Which of the following should the phlebotomist identify when documenting a glucose level of 50 mg dL

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Forest JC, Garrido-Russo M, LeMay A, Carrier R, Dube JL. Reference values for the oral glucose tolerance test at each trimester of pregnancy. Am J Clin Pathol. 1983 Dec; 80(6):823-831. 6356879

Hare JW. Gestational diabetes mellitus. Levels of glycemia as management goals. Diabetes. 1991 Dec; 40(Suppl 2):193-196. 1748258

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The blood drawing station is designed to obtain blood samples to test for important risk factors for cardiovascular disease and for other blood parameters of interest to CARDIA. It is potentially a difficult station because of the anxiety caused by blood drawing and the potential for injury. However, if carefully and professionally done, it provides a positive, safe experience for the participant. The blood drawing station will take approximately 10 minutes of the participant’s time.

This protocol was designed as a training manual for the phlebotomist at the clinical centers.

The following tests are planned for the Year 10 exam:

1. PLASMA LIPIDS and LIPOPROTEINS: total cholesterol, triglycerides, and HDL cholesterol;
2. SERUM INSULIN (fasting in all participants and 2 hour post-glucose load in those participating in the oral glucose tolerance test or OGTT);
3. SERUM GLUCOSE (fasting in all participants and 2 hour post-glucose load in those participating in the OGTT);
4. SERUM CHEMISTRIES: creatinine, GGT, and uric acid; and
5. PLASMA VITAMIN C.

The methods of measurement for each of these tests is found in the CARDIA Laboratory Operations Manual and the rationale for assessment is found in the CARDIA Protocol.

The Lipid Research Clinic’s Manual of Operations identifies several factors which may influence lipid and lipoprotein values. The following procedures are designed to standardize sample collection.

1. Blood should be drawn with the participant in a seated position. The reclining position should be allowed only in extreme circumstances (e.g., history of fainting).
2. Elevations of 2–5% in lipid measures are observed after two minutes of continuous venous occlusion. Therefore, a total tourniquet time of less than two minutes is required.
3. The state of fasting clearly influences parameters such as triglycerides. CARDIA participants should have fasted for 12 hours prior to blood drawing. However, blood will be drawn on all participants, regardless of fasting status. (NOTE: Participants fasting less than eight hours will be excluded from the OGTT.)
4. Participants are instructed to avoid vigorous exercise on the day of their examination.
5. No particular restriction need be made for smoking since the blood drawing station will be approximately one half hour after clinic arrival.
6. Blood drawing should occur early in the CARDIA visit but following the blood pressure measurement. It must precede the snack. (NOTE: Participants participating in the OGTT cannot have a snack until after the second blood draw.)

Site: The blood drawing should take place in an isolated room or one enclosed by dividers. Temperature should be 65–75° F. There should be no direct sunlight. The recliner should be placed in a space sufficient to allow the chair to fully recline. The chair may be elevated on a wooden box to facilitate comfort of the phlebotomist when drawing the blood.

The room should be equipped with all the necessary supplies. A table or counter should be prepared with the materials and vials needed for blood handling and aliquoting. The centrifuge, refrigerator and freezer should be nearby.

The Daily Temperature Record must be completed each day the clinic is in operation. Freezer, refrigerator and room temperatures should be recorded. The expected temperatures are: freezer: −75° to −65°C; refrigerator: 35° to 40°F; room: 65° to 75°F.

Staff: This station is staffed with a technician with documented class time and experience in phlebotomy. Certification will occur at the centralized training session. Recertification will occur once as requested by the Coordinating Center, and will be authorized by the clinic supervisor or medical director. The technician should be properly attired in a white laboratory coat. Long hair should be tied back. Gloves should be used at all times while handling blood specimens.

All participants will have 41 ml of blood drawn, using the following vacutainers:

• one 10-ml lavender top (EDTA) vacutainer tube;
• one 13-ml red top (SST) vacutainer tube;
• one 3-ml gray top (serum) vacutainer tube (containing lithium iodoacetate);
• one 10-ml lavender top (EDTA) vacutainer tube; and
• one 5-ml lavender top (EDTA) vacutainer (foil wrapped).

Participants selected for quality control will have an additional 16 ml of blood drawn, using the following vacutainers:

• one 7-ml lavender top (EDTA) vacutainer tube;
• one 6-ml red top (SST) vacutainer tube; and
• one 3-ml gray top (serum) vacutainer tube (containing lithium iodoacetate).

Participants included in the OGTT will have an additional 5.5 ml of blood drawn (2 hours after a glucose load), using the following:

• one 2.5-ml red top (SST) vacutainer tube; and
• one 3-ml gray top (serum) vacutainer tube (containing lithium iodoacetate).

Participants selected for quality control for OGTT will have an additional 5.5 ml of blood drawn, using the following:

• one 2.5-ml red top (SST) vacutainer tube; and
• one 3-ml gray top (serum) vacutainer tube (containing lithium iodoacetate).

