
Post-Hemanext processing data collection and sample processing are as 
follows:   
 
 
 
 
 

 
Study Protocol 
Protocol : 
CLIN-0001 
161 First St, Suite 1A 
Cambridge MA 02142 USA 
Clinical Investigation to Evaluate the New Health 
Sciences Hemanext® Oxygen Reduction System 
for Leukoreduced Red Blood Cells with CP2D 
Anticoagulant and AS-3 Additive - Pivotal Trial 
Date: 
28 Nov 2017 
Author:  Michael Dioguardi 
 
 
Page 39 of 65 
 
 
• 
Sampling date and time        
 
 
 
• 
Sampling room temperature       
 
 
 
• 
Pre-sample Gross Weight       
 
 
 
• 
Post-sample gross weight       
 
 
 
• 
CBC      
 
 
 
 
 
  
o WBC Count  
o RBC Count  
o MCV  
o MCH  
o MCHC  
o RDW  
o PLT Count  
o Total Hemoglobin  
o Hematocrit  
• 
Spun Hematocrit   
 
 
 
 
 
 
• 
Supernatant Hemoglobin   
• 
Time RBC placed in refrigerated storage  
 
 
 
 
  
 
For the purpose of this study: 
• 
For the procedural IFU confirmation, a response indicative of a 
deviation from the IFU will result in a prompt to record a protocol 
deviation in a separate Comments eCRF or other log. 
 
 
6.2.16.11 LR-RBC Bacterial Culture Samples (ONLY for units intended for in 
vivo autologous transfusion) 
5-7 days prior to infusion, units intended for transfusion as part of the in 
vivo cohort must be cultured.  Units evaluated as “+” will not be 
transfused.  Items in bold, underlined blue font refer specifically to in 
vivo cohort-specific data and analyses.  
• 
Sample date 
• 
Pre-sample unit gross weight  
• 
Post-sample unit gross weight 
• 
Outcome of bacterial culture (+/-) 

 
Study Protocol 
Protocol : 
CLIN-0001 
161 First St, Suite 1A 
Cambridge MA 02142 USA 
Clinical Investigation to Evaluate the New Health 
Sciences Hemanext® Oxygen Reduction System 
for Leukoreduced Red Blood Cells with CP2D 
Anticoagulant and AS-3 Additive - Pivotal Trial 
Date: 
28 Nov 2017 
Author:  Michael Dioguardi 
 
 
Page 40 of 65 
 
 
• 
If “+”, then date study donor notified not to return for in vivo 
study. 
 
6.2.16.12  21-day Analyses 
On day 21 post baseline, RBC units will be analyzed.    
21-day RBC data point collection and analyses are as follows: 
• 
Sampling date and time        
 
 
 
• 
Sampling room temperature       
 
 
 
• 
Pre-sample Gross Weight       
 
 
 
• 
Post-sample gross weight       
 
 
 
• 
CBC      
 
 
 
 
 
  
o WBC Count  
o RBC Count  
o MCV  
o MCH  
o MCHC  
o RDW  
o PLT Count  
o Total Hemoglobin  
o Hematocrit  
• 
Spun Hematocrit   
 
 
 
 
 
 
• 
RBC morphology 
• 
Supernatant Hemoglobin  
• 
ATP  
• 
2,3-DPG  
• 
SO2 
• 
pCO2 
• 
HCO3   
• 
pH 
• 
Potassium  
• 
Sodium 
• 
Glucose  

 
Study Protocol 
Protocol : 
CLIN-0001 
161 First St, Suite 1A 
Cambridge MA 02142 USA 
Clinical Investigation to Evaluate the New Health 
Sciences Hemanext® Oxygen Reduction System 
for Leukoreduced Red Blood Cells with CP2D 
Anticoagulant and AS-3 Additive - Pivotal Trial 
Date: 
28 Nov 2017 
Author:  Michael Dioguardi 
 
 
Page 41 of 65 
 
 
• 
Lactate  
• 
RBC Morphology 
• 
Time sample returned to refrigeration      
  
 
6.2.16.13  LR-RBC 42-day Analyses 
 
 
 
On day 42 post baseline, RBC units will be analyzed.    
 
 
 
42-day RBC data point collection and analyses are as follows:   
• 
Unit Sample date and time      
 
 
 
• 
Pre-sample Gross Weight       
 
 
 
• 
Post-sample gross weight       
 
  
• 
CBC      
 
 
 
 
 
 
o WBC Count  
o RBC Count  
o MCV  
o MCH  
o MCHC  
o RDW  
o PLT Count  
o Total Hemoglobin  
o Hematocrit 
• 
Supernatant Hemoglobin        
 
 
  
• 
Spun Hematocrit      
• 
SO2 
• 
pCO2 
• 
HCO3   
 
 
 
 
• 
pH  
• 
Potassium 
• 
Sodium 
• 
Glucose  
• 
Lactate 
• 
ATP  

 
Study Protocol 
Protocol : 
CLIN-0001 
161 First St, Suite 1A 
Cambridge MA 02142 USA 
Clinical Investigation to Evaluate the New Health 
Sciences Hemanext® Oxygen Reduction System 
for Leukoreduced Red Blood Cells with CP2D 
Anticoagulant and AS-3 Additive - Pivotal Trial 
Date: 
28 Nov 2017 
Author:  Michael Dioguardi 
 
 
Page 42 of 65 
 
 
• 
2,3-DPG  
• 
ATP Rejuvenation 
• 
2,3-DPG Rejuvenation 
• 
RBC Morphology 
• 
Time sample returned to refrigeration      
 
 
 
 
6.2.16.14  RBC in vivo recovery and survival (ONLY for units selected for the in 
vivo study) 
Items in bold, underlined blue font refer specifically to in vivo 
cohort-specific data and analyses.  
Donors participating in the in vivo cohort will have an additional 2-4 weeks 
between donations to ensure washout from prior in vivo survival 
procedures. 
• 
Double radiolabel % recovery 
o 24 hours (+/- 1hour) 
• 
Double radiolabel % survival- 28days 
o 24 hours (+/- 1hour) 
o 48 hours (+/- 4 hours) 
o 72 hours (+/- 8 hours) 
o 1 week (+/- 1 day) 
o 2 week (+/- 1 day) 
o 3 week (+/- 1 day) 
o 4 week (+/- 1 day) 
• 
RBC Microparticles 
• 
RBC Metabolomics panel 
• 
Pregnancy test  
• 
Bacterial Culture  
 
6.2.16.15  Blood Product Disposition 
Each whole blood unit collected in the study will generate one unit of LR-
RBC. All LR-RBC units will be stored by the site after completion of the 
study until written consent to discard the units is obtained from the 
Sponsor.  Written consent to discard blood products will not be required 
for units with a confirmed positive infectious disease result.   All Plasma 
units will be discarded. 

