Blood donor screening: Laboratory testing

INTRODUCTION — Laboratory testing of donated blood prior to transfusion is intended to ensure that recipients receive the safest possible blood products.

This review will discuss the various protocols in place for the laboratory testing of donated blood, with emphasis on screening for infectious agents. Specific discussions of transfusion-transmitted infections are presented separately. (See "Epidemiology and transmission of hepatitis C virus infection" and "Transfusion-transmitted bacterial infection".)

Additional safety measures related to blood donor screening are presented separately. (See "Blood donor screening: Overview of recipient and donor protections" and "Blood donor screening: Medical history".)

OVERVIEW OF LABORATORY TESTING — Prior to 1985, only two infectious disease screening assays were performed on donated blood. Many additional assays have subsequently been introduced into routine blood screening; several of these assays have undergone significant modifications to increase sensitivity and specificity (these revised assays are designated by a change in name, or generation or version number) [1]. Minipool nucleic acid testing (NAT) was added to routine serologic screening in 1999. As the risk for transfusion transmission of additional infectious agents has been recognized, testing has been added and/or removed as needed (eg, for Zika virus). (See 'Zika virus' below.)

Testing of the donated unit consists of determining the ABO blood group, RhD typing, and testing for red blood cell (RBC) antibodies. In some cases, a more extensive RBC antigen profile is obtained. Historically this was done using serologic (antibody-based) methods, referred to as extended RBC phenotyping, but genomics technology (referred to as extended RBC genotyping) may also be used [2].

Infectious disease screening is performed for a number of organisms that are summarized in the table (table 1) and discussed in the sections below [3]:

●Human immunodeficiency virus (HIV)-1 and HIV-2 (see 'HIV-1 and HIV-2' below)

●Human T-lymphotropic virus (HTLV)-I and HTLV-II (see 'HTLV-I and HTLV-II' below)

●Hepatitis C virus (see 'Hepatitis C virus' below)

●Hepatitis B virus (see 'Hepatitis B virus' below)

●West Nile virus (see 'West Nile virus' below)

●Treponema pallidum (syphilis) (see 'Syphilis' below)

●Trypanosoma cruzi (Chagas disease, only required to be performed on the first donation by a particular donor) (see 'Chagas disease' below)

In addition to these tests, some donated units are tested for Babesia microti (see 'Babesia microti' below) and some for cytomegalovirus (CMV) antibodies. (See 'Cytomegalovirus' below.)

Many apheresis platelet units are tested for bacterial contamination by an automated culturing technique initiated 24 hours after collection, and whole-blood-derived platelets undergo bacterial testing with point-of-release immunoassays. As an alternative to bacterial testing, apheresis platelet units may undergo a pathogen inactivation process. In 2019, the US Food and Drug Administration (FDA) issued guidance for platelet products that includes measures to reduce the risk of transfusion-transmitted infections, as discussed separately. (See 'Bacteria' below and "Platelet transfusion: Indications, ordering, and associated risks", section on 'Strategies for reducing bacteria and other pathogens'.)

All infectious disease screening assays must be negative to release the blood unit or its components to hospitals for transfusion.

This testing has resulted in very low risk of infectious disease from transfused blood components, as summarized in the table (table 2).

Testing of donated blood in the United States occurs at large centralized laboratories to ensure compliance with Good Manufacturing Practices (cGMP) of the US Food and Drug Administration (FDA) and which use expensive high throughput automated equipment, minimizing the chance for human error.

Specific testing protocols can vary by country and sometimes by region (see 'Babesia microti' below). As an example, donated blood in the United States is not routinely screened for evidence of infection with human parvovirus B19, a respiratory virus that can cause fifth disease in children and red cell aplasia in individuals with chronic hemolytic anemia (eg, sickle cell disease). In contrast, all Japanese Red Cross Blood Centers have screened for parvovirus B19 since 1997 using immunoassays or NAT [4]. Some countries only use serologic testing (without NAT). (See "Virology, epidemiology, and pathogenesis of parvovirus B19 infection" and "Clinical manifestations and diagnosis of parvovirus B19 infection".)

LABORATORY TESTING PROTOCOLS

Immunoassays for antibodies or pathogen antigens — The infectious disease screening assays for antibodies to pathogenic organisms (which demonstrate host response to an infection) and antigens from these organisms historically used the enzyme-linked immunosorbent assay (EIA) method, which has generally been replaced by a similar chemiluminescence enzyme immunoassay method using the following general testing protocol:

●If the initial sample readout is below the cutoff, the sample is classified as negative for that agent.