The following steps outline the procedures for blood drawing:

1. Wash hands at the beginning of the work day and as needed after processing specimens.
2. Greet the participant. Confirm that you have the correct envelope of ID labels. Locate the phlebotomy form (Form 5) in the participant’s form set and check that it has been properly labeled. Fill in the phlebotomist ID (first draw) on page 6 of the form. Ask the participant question 1 (whether he/she has been ill in the past 24 hours), question 2 (the time and date he/she last ate) and question 3 (whether he/she has any bleeding disorders). If the participant reports a history of bleeding disorders, the participant should be sampled under the supervision of a physician.
3. Explain the procedure. An example might be: “I am going to be drawing some blood from the vein in your arm. The purpose of this is to check levels of blood fats such as cholesterol. I will be taking five tubes -- about three tablespoons of blood. Are there any questions?” (NOTE: The number of tubes and the total amount of blood drawn will vary depending on whether the participant is selected for QC. Also, all of the clinics will draw additional blood for ancillary studies.)

   If the participant asks when he/she will receive the results of this test, direct the participant to ask the Clinic Coordinator at the exit interview.

4. With jackets removed, have the participant sit upright in the recliner with the sleeves rolled up to expose the antecubital fossa (elbow). The recliner should be in a complete upright position.

   Only when the participant has indicated that he/she has a history of fainting, should the technician recline the participant for the whole procedure. The technician may recline the participant by: (1) pulling the lever forward; (2) pulling up and forward on the arm rest; or (3) pushing back smoothly on the chair back. This reclining position is an exception to the protocol and should only be allowed in extreme circumstances. Its use is left to the discretion of the technician. It may be helpful to reassure an apprehensive participant that he may recline following the blood drawing.

   The chair position should not be changed from sitting to reclining during the blood drawing. Try to draw as much blood as possible when the participant is in the upright position.

