appendix c standard operating procedures
calibration - linearity - amr

8.2 Harvesting and Processing Prescription:
8.2.1 F-01 Harvest and Processing Prescription is to be completed and signed by the patient's physician indicating donor and recipient information, mobilization type, processing and storage requirements and collection endpoint.  
8.3 Venous Access:
Peripheral or central venous access will be determined prior to harvest by the 8.3.1 collection staff and the BMT Nurse  Coordinators. This information will be documented on F-01 Harvest and Processing Prescription. The nurse coordinators will arrange catheter placement, if needed, prior to collection. (Refer to section on Central Line Placement, procedure III-6 of BMT Program Clinical SOP's.)
8.4 Patient Information:
The use of ACDA during the apheresis procedure will chelate the patient's 8.4.1 calcium and reduce the ionized and serum calcium level. Therefore, it is necessary to instruct the patient to increase their intake of calcium products to help prevent hypocalcemia. Possible reactions to the collection procedure are:
cold/chills, tingling or numbness, vibration or heaviness in chest, feeling lightheaded, increased pulse rate, shallow respiration, excess perspiration, hives, rash and tissue swelling.  Calcium Gluconate given IV and blankets often decreases the effects of hypocalcemia. The order for calcium gluconate is documented in the patient's electronic medical record.  
8.5 Consent:
8.5.1 Prior to starting the procedure, an informed consent form must be signed by the HPC or MNC APHERESIS USING THE COBE SPECTRA - Version: 1.9. Index: C-01. Printed: 12-Jan-2015 09:09 Authorised on: 09-Jan-2015. Authorised by: Sunii Abhyankar. SOP Unique Reference: 822-51459702. Due for review on: 09-Oct-2016 Author(s): Kathleen Gailard, Dean Merkel Page 9 of 16 12-Jan C01. Printed:
1.9. Index:
APHERESIS USING THE COBE SPECTRA - Version:
6.  
7.  
donor. Donor consent must be obtained prior to the initiation of the recipient's myeloablative treatment. In the case of a minor donor (under the age of 18 years), it is necessary to attain consent from the minor's parent or legal guardian.
appendix c standard operating procedures
8.6 health assessment

8.6.1 8.6.2 Before each apheresis procedure is started, an interim health assessment should be performed by the apheresis staff. This is to monitor the patient's general health, white blood count, hemoglobin, and platelet count and to document any changes in health and medications since the last visit. Documentation of Health Assessment is in the patients electronic medical record.  
The donor evaluation criteria for contacting the BMT physician or ARNP
prior to or during the procedure is:
8.6.2.1 Systolic blood pressure of <90 or >180 8.6.2.2 Diastolic blood pressure of <50 or >105 8.6.2.3 Pulse of <50 or >120 8.6.2.4 RR >28 8.6.2.5 SaO2 <90 8.6.2.6 Temperature of >101F or 38.3℃
8.6.2.7 Obvious symptoms of infection or inflammation at catheter site 8.6.2.8 Platelet count of <50 8.6.2.9 Hemoglobin of <8.0 Vital signs are performed at the completion of each procedure and at any time during the procedure, if needed.  
The in-patient staff BMT physician performs a daily patient H & P evaluation of the donor and evaluates the prior day's collection results, if applicable.  
After each collection procedure, a CBC is performed. Platelet and RBC
transfusions are given per BMT transfusion guidelines. The determination of completion of collections is made by the BMT physician upon notification of the collection results by the Cell Processing lab staff. The donor is advised to follow-up with the BMT Clinic and their BMT Nurse Coordinator with any questions or concerns regarding post procedural care.  
8.6.3 8.6.4  
8.6.5 8.6.6 8.6.7
appendix c standard operating procedures
8.7 calcium replacement 8.7.1

8.7.2 Calcium Gluconate is ordered and infused throughout procedure.  
Rate may be titrated up if patient is having any symptoms of hypocalcemia.
appendix c standard operating procedures
8.8 blood type check 8.8.1

Blood type checks on a peripheral sample are required for the first Allogeneic collection, but are performed on all Autologous and Allogeneic collections. A blood type is performed on the peripheral blood sample when the donor is being harvested. This is to confirm previous blood bank typing or to establish a blood bank record. Any discrepancy is handled by the technologists and must be brought to the attention of the collection staff. Daily blood type validations are confirmed by the Cell Processing Lab.  
8.9 Product:
8.9.1 Labeling: Product must be labeled as outlined in G-03 Identification Protocol.  Examples of labels are attached. 8.9.1.1 HPC bag will be labeled with the following information:
8.9.1.1.1 Unique identifier 8.9.1.1.2 Proper name of the product 8.9.1.1.3 Recipient name and identifier if applicable HPC or MNC APHERESIS USING THE COBE SPECTRA - Version: 1.9. Index; C-01. Printed; 12-Jan-2015 09:09 thorised on: 09-Jan-2015. Authorised by: Sunil Abhyankar. SOP Unique Reference: 822-51459702. Due for review on: 09-Oct-2016 Author(s); Kathleen Gaillard, Dean Merkell,
. Printed:
C-01, Date and time collection ended 8.9.1.1.4 Approximate volume of collection 8.9.1.1.5 Name and volume or concentration of anticoagulant 8.9.1.1.6 8.9.1.1.7 Name and volume or concentration of other additives 8.9.1.1.8 Donor identifier and donor name if applicable 8.9.1.1.9 Identity and address of collection facility 8.9.1.1.10 Recommended storage temperature 8.9.1.1.11 If applicable, Biohazard labels per G-03 Identification Protocol.  
8.9.1.1.12  If applicable, Statement "For autologous use only" if autologous or "For use by Intended Recipient Only" if alogeneic.  
8.9.2 Product Storage 8.9.2.1 Products are to remain hanging on the instrument stored at room temperature until picked up by the Cell Processing Lab staff.  
8.9.2.2 Products are to be picked up prior to the patient leaving the room.  
8.9.2.3 Product label/patient identification is again verified and is documented on the cell processing worksheet prior to transport of the product to the cell processing lab.  
8.9.2.4 If a delay is anticipated, the apheresis staff may verify label/patient identification and transport of the product to the cell processing lab on completion of procedure.  
Transportation:
8.9.3.1 Product must be transported to the Processing Laboratory as outlined in G-09 Transportation Policy for HPC within the Facility.  
8.9.3.2 HPC's will be transported from the apheresis room to the cell processing laboratory at ambient temperature.  
8.9.3.3 Sealing - The collection container should be sealed in a manner that minimizes the risk of cell loss and microbial contamination. Seal with heat sealer. Heat seal a minimum of three times.  
8.9.3.4 Secondary container (cover bag) -The primary container should be placed in a secondary plastic bag and sealed to prevent leakage. Use zip-lock bags or other plastic bag that can contain any leakage and prevent exposure.  
8.9.3.5 Outer container (small cooler) - The outer container should be made of material adequate to withstand leakage of contents, shock, and other incidents to ordinary handling in transportation. Use a small Igloo cooler
(or equivalent) from the Cell Processing lab to transport the components.  
Expiration date of fresh products:  Fresh products are best used within 72 8.9.4 hours from time of completion of harvest. The patient's physician must approve release of the product if this time is exceeded.  
8.9.5
appendix c standard operating procedures
release and exceptional release

8.9.5.1 Criteria for product release Product label must be reviewed by two staff upon delivery of 8.9.5.1.1 poduct.  
Product must be labeled appropriately as described in G-03 8.9.5.1.2 Identification Protocol.  
8.9.5.1.3 Product label must be complete. Biohazard positive labeling must be present as described in G-
8.9.5.1.4 HPC or MNC APHERESIS USING THE COBE SPECTRA - Version: 1.9. Index: C-01. Printed: 12-Jan-2015 09:09 Authorised on: 09-Jan-2015. Authorised by: Sunil Abhyankar. SOP Unique Reference: 822-51459702. Due for review on: 09-Oct-2016.  
Author(s): Kathleen Gaillard, Dean Merkel Page 11 of 16
: 12-Jan-
C-01. Printed:
Version: 1.9, Index; 8.9.3 03 Identification Protocol.  
Product must not exceed expiration date.  
8.9.5.1.5 8.9.5.2 Exceptional release 8.9.5.2.1 8.9.5.2.2 If criteria for release are not verified, F-12 Deviation Form must be completed as per G-05 Deviation from Policy-
Procedure.  
The patient's physician must be notified before processing or infusion.  The decision to release/accept the unit is to be made by the attending physician.  
8.10 Procedure Duration, Objectives, & Acceptable Endpoints:
8.10.1 Each collection will be continued until prescribed blood volumes have been processed.  Extended collections may be performed if requested by the patient's transplant physician. A BMT physician will see the patient daily and monitor the patient's collections. A physician will be available by pager to respond to any patient problems or patient concerns.  
8.10.2  The objective of the Leukapheresis procedure is to harvest an adequate number of stem cells to accomplish adequate engraftment of the hematopoietic system for the HPC transplant recipient after myeloablative therapy. The collections should result in a viable, microbial free, CD34 enriched peripheral mononuclear poduct.  
8.10.3 See prescription for individual collection end points.  
8.10.4 If collection and processing end-points are not met, the transplant physician will be notified and remedial plans will be documented in the patient's medical record. The transplant physician or the nurse clinician will communicate with the patient concerning results.
appendix c standard operating procedures
review of collection records

8.11.1  Each collection day, the patient's procedure flowsheet is reviewed by the BMT
Apheresis Manager for accuracy and completion. Review is documented on the back of the sheet.  
8.11.2  Each collection day, the in-patient staff physician performs a patient evaluation and reviews the previous day's collection. This is documented in the patient's electronic medical record.  
8.11.3  At the completion of all collections, the BMT staff physician will review the summary of all the collections and documents on F-04 Stem Cell Data Summary Sheet- Allogeneic or F-05 Stem Cell Data Summary Sheet-Autologous whether the processing end-point was met. If the end-point was not met, remedial action taken is to be documented on the summary or in the patient's electronic medical record.  
8.11.4 .
appendix c standard operating procedures
8.12 blood warmer

8.12.1  The fluid warming set is a single use sterile device, designed to warm blood and intravenous fluid and deliver them the patient's intravenous return site at normothermic temperatures. Refer to C-05 Astotherm Blood Warmer Use with Spectra for instructions.
appendix c standard operating procedures
8.13 adverse reactions

8.13.1  F-09 Harvest - Problem and Adverse Reactions Form must be completed noting any problems or adverse reactions encountered during the harvest. All actions taken and follow-up must be documented on this form. F-09 Harvest - Problem HPC or MNC APHERESIS USING THE COBE SPECTRA - Version: 1.9, Index; C-01. Printed; 12-Jan-2015 09:09 prised on: 09-Jan-2015. Authorised by; Sunii Abhyankar. SOP Unique Reference: 822-51459702. Due for review on: 09-Oct-2016 Author(s): Kathloon Gallard, Dean Merkel Page 12 of 16 1.9. Index: C-01. Printed: 12-Jan-
SING THE COBE SPECTRA
8.11 and Adverse Reactions are to be reviewed by the BMT staff physician within 48 hours and filed in the patient's chart.  
8.14
appendix c standard operating procedures
disposables and fluid records

8.14.1 All disposables and fluids are sterile and checked for integrity prior to use.  
Records of all lot numbers and expiration dates of all disposables and fluids are recorded on procedure flowsheet.  
8.15 Donor Follow-up:
8.15.1 All donors are given post donation instructions.  
8.15.2 For all autologous donors: if the patient is in need of care post-collection, they are followed and treated in the BMT clinic. The patient is referred back to the BMT clinic for follow-up regarding any post apheresis concerns or complications.  
8.15.3  Follow-up of allogeneic donors is done by the apheresis staff 2-5 days post collection.  
Documentation is on F-42 Allogeneic Donor Post Collection Follow8.15.3.1 up and placed into the follow-up call binder.  
8.15.3.2 Follow-up calls are logged in the patient collection log spreadsheet.  
8.16 Biohazard Waste:
8.16.1 Biohazard waste must be placed into the appropriate biohazard container, (eg, plastic boxes for sharps, red bags for tubing, etc.) All biohazard waste is disposed of according to KU policy.  
8.17 Backup Instrument:
8.17.1  If a backup or extra Spectra is needed due to an instrument malfunction or multiple patients, contact the Inpatient Dialysis Department (8-6069) to request the use of one of their instruments.  
8.18
appendix c standard operating procedures
disaster preparedness

8.18.1 Refer to SOP G-15 Emergency and Disaster Preparedness 8.19 Cleaning and Sanitation 8.19.1 Environmental Services cleans the floors and removes trash on a daily basis.  
The COBE Spectra exterior surfaces and the patient's chairs are to be disinfected with Germicidal wipes or other hospital approved and provided cleaner after each procedure.
appendix c standard operating procedures
9. references