●If the sample readout exceeds that of the cutoff, the result is classified as initially reactive.

●All initially reactive samples are retested in the same assay system in duplicate, usually on the next working day. If one or both of the duplicate tests are also reactive the sample is classified as repeat reactive and is, by definition, positive. A positive screening test results in the destruction of the unit. If both repeat test results are negative, the initially reactive result is equivalent to a negative result and the unit is released from quarantine and made available for transfusion.

Such an approach is scientifically sound due to known problems with nonspecific binding, resulting in initially reactive immunoassay test results that cannot be duplicated on careful repeat testing.

Nucleic acid testing (NAT)

Minipool testing — Beginning in 1999, blood collection centers implemented minipool nucleic acid testing (NAT) technology for detection of hepatitis C virus (HCV) and HIV nucleic acids (RNA) in donated blood. Since 2009, blood centers have replaced the dual assay for HIV and HCV nucleic acids with a triplex assay that detects hepatitis B virus (HBV) DNA and HIV and HCV RNA. West Nile virus (WNV) NAT was added in 2003 and Zika virus NAT in 2016 (subsequently the requirement for Zika virus was removed) [5].

For logistical and cost reasons, NAT in the United States is performed on pools (designated minipool NAT or MP-NAT) of samples from 6 to 16 blood donors. There are two major methods in use: polymerase chain reaction (PCR) and transcription-mediated amplification (TMA). If a positive result is obtained on a pool of samples, it is necessary to determine which of the three viruses (HIV, HCV, or HBV) is present (this may require testing by an additional discriminatory assay); also, further testing is performed to establish which donation in the pool was the source of the positive result. The positive unit is discarded and all the negative units in the pool are used for transfusion.

Individual donation testing — NAT of individual samples (ID-NAT) is more expensive and time-consuming than MP-NAT, but ID-NAT results in higher assay sensitivity. As an example, it has been estimated that the time period in which transfusion of a blood component will cause infection (referred to as the "window period" and estimated to occur at a concentration of 1 viral copy per 20 mL) and its detection by NAT can be shortened from 9.0 to 5.6 days for HIV and from 7.4 to 4.9 days for HCV by replacing MP-NAT with ID-NAT [6,7]. This resultant shortening of the window period by conversion to ID-NAT has been estimated to result in an approximately 50 percent reduction in the very small risk of developing HIV or HCV infection from blood transfusion (table 2). However, taking MP-NAT as a baseline, the marginal cost of introducing ID-NAT has been estimated to exceed USD $12 million per quality-adjusted life-year [8]. This low cost-effectiveness has resulted in the continued use of MP-NAT for simultaneous detection of HIV, HCV, and HBV.

The situation with regard to ID-NAT for WNV is different, as it has been demonstrated that a significant proportion of WNV RNA-positive units will be missed by MP-NAT due to the low viral titers present in WNV infection. In the United States, blood collectors have adopted a more cost-effective strategy of targeted ID-NAT, implementing this test if and when the WNV infectivity exceeds a defined threshold in a given geographic region [9]. Most blood centers request that their testing laboratory implement ID-NAT after the detection of one or two MP-NAT positive donations and then revert back from ID-NAT to MP-NAT when the region has no ID-NAT reactive donations for at least 14 days [10,11]. (See 'West Nile virus' below.)

In August 2016, the US Food and Drug Administration (FDA) recommended national Zika virus ID-NAT be implemented. This extremely precautionary measure was taken due to the potentially severe effects of Zika virus infection during pregnancy and concerns that donor history questions (previously implemented in March 2016) would be insufficient to completely address the risk of transfusion-transmission. In July 2018, due to the significant decrease in cases of Zika virus infection, the FDA issued new guidance that recommended MP-NAT testing could be performed if reflexed to targeted ID-NAT using a strategy similar to that for WNV; This guidance was removed in May 2021 when cases of Zika infection were no longer recorded, making it unnecessary to continue Zika testing. (See 'Zika virus' below.)

