SITE PROCEDURES BLOOD SPECIMENS Blood Specimen Collection Supplies: Red Top Tubes Plain Cap Tubes Blood Specimen Labels 1. The local phlebotomy team should collect a minimum of 4.5 ml of child s blood at any time during hospital admission in a red top tube. For participants who have not had a CBC with differential done during the preadmission or admission blood testing, an additional 0.5 ml of blood will be collected in a purple top tube at any time during the hospital admission but preferably during the ED visit or first 24 hours of hospitalization. 2. If the required blood volume is not acquired through the blood draw sites should schedule another blood draw. If after other attempts, the acquired volume is still not collected, sites should contact Ashley Sullivan (afsullivan@partners.org 617-724-9712) about the possibility of a waiver for the enrolled subject. 3. At the discretion of investigator and parent/legal guardian, EMLA cream (lidocaine 2.5% and prilocaine 2.5%) will be applied to the skin to prevent pain associated with needle insertion 4. The blood specimen should be centrifuged at 3000 RPM at room temperature until the serum is fully separated (approximately 10 minutes.) 5. The serum will be siphoned off and placed into one tube and all of the remaining specimen (the pellet) will be transferred to a separate tube. (Please do NOT siphon too close to the top of the pellet when extracting the serum as the thin layer of white blood cells contains the DNA for testing.) 6. Both the serum and the and pellet tubes will be labeled, and stored at 70 o C or 80 o C. It is recommended that the serum be stored in the clear top tubes and the pellet in the blue top tubes provided for easier identification, but please note that the tubes are identical except for the color of the tops. 7. Verify that the study id printed on the label(s) is correct. Then, the blood volume, time (hh:mm) and date (mm/dd/yyyy) of collection should be written on the blood sample labels and entered into the REDCap blood specimen collection form. 8. For all participants, both the serum and pellet will be stored. However, for participants who did not agree to the genetics portion of the study, the pellet will NOT be shipped to the University of Arizona, but rather will be shipped to the Massachusetts General Hospital for possible future use consistent with the main consent. Labeling by Sites Supplies: Blood Serum or Blood Pellet Specimen Label Blood Serum or Blood Pellet Shipment List
1. After verifying the study id printed on the labels is correct, write the date (mm/dd/yr) and time (hh:mm) of sample collection on the Blood Serum/Pellet Labels. 2. Place the Blood Serum/Pellet Label on the appropriate transport tube. 3. Wrap a strip of clear tape around the whole tube, over the label, and atop itself to prevent the label from falling off. 4. Record the date (mm/dd/yr) and time (hh:mm) of sample collection and the sample ID number in the REDCap blood serum/pellet specimen collection form. 5. Record the sample ID number, collection date and collection time of each sample on the Blood Serum or Blood Pellet Shipment List (Do NOT check off last column of the Shipment List; this will be done when all samples are ready to be shipped). 6. Store sample in specimen biohazard bag in 80 C freezer (please refer to Blood Pellet/Serum Storage and Shipping Instructions for proper storage technique). Storage at Sites Supplies: 4º Refrigerator -80 C freezer (freezer must NOT be warmer than 70 C) Biohazard bag or box 1. Once sample has been transferred to the cryovials, place in the specimen biohazard bag. 2. Place the specimen biohazard bag on ice for transport to an -80 C freezer. 3. Walk specimen(s) to your local microbiology lab. 4. Store samples at 4 o C within one hour of specimen collection and freeze at 80 o C within 24 hours of collection. 5. Each specimen is stored in its specimen biohazard in the 80 C freezer until samples are ready to be shipped. Shipping of Serum to Massachusetts General Hospital (MGH) Note: Only lab personnel with IATA certification may package and ship the serum specimens. Site PIs should be present during the specimen mailing process to ensure that all specimens are included in the mailing, to give the necessary supplies to the person preparing the package, and to include the completed Blood Serum shipment list in the mailing. Specimens will be shipped one time during each recruiting season (May). The EMNet Coordinating Center will determine which sites ship during which week. Mindful of holidays, specimens should be sent only on Mondays, Tuesdays, or Wednesdays so that the specimens do not arrive on a Saturday, Sunday, or holiday. Prior to shipment, the shipment list should be matched against the serum specimen collection form in REDCap to ensure that all specimens are accounted for.
