Infectious Disease, HLA and ABO Donor Qualification Testing
Trypanosoma cruzi (anti-T. cruzi) (implemented in January 2007)
The parasite Trypanosoma cruzi Is the causative agent of Chagas disease, a serious illness. The agent is endemic in Latin America, but approximately 20 reports of transmission by blood transfusion have been published worldwide. The Red Cross blood donations are screened using the Ortho T. cruzi Enzyme-Linked Immunosorbent Assay (ELISA) for the qualitative detection of antibodies to T. cruzi in human serum or plasma samples. An FDA-licensed enzyme strip immunoassay (ESA) is used for confirmatory testing. The FDA has approved a T. cruzi reentry algorithm requiring a follow-up sample testing nonreactive by the two FDA-licensed screening tests and the ESA. Although T. cruzi can be transmitted by blood transfusion, to date, the Red Cross has not identified any recipients infected by blood components from screen-negative donors or screen-negative donors who subsequently tested positive (seroconverted). All reports of transfusion transmission in the United States and Spain (another country with a high number of residents having lived in endemic areas of the Americas) have been from unscreened whole blood, platelets (including leukoreduced and irradiated), except from one red cell case, or from whole blood from unscreened donors in Latin America. None of these transfusion transmissions (n=11 combined for the US and Spain from 1987-2011) were associated with an incident or autochthonous donor infection. Because T. cruzi is not endemic in the US without any incident cases in blood donors, the Red Cross (and all US blood centers) are allowed to test donors only once. The frequency of detecting a positive donor is about 1 per 15,000 first-time donations screened.
Hepatitis B virus (HBV) Surface Antigen (HBsAg) (implemented in 1971), Hepatitis B Core Antibody (Anti-HBc) (implemented in 1986), HBV DNA Nucleic Acid Testing (NAT) (implemented in June 2009)
HBV DNA and HBsAg are the first viral markers to circulate in an individual infected with HBV. Anti-HBc appears in the blood of individuals infected with HBV one to four weeks after the appearance of HBsAg, and at the onset of symptoms for those adults who develop symptoms (5% or less). The tests used for blood donor screening are the GS (Genetic Systems’ distributed by Ortho) HBsAg EIA 3.0, a qualitative ELISA for the detection of HBsAg, and the Ortho HBc ELISA for the qualitative detection of antibodies to HBV core antigen (anti-HBc) in human serum and plasma samples. An FDA licensed triplex NAT using transcription-mediated amplification was introduced by the Red Cross in June 2009. The assay detects HBV DNA, HIV RNA, and HCV RNA. Testing is performed in mini-pools (MPs) of 16 samples; components of reactive MPs are tested individually to identify the reactive donations, followed by virus-specific testing to determine the virus responsible for the sample's reactivity. Specific antigen neutralization is used for confirmation of HBsAg reactivity; HBsAg-reactive samples that are HBV DNA reactive do not require further testing by neutralization. If neutralization testing is positive and MP-NAT is nonreactive, or the donation sample is anti-HBc-reactive and HBsAg and MP-NAT are nonreactive, further testing is performed by HBV individual donation NAT. False-positive donors for any HBV marker may be reentered. The per-unit risk of HBV infection through blood transfusion (chance of releasing an infectious blood component) is approximately 1 per million units screened using published data through 2019, and this number has remained stable. NAT has reduced the window-period from HBV infection to detection by about 12 days. This leaves an approximate period of 2 to 3 weeks when an infected donor may not be detected by blood donation screening. The frequency of detecting an active HBV infection in a blood donor is about 1 per 12,000 donations screened.