DONOR NOTIFICATION AND ELIGIBILITY

Confirmatory testing — Because all transfusion-transmitted infectious agents are of low prevalence in the asymptomatic donor population, the predictive value of a positive screening test is also low [12]. Thus, since a positive screening result will usually be a false positive result, it is essential that screening tests be followed with a more specific supplementary or confirmatory test (US Food and Drug Administration licensed) prior to notifying donors of their test results. Results of supplemental testing may indicate that the donor is infected, is false positive and is not infected, or may be inconclusive (indeterminate) [13]. In the case of reactivity by nucleic acid testing (NAT) in the absence of positive serological assays, further NAT testing of an alternate sample source (eg, the plasma component bag) or follow-up sampling of the donor is needed to confirm the test results.

Notification and counseling — Blood centers have developed notification and counseling procedures for each type of result for each tested agent [13-15]. Most notifications, other than for HIV-seropositive donors, are done by letter. In addition to receiving the information, the donor is instructed to telephone an appropriate person at the blood center to discuss his or her test results. The basic principles of donor notification for confirmed seropositive donors involve providing information to the donor accurately, confidentially, as quickly as possible, and in a manner that alleviates anxiety and promotes understanding. Seropositive (or NAT positive) donors need to be informed of their ineligibility as donors, the medical significance of their test result, the need to see a physician (if appropriate), and the possible modes of transmission of the agent. Because notification of indeterminate donors carries uncertainty about their infection status, the counseling process is difficult and often results in donor anxiety [13,15].

Eligibility for future donation — Decisions as to the future eligibility of the donor must also be made [14]. HIV- and hepatitis C virus (HCV)-positive donors are ineligible for future donation, regardless of confirmatory test results. (See 'Viruses' below.)

In contrast, donors with repeat reactive anti-HBc (hepatitis B core antibody) results or with positive anti-HTLV EIA results not confirmed by supplemental testing are allowed by US Food and Drug Administration (FDA) regulations to continue donating until a second such occurrence (see "Hepatitis B virus: Screening and diagnosis in adults"). Donors who have been deferred for two reactive anti-HBc results can be reinstated ("re-entered") if the blood center follows the FDA prescribed retesting protocol.

INFECTIOUS DISEASE SCREENING AND SURVEILLANCE — A United States national system (the Transfusion-Transmissible Infection Monitoring Systems [TTIMS]) that tabulates HIV, hepatitis C virus (HCV), and hepatitis B virus (HBV) testing data and donor demographic data from the largest United States blood collection organizations (accounting for approximately 60 percent of United States blood collections) was established late in 2015 [16].

This system has provided (and continues to provide) an important source of data concerning prevalence and incidence of blood donor infections sorted by demographic characteristics and geographic region. It also allows for monitoring of trends over time, which in turn can be correlated with changes in donor eligibility criteria or donor demographics. One general conclusion from the first four years of TTIMS data is that residual risks of transfusion-transmission for the three major viruses (HIV, HCV, and HBV) have been decreasing or have remained constant over time [17]. In addition, TTIMS data as well as a decade of data from the American Red Cross show that the prevalence of these infections in persons who are first-time blood donors continues to be substantially higher than in repeat blood donors (persons who have previously donated blood and have previously tested negative for these viruses) [17,18].

The following sections describe the transfusion-transmitted viral, bacterial, and parasitic infections for which routine blood donor screening occurs in the United States.

Viruses — Potential transfusion-transmitted viral infections for which blood is screened include HIV, HTLV-I and -II, hepatitis B and C viruses, West Nile virus, and for some units, cytomegalovirus (CMV).

Information on the novel coronavirus (severe acute respiratory syndrome coronavirus 2 [SARS-CoV-2]) is discussed below. (See 'Emerging infectious disease agents' below.)

HIV-1 and HIV-2 — HIV is a lentivirus, a type of retrovirus that stores its genetic information as RNA. Infection with HIV causes acquired immunodeficiency syndrome (AIDS), due to progressive depletion of CD4 T cells. The first cases of AIDS were described in 1981, with subsequent expansion of cases around the world. The modes of transmission include sexual, perinatal, and bloodborne, principally via sharing of needles among people who inject drugs. (See "Global epidemiology of HIV infection" and "Acute and early HIV infection: Pathogenesis and epidemiology".)

Transmission by through blood products obtained from viremic individuals was noted in the early years of the pandemic. Historically (prior to 1985, when HIV testing was first instituted), transfusion of a product from an individual with HIV had a high probability (90 percent) of transmitting HIV. Rates of transfusion-transmitted HIV were equivalent from red blood cells (RBCs) stored for <21 days, platelets, or plasma. (See "Blood donor screening: Medical history", section on 'HIV'.)

