ATTACHMENT A

NOTE: The Diabetes Mellitus SOPs were revoked and redetermined in July 2011, as RMA instruments 89 and 90.

As a result of this change, no further serum dioxin level testing in diabetes claims should be arranged.  Please refer to SoP Bulletin 152 (July 2011) for guidance.

B29Nov99 (Dioxin Procedures DM SOP).doc  Page 1 of 8

BLOOD COLLECTION CENTRES

FOR THE PURPOSES OF SERUM DIOXIN ANALYSIS

NSW PaLMS Transfusion Service Out-Patient Clinic 9, Ground Floor Royal North Shore Hospital Pacific Highway. ST LEONARDS  NSW 2065 *     *     * Appointments Contact: Fiona Craig on (02) 9926 7798 [Appointments available Wed 1:00–4:30 only]	QLD Sullivan & Nicolaides Pathology Out-Patient Clinic, Ground Floor Greenslopes Private Hospital Newdegate St. GREENSLOPES *     *     * Appointments Contact:    (07) 3394 7721 [Appointments available Mon, Tues, Wed only]
VIC Central Pathology Collection Centre Suite 8, 1st floor St Vincent's Pathology 55 Victoria Pde FITZROY  VIC 3065 *     *     * Appointments Contact: Judy Mietzke on (03) 9288 3974 [Appointments available Mon, Tues, Wed only]	VIC Clinical Pathology – Blood Collection Svce Level 3, Harold Stokes Building Austin & Repatriation Medical Centre Burgundy St (Outpatients' Entrance) HEIDELBERG  VIC 3084 *     *     * Appointments Contact: Jo Cummins on (03) 9496 5472 [Appointments available Mon, Tues, Wed only]
WA St John of God Pathology (WA) Suite 9 Hollywood Specialist Centre 95 Monash Rd NEDLANDS  WA 6009 *     *     * Appointments Contact:    (08) 9386 9481 [Appointments available Mon, Tues, Wed only]	SA Haematology Day Centre Level 7, East Wing Royal Adelaide Hospital North Tce ADELAIDE  SA 5000 *     *     * Appointments Contact:   (08) 8222 5632 [Appointments available Mon, Tues, Wed only]

WHOLE BLOOD COLLECTION AND PROCESSING FOR DIOXIN ANALYSIS

[23/9/99 Version]

NB: UNIVERSAL PRECAUTIONS SHOULD BE OBSERVED AT ALL TIMES WHEN COLLECTING AND HANDLING BODILY FLUIDS

The following pages will describe the blood collection procedure and blood separation procedure for the analyses of dioxin and other analytes associated with it.

Materials provided by ESR:

(1)250mL Glass jars and lids

(2)5mL plastic tubes with push-fit stopper

(3)Parafilm for sealing 5mL tube

(4)Pre-printed labels for serum samples (bottles and tubes)

(5)Pre-printed labels for vacutainer tubes and blood bags

(6)Sample Submission forms

(7)Medical Laboratory (Wellington) submission form for lipids analysis

(8)Courier Consignment Notes for sending samples to ESR (Orange TNT forms)

(9)Sample packaging (plastic jar inside cardboard box) complying to IATA requirements for transport of biological materials

Stocks of bottles, labels and forms are held at ESR's Australian Office in Melbourne, and can be obtained from:

ESR

Australian Office

Level 5, 470 Collins St

Melbourne

Vic 3000

Phone:  (03) 9614 7611

Fax: (03) 9614 7633

If you are unable to contact Stuart Glen and need sample bottles etc. urgently, please contact either:

Cathy Bremner (Hatlar Environmental, Melbourne)

Phone  (03) 9629 5300

or

Dr Bob Symons (ESR, Wellington Science Centre, New Zealand)

Phone  tollfree 1800 123 444 (PIN 2846)

Please allow 24 hours for delivery of sample containers to your laboratory from ESR Melbourne and 48-72 hours from ESR New Zealand

Blood Collection using 600mL blood collection bags

I.Materials Needed Per Participant.

(1)Two alcohol swabs

(2)Tincture of iodine solution swab

(3)Gauze, sterile, individually wrapped, 2” x 2”

(4)Tourniquet

(5)600ml blood collection bag WITHOUT anticoagulant with attached 16g needle OR 600 ml bag with needle adapter.

