Donation testing

Abstract

The accurate testing of blood donations plays a vital role in the provision of safe blood for transfusion. This section covers testing that should be carried out on every unit of blood donated. All donations should be routinely tested for the ABO and Rh blood groups, screened for red cell antibodies and should be tested for transfusion transmissible infections (TTIs) including HIV, hepatitis B and C and syphilis. Anomalous test results should be investigated and resolved before blood is deposited in available stock. A range of systems, equipment and techniques are available for both the red cell serological testing and the testing for TTIs. Fully automated computerized systems are used for all aspects of the testing in large-scale testing. In some organizations manual techniques may be used, e.g. for red cell serology tests, test tubes, microplates or microcolumns, (gel technologies) are used. Anticoagulated samples are required when testing is automated. Clotted samples are suitable for blood grouping performed manually. Each donation must have a unique identification number see Section 9: Blood collection). When the donation specimens taken from the blood donors at the time of collection arrive at the testing laboratory, they should be correlated with the respective donations to check that the batch of blood donations received relates exactly to the accompanying specimens. Specimens should be stored at +4°C ± 2°C and tested as soon as possible, and within the time limits specified in test or kit manufacturer's instructions. By the end of this section, the student should be able to describe the testing of blood donations, with reference to blood groups and transfusion transmissible infections. The student should also be able to discuss algorithms used for the retesting of donations initially reactive for TTIs. These objectives are covered in the following: Red cell serology testing ABO and Rh typing red cell antibody screening ABO antibody strength (haemolysing potential) red cell antigen screening automated testing for red cell serology Transfusion transmissible infections Testing strategies human immunodeficiency virus hepatitis B hepatitis C confirmatory tests window periods algorithms Other transmissible infections syphilis malaria Overview of additional transfusion transmissible infections HTLV-I and -II Chagas’ disease cytomegalovirus West Nile virus emerging pathogens Look-back procedure. For the purpose of this publication the tests described in this section are based on manual testing. The principles however, apply to the testing in general. An overview of automation will be provided at the end of this subsection. The ABO and Rh typing should be carried out on every donation. Results should be compared with previous results if possible, and anomalies brought to the immediate attention of the person in charge for investigation and resolution. Saline methods are used to perform ABO grouping tests. Both forward and reverse grouping is performed. The results of forward and reverse grouping must correlate for the result to be acceptable. Reagent antisera should be standardized for both rapid spin and long incubation techniques, as a rapid spin technique is not practical when dealing with large numbers of specimens. Therefore, batches of tests may be incubated at room temperature for about an hour (depending on manufacturer's instructions) before reading. The ABO grouping result for a repeat donor should be compared with the result from the previous donation to check that it correlates, and action taken if it does not. In the case of an anomaly, the senior technologist should be responsible for investigating and resolving the problem. If the anomaly cannot be resolved, the donation should not be used. Group A or AB individuals, lacking the A1 antigen, sometimes develop anti-A1, usually as a cold antibody. When the A antigen is very weak and is not readily detectable on initial testing, even when using avid blood grouping reagents, the presence of anti-A1 in the donor serum/plasma may complicate the interpretation of the ABO blood group. On repeat testing of anomalous groups, it is therefore preferable to use both group A1 and A2 reagent red cells, together with the reagent group B cells. Using both group A1 and A2 reagent red cells would enable clarification of subgroups of A as shown in Table 10·1. A very weak subgroup of A, with anti-A1 in the serum/plasma may be mistaken for a group O if the weak agglutination is not detected in forward ABO grouping, and group A1 and B reagent red cells are used for the reverse grouping. However, an anomalous grouping result is noted when group A2 and B reagent red cells are used instead. The use of group A2 reagent red cells alerts the technologist to the fact that further testing is needed to resolve the ABO group, and prevents the incorrect interpretation of the blood group as group O. Similarly, if the very weak AB is not detectable in forward ABO grouping, and anti-A1 is present in the serum/plasma, it appears as group B when group A1 and B reagent red cells are used, and gives an atypical grouping result when group A2 and B reagent red cells are used instead. Reverse or serum grouping is indispensable as a means of confirming the forward or cell grouping of blood donations. Alloagglutinin tests should be read blind with no preconceived idea of what results should be. This also applies to records of ABO groups – the technologist reading the test should not have knowledge of the previous ABO group at the time of reading. Every atypical result should be investigated, including weak agglutination results where strong agglutination should be apparent. Once the ABO group of a donor has been confirmed, his/her record should be appropriately documented for future reference. Unusual or anomalous ABO types on donations need to be resolved before blood may be labelled and placed in available stock. With problematical groups, a