women`s interagency hiv study section 29: pbmc iqa protocol
Contents
1. WOMEN S INTERAGENCY HIV STUDY SECTION 29 PBMC IQA PROTOCOL NOTE Should any WIHS site choose a specimen processing laboratory that is not already approved for participation in the DAIDS Virology or Immunology Quality Assurance Programs VQA IQA the site PI must contact the WIHS Program Official named in the Notice of Award in order to initiate the approval process Participation in these programs is not automatic and the addition of new labs has to be pre approved by the official who oversees the VQA and IQA contract resources at DAIDS PART 1 INFORMATION AND SPECIMEN COLLECTION A PURPOSE The proper storage of viable peripheral blood mononuclear cells PBMCs is critical to the research efforts of the WIHS Specimens are batched during a core visit and saved locally or at the national repository These specimens may be used for assays performed months to years after initial freezing and storage The purpose of this program is to assess the ability of laboratories to process and store viable PBMC for use in various immunologic and virologic assays B BACKGROUND AND SIGNIFICANCE Information in this protocol was assembled from IQA the DAIDS Virology Manual and ACTG guidelines The DAIDS PBMC Cryopreservation Evaluation Program s assessment is accomplished by comparing the viability of PBMC s and the viable cell yield before freezing and after thawing Proper processing and freezing are critical components in th2. The tested packaging must bear the UN specification markings as required by the IATA regulation 6 The packages containing infectious substances must contain an itemized list of contents enclosed between the secondary and the outer packaging WIHS Manual of Operations 04 01 2014 Page 29 8 of 25 7 The package containing the infectious substances must be marked durably and legibly on the outside of the packaging with the NAME and TELEPHONE NUMBER OF A PERSON RESPONSIBLE FOR THE SHIPMENT The samples must be shipped frozen in DRY ICE Therefore a DRY ICE label must be placed on the outside of the secondary packaging When dry ice is used then the outer container must permit the release of carbon dioxide gas The primary receptacle must maintain its containment integrity at the temperature of the refrigerant being used as well as temperature and pressure of air transport to which the primary receptacle could be subject if the refrigerant were to be lost 9 Samples are to be shipped to the following address Raul Louzao IQA Program Manager Duke Human Vaccine Institute IQAC Lab GSRB 2 Room 4054 210 Research Drive Durham NC 27710 Tel 919 684 5861 Fax 919 681 8251 Email raul louzao duke edu 2 MARKING AND LABELING 1 The shipping laboratory shipper is responsible for all necessary marking and labeling of each package of dangerous goods Each package must be of a size where there is adequate space to affix all
3. 50 mL conical tube and is greater than 1 mm thick add 4 mL of medium If pellet does not cover the entire bottom of the 50 mL conical tube and is less than 1 mm thick add 2 mL of medium Experience from some ACTG laboratories has shown that acceptable viability is obtained if the cell pellet is resuspended in 1 to 4 mL of cold freezing medium provided that the cells are counted and dispensed into freezing vials within 15 minutes Due to the variability in Total Cell Viable Recovery performed by IQA participating labs should pay particular attention to their cell counting methods Please follow the methods described below Count and record the number of viable PBMCs per mL WIHS Manual of Operations 04 01 2014 Page 29 5 of 25 it 8 1 Pipette 10 uL of PBMC suspension into a 0 5 mL microcentrifuge tube Add 90 uL of 0 4 Trypan Blue stain making a 1 10 dilution final concentration of Trypan Blue is 0 36 Mix carefully to avoid aerosol formation Dilution Factor 90ul Trypan Blue 10u1 PBMC suspended in cell media not freezing media 10 10 ul PBMC 8 2 Load the mixture Trypan Blue PBMC s onto the Hemacytometer chamber until the area under the cover slip is sufficiently filled Labs should only use cover slip that is designed specifically for the Hemacytometer Allow the cell suspension to settle in the hemacytometer for 5 10 minutes before counting This will allow the cells to truly settle Also cells will
4. II for Blood Drawing Protocol 1 SCHEDULING SPECIMEN COLLECTION Coordination and laboratory staff should review the guidelines Appendix B three weeks prior to the shipment date Please make arrangements to obtain blood from your two volunteers two weeks before the shipment deadline Appendix D Ideally your volunteers should come in on the same date and time so that a single freeze can be done If this is not possible handle the samples in the same manner one that preserves the viability of the cells so that handling does not introduce differences between the two samples COLLECTION From each donor obtain 20ml of blood two 10ml tubes by standard venipuncture technique WIHS method See Section 10 ACTG method ACD yellow top should be used as the anticoagulant Isolate PBMC s from each donor by Ficoll Hypaque separation After separation determine the cell yield and viability by trypan blue dye exclusion Make sure to count at least 100 cells for these determinations F PUBLISHED CRYOPRESERVATION STUDIES 1 Bohbot A Lioure B Faradji A Schmitt M Cuillerot JM Laplace A Oberling Positive selection of CD34 cells from cryopreserved peripheral blood stem cells after thawing technical aspects and clinical use Bone Marrow Transplant 1996 Feb 17 2 259 64 Farzadegan H Imagawa D Gupta P Lee MH Jacobson L Saah A Grovit K Rinaldo CR Jr Polk BF The effect of fresh lymphocytes on increased sensitivity of H
5. be fully exposed to the Trypan Blue and marginally viable cells will succumb to the Trypan Blue This extended waiting period will provide a more accurate cell count Count the 4 large corner squares see diagram below Viable PBMCs will be clear nonviable PBMCs will be blue Record Viable and Non Viable Cells on the Freezing Form Appendix C or F a Pa LOL RE ae ot cclis in thea gt R E Al sae aes corner 1mm squares SQUARE Include cells that touch TT either the top line or left ERAH TERE vertical perimeter line tt of any corner square f HE H f E Do NOT count any HEHH i MIDDLE He that ae sac jis H ia sa line or right H perimeter line oo ses Sous gues of any corner square ai Iili FN 8 3 Calculate the number of PBMC mL 10 volume conversion factor to 1 mL 10 specimen dilution factor PBMC mL PBMC in all four squares 10 x 10 aN WIHS Manual of Operations 04 01 2014 Page 29 6 of 25 example 88 X 10 2 2 X 10 PBMC mL 4 8 4 To calculate Cell Viability Viability Number of Viable Cells Counted Total Number of Cells Counted 8 5 Total Viable Cell Count Total Number of Viable Cells in 4 squares X 10 X 10 Dilution Factor X Vol of original Cell suspension 9 10 11 12 4 Numter of squares counted Automated counting may also be used Follow manufacturer s instructions Cell concentration for cryopreservat