9.1 Gee, AP: Bone Marrow Processing and Purging: A Practical Guide. Boca Raton, FL. CRC
Press 1991 9.2 Cobe Spectra Apheresis System Operator's Manual. Lakewood, CO. Terumo, BCT
10. APPENDICES: FORMS - LOGS - WORKSHEETS:
F-01 Harvest and Processing Prescription 10.1 F-09 Harvest - Problem and Adverse Reactions Form 10.2 F-12 Deviation Form 10.3 F-42 Allogeneic Donor Post Collection Follow-up 10.4 Document Control History 10.5 HPC or MNC APHERESIS USING THE COBE SPECTRA - Version: 1.9. Index: C-01. Printed: 12-Jan-2015 09:09 Authorised on: 09-Jan-2015. Authorised by: Sunil Abhyankar. SOP Unique Reference: 822-51459702. Due for review on: 09-Oct-2016 Author(s): Kathleen Gaillard, Dean Merkel Page 13 of 16 ESIS USING THE COBE SPECTRA - Version: 1.9. Index; C-01. Printed: 12-Jan-
Appendix: SOP HPC or MNC APHERESIS USING THE COBE SPECTRA
appendix c standard operating procedures
appendix links

Please note: links are only correct at time of printing Linked to Controlled Document Printed: 12-Jan-2015 09:09 COL
Version: 1.9. Index:
I USING THE COBE SPECTRA
- SOP: G-03: Identification Protocol v2.0 (Superseded)
- SOP: P-21: Microbial Contamination v1.3 (Superseded)
- SOP: G-09: Transportation of Non-Frozen Biood and Marrow Products to and from an Outside Institution v1.7 (Superseded)
- Policy: G-15: Emergency and Disaster Preparedness Policy vl.O (Authorised)
- Policy: G-05: Deviations Policy v1.5 (Authorised) - SOP: C-05: Astotherm Blood Warmer Use v1.3 (Authorised)
- Document: F-01: F-01 Harvest and Processing Prescription v1.9 (Authorised)
- Document: F-09: F-09 Apheresis - Problem and Adverse Reaction Form v1.8 (Authorised)
- Document: F-12: F-12 DEVIATION FORM v1.4 (Authorised)
- Document: BMT Apheresis & Cell Processing Lab 511: F-42 Allogeneic Donor Post Collection Follow-up v1.0 (Authorised)
- SOP: C-01: Leukapheresis Procedure v1.6 (Superseded) - SOP: G-03: Identification Protocol v2.1 (Superseded) - SOP: P-21: Microbial Contamination v1.4 (Authorised)
- SOP: G-03: Identification Protocol v2.2 (Authorised)
- SOP: G-09: Transportation of Non-Frozen Blood and Marrow Products to and from an Outside Institution v1.8 (Authorised)
- SOP: C-01: Leukapheresis Procedure v1.8 (Superseded)
appendix c standard operating procedures
document revision history

Superseded on 09-Jan-2015 16:53 by Sunil Abhyankar Version 1.8 superseded by version 1.9 Authorized on 09-Jan-2015 16:53 by Sunil Abhyankar Authorized version 1.9 - Revised SOP. The following users will be notified when a review is due for this document: Dean Merkel Authorisation Requested on 09-Jan-2015 16:43 by Shaun Dejarnette Authorisation request sent to Sunil Abhyankar by Shaun Dejarnette on 09-Jan-2015 16:43.  
Draft Created on 09-Jan-2015 13:42 by Dean Merkel Reason: Removed the run worksheet and added EMR documentation.  
HPC or MNC APHERESIS USING THE COBE SPECTRA - Version: 1.9. Index; C-01. Printed; 12-Jan-2015 09:09 Authorised on: 09-Jan-2015. Authorised by: Sunil Abhyankar. SOP Unique Reference: 822-51459702. Due for review on: 09-Oct-2016.  
Author(s): Kathleen Gaillard, Dean Merkel Page 14 of 16 Appendix: SOP HPC or MNC APHERESIS USING THE COBE SPECTRA
appendix c standard operating procedures
authorised on 16-aug-2014 2320 by

Authorised version 1.8 - Additional changes due to FACT. . The following users will be notified when a review is due for this document: Shaun Dejarnette, Dean Merkel
appendix c standard operating procedures
authorisation requested on 15-aug-2014 1315

Authorisation request sent to Sunil Abhyankar by Shaun Dejarnette on 15-Aug-2014 13:15.
appendix c standard operating procedures
authorisation requested on 15-aug-2014 1314

Authorisation request sent to Sunil Abhyankar by Shaun Dejarnette on 15-Aug-2014 13:14.
appendix c standard operating procedures
peer review requested on 15-aug-2014

Peer Review tasks were assigned to the following users: Shaun Dejarnette.  
This review is to be completed by 15-Aug-2014
appendix c standard operating procedures
authorised on 14-aug-2014 1527 by

Authorised version 1.7 - SOP updated post FACT inspection to include critical reagents, supplies etc. and product storage time in Apheresis. The following users will be notified when a review is due for this document: Shaun Dejarnette, Dean Merkel
appendix c standard operating procedures
authorisation requested on 14-aug-2014 1328

Authorisation request sent to Sunil Abhyankar by Shaun Dejarnette on 14-Aug-2014 13:28.  
HPC or MNC APHERESIS USING THE COBE SPECTRA - Version: 1.9. Index; C-01. Printed: 12-Jan-2015 09:09 Authorised on: 09–Jan-2015. Authorised by; Sunii Abhyankar, SOP Unique Reference; 822-51450702. Due for review on: 09-Oct-2016.  
Author(s); Kathloen Gallard, Dean Merkell, Page 15 of 16  
  
Appendix: SOP HPC or MNC APHERESIS USING THE COBE SPECTRA
appendix c standard operating procedures
review task completed on 14-aug-2014

Shaun Dejarnette completed task, "Review complete - no changes needed."
Peer Review Requested on 14-Aug-2014 13:20 by Dean Merkel Peer Review tasks were assigned to the following users: Shaun Dejarnette.  
This review is to be completed by 15-Aug-2014
appendix c standard operating procedures
draft created on 14-aug-2014 1317

Reason: Updates to procedure post FACT audit.  
Updated critical supplies reagents equipment.  
Updated storage time in apheresis.  
Authorised on 13-Jan-2014 17:34 by Sunil Abhyankar Authorised version 1.6 - . The following users will be notified when a review is due for this document:
Shaun Dejarnette, Dean Merkel Authorisation Requested on 07-Jan-2014 16:56 by Shaun Dejarnette Authorisation request sent to Sunil Abhyankar by Shaun Dejarnette on 07-Jan-2014 16:56.  
Review Task Completed on 07-Jan-2014 16:10 by Shaun Dejarnette Shaun Dejarnette completed task, "Reviewed and okayed." Creation on 19-Nov-2013 11:09 by Kathleen Gaillard New SOP created Peer Review Requested on 19-Nov-2013 11:09 by Kathleen Gaillard Peer Review tasks were assigned to the following users: Shaun Dejarnette. This review is to be completed by 27-Nov-2013.  
HPC or MNC APHERESIS USING THE COBE SPECTRA - Version: 1.9. Index; C-01. Printed: 12-Jan-2015 09:00 Authorised on: 00-Jan-2015. Authorised by: Sunil Abhyankar. 90P Unique Reference: 822-51459702. Due for review on: 00-Oct-2016.  
Author(s); Kathloan Gailard, Dean Market Page 16 of 16 DEPARTMENT OF PATHOLOGY AND LABORATORY MEDICINE
Title:
DETERMINATION OF PERIPHERAL CD34 COUNT  
| Index:            | C-07                                |
|-------------------|-------------------------------------|
| Author:           | Kathleen Gaillard and Dean Merkel   |
| Department Name:  | BMT Apheresis & Cell Processing Lab |
| Category:         | BMT COLLECTION                      |
| Document Type:    | SOP                                 |
| Version:          | 1.2                                 |
| Document Status:  | Authorised                          |
| Authorized By:    | Sunil Abhyankar                     |
| Date Authorized   | 13-Jan-2014                         |
| Date Printed:     | 18-Mar-2015 14:29                   |
| Location Of Copy: | BMT Apheresis                       |
| Distributed to:   | KU Hospital                         |  
  
DETERMINATION OF PERIPHERAL CD34 COUNT  - Version: 1.2. Index: C-07. Printed: 18-Mar-2015 14:29 Athorised on: 13- Jan-2014. Authorised by: Sunil Abhyankar. SOP Unique Reference: 822-53040439. Due for review on: 08-Oct-2016 Author(s); Kathleen Gaillard, Dean Merkel 1.2. Index: C-07. Printed; 18-Mail
appendix c standard operating procedures
c-07 determination of absolute peripheral

1.   PRINCIPLE - CLINICAL SIGNIFICANCE
1.1. The peripheral blood CD34 cell percentage may be predictive of the total yield of hematopoietic progenitor cells and may be used to determine when to initiate apheresis.  
1.2. Absolute CD34+ cell content of the peripheral blood is determined by performing a CD34 flow analysis on a peripheral blood sample and multiplying the percent CD34(+)
by the peripheral blood WBC count.  
2. SPECIMEN:
2.1. Peripheral Blood 3.   REAGENTS - SUPPLIES - EQUIPMENT: n/a 4.  QUALITY CONTROL: n/a 5.   PROCEDURE
2015 C-07. Printed: 18-Mar-  
  
6.   CALCULATIONS - INTERPRETATION - RESULT REPORTING - NORMAL VALUES -  
CRITICAL VALUES: n/a
7.   CALIBRATION - LINEARITY - AMR - CALIBRATION VERIFICATION: n/a 8.   PROCEDURE NOTES - LIMITATIONS
8.1. Suggested peripheral absolute CD34 Count / μ l for a successful collection is >20.  
8.2. Counts from 10 - 20 are acceptable to initiate collection.  
8.3. Collections from patients with counts <10 may be poor. However, in patients that have received large doses of chemotherapy previously, counts of 5 –10 may be the highest value reached and may be acceptable to initiate collection. A BMT physician should be consulted in this circumstance.
appendix c standard operating procedures
9. references n/a

10. APPENDICES: FORMS - LOGS - WORKSHEETS:
Document Control History 10.1.  
  
Index:
1.2.1 COUNT - Version
appendix c standard operating procedures
appendix document revision history

Document Reviewed on 05-Jan-2015 16:02 by Dean Merkel Review date set to 08-Oct-2016 - Biennial review.. The following users will be notified when a review is due for this document:
Dean Merkel.  
This document was originally due for review on 09-Jan-2015.
appendix c standard operating procedures
review task completed on 05-jan-2015

Sunil Abhyankar completed task, "Reviewed SOP "
Peer Review Requested on 05-Jan-2015 13:38 by Dean Merkel Printed:
Peer Review tasks were assigned to the following users: Sunil Abhyankar.  
This review is to be completed by 10-Jan-2015 00000 Authorised on 13-Jan-2014 17:20 by Sunil Abhyankar Authorised version 1.2 - . The following users will be notified when a review is due for this document:
Shaun Dejarnette, Dean Merkel COM
Authorisation Requested on 09-Jan-2014 13:16 by Shaun Dejarnette Authorisation request sent to Sunil Abhyankar by Shaun Dejarnette on 09-Jan-2014 13:16.  
Review Task Verified on 09-Jan-2014 13:16 by Shaun Dejarnette Shaun Dejarnette verified review task: ** Notes: Reviewed and approved.  
Review Task Completed on 09-Jan-2014 13:16 by Shaun Dejarnette Shaun Dejarnette completed task, "Reviewed and approved."
appendix c standard operating procedures
peer review requested on 09-jan-2014

Peer Review tasks were assigned to the following users: Shaun Dejarnette.  
This review is to be completed by 10-Jan-2014 DETERMINATION OF PERIPHERAL CD34 COUNT  - Version: 1.2. Index; C-07. Printed: 18-Mar-2015 14:29 Authorised orc 13-Jan-2014. Authorised by: Sunii Abhyankar. SOP Unique Reference: 822-53040439. Due for review orc 08-Oct:2016 Author(s); Kathleen Gaillard, Dean Merkell Page 2 of 3  
  
Appendix: SOP DETERMINATION OF PERIPHERAL CD34 COUNT
appendix c standard operating procedures
change request approved on 09-jan-2014