 
Study Protocol 
Protocol : 
CLIN-0001 
161 First St, Suite 1A 
Cambridge MA 02142 USA 
Clinical Investigation to Evaluate the New Health 
Sciences Hemanext® Oxygen Reduction System 
for Leukoreduced Red Blood Cells with CP2D 
Anticoagulant and AS-3 Additive - Pivotal Trial 
Date: 
28 Nov 2017 
Author:  Michael Dioguardi 
 
 
Page 43 of 65 
 
 
 
7.0 
Study Risks and Safety Reporting 
7.1 
Analysis of Risks  
7.1.1 Test System 
The IP is regulated as an investigational device and as such, risks associated 
with the use of the IP are those expected with manual whole blood donation.  
See sections 7.1.3 and 7.2.2.5 for more information regarding risks associated 
with manual whole blood donation. 
 
7.1.2 Control System 
Refer to the Precautions, Note(s), and Warnings in the Leukotrap® IFU. See 
sections 7.1.3 and 7.2.2.5 for more information regarding risks associated with 
manual whole blood donation. 
 
7.1.3 Risk Assessment of Whole Blood Donation 
The risks in the manual whole blood donation for this study are the same as any 
manual whole blood donation.  The study donor may experience a vasovagal 
reaction. These occur in two to four percent (2-4%) of blood donations, 
regardless of the type.  Very rarely, these progress to syncope and/or seizure 
activities (1:1000).  In addition, as with any blood collection procedure, there is 
the risk of hematoma formation.  Whole blood collections will be performed by 
trained staff at each site that will recognize the early signs of an adverse reaction 
and will discontinue the collection and administer appropriate aid to minimize the 
severity of a reaction under the direction of the Principal Investigator or designee.  
See section 7.2.2.5 for a list of expected adverse events. 
 
7.1.4 Anemia 
Current blood collection standard practice defines a maximum whole blood 
donation for a 110 pound donor within an eight week period as a single unit.  
Typically, the donor’s body replenishes the fluid lost from donation within 24 
hours, and replaces the lost red blood cells within two weeks. 
With the exception of those study donors who participate in the in vivo cohort, 
donors will adhere to the collection standard practice.  For the in vivo cohort 
participants who will have an additional ≤110 mL of whole blood drawn as part of 
the RBC recovery assessment at 6 weeks following each donation, they are still 
well below the two unit volume of red blood cells that would be donated via red 
cell apheresis which may occur every 16 weeks. 

 
Study Protocol 
Protocol : 
CLIN-0001 
161 First St, Suite 1A 
Cambridge MA 02142 USA 
Clinical Investigation to Evaluate the New Health 
Sciences Hemanext® Oxygen Reduction System 
for Leukoreduced Red Blood Cells with CP2D 
Anticoagulant and AS-3 Additive - Pivotal Trial 
Date: 
28 Nov 2017 
Author:  Michael Dioguardi 
 
 
Page 44 of 65 
 
 
To maintain study donors’ safety relative to the donation of whole blood for the 
purpose of this study, Hct or Hgb levels and other applicable inclusion/exclusion 
criteria will be confirmed prior to each donation. 
 
7.1.5 Risk to Those Conducting the Research 
Chosen investigative sites all have long experience in blood collection, storage 
clinical trials and in vitro studies.  All participating staff at the study sites has had 
training and experience in the handling of potentially biohazardous materials and 
has been offered hepatitis B vaccine.  All phlebotomies are performed in a well-
equipped area and only staff with phlebotomy training and experience will 
perform the whole blood collections.  Each of the sites has in-place SOPs for 
drawing whole blood units and producing and testing blood and blood products.  
Site staff members are required to have documented SOP training relative to 
study-related activities.  Study donors will be screened with the same criteria 
used for whole blood donation. 
Site staff performing the in vivo portion of the study will be exposed to the 
radiation attendant to performing the in vivo measurement procedures.  Site staff 
involved in performing the radiolabeled red cell recovery procedures has been 
trained in handling radioactive materials and wear radiation collection badges 
that are periodically evaluated.  Each investigative site has more than 10 years of 
experience in the performance of 51Cr and 99mTc radiolabeled red cell recovery 
procedures and has SOPs in place for the performance of all required 
procedures.  These procedures are well recognized as being safe.   
 
7.1.6 Radiation Exposure 
Subjects participating in the in vivo portion of the trial will be exposed to the 
radiation concomitant to the in vivo measurement procedures.  Approximately 15-
20 µCi of radioactivity will be administered to in vivo subjects in the autologous, 
radiolabeled packed RBC + whole blood mixture one on day 42.  Red cells 
labeled with 51Cr are frequently used in immunohematology to assess the 
survival of either allogeneic or autologous red cells.  99mTc labeled red cells are 
used to measure total blood volume.  Each investigative site is experienced in 
the performance of 51Cr and 99mTc radiolabeled red cell recovery and survival 
procedures.  These procedures are well recognized as being safe.  Standard 
procedures at both sites for radiolabeled red cell recovery measurement include 
a determination of the radiation exposure history of each subject that will 
participate in radiolabeled red cell recovery procedures to ensure that, with the 
addition of the expected exposure in the study, the subject total exposure will not 
exceed legal limits.  In addition, site procedures are expected to minimize the 
subject radiation dose in the study.  There are no known adverse events 
associated with the low radiation dose that will be administered in this study.  
Female in vivo subjects must have a negative pregnancy test prior to each 
reinfusion, be postmenopausal (> 1 year), or surgically sterile.   
 

 
Study Protocol 
Protocol : 
CLIN-0001 
161 First St, Suite 1A 
Cambridge MA 02142 USA 
Clinical Investigation to Evaluate the New Health 
Sciences Hemanext® Oxygen Reduction System 
for Leukoreduced Red Blood Cells with CP2D 
Anticoagulant and AS-3 Additive - Pivotal Trial 
Date: 
28 Nov 2017 
Author:  Michael Dioguardi 
 
 
Page 45 of 65 
 
 
7.1.7 Receiving Allogeneic Blood 
Subjects participating in the in vivo portion of the study will receive a reinfusion of 
a small aliquot of their own (autologous) radiolabeled red cells.  The possibility 
exists that in the case of protocol deviation, the study donor could receive 
allogeneic (from a different donor) red cells.  Possible adverse events that could 
occur from the infusion of allogeneic blood include: 
• 
Infectious disease transmission 
• 
Hemolytic reaction to non-compatible red cells  
 
Each clinical site has more than 15 years’ experience in performing autologous, 
in vivo, radiolabeled red cell recovery and survival procedures and has current 
standard procedures that include redundant subject identification procedures to 
ensure that only autologous blood is reinfused. 
 