6. the middle left of the screen scroll down the list and double click on the IQA Cryopreservation and Viability row Repeat steps 2 3 for each specimen Click the Save button on the toolbar m mgg 40S f if a 4 023701000042 5000000 00 300000000 CEL A 029701000042 5000000 00 3000000 00 CEL 5000000 00 ELLEN pg CEL 029701000042 ep N ZA 0 00 i Assay 10 00 UL 39001 0 0 20010928 38 IQA Cryopreservation and Viability 39_ TUNEL Assay 2000000 00 CEL 0 E e Oe ee T 1 IQA Cryopreservation and Viability No BLD HEP 2 IQA Cryopreservation and Viability 2000000 00 CEL BLD IQA Cryopreservation and Viability 200000000 CEL BLD IQA Cryopreservation and Viabilit 2000000 00 CEL BLD T Test not run Figure 1 2 Ordering the IQA Cryopreservation and Viability Assay Designing the Custom Labels for IQA specimens Custom labels will need to be designed for the IQA group that includes the following fields Donor patient ID LDMS specimen number volume and volume unit specimen date and derivative type Pa ol al a Select the Labels from the Tools menu Select the blank row from the Format combo box Click on the Add button on the toolbar Type IQA in the Label Name dialog box and click OK Select IQA from the Format combo box Click on a row in the Description grid to select a label type Click the checkbox next to each data item that should be included on the label and enter a row and column for each item
7. with IATA Packing Instructions 650 There must be two copies of the declaration presented to the shipping carrier i e FedEx The carrier holds one of these copies and the other copy is forwarded with the shipment to its destination A Shippers Declaration is NOT required and should NOT be used A class 6 hazard label is NOT required and should NOT be used An Air Eligible label label with the airplane is required for specimens being shipped by air mandatory January 2004 Your shipments will still be considered Dangerous Goods and you will need to check the Yes Shipper s Declaration not required box in section 6 of a Federal Express Air Waybill in response to the question Does this shipment contain dangerous goods As a shipment containing Dangerous Goods the 24 hour Emergency Response contact number will still be required on the outside of the package and recipient notification must also be made prior to releasing the package to a carrier 4 PRIOR ARRANGEMENTS 1 Dangerous Goods Regulations 1 3 3 1 covers special arrangements that a shipper of dangerous goods must comply with The shipper must notify the recipient that a dangerous goods package is being shipped to The shipper must provide a telephone number that is available to be answered 24 hours a day in case of an emergency The name and title of the responsible person for the package must be printed or stamped The date and place and signature by the re
8. 7 Collection of Samples by Sites December 1 Shipment of Cryo Samples to IQA July SM TW TF S 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 August SMTWT F S 2 4 5 6 7 8 9 11 12 13 14 15 16 q8 19 20 21 22 25 26 27 28 29 8 9 10 11 12 15 16 17 18 19 22 23 24 25 26 29 30 October SMTWTFF S 123 4 5 6 7 8 9 10 Il 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 November SM TW TF 23 4 5 6 7 16 17 18 19 20 21 S 1 8 9 10 11 12 13 14 15 22 23 24 25 26 27 28 29 December SMTWTFF S q 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 WIHS Manual of Operations 04 01 2014 Page 29 17 of 25 APPENDIX E LDMS INSTRUCTIONS IQA Cryopreservation and Viability Panel Documentation This document describes the steps that must be taken in the LDMS to complete the IQA Cryopreserbation and Viability Panel It is divided into two sections one for the sending lab and another for the IQA lab Please follow the instructions as they appear in the document For additional assistance please call LDMS User Support at 716 834 0900 x311 Sending Labs Entering the IQA specimens To begin you must first enter the samples into the LDMS and create cell aliquots Click on the Specimen Management button on the LDMS toolbar Select IQA from the group combo box Enter the donor
9. Click the Save button on the toolbar This custom label is now available for use WIHS Manual of Operations 04 01 2014 Page 29 19 of 25 Label 1 1 75 g 1 Labels Full Sheet Hawes Date Label 2 1 75 5 Labels Full Sheet IDA z M ID 1 PID Label 3 1 x 5 8 Labels Full Sheet LI 1D 2 Frotocol Label 1B iingie Column Ll ID 3 SID el 2 i els a Tr tire Late L OFID Label 3B if bels Single Column F Other Speci Label 4 1 5 x 75 CryoLabels Full Sheet LI Primary Label 5 1 28 x 5 CryoLabels Full Sheet 5 v Spec Date Label 6 15 16 x 15 16 Labels Full Sheet T Group All Ll Spec Time Label 3C 1 x 5 8 Full Sheet Laser v Specimen ID Label 3D 1 5 8 Full Sheet 5 column 6 Sub Add Der 3 8 6 Label 6B 15 16 x 15 16 6 column j E TA 8 7 IOO Time Time Unit VID Unit VID Value Vol Units Volume Label 6C 15 16 x 15 16 3 column Laser le Label 7 2 x 4 Half Sheet Label 8 1 x 3 4 Single Column Label 8B 1x 3 4 Full Sheet Laser Il ef Label3 7 8 x 7 8 Full Sheet 6 Label Maruti RIOR SpecID from eS a ee ste gt Spec ID to Harvest Date 7a Boc Date o3 26 2001 La saan at n E eee 1 Group Pe r i Figure 1 3 Designing a custom IQA label Generating Labe
10. IV 1 isolation a multicenter study J Acquir Immune Defic Syndr 1990 3 10 981 6 Fowke K Behnke J Hanson C Shea K Cosentino LM Apoptosis a method for evaluating the cryopreservation of whole blood and peripheral blood mononuclear cells J Immun Meth 2000 24 139 144 Gjerset G Nelson KA and Strong DM Methods for cropreserving cells In Manual of Clinical Laboratory Immuniology NR Rose EC de Macario JL Fahey H Friedman and GM Penn eds 4 ed American Society for Microbiology Washington DC 1992 61 67 Lee TH Sheppard HW Reis M Dondero D Osmond D Busch MP Circulating HIV 1 infected cell burden from seroconversion to AIDS importance of postseroconversion viral load on disease course J Acquir Immune Defic Syndr 1994 Apr 7 4 381 8 Milson TJ Jr Keller RH The variable effect of cryopreservation on peripheral blood mononuclear populations J Clin Lab Immunol 1982 Apr 7 3 205 13 Paul MO Tetali S Pahwa S Effective use of frozen donor peripheral blood mononuclear cells for human immunodeficiency virus type isolation from vertically infected pediatric patients J Clin Microbiol 1994 May 32 5 1379 82 WIHS Manual of Operations 04 01 2014 Page 29 2 of 25 8 Pratt Riccio EK Neves I Banic DM Coret Real S das Gra as Alecrim M Morgado M Daniel Ribeiro CT Ferreira da Cruz MF Cryopreservation of peripheral blood mononuclear cells does not significantly affect the levels of spontaneous apoptosis aft