5. Palpate and trace the path of veins several times with the index finger. Unlike veins, arteries pulse, are more elastic, and have a thick wall. Thrombosed veins lack resilience, feel cord-like, and roll easily. If superficial veins are not readily apparent, have participant close fist or lower the extremity over the arm of the chair to allow the veins to fill to capacity.
6. Identify the best available vein.
7. A tourniquet is used to increase venous filling. It makes the veins more prominent and easier to enter. PRECAUTIONS WHEN USING A TOURNIQUET: The tourniquet should be on the arm for the shortest time possible. Never leave the tourniquet on for longer than two (2) minutes. To do so may result in hemoconcentration and a variation in blood test values. If a tourniquet must be applied for the preliminary vein selection, it should be released and reapplied after a wait of two minutes. If the patient has a skin problem, put the tourniquet over the participant’s shirt or use a piece of gauze or paper tissue so as not to pinch the skin.
   1. Wrap the tourniquet around the arm 3 to 4 in. (7.5 to 10.0 cm) above the venipuncture site.
   2. Tuck the end under the last round.
   3. If a velcro tourniquet is used, adhere the tabs to each other.
8. Cleanse the venipuncture site:
   1. Remove alcohol prep from its sterile package.
   2. Cleanse the vein site with a circular motion from the center to the periphery.
   3. Allow the area to dry to prevent possible hemolysis of the specimen and a burning sensation to the patient when the venipuncture is performed.
   4. If venipuncture becomes difficult, the vein may need to be touched again with your hand. If this happens, the site should be cleansed again with alcohol.
9. Use of evacuated tubes (vacutainers):
   1. Thread the appropriate 21-gauge 1.5″ multidraw needle into the needle holder until it is secure.
   2. Insert the first blood collection tube (10-ml lavender top) into the holder and onto the needle up to the recessed guideline on the needle holder. Do not push the tube beyond the guideline, as premature loss of vacuum may result. The tube will retract slightly. Leave it in this position.
   3. If a tube has been previously or unsuccessfully used, it should be discarded in a proper container, not re-used.
   4. Inspect the tip of the needle visually to determine that it is free of hooks at the end of the point, and that its opening is clear of any small particles that would obstruct the flow of blood. The needle must be sterile. Do not use a needle from a package which is broken or contaminated in any way.
10. Perform the venipuncture:
    1. Grasp the participant’s arm firmly, using your thumb to draw the skin taut. This anchors the vein. The thumb should be 1 or 2 in. (2.5 or 5.0 cm) below the venipuncture site.
    2. With the needle bevel upward, enter the vein in a smooth continuous motion.
    3. Make sure the participant’s arm is in a flat or downward position while maintaining the tube below the site when the needle is in the vein. It may be helpful to have the participant make a fist with the opposite hand and place it under the elbow for support.
    4. Grasp the flange of the needle holder and push the tube forward until the butt end of the needle punctures the stopper, exposing the full lumen of the needle.
    5. Remove the tourniquet after blood is flowing. (If necessary, the tourniquet can remain on the arm until the last tube has started to fill.) Once the draw has started, do not change the position of the tube until it is withdrawn from the needle. During the procedure, try not to allow the contents of the tube to contact the stopper.
    6. Keep constant, slight forward pressure (in the direction of the needle) on the end of the tube. This prevents release of the shut-off valve and stopping of blood. Do not vary pressure nor reintroduce pressure after completion of the draw.
    7. Fill the vacutainer tube as completely as possible, i.e., until the vacuum is exhausted and blood flow ceases. A partially filled lavender top tube should be avoided if possible, because the higher resulting final concentration of anticoagulant will affect lipid values.
    8. When the blood flow ceases, remove the tube from the holder. The shutoff valve recovers the point, stopping blood flow until the next tube is inserted (if necessary).
    9. Immediately and thoroughly mix the contents of ALL tubes by gently inverting eight (8) times. To prevent hemolysis, avoid jarring or shaking the tube. Put the tube into a rack or jacket pocket; do not lay on table.
    10. To obtain additional specimens, insert next tube (13-ml red top vacutainer followed by 3-ml gray top vacutainer, 10-ml lavender top vacutainer, and 5-ml lavender top vacutainer wrapped with aluminum foil) into holder and repeat procedure from #10d.
11. If a blood sample is unobtainable:
    1. Change the position of the needle. If it has penetrated into the vein too far, pull it back a bit. If it has not penetrated far enough, advance it farther into the vein. Rotate needle half a turn.
    2. Try another tube. The tube in use may not have had sufficient vacuum.
    3. Loosen the tourniquet. It may have been applied too tightly, thereby stopping the blood flow. Reapply the tourniquet loosely. If the tourniquet is a velcro type, quickly release and press back together. Be sure however, that the tourniquet remains on for no longer than two minutes at a time.
    4. Probing is not recommended. This is painful to the participant. In most cases, another puncture in a site below the first site is advised.
    5. A syringe and needle may be used instead of a vacutainer, particularly in women, whose veins are oftentimes smaller.
    6. Do not attempt a venipuncture more than twice. Ask the participant if they would be willing to have your supervisor attempt to draw the specimen.
    7. If no staff member is able to obtain a blood sample, indicate this on the form (question 4). If a participant refuses to have blood drawn, this should also be indicated on the form (question 4).
12. Ask participant to open his/her hand after enough blood has been collected.
13. To remove the needle, lightly place clean gauze over venipuncture site. Remove the needle quickly and immediately. Apply pressure to the site with a pad. Discard needle into needle box.
14. Have the participant hold the pad firmly for one to two minutes to prevent a hematoma.
15. Bandage the arm.
    1. Under normal conditions:
       1. Slip the gauze pad down over the site, continuing mild pressure.
       2. Apply an adhesive or gauze bandage over the venipuncture site after making sure that blood flow has stopped.
    2. If the patient continues to bleed:
       1. Apply pressure to the site with a gauze pad. Keep the arm elevated until the bleeding stops.
       2. Wrap a gauze bandage tightly around the arm over the pad. An ice pack may be placed over the pad for a short time to arrest the bleeding.
       3. If bleeding persists longer than five minutes, the supervisor should be alerted. Continue pressure on the site as long as necessary to stop the bleeding.
       4. Tell the patient to leave the bandage on for at least 15 minutes.
16. If participant is participating in the oral glucose tolerance test (OGTT), follow directions for OGTT below. If participant has refused the OGTT, indicate this on the form (question 11), thank the participant and take him/her to the next station.
17. PRECAUTIONS IF A PARTICIPANT FEELS FAINT OR LOOKS FAINT FOLLOWING THE BLOOD DRAWING:
    1. Have the person remain in the recliner, recline the chair if necessary or have him/her sit with their head between their knees.
    2. Take an ampule of smelling salts and crush it and wave it under the person’s nose for a few seconds.
    3. Provide the person with a basin if he/she feels nauseous.
    4. Have the person stay reclined until the color returns and he/she feels better.
    5. Place a cold wet rag on the back of the person’s neck.
    6. If the person continues to feel sick, take a blood pressure and pulse reading. Contact a medical staff member who will advise you on further action.
18. HANDLING PARTICIPANTS WHO ARE EXTREMELY APPREHENSIVE ABOUT HAVING BLOOD DRAWN: Do not, under any circumstances, force the participant to have blood drawn. It is sometimes best to let the participant go on with another part of the visit, and have them return when the phlebotomist has more time to talk with the person. (However, the participant should not be given a snack until a final decision regarding the blood draw has been reached.) It also helps to have the participant recline in the blood drawing chair just so the phlebotomist can check the veins in the participant’s arms, without actually drawing blood. If the participant has “good veins” the phlebotomist can reassuringly say, “Oh, you have good veins; there should be no problem.”

    If the participant refuses to have any blood drawn, Form 5 should be completed as follows:

    1. record the exam date and the phlebotomist ID (first draw) on page 6;
    2. leave the first three questions blank; and
    3. record “no” for question 4 (“Was any blood drawn?”) and record “refused” for the reason.
19. CAUTION IN HANDLING BLOOD SAMPLES: Improper handling of blood collection tubes and needles is extremely dangerous. All blood samples should be handled as though the participant has a blood-borne transmissible disease (i.e., universal precautions). Therefore, the following procedures should be observed.
    1. Needles must be disposed of in proper disposal containers.
    2. Avoid contact with plasma. Gloves should always be worn when handling samples.
    3. If the phlebotomist accidentally sustains a contaminated needle stick, the wound should be thoroughly cleansed with soap and water. The medical director should be notified immediately.
    4. All used vacutainer tubes are to be placed in a biohazard box (i.e., sharps container). Blood products are to be placed in biohazard bags for disposal.

