summarySpecimens used for HIV testingSelecting specimens Advantages and disadvantages of serum and plasma Advantages and disadvantages of whole blood Advantages and disadvantages of dried blood spots (DBS) Advantages and disadvantages of oral fluids
Collecting,
processing and storing blood specimens Collecting and storing DBS Collecting and storing oral fluids Labelling and logging specimens
Many types of specimens can be used for biological surveillance of HIV: plasma, serum, whole blood, DBS and oral fluid. The choice of specimen collected depends on the logistics, populations and sites selected, and the HIV
testing strategy and algorithm. Specimens must be collected, tested and stored in an appropriate manner in order to obtain accurate and reliable results. For serosurveillance activities, specimens are usually collected and stored prior to HIV testing at a
regional or national laboratory. Serum, plasma and DBS can be stored and tested at a later date; specifications for storage will depend on the type of specimen collected. Specimens not tested on site at the local level will need to be transported to a regional or national laboratory for testing. The methods by which specimens are transported will depend on the country’s infrastructure. Few countries may have courier systems linking health-care facilities and laboratories. More frequently,
the field surveillance staff members themselves transport the specimens from the local to the national laboratory. However, other options such as public transportation can be explored. 3.1. Selecting specimens3.1.1. Advantages and disadvantages of serum and plasmaHIV testing of serum and plasma, which can be collected by venepuncture (see
section 3.2), have the following advantages and disadvantages: AdvantagesHave higher concentrations of HIV antibodies than oral fluids19 Have
the potential for additional routine testing (e.g. syphilis, hepatitis B, hepatitis C) from a single specimen Have the potential for special studies (e.g. testing for recent infections, HIV typing [HIV-1 vs HIV-2], HIV subtyping, antiretroviral [ARV] resistance) Are easy to collect and test in clinical settings with a trained phlebotomist and a laboratory
DisadvantagesRequire invasive collection technique
Require skilled staff (for collecting and processing serum or plasma) Compared with oral fluid, require more equipment (e.g. needles, tubes or lancets) and biohazard waste facilities Serum or plasma are difficult to collect in non-clinical settings as venepuncture is required Pose a greater risk to health-care workers and staff through inadvertent exposure, both because of higher HIV concentrations and the use of sharp collection devices
3.1.2. Advantages and disadvantages of whole bloodWhole blood, which can be collected by venepuncture or finger-stick (see section 3.2), has the following advantages and disadvantages in HIV testing: AdvantagesHas higher concentrations of HIV antibodies than oral
fluids19 Has the potential for additional routine testing (e.g. syphilis, hepatitis B, hepatitis C) from a single specimen Is easy to collect and test in clinical settings (with trained phlebotomist, if venous whole blood) Is easy to
collect in non-clinical settings (if finger-stick)
DisadvantagesRequires invasive collection technique Requires skilled technician (for collection) Compared with oral fluid, requires more equipment (e.g. needles, tubes or lancets) and biohazard waste facilities Difficult to collect in non-clinical settings if venepuncture is required Poses a greater risk to health-care workers and
technicians through inadvertent exposure, both because of higher HIV concentrations and the use of sharp collection devices
3.1.3. Advantages and disadvantages of dried blood spotsDried blood spots can be prepared by collecting venous whole blood or finger-stick whole blood and dropping an amount onto a filter-paper. DBS have the following advantages and disadvantages: AdvantagesAre easy to collect in
a clinical or non-clinical setting, depending on whether venepuncture is available Do not require a centrifuge or other equipment for processing the blood specimen Once dried, can be stored at room temperature for a short period of time Can be transported easily to the central laboratory for further testing Facilitate testing for
prevalence, incidence and special studies such as resistance testing
DisadvantagesRequire specific filter-paper (see
Section 3.2) for preparation Modified procedure required for DBS elution and HIV testing Potential for less accuracy (false-positives and/or false-negatives) if the test is not optimized for DBS Make the testing process more lengthy as an elution step needs to be performed Limited number of tests validated for use with DBS specimens
3.1.4. Advantages and disadvantages of oral fluidOther specimens besides blood and blood products can be used for HIV testing. For linked testing, where informed consent must be obtained, oral fluid may be used. AdvantagesDoes not require a trained laboratory technician for specimen collection and
processing, can be collected by a trained health worker Does not require contact with possibly contaminated laboratory materials, e.g. used needles or lancets that need biohazard waste facilities for sharps disposal Can be collected in a variety of field settings, including non-clinical settings Collection of oral fluid may be more acceptable to hard-to-reach populations than specimen collection requiring venepuncture or finger-stick. Therefore, a
greater percentage of the target population may agree to be tested.