11. e process of storing viable PBMC s As such all WIHS labs that prepare and store viably frozen PBMC s from subjects enrolled in WIHS studies must participate in this ongoing program Every three months frozen PBMC must be shipped from your laboratory to the NIAID Immunology Quality Assessment IQA contractor The contractor determines the percentage of viable cells and viable cell yield for each sample received from each lab The data is reported back to the labs and to the following committees Executive Committee Lab Working Group Based on criteria for acceptable performance a lab is deemed capable or not capable to freeze viable PBMCs If any lab demonstrates poor performance in its ability to freeze viable PBMCs the IQA will work with the lab to try to determine what the problem may be C IRB APPROVAL Participation in the PBMC Cryopreservation Evaluation Program is dependent on local IRB approval Sites can use the attached sample consent form in Appendix A to aid in preparation of the IRB application D ELIGIBILITY OF DONORS Identify and obtain consent for donation of 2 10mL tubes from 2 participants One donor must be a healthy HIV seronegative volunteer The second donor must be an HIV seropositive volunteer with an absolute CD4 count of greater than 200 cells per mm and Viral Load less than 5000 copies ml WIHS Manual of Operations 04 01 2014 Page 29 1 of 25 E SPECIMEN COLLECTION Refer to Section 10 Part
12. e the total viable cell count 1018 lndicate the viability of the specimen before freezing What was the date the specimen was frozen Aone i What is the viable cell recovery se Indicate the number of vials frozen Comments Indicate total viable cell count per vial aa EASON TE Indicate the volume per vial Indicate original volume of the specimen drawn lndicate the mast current CO4 absolute number Data Entered by UL Figure 1 7 Entering IQA findings for Cryopreservation and Viability samples Deleting Extra Aliquots from the Pending List After the IQA lab s findings have been entered for each aliquot tested there may be extra aliquots that have been sent and have been ordered for the IQA Cryopreservation and Viability Assay To remove these samples from appearing in the pending list for the assay please complete the steps listed below 1 Click the Specimen Management button on the LDMS toolbar 2 Click the Browse button on the toolbar 3 Type in the specimen number you wish to delete 4 Click on the Run button click on a row to highlight a specimen and then click the Select button 5 Click on the Test Setup tab and click on the aliquot listed in the lower grid that you want to delete the test information A No should appear in the Done column 6 Click the Delete button on the toolbar 7 Click Yes in the Delete Test message box that appears 8 Repeat steps 2 7 for each test that you wish t
13. each donor obtain 20m1 of blood two 10ml tubes by standard venipuncture technique ACD EDTA or Heparin may be used as the anticoagulant Isolate PBMC s from each donor by Ficoll Hypaque separation After separation determine the cell yield and viability by trypan blue dye exclusion Make sure to count at least 100 cells for these determinations Use the Cryopreservation method described in Section 29 Part II B for all freezes LDMS IQA Cryopreservation and Viability samples should be logged in resulted and shipped through the LDMS Please follow the steps outlined below to ensure that the data has been entered properly For additional information please reference the LDMS User Manual sections listed for each step The LDMS manual is available online at http www fstrf org ACTG ldmsweb manual ldms_manual html Step 1 Log the samples into Specimen Management Samples must be logged into the Specimen Management module as group IQA with the PID or donor identifier entered into the ID1 field For each aliquot enter the cell count for the volume with CEL as the derivative and the volume unit The IQA Cryopreservation and Viability assay should be ordered in the Test Setup tab of Specimen Management for each aliquot Please see LDMS User Manual pgs 5 5 5 11 5 22 5 23 Step 2 Generate Labels for IQA Specimens Use the Labels module to create custom labels for the IQA specimens that will print out the ID1 LDMS specimen cell vol
14. ear Cells Clin Diagn Lab Immunol Jul 2000 7 4 714 716 15 Weinberg A Betensky RA Zhang L Ray G Effect of Shipment Storage Anticoagulant and Cell Separation on Lymphocyte Proliferation Assays for Human Immunodeficiency Virus Infected Patients Clin Diagn Lab Immunol Nov 1998 5 6 804 807 PART II PROCESSING FREEZING THAWING AND SHIPPING It is preferable that WIHS labs use CPT for collection as well as the extraction and freezing methods stated in Section 10 However some WIHS labs are also ACTG labs and would be required to send samples drawn from another tube or method In the second instance if possible please send two samples using the WIHS protocol and the ACTG protocol Labs are advised to keep a parallel vial of each donor to check the report from the IQA contractor A EQUIPMENT AND SUPPLIES FOR PROCESSING ACTG 1 Gloves latex vinyl nitrile 2 Lab coat or protective gown 3 Anticoagulated blood EDTA ACD or HEP 2 10 mL tubes 4 Ficoll density gradient solution density 1 077 sterile and endotoxin tested Label container with date after opening The shelf life for Ficoll is 6 months after opening WIHS Manual of Operations 04 01 2014 Page 29 3 of 25 10 11 12 13 14 15 16 17 18 19 20 21 22 However discard if manufacturer s expiration date occurs before this 6 month period It is best to purchase small volumes of this reagent and replace frequently Examples Fico