Supplies: Sterile gloves Biohazard bag(s) Absorbent material Blood Serum Shipment List (completed) Envelope for Blood Serum Shipment List 5 lbs (2.27 kgs) dry ice (small pellets rather than large blocks) Sample box Styrofoam box (to hold dry ice and sample box) Cardboard shipping box (to hold Styrofoam box) Biohazard sticker or biohazard bag (for top of Styrofoam box lid) Exempt Human Specimen label Dry Ice shipment label 1. Put on sterile gloves and remove samples from freezer. 2. If each sample is not already in a biohazard bag, place the blood serum sample in a biohazard bag (only ONE sample per bag) with absorbent material, while noting on the Blood Serum Shipment List their inclusion (At this time, the last column of the Shipment List should be completed). 3. Place the biohazard bag(s) in sample box(es) 4. Add bubble wrap or foam padding around the bags for cushioning and put the lid on the sample box. 5. Place the sample box(es) in the Styrofoam box. 6. Completely surround the box with samples with the 5 lbs (2.27 kgs) of dry ice. 7. Insert padding around the sample box(es) if there is additional space in the Styrofoam box. 8. Put lid on Styrofoam box and place into cardboard box. 9. Affix an orange biohazard sticker (or a bag that is pre-labeled with the universal biohazard symbol) to the Styrofoam box lid if one is not already present. 10. Put completed Blood Serum Shipment List in an envelope and place on top of Styrofoam lid. Keep a copy of the Shipment List for your files. 11. Seal cardboard box and affix the completed dry ice label. 12. On the FedEx label, be sure to check off Dry Ice and note the weight of dry ice. Also affix the Dry Ice label, which is required to be on the box and also notes the weight of the dry ice. 13. Attach Exempt Human Specimen label near the Dry Ice label. Ship samples (overnight) to: Serum specimens must be shipped (overnight) to: Carlos A. Camargo, M.D. Attention: Ashley Sullivan Massachusetts General Hospital 326 Cambridge Street, Suite 410 Boston, MA 02114 Tel: 617-726-5276 FedEx is the preferred carrier for blood shipments. Please use the FedEx Priority Overnight service.
On the day that the specimens are sent, the site PI should email both afsullivan@partners.org and ccamargo@partners.org the FedEx tracking number for the mailing. Shipping of Pellet to University of Arizona Note: Only lab personnel with IATA certification may package and ship blood pellet specimens. Site PIs should be present during the specimen mailing process to ensure that all specimens are included in the mailing, to give the necessary supplies to the person preparing the package, and to include the completed Blood Pellet shipment list in the mailing. Specimens will be shipped one time during each recruiting season (May). The EMNet Coordinating Center will determine which sites ship during which week. Mindful of holidays, specimens should be sent only on Mondays, Tuesdays, or Wednesdays so that the specimens do not arrive on a Saturday, Sunday, or holiday. Prior to shipment, the shipment list should be matched against the pellet specimen collection form in REDCap to ensure that all specimens are accounted for. Supplies: Sterile gloves Biohazard bag(s) Absorbent material Blood Pellet Shipment List (completed) Envelope for Blood Pellet Shipment List 5 lbs (2.27 kgs) dry ice (small pellets rather than large blocks) Sample box Styrofoam box (to hold dry ice and sample box) Cardboard shipping box (to hold Styrofoam box) Biohazard sticker or biohazard bag (for top of Styrofoam box lid) Exempt Human Specimen label Dry Ice shipment label 1. Put on sterile gloves and remove samples from freezer. 2. If each sample is not already in a biohazard bag, place each blood pellet sample in a biohazard gab (only ONE sample per bag) with absorbent material, while noting on the Blood Pellet Shipment List their inclusion (At this time, the last column of the Shipment List should be completed). 3. Place the biohazard bag(s) in the sample box(es) 4. Add bubble wrap or foam padding around the bags for cushioning and put the lid on the sample box. 5. Place the sample box(es) in the Styrofoam box. 6. Completely surround the box with samples with the 5 lbs (2.27 kgs) of dry ice. 7. Insert padding around the sample box(es) if there is additional space in the Styrofoam box. 8. Put lid on Styrofoam box and place into cardboard box. 9. Affix an orange biohazard sticker (or a bag that is pre-labeled with the universal biohazard symbol) to the Styrofoam box lid if one is not already present. 10. Put completed Blood Pellet Shipment List in an envelope and place on top of Styrofoam lid. Keep a copy of the Shipment List for your files.