Kathleen Gaillard approved change request: "Re-evaluate righ-sided margins - text runs off the page.  
Change Requested on 07-Jan-2014 17:20 by Shaun Dejarnette Shaun Dejarnette requested a change: "Re-evaluate righ-sided margins - text runs off the page.  
Creation on 19-Nov-2013 13:19 by Kathleen Gaillard New SOP created 14:29 2015-2015 Printed: 18-Mar C07.  
. CD34 COUNT
Peer Review Requested on 19-Nov-2013 13:19 by Kathleen Gaillard Peer Review tasks were assigned to the following users: Shaun Dejarnette.  
This review is to be completed by 27-Nov-2013 DETERMINATION OF PERIPHERAL CD34 COUNT  - Version: 1.2. Index: C-07. Printed: 18-Mar-2015 14:29 Authorised on: 13-Jan-2014. Authorised by: Sunii Abhyankar. SOP Unique Reference: 822-53040439. Due for review on: 08-Oct-2016 Author(s): Kathleen Gailard, Dean Merkel Page 3 of 3 DEPARTMENT OF PATHOLOGY AND LABORATORY MEDICINE
Title:
Processing and Preparation of HPC or MNC, Apheresis for Freezing  
| Index:            | P-01                                |
|-------------------|-------------------------------------|
| Author:           | Kathleen Gaillard and Dean Merkel   |
| Department Name:  | BMT Apheresis & Cell Processing Lab |
| Category:         | BMT Processing                      |
| Document Type:    | SOP                                 |
| Version:          | 1.6                                 |
| Document Status:  | Authorised                          |
| Authorized By:    | Sunil Abhyankar                     |
| Date Authorized   | 09-Jan-2015                         |
| Date Printed:     | 12-Jan-2015 09:23                   |
| Location Of Copy: | BMT Apheresis                       |
| Distributed to:   | KU Hospital                         |  
  
Processing and Preparation of HPC or MNC, Apheresis for Freezing - Version: 1.6. Index; P-01. Printed: 12-Jan-2015 09:23 uthorised on: 09-Jan-2015. Authorised by: Sunil Abhyankar. SOP Unique Reference: 822-51459940. Due for review on: 09-Oct-2016 Author(s): Kathleen Gallard, Dean Merkel 1.6. Index: P-01. Printed: 12-Jan-2 MNG,
appendix c standard operating procedures
p-01 processing & preparation of

1.   PRINCIPLE - CLINICAL SIGNIFICANCE
1.1. The peripheral hematopoietic progenitor cells (HPCs) or mononuclear cells (MNCs) are collected and processed for freezing. The goal in this procedure is to concentrate the mononuclear cells collected from a leukapheresis procedure and safely cryopreserve them while maintaining their viability and integrity.  Cryopreservation is required to preserve the viable hematopoietic progenitor cells for later transplant following myeloablative or submyeloablative therapy or MNCs for other therapies.  
1.2. Dimethylsulfoxide (10%) is the cryoprotectant used to reduce the freeze-thaw injury to these clls.  
1.3. Cryopreservation specific variables including the concentration of cells frozen, concentration of cryoprotectant, electrolyte medium, and the cryopreservation rate must be consistent to properly preserve cells.  
2. SPECIMEN
2.1. Hematopoietic Progenitor Cells or HPC, Apheresis 2.2. Mononuclear Cells or MNC, Apheresis 3.   REAGENTS - SUPPLIES - EQUIPMENT
3.1. Centrifuge with large buckets to handle 600 ml transfer packs 3.2. Balance 3.3. Expresser 3.4. Laminar flow hood 3.5. Blood tube sealer 3.6. Control rate freezer with temperature probe 3.7. Freezer canisters and frames 3.8. Freezer racks 3.9. Liquid (nitrogen) storage container Cryopreservation freezing bags: 50,250,500 ml size (Origen or Miltenyi)
3.10.  
Dimethyl sulfoxide (DMSO)
3.11.  
Plasmalyte A
3.12. 3.13.  
600 mL transfer packs 400 mL transfer packs 3.14.  
140 cc, 60 cc & 3 cc syringes 3.15.  
Sterile over-wrap bag 3.16.  
3.17.  
Centrifuge bag "4R4424" Hemostats, scissors, alcohol & betadine pads and marking pens 3.18.  
Female coupler adapters 3.19.  
Gloves and 18 gauge blunt fill needles 3.20.  
3.21.  
Liquid nitrogen 3.22.  
Sample site couplers Sterile culture tubes 3.23.  
Microbial bottles (aerobic and anaerobic)
3.24.
appendix c standard operating procedures
4. quality control n/a

5. PROCEDURE
5.1. Use sterile technique throughout procedure.  Perform all procedures with a biosafety cabinet.  
Processing and Preparation of HPC or MNC, Apheresis for Freezing - Version: 1.6. Index; P-01. Printed; 12-Jan-2015 09:23 Authorised on: 09-Jan-2015. Authorised by: Sunii Abhyankar. SOP Unique Reference: 822-51459940. Due for review on: 09-Oct-2016.  
Author(s): Kathleen Gaillard, Dean Merkel Page 1 of 8 Printed: 12-Jan-2015.  
P-01.  
1.  
tion of HPC or MNC, Apheresis for Freezing - 1 Spike collection bag port with a coupler and remove 2 ml of product with a 3ml syringe.  Mix cells well in the bag before removing the 2 ml.  Dispense 0.5 ml of the cells from the syringe into microbial culture each bottle for sterility test. Add remaining cells to a culture tube for cell count, viability and flow cytometry assays.  The cell count is done in the Hematology laboratory according to their current method.  
5.2. Label the appropriate number of freezer bags and canisters with patient name, patient number, patient DOB, date, product id \#, contents and any other requirements as outlined in the Identification Protocol (G-03). All label checks must be performed by 2 staff prior to product transfer.  
5.3. QUARANTINE: Products not meeting eligibility requirements should be stored in a designated quarantine area of the isothermal cryo-storage tanks. Quarantine areas are designated on diagrams posted on the front of the tanks. An inventory list of all quarantine products can be accessed and printed for review.  
Note: see Analyzing Components section at the end of the procedure.  
5.4. Transfer stem cell collection into a transfer pack.  Heat seal and remove the excess tubing.  
5.5. The products are double bagged for centrifugation. Place the transfer pack into a sterile overwrap bag. Then place this into a "4R4424" centrifuge bag.  
5.6. Balance the cell product in the Hettich 460RS centrifuge and centrifuge at 2420 rpm for 10 minutes or the Jouan at RPM of 2522) for 10 minutes.  The temperature should be set at 20 degrees centigrade.  
5.7. During the centrifugation time mix the 10 ml of DMSO and the 30 ml of Plasmalyte A.  This can be done through one of the middle ports in the 500 ml cryo-freezer bag that has a 60 ml syringe attached.  The plunger is removed from the syringe and the syringe can be used as a measuring device. Close the port with its clamp. Verify lot numbers on the daily processing reagent log.  
5.8. Chill mixture in monitored BB refrigerator or between cool packs.  
It is desired to have a final concentration of cells in the cryo-bag of <3.0 x 10®
5.8.1.  
cells/ml. If the nucleated cell count is >1.5 x 10¹⁰ and <3.0 x 10¹⁰, use the above volumes of mixture. This will give a final cell concentration of <3.0 x 10' cells/ml.  
lf nucleated cell count is >3.0 x 10¹⁰, double the mixture and divide the 5.8.2.  
products to be frozen into 2 - 500 ml cryo-bags. If the cell count is >6.0 x 10 10 ,
triple the mixture and divide the products into 3 - 500 ml cryo-bags, etc.  This will give a final cell concentration of <3.0 x 10 8 cells/ml.  
5.8.3.  
Optional: Use the table below to determine final volume per bag, volume of reagents and size of cryo-bags to use for various total cell counts.  
| Total Cell Count              | DMSO             | Plasma   | Cells &   | Final      | Bag Type   | Final     |     |
|-------------------------------|------------------|----------|-----------|------------|------------|-----------|-----|
| mis                           | Lyte A           | Plasma   | Volume    | & Number   | Volume     |           |     |
| mis                           | mis              | mis      | of Bags   | mls/bag(s) |            |           |     |
| <1.5 x 10 10                  | 15               | 30       | 50        | 1 - 250ml  | 50         |           |     |
| 2.5                           | 45               | 75       | 75        |            |            |           |     |
| >1.0 x 10 10                  | and <2.0 x 10 10 | 7.5      | 1 - 500ml |            |            |           |     |
| >1.5 x 10 "                   | and <3.0 x 10 ₪  | 10       | 30        | 60         | 100        | 1 - 500ml | 100 |
| >3.0 x 10 10 and <6.0 x 10 10 | 2 - 500ml        |          |           |            |            |           |     |
| 20                            | 60               | 120      | 200       | 100        |            |           |     |
| >6.0 x 10 10 and <9.0 x 10 10 | 90               | 180      | 300       | 3 - 500ml  | 100        |           |     |
| Optional                      |                  |          |           |            |            |           |     |
| >3.0 x 10 10                  | 15               | 45       | 150       | 2 - 250ml  | 75         |           |     |
| ' and <4.5 x 10 "             | 90               |          |           |            |            |           |     |  
5.9. 5.10.  
Also during this time the freezer chamber should be pre-cooled. The instructions for using the freezer are in the procedure on Controlled Rate Freezing.  
Fill out the completion of processing label and canister per Identification Protocol.  
Processing and Preparation of HPC or MNC, Apheresis for Freezing - Version: 1.6. Index; P-01. Printed: 12-Jan-2015 09:23 sthorised on: 09-Jan-2015. Authorised by: Sunil Abhyankar. SOP Unique Reference: 822-51459940. Due for review on: 09-Oct-2016.  
Author(s); Kathleen Gallard, Dean Merkel Page 2 of 8 5.11. Fill out the stem cell processing worksheet with the necessary information.  
When the product has finished centrifugation, attach a transfer pack to a port.  Using a 5.12.  
plasma expresser in a biosafety cabinet, expel most of the plasma until approximately 20-30 mls of specimen is left. Clamp line with hemostat. Mix the cell and plasma remaining in the bag thoroughly.  
Spike a port with a plasma transfer coupler and attach a 60 or 140 cc syringe. Draw out the 5.13.  
cells and plasma mixture.  Unclamp hemostats to the plasma bag and rinse out bag with approximately what is needed to fill the syringe to 62 mls.  
5.14.  
The Plasmalyte A & DMSO mixture should be pre-cooled until the concentrated cells are available.  
Pre-cool the cell product with refrigerated cool packs prior to addition of cells to the pre5.15.  
cooled freeze medium. The syringe of the concentrated cells is added to the labeled freezer bag containing the DMSO and Plasmalyte A mixture through the second port in the center of the bag and mix well.  
Let the syringe remain attached to the bag. After mixing the contents well, remove all the air 5.16.  
in the bag.  
5.17.  
NOTE: It is important not to delay freezing after the cells have been added to the DMSO mixture.  If a delay is over 10 minutes, place the cells-DMSO mixture on cold packs.  DMSO may be toxic to the cells at room temperature.  
5.18.  
Approximately 0.5 mls of this mixture is to remain in the cryobag pigtail and remain attached to the bag to be frozen with the stem cell product.  These may be used for further testing if desired.  
5.19.  
Sterility checks are performed at this time.  Aseptically add approximately 0.25 - 0.5 ml to each microbial culture bottle for POST PROCESSING sterility.  
The bag is then heat sealed 2-3 times near the base. Check bag for leaks and place in the 5.20.  
labeled metal canister.  The canister is then placed in the freezer with the sensor probe in place.  
5.21.  
Control Rate Freezer(CRF)
5.21.1. Input patient and product data into the CRF.  
5.21.2. Cool down the freezing chamber before putting in the sample.  The controller will hold the freezer at a constant temperature until ready to start freezing the sample 5.21.3. Pre-cool the freezer CBS
Enter the patient information and start pre-cool as described in the Control rate 5.21.3.1.  
freezing procedure (P-08 CONTROLLED RATE FREEZING AND STORAGE).  
Open precooled freezing chamber and place the canisters in the freezing 5.21.3.2.  
chamber with temperature probe inserted in the center hole of the canister in cntact with the product.  
Close chamber door. Press "continue" and the freeze program will continue.  
5.21.3.3. 5.21.3.4.  
The program will run without assistance until sample temperature reaches -80°C.  
5.22.  
End of Freeze 5.22.1. When the product reaches -80 degrees Celsius it is ready to be put in liquid nitrogen storage tank at <160℃. Press the stop keypad and transfer the cryopreserved product to the storage tank.  Quarantine products should be stored in designated locations.  
5.22.2. Record the starting and stopping times of the freeze on the processing worksheet.  
5.22.3. The freezer can be thawed using the warm program. This is not necessary unless the freezer is to be used again shortly.  
5.22.4. The control rate freezing curve should be reviewed for the proper freeze and electronically saved with the patients name, date and type of specimen. Document review on the processing worksheet. Any disruption in the freezing or abnormal freezing curve should be documented on a Deviation Form.  
5.22.5. An alphabetical inventory system, see Cryopreserved HPC Inventory Procedure (P-08).  
Processing and Preparation of HPC or MNC. Apheresis for Freezing - Version: 1.6. Index; P-01. Printed; 12-Jan-2015 09:23 Authorised on: 09-Jan-2015. Authorised by: Sunil Abhyankar. SOP Unique Reference: 822-61459940. Due for review on: 09-Oct-2016.  
Author(s): Kathleen Gallard, Dean Merkell Page 3 of 8 Version: 1.6. Index: P-01. Printed: 12-Jan-2015 1 Patient's information should be entered into this system after their components are processed.  The assigned inventory numbers, tank number and date should be entered into this system by the patient's name, social security and medical record number.  The reinfusion dates should be documented when the specimen is returned to the patient.  
5.23.
appendix c standard operating procedures
analyzing components