7.1.8 Study Donor Benefits 
Study donors will not receive any direct benefit from study participation. 
 
7.2 
Adverse Events 
7.2.1 Monitoring 
This multiple site study is intended to enroll healthy individuals to serve as study 
donors.  Study donors’ study activity will be limited to the donation of two units of 
blood and that the active study duration is expected to be less than 24 hours for 
each unit.  As such, a Data Safety Monitoring Board (DSMB) will not be 
appointed.  Each site will serve as its own primary safety monitor responsible for 
identifying any adverse events, providing any necessary immediate medical care 
for a study-related adverse event and the accurate and timely reporting of any 
adverse events.   
Study-related adverse events are defined as those study participant-reported 
adverse events occurring within 24-hours of donation and/or transfusion.  Non-
study-related adverse events outside of these periods will not be recorded unless 
they are of a nature to impact study participant evaluability, safety or data 
validity. 
Reports for events determined by the investigator to be possibly or definitely 
related to study participation and reports of events resulting in death must be 
promptly verbally reported to the sponsor within 24 hours, formally reported to 
the sponsor within 72 hours, and forwarded to the relative site’s IRB within the 
timing dictated by the respective IRB (typically within 3 days).    
 
 

 
Study Protocol 
Protocol : 
CLIN-0001 
161 First St, Suite 1A 
Cambridge MA 02142 USA 
Clinical Investigation to Evaluate the New Health 
Sciences Hemanext® Oxygen Reduction System 
for Leukoreduced Red Blood Cells with CP2D 
Anticoagulant and AS-3 Additive - Pivotal Trial 
Date: 
28 Nov 2017 
Author:  Michael Dioguardi 
 
 
Page 46 of 65 
 
 
7.2.2 Definitions 
The following definitions are based on 21 CFR 314.80, ISO 14155-2011 Clinical 
investigation of medical devices for human subjects, MEDDEV 2.7/3 Guidelines 
on Clinical Investigations: Serious Adverse Event Reporting under Directives 
90/385/EEEC and 93/42/EEC and ICH E6 Good Clinical Practice: Consolidated 
Guidance. 
 
7.2.2.1 
 Adverse Event (AE) 
An AE is defined as any untoward medical occurrence, unintended 
disease or injury or untoward clinical sign (including abnormal laboratory 
finding) in a study donor while enrolled in the clinical investigation, 
whether or not considered investigational product- related. 
Study-related adverse events are defined as those study participant-
reported adverse events occurring within 24-hours of donation and/or 
transfusion.  Non-study-related adverse events outside of these periods 
will not be recorded unless they are of a nature to impact study participant 
evaluability, safety or data validity. 
 
7.2.2.2 
 Serious Adverse Event (SAE) 
An adverse event is defined as serious, if it:  
• 
led to death; 
• 
led to a serious deterioration in the health of the patient that: 
• 
resulted in a life-threatening illness or injury, 
• 
resulted in a permanent impairment of a body structure or a body 
function, 
• 
required in-patient hospitalization or prolongation of existing 
hospitalization, 
• 
resulted in medical or surgical intervention to prevent permanent 
impairment to a body structure or a body function; 
• 
led to fetal distress, fetal death, congenital abnormality or birth 
defect. 
 
The term “life-threatening” is an event that places a patient or study donor 
at immediate risk of death from the event as it occurred; it does not 
include an event that, had it occurred in more severe form, might have 
caused death. 
Important medical events that may not result in death, be life-threatening, 
or require hospitalization may be considered a serious adverse event 

 
Study Protocol 
Protocol : 
CLIN-0001 
161 First St, Suite 1A 
Cambridge MA 02142 USA 
Clinical Investigation to Evaluate the New Health 
Sciences Hemanext® Oxygen Reduction System 
for Leukoreduced Red Blood Cells with CP2D 
Anticoagulant and AS-3 Additive - Pivotal Trial 
Date: 
28 Nov 2017 
Author:  Michael Dioguardi 
 
 
Page 47 of 65 
 
 
when, based upon appropriate medical judgment, they may jeopardize 
the patient or subject and may require medical or surgical intervention. 
“Non-serious adverse events” are all events that do not meet the criteria 
for a “serious” adverse event. 
All serious and/or unexpected AEs shall be reported to NHSi within 1 
business day of learning of their occurrence.  They shall be reported by 
telephone to a study monitor or other designated individual and recorded 
within the EDC system on the provided AE electronic form.  Additional 
documentation requested by NHSi will be e-mailed or faxed to NHSi 
within 1 business day following the request. 
 
7.2.2.3 
 Unexpected Adverse Event 
AE’s for which the nature or severity of which is not consistent with the 
applicable product information (e.g., Investigator's Brochure or IFU) or 
been previously identified in the risk analysis report. This includes events 
that may be symptomatically and pathophysiologically related to an event 
listed in the labeling, but differ from the event because of greater severity 
or specificity.  
The investigator must determine if the AE was related to the 
investigational product using the following scoring system: 
• 
Unrelated 
The AE is clearly NOT related to the investigational 
product. The AE has no temporal relationship to 
the administration or use of the investigational 
product, follows no known or suspected pattern of 
response, and an alternative cause is present.  
• 
Unlikely 
related 
The AE is unlikely to be related to the 
investigational product. The AE has a temporal 
relationship to the administration or use of the 
investigational product, but follows no known or 
suspected pattern of response, and an alternative 
cause is present.  
• 
Possibly 
related 
There is a reasonable possibility that the event 
may have been caused by or is linked in a 
significant way to the research; the AE has a 
temporal relationship to the administration or use 
of the investigational product, follows a suspected 
pattern of response, but an alternative cause is 
present.  

 
Study Protocol 
Protocol : 
CLIN-0001 
161 First St, Suite 1A 
Cambridge MA 02142 USA 
Clinical Investigation to Evaluate the New Health 
Sciences Hemanext® Oxygen Reduction System 
for Leukoreduced Red Blood Cells with CP2D 
Anticoagulant and AS-3 Additive - Pivotal Trial 
Date: 
28 Nov 2017 
Author:  Michael Dioguardi 
 
 
Page 48 of 65 
 
 
• 
Probably 
related 
The adverse event is likely related to the 
investigational product. The AE has a temporal 
relationship 
to 
the 
administration 
of 
the 
investigational product or research intervention, 
follows a known or suspected pattern of response, 
but an alternative cause may be present.  
• 
Definitely 
related 
The AE is clearly related to the investigational 
product or research intervention. The adverse 
event has a temporal relationship to the 
administration of the investigational product or 
research intervention, follows a known pattern of 
response, and no alternative cause is present.  
 
Each AE should be evaluated as either expected or unexpected. If the 
risks for entering the study described in the informed consent, clinical 
protocol or risk analysis reflect the severity or specificity of the AE, then it 
was expected. If not, then the AE is unexpected.  
 
7.2.2.4 
 Adverse Event Severity 
The investigator shall determine the AE Severity using the following 
scoring system: 
• 
“Mild” – An adverse event which is easily tolerated by the patient, 
causing minimal discomfort and not interfering with everyday 
activities. 
• 
“Moderate”- An adverse event, which is sufficiently discomforting 
to interfere with normal everyday activities. 
• 
“Severe”- An adverse event, which is incapacitating and prevents 
normal everyday activities. 
 