DisadvantagesMay require special collection devices Currently available testing technologies used for oral fluid specimens are limited but additional new tests are being validated. Cannot be used to perform additional testing for special studies (e.g. testing for recent infections, HIV subtyping, ARV resistance) Same specimen
cannot be used to confirm initial reactivity with a second test; therefore, a second specimen must be taken, i.e. whole blood, serum, plasma for further testing (this is specific to the OraQuick HIV rapid test as the oral fluid collection device and test are one and the same) Should not be used for confidential linked testing (i.e. with the return of results to the
individual)
recommendationBlood (serum, plasma, DBS) is the preferred specimen for testing because it has a higher concentration of HIV antibodies than oral fluid. It also allows for additional testing, including for syphilis, hepatitis B and hepatitis C, and for special studies of HIV type and subtype, and ARV resistance. 3.2. Collecting, processing and storing blood, serum
and plasma specimensbloodBlood needed for an HIV test can be collected either by venepuncture (whole blood, serum, plasma) or by finger-stick (whole blood). 3.2.1. Processing blood collected by venepunctureTo collect blood by venepuncture, follow local clinical or laboratory procedures. See the Appendix and
Section 6.3 for information on safety procedures. The following steps are recommended for processing blood collected by venepuncture20: Collect up to 10 ml of blood from the patient’s
vein into a sterile 10 ml tube. For serum, blood is collected in a red-top tube (without anticoagulants). For plasma, blood is collected in a purple-top tube (with anticoagulants, e.g. EDTA). For safety reasons, the use of an evacuated blood collection system (e.g, Vacutainer® tube) is recommended. (Note: Obtaining an additional tube of blood during routine blood collection solely for the purpose of unlinked anonymous testing
is considered unethical and is not advised.)
If the blood specimen will not or cannot be processed immediately (e.g. no centrifuge is available), collect the blood in a red-top tube and allow it to stand at room temperature for at least 20–30 minutes, and then remove the serum. Usually, plasma takes longer (~1 hour) to separate without centrifugation than serum collected in a red-topped tube. Process (see Step #3) and test within 24 hours to avoid haemolysis of the
specimen. Centrifuge the specimen to separate the serum (without EDTA) or plasma (with EDTA). If blood is collected for serum, allow the blood to stand for at least 20–30 minutes so that a clot forms before the specimen is centrifuged. In general, the specimen should be centrifuged at 300–400 g or 1200–1500 rpm for 10
minutes.21 After the specimen is centrifuged or has had time to separate, use a clean plastic pipette (do not pour) to remove an aliquot of 0.5–2.0 ml off the top layer. Transfer this to another sterile labelled tube (plastic, not glass) or cryovial (1.5–2.0 ml with
a screw cap) and tighten the cap. The specimen is ready for storage and testing.
3.2.2. Storing serum and plasma collected by venepunctureTo store serum and plasma, consider the following: 22,
23 Make sure the cap is tight on the labelled cryovial or plastic tube. (Do not use glass tubes for storing specimens.) Place the cryovials in a cardboard freezer box with a partitioned insert. If the specimens are to be transported to the testing
laboratory, store the specimens at 4–8 °C for up to a maximum of 1 week. For longer-term storage, the specimens should be frozen at –20 °C or below. Pack the specimens upright in a cooler containing cold packs for transport to the testing facility. Limit the number of freeze/thaw cycles because it may impact the HIV test results and subsequent additional testing.
3.2.3. Collecting blood by finger-stickBlood collected
by finger-stick can be used to perform rapid tests or make DBS on filter-paper. A DBS may be preferred in rural and non-clinical settings, which often do not have trained phlebotomists or laboratory facilities with appropriate equipment (e.g. centrifuges). To obtain a finger-stick specimen, massage the finger (preferably the middle or ring finger), which will cause blood to accumulate at the tip of the finger. Cleanse the finger pad (not just the tip or side of the
finger) with 70% isopropyl (rubbing) alcohol. Wipe away the alcohol with a sterile gauze pad. Use a sterile lancet to firmly prick the finger pad. Wipe the first drop of blood off the finger with sterile gauze before collecting subsequent blood using a collection device to place on the rapid test device or on the filter-paper for the DBS. If the original puncture is
inadequate, the same site should not be reused; another site or finger should be used. Avoid milking or squeezing the puncture as this may cause haemolysis of the specimen and could invalidate the test result.19 The ear lobe may be pricked instead of the finger.
