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Specimens used for HIV testing
summary
Selecting 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.
HIV testing of serum and plasma, which can be collected by venepuncture (see section 3.2), have the following advantages and disadvantages:
Have higher concentrations of HIV antibodies than oral fluids 19
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
Require 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
Whole blood, which can be collected by venepuncture or finger-stick (see section 3.2), has the following advantages and disadvantages in HIV testing:
Has higher concentrations of HIV antibodies than oral fluids 19
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)
Requires 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
Dried 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:
Are 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
Require specific filter-paper ( 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
Other 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.
Does 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.
May 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)
recommendation
Blood (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.
Blood needed for an HIV test can be collected either by venepuncture (whole blood, serum, plasma) or by finger-stick (whole blood).
To 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.)
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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&#;400or &#; 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.
To 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.
Blood 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.
Blood 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.
For 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.
Oral 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.
The 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 ).
A 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 , ).
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 ).
The study included 150 participants from Kenya (n = 49), Malawi (n = 47), and South Africa (n = 54), resulting in 33 participants using an oral test (O1) and 117 using a fingerstick test (FS1 = 29, FS2 = 29, FS3 = 30, FS4 = 29) (Table 2). Errors were common. Less than 25 % of all users correctly performed all steps (39.4 % O1, 24.1 % FS1, 20.7 % FS2, 13.3 % FS3, and 24.1 % FS4), and 47.3 % of participants conducted multiple errors. Data have been analyzed to identify test attributes that performed well or need improvement and can be used to inform TPP development.
Table 2 Participant demographicsFull size table
Video observation revealed that 88 % of participants reviewed the instructions before, during, and after testing. Many referred to them step-by-step. For each test, most participants (61.3&#;92.3 %, depending on the test) used only one kit (Table 3). Of those who opened a second test kit, some did so to use another lancet, and others redid the test, stating that they wanted to repeat incorrect steps. The inclusion of pictorial and written instructions was considered helpful by the respondents.
Table 3 Participants conducting steps correctlyFull size table
&#;They&#;re good, especially because of the photographs&#;&#; (Kenya, Female, FS4)
Local translations were also valued.
&#;You cannot say this to somebody who has never gone to school &#; unless you put it in [their] mother tongue, I bet this is only meant for people who are educated.&#; (Kenya, Female, FS4)
Qualitative interviews revealed that some participants felt instructions pertaining to specific test components or procedures were unclear. Test components not described in the test instructions, such as bandages and desiccant sachets, caused confusion and errors. Errors included participants adding the desiccant sachet contents to the test liquid or onto the sample collection device (n = 3), and recording time on the bandage instead of the space provided (n = 1).
&#;[The step] was a bit confusing, because at first I didn&#;t know if I should remove the [cap] on the test or pour over it. Because there is no instruction for that step. Even the picture doesn&#;t show.&#; (South Africa, Male, FS3)
Less than half (39.4 %, n = 13/33) of the participants collected the oral sample correctly. Errors included using the wrong end of the collection device (n = 2); swabbing only one side of the mouth or only the upper or lower gum (n = 11); swabbing the incorrect area of the mouth (n = 9), such as teeth, tongue, or cheek; or swabbing incorrectly (e.g., like brushing teeth). Some dipped the swab in the test liquid before swabbing (n = 3). Participants explained that their confusion was generally due to unclear instructions, misunderstanding language, and complicated procedures.
&#;Eh It was difficult because I couldn&#;t understand the word &#;swab,&#; I couldn&#;t understand what was required of me&#;&#; (South Africa, Male, O1)
&#;They were not difficult, except step number eight which is about swabbing of the gums, it was difficult to follow the instructions and the picture wasn&#;t all that clear.&#; (Malawi, Male, O1)
Only 60.7 % (n = 71/117) of participants were able to prick their finger with the lancet and produce a drop of blood. Videos showed participants examining the lancet in detail. Others unintentionally triggered the lancet before pricking (n = 20), and 43.6 % of participants pricked themselves more than once. Some utilized a second lancet from another test kit or provided separately (n = 47), including 24 who opted for the generic blade-style lancet; two were observed reaching for personal items (e.g., safety pin or razor blade) to try to prick themselves. A few participants (n = 6) were observed conducting the process with greater ease with a second lancet. Field note observations showed that some participants indicated a great deal of frustration with pricking their finger (including two abandoning the test after failing). Few participants (n = 3) expressed reluctance or fear of pricking.
Exit interviews revealed that participants did not know how to use the triggered (touch sensitive or button) lancets.
&#;It was difficult to use the lancet from the testing device, even though the picture was showing how to do it&#; I didn&#;t figure [it] out.&#; (Malawi, Male, FS4)
Some participants felt that the provided instructions alone were inadequate to guide them through the steps.