7.2.2.5 
 Adverse Event Expectedness 
Expected AEs for this study would include the following: 
• 
Vasovagal response 
• 
Thirst 
• 
Arm pain 
• 
Hives / Itching 
• 
Hypotension 
• 
Tingling 
• 
Unusual taste 
• 
Bruising / Hematoma 

 
Study Protocol 
Protocol : 
CLIN-0001 
161 First St, Suite 1A 
Cambridge MA 02142 USA 
Clinical Investigation to Evaluate the New Health 
Sciences Hemanext® Oxygen Reduction System 
for Leukoreduced Red Blood Cells with CP2D 
Anticoagulant and AS-3 Additive - Pivotal Trial 
Date: 
28 Nov 2017 
Author:  Michael Dioguardi 
 
 
Page 49 of 65 
 
 
• 
Arterial puncture 
• 
Nerve injury 
• 
Iron depletion 
• 
Weakness / Lightheadedness  
• 
Pallor / Sweating / Chills / Febrile reaction 
• 
Shortness of breath / Hyperventilation 
• 
Nausea / Vomiting / Abdominal cramps 
• 
Syncope - transient loss of consciousness 
• 
Headache 
• 
Tiredness 
• 
Severe reactions – convulsions/ Chest pains / Cardiac arrest 
 
Study donors participating in the in vivo portion of the study will receive 
an autologous, radiolabeled RBC transfusion.  The radiation exposure is 
very small and the level used in this trial has not been found to cause 
cancer. These study donors would be further subject to experiencing the 
following expected AEs in addition to those listed above: 
• 
Infection from bacterial contamination 
 
Each AE will be evaluated to determine if it meets serious criteria. 
 
8.0 
Study Sites 
8.1 
Study Sites 
Three clinical sites will participate in this study.  American Red Cross Mid-Atlantic 
Research Facility (Site A) and Hoxworth Blood Center (Site H) and Blood Center of 
Wisconsin (Site B) will all participate as primary research sites.   
 
8.2 
Central Laboratory 
Analyses of blood products will occur at the laboratories associated with each of the 
clinical sites. 
 
9.0 
Data Management 
9.1 
Case Report Forms 
All original source data will be contained in original laboratory printouts, site laboratory 
notebooks, site data sheets and/or study-specific source documents.  Data transmittal to 
the Sponsor will be accomplished using electronic Case Report Forms (eCRFs). Original 
source data will not be entered directly on the eCRFs.  Original data is to be transcribed 

 
Study Protocol 
Protocol : 
CLIN-0001 
161 First St, Suite 1A 
Cambridge MA 02142 USA 
Clinical Investigation to Evaluate the New Health 
Sciences Hemanext® Oxygen Reduction System 
for Leukoreduced Red Blood Cells with CP2D 
Anticoagulant and AS-3 Additive - Pivotal Trial 
Date: 
28 Nov 2017 
Author:  Michael Dioguardi 
 
 
Page 50 of 65 
 
 
from source data records to the eCRFs by site study staff.  Data entered into the eCRFs 
will then be verified against the source data during Sponsor on-site monitoring visits.  At 
the study site visits the verified and corrected CRFs must be reviewed and electronically 
signed by the respective Principal Investigator to finalize the data.  Investigative site 
study data and records for this protocol are subject to monitoring or inspection by the 
Sponsor, FDA and relevant state or federal government agencies. 
 
9.2 
Monitoring 
Monitoring will be conducted by the sponsor or delegate according to clinical monitoring 
SOPs to assure that the protocol is followed, any adverse events are reported, data 
collected are scientifically sound, and that the study is conducted in compliance with 
federal and ICH guidelines for GCP.  On-site monitoring visits will occur at study 
initiation, during the study performance as needed (minimum one interim visit) and at the 
study close. 
 
9.3 
Protocol Deviations 
Sites are required to perform the study as described in the protocol and informed 
consent.  All protocol deviations are to be documented in source records and transcribed 
to the appropriate eCRF page with a description of the deviation and reason(s) for the 
deviation. 
Any protocol deviation that impacts study donor safety or the scientific integrity of the 
study must be reported within 24 hours of notification to the Sponsor, and within 48 
hours to the site IRB where the deviation occurred. 
Issuance of a revised protocol is required for a protocol deviation/modification that 
substantially affects the design of the study or study donor safety (including but not 
limited to a change in, principal investigator, inclusion/exclusion criteria, number of study 
donors to be enrolled, study sites or study procedures).  Each protocol addendum must 
be signed by the Sponsor and the principal investigators and be submitted for local IRB 
review and approval before implementation of the protocol addendum.  The study can 
continue under the previous protocol during this process, if appropriate.  Once the 
Sponsor and principal investigator signature, IRB approval has been obtained, 
performance of the protocol addendum can begin at that site. 
Minor deviations such as a missed measurement must be documented as described but 
do not require immediate reporting to the IRB.  Minor deviations will be reported to local 
IRB in continuing review reports and the study final report. 
 
10.0 Data Use 
10.1 
Test Report 
A final report will be prepared by NHSi and be reviewed by the study sites.  This final 
report will include data reported on all study donors enrolled including cases where they 

 
Study Protocol 
Protocol : 
CLIN-0001 
161 First St, Suite 1A 
Cambridge MA 02142 USA 
Clinical Investigation to Evaluate the New Health 
Sciences Hemanext® Oxygen Reduction System 
for Leukoreduced Red Blood Cells with CP2D 
Anticoagulant and AS-3 Additive - Pivotal Trial 
Date: 
28 Nov 2017 
Author:  Michael Dioguardi 
 
 
Page 51 of 65 
 
 
are excluded from the total evaluable study donors.  This report will be made available to 
all persons responsible for the conduct of the study.   
 
10.2 
Confidentiality 
Confidentiality of each study donor shall be preserved in the report and any publication 
of the clinical investigation data. 
 
10.3 
Positive TTD Results – Result Reporting Requirements and Unit Disposition 
In the event of a positive TTD testing report or other significant abnormality found during 
subject testing, the subject will be confidentially informed by the site principal 
investigator in person and/or by mail.  The result will be explained with tact and 
understanding.  A written record of the finding will be provided to the subject’s personal 
physician with permission of the subject.  The subject may be referred to an appropriate 
health care source.  At each site positive infectious disease results are required to be 
reported to the appropriate state agencies.  Subjects, and their blood products, yielding 
a positive infectious disease result or displaying another significant abnormality will be 
discontinued from the study and replaced with a new subject.  Note that some form of 
documentation must exist within the study records and/or other site records that 
demonstrate that these requirements have been fulfilled. 
 
10.4 
Publication 
Publication of the trial results is subject to the mutual agreement of each research site 
and the sponsor.   
 