5.23.1. Testing: All samples for lab testing must be labeled with the donor's name, MR\#, date, and product identifier.  
5.23.2. Cell Count An aliquot of the peripheral stem cell collection is taken to the Hematology lab 5.23.2.1.  
and for a cell count.  The WBC count result of the sample is multiplied by the milliliters of the collection bag after apheresis.  This will give the total nucleated cell count for the harvest.  A smear is made and Wright stained. A differential is performed and the total mononuclear cell count is calculated.  All documentation of results and calculations are made on the processing worksheet.  
5.23.3. Flow Analysis An aliquot of the peripheral cell collection is taken to the Flow Cytometry Lab 5.23.3.1.  
where a CD34 or other flow analysis is performed.  
CD34 or other flow analysis is performed and the result is given as a percentage 5.23.3.2.  
of the total cells.  This value is multiplied by the total nucleated cell count and the total CD34+ or CD3+ cell count is determined for that harvest.  
Divide this result by the patient or recipient's weight in kilograms and the cells /
5.23.3.  
kilogram is calculated.  Enter the CD34 results into the Mysis lab computer system.  It is ordered and resulted as the test code CD34.  
5.23.4. Microbial Detection A sample (0.25 - 0.50 mls) of the collection is added to a microbial vial for sterility 5.23.4.1.  
testing.  The microbial vial is placed in an incubator at 37 degrees Celsius for a maximum of 14 days. The cultured samples are continually monitored for microbial growth. All cultures are sent to and processed by the Microbiology lab for tested for organism presence, identification, and sensitivity.  
5.23.4.2.  
The BMT physician is notified immediately of the positive culture.  Any further action with the patient will be dictated by the physician. Complete a deviation form for any positive cultures.  
5.23.4.3.  
All results and physician notification is documented in the lab computer. This information should also be entered into the patient inventory of the freezer inventory book under the comment section.
appendix c standard operating procedures
6. calculations - interpretation -

6.1. Flow Analysis:
6.1.1.   CD34% on the PSC collected sample: >0.10% 6.1.2. Percent recovery of CD34+ cells on a selected manipulated sample: >20%
6.2. Microbial Detection: 100% non-contaminated Notify the BMT physician immediately of the positive culture. Contaminated 6.2.1.  
products do not necessarily need to be destroyed. Decisions regarding the disposition of culture positive HPC's must be made by patient's transplant physician after considering the type of contamination and the potential benefits and the risks from the use of the product. Complete a Deviation Form on positive cultures.  
6.3. Viability: viability checks: >90% viable 6.4. Cell counts:
rocessing and Preparation of HPC or MNC. Apheresis for Freezing - Version: 1.6. Index; P-01. Printed; 12-Jan-2015 09:22 Athorised on: 09-Jan-2015. Authorised by; Sunil Abhyankar, SCP Unique Reference; 822-51459940. Due for review on: 09-Oct-2016 Author(s); Kathleen Gaillard, Dean Merkel Page 4 of 8 s for Freezing - Version: 1.6. Index: P-01. Printed: 12-Jan-2015 (
HC or MNC, Aphere  
6.4.1. Cell counts are useful in determining the absolute CD34 count, but there are no expected results. Total nucleated cell count results generally range from 1.0 x 10 10 to 10.0 x 10 10 cells.
appendix c standard operating procedures
7. calibration - linearity -

8.1.1. Products may be stored until processing. Components may be kept overnight for next day processing. Except for components held for CD34 selection, processing should be performed within 24 hours. Components held for CD34 selection should be processed within 36 hours, but may be longer in the event of an international product. All components must be stored between 2°-8°C in the Ste m Cell Processing lab's monitored blood bank refrigerator.  
8.2. Disposables and Freezing Mixture Records:
8.2.1. All disposables and fluids are sterile and checked for integrity prior to use. Record all disposables and fluids used on the processing worksheet. Lot numbers and expiration dates of all disposables and fluids will be listed on the daily supply log.  
Only validated freezing bags designed for HPC freezing and storage are to be used.  
8.2.2.  
8.3. DMSO:
8.3.1.  
The most widely used cryoprotectant is dimethyl sulfoxide (DMSO), although it has not been formally been approved by the FDA for this use. DMSO is cited in medical literature as the most common freezing mixture for cryopreserving hematopoietic cells.  
Adverse reactions to this agent can occur, particularly in the case of HPC transplants, when large volumes are infused. It is recommended that only the purest contaminantfree DMSO be used as a reagent in the preparation of a freezing mixture 8.4. Plasmalyte A:
8.4.1. Used as a buffered fluid replacement medium for cryopreservation.  
8.5. Biohazard Waste:
8.5.1. All hazardous materials must be disposed of as directed by the institution's hazardous waste plan. Biohazard waste should be placed into the appropriate biohazard container,
(eg, plastic boxes for sharps, red bags for tubing, etc.).
appendix c standard operating procedures
8.6. note products must never

8.7. NOTE: Document any encountered deviations, processing problems, or discrepancies on F-12 Deviation Form. This completed form should be brought to the attention of the Medical Director for review. A copy of the completed form should be placed into the patient's Cell Processing chart for future reference in the event of a component delay or failure.  
8. NOTE: All units will be kept indefinitely unless patient expires. Units from expired patients will be discarded per BMT physician's approval by the hospital's biohazard waste removal procedure.  Refer to P-15 Cryopreserved Hematopoietic Progenitor Cells Disposal.
appendix c standard operating procedures
8.9. limitations

8.9.1. Final cell concentration of product to be cryopreserved should be <3.0 x 10 8 cells/ml.  
Processing and Preparation of HPC or MNC, Apheresis for Freezing - Version: 1.6. Index; P-01. Printed; 12-Jan-2015 09:23 Authorised on: 09-Jan-2015. Authorised by: Sunil Abhyankar. SOP Unique Reference: 822-51459940. Due for review on: 09-Oct-2016.  
Author(s); Kathleen Gailard, Dean Merkel Page 5 of 8 on of HPC or MNC, Apheresis for Freezing - Version: 1.6. Index; P-01. Printed; 12-Jan-2015 09:23 8.9.2. Toxicity has been reported to be associated with the use of DMSO cryoprotectant.  
Reducing the amount of DMSO required is desirable. By reducing the volume of component to be frozen, we are reducing the volume of cryoprotectant needed.  
8.9.3. All ABO discrepancies must be resolved prior to release of products and the staff physician must be notified.
appendix c standard operating procedures
9. references

9.1. Gorin NC: Cryopreservation and storage of stem cells, in Areman, Deeg, Sacher (eds): Bone Marrow and Stem Cell Processing: A Manual of Current Techniques. Philadelphia, PA. Davis, 1992.  
9.2. Gee, AP: Bone Marrow Processing and Purging: A Practical Guide. Boca Raton, FL. CRC
Press 1991.  
9.3. Hematopoietic Cell Transplantation, Second Edition, E.D. Thomas, K.G. Blume, S.J. Forman, Blackwell Science, Inc., Malden, MA, 1999.  
10. APPENDICES: FORMS - LOGS - WORKSHEETS
10.1.  
F-02 Peripheral Stem Cell Processing Worksheet 10.2.  
F-12 Deviation Form 10.3.  
Document Control History Processing and Preparation of HPC or MNC, Apheresis for Freezing - Version; 1.6. Index; P-01. Printed; 12-Jan-2015 09:23 Authorised on: 09-Jan-2015. Authorised by: Sunil Abhyankar. SOP Unique Reference: 822-51459940. Due for review on: 09-Oct-2016 Author(s): Kathleen Gaillard, Dean Merkel Page 6 of 8 sion: 1.6. Index; P-01. Printed: 12-Jan-2015 09:23 Appendix: SOP Processing and Preparation of HPC or MNC, Apheresis for Freezing
appendix c standard operating procedures
appendix links

Please note: links are only correct at time of printing Linked to Controlled Document
- SOP: P-21: Microbial Contamination v1.4 (Authorised)
- SOP: P-06: Preparation of Bone Marrow for Freezing v1.6 (Authorised)
- SOP: P-01: Processing and Preparation of HPC, Apheresis for Freezing v1.5 (Superseded)
12-Jan-2015 09:23
appendix c standard operating procedures
document revision history

Superseded on 09-Jan-2015 17:00 by Sunil Abhyankar
. Printed:
Version 1.5 superseded by version 1.6 P01 Index:
appendix c standard operating procedures
authorized on 09-jan-2015 1700 by

Authorized version 1.6 - Please review minor revisions. The following users will be notified when a review is due for this document: Dean Merkel Authorization Requested on 09-Jan-2015 16:59 by Dean Merkel Authorization request sent to Sunil Abhyankar by Dean Merkel on 09-Jan-2015 16:59.  
Authorisation Requested on 09-Jan-2015 14:32 by Shaun Dejarnette Authorisation request sent to Sunil Abhyankar by Shaun Dejarnette on 09-Jan-2015 14:32.  
Draft Created on 09-Jan-2015 13:30 by Dean Merkel Reason: Added MNCs and other flow assays Authorised on 13-Jan-2014 17:21 by Sunil Abhyankar Authorised version 1.5 - . The following users will be notified when a review is due for this document:
Shaun Dejarnette, Dean Merkel Authorisation Requested on 09-Jan-2014 12:38 by Shaun Dejarnette Authorisation request sent to Sunil Abhyankar by Shaun Dejarnette on 09-Jan-2014 12:38.  
Processing and Preparation of HPC or MNC, Apheresis for Freecing - Version: 1.6. Index; P-01. Printed; 12-Jan-2015 09:23 Authorised on: 09-Jan-2015. Authorised by: Sunii Abhyankar, SOP Unique Reference: 822-51459940. Due for review on: 09-Oct-2016.  
Author(s); Kathleen Gaillard, Dean Merkell Appendix: SOP Processing and Preparation of HPC or MNC. Apheresis for Freezing Review Task Completed on 09-Jan-2014 12:37 by Shaun Dejarnette Shaun Dejarnette completed task, "Reviewed and approved."
Creation on 26-Dec-2013 13:43 by Kathleen Gaillard New SOP created Peer Review Requested on 26-Dec-2013 13:43 by Kathleen Gaillard Peer Review tasks were assigned to the following users: Shaun Dejarnette.  
This review is to be completed by 03-Jan-2014 1.6. Index: P-01. Printed: 12-Jan Processing and Preparation of HPC or MNC, Apheresis for Freezing - Version: 1.6. Index; P-01. Printed; 12-Jan-2015 09:23 Authorised on 09-Jan-2015. Authorised by: Sunil Athyankar. SOP Unique Reference: 822-51450940. Due for review on 00-Oct-2016 Author(s): Kathleen Gallard, Dean Merkel Page 8 of 8
appendix c standard operating procedures
appendix d guidance for use

A) Heparin may be used as an anticoagulant additive to ACDA on patients having a stem cell collection procedure in certain instances. Guidelines for heparin usage are to be determined on each patient by CD34 assessment and a clinical assessment. 1) For patients on anticoagulant therapy, i.e., Coumadin, Lovenox, etc, do not use heparin as an anticoagulant.  
2) For patients with heparin induced antibodies, do not use heparin as an anticoagulant.  
3) All large volume collections procedures are dependent upon patient's tolerance for procedure and other clinical/medical conditions, i.e., platelet counts, hypervolemia, dialysis, etc.
appendix c standard operating procedures
circulating peripheral cd34 count and

A) Patients needing cells for one or multiple peripheral stem cell transplants. 2.5 x x106 CD34/Kg is the desired goal for each transplant.  
1) If the CD34 count is **less than 5 cells/ul**, do not perform collection a. Give 10 mcg/kg G-CSF now.  
b. Administer Mozobil® that evening.  
c. Perform another peripheral CD34 count on the following morning.  
d. If on **second day the CD34 count is less than 5 cells/ul**, do not perform collection.  
e. The physician is to advise on the plan which may include the continuation of Mozobil
®.  
2) If the CD34 count is **greater than 5 and less than 10 cells/ul**, do not perform collection a. Give G-CSF now.  
b. Administer Mozobil® that evening.  
c. G-CSF will also be given 1 hour pre-collection the following morning, if the pre-collection WBC count is < 65K.  
d. Perform a larger volume collection (approximately 5 - 6 blood volumes)
utilizing heparin/ACDA as the anticoagulant. Each blood volume processed can reduce the patient's platelet count by 10%.  
3) If the CD34 count is **greater than 10 cells/ul,** Perform collection as in 2)d. above.  
4) The provider has the discretion to perform a collection outside of this algorithm.
appendix c standard operating procedures
post-collection results and mozobil usage

