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1、ACTN Laboratory Technologist Committee Version 1.1 ACTN Dried Blood Spots Procedure 11 March 2009 1 DRIED BLOOD SPOTS CARD COLLECTION, PROCESSING, AND STORAGE PROCEDURES 1. Rational 1.1 Dried blood spots (DBS) have been used for years for HIV-1 RNA, DNA, Genotyping, and other diagnostic assays. Drie
2、d blood spots can be used as an easy and inexpensive means for the collection and storage of peripheral blood specimens from infants, children and adults in settings where collection and storage of plasma is not optimal. Dried blood spots use a small amount of peripheral blood on protein saver filte
3、r paper cards from infant heel pricks, or finger sticks from adults and require less skill and supplies than venous blood draws. The cost and difficulties of cold- chain shipping of plasma samples is greatly reduced by the use of dried blood spots and they can be shipped as non-dangerous goods. 2. R
4、EAGENTS AND MATERIALS 2.1 Materials for Heel Stick Method 2.1.1 Unistick 2 device (Fisher Scientific #22-0227) 2.1.2 Alcohol swab 2.1.3 Dry sterile gauze pad 2.1.4 Whatman Protein Saver Card #903 (Whatman 10534612 Fisher Scientific # NC9307519) 2.1.5 Gas impermeable storage bag (Fisher Scientific# N
5、C9307519) 2.1.6 Desiccant pack (Whatman 10548234-Fisher Scientific# 50853571) 2.1.7 Humidity indicator Cards (Multisorb Des Manufacture # MS200032 Fisher Scientific # NC9511648) 2.1.8 Whatman card drying rack, (VWR catalogue # 89015-592) 2.1.9 Gloves preferably powder free 2.1.10 Water proof marker
6、2.2 Materials for Venous Blood Method Using Vacutainer Tubes 2.2.1 Vacutainer Evacuated Blood Collection Tubes 2.2.2 Alcohol swab 2.2.3 Tourniquet 2.2.3 Bandage/Plaster 2.2.4 Vacutainer Needle 2.2.5 Vacutainer Needle Holder 2.2.6 Whatman Protein Saver Card #903 (Whatman 10534612 Fisher Scientific #
7、NC9307519) 2.2.7 Gas impermeable storage bag (Fisher Scientific# NC9307519) 2.1.8 Desiccant pack (Whatman 10548234-Fisher Scientific# 50853571) ACTN Laboratory Technologist Committee Version 1.1 ACTN Dried Blood Spots Procedure 11 March 2009 2 2.1.9 Humidity indicator Cards (Multisorb Des Manufactur
8、e # MS200032 Fisher Scientific # NC9511648) 2.1.10 Whatman card drying rack (VWR catalogue # 89015-592) 2.1.11 Gloves preferably powder free 2.1.12 Water proof marker 3 Biosafety 3.1 It is essential that universal precautions be taken while working with these specimens. Dried blood spots on filter p
9、aper are not considered to be a biohazard. However, you should wear gloves and a lab coat/gown at all times, even with DBS, to ensure safe handling of samples. If you should tear a glove, remove the torn one and replace it immediately. If a needle puncture should occur, notify the lab manager or pro
10、ject coordinator immediately so that appropriate actions may be taken. ACTN Laboratory Technologist Committee Version 1.1 ACTN Dried Blood Spots Procedure 11 March 2009 3 4. SPECIMEN COLLECTION 4.1 Heel stick Procedure The Unistick 2 device has a penetration depth of 1.8 mm and a convex tip for accu
11、rate positioning that was designed to eliminate the risk of accidental needle stick injury or cross-infection as well as reduce the risk of osteomyelitis of the heel. Preferably, the baby should be in a supine position with the knee at the edge of the table or the baby may be held in the mothers arm
12、s. This position allows for the foot to hang lower than the torso, improving blood flow. Prepare the Unistick 2 device by depressing the pink plunger until it clicks. Twist the plunger until it breaks off and remove it from the device. The Unistick 2 device is now ready to trigger. When the baby is
13、in an acceptable position for this procedure, clean the incision area of the heel with an alcohol pad and allow the heel to air dry. Do not touch the incision site or allow the heel to come into contact with any non-sterile item or surface. Wrap your index finger around the base of the heel and your
14、 thumb around the ankle. See figure 1.0 below The Unistick 2 heel-stick device is properly positioned against the heel and triggered. The first drop of blood is wiped away using a dry sterile gauze pad. The blood is then allowed to flow to be collected on the dried blood spot card. To do this, gentl
15、y touch the filter paper card to the blood spot. Allow the card to absorb the blood until the circle is full. You should be able to obtain 5 spots of blood on a card. You may need to squeeze the heel to obtain more blood; however, do not milk the heel as it mixes interstitial fluid with the blood. O
16、nce all 5 spots are obtained, gently press a sterile pad to the incision site until bleeding has ceased. The babys heel must then be monitored for late bleeding and inflammation. Placing a sterile pad over the wound will help prevent a hematoma from forming. Note: Bandaging the babys foot is a contr