(6)17g AV fistual needle OR 19g butterfly needle that can be attached to  bag with needle adapter

(7)Fenwal centrifuge bag (may not be required if a dry-wall collection bag is used)

(8)300 ml transfer pack

(9)Plasma extractor

(10)Plasma transfer set

(11)Hand scale or vacuum assist device

(12)Latex gloves

(13)Tubing Clamps

(14)Hemostats

(15)Pre-printed labels

(16)Bandaid

II.General Considerations

VERY IMPORTANT – Blood collection for serum differs in two ways from standard blood collection for plasma:

(1)Collection bags must have NO anticoagulant

(2)The blood pack must be allowed to clot at room temperature for 3-6 hours and be placed in a refrigerator  overnight.  The next morning the pack must be moved from the refrigerator and stand at room temperature for 1-2 hours before centrifugation to separate serum.

Blood shall be collected from donors by trained phlebotomists (or other qualified person) working under the standard work practices for your laboratory.  Blood collection must be made by aseptic methods, using a sterile, closed system, and a single venipuncture.  If more than one skin puncture is needed, another collection bag with new needles must be used.

NB: It is important to note that blood collection practices may vary slightly from laboratory to laboratory. It is important that each participating collection laboratory operates under it's standard work practices for the extraction of the quantity of blood specified in this protocol. The critical step in the process is the preparation of the sample for analysis (extraction of the serum), not the blood collection from the patient. Therefore variations to the following collection protocol are allowed, but must be fully documented.

III.Preparation of Venipuncture Site

(1)blood will be drawn form a large, firm vein in an area free from skin lesion.

(2)Apply the tourniquet, select a vein and release tourniquet

(3)Prepare as follows:

(a)Scrub a site approximately 3 inches in a diameter over the selected vein for 30 seconds

(b)Remove any residual soap with one of the alcohol swabs using a spiral motion going from the center of the outer edge of the cleaned area

(c)Apply the iodine starting in the center of the cleaned area and work outward using a circular motion.  Allow to air dry.

(d)Remove the iodine residue with an alcohol swab using the same motion as for the soap.

(e)Cover the sire with sterile gauze until ready to perform the venipuncture.  The prepared site must not be touched after its has been cleaned.

IV.Blood Collection

(1)Check the bag for possible defects

(2)The bag may be filled by gravity or vacuum filled.  If the bag is filled by gravity, a hand scale should be used (not supplied).  If the bag is vacuum filled, a vacuum assist device will be used to monitor volume.

(3)The bag should be appropriately labeled and the label for “Collection Bag” should be placed on the bag prior to venipuncture.

(4)Prepare the skin site, reapply the tourniquet and perform the venipuncture.

(5)Once the needle is firmly in place in the vein, open any clamps applied to the tubing and ensure that blood flow has been established If vacuum assist is used, it should be activated at this point.

(6)The tubing may be taped to hold the needle in place while the blood filling the bag.

(7)Fill the bag with 300-350mls of blood.  Remove tourniquet.

(8)The tubing to the bag should be clamped and the needle may be removed from the participant's arm.  Cover the wound with sterile gauze and have participant raise the arm straight into the air while holding pressure over the wound.  This should be done for 25 minutes to prevent hematoma.

(9)After 15 minutes a bandage should be applied over the puncture site.

V.Care of the participant

(1)Patient care should be as per the laboratory's standard practices for the extraction of this volume of blood.

VI.Blood processing and Separation

NB: THIS IS THE CRITICAL PART OF THE PROCESS

Step 1:

ALLOW THE BLOOD TO CLOT 3-6 HOURS AT ROOM TEMPERATURE AND REFRIGERATE OVERNIGHT BEFORE CENTRIFUGATION.

Step 2:

(1)A Beckman Centrifuge Model #J-6M (or equivalent) should be used.

(2)The blood bag should be placed inside one of the Fenwal centrifuge bags (this may not be required depending on the type of collection bag used). Be sure to balance properly.