Hepatitis C virus (HCV 3.0) Antibody testing (implemented in December 1990) and Nucleic Acid Testing (NAT) (implemented in September 1999)
HCV is the causative agent for most, if not all, blood-borne non-A, non-B hepatitis. The test used for blood donor screening is the Ortho HCV ELISA for the qualitative detection of antibodies to HCV antibodies (anti-HCV) in human serum or plasma samples. Duplex NAT was introduced for HIV/HCV RNA detection in September 1999 and updated to include the detection of HBV DNA in June 2009 (see above). Donors who test HCV-antibody reactive, but NAT nonreactive by routine testing in MPs of 16 are further tested individually for HCV RNA by NAT. If nonreactive on the specific individual HCV NAT assay, the sample is further tested by another FDA-licensed HCV-antibody screening test to determine if the antibody reactivity is specific. Donors who test anti-HCV and HCV NAT reactive do not require further testing. False-positive donors by either antibody or HCV NAT may be reentered. The last published figure (data through 2019) for the per-unit risk of HCV infection through blood transfusion (chance of releasing an infectious blood component) is less than 1 per 2 million units screened, but currently much lower due to the availability of direct-acting, highly active anti-virals that continue to decrease the frequency of HCV NAT-reactive donors. NAT closes the window period between infection and the detection of antibodies for those infected with HCV by about 50 to 60 days. This leaves an approximate period of about 1 week when an infected donor may not be detected by blood donation screening. The frequency of detecting a positive donor is about 1 per 5,000 donations screened.
Human Immunodeficiency viruses, Types 1 and 2 (HIV 1,2) Antibody testing (implemented in March 1985) and Nucleic Acid Testing (NAT) (implemented in September 1999)
Blood donation screening for HIV-1, the causative agent of AIDS began with antibody testing in 1985. Many improvements in testing have occurred, including the detection of a second HIV agent (HIV-2 in 1992). The test used for blood donor screening is the GS (Genetic Systems’ distributed by Ortho) HIV-1/HIV-2 PLUS O EIA for the simultaneous qualitative detection of anti-HIV 1 (groups M and O) and/or HIV-2 in human serum or plasma. A duplex NAT was introduced for HIV/HCV RNA detection in September 1999 and updated to include the detection of HBV DNA in June 2009 (see above) and HIV-2 RNA detection in July 2020. Donors who test antibody reactive are further evaluated by additional tests to confirm the presence of HIV antibodies and to differentiate HIV-1 from HIV-2 antibodies. Such tests have included an HIV western blot, an HIV-2 enzyme-linked immunoassay (EIA), and an HIV-1 and HIV-2 rapid test for viral differentiation (all FDA licensed), but now with the elimination of the western blot and HIV-2 EIA, only the rapid test (Genetic Systems’ GEENIUS) is used. Donors who test anti-HIV-1/HIV-2 and HIV NAT reactive are not further tested. Currently only the rapid test is performed on HIV-1/HIV-2 antibody-reactive donations that test NAT negative to confirm antibody reactivity and differentiate HIV-1 from HIV-2 antibodies. Donors testing falsely positive by either antibody or HIV NAT may be reentered. The last published per-unit risk of HIV infection through blood transfusion (chance of releasing an infectious blood component) including data through 2019 is less than 1 per 2 million units screened but continues to decrease. NAT closes the window period between infection and antibody detection for those infected with HIV by about 2 weeks. This leaves an approximate period of 7 to 10 days when an infected donor may not be detected by blood donation screening. Since the first implementation of HIV NAT in 1999, only 5 transfusion-transmitted HIV infections have been documented (from 6 infected donors). The frequency of detecting HIV-1 in a blood donor is about 1 per 33,000 donations screened. However, detecting HIV-2 in a blood donor is extremely rare at 1 per 57 million donations, with only 5 such infected donors ever identified since HIV-2 screening began in 1992.
Human T-Lymphotropic virus (HTLV-1/2) (implemented in 1998)
HTLV-1 and HTLV-2 are human retroviruses. While HTLV-1 has been associated with neoplastic conditions and various demyelinating disorders, HTLV-2 is not yet proven unequivocally to be of significant clinical concern. The test used for blood donor screening is the Avioq HTLV-1/2 Microelisa system (distributed by Ortho) for the qualitative detection of antibodies to HTLV-1 and HTLV-2 in human serum or plasma samples. Donors who test reactive for anti-HTLV-1/2 are further tested using an FDA licensed western blot to determine if antibodies are present. The western blot confirms infection and differentiates between HTLV-1 and HTLV-2 (with HTLV-2 slightly more common than HTLV-1). There are no nucleic acid tests (NAT) available for HTLV-1/2. False-positive donors by antibody may be reentered, except those that were confirmed positive by various tests used before the availability of an FDA-licensed western blot. The frequency of detecting an infected donor is 1 per 50,000 donations screened.