15. ed Manual Total Viable Cell Yield Total of PBMC x 10 without Trypan Blue Viability I Automated Specify Viability Method Used Trypan Blue Other Specify Freezing Date HHHH Volume per Vial Imi jms mmm d d y y y y per vial times Number of Vials Frozen _ Total Viable Cell Count per Vial T lx 10 10 million Shipping pate 11 Number of Vials Shipped Ra mmm d d y y y y Date of Thaw 1 C m mm d d Should be volume y y WIHS Manual of Operations 04 01 2014 Page 29 16 of 25 January SM TW TF S 1 2 3 4 5 6 7 8 9 10 I1 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 February SM T WT F 4 5 6 11 12 13 18 19 20 525 26 27 N o mlu March SM TW TF S 1 4 5 6 11 12 13 18 19 20 25 26 27 May SM TW TF 1 2 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 9 20 21 22 23 24 25 26 27 28 29 30 31 APPENDIX D SAMPLE IQA TIMELINE 2003 IQA Cryopreservation Project Timeline January 27 Notification by IQA February 24 Collection of Samples by Sites March 10 Shipment of Cryo Samples to IQA April 28 Notification by IQA May 19 Collection of Samples by Sites June 2 Shipment of Cryo Samples to IQA July 14 Notification by IQA August 18 Collection of Samples by Sites September 8 Shipment of Cryo Samples to IQA October 20 Notification by IQA November 1
16. elect the IQA samples in the Specimens Found screen and click the Add to Run button Enter data or select responses for each of the fields on the Result screen Click the Add button on the toolbar to save the record Use the VCR buttons to scroll to the next record Repeat steps 9 12 for all remaining samples Assau Name 0A Dyspressr janan ant Viability Specimen Type Spec Id fozsvor apanga Primary eto Additive HEP Derivative CEL Sub AND NVA Spec Date asv28r 2001 Rund fi 8041 Seq tt fi M 99001 What was the total cell yield of the specimen alter separation so ik 10 6 Indicate the viability of the specimen betore freezing a _ What was the date the specimen was frozen 09 28 2001 In Indicate the number of vials frozen Indicate total viable cell count per vial L Indicate the volume per vial Indicate the mast current CD4 absolute number Wete Results obtained on this specimen Yes C No Indicate HIY Status l Negative What was the date of blood separation og 2ev2001 Indicate original volume of the specimen drawn ml x 106 Data Entered by MC ml tama Figure 1 5 Entering IQA Cryopreservation and Viability Results WIHS Manual of Operations 04 01 2014 Page 29 21 of 25 Creating a Shipping Disk and Manifest Report Once the samples have been resulted in the Assay Module the last step for the sending lab is to create a shipping disk and manifest repo
17. er 24 h culture Cryobiology 2002 45 127 134 9 Reimann KA Chernoff M Wilkening CL Nickerson CE Landay AL ACTG Preservation of Lymphocyte Immunophenotype and Proliferative Responses in Cryopreserved Peripheral Blood Mononuclear Cells from Human Immunodeficiency Virus Type 1 Infected Donors Implications for Multicenter Clinical Trials Clin Diagn Lab Immunol May 2000 7 3 352 359 10 Sleasman JW Leon BH Aleixo LF Rojas M Goodenow MM Immunomagnetic Selection of Purified Monocyte and Lymphocyte Populations from Peripheral Blood Mononuclear Cells following Cryopreservation Clin Diagn Lab Immunol Nov 1997 4 6 653 658 11 Strong DM Ortaldo JR Pandolfi F Maluish A Herberman RB Cryopreservation of human mononuclear cells for quality control in clinical immunology I Correlations in recovery of K and NK cell functions surface markers and morphology J Clin Immunol 1982 Jul 2 3 214 21 12 Tollerud DJ Brown LM Clark JW Neuland CY Mann DL Pankiw Trost LK Blattner WA Cryopreservation and long term liquid nitrogen storage of peripheral blood mononuclear cells for flow cytometry analysis effects on cell subset proportions and fluorescence intensity J Clin Lab Anal 1991 5 4 255 61 13 Venkataraman M Effects of Cryopreservation on Immune Responses Cryobiology 1996 33 581 588 14 Weinberg A Zhang L Brown D Erice A Polsky B Hirsch M Owens S Lamb K Viability and Functional Activity of Cryopreserved Mononucl
18. g Examples Sarstedt cat 72 694 005 Corning cat 430489 Cryo Labels specific for use in freezing and liquid nitrogen Examples Cryotags Cryobabies 1 50 x 0 75 Cat LCRY 1200 Shamrock 5 8 x 1 satin cloth labels cat ACTG SCPF Pioneer 1 75 x 0 5 cat 710 CILS 9100 labels Laminar flow hood minimum class 2 Type A Biosafety hood Centrifuge with horizontal rotor with speeds up to 1800 X g and equipped with aerosol safe canisters Hemacytometer and microscope Nalgene Mr Frosty Nalgene Cryo 1 C Freezing container Nalgene cat 5100 0001 Curtis Matheson Scientific cat 288 383 or Fisher Scientific cat 15 350 50 or Cryomed Freezing Chamber Gordinier Electronics WIHS Manual of Operations 04 01 2014 Page 29 4 of 25 23 24 25 20 C freezer 70 C freezer Liquid nitrogen storage tank with LN2 rated boxes with holes to allow LN2 drainage Note storage of single vials in canes is not recommended due to safety concerns submersion in liquid phase and possible damage to the affixed labels PROCESSING AND FREEZING PROCEDURE ACTG These instructions assume the lab uses LDMS and the ACTG method Sites who do not use LDMS can also participate 1 Label cryovials with LDMS derived specimen number WIHSID protocol date of collection VID cell count and LDMS specimen code The label should be placed on the tube so that the contents are visible Cryovials may be chilled in t
19. h you are otherwise entitled Otherthan non participation in the research available altematives include Since this isnot a treatment study there is no altemative except not to participate and have blood tests done by your primary care doctorata Clinical laboratory 6 All reasonable measures to protect the confidentiality of yourrecordsand your identity will be taken Your identity will not be revealed in any publication that may result from this study The confidentiality of all study related recordswillbe maintained in accordance with State and Federallaws There isa possibility that your medical research record including identifying information may be inspected and photocopied by officials of the Federal or State govemment agenciesand the University Human StudiesCommittee and the National Institutes of Health 7 If you have any questionsorconcemsregarding this study orif any problemsarise you may callthe Principal Investigatorat You may also ask questionsorstate concems regarding your rights asa research subject to Dr Chairman of the University s Institutional Review Board at 8 University investigatorsand theircolleagueswho provide servicesat University Medical Center hospitals and facilities recognize the importance of your contribution to research studies that are trying to improve medical care University investigators and their staffs will make every effort to minimize control and treat any complications that may arise a