11. Seal cardboard box and affix the completed dry ice label. 12. On the FedEx label, be sure to check off Dry Ice and note the weight of dry ice. Also affix the Dry Ice label, which is required to be on the box and also notes the weight of the dry ice. 13. Attach Exempt Human Specimen label near the Dry Ice label. For blood pellets from participants that agreed to the genetic portion of the study, ship samples (overnight) to: Fernando Martinez, M.D. Arizona Respiratory Center University of Arizona Keating Building 1657 East Helen Street Tucson, Arizona 85721-0240 Tel: 520-626-7670 FedEx is the preferred carrier for blood shipments. Please use the FedEx Priority Overnight service. Pellet specimens can NOT be shipped via USPS. On the day that the specimens are sent, the site PI should email both afsullivan@partners.org and fernando@arc.arizona.edu the FedEx tracking number for the mailing. For blood pellets from participants that did not agree to the genetic portion of the study, ship samples (overnight) to: Carlos A. Camargo, M.D. Attention: Ashley Sullivan Massachusetts General Hospital 326 Cambridge Street, Suite 410 Boston, MA 02114 Tel: 617-726-5276 FedEx is the preferred carrier for blood shipments. Please use the FedEx Priority Overnight service. On the day that the specimens are sent, the site PI should email both afsullivan@partners.org and ccamargo@partners.org the FedEx tracking number for the mailing.
SAMPLE BLOOD SERUM SHIPMENT LIST Site # Sample ID# Date Collected (mm/dd/yr) Time Collected (hh:mm) Included in Shipment? 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
SAMPLE BLOOD PELLET SHIPMENT LIST Site # Sample ID# Date Collected (mm/dd/yr) Time Collected (hh:mm) Included in Shipment? 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
Laboratory Evaluations/Assays Blood specimens: Allergen-Specific IgE, Total IgE, 25(OH)D, Genetic testing The CBC and differential will be performed locally at each hospital. The 25(OH)D will be tested by Massachusetts General Hospital. The Allergen Specific Immunoglobulin E (ASIgE) and Total IgE testing will be performed by Mayo Medical Laboratories. The genetic testing will be performed by the Arizona Research Laboratory at the University of Arizona. ASIgE The selection of items parallels the mapi allergy testing as much as possible (1). Because the mapi was performed on older children, skin prick testing was utilized. With our younger cohort, we will use ASIgE to help evaluate for major aeroallergens and food allergens; an approach agreed upon by the first author of the mapi manuscript (personal communication with Dr. Theresa Guilbert, October 2008). This test will be performed using a Fluorescence Enzyme Immunoassay. (Package insert: ImmunoCAP System Specific IgE FEIA, Uppsala, Sweden Rev February 2005) Total IgE This test will also be performed using a Fluorescence Enzyme Immunoassay. (Package insert: Phadia CAP System IgE FEIA. issued August 2000, revised March 2006) Serum 25(OH)D Serum 25(OH)D measurement will be determined by radioimmunoassay (DiaSorin, Stillwater, MN). The limit of detection is 1.5 ng/ml. All specimens will be run in duplicate and the values averaged. Total (intra-assay and inter-assay) coefficients of variation for control values of 14.4 and 54.7 ng/ml were 8.5% and 13.2%, respectively. We ve chosen testing with RIA for this study population (instead of liquid chromotographytandem mass spectroscopy) because of the presence of C-3 epimers of 25(OH)D in neonates and young infants. Genetic testing: Tissue Bank In accordance with the tissue bank consent form, pellet specimens, which remain after centrifuging the blood and extracting the serum are being taken for the purpose of establishing a DNA biorepository to examine the possible genetic causes of severe bronchiolitis, recurrent wheezing, asthma and related concepts (see Sample Tissue Bank Consent Form, Appendix B2.) This testing will be performed under the future use clause of the main consent form (see Sample Consent Form, Appendix B1.) 1. Guilbert TW, Morgan WJ, Zeiger RS, Mauger DT, Boehmer SJ, Szefler SJ, et al. Longterm inhaled corticosteroids in preschool children at high risk for asthma. N Engl J Med2006 May 11;354(19):1985-97.