sample from the pilot tube of the donation should also be tested. On rare occasions when the blood type cannot be interpreted with confidence, the donation should not be used and the anomaly may need to be resolved at the time of the next donation. Table 10·2 shows an example on an anomalous ABO grouping result. Even though it appears from the forward grouping that the result is group A, the group cannot be interpreted as the reverse grouping does not confirm the group A type. Blood grouping records for repeat donors should be consulted to check that the group on record is the same as the group for the current donation. If it is not, then the pilot tube of the blood unit should be grouped. If this differs from the group on the specimen, then a switch is confirmed. However, if the group using the pilot tube is the same as the group using the specimen, then the group on record may be incorrect, or the current donation misidentified. The implications of such errors could be far reaching, and results on other units of blood donated at the same session, put into doubt. The person in charge is responsible for carrying out an investigation in such cases. If there is no conclusive resolution, the blood cannot be used. Donations are separated into two Rh types: Rh-positive and Rh-negative. Rh typing results that indicate a weak D or D variant should also be classified Rh positive. This is to ensure that Rh-negative recipients do not receive blood containing the D antigen. If Rh-negative patients are transfused with Rh-positive red cells, this could stimulate the production of anti-D antibodies. This may subsequently have serious implications for Rh-negative patients, such as those mentioned here: Anti-D antibodies present in an Rh-negative woman, pregnant with an Rh-positive fetus, could lead to haemolytic disease of the fetus and newborn. Anti-D antibodies present in an Rh-negative patient transfused with Rh-positive blood in an emergency, or in error, could cause a haemolytic transfusion reaction. Rh typing results on repeat donors should be compared with the results from previous donations to check that they are the same, and action taken if they are not. In the case of an anomaly, the person in charge should be responsible for investigating and resolving it. If it cannot be resolved, the donation should not be used. Such donations should be flagged so that further testing may be carried out at the donor's next donation to investigate the anomaly. Rh typing is usually carried out using IgM (saline reacting) commercial reagents. Donors should be typed with a minimum of one selected monoclonal anti-D reagent and negative results should be confirmed using a second appropriate reagent, which could be an anti-D blend reagent, second phase or a saline reactive monoclonal reagent known to detect weak D. If the initial Rh typing is negative and additional testing using the indirect antiglobulin test (IAT) is positive, further testing should be performed to determine whether the red cells were sensitized prior to testing, in which case the result would be invalid. This may involve performing a direct antiglobulin test (DAT) on the donor red cells, or repeating the test with the appropriate typing control according to manufacturer's instructions. Table 10·3 gives examples of additional testing carried out to determine the Rh type. Depending on the anti-D reagents in use, the original test may be taken to the IAT phase or the test may be repeated including the first saline phase. There are a large number of variations in procedure depending on the source of the monoclonal anti-D selected for use in the laboratory. It is important to follow the manufacturer's instructions/laboratory protocol. Positive and negative controls should be performed on all reagents used, in parallel with every batch of tests, or at least once a day at the same time of the day, for standard ABO and Rh typing reagents. If the reagents perform as expected, this will provide the assurance that the reagents meet sensitivity and specificity requirements and that test results produced using the reagents can be accepted. Any reagent, in which controls show anomalous results, should be removed from circulation to avoid its continued use until an investigation has been carried out by the technologist in charge, to resolve the problem. When faulty reagents are used for testing, all results for that batch are invalid. Irregular red cell antibodies in donor plasma may have an adverse effect on a recipient with the corresponding antigen especially when whole blood or plasma components are transfused. Plasma containing strong irregular antibodies may not be suitable for the preparation of fresh frozen plasma or for transfer to a fractionation facility for the preparation of plasma derivatives. A set of reagent group O screening cells is used to test for irregular red cell antibodies, as described in Section 4: Principles of laboratory techniques. When large numbers of specimens are tested manually, it may not be feasible to use the IAT method, which may be the most sensitive but is also the most labourious. There is a variety of different techniques for automated systems depending on the instrument in use. These include the use of bromelin-treated cells, gel cards or solid phase technology. When the red cell antibody screen is positive, antibody identification should be performed to determine antibody specificity. Suitable records should be kept for donors with antibodies so that they are recognized at each donation and antibodies of known specificity are not re-identified at every donation. Donations showing the presence of cold autoantibodies, which are not of clinical significance, should be tested to ensure that the cold autoantibodies do not mask a clinically significant antibody. Whole blood donations with strong autoantibodies should not be transfused to patients undergoing hypothermia. To detect potentially harmful anti-A and anti-B, specimens