17、oversial issue because of skin sensitivity and potential bandage aspiration. However, the incision should be noted by the primary care nurse to ensure that the heel can be monitored for bleeding and inflammation. A bandage is not necessary as long as the bleeding has stopped before the child leaves
18、the clinic. Hold the Protein Saver 903 Card without touching the filter paper (See figure 2 below). Care should be taken to not touch the DBS circle once blood is applied. Allow the blood spot to air dry, with out card flap over spots, in a clean dry place (see drying racks Figure 3) that is protect
19、ed from rodents or insects and direct sunlight for at least 4 hours (drying overnight may be necessary in areas with higher humidity). Place ACTN Laboratory Technologist Committee Version 1.1 ACTN Dried Blood Spots Procedure 11 March 2009 4 the card in a gas impermeable bag with a desiccant pack and
20、 humidity indicator card. (See Figure 4). Note: Do not use electric dryer or oven to dry cards. Store no more than one card per bag. Do not heat, stack or allow DBS to touch other surfaces during the drying process. Once in the bag spots can be stored at room temperature, about 15-30o C or - 20 to -
21、70 o C according to protocol MOPS and/or LPC until shipped to the processing or receiving laboratory. Figure 1. Heel stick Placement and Device Figure 2. Holding the Protein Saver 903 Card without touching the filter paper Field Cod ACTN Laboratory Technologist Committee Version 1.1 ACTN Dried Blood
22、 Spots Procedure 11 March 2009 5 Figure 3. Whatman Protein Saver 903 Card Drying Racks and Common drying rack ACTN Laboratory Technologist Committee Version 1.1 ACTN Dried Blood Spots Procedure 11 March 2009 6 Figure 4. DBS Card in a gas impermeable bag with a desiccant pack and humidity indicator c
23、ard ACTN Laboratory Technologist Committee Version 1.1 ACTN Dried Blood Spots Procedure 11 March 2009 7 4.2 Collection of Dried Blood Spot from Finger Stick 4.2.1 Clearly label the Protein saver 903 Card with Patient ID and Date, Protocol, Visit ID. 4.2.2 Disinfect selected site and prick using lanc
24、et/needle. 4.2.3 Uniformly saturate entire circle by quickly and gently touching NOT PRESSING the puncture site to the filter paper. Dont touch the Whatman Protein saver 903 Card at any stage of collection. See examples of good and bad spots Section 7.0 4.2.4 After collecting 5 dried blood spots, cl
25、ean the site and leave it un-bandaged. See Figure 5 Figure 5. Collection of DBS from Finger Stick 4.3 Collection of Venous blood using EDTA tube (other tubes as determined by protocol team) 4.3.1 Vacutainer Evacuated Blood Collection Tubes these tubes are designed to be filled with a predetermined v
26、olume of blood by vacuum. The rubber stoppers are color-coded according to the additive that the tube contains. Collect 1 EDTA (purple top) tube at each time point required. Total volume required per card will be .250mL or 5 X .050mL per spot. 4.3.2 The tubes should be labeled with the patient ID nu
27、mber, the date of collection, and the study and visit identifier. Fill the blood collection tube to the recommended volume so the anticoagulant is at the proper dilution. Gently invert the tube (5-10 times) to mix the blood thoroughly. After the blood is completely mixed, remove the cap, and take 50
28、 micro liters of the ACTN Laboratory Technologist Committee Version 1.1 ACTN Dried Blood Spots Procedure 11 March 2009 8 whole blood and apply to a single spot. Repeat four additional times to fill all five spots on the card. This transfer of blood should be performed with a pipette and a disposable
29、 tip. Care should be taken to not touch the DBS circle once blood is applied. Allow the blood spot to air dry, with out flap over spots , in a clean dry place that is protected from rodents or insects and direct sunlight, for at least 4 hours (overnight may be necessary in areas with higher humidity
30、), Do not heat, stack or allow DBS to touch other surfaces during the drying process 4.3.3 Tuck the flap of the Protein saver 903 card as indicated on the card. 4.3.4 Insert folded card into sealable gas impermeable zip lock bag containing desiccant packs and humidity indicator. Store no more than o
31、ne card per bag. 4.3.5 Seal zip lock bag and store at room temperature (15-30C) until ready to ship/transport. Store the DBS bag according to protocol MOPS and/or LPC until shipped to the processing or receiving laboratory. The protocol team will notify sites when to ship samples to the receiving la
32、boratory. 5.0 Sample Chain of Custody 5.0.1 It is essential that each sample be logged into the LDMS. If DBS are prepared in the clinic and transported to the lab, immediately compare the inventory from the clinic to the specimens received. If there are ANY discrepancies, please notify the lab manag
33、er immediately as action needs to be taken to try and recover samples. 6.0 Labeling 6.1 Cards prepared using the venous blood methods should be logged into the LDMS with the primary type=BLD, additive=EDT (or appropriate additive if a different tube type is received) and derivative=DBS. Cards prepar