A) If on the first day of collection the collected product contains LESS THAN 65% of the desired dose, use Mozobil® that evening. G-CSF will also be given 1 hour pre-collection the following morning if the WBC count is <65,000.  
B) **Goal 65% of goal**
2.5 x 106 CD34/Kg (<1.63 x 106 CD34/Kg) 5.0 x 106 CD34/Kg (<3.25 x 106 CD34/Kg) 7.5 x 106 CD34/Kg (<4.88 x 106 CD34/Kg)
appendix c standard operating procedures
mozobil dosage and administration

1) Notify BMT Pharmacist. To reach correct pharmacist call BMT Triage Nurse at 913-588-9821. The triage nurse will direct the call to the pharmacist.  
2) BMT Pharmacist to consult and write order for use of Mozobil®.  
3) Repeat Mozobil® dose up to 4 consecutive days.  
4) Select dose based on 0.24 mg/kg actual body weight (0.012 X patient's actual body weight in kg = volume to be administered in mL).  
5) Maximum dose is 24 mg or 1 vial. (1.2 mL of a 20 mg/mL solution). 6) Administer by subcutaneous injection approximately 10 - 14 hours prior to initiation of apheresis.  
7) Renal impairment: If creatinine clearance is ≤ 50 mL/min, decrease dose by one-third to 0.16 mg/kg.
appendix c standard operating procedures
adverse reactions

A) Most common adverse reactions (≥ 10%): diarrhea, nausea, fatigue, injection site reactions, headache, arthralgia, dizziness, and vomiting.
appendix e standard operating procedures
1. principle/clinical significance

A. Principle This procedure facilitates antibody access to intracellular structures and leaves the morphological scatter characteristics of the cells intact.  There are several antigens that are expressed predominantly within the cell that have excellent sensitivity and specificity for lymphoid or myeloid differentiation.  Nuclear terminal deoxynucleotidyl transferase
(nTdT), myeloperoxidase (MPO), BCL-2, Zap-70, CD22, CD3, CD79a, pan-Cytokeratin, and immunoglobulin determination are commonly tested using this procedure.  
B.  Clinical Significance Intracellular markers are useful in the identification and classification of acute leukemias, and in the prognostication of chronic lymphocytic leukemia.  
nTdT marks immature B and T cells
.  
.  
cyCD3 marks immature T cells cyCD22 marks immature B cells
.  
MPO marks myeloid cells
.  
cyCD79a marks immature and mature B cells
.  
pan-Cytokeratin marks small cell carcinomas
. .  
Cytoplasmic immunoglobulin marks plasma cell neoplasms ZAP-70 is a prognostic indicator in chronic lymphocytic leukemia
.  
.  
BCL-2 is differentiates between reactive hyperplasic and follicular lymphoma.  
2. SPECIMEN
A. Specimen Type 1. Peripheral Blood¹.²
- EDTA specimens are viable up to 24 hours at room temperature.  
Heparin specimens are viable up to 48 hours at room temperature.  
.  
ACD specimens are viable up to 72 hours at room temperature.  
.  
Transport specimen in biohazard transport bag and hold at room temperature
.  
(18-25°C).  
Do not refrigerate.  
.  
2. Bone Marrow Aspirate 1-2mL of bone marrow in a heparinized syringe or collection tube at room
.  
temperature Transport specimen in biohazard transport bag and hold at room temperature
.  
(18-25°C).  
- Do not refrigerate.  
3.   Bone Marrow Biopsies If bone marrow procedure was a dry tap, the bone marrow biopsy can be
.  
processed for flow.  
Place into RPMI or saline and transport in a biohazard transport bag and hold at
.  
room temperature (18-25°C).  
If biopsy is in saline, replace the saline with RPMI immediately and hold until
.  
processing.  
Do not refrigerate.  
.  
4. Fluids - CSF, pleural, bronchial lavage, etc.  
Natural cellular suspension or supplemented with heparin in a collection or test
.  
tube.  
5.  
Transport specimen in a biohazard transport bag and hold at room temperature
.  
(18-25°C).  
Do not refrigerate.  
.  
Tissue - lymph node, spleen, other soft tissue, etc.  
Tissue or cellular suspension held in supplemented RPMI.  
.  
Note: Mince tissue and express through wire mesh screen to dislodge cells.  
.  
Transport specimen in biohazard transport bag and hold at 2-8°C.  
.  
Post Stem Cell Apheresis Collection Product (PSC).  
6.  
B.  Specimen Rejection Incomplete data or patient identification on label.  Pathologist will be consulted.  
1.  
Test will be canceled, if appropriate, in LIS and clinician will be notified.  
Improperly collected specimen:  clotted, auto-lysed, cracked or leaking tube.  
2.  
Indicate if clot present.  Pathologist will be consulted.  Test will be canceled, if appropriate, in LIS and clinician will be notified.  
3.  
Quantity not sufficient to perform test.  Pathologist will be consulted.  Test will be canceled, if appropriate, in LIS and clinician will be notified.  
Peripheral blood 4.  
EDTA specimens greater than 24 hours old.  
.  
Heparin specimens greater than 48 hours old.  
.  
ACD specimens greater than 72 hours old.  
.  
If peripheral blood is greater than the designated acceptable range, then a viability will be performed and reported.  A text box will be added to the FCS Express layout informing the pathologist of the viability and age of the specimen.  
The reporting is at the discretion of the pathologist.  
Tissue and Body Fluid specimens that have a suboptimal viability of < 50%.  A
5.  
text box will be added to the FCS Express layout informing the pathologist of the viability and reporting is at the discretion of the pathologist.  
6.  
CSF specimens greater than 4 days old will have a text box added to the FCS
Express layout informing the pathologist of the age of the specimen and reporting is at the discretion of the pathologist.  
Inappropriate orders:  The ordering physician will be contacted to verify the correct order and documentation will be included when the test is canceled.  
3. REAGENTS/SUPPLIES/EQUIPMENT
A.  Reagent Handling and Storage 1. Conjugated Monoclonal Antibodies Store at 2-8°C.  
.  
Do not freeze.  
.  
Stable until the expiration listed on the bottle.  
.  
Use according to manufacturer's instructions or validated procedure.  
.  
Contains sodium azide to inhibit bacteria growth. Chronic exposure has shown to
.  
have mutagenic effects. PPE should be worn at all times. Wash thoroughly after handling.  
Sodium azide is harmful if swallowed. Wear protective clothing. If swallowed,
.  
seek medical advice immediately and show container or label. Contact with acid liberates very toxic gas. Azide compounds should be flushed with large volumes of water during disposal to avoid deposits in lead or copper plumbing where explosive conditions can develop.  
2. Fix and Perm Cell Permeabilization Reagents Invitrogen Corporation, Cat\# GAS-003
.  
Store at room temperature.  
.  
.  
.  
Stable until expiration on kit.  
Reagent A contains Formaldehyde.  
Precautions:  Formaldehyde is toxic, allergenic and a suspected carcinogen.  
.  
Avoid contact with eyes, skin and clothing.  
In case of contact, rinse immediately with plenty of water and contact physician.  
.  
RPMI 5% FBS with Heparin 3.  
Cardinal Health, Catalog No. M7201-56
.  
Store at 2-8°C,
.  
Fetal Bovine Serum (FBS) - Sigma Aldrich, Catalog No. F4135-500mL
.  
When received, thaw and aliquot out 50mL conical tubes of FBS. Label and
.  
refreeze. Store in freezer until outdate.  
Penicillin/Streptomycin - Sigma Aldrich, Catalog No. P0781-20mL
.  
Store in Freezer until outdate.  
.  
Heparin - 1,000 USP units/mL, 2mL vial
.  
Store at room temperature.  
.  
.  
Obtain from Bone Marrow bench.  
Prepare using sterile technique; remove 35mL of RPMI and add 25mL of thawed
.  
FBS, 10mL of thawed Pen/Strep solution, and one vial of heparin to the RPMI. Store media in refrigerator. Stable for 6 months or until pH has changed. pH change is indicated when the salmon colored media has changed to a yellow or pink solution.  
Store at 4°C for 6 months.  
.  
May cause irritation to skin and eyes. Wash with large amounts of water (eye
.  
wash). Call a physician if necessary.  
Use appropriate PPE:  gloves, lab coat, and protective eye wear.  
.  
Dulbecco's Phosphate Buffered Saline (PBS) (10X)
4.  
Thermo Scientific, Catalog No. SH30378.02
.  
Store 15-30°C.  
.  
Stable until the expiration listed on the bottle.  
.  
Dilute to 1X prior to use with DI water.  
.  
pH the 1X solution with pH indicator strips. It should fall within the range of 7.2-
.  
7.4. Adjust as necessary with 1N NaOH or 1N HCl.  
Expiration is one month from date of preparation.  
.  
.  
Irritant to eyes, respiratory system, and skin. In case of contact, rinse immediately with plenty of water.  
5.  
RBC Lysis Buffer (10X)
BioLegend, Catalog No. 420301.  
.  
.  
Store at 2-8°C.  
Stable until the expiration listed on the bottle.  
.  
Measure 100mL of RBC Lysis Buffer. QS to 1L with DI water.  
.  
pH the 1X solution with pH indicator strips. It should fall within the range of 7.1-
.  
7.4. Adjust as necessary with 1N NaOH or 1N HCl.  
.  
Warm the 1X solution to room temperature prior to use.  
Stable for one month.  
.  
6. 1N NaOH
Sigma, Catalog No. S2770
.  
Store at room temperature.  
.  
Irritant to eyes, respiratory system, and skin. In case of contact, rinse
.  
immediately with plenty of water and contact physician.  
7.  
1N HCI
Sigma, Catalog No. H9892
.  
Store at room temperature.  
.  
Irritant to eyes, respiratory system and skin. In case of contact, rinse immediately
.  
with plenty of water and contact physician.  
BD FACS Flow 8.  
BD Biosciences, Catalog No. 342003
.  
Store at room temperature.  
.  
Stable until the expiration date listed on box.  
9.   BD FACS Clean BD Bioscience, Catalog No. 340345
.  
Store at room temperature.  
.  
Stable until the expiration date listed on box.  
.  
10. DI Water - Reagent Grade Type 1 Water Ricca Chemical Co., Catalog No. 9150-5
.  
Store unopened until expiration date listed on box.  
.  
After opening, use within 30 days.  
.  
Store at room temperature.  
.  
1. Bleach Concentrate (6% sodium hypochlorite)
.  
Store at room temperature Corrosive: Irritant to respiratory tract, skin, and eyes (flush with plenty of water –
.  
use eye wash/safety shower). Ingestion causes nausea and vomiting. Call a physician.  
Use appropriate PPE: glove, lab coat, and protective eye wear.  
.  
12. Bleach-Rite Fisher Scientific, Catalog No. 14-412-53.  
.  
Store at room temperature. Do not expose to excessive heat or sunlight or UV
.  
light or else bleach efficacy may deteriorate. 1:10 dilution of bleach and detergent.  
.  
Stable until expiration date.  
.  
Irritant to eyes. Rinse with copious amounts of water for 15-20 minutes.  
.  
Use appropriate PPE: glove, lab coat, and protective eye wear.  
.  
B.  Materials and Equipment 12x75mm polystyrene test tubes 1.  
2. Pipettes (20uL, 200uL, 1000uL)
Pipette tips 3.  
Repeat pipettor 4.  
Disposable transfer pipettes - large and small tipped 5.  
6.  
Test tube rack 17x100mm test tubes 7.  
Petri dish 8.  
Tissue sieve 9.  
10. 10mL syringe plunger 1. Scalpel and/or surgical scissors 12. 100um cell strainer 13. pH indicator strips 14. Centrifuge 15. Vortex mixer 16. Timer 17. Mechanical rocker 18. BD FACS Lyse Wash Assistant - Serial No. K14610025 19. BD FACSCanto II - Serial No. V96300795, Serial No. V33896201741 Laser safety:  The FACSCanto II flow cytometer is a Class I laser product. The
.  
laser is fully contained within the structure and calls for no special work area safety requirements. Nevertheless, regulations require that a warning be posted to avoid tampering with the instrument. The protective shields surrounding the laser source should never be removed or altered except by the technical service personnel. Removal of any of these safety shields would place the operator at risk of scattered or direct exposure to laser radiation.
appendix e standard operating procedures
4. quality control