Participants from all centers will be recruited for the oral glucose tolerance test (OGTT). The OGTT will provide information on whether the participant is diabetic or has impaired glucose tolerance.

A participant will be informed about this test prior to signing the informed consent form. Only those participants whose appointment is earlier than 10:00 A.M. will be offered the opportunity to participate in the test.

1. If a participant is recruited for the OGTT, explain that the test requires a fasting blood draw and one additional blood draw two hours after consuming a glucose solution. Ask the participant to sign the consent form.
2. If the participant agrees to the OGTT, indicate it on the scheduling sheet.
3. If a participant checks in after 10:00 A.M., indicate to the participant that because glucose values may vary considerably throughout the day, the second blood draw may provide an inaccurate value if it is done after 12:30 P.M. Therefore, we will only measure the fasting glucose.
4. Make certain that the pulmonary function test will be completed after the intake of glucose solution and at least 30 minutes before the assigned time for the second blood draw.
5. Make certain that the participant returns to the phlebotomy station 10 minutes before the assigned time for the second blood draw.

1. Indicate whether participant agreed to participate in the OGTT (question 11).
2. Determine whether the first blood draw occurred before 10:30 A.M. by referring back to question 6 on page 1. If the blood was drawn after 10:30 A.M., mark “yes” for question 12 on page 3; the participant is excluded from the test.
3. Determine whether the participant fasted for at least eight hours by referring back to question 2 on page 1 (question 13). If the participant fasted less than eight hours, he/she is excluded from the OGTT.
4. Ask participant:
   1. whether he/she has ever been told by a physician that he/she is diabetic (question 14). If the answer is “yes”, ask whether he/she is taking insulin and/or oral drugs for diabetes. If the participant is taking insulin and/or oral drugs, exclude from the OGTT.
   2. whether he/she is using steroids (e.g., prednisone) for any reason (question 15). If the answer is “yes”, exclude from the OGTT.
   3. whether she is pregnant (question 16). (If participant is male, do not ask question 16, but do mark the “not applicable” response.) If the participant is pregnant or is not sure whether she is pregnant, exclude her from the OGTT.

      Thus, the following participants should be excluded from the OGTT: (1) those arriving at the clinic after 10:00 A.M.; (2) those who did not fast for at least eight hours; (3) those who are diabetic and take insulin and/or oral drugs; (4) those using steroids; and (5) those who are pregnant (or may be pregnant).

5. If the time for the first blood draw is prior to 10:30 A.M., administer 75 gm glucose solution (TRU GLU) within a period not to exceed 5 minutes. Encourage the participant to drink the solution as quickly as possible, and make certain that the participant drinks the entire volume.
6. Record the time that the participant started the glucose drink (question 17).
7. Record the time that the participant completed the glucose drink (question 18). (NOTE: Do not exclude participant from the OGTT if the time for completing the glucose solution exceeds five minutes.)
8. Record the expected time of the second blood draw on the form (question 19). Record the time that you want the participant to return to the phlebotomy station (10 minutes prior to the expected blood draw) on an adhesive label and ask the participant to stick the label on the left side of his/her chest.
9. Inform the Clinic Coordinator of the time for the second blood draw to ensure that the pulmonary function test will be completed at least 30 minutes before the assigned time for the second blood draw. (NOTE: It is preferable to perform pulmonary function after the second blood draw, if possible.)
10. Instruct the participant to refrain from smoking and heavy physical activity and to return to the phlebotomy station at the assigned time for the second draw.
11. When the participant returns to the phlebotomy station, check the expected time of the second blood draw (question 19).
12. Record the time of the completion of the pulmonary function test (question 20).
13. If the participant vomits after drinking the glucose solution (at any time prior to the second blood draw), stop the OGTT and indicate it on the form (question 21).
14. If you are unable to draw blood for the OGTT sample, indicate this on the form (question 22).
15. Within (±) 5 minutes of the expected time for the second blood draw, draw 3 ml blood into the gray top vacutainer for plasma glucose and 2.5 ml blood into the red vacutainer for plasma insulin, following instructions for blood drawing outlined above. Record the time of the second blood draw (question 23).

1. Record the time that the pulmonary function test was completed on a piece of paper and have the participant give it to the phlebotomist (or give the time directly to the phlebotomist).
2. If the pulmonary function test occurs after the second blood draw, give the time the test was completed directly to the phlebotomist.

The proper handling of the collected samples is critical because deviation from the protocol can significantly affect the parameter being measured in the blood. It is particularly important that time deadlines in handling be observed and that samples not be left open to the atmosphere longer than necessary. A total of ninety (90) minutes is permitted between blood drawing and final placement of samples in the freezer, so schedule your time accordingly.