20. he freezer for at least 30 minutes prior to cell freezing Prepare the freezing medium 90 fetal bovine serum 10 DMSO chill on ice or place in refrigerator at 4 C for at least 30 minutes Freezing medium may be stored up to one week Experience from some ACTG laboratories has shown that larger volumes can be prepared aliquotted and stored at 20 C for up to one year Centrifuge the whole blood tubes at 400 X g for 10 minutes at room temperature Remove the plasma layer and save Separate the PBMCs using Ficoll Follow Ficoll manufacturer s instructions with respect to blood Ficoll ratios and centrifugation time and speed Centrifuge at room temperature with NO brake After centrifugation transfer the cloudy interface layer PBMC layer into appropriately labeled 50 mL 15 mL if pediatric sample sterile centrifuge tubes by carefully aspirating the cells with a sterile transfer pipette Avoid aspirating the Ficoll solution by maintaining the pipette tip above the cell layer and SLOWLY drawing the cells up into the pipette Wash PBMCs by diluting the PBMC layer solution with at least an equal volume of PBS or HBSS Centrifuge the cells at 200 400 X g for 10 minutes at room temperature Discard supernatant A second wash is recommended When ready to count the PBMCs resuspend them in 1 to 4 mL of complete medium depending on the size of the cell pellet For example if the PBMC pellet covers the entire bottom of the
21. ion can be adjusted to 5 10 x 10 viable cells mL per cryovial If cells were resuspended in complete medium for counting purposes centrifuge the cells before resuspending them in cold freezing medium Dispense 0 5 mL aliquots of the cell suspension into cryovials Be sure that the cryovial caps are securely tightened Immediately place the cryovials in a slow freeze container e g Mr Frosty and place the container in a 70 C freezer for 4 to 24 hours Alternatively place the cryovials into controlled rate LN2 freezing chamber Cryomed Freezing Chamber NOTE To prepare the Mr Frosty e Remove the high density polyethylene vial holder and foam insert from the polycarbonate unit e Add 250 mL of 100 isopropyl alcohol to the fill line DO NOT OVERFILL Avoid slopping the isopropyl alcohol on the labels because the alcohol can cause the ink to run e Replace alcohol after every fifth use and document this reagent change e Replace foam insert and vial holder e Place cryovials containing sample into holes in vial holder e Close Mr Frosty and place in 70 C freezer e Pre chilling Mr Frosty in the refrigerator 4 C prior to use does not affect cell viability ACTG experience After 4 to 24 hours in a 70 C freezer transfer the cryovials into a LN2 rated box and place the box into vapor phase liquid nitrogen 135 C for long term storage Avoid liquid phase storage due to safety concerns and to preve
22. ll Paque Amersham Pharmacia cat 17 1440 02 Sigma Histopaque 1077 Hybri Max cat H8889 Sterile Phosphate Buffered Saline PBS Ca free and Mg free or Sterile Hanks Balanced Salt Solution HBSS Observe manufacturer s outdate Label bottle with open date use opened bottle within three months Fetal Bovine Serum FBS heat inactivated at 56 C for 30 minutes mix larger volumes several times while inactivating Sterile Complete RPMI 1640 medium Supplement RPMI with L glutamine 2 mM final concentration 100 units Pen mL 100ug Strep mL and 10 fetal bovine serum Medium may be filter sterilized after addition of supplements Dimethyl Sulfoxide DMSO Store at room temperature DMSO must be fresh and sterility maintained The shelf life for DMSO is 6 months after opening Label with the date upon opening Example Hybrimax Sigma cat D2650 0 4 Trypan Blue solution Store at 18 to 25 C Filter as needed Observe manufacturer s outdate Example Sigma T81540 100mls Sterile conical centrifuge tubes 50 mL and 15 mL Microcentrifuge tube for cell counting 0 5 mL Sterile pipettes graduated and transfer Pippetting device Sterile pipette tips Micropipettors of various volumes Sterile Cryopreservation Vials cryovials 1 8 to 2 mL with screw cap external threads and O rings a NOTE Some cryovials are unacceptable for use in liquid nitrogen Please check the manufacturer s recommendations before usin
23. ls for IQA specimens Once your custom label is designed you can then print out labels for each of your aliquots 1 In the Labels module select IQA from the Format combo box and select your label type from the Description grid Select group from the Field combo box from the Operator combo box and IQA from the Value combo box Click the Add button beneath these combo boxes to add your search criteria Select the received date for your samples Click the Execute button on the toolbar Click the Print button on the Crystal Reports toolbar to print your labels N Mie w vii 029000004 Se HJ CEL O20 CEL Figure 1 4 Custom IQA label WIHS Manual of Operations 04 01 2014 Page 29 20 of 25 Completing the IQA Cryopreservation and Viability Data Entry Screen Prior to shipping the samples to the IQA lab each aliquot must be resulted in the Assay module eee D 10 Click the Report button on the toolbar to print a patient report 12 13 11 Click on the Assay button on the LDMS toolbar Click on the plus sign next to Immunology Category Click on the IQA Cryopreservation and Viability row Click on the New Run Not Setup radio button Click the Select button on the Assay Selection screen Enter the specimen received date in the From calendar combo box You may also generate a pending specimen report from this tab by clicking the Report button on the toolbar Click the Find Specimens button S
24. n the Assay Selection screen Enter the specimen received date in the From calendar combo box You may also generate a pending specimen report from this tab by clicking the Report button on the toolbar Click the Find Specimens button Select the IQA samples in the Specimens Found screen and click the Add to Run button Enter data or select responses for each of the fields on the Result screen The left side of the result screen displays the sending lab s specimen information These fields are grayed out and the results can not be changed Click the Add button on the toolbar to save the record Click the Report button on the toolbar to print a patient report 12 13 Use the VCR buttons to scroll to the next record Repeat steps 9 12 for all remaining samples WIHS Manual of Operations 04 01 2014 Page 29 23 of 25 Assad Name 104 Civeprezer vation and Viabilito Assay Selection Filters Criteria Specimens Found Results a ID3 0 99001 ID2 Specimen Type Spec Id fozsvoronnn42 Primary ato Additive HEP Derivative CEL Sub A D N A Spec Date 097287200 Runid fem segt F E Were Results obtained on this specimen yee Ki fs Specify reason F Iridicate HI Status j Neive What was the date of blood separation Datespecimen Thawed 0322872001 La Indicate the viability of the sample after Z What was the total cell yield of the specimen alter separation thawing Indicat