10.5 
Anticipated Study Schedule 
IRB submission: 
 
September, 2017 
Procedures start date: 
November, 2017 
Enrollment completed: 
May, 2018 
Test report completed: 
July, 2018 
 
11.0 Responsibilities 
Sponsor/promoter: 
 
NHSi Corporation 
 
 161 First St., Suite 1A 
 
 Cambridge, MA 02142 
 
 

 
Study Protocol 
Protocol : 
CLIN-0001 
161 First St, Suite 1A 
Cambridge MA 02142 USA 
Clinical Investigation to Evaluate the New Health 
Sciences Hemanext® Oxygen Reduction System 
for Leukoreduced Red Blood Cells with CP2D 
Anticoagulant and AS-3 Additive - Pivotal Trial 
Date: 
28 Nov 2017 
Author:  Michael Dioguardi 
 
 
Page 52 of 65 
 
 
12.0 References 
1) Davey, R.J. Red Cell Radiolabeling in Transfusion Medicine.  In: Davey RJ, Wallace ME, 
eds.  Diagnostic and investigational uses of radiolabeled blood elements. Arlington, VA: 
AABB, 1987:93-114.  
2) Heaton, W.  Evaluation of Post Transfusion Recovery and Survival of Transfused Red 
Cells.  Transfus Med Rev 1992:6:153-169. 
3) Thomas, S.  Ambient overnight hold of whole blood prior to the manufacture of blood 
components.  Transfus Med. 2012 Dec;20(6):361-8. 
4) Wilsher C, Garwood M, Sutherland J, Turner, C and Cardigan R. The effect of storing whole 
blood at 22°C for up to 24 hours with and without rapid cooling on the quality of red cell 
concentrates and fresh-frozen plasma. Transfusion. 2008, Nov:4(11):2338-2347. 
 
13.0 
Appendices 
13.1 
Study Flow Chart 
Highlevel flowsheet 
Pivotal Crossover.pdf 
 
 
 
 
 
 
13.2 
Control Product/Leukotrap System – Instructions for Use 

 
Study Protocol 
Protocol : 
CLIN-0001 
161 First St, Suite 1A 
Cambridge MA 02142 USA 
Clinical Investigation to Evaluate the New Health 
Sciences Hemanext® Oxygen Reduction System 
for Leukoreduced Red Blood Cells with CP2D 
Anticoagulant and AS-3 Additive - Pivotal Trial 
Date: 
28 Nov 2017 
Author:  Michael Dioguardi 
 
 
Page 53 of 65 
 
 
 

 
Study Protocol 
Protocol : 
CLIN-0001 
161 First St, Suite 1A 
Cambridge MA 02142 USA 
Clinical Investigation to Evaluate the New Health 
Sciences Hemanext® Oxygen Reduction System 
for Leukoreduced Red Blood Cells with CP2D 
Anticoagulant and AS-3 Additive - Pivotal Trial 
Date: 
28 Nov 2017 
Author:  Michael Dioguardi 
 
 
Page 54 of 65 
 
 
Leukotrap® WB System 
 
CP2D/AS-3 Blood Bag Unit with In-Line WBF Filter* and Sampling 
System 
 
For collection of blood and preparation of red blood cells and plasma with pre-storage 
leukocyte reduction. 
Use aseptic technique. Note: Platelet concentrates are not intended to be made with this 
product. 
Instruction for Use for Systems Containing a Y Sampling Site (YSS) or Sample Diversion 
Pouch (with or without a pre-attached SampLok® Vacuum Tube Holder) - see unit foil 
package label for specific product description being used. 
Precaution: Use only if solutions are clear. Sterile, nonpyrogenic fluid path. Sterilized by steam.  
Warning: Failure to achieve and maintain a closed system during processing would result in a 
product that must be transfused within 24 hours. 
Rx only. This product is free of natural rubber latex. 
 
I.  Blood Collection Instructions for Systems Containing a Y Sampling Site (YSS) 
Only 
1. Load blood agitation device or suspend blood bag on donor scale and adjust donor scale to 
desired collection gross weight as per manufacturer's instructions. 
2. Clamp donor tubing between DonorCare® Needle Guard (DCNG) and Y Sampling Site. 
3. Secure donor tubing above the Y connector and disinfect site of phlebotomy. 
4. If using blood pressure cuff, inflate to not more than 60 mm Hg. 
5. Remove donor needle cover and accomplish phlebotomy. 
6. Release clamp and ensure there is blood flow. Reduce pressure as required. 
7. Slide the DCNG midway over the needle hub and securely tape DCNG to the donor's arm as 
close to the top of the DCNG as possible.  
Note: If blood flow is slow, slide DCNG away from the needle hub, adjust and re-engage DCNG. 
If repeated needle adjustment is necessary, slide DCNG away from the needle hub and re-
engage at the end of blood collection. 
8. Collect appropriate volume of blood into collection bag, as indicated on packaging. 
Note: Mix blood and anticoagulant frequently during collection, for example, once every 45 
seconds, and immediately after collection. If blood agitation device is used, follow 
manufacturer's operating instructions. 
9. After required amount of blood has been collected, seal donor tubing between Y Sampling 
Site and collection bag. 

 
Study Protocol 
Protocol : 
CLIN-0001 
161 First St, Suite 1A 
Cambridge MA 02142 USA 
Clinical Investigation to Evaluate the New Health 
Sciences Hemanext® Oxygen Reduction System 
for Leukoreduced Red Blood Cells with CP2D 
Anticoagulant and AS-3 Additive - Pivotal Trial 
Date: 
28 Nov 2017 
Author:  Michael Dioguardi 
 
 
Page 55 of 65 
 
 
** Note: If pre-filtration quality control is to be performed, leave an adequate length (~10 inches) 
of QC tubing containing anticoagulated blood attached to the collection bag. 
10. For blood sampling, remove the Y Sampling Site needle cover. Ensure the protective sheath 
is in place over the sampling needle. 
11. Fasten the vacuum tube holder on to the base of the sampling needle. 
12. Collect blood samples into vacuum tubes. 
13. Ensure the vacuum tubes are centered within the vacuum tube holder. 
14. Maintain forward pressure on the vacuum tubes during sample collection.  
Note: After the last tube is collected, it is recommended that the vacuum tube holder be left in 
place. 
15. After blood samples are collected, clamp donor tubing between the Y Sampling Site and as 
close to the DCNG as possible. 
16. Release any remaining pressure from the donor's arm. 
17. DCNG must be held stationary while the needle is withdrawn into it. While holding sides of 
DCNG near the front, grasp the tubing below the clamp and pull the needle into the DCNG until 
it locks into place, and the needle hub engages the bottom of the DCNG. 
18. Insert the DCNG into the vacuum tube holder.  
Note: It is recommended that the DCNG be inserted securely into the vacuum tube holder, prior 
to discarding. 
19. Seal donor tubing adjacent to DCNG.** Detach and discard needle, DCNG, Y Sampling Site 
and tubing.** 
20. Strip tubing between seal and collection bag. 
21. Continue to "Filtration Instructions”, Section IV, Step 1. 
 