34、ed using the heel stick method should be logged into the LDMS with the primary type=BLD, additive=NON, and derivative=DBS. The number of aliquots created will be based on the number of whole spots per card. For each complete spot create one 50 micro liter aliquot using the DBS derivative in the LDMS
35、. If a spot is not complete make a best estimate as to the volume the spot contains. One LDMS label containing the protocol (ACTG/IMPAACT) required identifiers (which include but are not limited to the patient ID, visit and sample dates and draw times) should be affixed to the card itself. Ensure th
36、at the label is FIRMLY AFFIXED to the card. For ease of handling it is also recommended to affix a label to the outside of the gas impermeable bag after storing the card as detailed below. ACTN Laboratory Technologist Committee Version 1.1 ACTN Dried Blood Spots Procedure 11 March 2009 9 7.0 Storage
37、 and Shipping 7.1 STORAGE AT ROOM TEMP DBS Protein saver 903 cards that will be shipped within 4 weeks. 7.1.1 Be sure the DBS Protein saver 903 card is completely dry before packing. 7.1.2 Close the cover of the Protein saver 903 card. 7.1.3 Insert the DBS Card into a gas impermeable plastic bag con
38、taining desiccant packet. 7.1.4 Be sure that the patient identifier and date (or LDMS generated label) are on the outside of the bag and on the DBS Protein saver 903 card. 7.1.5 Seal the plastic bag. 7.1.6 Place sealed bag containing the DBS Protein saver 903 card in a clean, dry area of lab with no
39、 exposure to direct sunlight, free of insects or rodents, and where ambient temperatures will not exceed 30oC. 7.1.7 Shipment of DBS Protein saver 903 cards stored at room temperature must occur within 4 weeks. Alternatively, cards should be placed in a refrigerator or freezer according to the proto
40、col specific instructions. 7.1.8 Check humidity indicators in bags frequently and change desiccant packs if indicator color changes from blue to pink. (See Figure 6) 7.2 STORAGE OF DBS FILTER PAPER CARDS AT 4-20oC DBS Protein saver 903 cards that are used for DNA, RNA, PK, Genotyping, etc. and that
41、will be shipped 4 weeks but less than 4 months. 7.2.1 Be sure the DBS Protein saver 903 card is completely dry before packing. 7.2.2. Close the cover of the DBS Protein saver 903 card. ACTN Laboratory Technologist Committee Version 1.1 ACTN Dried Blood Spots Procedure 11 March 2009 10 7.2.3 Insert t
42、he DBS Protein saver 903 card into a gas impermeable plastic bag containing desiccant packet. 7.2.4. Be sure that the patient identifier and date (or LDMS generated label) are on the outside of the bag and on the DBS Protein saver 903 card. 7.2.5. Seal the plastic bag. 7.2.6. Place sealed bag contai
43、ning DBS Protein saver 903 card in designated area of -20oC freezer until shipment is requested. 7.2.7. If bagged DBS Protein saver 903 cards are to be shipped on dry ice, they should be placed in shipping box containing dry ice directly from freezer. 7.2.8. If bagged DBS Protein saver 903 cards are
44、 to be shipped at ambient temperature. Remove bag containing DBS Protein saver 903 card and allow to equilibrate to room temperature prior to shipping. Check desiccant to be sure it is still functional (note color) and replace desiccant if necessary. 7.3 STORAGE OF DBS Protein saver 903 card AT -70o
45、C - DBS Protein saver 903 cards that are used for DNA, RNA, PK, Genotyping, etc. and that will be shipped 4 months. 7.3.1. Be sure the filter paper is completely dry before packing. 7.3.2. Close the cover of the filter paper card. 7.3.3. Insert the DBS Protein saver 903 card into a gas impermeable p
46、lastic bag containing desiccant packet. 7.4.4. Be sure that the patient identifier and date (or LDMS generated label) are on the outside of the bag the DBS Protein saver 903 card. 7.4.5. Seal the plastic bag. 7.4.6. Place sealed bag containing DBS Protein saver 903 card in designated area of -70oC f
47、reezer until shipment is requested. 7.4.7. If bagged DBS Protein saver 903 cards are to be shipped on dry ice, they should be placed in shipping box containing dry ice directly from freezer. It is recommended that desiccant packs be checked before shipping and replaced if color has changed. ACTN Lab
48、oratory Technologist Committee Version 1.1 ACTN Dried Blood Spots Procedure 11 March 2009 11 7.4.8. If bagged DBS Protein saver 903 cards are to be shipped at ambient temperature, remove bag containing DBS Protein saver 903 card and allow to equilibrate to room temperature prior to shipping. Check t
49、o be sure desiccant packs are still functional (note color) and replace if necessary. Figure 6. Example of Humidity Indicator 7.5 Shipping of DBS Protein Saver 903 Cards 7.5.1 Dried Blood Spots are classified as Exempt Biological Specimens according to ICAO and IATA shipping regulations. DBS should be shipped by overnight or second day courier services. DB