The recently published per-unit risk of transfusion-transmitted HTLV-1/2 (2020-2021) is 1 per 2.8 million units screened and when coupled with successful leukoreduction (LR), since HTLV is a cell-associated virus, is 1 per 3.3 billion (assumes a 0.085% LR failure rate). The low residual risk and low frequency of significant clinical disease have led to the question of whether screening each donation is necessary versus testing donors only once (as done for anti-T. cruzi).
Syphilis (Treponema pallidum) (implemented in the 1950's)
Screening for syphilis is performed using a qualitative test that detects the presence of antibodies to the spirochete (corkscrew-shaped bacterium), Treponema pallidum, by an automated agglutination assay based on specific pattern recognition. Confirmation is performed using another serologic test for total antibodies, an EIA, as well as a test for reagin (a protein-like substance that is present during acute infection and for several months following resolution of infection). Reagin testing was the first screening method used but was replaced by antibody testing in 1995. No cases of transfusion-transmitted syphilis have been recorded in more than 50 years. False-positive donors for syphilis may be reentered.
Zika virus (ZIKV) Nucleic Acid Testing (NAT) (implemented in June 2016 – discontinued in May 2021)
ZIKV, a flavivirus, closely related to dengue viruses, resulted in an epidemic in the Americas during 2016-2017. ZIKV usually causes mild symptoms such as: fever, skin rash, conjunctivitis, muscle and joint pain, or headache. More severe neurological complications have been documented, such as Guillain-Barré syndrome and microcephaly or other congenital brain abnormalities in the developing fetus of an infected pregnant female. ZIKV was first identified in rhesus monkeys in the Zika forest of Uganda in 1947. More recently, it expanded across the South Pacific islands into South America, Central America and the Caribbean with limited transmission in 2015-2016 in the US (Florida and Texas). The virus is most frequently transmitted to humans through mosquito bites; but may also be transmitted by sexual contact, laboratory acquisition or rarely by blood transfusion. ZIKV nucleic acids (RNA) are detected using a nucleic acid test (NAT) similar to that of West Nile virus (see below). Investigational NAT in mini-pools (MPs) of 16 was introduced in June 2016 in 5 southeastern states, but as required by FDA, expanded to all US donations (first as individual donations and by January 2019 in MPs using the Grifols FDA-licensed assay). NAT-reactive donations were further tested by repeat NAT and antibody testing to confirm infection. During the peak of the outbreak in 2016-2017, a rate of 1 per 950,000 was obtained (all positives with naturally occurring infection had their exposure outside of the US or in Florida); our last confirmed-positive donation was in March 2018 (exposure in Cuba). Since 2018, there have been no reports of local transmission in the US, and since 2019, none reported in US territories. No suspect transfusion-transmission investigations were reported during the period when ZIKV NAT was performed. Donors testing ZIKV NAT reactive were eligible to donate 120 days after their ZIKV NAT-reactive result. In May 2021 the FDA approved the discontinuation of ZIKV NAT for all routine blood donor donations. The Red Cross discontinued ZIKV NAT in June 2021 due to low donor yield and the end of the outbreak.
West Nile virus (WNV) Nucleic Acid Testing (NAT) (implemented in June 2003)
WNV is a flavivirus commonly found in many areas of the world, including West Africa, Europe, and the Middle East. The virus, generally transmitted to humans through mosquito bites, was first detected in the US in 1999 in New York and reached epidemic proportions in 2002. In the same year, WNV was reported to be transmitted by blood transfusion and organ transplants. WNV RNA is detected by an FDA licensed NAT assay similar to that used for HIV, HCV, and HBV (and previously, Zika virus). NAT-reactive donations are further tested by repeat NAT and antibody to confirm infection. Following the introduction of blood donation screening in 2003 through the end of 2018, there have been 15 cases of documented WNV-transfusion transmission from screened blood; all are believed to be due to donations having very low levels of virus. This translates to a risk of about 1 per 84 million donations for the Red Cross overall (or 1 per 35 million during the summer transmission season). Testing by NAT occurs in MPs of 16 like that done for HIV-1, HCV, and HBV; however, in addition, measures are in place to reduce the risk of transmissions from donors with low concentrations of virus that would occur from donors having early or late infection during WNV local outbreaks. To increase sensitivity, screening is converting from testing donations in MPs to testing donors individually in areas of on-going outbreaks. Since WNV NAT began in June 2003 through the end of 2022, the Red Cross has detected 2,797 WNV-infected donors. Donors testing WNV NAT reactive are eligible to donate again after 120 days.