II.  Blood Collection Instructions for Systems Containing a Sample Diversion 
Pouch with or without a pre-attached Samplok® Vacuum Tube Holder 
When using systems with pre-attached SampLok vacuum tube holder, follow instructions as 
noted below, but refer to Section III when indicated to do so. 
1. Load blood agitation device or suspend blood bag on donor scale and adjust donor scale to 
desired collection gross weight as per manufacturer's instructions. 
2. Clamp donor tubing between DonorCare Needle Guard (DCNG) and Sampling Site. 
3. Secure donor tubing above the Y connector and disinfect site of phlebotomy. 
4. If using blood pressure cuff, inflate to not more than 60 mm Hg. 
5. Remove donor needle cover and accomplish phlebotomy. 
6. Release clamp and ensure there is blood flow. 

 
Study Protocol 
Protocol : 
CLIN-0001 
161 First St, Suite 1A 
Cambridge MA 02142 USA 
Clinical Investigation to Evaluate the New Health 
Sciences Hemanext® Oxygen Reduction System 
for Leukoreduced Red Blood Cells with CP2D 
Anticoagulant and AS-3 Additive - Pivotal Trial 
Date: 
28 Nov 2017 
Author:  Michael Dioguardi 
 
 
Page 56 of 65 
 
 
7. Slide the DCNG midway over the needle hub and securely tape DCNG to the donor's arm as 
close to the top of the DCNG as possible.  
Note: If blood flow is slow, slide DCNG away from the needle hub, adjust and re-engage 
DCNG. If repeated needle adjustment is necessary, slide DCNG away from the needle hub and 
re-engage at the end of blood collection. 
8. The donor blood will be automatically diverted to the sample diversion pouch. Once the 
sample diversion pouch is filled, close clamp immediately on tubing between the sample 
diversion pouch and Y connector.  
Warning: To avoid risk of air embolism to donor, do not squeeze sample diversion pouch while 
tubing is open. 
9. Open snap-open closure between the Y connector and the collection bag to initiate blood 
collection. Reduce pressure as needed. 
10. Permanently seal tubing between the sample diversion pouch and the Y connector to 
maintain sterility of the system prior to collecting blood samples. 
** Note: When using systems with a pre-attached SampLok vacuum tube holder, go to Section 
III. 
11. For blood sampling, remove the Sampling Site needle cover. Ensure the protective sheath is 
in place over the sampling needle. 
12. Fasten the vacuum tube holder on to the base of the sampling needle. 
13. Position the sample diversion pouch downwards so that the air rises to the top of the pouch 
and away from the vacuum tube holder.  
Note: Drawing air into the vacuum tube may cause hemolysis. 
14. Collect blood samples from the sample diversion pouch within approximately four minutes to 
avoid possible clot formation. 
15. Ensure the vacuum tubes are centered within the vacuum tube holder during sample 
collection. 
16. Maintain forward pressure on the vacuum tubes during sample collection.  
Note: After the last tube is collected, it is recommended that the vacuum tube holder be left in 
place. 
17. Collect appropriate volume of blood into collection bag as indicated on packaging. 
Note: Mix blood and anticoagulant frequently during collection, for example, once every 45 
seconds, and immediately after collection. If blood agitation device is used, follow 
manufacturer's operating instructions. 
18. After required amount of blood has been collected, seal donor tubing between snap-open 
closure and collection bag. 
** Note: If pre-filtration quality control is to be performed, leave an adequate length (~10 inches) 
of QC tubing containing anticoagulated blood attached to the collection bag. 
19. Clamp donor tubing between the Y connector and DCNG as close as possible to the DCNG. 

 
Study Protocol 
Protocol : 
CLIN-0001 
161 First St, Suite 1A 
Cambridge MA 02142 USA 
Clinical Investigation to Evaluate the New Health 
Sciences Hemanext® Oxygen Reduction System 
for Leukoreduced Red Blood Cells with CP2D 
Anticoagulant and AS-3 Additive - Pivotal Trial 
Date: 
28 Nov 2017 
Author:  Michael Dioguardi 
 
 
Page 57 of 65 
 
 
20. Release any remaining pressure from donor’s arm. 
21. DCNG must be held stationary while the needle is withdrawn into it. While holding sides of 
DCNG near the front, grasp the tubing below the clamp and pull the needle into the DCNG until 
it locks into place, and the needle hub engages the bottom of the DCNG. 
22. Insert the DCNG into the vacuum tube holder, if desired.  
Note: It is recommended that the DCNG be inserted securely into the vacuum tube holder, prior 
to discarding. 
23. Seal donor tubing adjacent to DCNG.** Detach and discard needle, DCNG, sample 
diversion pouch and tubing.** 
24. Strip tubing between seal and collection bag. 
25. Continue to "Filtration Instructions”, Section IV, Step 1. 
III.  When Using Systems with a Pre-attached Samplock® Vacuum Tube Holder 
1. To collect blood samples, open lid from SampLok vacuum tube holder. 
2. Open snap-open closure between sample diversion pouch and SampLok vacuum tube 
holder. 
3. Position the sample diversion pouch downwards so that the air rises to the top of the pouch 
and away from the SampLok vacuum tube holder.  
Note: Drawing air into the vacuum tube may cause hemolysis. 
4. Collect blood samples from the sample diversion pouch into vacuum tubes within 
approximately four minutes to avoid possible clot formation. 
5. Ensure the vacuum tubes are centered within the SampLok vacuum tube holder during 
sample collection. 
6. Maintain forward pressure on the vacuum tubes during sample collection. 
7. The lid may be closed on the SampLok vacuum tube holder after sample collection. 
8. Return to Section II, Step 17.  
Note: When collection of unit is complete, and the donor needle is engaged in the DCNG, open 
the lid of the SampLok vacuum tube holder and insert the DCNG into the holder. Twist until it 
locks into place. An audible click will confirm that it is locked. 
IV.  Filtration Instructions 
1. Filter and process whole blood within 72 hours of collection. 
2. Mix whole blood/anticoagulant thoroughly. 
3. Place empty red cell storage bag on a horizontal surface. 
4. Ensure cap of blood recovery vent above the filter is tightly closed. 
5. Hang whole blood bag up to 60 inches (1.52 meters) above empty red cell storage bag and 
ensure filter is vertical.  