25. ng Investigator when informed consent responsibility is entrusted to a designee See HSC Guidelineson Who May Obtain Consent to Participate in Research Activities This form is valid only if the Institutional Review Board s current stamp of approval is shown below WIHS Manual of Operations 04 01 2014 Page 29 13 of 25 APPENDIX B SAMPLE PBMC QA REMINDER The IQA contractor will send a formal version of this memo to participating sites prior to all scheduled shipments This QA program for preparation of viably frozen PBMC is required quarterly of ALL labs processing and freezing PBMC Sites will review this reminder three weeks prior to each quarterly shipment cycle stated in the IQA timeline Obtain Samples Identify and obtain consent from 2 blood donors One donor must be a healthy HIV seronegative volunteer The second donor must be an HIV seropositive volunteer with an absolute CD4 count of greater than 200 cells per mm and Viral Load less than 5000 copies ml Refer to Section 29 Appendix 1 for an IRB sample Please make arrangements to obtain blood from your two volunteers two weeks before the shipment deadline see the IQA timeline Ideally your volunteers should come in on the same date and time so that a single freeze can be done If this is not possible handle the samples in the same manner one that preserves the viability of the cells so that handling does not introduce differences between the two samples From
26. nt possible problems with label adhesion failure Record results for each sample on the Freezing Form in Appendix C or F It is important for sites to write down the correct Total Viable Cell Count per vial on the Data form Include handwritten calculations for Viability and Cell Count for one of the samples vials being submitted to the IQA WIHS Manual of Operations 04 01 2014 Page 29 7 of 25 C THAWING PROCEDURES NOTE Be sure to wear a full face shield during the thawing procedure because cryovials containing liquid nitrogen have been known to explode 1 5 Remove the cryovials from the liquid nitrogen container to a 37 C water bath If liquid nitrogen has seeped into the cryovial loosen the cap slightly to allow the nitrogen gas to escape during thawing Hold the cryovial in the surface of the water bath with an occasional gentle flick during thawing Do not leave the cryovial unattended during the thawing process It is important for cell viability that the cells are thawed and processed quickly thawing only takes a few seconds When a small bit of ice remains in the cryovial transfer the cryovial to the biosafety hood Dry off the outside of the cryovial and wipe with an alcohol solution before opening the vial to prevent contamination Quickly transfer the thawed cell suspension from the cryovial to a 15 mL conical centrifuge tube containing 10 mL of chilled Complete RPMI medium contains 10 fetal bovine se
27. number or PID in the ID1 field Select the specimen and receive dates Select BLD as the primary type and select either ACD EDT or HEP as the additive type Enter 20 ml as the primary volume Select 4 for the number of aliquots 5 for volume CEL for units and CEL for derivative and the click the add button on the side of the aliquot grid 8 Click the Add button on the LDMS toolbar 9 Click the Enroll button on the dialog box that appears ee ae ee NOTE Repeat the above steps for the second blood donor User Defined IDs D 99001 ID2 Spec Date 09728 2001 Las ee se P SE Bature Derivative A mpor Culture Derivative Bee Date 9 25 2001 Les Lime __ Evpait 0 Import date Hot Tubes Ermat Type BLD Blood Whole 17 Other Spec ID Time aaj Delete EE 2pecimen a 02901000042 BLO HEF i gt 20 00 ae Aliquot Imi fe Units Detvativel 7 Sub Add Der aj Other ct Prot y oume tE Dither Spec x a 023 01000042 BLO gt Sr EE Ae 104 399001 2 029701000042 BLD m N A a 5 00 CEL 194 99001 3 02901000042 BLD HEP CEL JN A x 5 00 CEL 1GA 99001 4 02901000042 BLD HEP CEL N A x 5 00 CEL 1Q4 99001 Figure 1 1 Logging IQA specimens in Specimen Management WIHS Manual of Operations 04 01 2014 Page 29 18 of 25 Ordering the IQA Cryopreservation and Viability assay Click on the Test Setup tab Click on a sample in the Specimens grid In the Tests grid in
28. o delete from your pending list NOTE Deleting the test will not remove the specimen record from the LDMS Specimen Management module WIHS Manual of Operations 04 01 2014 Page 29 24 of 25 APPENDIX F WIHS FREEZING FORM WIHS FREEZING ASSESSMENT PROGRAM IQA Cryopreservation and Viability Data Entry Form LDMS ster Lab Site TTT 1 Site Name Name of Contact Contact Telephone Contact Email Directors Email Name of Lab Director Director Telephone m m m d d y y y y HIV status _ Positive Negative Volume of Blood Draw I frs Method Ficoll CPT Other Most Current CD4 Avsoute mm Anticoagulant used ACD EDTA Heparin Date of Blood Separation CIJ LIT mmm d d y y y y Cell Counting Method Used Manual Total Viable Cell Yield Total of PBMC xio without Trypan Blue Viability TMA Automated Specify Viability Method Used Trypan Blue Other Specify Freezing Date CIT LI J voumepervirmi mis m m m d d y y y y Number of Vials Frozen _ Total Viable Cell Count per Vial Ik 10 10 million shiping Date _ CIT TT Number of Vials Shippea m m m d d y y y y Date of thaw Tf Percent Viable Cell Recovery 1 m m m d d y y y y Total Viable Cell Count T x 10 viability Cell Counting Method Used Manual L with Trypan Blue Viability Method Used Trypan Blue Oo without Trypan Blue WIHS Manual of Operati
29. ons 04 01 2014 Page 29 25 of 25 with Trypan Blue _
30. rcumstances Include in your message the WIHS site name number and the specific lab unable to participate Please Note Labs are required to freeze 4 vials on each Donor specifically for this QA program Two vials from each Donor to be shipped to the IQA and 2 vials to remain at the lab site The conc per vial of Cryopreserved PBMC s is 10 x 10 ml in 0 5ml aliquots Therefore a total of 5 x 10 Viable cells per VIAL Both viability and viable cell recovery will be assessed by the IQA after thawing Therefore an accurate count of total viable cells in each vial is important For instructions on cell counting methodology refer to Section 29 Part II B 9 Make sure label states Donor LDMS Specimen cell Volume Date Der CEL WIHS Manual of Operations 04 01 2014 Page 29 15 of 25 APPENDIX C ACTG FREEZING FORM ACTG FREEZING ASSESSMENT PROGRAM IQA Cryopreservation and Viability Data Entry Form LDMS site Lab seel Site Name Name of Contact Contact Telephone Contact Email Directors Email Name of Lab Director Director Telephone ponornumber _ Date of Blood Draw CITT mmm d d HIV Status __ Positive Negative Volume of Blood Draw T mis Most Current CD4 Absolute mm Anticoagulant used _ ACD Heparin Jeota Date of Blood Separation CIJ 1T mmm d d y y y y yyy y with Trypan Bue _ Cell Counting Method Us