1. Immediately after drawing the sample, affix one of the large plain white labels with the participant’s ID to each of the five blood tubes. This is necessary to identify which tubes belong to which participant.
2. Immediately place the three lavender top tubes (including the one wrapped in aluminum foil) into a container of water and ice. Cool the samples on water and ice for 30 minutes but not more than 60 minutes. (NOTE: Remove the aluminum foil from the 5-ml vacutainer before placing it in the centrifuge. Replace the foil when the vacutainer is removed from the centrifuge.)
3. Place the red top tube in the test tube rack. Allow the sample to clot by standing for at least 30 minutes but not more than 60 minutes at room temperature (65–75°F).
4. Place the gray top tube in the test tube rack. Allow the sample to clot by standing for at least 45 minutes but no more than 60 minutes at room temperature (65–75°F).
5. Do not let the samples stand in direct sunlight or at extreme temperatures.

1. Immediately after drawing the sample, affix one of the large plain white labels with the participant’s ID to each of the two blood tubes. This is necessary to identify which tubes belong to which participant.
2. Place the red top tube in the test tube rack. Allow the sample to clot by standing for at least 30 minutes but not more than 60 minutes at room temperature (65–75°F).
3. Place the gray top tube in the test tube rack. Allow the sample to clot by standing for at least 45 minutes but no more than 60 minutes at room temperature (65–75°F).

1. To pre-cool the centrifuge, close and lock the cover, set the TEMPERATURE control at 4°C, set the SPEED control to 1000 RPM, set the TIMER control to 20 minutes, and push the RUN button. If the chamber is already cool, begin operation at #2. (NOTE: The shields should be refrigerated if they are removed from the centrifuge, so that they will be chilled prior to spinning blood.)
2. Set the POWER switch to ON.
3. Balance the load.
   1. Place opposite shields containing filled vacutainers on the balance.
   2. To the lighter centrifuge shield, add water (or 50/50 water/alcohol) around the vacutainers, but no closer to the top of the container than 1/4 inch. Add same solution to the other centrifuge shield until the two are balanced.
4. Load the rotor, being careful to place balanced shields directly opposite each other.
5. Set the TEMPERATURE control at 4°C. Set the SPEED control at 3,000 RPM (1,500 g, 5.0″ rotating radius). Set the TIMER to 20 minutes. Set the BRAKE control to the MAX position.
6. Close cover and lock.
7. Push the RUN button to begin the run cycle. Record the time the samples were placed in the centrifuge in the TIME SPUN section on the form.
8. The centrifuge will stop automatically at the preselected time and the stop indicator will light. Remove samples one at a time.
9. Consult the Centrifuge Service Manual for other guidelines and for troubleshooting.

The five vacutainers for the core blood components will be aliquoted into 27 vials for clinical tests and storage. The two vacutainers for the OGTT blood components will be aliquoted into 2 vials for clinical tests. Refer to “Labeling Scheme” (below), “Aliquoting Scheme” (next section) and “Blood Flow Chart” (Appendix D) for assistance in this procedure.

The phlebotomist should prepare the work area by laying out the plastic transfer pipettes and aliquoting vials and tubes. Affix the small ID labels to each specimen vial as indicated in the following diagram and table. DO NOT DEVIATE FROM THE LABEL COLORS SPECIFIED FOR EACH SAMPLE, ESPECIALLY FOR THE STORAGE VIALS.

All labels should be placed vertically rather than horizontally to maximize the laboratory’s ability to read the bar codes on the labels. The center of the label should be covered horizontally with low temperature freezer tape. The tape should completely encircle the tube and overlap slightly. The corresponding caps should be nearby. Place these vials in the aliquoting board in the order in which they are to be filled.

DIAGRAM FOR LABEL PLACEMENT

1. 10-ml lavender top tube: After centrifuging, the plasma should be promptly separated from the cells. Using the plastic transfer pipette, transfer 3 ml of plasma into the red-labeled 8-ml vial. There should be approximately 2 ml of plasma left. Pipette 0.5 ml aliquots into each of four of the white-labeled cryovials for storage.
2. 13-ml red top tube: After centrifuging, the gel in the tube should lie between the serum and clot; if not, re-centrifuge for an additional 10 minutes. Use the stopper remover to extract the red rubber stopper, and dispose of stopper in removed. To do this, transfer 1 ml to the blue-labeled 2-ml cryovial, 2 ml to the green-labeled 2-ml cryovial and pipette 0.5-ml aliquots into each of the 6 yellow-labeled cryovials using a separate pipette than the one used to pipette plasma from the lavender tubes.
3. 3-ml gray top tube: After centrifuging, the serum should form a layer above the clot; if not, re-centrifuge for an additional 10 minutes. Serum should be promptly removed. To do this, transfer 1 ml to the orange-labeled 2-ml cryovial using a separate pipette than the one used to pipette plasma from the lavender top tubes.
4. 10-ml lavender top tube: After centrifuging, the plasma should be promptly separated from the cells. Pipette the plasma in 0.5-ml aliquots into the remaining 7 white-labeled cryovials for storage using the pipette used for plasma transfer.
5. 5-ml lavender top tube wrapped w/foil: Pipette 1.5 ml of metaphosphoric acid solution (see Appendix B for ingredients and mixing instructions) into each of the four Wheaton vials prior to pipetting the plasma into the vials. After centrifuging, the plasma should be promptly separated from the cells. Pipette the plasma in 0.5-ml aliquots into each of the 4 teal-labeled Wheaton vials. Use the crimper to secure the stoppers and seals of the Wheaton vials.
6. Red cell packs from the two 10-ml lavender top tubes: Gently pour the red cell packs from each of the 10-ml lavender tubes into the gray-labeled 50-ml conical tubes.