may be tested for haemolysins or for high titre anti-A and/or anti-B antibodies. High titre antibodies can be detected by using a single dilution (e.g. 1 in 100, in saline). At this dilution strong alloagglutinins should still be able to agglutinate group A1 and B reagent red cells. In the haemolysin test, complement is triggered by the reaction of donor immune anti-A and/or immune anti-B with reagent red cells, causing the red cells to haemolyse. It is important that donors with haemolytic or high titre anti-A and/or anti-B are detected so that their blood/plasma is not used for heterologous group transfusions. Group A1B (or A1 and B) reagent red cells are used for the selected tests as they contain the strongest A (A1) and B antigens to react with either anti-A or anti-B donor alloagglutinins. When used for haemolysin tests, reagent red cells may have to be washed to remove preservative fluid if it is anti-complementary. Plasma samples are unsuitable for haemolysin tests, as anticoagulants are anti-complementary. Time also negatively affects complement. Provided that serum is used and the sample is less than 24 hours old, it is not necessary to add complement from an external source. When testing specimens older than this, the addition of extraneous complement is required. Without active complement, haemolysins are indistinguishable from non-haemolysing alloagglutinins. Donations identified as containing potentially harmful anti-A and anti-B, either by titration or haemolysin test, are labelled ‘high titre’ for homologous group transfusion only. Other donations, that do not demonstrate high titre/haemolysing alloantibodies, are labelled ‘low titre’. As an ongoing proactive measure, selected donations (e.g. group O donations) may be screened for additional red cell antigens, such as those within the Rh and Kell systems. This may be carried out by using reagents with specificities such as anti-C, anti-c, anti-E, anti-e and anti-K. Results of extended antigen screening should be added to the records, and subsequent donations from the same donor flagged so that the donations can be identified from available stock, and selected if they fit the type required to resolve a compatibility problem. This avoids time consuming screening that may be needed at the time of crossmatch, to find compatible blood. Alternately the donors may later be traced to donate blood for patients with antibodies but there is a time delay between calling the donor to give blood, and the fully tested donation being available for crossmatch and issue. Ongoing screening of donations may be performed to find donors who lack high incidence antigens so that rare donations identified in this way may be stored frozen in glycerol in a low temperature freezer for future use. When a patient with antibodies to a high frequency antigen requires blood, suitable blood can be requested from the rare donor registry. The blood will need to be thawed, and deglycerolized prior to crossmatch and issue. All the tests described previously may be fully automated or partially automated according to the systems and modules selected. For information on automation, see Section 4: Principles of laboratory techniques. Automated testing of all donations in batches to perform the red cell serology tests is much quicker than manual testing and suits laboratories that handle a large number of samples. (The time taken to complete a batch of tests and be ready to start the next batch is sometimes referred to as turnaround time.) Most automated machines require special reagents with optimum reaction temperatures and techniques. It is important to note that some reagents require dilution before loading into the machine, and manufacturer's instructions should be closely followed. Automation saves time as the machine is programmed to rapidly interpret all standard reaction patterns for the tests. This process categorizes the majority of donations into one of the four ABO groups (A, B, O or AB), one of the two Rh types (D+ or D−) and identifies antibody screen positive and high titre donations. The computer program linked to the automation set-up should be designed to detect and flag anomalous results so that they can be investigated further. Automated machines require the inclusion of specific quality control samples of known ABO and Rh type, in the batch being tested, to ensure that all reagents are working correctly. Controls should be included in every batch and when large numbers of specimens are tested, after a certain number of tests (such as every 200 tests) and should always be repeated when reagents are replaced or new dilutions made. When automated control results fail it is important to establish the root cause of the failure. Each laboratory should have a troubleshooting procedure in place applicable to the specific instrument in use. The training and operating manuals should provide such information. Problems with automated machines may need to be resolved by technical personnel from the supplier or manufacturer of the machine. Common problems may, however, be identified by suitably trained technologists working with the machine in the laboratory, but it is critical that qualified and experienced support from the supplier or manufacturer is readily available. When blood grouping is automated, technologists should regularly perform manual testing, so if the machine fails and is not operational for an extended period, they retain competency in manual techniques and are able to continue testing blood donations. Testing for TTIs is subject to ongoing change and improvement, as additional and more sensitive tests and automated test systems become available or new risks of possible infections are identified. The interpretation of results and the strategy for donor deferral or exclusion as well as the way in which donors are notified of anomalous or reactive results, is dynamic. In this section, only basic information is provided. Every donation of blood