A.  BD Cytometer Setup and Tracking Beads - Catalog No. 641319.  
Store the bead vial at 2-8°C
.  
Protect from light.  
.  
Do not freeze.  
.  
Dilute one drop of CS&T Beads with 450ul of BD FACSFlow reagent.  
B.  Leukemia/Lymphoma QC panel - CD-Chex Plus Stored at 2-8°C. Do not freeze.  
.  
Once open, tubes are good for 30 days.  
.  
A positive control panel is run twice a month.  Control results should fall within control
.  
ranges.  If control(s) are out of range, the test system must be evaluated for source of error. Determine cause by evaluating and troubleshooting. Repeat, remake, and/or check them. Note in the "Comments" section of the CD-Chex Plus QC Log any corrective action that has occurred.  
All control specimens must be tested in the same manner and by the same personnel
.  
as patient samples.  
QAP monthly report compares KU data to QC data reported by peer lots. Results
.  
reviewed against group results for precision accuracy. Investigate when SDIs and CV are >2.0.  
C.  BD CompBeads - Anti-Mouse Ig/k Negative Control Compensation Particles Set.  This panel is run weekly or whenever there has been service on the instrument.  
D. Gating Controls:  Population of interest by light scatter and anti-leukocyte antibody
(CD45).  
E.   pH Control Log - pH checks of reagents should be recorded on the monthly pH control log.
appendix e standard operating procedures
5. procedure

A. Label each tube with the antibodies per panel, along with two patient identifiers.  
B. Pipette appropriate volume of adjusted cell suspension into each tube (50uL).  
C. Pipette appropriate volume of the conjugated antibodies directed to the cell surface markers of interest into the appropriate tubes.  
D. Vortex and incubate for 15 mins in the dark at room temperature.  
E.  Add 100uL of Reagent A (Fixative Medium).  
F. Vortex each tube for 2-3 seconds and incubate at room temperature for 15 minutes.  
G.  Wash once in 2 mL of PBS.  
H. Centrifuge the tubes for 3 mins at 400xG or 1600 rpms and decant supernatant.  
Vortex until cell pellet is fully resuspended.  
1.  
J. Add 100uL of Reagent B (Permeabilization Medium) and recommended volume of the conjugated intracellular antibodies.  
K. Vortex 1-2 seconds and incubate for 20 minutes at room temperature in the dark.  
Wash twice in 2 mL of PBS.  
L.  
M. Centrifuge for 3 minutes at 400xG or 1600 rpms and decant supernatant. N. Resuspend cells in 0.5mL of PBS.  
O.  Store at 2-8°C in the dark until panel is run.  
P. Acquire data on the BD FACSCanto II with the DIVA software.  
Q. Analyze data with the FCS Express software off-line.  
6.   CALCULATIONS/INTERPRETATION/RESULT REPORTING/NORMAL VALUES/CRITICAL
VALUES
A. The results are analyzed by the flow cytometry technologists and delivered to the pathologist for interpretation.  The pathologist will complete the final report and notify the physician if indicated.  
7.   CALIBRATION/LINEARITY/AMR/CALIBRATION VERIFICATION
A. Instrument calibration is performed by BD Technical Service on a semi-annual basis. B. Application Settings are performed after every major maintenance or service issue and performed after a new CS&T baseline is established.  
C. Compensation is performed on weekly basis.
appendix e standard operating procedures
8. procedure notes/limitations

A. There are multiple components within the reagent kit.  The laboratory only uses within kit lot components of reagents unless otherwise specified by the manufacturer.  
b.  DO NOT USE REAGENTS IF A PRECIPITATE FORMS OR DISCOLORATION
OCCURS.  
C. Reagents are stable for the period shown on the package label when stored as directed.  
D. If the CS&T beads do not pass, the instrument should not be operated. Refer to the troubleshooting information in the BD Cytometer Setup and Tracking Application Guide.  
When performing a Zap-70, if a defined population of T-cells is not present, or if a Zapi 70 negative population is not present, a patient control must be done.  
F. If instruments are inoperable:
1. For analyzer problems, call BD Bioscience at 1-877-232-8995 and log the problem in the Action Log with a reference number.  
2. If one instrument is down, use the other instrument only and contact technical service.  
If both instruments are down the hematology supervisor or medical staff on call for 3.  
hematology should be notified at this time.  
All testing should be sent to St. Luke's by courier.   Call the Flow Lab at St. Luke's at 4.  
816-932-6265 to let them know we are sending specimens.  
Have St. Luke's fax flow cytometry requisition with indication of necessary 5.  
information to be filled in.  
Use St. Luke's courier until 7:00 pm (816-932-3850).  After hours use KU's courier, 6.  
Quicksilver (321-5959).  
Follow reference lab's protocol for collection and handling of specimens.  
7.
appendix e standard operating procedures
9. references

1. Stewart, C. C., Nicholson, J.K.A. "Immunophenotyping." Page 181-182.  New York:  A
John Wiley & Sons, Inc., 2000. Print.  
2. Hawley, T. S., Hawley, R. G. "Flow Cytometry Protocols." Third Edition. Pages 297 and 301.  New York:  Humana Press.  Print.  
3. Keren, D F.., McCoy Jr., J. Phillip, Carey, J. L. "Flow Cytometry in Clinical Diagnosis."
4 th Edition, ASCP Press, 2007.  
4. Gorczyca, W. "Flow Cytometry in Neoplastic Hematology." United Kingdom: Taylor &
Francis.  
5.   Cornfield, D; Liu, Z; Gorczyca, W; Weisberger, J. "The potential role of flow cytometry in the diagnosis of small cell carcinoma."
6. Archives in Pathology & Laboratory Medicine, Vol. 127, April 2003.  
7. Invitrogen Corporation - Fix & Perm Cell Permeabilization Reagent package Insert. 8.   BD FACSCanto II Instructions For Use, \#644450, April 2009.
appendix e standard operating procedures
10.appendices

A. Attachments 1. pH Log B.  Bench Manual 1. Flow Cytometry panels
appendix f multiparametric flow cytometry
multiple myeloma (mm)-minimal residual disease

2015-IIT-BMT-MM-AutoSCT Multiparametric Flow Cytometry (MFC) - Validation Model - MM-MRD
MM MRD Panel  
| MM MRD Panel   |      |          |          |                     |     |       |         |          |
|----------------|------|----------|----------|---------------------|-----|-------|---------|----------|
| Tube #         | Name | FITC     | PE       | PerCP5.5     PE-Cy7 | APC | AC-H7 | v450-PB | v500c    |
| l              | M1   | 27       | 17       | 138                 | 19  | 38    | 45 56   | 20 BV510 |
| MM             |      |          |          |                     |     |       |         |          |
| [2             | 81   | 28       | 138      | 38                  | 45  |       |         |          |
| 4              | cyMM | cy Kappa | cyLambda | 138                 | 19  | 38    | 45      |          |
appendix f multiparametric flow cytometry
appendix g 8-color leukemia /

Immunophenotypic analysis involves the identification and classification of the cells by the presence of surface antigens or intracellular antigens, which are identified by fluorochrome-labeled monoclonal antibodies directed against those antigens. Panels of antibodies are utilized to determine the origin and stage of differentiation of hematopoietic neoplasms including acute and chronic leukemias and lymphomas.
appendix f multiparametric flow cytometry
b. clinical significance

Immunophenotypic analysis by flow cytometry is routinely utilized for diagnostic and therapeutic decision making for types of acute and chronic leukemias and lymphomas.
appendix f multiparametric flow cytometry
2. specimen a. specimen type

1. Peripheral Blood1,2
- EDTA specimens are viable up to 24 hours at room temperature.  
- Heparin specimens are viable up to 48 hours at room temperature.  
- ACD specimens are viable up to 72 hours at room temperature. - Transport specimen in biohazard transport bag and hold at room temperature
(18-25oC).  
- Do not refrigerate.  
2. Bone Marrow Aspirate
- 1-2mL of bone marrow in a heparinized syringe or collection tube at room temperature
- Transport specimen in biohazard transport bag and hold at room temperature
(18-25oC).  
- Do not refrigerate.  
3. Bone Marrow Biopsies
- If bone marrow procedure was a dry tap, the bone marrow biopsy can be processed for flow.  
- Place into RPMI or saline and transport in a biohazard transport bag and hold at room temperature (18-25oC).  
- If biopsy is in saline, replace the saline with RPMI immediately and hold until processing.  
- Do not refrigerate.  
4. Fluids - CSF, pleural, bronchial lavage, etc.  
- Natural cellular suspension or supplemented with heparin in a collection or test tube.  
- Transport specimen in a biohazard transport bag and hold at room temperature
(18-25oC).  
- Do not refrigerate.  
5. Tissue - lymph node, spleen, other soft tissue, etc.  
- Tissue or cellular suspension held in supplemented RPMI. - Note: Mince tissue and express through wire mesh screen to dislodge cells.  
- Transport specimen in biohazard transport bag and hold at 2-8oC.1 6. Post Stem Cell Apheresis Collection Product (PSC).
appendix f multiparametric flow cytometry
b. specimen rejection

1. Incomplete data or patient identification on label. Pathologist will be consulted.  
Test will be canceled, if appropriate, in LIS and clinician will be notified.  
2. Improperly collected specimen: clotted, auto-lysed, cracked or leaking tube.  
Indicate if clot present. Pathologist will be consulted. Test will be canceled, if appropriate, in LIS and clinician will be notified.  
3. Quantity not sufficient to perform test. Pathologist will be consulted. Test will be canceled, if appropriate, in LIS and clinician will be notified.  
4. Peripheral blood
- EDTA specimens greater than 24 hours old. - Heparin specimens greater than 48 hours old.  
- ACD specimens greater than 72 hours old.  
If peripheral blood is greater than the designated acceptable range, then a viability will be performed and reported. A text box will be added to the FCS Express layout informing the pathologist of the viability and age of the specimen. The reporting is at the discretion of the pathologist.  
5. Tissue and Body Fluid specimens that have a suboptimal viability of < 50%. A
text box will be added to the FCS Express layout informing the pathologist of the viability and reporting is at the discretion of the pathologist.  
6. CSF specimens greater than 4 days will have a text box added to the FCS
Express layout informing the pathologist of the age of the specimen and reporting is at the discretion of the pathologist.  
7. Inappropriate orders: The ordering physician will be contacted to verify the correct order and documentation will be included when the test is canceled.
appendix f multiparametric flow cytometry
3. reagents/supplies/equipment

A. Reagent Handling and Storage 1. Conjugated Monoclonal Antibodies
- Store at 2-8oC.  
- Do not freeze. - Stable until the expiration listed on the bottle.  
- Use according to manufacturer's instructions or validated procedure. - Contains sodium azide to inhibit bacteria growth. Chronic exposure has shown to have mutagenic effects. PPE should be worn at all times. Wash thoroughly after handling.  
- Sodium azide is harmful if swallowed. Wear protective clothing. If swallowed, seek medical advice immediately and show container or label. Contact with acid liberates very toxic gas. Azide compounds should be flushed with large volumes of water during disposal to avoid deposits in lead or copper plumbing where explosive conditions can develop.  
2. Propidium Iodide
- Sigma, Catalog No. P4170
- Store at 2-8oC.  
- Dissolve 0.005g into 500mL PBS. Store at 2-8oC.  
- Prepared PI is stable for 2 years.  
- PI is known to show possible mutagenic effects. PPE should be worn at all times.  
Wash thoroughly after handling.  
3. RPMI 5% FBS with Heparin
- Cardinal Health, Catalog No. M7201-56
- Store at 2-8oC.  
- Fetal Bovine Serum (FBS) - Sigma Aldrich, Catalog No. F4135-500mL
- When received, thaw and aliquot out 50mL conical tubes of FBS. Label and refreeze. Store in freezer until outdate.  
- Penicillin/Streptomycin - Sigma Aldrich, Catalog No. P0781-20mL
- Store in Freezer until outdate.  
- Heparin - 1,000 USP units/mL, 2mL vial
- Store at room temperature. - Obtain from Bone Marrow bench.  
- Prepare using sterile technique; remove 35mL of RPMI and add 25mL of thawed FBS, 10mL of thawed Pen/Strep solution, and one vial of heparin to the RPMI. Store media in refrigerator. Stable for 6 months or until pH has changed. pH change is indicated when the salmon colored media has changed to a yellow or pink solution.  
- Store at 4oC for 6 months.  
- May cause irritation to skin and eyes. Wash with large amounts of water (eye wash). Call a physician if necessary.  
- Use appropriate PPE: gloves, lab coat, and protective eye wear.  
4. Dulbecco's Phosphate Buffered Saline (PBS) (10X)
- Thermo Scientific, Catalog No. SH30378.02
- Store 15-30oC.  
- Stable until the expiration listed on the bottle. - Dilute to 1X prior to use with DI water.  
- pH the 1X solution with pH indicator strips. It should fall within the range of 7.27.4. Adjust as necessary with 1N NaOH or 1N HCl.  
- Expiration is one month from date of preparation. - Irritant to eyes, respiratory system, and skin. In case of contact, rinse immediately with plenty of water.  
5. RBC Lysis Buffer (10X)
- BioLegend, Catalog No. 420301.  
- Store at 2-8oC.  
- Stable until the expiration listed on the bottle.  
- Measure 100mL of RBC Lysis Buffer. QS to 1L with DI water.  
- pH the 1X solution with pH indicator strips. It should fall within the range of 7.17.4. Adjust as necessary with 1N NaOH or 1N HCl.  
- Warm the 1X solution to room temperature prior to use.  
- Stable for one month.  
6. 1N NaOH
- Sigma, Catalog No. S2770
- Store at room temperature.  
- Irritant to eyes, respiratory system, and skin. In case of contact, rinse immediately with plenty of water and contact physician.  
7. 1N HCl
- Sigma, Catalog No. H9892 - Store at room temperature.  
- Irritant to eyes, respiratory system and skin. In case of contact, rinse immediately with plenty of water and contact physician.  
8. BD FACS Flow
- BD Biosciences, Catalog No. 342003 - Store at room temperature.  
- Stable until the expiration date listed on box.  
9. BD FACS Clean
- BD Bioscience, Catalog No. 340345 - Store at room temperature. - Stable until the expiration date listed on box.  
10. DI Water - Reagent Grade Type 1 Water
- Ricca Chemical Co., Catalog No. 9150-5
- Store unopened until expiration date listed on box.  
- After opening, use within 30 days. - Store at room temperature.  
11. Bleach Concentrate (6% sodium hypochlorite)
- Store at room temperature. - Corrosive: Irritant to respiratory tract, skin, and eyes (flush with plenty of water -
use eye wash/safety shower). Ingestion causes nausea and vomiting. Call a physician.  
- Use appropriate PPE: glove, lab coat, and protective eye wear.  
12. Bleach-Rite
- Fisher Scientific, Catalog No. 14-412-53.  
- Store at room temperature. Do not expose to excessive heat or sunlight or UV
light or else bleach efficacy may deteriorate.  
- 1:10 dilution of bleach and detergent. - Stable until expiration date.  
- Irritant to eyes. Rinse with copious amounts of water for 15-20 minutes. - Use appropriate PPE: glove, lab coat, and protective eye wear.  
B. Materials and Equipment 1. 12x75mm polystyrene test tubes 2. Pipettes (20uL, 200uL, 1000uL) 3. Pipette tips 4. Repeat pipettor 5. Disposable transfer pipettes - large and small tipped 6. Test tube rack 7. 17x100mm test tubes 8. Petri dish 9. Tissue sieve 10. 10mL syringe plunger 11. Scalpel and/or surgical scissors 12. 100um cell strainer 13. pH indicator strips 14. Centrifuge 15. Vortex mixer 16. Timer 17. Mechanical rocker 18. BD FACS Lyse Wash Assistant - Serial No. K14610025 19. BD FACSCanto II - Serial No. V96300795, Serial No. V33896201741
- Laser safety: The FACSCanto II flow cytometer is a Class I laser product. The laser is fully contained within the structure and calls for no special work area safety requirements. Nevertheless, regulations require that a warning be posted to avoid tampering with the instrument. The protective shields surrounding the laser source should never be removed or altered except by the technical service personnel. Removal of any of these safety shields would place the operator at risk of scattered or direct exposure to laser radiation.
appendix f multiparametric flow cytometry
4. quality control