   NOTE: According to OSHA guidelines, all vacutainers should be re-capped after the plasma or serum has been aliquoted. Dispose of capped vacutainers in a biohazard box (i.e., sharps container).

1. The Clinic Coordinator will identify the QC participants. Labels for the core QC participants are enclosed in a small envelope. The envelopes should be opened consecutively beginning with envelope #1. Label each of the three vacutainers (7-ml lavender, 6 ml red and 3 ml gray) with a large white label and also affix a large white label to page 3 of the phlebotomy form (next to the question “Is participant quality control?”). The red-striped label should be placed on the 8-ml screw-top tube. The blue-striped label should be placed on a 2-ml cryovial, the green-striped label on a 3.5-ml vial, and the orange-striped label on a 2-ml cryovial.
2. 7-ml lavender top tube: After centrifuging, the plasma should be promptly separated from the cells. Using the plastic transfer pipette, transfer 3 ml of plasma into the red-labeled 8-ml screw-top tube.
3. 6-ml red top tube: After centrifuging, the gel in the tube should lie between the serum and clot; if not, re-centrifuge for an additional 10 minutes. Serum should be promptly removed. To do this, transfer 1 ml to the blue-labeled 2-ml cryovial and 2 ml to the 3.5-ml green-labeled vial, using a separate pipette than the one used to pipette plasma from the lavender top tube.
4. 3-ml gray top tube: After centrifuging, the serum should form a layer above the clot; if not, re-centrifuge for an additional 10 minutes. Serum should be promptly removed. To do this, transfer 1 ml to the orange-labeled 2-ml cryovial using a separate pipette than the one used to pipette plasma from the lavender top tube.
5. Take one of the three 0.5 ml aliquots labeled with teal-striped labels (intended for Vitamin C storage samples) and re-label with a teal-stripe QC label. (NOTE: This was done to avoid an additional tube being drawn on the QC participant.)

1. The Clinic Coordinator will identify the QC participants. (NOTE: The OGTT QC participant should not be the same participant used for core QC measurements.) Labels for the OGTT QC participants are enclosed in a small envelope with “OGTT” written on the outside of the envelope. The envelopes should be opened consecutively beginning with envelope #1. Label each of the two vacutainers (2.5 ml red and 3 ml gray) with a large white label and also affix a large white label to page 6 of the phlebotomy form (next to the question “Is participant quality control for OGTT?”). The lavender-striped label should be placed on the on a 2-ml cryovial and the pink-striped label on a 2-ml cryovial.
2. 2.5-ml red top tube: After centrifuging, the gel in the tube should lie between the serum and clot; if not, re-centrifuge for an additional 10 minutes. Serum should be promptly removed. To do this, transfer 1 ml to the lavender-labeled 2-ml cryovial, using a separate pipette.
3. 3-ml gray top tube: After centrifuging, the serum should form a layer above the clot; if not, re-centrifuge for an additional 10 minutes. Serum should be promptly removed. To do this, transfer 1 ml to the pink-labeled 2-ml cryovial using a separate pipette.

1. The plastic transfer pipettes are graduated in 0.25 ml increments. The approximate capacity is 1 ml. Two pipettes should be used for each participant, one to transfer plasma and one to transfer serum.
2. Be careful not to disturb cells when drawing plasma into a pipette. If cells mix with the plasma, re-centrifuge the sample to insure obtaining the maximum amount possible. When using pipettes, avoid drawing red cells into the bulb.
3. DO NOT OVERFILL VIALS. Room is needed for expansion during freezing.
4. Priority of filling vials when an inadequate amount of blood is drawn:
   1. 8-ml screw-top tube for lipids (plasma)
   2. 2-ml cryovial for insulin (serum)
   3. 3.5 ml vial for serum chemistries (serum)
   4. 2-ml cryovial for glucose (serum)
   5. 2-ml cryovials for storage samples (plasma and serum)
   6. 2-ml Wheaton vials for Vitamin C (plasma).
5. Dispose of blood tubes in biohazard box (i.e., sharps container), re-capped with their original stoppers.
6. Seal or cap all vials immediately; specimens should not be allowed to stand open.

1. All vials are to be frozen immediately; do not allow vials to sit out. Freezer temperature should be −70°C.
2. Record time of freezing on the phlebotomy form. Samples must be placed in the freezer within 90 minutes of blood drawing.
3. Update the shipping forms daily as you store the samples. All vials should be stored in storage boxes which will also hold the specimens when they are shipped to the laboratory.
4. Quality control samples should be stored in a separate section of the freezer so that samples are shipped one month after the shipment of the regular specimens.
5. Different boxing procedures are necessary for those samples which are to be analyzed, as opposed to those samples intended for long-term storage.