31. rt to send with the frozen aliquots to the IQA lab 1 PrOD PSO 9 10 11 12 13 14 15 16 Click on the Shipping icon on the LDMS toolbar Click on the Setup Shipment tab to search for your samples Select IQA from the Group combo box Select Received Date from the Type combo box and enter the received date into the ID edit box Click the arrow button to move the criteria into the next grid Click the Execute button on the toolbar Select two aliquots from each blood donor by clicking on the rows to highlight them Click on the Shipment Destination tab and select lab 213 Immunology Quality Assessment Lab from the Lab combo box Select a contact from the Contact Person combo box Select a contact from the Contact Sending Lab combo box Click the Add button on the toolbar to batch the shipment In the View Shipment screen select your batch and click on the Manifest Report button Print the Box Map report if applicable Right click on the selected batch and select Create Diskette from the menu Click OK to view the storage report or click Cancel to continue Select your disk drive and select LDMS format Click OK on the dialog box that appears and write the shipping batch number on the diskette IQA Lab LDMS 213 Importing IQA samples OO SION ON oS a Click the Shipping button on the LDMS toolbar Click the Import tab Click on the Shipment Location button and select the floppy drive on your PC En
32. rticipation in this blood donor program in the amount of X to X The amount you receive is dependent on the amount of time required to complete the pre screen visit and the blood donation Screening visit X enrollment Up to one hour of time Blood Donation X 3 There are certain risks and discomforts that may be associated with this research They include Asa blood donor you should understand that there may be some risks and side effectsassociated with donating blood Drawing blood from a vein can cause local pain bruising or bleeding from where the needle enters the skin There isalso a minimal risk of inflammation or infection of the vein decreased blood pressure dizziness or fainting that Can occur during orafter you donate blood 4 The possible benefits to you and society from this research are Asa blood donor you will receive no therapeutic benefit from this study However the improvement in the quality of the blood studies being performed in the laboratories throughout the United States will benefit all HIV infected patientswho need to have these blood studiesdone aspart of their routine medical care and participation in clinical drug trials 5 Your participation is voluntary and you may choose not to participate in this research study or withdraw your consent at any time Your choice will not at any time affect the commitment of yourhealth care providersto administercare and there willbe no penalty orlossof benefitsto whic
33. rum Centrifuge at room temperature at 200 400 X g for 10 minutes Remove the supernatant without disturbing the cell pellet Wash once with 10 mL of room temperature PBS and centrifuge at room temperature at 200 400 X g for 10 minutes Gently resuspend the PBMCs in the appropriate medium for the assay to be performed Determine the cell number and percent viable cells D SHIPPING A WIHS and or ACTG Freezing Form must accompany all samples The anticoagulant and method should be clearly marked Please see Appendix D for the current shipping timeline Sites using LDMS should consult Appendix E instructions to prepare samples 1 IQA SHIPPING INSTRUCTIONS 1 Samples must be frozen in 1 8 mL cryovials 2 Samples must be labeled with all the proper identification Donor ID Sample date frozen etc 3 Samples must be put into a watertight primary receptacle cryovial Then samples must be place into a watertight secondary packaging sealed Styrofoam container 4 Place an absorbent material between the primary receptacle and the secondary packaging The absorbent material must be sufficient to absorb THE ENTIRE contents of ALL primary receptacles If multiple primary receptacles are placed in a secondary packaging they must be wrapped individually to ensure that contact between them is prevented 5 A sturdy outside packaging container constructed of corrugated fiberboard must be used The minimum size is 7 x 4 x 2
34. sa result of this research If you believe that you are injured asthe result of the research question being asked in the study please contact the WIHS Manual of Operations 04 01 2014 Page 29 12 of 25 Principal Investigator and or the Chairman of the Institutional Review Board asstated in item 7 Washington University reservesthe right to make decisionsconceming payment for medicaltreatmentforinjunessolely and directly relating to yourparticipation in biomedical or behavioral research 9 You will be informed of any significant new findings developed during the course of participating in thisresearch that may have a bearing on yourwillingnessto continue in the study The investigator may withdraw you from this research if circ umstancesarise which warrant doing so I have read this consent form and have been given the opportunity to ask questions will also be given a signed copy of this consent form for my records hereby consent to my participation in the research described above tited ACTG 001 Blood Donation for the Immunology Quality Assessment Program Parent orlegal guardians signature on Date Participant s Signature Date participants behalf if participant is less than 18 years of age ornot legally competent Blood drawing only Less than 17 years of age Informed Consent provided by Relationship to Child Signature of Principal Investigator or DesigneeDate Signature of Principal Investigator or Collaborati