(3)The bags will be centrifuged for 15 minutes at the RPM's necessary to attain a force of 5325 x g at 4-10°C.  The formula to calculate the number of RPM necessary to attain 5325 z g is: RPM = 9450 divided by the square of (R), where R is the distance in cm from the center of rotation to the bottom of a test tube when it is extended in the centrifuge head,  (For example, when R = 20.5cm, RPM = 4500).

(4)Place the centrifuge bag of blood on the plasma extractor.  Using the 300ml-transfer bag, spike the collection bag using the coupler at the end of the transfer bag.  It is acceptable to transfer some of the red cells along with the serum since the transfer bag will also be centrifuge.

(5)Place clamps on the tubing to the transfer bag 1-inch apart and cut between the clamps.

(6)Centrifuge the transfer bag containing the serum using the same procedure above.

(7)Two aliquots will be made for each serum specimen. Using pre-printed labels for each participant, label each of the containers as follows:

Priority	Size/type Container	Container Label
1.	250mL glass jar	“SERUM DIOXIN-S1”
2.	5mL plastic tube	“SERUM DIOXIN –S2”

(8)Use a ballpoint pen to add the date collected and your initials to the labels on all containers.

(9)Place the transfer bag containing the serum being careful not to disturb the red cells on the plasma extractor.  A plasma transfer set will be used to transfer the serum to the two containers listed above.

(a)Close the roller clamp on the tubing of the plasma transfer set and insert the white plastic coupler into one of the ports on the transfer bag to break the seal.

(b)Release the handle on the plasma extractor and dispense the serum using the needle end of the plasma transfer set into the bottom of the bag.

(c)Be sure that labels are on each bottle and all patient details match.

(d)Place the caps and/or stoppers back on each of the bottles

(e)Parafilm (or similar sealing tape) should be applied to the 5mL tube to stop any leakage.

(f)Record details on the ESR Sample Submission Form and note in the comments any unusual conditions which affected the specimen, for example: Turbidity, haemolysis (red tinge), low volume, delay in freezing etc.

(g)Fill in the patient details on the Medical Laboratory form.

(h)Store samples at -20°C (or colder) until shipment.

(i)Before shipment, check caps on the large bottles and re-tighten if necessary.

Blood Collection using Vacutainer Tubes

I.Materials needed per participant:

(1)Gauze Sponges

(2)Alcohol Wipes

(3)Bandaid

(4)Paper Tape

(5)10 mL non siliconized glass vacutainer tubes (15 per participant)

(6)19g ¾” butterfly with multiple sample luer adaptor

(7)Preprinted labels

(8)Tourniquet

(9)Vacutainer Holder

(10)Storage Boxes

II.Venipuncture Procedures

NB: It is important to note that blood collection practices may vary slightly from laboratory to laboratory. It is important that each participating collection laboratory operates under it's standard work practices for the extraction of the quantity of blood specified in this protocol. The critical step in the process is the preparation of the sample for analysis (extraction of the serum), not the blood collection from the patient. Therefore variations to the following collection protocol are allowed, but must be fully documented.

The only critical step in the blood collection process using vacutainers is that the tubes MUST NOT BE INVERTED after the sample has been collected.

(1)Lay out blood collection supplies.

(2)Located the puncture site.  Hold with 2 fingers on one side of the “alcohol wipe” so that only the other side touches the punctured site.  Wipe the area in a circular motion beginning with a narrow radius and moving outwards so as not to cross over the area already cleaned.  Repeat with a second alcohol wipe.

(3)Locate vein and cleanse in manner previously described, then apply the tourniquet.  If necessary to feel the vein again, do so: but after you feel it cleanse with alcohol prep again, and dry with a sterile gauze square.

(4)Fix the vein by pressing down on the vein about 1 inch below the proposed point entry into the skin and pull the skin taut.

(5)Approach the vein in the same direction the vein is running, holding the needle so that a 15° angle with the examinees arm.

(6)Grasping the butterfly wings with bevel facing up, push the needle firmly and deliberately into the vein.  Tape the needle in place to allow both hands to be free for collecting the tubes of blood.  Activate the vacuum collection tube.  If the needle is in the vein, blood will flow freely into the tube.  If no blood enters the tube, probe for the vein until entry id indicated by blood flowing into the tube.