 
Study Protocol 
Protocol : 
CLIN-0001 
161 First St, Suite 1A 
Cambridge MA 02142 USA 
Clinical Investigation to Evaluate the New Health 
Sciences Hemanext® Oxygen Reduction System 
for Leukoreduced Red Blood Cells with CP2D 
Anticoagulant and AS-3 Additive - Pivotal Trial 
Date: 
28 Nov 2017 
Author:  Michael Dioguardi 
 
 
Page 58 of 65 
 
 
Note: The maximum head height should be 60 inches (1.52 meters) for whole blood filtered at 
room temperature and at 1-6 °C. 
6. Open snap-open closure of whole blood bag. Priming will occur automatically by gravity. 
7. After blood fills one side of the air vent below the filter, open snap-open closure immediately 
below the air vent to begin filtration. 
8. Allow blood to filter by gravity.  
Note: Do not apply mechanical or manual pressure to increase flow rate. Filtration is complete 
when whole blood bag is empty. 
9. Remove cap from blood recovery vent above the filter and allow the upstream side (non-
printed side) of the filter to drain. 
10. When the upstream side of the filter is empty, clamp tubing below the filter.  
Note: Filtration times can be influenced by collection and processing conditions and biological 
variability of donors. Experimental data with some filter products indicate that a prolonged 
filtration can be an indication of sub-optimal leukocyte reduction. 
11. Seal tubing just below the snap-open closure.** 
12. Detach and discard collection bag and filter. 
** Note: Do not strip tubing prior to sealing the tubing distal to the filter. If it is desired to strip 
blood from numbered tubing, do so only after tubing has been sealed close to the snap-open 
closure and detached. 
13. If desired, seal at or adjacent to "X” marks on the tubing to provide numbered segments of 
anticoagulated blood for typing or crossmatching. 
** Notes: If it is necessary to strip blood from numbered tubing for re-suspension, care should 
be taken when stripping is performed. Increased (mechanical) hemolysis has been associated 
with stripping when blood is cold and has a higher hematocrit. Do not strip forcefully or 
frequently against a snap-open closure. 
V.  Processing Instructions 
1. Load filtered blood and remaining bags into centrifuge cup, ensuring that the tubing stays in 
the top half of the cup. 
2. Centrifuge at appropriate conditions to produce desired components. 
3. Carefully remove the unit from the centrifuge and place the red cell storage bag in the plasma 
expressor. 
4. Clamp tubing to extra satellite bags, if present. 
5. Gently apply expressor pressure. 
6. Open snap-open closure to satellite bag and express plasma. 
7. After plasma is expressed, clamp tubing between red cell storage bag and Y connector, and 
release expressor pressure. 
8. Clamp tubing between the Y connector and plasma bag. 

 
Study Protocol 
Protocol : 
CLIN-0001 
161 First St, Suite 1A 
Cambridge MA 02142 USA 
Clinical Investigation to Evaluate the New Health 
Sciences Hemanext® Oxygen Reduction System 
for Leukoreduced Red Blood Cells with CP2D 
Anticoagulant and AS-3 Additive - Pivotal Trial 
Date: 
28 Nov 2017 
Author:  Michael Dioguardi 
 
 
Page 59 of 65 
 
 
9. Hang AS-3 bag above red cell storage bag and remove clamp from tubing to the red cell 
storage bag. 
10. Open snap-open closure on the bag containing the AS-3 additive solution and transfer to the 
bag containing the red cells.  
Note: AS-3 solution should be added to the packed red blood cells immediately after removal of 
plasma. Transfer AS-3 solution under one of the following processing conditions: 
a. within 8 hours of collection if whole blood is held at room temperature. 
b. within 72 hours of collection if whole blood is refrigerated. 
11. Seal tubing and detach the bag containing packed red cells, and set aside plasma for further 
processing. 
** Note: If quality control is to be performed on post filtration sample, leave an adequate length 
(~10 inches) of tubing attached to the bag containing red cells. 
12. Gently mix packed red cells and AS-3 solution. 
13. Store CP2D/AS-3 preserved red blood cells at 1—6 °C for up to 42 days and use as 
indicated.  
Note: If AS-3 is not used, whole blood or red blood cells in CP2D alone may be stored at 1-6 °C 
for up to 21 days. 
 
147126927, issued Jul. 2010 
 
* WBF2 
** During processing, always observe the following precautions: 
1. Sealing should be done in a manner that avoids fluid splatter. 
2. Always dispose of blood-contaminated products in a manner consistent with established 
BIOHAZARD safety procedures. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

 
Study Protocol 
Protocol : 
CLIN-0001 
161 First St, Suite 1A 
Cambridge MA 02142 USA 
Clinical Investigation to Evaluate the New Health 
Sciences Hemanext® Oxygen Reduction System 
for Leukoreduced Red Blood Cells with CP2D 
Anticoagulant and AS-3 Additive - Pivotal Trial 
Date: 
28 Nov 2017 
Author:  Michael Dioguardi 
 
 
Page 60 of 65 
 
 
13.3 
Investigational Product/Hemanext RBC Processing System – Instructions for Use 
Hemanext™ RBC Processing System 
 
CAUTION – Investigational Device. Limited by Federal (or United States) Law to 
Investigational Use. 
 
 
DEVICE DESCRIPTION: 
Blood container set used to process and store CP2D/AS-3 Red Blood Cells, Leukocytes Reduced, and O2/CO2 
Reduced.   
 
Each system consists of: 
• 
One Oxygen Reduction Bag (ORB): empty oxygen impermeable container for processing of 
CP2D/AS-3 Red Blood Cells, Leukocytes Reduced. 
• 
One Hemanext Storage Bag (HSB): empty oxygen impermeable container for storage of 
CP2D/AS-3 Red Blood Cells, Leukocytes Reduced, and O2/CO2 Reduced. 
• 
One LR RBC blood line  
• 
Three flow control blood line clips 
 

 
Study Protocol 
Protocol : 
CLIN-0001 
161 First St, Suite 1A 
Cambridge MA 02142 USA 
Clinical Investigation to Evaluate the New Health 
Sciences Hemanext® Oxygen Reduction System 
for Leukoreduced Red Blood Cells with CP2D 
Anticoagulant and AS-3 Additive - Pivotal Trial 
Date: 
28 Nov 2017 
Author:  Michael Dioguardi 
 
 
Page 61 of 65 
 
 
 
Figure 1: Hemanext RBC Processing System 
 
 
LR RBC blood line 
Clip 
Over sleeve 
Oxygen  
Indicator 
Oxygen Reduction Bag (ORB) 
Hemanext Storage Bag (HSB) 

 
Study Protocol 
Protocol : 
CLIN-0001 
161 First St, Suite 1A 
Cambridge MA 02142 USA 
Clinical Investigation to Evaluate the New Health 
Sciences Hemanext® Oxygen Reduction System 
for Leukoreduced Red Blood Cells with CP2D 
Anticoagulant and AS-3 Additive - Pivotal Trial 
Date: 
28 Nov 2017 
Author:  Michael Dioguardi 
 
 
Page 62 of 65 
 
 
INDICATIONS FOR USE: 
 
Blood container set used to process and store Red Blood Cells Leukocytes Reduced, O2/CO2 Reduced. 
The Hemanext system is intended to process and store CP2D/AS-3 Red Blood Cells, Leukocytes Reduced that have 
been prepared within the standard 8-hour hold time. The Red Blood Cells will be processed using the Hemanext 
system within 12 hours of collection.  The Hemanext RBC Processing System limits the O2 and CO2 levels in the 
storage environment. Red Blood Cells Leukocytes Reduced, O2/CO2 Reduced may be stored for up to 42 days at 1-
6°C.  The Hemanext RBC Processing System is used for volumes no greater than 350 ml of LR RBC.   
  