   Samples foranalysis (red-, blue-, green-, orange- or teal-striped labels): Place these samples in the freezer in separate boxes by label color and vial size. This will aid in specimen retrieval at shipping time.

   Within a box, place the samples in order of the date drawn, from the back of the box to the front. Begin at the sample position at the upper left corner of the box and place successive samples moving to the right and down, as you would read a book. (NOTE: Three of the four teal-labeled Wheaton vials will be shipped to Solomon Park for storage; quality control participants will have only two teal-labeled Wheaton vials shipped to Solomon Park.)

   Samples intended forlong-term storage (white or yellow labels): The Solomon Park storage facility has requested that all of each participant’s plasma (white label) and serum (yellow label) samples be stored and shipped together. Each storage box has 100 spaces for samples. Therefore, since each participant should have 17 specimens, all of the specimens for five participants can be stored in one box. DO NOT SPLIT A PARTICIPANT’S SAMPLES ACROSS TWO BOXES.

   Before you begin packing a box, wrap a length of freezer tape around the outside corners of the box’s lid, in order to reinforce it. When fully packed, the box may be slightly crowded.

   Within a box, pack the samples in order of the date drawn, from the back of the box to the front. For each participant, pack the plasma samples first, then the serum samples. Begin at the first sample position on the first row of the box and place successive samples moving to the right and down, as you would read a book. Leave the far right sample positions on each row empty.

   If a participant has a full set of storage samples (11 plasma vials and 6 serum vials), leave an empty space between the last plasma vial and first serum vial (Figure 1). In this figure, all participants have a full complement of vials.

Figure 1.

Packing diagram for Solomon Park storage specimens. “P” indicates a plasma vial, “S” indicates a serum vial, “▪” indicates an empty space.

If a participant has 10 or fewer plasma samples, begin filling the participant’s first row at the first sample position, as above, placing up to 9 plasma vials. If present, place the tenth sample in the first sample position of the participant’s second row. As above, begin placing the serum samples at the fourth sample position of the participant’s second row (Figure 2).

Figure 2.

Packing diagram for Solomon Park storage specimens. “P” indicates a plasma vial/“S” indicates a serum vial, “▪” indicates an empty space.

In Figure 2, participant #1 has 10 plasma samples and 6 serum samples. Participant #2 has 5 plasma samples and 6 serum samples. Participant #3 has no plasma samples (note the empty row) and 6 serum samples. Participant #4 has 11 plasma samples and 3 serum samples. Participant #5 has 11 plasma samples and no serum samples.

When you finish packing a participant’s samples, place the remaining white label for each participant in a column toward the left side of the TOP of the lid of the storage box and write beside the labels in ink the number of plasma samples enclosed for the respective IDs. Place one of the remaining yellow labels for each participant to the right of the center of the lid and write beside them in ink the number of serum samples enclosed for the respective IDs. (NOTE: The remaining two yellow labels for each participant will be used for the urine samples for measurement of albumin and creatinine. Please provide these labels to the laboratory technician processing the urine specimen.) The ID labels should be placed on the lid in the same order that the corresponding samples are packed in the tray (Figure 3).

If a white label or a yellow label for the participant’s samples is not available, write the full 12-digit ID, in ink, in the location the label should occupy.

When a box has been filled, the labels affixed and the number of storage samples (plasma and serum) recorded for each participant, place a vertical strip of freezer tape over each column of labels on the top of the box. This will prevent the labels from falling off during storage.

Figure 3.

Placement of participant ID labels and sample counts on the top of the lid of the Solomon Park storage box.

Samples are transported as outlined:

Monthly shipments will occur on Wednesdays. Birmingham will ship on the first Wednesday of the month, Chicago on the second Wednesday, Minneapolis on the third Wednesday, and Oakland on the fourth Wednesday.

The first shipping date for Birmingham and Chicago will be June 14, 1995 and for Minneapolis and Oakland, June 21, 1995. The regular shipping schedule will be followed from that point on.

Quality control samples should be shipped to the respective laboratory one month after the shipment of the regular specimens. It is extremely critical that this guideline be followed throughout the exam cycle. Quality control samples should be placed with analysis samples in the appropriate shipping box (e.g., QC samples with a red label should be placed in the boxes with analysis samples being shipped to NWLRL).

Packaging and shipping of frozen samples is the responsibility of each center.