35. sponsible individual for the packaged may be printed or stamped WIHS Manual of Operations 04 01 2014 Page 29 10 of 25 APPENDIX A SAMPLE IRB CONSENT Form 2 02 99 Human Studies Committee INFORMED CONSENT FOR PARTICIPATION IN RESEARCH ACTIVITIES Participant HSC Approval Number Principal Investigator PI s Phone Number Title of Project Bood Donation for the Immunology Quality Assessment Program This consent form may contain words that you do not understand Please ask the study doctororthe study staff to explain any wordsor information that you do not cleanly understand You are being asked to participate in a blood donor program forthe Immunology Quality Assessment Program IQA under the local direction of Dr X 1 You are invited to participate in a research study conducted by Dr and or colleagues The overall purpose of this research is to measure the quality of blood tests being done by our laboratories by sending a blood specimen from two donorsto a central testing lab which will compare its findingsto ourlabsto make sure that our laboratory reports the corect results Thistype of testing for quality will help assure that HIV infected patients throughout the United States have accurate immune blood tests that monitor their disease We are one of many laboratories throughout the United States that perform immune blood tests to follow the course of HIV disease This study will enroll approximately 8 12 people This study
36. ter the batch number from the Shipping Manifest in the Batch Number edit field Click on the Import button Select LDMS as the shipping disk format Click Yes on the Test Setup dialog box Click OK on the Success dialog box WIHS Manual of Operations 04 01 2014 Page 29 22 of 25 View Shipment Setup Shipment Shipment Destination Shipment Location Batch Number 435 IMPORT a Jozsvo1 o00042 BLD CEL HEP 09 28 2001 5e 06 SAT Satisfactory No a 029701000042 BLD CEL HEP 09 28 2001 5e 06 SAT Satisfactory Yes 3 029v01000042 BLD CEL HEP 09 28 2001 5e 06 SAT Satisfactoy Yes BLD CEL HEP 09 28 2001 __5e 06 SAT Satisfactory Yes a 029v01000042 Continue Cancel Figure 1 6 Importing an IQA shipping disk NOTE Your imported IQA samples will automatically show up in Specimen Management with the imported box checked the imported date the IQA Cryopreservation and Viability test ordered and the sending labs results available in the Assay Module Completing the IQA Cryopreservation and Viability Data Entry Screen After the samples have been imported and tested you need to go into the Assay module and enter your findings for each sample 1 SN Ot da bo I 10 11 Click on the Assay button on the LDMS toolbar Click on the plus sign next to Immunology Category Click on the IQA Cryopreservation and Viability row Click on the New Run Not Setup radio button Click the Select button o
37. the required markings and labels For each package the shipper must make sure all relevant markings and labels are in the correct location and any irrelevant markings and labels are to be removed There are two different kinds of labels that are required for shipment a HAZARDS LABELS identifies the class of dangerous goods and must bear the class or division number in the bottom corner of the label b HANDLING LABELS identifies any special handling instructions that apply to the class of dangerous goods The following labels are required for the shipment of dangerous goods and must be marked durably and legibly on the outside of the package a PROPER SHIPPING NAME of the contents in the package and the corresponding UN NUMBER S or ID NUMBERS b A class 9 hazard label and label stating UN1845 DRY ICE kg is required for specimens shipped on dry ice There is a maximum of 200kg per package c NAME and TELEPHONE NUMBER of the person responsible for the shipment must be available to answer 24 hours of the day in case of an emergency regarding the package s shipped WIHS Manual of Operations 04 01 2014 Page 29 9 of 25 3 SHIPPING DOCUMENTATION The shipper is responsible for the completion of documentation for each shipment containing dangerous goods as defined by IATA Paperwork i e Air Waybill Commercial Invoices and the box should include the phrase Diagnostic Specimens Packed in compliance
38. ume specimen date and derivative type for each label Please see LDMS User Manual pages 11 5 11 10 11 11 Step 3 Complete the IQA Cryopreservation and Viability Data Entry Screen Go into the Assay module and select the IQA Cryopreservation and Viability assay from the Immunology tests that are listed Select a new run and gt search for your specimens in the Filters Criteria tab Select your specimens and complete all fields on the Results tab Print out patient reports for your records Please see LDMS User Manual pages 8 4 8 8 8 77 Step 4 Create a Shipping Disk and Manifest Report In the Shipping module search for your IQA samples and create a shipping disk to be sent to the IQA Lab 213 Print out and include a copy of the manifest report in your shipment Please see LDMS User Manual pages 7 3 7 5 7 11 7 17 7 23 WIHS Manual of Operations 04 01 2014 Page 29 14 of 25 If you have any questions using the LDMS program please contact LDMS user support at 716 834 0900 ext 311 or email dmc ldms usersupport fstrf org Shipping Instructions Make arrangements to ship the samples as stated in Section 29 Part II D on Monday Tuesday or Wednesday of the week of two weeks before specified ship date If your site is not going to be able to obtain samples for this iteration of the program please send a message to Mr Raul Louzao raul louzao duke edu two weeks before the scheduled ship date with an explanation of your ci
39. will last seven years Your participation however will be limited to one blood donation per year 2 Your participation will involve Screening If you decide to participate asa blood donorin this study and sign this consent you will have testsand examinations done before you donate any blood This is being done to be make sure that you will be able to donate blood without ham to you These tests and proceduresinclude measuring blood pressure body temperature heart and breathing rate Yourhemoglobin level may about 2 teaspoon of blood be measured to make sure that you are not too anemic having low numbers of red cells to participate in this program without affecting your health Blood Donation Once it isdetermined that you are able to participate in this project an appointment willbe made forthe days that your blood will be drawn Blood willbe obtained by inserting a needle in youram vein the amount of blood to be taken will vary but will not exceed 100 ml 6 tablespoons You willbe allowed to donate no more than once a yearand will receive payment foreach pre screen visit and blood donation The Medical Director will be available to discuss any screening test results with you and will share these results with your physician if you approve WIHS Manual of Operations 04 01 2014 Page 29 11 of 25 Financial consideration Your ability to pay will not affect your participation in this study You will receive payment for your pa
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