A. BD Cytometer Setup and Tracking Beads - Catalog No. 641319
- Store the bead vial at 2-80C
- Protect from light. - Do not freeze.  
- Dilute one drop of CS&T Beads with 450ul of BD FACSFlow reagent.  
B. Leukemia/Lymphoma QC panel - CD-Chex Plus
- Stored at 2-8oC. Do not freeze.  
- Once open, tubes are good for 30 days.  
- A positive control panel is run twice a month. Control results should fall within control ranges. If control(s) are out of range, the test system must be evaluated for source of error. Determine cause by evaluating and troubleshooting. Repeat, remake, and/or check them. Note in the "Comments" section of the CD-Chex Plus QC Log any corrective action that has occurred.  
- All control specimens must be tested in the same manner and by the same personnel as patient samples.  
- QAP monthly report compares KU data to QC data reported by peer lots. Results reviewed against group results for precision accuracy. Investigate when SDIs and CV are >2.0.  
C. BD CompBeads - Anti-Mouse Ig/k, Negative Control Compensation Particles Set. This panel is run weekly or whenever there has been service on the instrument.  
D. Gating Controls: Population of interest by light scatter and anti-leukocyte antibody
(CD45).  
E. pH Control Log - pH checks of reagents should be recorded on the monthly pH control log.
appendix f multiparametric flow cytometry
5. procedure

A. Manual Method:
1. Concentrate cellular elements of the specimen by transferring an aliquot (1-2mL) of well-mixed sample to a 17x100mm tube.  
- If BM specimen, break up spicules with a small tipped pipette.  
- If tissue specimen, use tissue sieve and rubber tipped syringe plunger to gently dissociate.  
2. Add 5% FBS RPMI and centrifuge at 400g or 1600rpms for 5 minutes to wash the cells.  
3. If tissue or fluid specimen, proceed to step 9.  
4. Aspirate the supernatant. 5. Add 10mL of 1X RBC Lysis Buffer to specimen and place on mixer for 10 minutes.  
6. Centrifuge at 400g or 1600 rpms for 5 minutes.  
7. Decant supernatant. Note: If incomplete lysis has occurred - repeat steps 4-6.  
8. Add PBS to specimen to wash cells. Centrifuge at 400g or 1600 rpms for 5 minutes. 9. Decant supernatant and resuspend the cells in 1 mL of 5% FBS RPMI. 10. Obtain a cell count using CountBright Beads (see appropriate procedure). Adjust the concentration to 10.0 x 106 WBC/mL (working solution).  
11. Pipette the appropriate antibodies into labeled tubes. 12. Add 100uL of working solution of cells to the antibody panel.  
- If setting up any MRD panel, use 200uL of working solution if the cell concentration is < 10.0 x 106 WBC/mL.  
13. Incubate the panel for 15 to 30 minutes in the dark at room temperature.  
14. If tissue or fluid specimen, add 2 mLs of RBC Lysis Buffer and incubate in the dark for 10 minutes. Centrifuge at 400g or 1600 rpms for 3 minutes. Decant tubes.  
15. Add 2 mL of PBS to each tube and vortex gently. 16. Centrifuge at 400g or 1600 rpms for 3 minutes.  
17. Decant supernatant and add 0.5 mL of PBS to each tube and gently vortex. 18. Remove the PI viability tube (if appropriate) and place 1 mL of Propidium Iodide into the PI tube. Vortex and place back into carousel rack. PI will incubate while the panel is running.  
19. Run the panel on the BD FACSCanto II using the Diva Software. Gate on the CD45 bright population and/or population of interest. Analyze data using FCS Express software.  
20. After analysis, tubes can be disposed of in a biohazard container. 21. Original specimen is kept at room temperature for 7 days.  
B. Lyse Wash Assistant Method (LWA):
1. Concentrate cellular elements of the specimen by transferring an aliquot (1-2 mL) of well-mixed sample to a 17 x 100mm tube.  
- if BM specimen, break up spicules with a small tipped pipette .  
- if tissue specimen, use tissue sieve and rubber tipped syringe plunger to gently dissociate.  
2. Add 5% FBS RPMI and centrifuge at 400g or 1600 rpm for 5 minutes to wash the cells.  
3. If tissue or fluid specimen, proceed to step 6.  
4. Aspirate the supernatant. 5. Add PBS to specimen to wash cells. Centrifuge at 400g or 1600 rpms for 5 minutes. 6. Decant supernatant and resuspend the cells in 1 mL of 5% FBS RPMI.  
7. Obtain a cell count using CountBright Beads (see appropriate procedure). Adjust the concentration to 10.0 x 106 WBC/mL (working solution).  
8. Pipette the appropriate antibodies into labeled tubes. 9. Add 100uL of working solution of cells to the antibody panel.  
- If setting up an AML/ALL MRD panel, use 200uL of working solution if the cell concentration is < 10.0 x 106WBC/mL.  
10. Gently vortex tubes and load the panel into a carousel rack.  
11. Place carousel on the LWA and close the lid.  
12. Make sure all reagent volumes are full.  
13. Select the *KU/incub/lyse/wash* protocol and then Run.  
14. When the LWA cycle is completed, open lid and remove carousel and place on the BD FACSCanto II.  
15. Remove the PI viability tube (if appropriate) and place 1 mL of Propidium Iodide into the PI tube. Vortex and place back into carousel rack. PI will incubate while the panel is running.  
16. Run the panel on the BD FACSCanto II using the Diva Software. Gate on the CD45 bright population and/or population of interest. Analyze data using FCS Express software.  
17. After analysis, tubes can be disposed of in a biohazard container. 18. Original specimen is kept at room temperature for 7 days.  
6. CALCULATIONS/INTERPRETATION/RESULT REPORTING/NORMAL VALUES/CRITICAL
VALUES A. The results are analyzed using FCS Express software, by the flow cytometry technologists and delivered to the pathologist for interpretation. The pathologist will complete the final report and notify the physician if indicated.
appendix f multiparametric flow cytometry
7. calibration/linearity/amr/calibration verification

A. Instrument calibration is performed by BD Technical Service on a semi-annual basis. B. Application Settings are performed after every major maintenance or service issue and performed after a new CS&T baseline is established.  
C. Compensation is performed on a weekly basis.
appendix f multiparametric flow cytometry
8. procedure notes/limitations

A. To ensure that immunoglobulin staining is intrinsic and not extrinsic, CD19 is stained in the same tube with Kappa and Lambda. Also, the specimen is washed with PBS prior to staining to alleviate non-specific binding.2 B. The laboratory uses antibodies appropriate for the clinical situation. Pathologists review as necessary.  
C. The use of Hyaluronidase has been validated in BAL's and fluids. If these specimens are thick and mucoid add a few crystals of Hyaluronidase (found in Hematology's freezer). Wait until the sample liquefies then proceed with above procedure.  
D. There are numerous variables that may affect the staining properties or the identification of specific cell populations and thus result in inaccurate results. These include the following: 1. Patient's disease state or therapy, such as immune-suppressive drugs, may affect the normal antibody staining characteristics.  
2. Purity of the gated cell population.  
3. Resolution between the positive and negative populations.  
4. Non-specific staining and auto-fluorescence. 5. Cell count of population of interest may be too low to collect significant results.  
E. If the CS&T beads do not pass, the instrument should not be operated. Refer to the troubleshooting information in the BD Cytometer Setup and Tracking Application Guide.  
F. If instruments are inoperable:
a. For analyzer problems, call BD Bioscience at 1-877-232-8995 and log the problem in the Action Log with a reference number.  
b. If one instrument is down, use the other instrument only and contact technical service.  
c. If both instruments are down the hematology supervisor or medical staff on call for hematology should be notified at this time.  
d. All testing should be sent to St. Luke's by courier. Call the Flow Lab at St. Luke's at 816-932-6265 to let them know we are sending specimens.  
e. Have St. Luke's fax flow cytometry requisition with indication of necessary information to be filled in.  
f. Use St. Luke's courier until 7:00 pm (816-932-3850). After hours use KU's courier, Quicksilver (321-5959).  
g. Follow reference lab's protocol for collection and handling of specimens.  
9. REFERENCES For this Appendix 1. Stewart, C. C., Nicholson, J.K.A. "Immunophenotyping." Page 181-182. New York: A
John Wiley & Sons, Inc., 2000. Print.  
2. Hawley, T. S., Hawley, R. G. "Flow Cytometry Protocols." Third Edition. Pages 297 and 301. New York: Humana Press. Print.  
3. Keren, D F.., McCoy Jr., J. Phillip, Carey, J. L. "Flow Cytometry in Clinical Diagnosis."
4th Edition, ASCP Press, 2007.  
4. BD Cytometer Setup and Tracking Application Guide for BD FACS Digital Flow Cytometers. BD Biosciences. com. 23-12881-00, Rev.01 10/2010.  
5. BD FACSCanto II Instructions For Use, \#644450, April 2009.  
6. BD Bioscience - BD FACSCanto II Operators Course Workbook, 23-10926-00, August 2009.  
7. BD Bioscience - BD FACS Lyse Wash Assistant User's Guide, 23-11113-00 Rev. A,
January 2010.
appendix f multiparametric flow cytometry
appendix h summaries of changes

- Previous Protocol Version 4.0 dated 04-06-2016 - Revised Protocol Version 5.0 dated 11-21-2016
appendix f multiparametric flow cytometry
general comments