1. The boxes with frozen samples must be placed upright in the supplied insulated shipping container and surrounded with dry ice pellets. At least 10 pounds of dry ice is necessary to keep the samples frozen for up to 48 hours; 15–20 pounds is recommended. (It is not necessary to place the boxes in ziplock plastic bags before placing in dry ice. The 50 ml conical tubes on the styrofoam base should be placed in large freezer bags for shipping.)
2. The two shipping forms (face sheet and contents sheet) and a self-addressed, stamped envelope must be enclosed or attached with each shipment. Instructions for the completion of these forms are appended to this document with a copy of the forms. Use a separate face sheet for each box of samples.
3. Samples must be shipped by air courier so that they arrive at the laboratory by 10:30 A.M. the following morning. The centers must request that the containers be returned as soon as possible by the laboratory at the center’s expense. Return need not be by air courier.
4. On the day of shipment, the primary phlebotomy technician should notify each laboratory that the samples have been shipped (to arrive by air courier by 10:30 A.M. the following day). (Due to differences in time zones, the technician may need to call the laboratory the morning that the shipment will be arriving.) If the shipment is not received by noon, the laboratory personnel should contact the Coordinating Center and the Clinic Coordinator of the pertinent clinical center immediately. This will permit the clinic to put a trace on the missing samples and, hopefully, locate them before thawing occurs.
5. Shipments MUST be made according to the shipping schedule.
6. Label each shipment with “CONTENTS TO REMAIN FROZEN”.
7. The storage vials are shipped in the storage boxes in which they have already been stored at the clinical center.
8. Samples must be frozen for at least twenty-four hours before they are packaged to be shipped.

In order to assess the reproducibility of the measurements made at the centralized laboratories, a split sample external surveillance program has been developed. The purpose of the program is to produce data which may be used: (1) to identify problems as they occur and to communicate this information to the clinic and laboratory for corrective action; and (2) to document the performance of the entire process, i.e. sample collection, preparation, handling, shipping and analytical performance.

The quality control procedures will consist of providing the laboratories with duplicate specimens, one sample labeled with the participant ID and the other with a fictitious ID. Duplicate samples will also aid in measuring “across assay” reproducibility. In order to accomplish this, the duplicate blood specimens must be split across shipments to the laboratories. Otherwise, the sample is analyzed in the same manner as all other specimens in that particular month.

Each of the clinical centers will draw the quality control tube on an approximate fourteen percent sample of the participants for the first three months (generally one out of every seven participants per week) and an approximate nine percent sample of the participants for the remainder of the exam cycle (generally one out of every 11 participants per week). The quality control participant will be identified at the time of the clinical center’s weekly phone call to the Coordinating Center. In the event that the additional sample could not be drawn on the identified participant, the phlebotomist should draw the quality control sample on the NEXT PARTICIPANT.

The participant need not be told that he/she is having extra blood drawn for quality control. The consent form covers the maximum amount of blood that could be drawn on any participant.

The volume of additional blood that will be drawn for the core quality control is: one 7-ml lavender top (EDTA) vacutainer tube for duplicate plasma lipid measures, one 6-ml red top (SST) vacutainer for duplicate serum insulin and serum chemistries measures, and one 3-ml gray-top tube for fasting serum glucose measures. The plasma is aliquoted into a red-labeled 8-ml screw-top tube. The serum from the red top vacutainer is aliquoted into a blue-labeled 2-ml cryovial and a green-labeled 3.5-ml cryovial.

The volume of additional blood that will be drawn for the OGTT quality control is: one 3-ml gray top tube and one 2.5-ml red top (SST) vacutainer for duplicate 2-hour serum glucose measures and 2-hour serum insulin measures. The serum is aliquoted into a pink-labeled 2-ml cryovial and a lavender-labeled 2-ml cryovial.

When an additional amount of blood is drawn for a quality control duplicate specimen, it is to be submitted with the following month’s shipment. That is, duplicate specimens should not be shipped with the original specimen; shipments of original and duplicate specimens must be separated by one month. Review the quality control shipping schedule in the preceding Transporting Samples section (VII).

Each clinical center should receive from the Coordinating Center individual results reports within 4–6 weeks following shipment of the specimens for analysis. Results will be forwarded for the following measurements: (1) lipids and lipoproteins (from Northwest Lipid Research Laboratory); and (2) fasting glucose (or OGTT) and serum chemistries (from Linco Research, Inc.).

When the participant results arrive at the clinical center, several items should be checked:

1. Indicate on your inventory forms that the results have been received to confirm that all specimens which were shipped were also analyzed.
2. Scan all reports for obvious laboratory errors.
3. Report any missing results or errors to the contact person at the laboratory and to the quality control person at the Coordinating Center.
4. File the report in the participant’s form set.

A sample of the results will be reviewed on a monthly basis at the Coordinating Center and periodically by the Quality Control Committee.

The clinical center is responsible for reporting specific laboratory test results to their participants. Participants with an LDL-cholesterol level of 160 mg/dl or greater will be referred to an appropriate medical care provider. Participants with a triglyceride level greater than 500 mg/dl will be referred. In addition, participants with a fasting glucose level of 140 mg/dl or greater (or a 2 hour post-load glucose of 200 mg/dl or greater) will be referred to an appropriate medical care provider. The clinical center may offer to re-evaluate the participant’s lipid and/or glucose levels for public relations purposes, as deemed necessary. Please contact the Coordinating Center for an ID to be used for the re-analysis sample. DO NOT USE THE PARTICIPANT’S CARDIA ID FOR THE RE-ANALYSIS SAMPLE. If repeat results are above the thresholds stated above, participants will be referred to an appropriate medical provider.