should be tested for the following TTIs: HIV Hepatitis B Hepatitis C Syphilis. Other transmissible infections that relate to the geographical area of the blood service should be included in TTI testing. Test methods for the detection of infectious markers include: Enzyme linked immunosorbent assay (ELISA) or enzyme immunoassay (EIA) Nucleic acid testing (NAT) Rapid tests. Rapid tests for viral markers may be used for various reasons, such as when a small number of tests are required. These rapid tests and their applications are not in the scope of this publication. When a TTI test on a blood donation is reactive, care must be taken as to how this result is confirmed, and to ensure that appropriate action is taken with regard to donor notification. Partly because of the need for sensitivity in testing systems, TTI screening may lead to falsely reactive results, and biological false positives do occur. Extreme care needs to be taken with follow-up action, including confirmatory testing on the donation. The confirmed reactivity to certain TTIs such as HIV, HBV and HCV leads to permanent exclusion of the donor, whereas the risk of other TTIs such as malaria, may have specified time deferrals. Each laboratory/service should have protocols in place for both initial and confirmatory testing. Western blot is not used routinely for screening blood donations for viral markers. It is a gel electrophoresis technique and requires specialized equipment. It is useful for confirmatory testing for HIV. The principles of the tests used for detection of TTIs are to be found in Section 4: Principles of laboratory techniques. Every donation should be tested for HIV-1 and HIV-2, using a test system such as EIA or NAT. Laboratory markers for HIV include anti-HIV, p24 antigen, and viral RNA. Every donation should be tested for hepatitis B, using a test system such as EIA, PCR or NAT. Laboratory markers for HBV include HBsAg and viral DNA. Every donation should be tested for hepatitis C, using a test system such as EIA or NAT. Laboratory markers for HCV include anti-HCV and viral RNA. When a result is reactive, it should be confirmed by retesting. This may be performed by repeating the original test and by including additional or different test systems. Confirmatory testing should be performed using a sample taken from the pilot tube of the blood bag or plasma sample from the blood bag if possible, to confirm that the specimen used relates to the donation. Confirmatory testing may also be performed using a second sample from the same donor, if this is available, in case the specimen used originally became contaminated in the laboratory or in transit. When a donation is TTI reactive, it must be embargoed until the confirmatory testing is complete and resolved. If the result is confirmed reactive, the donation cannot be used for transfusion. It must be discarded using the appropriate disposal protocol/methods and according to local regulations for the disposal of biohazardous material. Discrepancies between initial test result and confirmatory test result must be fully investigated and resolved before the final decision is made regarding the donation. Any donation that is reactive, in screening and confirmatory testing, must not be transfused. The information on all reactive units must be referred to the appropriate department for authorized donor follow-up (for more information, see Section 9: Blood collection). The window period refers to the period of time between contracting an infection and the appearance of detectable antibodies or other markers in the bloodstream. It is a latent period of infection (immunosilence), as laboratory tests for markers of the infection are non-reactive. A blood donation given during the window period will test negative, be classified safe for use and be placed in available stock yet may be infectious. As window period donations may transmit infection, the donor questionnaire and donor interview carried out prior to acceptance of a donor to give blood, are very important. A screening process using an interview and a questionnaire should detect individuals at risk of an infection, so that they are excluded from donation. The biological attributes of host–virus interaction, replication times, infectivity dose, natural history of infection, serum volume used for in vitro testing, and the relative sensitivity of each assay employed to detect an infection present in a blood donor are highly variable. As illustrated in Table 10·4, the lag time (cumulative window period) for ability of each serological test to detect an infection present in a donor's blood is based on the window period data for HIV, HBV and HCV published in 2007 by Busch (see reference related to Table 10·4). Thus, the lag time for anti-HIV to detect HIV infection can be as long as 190 days, for HBsAg test to detect HBV infection as long as 44 days, and for anti-HCV to detect HCV infection as long as 58 days. This lag period gets truncated by direct tests for viral gene amplification with NAT. The EIA for viral antigens and antibodies are indirect tests of measuring host response to an infection, but NAT testing is the most direct method of screening blood for preventing transfusion transmitted viral infections. In the context of TTIs, an algorithm is the term used for a sequence of steps that is documented and followed in order, when a donation is initially found to be reactive. Depending on the TTI concerned, the algorithm is unique. For example, the steps taken when a sample is HIV reactive may not be the same as steps taken when the sample is syphilis or malaria reactive. However, all reactive tests should be repeated, as screening tests should not be interpreted without confirmatory testing. Many TTI test systems rely on cut-off values to determine whether a result is reactive or not, so some test results may fall close to a range of uncertainty (i.e. ‘indeterminate’ result). Whenever possible, it is preferable to confirm