(7)For collection, loosen the tourniquet immediately after blood flow is established and release entirely as the last tube fills.  Collect all 15 and label with the patient name.

(NB: DO NOT INVERT THE TUBES.  PLACE UPRIGHT TO AVOID ANY CONTACT WITH THE RUBBER STOPPER WHILE CLOTTING)

(8)After the last tube has filled, withdraw the needle with a swift backward motion.  When the needle is out of the arm, press gauze firmly on the puncture.  Heavy pressure as the needle is being with drawn should be avoided because it may cause the sharp point of the needle to cut the vein.

(9)Have the examinee raise their arm (not bend it) and continue to hold the gauze in place for several minutes.  This will help prevent haematomas.

(10)Label all tubes with the preprinted labels provided (marked “For Vacutainers”), and use a ballpoint pen to add the date collected and your initials to the label.

(11)Place a Bandaid on the subject's arm.

III.WHOLE BLOOD PROCESSING PROCEDURE

Store upright (VERY IMPORTANT!) and allow 20-30 minutes for these tubes to clot before centrifugation.

IV.SERUM PROCESSING PROCEDURE

Materials and Equipment Needed per Participant

(1)Transfer Pipette (1) – can be either glass or plastic (glass pasteur pipette is preferable)

(2)200 mL glass jar with screw cap (1)

(3)5 mL plastic tube with stopper (1)

(4)Pre-printed Labels

(5)Racks

(6)Centrifuge

(7)Freezer (-20°C)

Processing

(1)After the blood has been allowed to clot at room temperature for 20-30 minutes, centrifuge the vacutainer tubes for 10 minutes at the rpm necessary to attain a force of 1000g.  To calculate the number of rpm necessary to attain 1000g, use the following formula:

Rpm = 9450/r

Where r is the distance in centimeters from the center of rotation to the bottom of a test tube when it is extended in the centrifuge head.

Example: for r = 16, rpm = approximately 2400

(2)Using the pre-printed labels for each participant, label each of the containers as follows:

Priority	Size/type Container	Container Label
1.	250mL glass jar	“SERUM DIOXIN-S1”
2.	5mL plastic tube	“SERUM DIOXIN –S2”

(3)Use a ballpoint pen to add the date collected and your initials to the labels on all containers.

(4)To maximise the amount of serum recovered from all of the vacutainer tubes, do the following:

(5)Attach a rubber bulb to one of the Pasteur pipettes (or use a clean plastic transfer pipette).

(6)Transfer all of the serum that is free of red cells from each tube to the 250mL glass jar.

(7)Any remaining serum left in the vacutainer tubes that may have become mixed with red cell should be transferred to a clean 10mL vacutainer tube.  One or two clean tubes may be needed to hold all of the remaining serum/red cell mix.

(8)Centrifuge for 10 minutes and transfer the clear serum to the glass jar containing the serum originally harvested.

(9)Swirl the serum in the jar gently and transfer 3-4mL into the 5mL tube.

(10)Place the screw caps on the 250mL jars and the stopper in the 5mL tube.  Seal the 5mL tube with Parafilm (or similar sealing film) to avoid leakage of the sample. Pace upright in a -20ºC (or lower) freezer and store at the same temperature until shipment to ESR.

(11)Record details on the ESR Sample Submission Form and note in the comments any unusual conditions which affected the specimen, for example: Turbidity, haemolysis (red tinge), low volume, delay in freezing etc.

(12)Fill in the patient details on the Medical Laboratory form.

(13)Store samples at -20°C (or colder) until shipment.

(14)Before shipment, check caps on the large bottles and re-tighten if necessary

Specimen Shipping

NB: Before shipment, check caps on the large bottles and re-tighten if necessary

All specimen containers should be returned to the boxes in which they were supplied.  Place each of the specimens inside the bubble bag and seal. Place some freezer blocks or bags in the plastic container with the bagged specimens to keep them frozen during transit.  Add any packing material needed to ensure samples will not break.

Ship by overnight courier to:

ESR

Australian Office

Level 5, 470 Collins St

Melbourne

Vic 3000

Phone:  (03) 9614 7611

Fax: (03) 9614 7633

NB: On the day a shipment is being made, please phone, fax or Email ESR to advise shipping details.