&#;I had a problem on how to use the lancet such that I pricked the wrong finger, we need to be taught how to use it otherwise people will be just pricking themselves several times.&#; (Malawi, Female, FS2)
&#;That thing for pricking the finger [lancet], it doesn&#;t show that there is something inside&#;that should be reviewed a little so that even if you can&#;t see it, you can tell that there is something that can prick you inside.&#; (Kenya, Female, FS2)
All fingerstick tests required the participant to collect blood into a pipette or capillary. Only 47 % (n = 55/117) were able do this according to instructions. Tests with a separate blood collection device (FS1 and FS3) resulted in 39 % (n = 23/59) of participants correctly collecting and transferring the sample to the test. Participants had difficulties with both the mechanics of using a pipette and a capillary tube. Some applied the sample directly to the test (n = 8), either after not being able to collect blood or because of incorrectly interpreting the instructions. Tests with sample collection integrated into the test cassette (devices FS2 and FS4) had better results, with 55.2 % (n = 32/58) of participants applying blood directly to the test correctly.
All of the tests in this study utilized a test liquid. The method for combining sample with test liquid varied. With a test that involved three different steps for adding test liquid (FS3), only 20.0 % (n = 6/30) of participants did this correctly. In contrast, when the test was added to the premeasured test liquid in one step (O1, FS2), this was performed correctly by 72.6 % of the participants (n = 45/62). Opening test liquid containers proved problematic. Two participants used a personal item or a generic lancet to cut or puncture sealed containers. Containers with pop-open tops presented challenges when participants attempted to unscrew (n = 6), resulting in five instances of spillage. For the FS2 that delivered test liquid automatically by pushing the test into a sealed cap (thereby eliminating the need to open the container or count drops), 41.4 % (n = 12/29) of participants were observed not pushing the test far enough to break the seal and adequately immerse it in the liquid.
Participants found that the steps involving the test fluid could be complex.
&#;Oh this watery like substance?&#;I saw it, but I got confused as to what shall I use it for.&#; (South Africa, Male, FS1)
Most participants (69.3 %) did not wait for the required time to read results, including a number in the exit interviews who explained that they did not do this as they knew they would not receive test results in this study. Participants who did time their test generally used the clock provided in the testing space; however, a few used their watch or mobile phones (n = 3).
Most participants identified negative and strong positive results correctly (Table 4). Negative test results were correctly interpreted 79.9 % of the time (n = 119/149). Strong positive results were correctly interpreted 78.7 % of the time (n = 163/207). Weak positive results with faint test lines were correctly interpreted as positive by only 26.7 % of the participants (n = 24/90). An invalid test is indicated by no lines or lack of a control line appearing on the test. Most invalid results were correctly interpreted (72.7 %, n = 258/355).
Table 4 Participants correctly identifying test result panelsFull size table
During results interpretation for FS1, which has three lines (control, HIV-1, and HIV-2), some participants perceived that a result of one line indicated a negative test result, two lines indicated a positive test result, and three meant the person was very ill.
&#;Firstly we all know that if there are two lines it means it is positive so here there are two lines and they say it is invalid, for a villager they cannot understand this, it doesn&#;t matter where the lines are but as long as there are two lines to many people that is positive, so they better look into that.&#; (Malawi, Female, FS2)
Another test did not explicitly label the locations of the test and control lines on the cassette (FS2). This test had the lowest frequency of correctly interpreted negative results (48.3 %, n = 14/29) and invalid results without a control line (41.4 %, n = 12/29). The highest percentage of participants (90.8 %) correctly identified the results of the flow-through test, which utilized different symbols for control and test.
Exit interview data from questionnaires revealed that more than half of participants using each test rated them as very easy or easy to use (Table 5). Over 80 % of users of all tests felt confident doing the tests and over 70 % felt confident reading results. More than 80 % of participants agreed that they were likely or very likely to use the test again if it were free and also indicated that they were willing to buy the test. There was a general sense of enthusiasm about the prospect of being able to undertake HIVST. Reasons provided in the qualitative interviews included time-savings, reduced fear of HIV status disclosure, and convenience.
Table 5 Participants&#; perceptions of testsFull size table
&#;&#;sometimes there are queues at clinics. And also I am afraid that people will see me in that queue and know that I came for HIV test, whereas at home it is easy and everything you do is your secret. At least you will only have to go to the clinic if you have the disease.&#; (South Africa, Female, FS2)
&#;It can be beneficial to your family and you as a person&#; you don&#;t need to plan a journey to go to the hospital. You test yourself there and then.&#; (Kenya, Male, FS4)
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