initially reactive results by using a different testing system, as well as by repeating the original screening test. The pilot tube from the donation is ideally used for follow-up testing, to check that blood bags and specimens were not switched at the time of donation. However, this step is more difficult if testing is performed at a central laboratory and blood donations retained in the peripheral blood bank. On retesting, confirmed reactive donations are discarded. If there is any doubt about the result on repeat testing, the donation should also be discarded. Algorithms developed by transfusion services are usually complex and individualized for each TTI. In this publication, however, a single algorithm is shown that generalizes and summarizes the suggested flow of actions when a donation is reactive for a TTI. Figure 10·1 is a guideline algorithm summarizing the sequence of actions that may be taken when a laboratory test is initially reactive for HIV, HBV or HCV. The algorithm does not describe the procedure to follow for a specific TTI. Guideline algorithm for TTI testing: minimum steps only – actual process may be complex. Of all the transfusion transmissible infections, HIV, HBV, HCV and syphilis are the most important with regard to universal screening of blood donations. However, because of the increase in international travel, other transmissible infections too, are more likely to be spread amongst communities in which they were previously not detected. Individuals who are particularly vulnerable due to a lack of immunity to an infection rarely encountered in their home environment become increasingly exposed to these infections as a result of travel. Syphilis is a global concern, and some detail on testing is therefore given. The same applies to malaria. Ref: WHO World Health report 2007: the deadly interaction: HIV/AIDS and other diseases. The interaction of HIV/AIDS with other infections diseases is an increasing public health concern. In sub-Saharan Africa, for example, malaria, bacterial infections and tuberculosis have been identified as the leading causes of HIV-related morbidity. HIV infection increases both the incidence and severity of clinical malaria in adults. In some parts of Africa, falciparum malaria and HIV infection represent the two most important health problems. One of the main reasons for screening donations for syphilis is because of its value as a surrogate test. Individuals with syphilis are more likely to be infected with other sexually transmitted infections such as HIV and hepatitis B. Serological tests for syphilis include: Rapid plasma reagin test (RPR) Treponema pallidum haemagglutination test (TPHA) Fluorescent Treponemal antibody absorption test (FTA). Transfusion services may decide to use either RPR or TPHA as a first-line screening test and retest reactives using FTA, which is less likely to give false positive results. However, if a confirmatory test is not performed, donors should not be notified solely on the result of a screening test, although reactive donations are not transfused. The donor record should be flagged (in code) to draw attention to the testing result when a subsequent donation is given. Serum/plasma from the donor is tested for antibodies to reagin and not for antibodies to T. pallidum. Reagin levels are raised in certain infectious conditions such as syphilis, and this causes an antibody response in the host. Reactive results are demonstrable as flocculation; a form of precipitation between antibody to reagin in the donor sample and the reagin reagent. The TPHA test is used to detect antibodies to T. pallidum. The test uses avian erythrocytes coated with antigenic components of the T. pallidum organism. This test can be automated and results produced are read either by the technologist or by machine. Some transfusion services may use an FTA test, which measures specific antibody for T. pallidum, to confirm screen reactives. However, once reactive, an individual remains reactive for life, so the test does not differentiate between infectious and non-infectious individuals. Serum/plasma from the donation sample is mixed with T. pallidum and after processing, an antihuman globulin reagent labelled with a fluorescent indicator is added. If antibodies are present in the sample, the labelled AHG will act as an indicator, and fluorescence will be detected when using a specially designed microscope or appropriate reader. The FTA is costly and requires a degree of technical skill to perform. Red cells, white cells, platelets and fresh plasma are all capable of transmitting malaria although red cells are the most likely to do so. Transfusion transmitted P. falciparum may cause severe malaria and lead to the death of the recipient. The effort to maintain a malaria-free blood supply is an ongoing challenge for many transfusion services for two reasons: malaria in asymptomatic carriers is difficult to detect due to the very low level of parasitaemia, although even a single parasite transfused into a recipient can cause infection; and donors from endemic areas often have circulating antibody as a result of partial immunity to malaria. There does not seem to be one ideal laboratory test system for the screening of blood donations, although evaluation of new test systems is ongoing. The options available to a transfusion service include the following: Deferral of donors at risk of carrying malaria Screening of all blood donations, or at-risk donations (at-risk donations are those taken from donors who could have been exposed to malaria) Concomitant administration of malaria prophylaxis to recipients of at-risk blood A combination of all of the above. The various screening tests available include the following: Screening for parasites found within the red cells Screening for antibodies to the malaria parasite Screening for the presence of malaria antigens Screening for the presence of plasmodial DNA. Examining blood smears directly for i