Babesia Antibody and Nucleic Acid Testing (NAT) (implemented with antibody in June 2012) and NAT only (implemented in May 2018 with the discontinuation of antibody)
Babesia is a red cell parasite endemic in the Northeastern and upper Midwestern regions of the US and responsible for the disease referred to as babesiosis. The parasite is naturally transmitted by the bite of an infected tick but may also be transmitted by blood transfusion. Infection may be asymptomatic or cause non-specific symptoms: fever, fatigue, headache, and muscle pain. In some cases, the disease can be severe, especially in those with a compromised immune system, hemoglobin-related diseases (such as sickle cell disease), and those lacking a spleen. The Red Cross began blood donation screening for Babesia microti, the species most common in the US, under an investigational protocol in June 2012 in the New England area and upper Midwest. Donors who consented to investigational screening were tested by two tests: an individual donation NAT assay (polymerase chain reaction, PCR) and an antibody assay, the latter using an indirect immunofluorescence assay (IFA). In May 2018, the Red Cross began testing whole blood samples using a NAT assay that detects the main four species of Babesia pathogenic to humans. The FDA-licensed assay that detects ribosomal RNA of the parasite following red cell lysis, significantly increased sensitivity and obviated the need for antibody testing. Because the lysis step releases thousands of Babesia-RNA copies, mini-pool testing can be performed (MPs of 16). If a reactive donation is identified, testing is repeated, and a sample is sent for antibody testing by IFA. In May 2019, the FDA released guidance to test all donations collected in the 14 states considered Babesia endemic or contiguous to an endemic state: Maine, Vermont, New Hampshire, Connecticut, Massachusetts, New York, New Jersey, Delaware, Rhode Island, Maryland, Pennsylvania, Virginia, Minnesota, and Wisconsin and in the District of Columbia. Red Cross implemented the guidance for testing in May 2020. Since then, the ARC tests about 1,600,000 donations for Babesia every year and identifies an average of 380 reactive donations. Babesia-positive donations have been identified in all 14 states, with the higher rates reported in Connecticut (1:1,581), Massachusetts (1:1,908), Maine (1:1,517), New Hampshire (1;3,944), New Jersey (1:1,2517), New York (1:3,715), and Pennsylvania (1:4,061). Reactive donors are eligible to donate again if they test NAT-nonreactive after a 2-year deferral period. Since the implementation of testing (either investigational or licensed), there have been no reported transfusion-transmitted babesia cases from tested blood. A small number of cases have been reported from states in which testing does not occur demonstrating that the range for the parasite is expanding.
Every unit of blood is tested to identify the donor's blood group (O, A, B or AB) and Rh type, and screened for atypical or unusual red cell antibodies.
Cytomegalovirus (CMV) Antibody Testing (implemented prior to 1985)
The current interventions for high-risk blood recipients who are at increased risk of CMV infection include leukoreduction and CMV antibody testing, neither of which has the capability of detecting free virus in plasma during the acute phase of infection when transmission risk is most likely to be present (i.e., the window period). However, little to no breakthrough CMV infection via transfusion has been documented since the implementation of universal leukoreduction, and hospitals both internationally and in the United States have dropped, and continue to drop, their requirements for CMV seronegative labeled blood with the use of universal leukoreduction. The major risk of CMV infection in low and very low birth weight infants (the major risk group) is from breast milk from infected mothers. The current risk of CMV transfusion transmission remains theoretical; such risk from the CMV DNA window period is undocumented and unknown.
CMV antibody testing is performed on a portion of blood donations, including all platelet donations, to meet hospital demand for CMV "safe" (seronegative) blood products. Antibody testing may be requested in addition to universal leukoreduction as intended for specific vulnerable patient populations. CMV-seronegative donations are labeled as negative.
Human Leukocyte Antigens (HLA) Antibody Testing (implemented in 2016)
HLA testing is performed on donations from female donors who indicate they have ever been pregnant (first-time donors) or donors who have had a pregnancy since their prior HLA-negative donation (repeat donors) as a mitigation for HLA-antibody-mediated Transfusion-Related Acute Lung Injury (TRALI). Donors who test HLA reactive by the FDA-licensed screening test for HLA class I and II antibodies are further tested to determine their ongoing suitability via Luminex using a validated cutoff for donor screening. Donors testing negative in duplicate can be reinstated..