CONTRAINDICATIONS: 
 
• 
Sickle Cell Trait blood cannot be used for Hemanext RBC processing. 
  
PRECAUTIONS: 
 
• 
The Hemanext RBC Processing System is not intended for manufacture of red blood cells using 
other additive solutions than AS-3. 
• 
Not intended for non-leukocytes reduced red blood cells. 
• 
Not intended for processing of apheresis red blood cells. 
• 
Dispose of used components using acceptable biohazard disposal methods. 
• 
Units must be used within 24-hours of opening individual packaging. 
• 
Studies have not been performed to allow irradiation and/or freezing of Hemanext red blood cells.   
• 
Studies have not been performed to allow cell washing.   
• 
Processing with Hemanext RBC Processing System must be completed with red cells at room 
temperature prior to refrigeration. 
 
 
INSTRUCTIONS FOR USE 
 
 
REMOVAL FROM PACKAGING: 
 
1) Remove Hemanext RBC Processing System from the individual package and inspect for damage.  
Warning: Do not use if package or disposable set is damaged.  
2) Smooth out any kinks observed in the tubing prior to processing.   
3) Close all 3 clips on the LR RBC, the ORB, and the HSB blood lines. 
4) Confirm the color indicators on the ORB and HSB are pink.   
Caution: If an indicator is purple, the product may not be used. 
 
HEMANEXT PROCESSING OF RED BLOOD CELLS: 
 
1) Confirm all clips are closed on blood lines. 

 
Study Protocol 
Protocol : 
CLIN-0001 
161 First St, Suite 1A 
Cambridge MA 02142 USA 
Clinical Investigation to Evaluate the New Health 
Sciences Hemanext® Oxygen Reduction System 
for Leukoreduced Red Blood Cells with CP2D 
Anticoagulant and AS-3 Additive - Pivotal Trial 
Date: 
28 Nov 2017 
Author:  Michael Dioguardi 
 
 
Page 63 of 65 
 
 
2) Ensure incoming CP2D/AS-3 Leukocytes Reduced RBC blood line is not segmented.   (Caution: 
unit must be no more than 350 ml) 
3) Sterile dock the CP2D/AS-3, Leukocytes Reduced RBC bag to the LR RBC blood line.   
4) Confirm that the O2 indicator of the ORB is pink.   
5) At room temperature (22+2°C), hang LR RBC bag at a head height of no greater than 72 inches. 
6) Open clip on LR RBC blood line and ORB blood line.  
7) Pinch the sterile connection on the LR RBC blood line to allow red blood cells to flow into the 
ORB.   
8) Transfer the LR RBC by gravity to the ORB.  
9) Close the clip on the ORB blood line. 
10) Move the clip on the Hemanext LR RBC blood line close to the Y connector.  Close the clip on 
the LR RBC blood line. 
11) Seal and separate the LR RBC blood line between the Y connector and the ORB. 
 
 
 
12) Transfer the Hemanext RBC Processing System to a flatbed agitator shelf at room temperature 
(22+2°C).   
13) When placing on the flatbed agitator shelf, be sure to place no more than two Hemanext RBC 
Processing Systems per shelf with the ORB parallel to the axis of motion and the HSB on top of 
the ORB as shown in the diagram below (see Figure 2). Note: ORB must lay flat on shelf surface 
and ensure HSB and tubing is placed completely within the shelf.   
 
 
      
 
 Figure 2 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

 
Study Protocol 
Protocol : 
CLIN-0001 
161 First St, Suite 1A 
Cambridge MA 02142 USA 
Clinical Investigation to Evaluate the New Health 
Sciences Hemanext® Oxygen Reduction System 
for Leukoreduced Red Blood Cells with CP2D 
Anticoagulant and AS-3 Additive - Pivotal Trial 
Date: 
28 Nov 2017 
Author:  Michael Dioguardi 
 
 
Page 64 of 65 
 
 
Note:  If flatbed agitator shelf is less 36 x 76 x 2.5 cm, please place one Hemanext RBC Processing System 
as shown in Figure 3 below. 
 
 
 
 
 
 
Figure 3 
 
 
 
 
14) Make sure the agitator shelf is fully closed.  If applicable, make sure the incubator doors are 
closed.  Note: limit the amount of times the agitator doors are opened and remain open during 
processing.    
15) Leave Hemanext RBC Processing System on the agitator shelf for 3 hours +/- 15 minutes for 
O2/CO2 reduction.    
16) After 3 hours, remove the Hemanext RBC Processing System from the agitator shelf.  Note: 
confirm that the O2 indicator in the ORB is pink.   
 
 
17) Hang ORB at head height of no greater than 72 inches. 
18) Open clip on the ORB blood line and the HSB blood line.  Transfer the LR RBC O2/CO2 reduced 
into the HSB. 
19) Once all volume has been transferred to the HSB, close the clip on the HSB blood line and heat 
seal the tubing immediately after the white over sleeve section. (Caution: Do not close clip on 
white over sleeve section. Do not heat seal tubing in the white over sleeve section) 
20) Remove the ORB and dispose accordingly. 
21) Segment bloodline as needed. 
22) Place HSB into cold storage of 1 to 6° C for up to 42 days after collection. 
23) When pulling from cold storage, confirm that the O2 indicator in the HSB is pink.   
 
STORAGE AND HANDLING 
• 
Do not use if:  

 
Study Protocol 
Protocol : 
CLIN-0001 
161 First St, Suite 1A 
Cambridge MA 02142 USA 
Clinical Investigation to Evaluate the New Health 
Sciences Hemanext® Oxygen Reduction System 
for Leukoreduced Red Blood Cells with CP2D 
Anticoagulant and AS-3 Additive - Pivotal Trial 
Date: 
28 Nov 2017 
Author:  Michael Dioguardi 
 
 
Page 65 of 65 
 
 
o Tamper evident package has been opened 
o Signs of deterioration are visible  
o The fluid path closures are loose or not intact 
o The package is damaged 
o The ORB, which contains RBC, is dropped on a hard surface (e.g. floor, lab bench, etc.) 
• 
Store at room temperature 
o Avoid exposure to excessive heat 
o Protect from freezing 
 
 
SYMBOL LEGEND 
 
Symbol for Manufacturer 
 
Symbol 
for 
Date 
of 
Manufacture 
 
Lot Number 
 
Symbol for Sterile Fluid Path 
 
Symbol for Single Use Only 
 
Symbol 
for 
Refer 
to 
Instructions for Use 
 
 
 
CONTACT INFORMATION  
 
New Health Sciences, Inc. 
161 First Street, Suite 1A  
Cambridge, MA 02142 
Phone:    781-866-9587 
fieldeval@newhealthsciences.com