3.2.4. Preparing and storing a dried blood spot for an HIV testBlood from a finger- or ear-lobe stick can be used to make DBS.24 Although finger-stick is the most typical method, DBS can also be obtained by using blood collected in a tube with an
anticoagulant.25 DBS have the advantage of being easy to transport, without the need for a cold chain. Apply blood directly from a finger or a pipette onto special filter-paper (Schleicher and Schuell Grade 903 filter paper). The paper may come with preprinted circles
that will contain approximately 100 µl blood when completely filled. If the paper does not have preprinted circles, place blood on the paper so that it makes a circle with a 1.5 cm diameter. Allow the blood to soak through and fill the entire circle. Caution: If the blood does not saturate the filter-paper, do not use that paper. Label the side of the filter-paper with a specimen reference code after the filter-paper is saturated with blood (circle is filled). Suspend
filter-paper strips containing the filled circles during the drying process to allow air to circulate around the paper. Stands for holding the strips are commercially available. However, strips may also be dried by placing them between two books (taping the edges of the strips to the books with sticky tape) on a table or a laboratory bench top so that the blood-containing part of the paper is not in contact with the surface of the table or laboratory bench top. Be sure not to get tape on the
blood spot. Let the blood spots air dry at room temperature for at least 4 hours (and for at least 24 hours in humid climates). Do not heat or stack blood spots, and do not allow them to touch other surfaces while they are drying. After blood spots have been adequately dried, wrap the strip in one sheet of glassine paper or plastic to prevent carryover of specimen from one sheet to another. Place the wrapped strips in a gas-impermeable bag (zip-lock
bag) with desiccant and humidity indicator cards. Approximately 20 strips may be placed in each bag. Bags may be kept at room temperature for up to 30 days and then stored at 4 °C for up to 90 days. If the DBS in their plastic bags are to be stored for more than 90 days, they should be maintained at –20
°C.25 Properly stored DBS have been shown to be stable for at least two years. The bags should be placed in a sturdy envelope for
shipment.26 If additional testing, such as resistance testing, is anticipated, DBS must be stored at –20 °C or below immediately after the DBS specimens are dry.
3.3. Collecting and storing oral fluid3.3.1. Collecting oral fluidFor specimen collection, follow test instructions as part of a standard operating procedure. Oral fluid collection devices are available and may be used, if indicated. Some rapid test devices contain an oral fluid collection pad at one end which facilitates collection and testing. See
Section 6.3 for information on safety procedures. Oral fluid can only be used with certain EIAs and rapid tests designed for oral fluid specimens, such as the OraQuick brand. Additional rapid tests using oral fluid are currently under field evaluation. The following are the general steps for collecting a specimen: Use a specially treated absorbent pad attached to a plastic stick
(usually provided by the test kit manufacturer). Collection procedures are specified by the manufacturers of collection devices, which must be carefully followed. Then place the pad into a vial containing a preservative solution (usually provided by the test kit manufacturer). If an oral fluid-specific rapid test (e.g. OraQuick) is performed, storage
and transport are not necessary.
Due to the complexity of the test, oral fluid specimens collected for EIAs are sent to a laboratory performing EIAs for analysis. 3.3.2. Storing oral fluidOral fluid specimens should be stored at 4 °C for a short period of time. They should be refrigerated during shipment. Specimens should be frozen (–20 °C or below) if stored for an extended period of time. Once thawed, they can be refrozen once. Consult the
test kit insert prior to testing for more specific storage information. 3.4. Labelling and recording collected specimens3.4.1. Labelling specimensThe plastic tube, cryovial or filter-paper containing the specimen must be labelled with a specimen code at the time of collection and processing. If labels are used, make sure the label is placed on the side of the tube, not on the cap. Pre-printed cryolabels
designed to adhere during freezer storage should be used when specimens are stored in cryovials. It is important that freezing does not affect the visibility of the printing on the label. Surveillance coordinators should provide the field staff responsible for specimen collection with a series of labels or permanent markers and the codes to be used. For unlinked anonymous testing, label the tube only with a new specimen code unlinked to personal identifying information (see
Figure 2.1). 3.4.2. Recording specimens and test resultsA separate laboratory logbook or line-listing for surveillance activities should be maintained to record HIV test results by the corresponding code. The logbook should be accessible only to laboratory and surveillance staff; it should be secured
in a locked drawer or cabinet when not in use to ensure confidentiality of the persons’ test results as well as their participation in surveillance activities (see Figures 2.1,
2.2). For unlinked anonymous testing, the logbook or line-listing should contain only the new specimen codes and corresponding HIV test results; no personal identifying information on the patients whose specimens are tested should be included. HIV test results can be matched by the new specimen code to the demographic information
abstracted earlier on the surveillance form (see Figure 2.1).
Step 1: Identify The Vein
The first step in drawing blood correctly is to identify the appropriate veins to puncture.
Which of the following actions should a phlebotomy technician take when performing a venipuncture using an evacuated tube system? The technician should position the needle in the same direction as the vein and at a 30 degree angle with the bevel up.
For which of the following reasons should a phlebotomy technician examine the Antecubital?
79) For which of the following reasons should a phlebotomy technician examine the antecubital areas in both of a patient's arms? The median cubital vein could be more accessible on one side than the other.
When collecting for a coagulation studies the phlebotomy technician should select what colored top tube?
Blood Culture
2. Light Blue Top (plasma): 3.2% sodium citrate. These tubes are used for coagulation tests and need to be completely filled to ensure the proper ratio of blood to anticoagulant.
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