This protocol version incorporates the protocol clarification letter dated 05-18-2016 (allows use of filgrastim biosimilar), removes an inclusion criterion, and includes administrative updates & clarifications.  
| Key Protocol Changes:                                                                                                                                                                                                                                                                                                                                                                                                                               |                                                   |               |
|-----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|---------------------------------------------------|---------------|
| Section                                                                                                                                                                                                                                                                                                                                                                                                                                             | Summary of Change                                 | Rationale     |
| Patients with increased  creatinine/kidney disease  would not be  contraindicated for stem  cell collection.  Bortezomib is the  preferred myeloma drug  for patients with kidney  disease and using  Bortezomib for  mobilization may be of  benefit for patients with  kidney disease. Patients  who meet all other  enrollment criteria but  have kidney disease  and/or increased  creatinine should not be  excluded from the clinical  trial. |                                                   |               |
| Removed Inclusion Criterion:                                                                                                                                                                                                                                                                                                                                                                                                                        |                                                   |               |
| 3.1.5                                                                                                                                                                                                                                                                                                                                                                                                                                               | "Creatinine - within normal institutional limits" |               |
| Added clarification for Treatment Group A & B: "Physician discretion will be used to advise on the  plan which may include the continuation of  Mozobil. The provider has the discretion of  collection outside the algorithm included in  Appendix C."                                                                                                                                                                                             |                                                   |               |
| 4.0                                                                                                                                                                                                                                                                                                                                                                                                                                                 | Clarification                                     |               |
| Added clarification for Adverse Event reporting: The study ends 30 days after the participant's last  protocol therapy (collection) for the purpose of AE reporting. If a reportable AE occurs within 30  days after a participant's collection, recording of  concomitant medications should continue until  resolution of the AE.                                                                                                                 | Clarification -                                   |               |
| The last day should be  the collection date  because not all patients  make it to AutoSCT.                                                                                                                                                                                                                                                                                                                                                          |                                                   |               |
| Throughout Throughout                                                                                                                                                                                                                                                                                                                                                                                                                               | Added that Filgrastim G-CSF biosimilar (Zarxio)   | Clarification |
| may/will be substituted for filgrastim                                                                                                                                                                                                                                                                                                                                                                                                              | (institutional standard)                          |               |  
| Administrative Protocol Changes:   |                                               |                        |
|------------------------------------|-----------------------------------------------|------------------------|
| Section                            | Summary of Change                             | Rationale              |
| All                                | Added new protocol version and date           | Protocol tracking      |
| Page 2                             | Updated biostatistician                       | Personnel change       |
| Appendix H                         | Added latest protocol changes to this section | Clarity/Consistency/QA |
| All                                | Formatting                                    | Administrative         |  
| Consent Form Changes:                                                    |                               |           |
|--------------------------------------------------------------------------|-------------------------------|-----------|
| Section                                                                  | Summary of Change             | Rationale |
| Administrative /                                                         |                               |           |
| Prevents maintenance when consent is  unaffected by protocol  amendments |                               |           |
| Header                                                                   | Removed protocol version/date |           |  
***Re-consent/notification of participants is not required.***
appendix f multiparametric flow cytometry
summary of changes for revised

- Previous Protocol Version 3.0 dated 03-14-2016 - Revised Protocol Version 4.0 dated 04-06-2016
appendix f multiparametric flow cytometry
general comments

This revised protocol version 4.0 dated 04-06-2016, incorporates the addition of one exclusion criterion, a note (repeated in several places in the section on adverse events) regarding planned hospital admission, and a study staff change as listed below:
- Patients who have received more than 6 months of lenalidomide (Revlimid®) therapy prior to stem cell collection are excluded from this study.
appendix f multiparametric flow cytometry
- note for this study

- Brea Lipe, MD was removed as a sub-investigator - Other minor version-related administrative protocol changes were also completed. - Changes to the consent form were completed for clarity of standard of care study processes related to stem cell collection and timing
- **It is our opinion, because the changes clarify study procedures undergone by the**
appendix f multiparametric flow cytometry
study participants any currently enrolled

the consent form after IRB approval.  
| Key Protocol Changes:                                                                                                                                                      |                                                                                                                               |                                                     |
|----------------------------------------------------------------------------------------------------------------------------------------------------------------------------|-------------------------------------------------------------------------------------------------------------------------------|-----------------------------------------------------|
| Section                                                                                                                                                                    | Summary of Change                                                                                                             | Rationale                                           |
| Excessive exposure to  Lenalidomide (Revlimid®)  prior to stem cell harvest is known to adversely affect  stem cell mobilization. Due  to that fact, heavily pretreated patients ( > 6  months of Lenalidomide  exposure) will not be  included in this trial.                                                                                                                                                                            |                                                                                                                               |                                                     |
| Added Exclusion Criterion –Patients who have  received more than 6 months of lenalidomide  (Revlimid®) therapy prior to stem cell collection are excluded from this study. |                                                                                                                               |                                                     |
| 3.2                                                                                                                                                                        | This added exclusion  increases participant  safety                                                                           |                                                     |
| Sections: 6.1.1 6.2.1 6.2.2 6.2.3 6.2.4                                                                                                                                    | Added the following note:  NOTE: For this study, planned hospital  admission will NOT be considered a Serious  Adverse Event. | Clarification of study  reporting for this disease. |
| (Repeated in multiple positions for clarity for  study team during clinical operations)                                                                                    |                                                                                                                               |                                                     |  
| Administrative Protocol Changes:                                      |                                                                                                                                                                                                                                            |                                                             |
|-----------------------------------------------------------------------|--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|-------------------------------------------------------------|
| Section                                                               | Summary of Change                                                                                                                                                                                                                          | Rationale                                                   |
| Title Page                                                            | Added information regarding current  protocol version                                                                                                                                                                                      | Clarity and Protocol tracking                               |
| Header                                                                | Updated to include current protocol version                                                                                                                                                                                                | Protocol Tracking/                                          |
| and date                                                              | Clarification/Version Control                                                                                                                                                                                                              |                                                             |
| Footer                                                                | Updated to include current protocol version                                                                                                                                                                                                | Protocol Tracking                                           |
| and date                                                              | /Clarification/Version Control                                                                                                                                                                                                             |                                                             |
| Contents                                                              | Updated page numbers and section titles                                                                                                                                                                                                    | Protocol consistency per changes                            |
| Table of                                                              | in current version                                                                                                                                                                                                                         |                                                             |
| Section 14.0                                                          | Added reference # 46, pertinent to added  exclusion criterion                                                                                                                                                                              | Clarity/Consistency                                         |
| References Appendix H - Summaries                                     | Added latest changes to this section                                                                                                                                                                                                       | Clarity/Consistency/QA                                      |
| of Changes ALL Consent Form Changes  Section Summary of Change        | Rationale                                                                                                                                                                                                                                  |                                                             |
| Header                                                                | Updated notation regarding protocol version  and date                                                                                                                                                                                      | Clarity for Study Team/Per SOPs                             |
| What is                                                               |                                                                                                                                                                                                                                            |                                                             |
| being tested                                                          | Changed "twelve" to "12"                                                                                                                                                                                                                   | Correction per grammar rules                                |
| in this study? What will I be  asked to do?  / Group B                | Added note that bortezomib doses are per                                                                                                                                                                                                   | Clarification/ Standard of Care                             |
| institutional standard                                                | treatment for this disease                                                                                                                                                                                                                 |                                                             |
| Added the word "NOTE" to notation                                     |                                                                                                                                                                                                                                            |                                                             |
| regarding 72 hours required between each  dose or bortezomib.         | Clarity/Emphasis                                                                                                                                                                                                                           |                                                             |
| Study                                                                 |                                                                                                                                                                                                                                            |                                                             |
| Treatment                                                             | Clarification/ Standard of Care  treatment for this disease                                                                                                                                                                                |                                                             |
| Added note that bortezomib doses are per  institutional standard      |                                                                                                                                                                                                                                            |                                                             |
| Study                                                                 |                                                                                                                                                                                                                                            |                                                             |
| Calendar Treatment  Period/ Day  0                                    | Calculation of days in window  was incorrect - now corrected. This window was previously  approved in Protocol version 3.0  added at that time per standard of  care for stem cell collection and  for Participant safety and  convenience |                                                             |
| Changed collection window days from " 4 to  8 days" to "8 to 12 days" |                                                                                                                                                                                                                                            |                                                             |
| Study                                                                 |                                                                                                                                                                                                                                            |                                                             |
| Procedures  and Tests /  Bortezomib                                   | Added note that bortezomib doses are per  institutional standard                                                                                                                                                                           | Clarification/ Standard of Care  treatment for this disease |
appendix f multiparametric flow cytometry
summary of changes for revised

- Previous Protocol Version 2.0 dated 02-11-2016 - Revised Protocol Version 3.0 dated 03-14-2016
appendix f multiparametric flow cytometry
general comments

- This revised protocol version 3.0 dated 03-14-2016, incorporates only the addition and clarification of the following:
o Clarification of Window for stem cell collection (Day 0 + 4 days)
o Clarification that mozobil will be used if needed for BOTH groups A and B (per Standard of Care (SOC)
o Addition of Appendix G - 8-Color Leukemia / Lymphoma Immunophenotyping Processing Procedure o Addition of Appendix H - Summaries of Changes
- Other administrative protocol changes related to this change, including template language and quality assurance updates, were also completed.  
- Changes to the consent form were completed for clarity of standard of care study processes related to stem cell collection timing
- **It is our opinion, because the changes clarify study procedures undergone by the**
study participants; any currently enrolled participants **must re-sign this version of** the consent form after IRB approval.  
| Section                                                                       | Summary of Change                                                                                                                                                          | Rationale                                                   |
|-------------------------------------------------------------------------------|----------------------------------------------------------------------------------------------------------------------------------------------------------------------------|-------------------------------------------------------------|
| Schema                                                                        |                                                                                                                                                                            |                                                             |
| Study Summary 1.4 - Paragraph 8 4.1 5.2 5.4                                   | Clarification of Window for stem cell collection  (Day 0 + 4 days) Clarification that mozobil will be used if  needed for BOTH groups A and B (per  Standard of Care (SOC) | Clarification / Participant  safety and convenience Clarity |
| Added Appendix G - 8-Color Leukemia /                                         |                                                                                                                                                                            |                                                             |
| Lymphoma Immunophenotyping Processing  Procedure                              | Per KUCC Quality  Assurance, going                                                                                                                                         |                                                             |
| forward, summaries of  changes should be  included in the protocol  document. |                                                                                                                                                                            |                                                             |
| 15.0                                                                          | Added Appendix H - Summaries of Changes                                                                                                                                    |                                                             |  
Key Protocol Changes for Revised Protocol Version 3.0 dated 03-14-2016:  
| 2016:                                                                                                                                                |                                                                                                        |                                     |
|------------------------------------------------------------------------------------------------------------------------------------------------------|--------------------------------------------------------------------------------------------------------|-------------------------------------|
| Section                                                                                                                                              | Summary of Change                                                                                      | Rationale                           |
| Added information and corrected section  header regarding previous and current                                                                       | Clarity and Protocol tracking                                                                          |                                     |
| protocol modifications                                                                                                                               |                                                                                                        |                                     |
| Title Page                                                                                                                                           |                                                                                                        |                                     |
| Header                                                                                                                                               | Updated to include current protocol version                                                            | Protocol Tracking/                  |
| and date                                                                                                                                             | Clarification/Version Control                                                                          |                                     |
| Footer                                                                                                                                               | Updated to include current protocol version                                                            | Protocol Tracking                   |
| and date                                                                                                                                             | /Clarification/Version Control                                                                         |                                     |
| Contents                                                                                                                                             | Updated page numbers and section titles                                                                | Protocol consistency per changes in |
| Table of                                                                                                                                             | current version                                                                                        |                                     |
| Consent Form Changes for Revised Protocol Version 3.0 dated 03-14-2016:                                                                              |                                                                                                        |                                     |
| Section                                                                                                                                              | Summary of Change                                                                                      | Rationale                           |
| Header                                                                                                                                               | Changed Amendment/Version  Information                                                                 | Auditing reference/version control  |
| What is being  tested in this  study?  Paragraph                                                                                                     |                                                                                                        |                                     |
| regarding Group B                                                                                                                                    | Clarification / 4 –day window                                                                          |                                     |
| clarification added per standard of  care for stem cell collection and  for Participant safety and  convenience                                      |                                                                                                        |                                     |
| Added that stem cell collection may  occur approximately eight to twelve  days after the second dose of  bortezomib. Moved and bolded note that both |                                                                                                        |                                     |
| Group A and B participants will receive  G-CSF and may have Mozobil if  needed                                                                       |                                                                                                        |                                     |
| What is being  tested in this  study?                                                                                                                | Clarification                                                                                          |                                     |
| What will I be  asked to do?  Group B                                                                                                                | Added that stem cell collection may                                                                    |                                     |
| occur up to 12 days after second dose  of bortezomib.                                                                                                | See rationale for "What is being  tested in this study?", above                                        |                                     |
| Group B - modified language in bullet 3  per the clarification to the window for  standard of care for stem cell collection.                         | See rationale for "What is being  tested in this study?", above                                        |                                     |
| Study Treatment                                                                                                                                      | Added bullet 4 to indicate mozobil will  be used for this group also, if needed                        | Clarification                       |
| Changed/expanded treatment-period  column headings (only) per the  clarification to the window for standard  of care for stem cell collection.       | Clarification and see rationale for  "What is being tested in this  study?", above.                    |                                     |
| Study Calendar                                                                                                                                       | Deleted "Day - 1" per the clarification to  the window for standard of care for  stem cell collection. | Consistency                         |
| Blood tests                                                                                                                                          |                                                                                                        |                                     |