The historical evolution of testing procedures has resulted in progressively lower risk of transmission, mostly due to a narrowing of the window period (the period during which a donor with HIV is potentially infectious via the blood transfusion route but has not yet developed any laboratory evidence of HIV infection).

Two HIV screening tests are performed on each donated unit:

●Antibody testing – HIV-1 antibody testing was first implemented in 1985 and has subsequently undergone significant improvements. In 1992, an assay (HIV-1/HIV-2 antibody test) with improved sensitivity was implemented; the increased sensitivity was due both to its ability to detect IgM in addition to IgG antibody, and its detection of HIV-2 antibody due to the addition of HIV-2 specific antigens [19]. Seroconversion following HIV infection occurs at approximately three weeks (22 days) [20].

Samples that test positive by the antibody screening assay (either an enzyme-linked immunosorbent assay [EIA] or a chemiluminescence enzyme immunoassay) are either confirmed as positive if they have a concurrently positive HIV-1 RNA result (see section immediately below) or are further tested by a US Food and Drug Administration (FDA)-licensed antibody confirmatory assay. Donors with confirmed positive results are counseled that they have been infected with HIV-1 (or in very rare cases, HIV-2); however, false positive confirmatory results can sometimes occur in the low prevalence setting of blood donor screening [21].

The window period with the first generation assays was estimated at 56 days; with second generation assays introduced in 1987, the window period was estimated at 42 days [22]. The Centers for Disease Control and Prevention (CDC) in the United States estimated that prior to antibody testing, 12,000 to 25,000 cases of transfusion-transmitted HIV may occurred [23]. After institution of antibody testing, the rate of transmission was reduced to 1 in 450,000 to 650,000 units [24-26].

●Nucleic acid testing (NAT) – HIV-1 RNA testing in minipools (MP) of 6 to 16 samples was introduced into routine blood donor screening in 1999/2000 [27]. The window period is approximately 11 days when blood is tested by MP-NAT and approximately 8 days when tested by ID-NAT [28]. The shorter window period with NAT compared with antibody testing occurs because HIV RNA appears earlier than p24 viral protein or anti-HIV antibody [6,29].

In the United States, during the first three years after MP-NAT was implemented (1999 to 2002), 12 HIV RNA positive, HIV antibody negative units were identified (1 in 3.1 million units tested) [30]. In 2011 to 2012, 13 units were identified (1 in 1.1 million) [16]. It has been estimated that the most common screening system in the United States detects approximately 500 HIV RNA copies/mL, 95 percent of the time, in each of the specimens entering the pool [6]. Estimates for the risk of transfusion-transmitted HIV infection range from 1 in 1.6 million to 1 in 2.3 million units in the United States (table 2) and 1 in 7.8 to 10 million units in Canada [17,18,31].

It is estimated that using individual donation NAT (ID-NAT) rather than MP-NAP would further shorten the window period to 8 days; however, implementation of this testing would be expected to detect only two to three additional HIV infectious units per year, resulting in an HIV risk of 1 in 3 to 4 million, with a marginal cost exceeding USD $12 million per quality associated life year [6,8]. HIV ID-NAT screening has not been used in the United States but has been implemented in some countries with very high HIV incidence (South Africa and countries in Southeast Asia) [32-34].

HIV-1 p24 antigen testing was introduced in 1996; it was subsequently discontinued and replaced by HIV NAT, as discussed above [26].

HTLV-I and HTLV-II — Each unit of blood is screened in a single assay that detects antibodies to either of the closely related retroviruses, human T cell lymphotrophic virus (HTLV)-I and HTLV-II. HTLV-I can rarely cause adult T cell leukemia-lymphoma; both HTLV-I and HTLV-II rarely cause HTLV-associated myelopathy (HAM) [35]. (See "Clinical manifestations, pathologic features, and diagnosis of adult T cell leukemia-lymphoma".)

Screening of donated blood for antibodies to HTLV-I began in late 1988 using HTLV-I viral lysate as antigen in an EIA format. Due to immunologic crossreactivity, antibodies to HTLV-II, a closely related retrovirus prevalent in United States blood donors, are often but not always detected by this assay. Consequently, in early 1998, the screening assay was improved by inclusion of HTLV-II antigens. As of 2016, screening test results are confirmed by an FDA-licensed confirmatory assay.

The estimate for the risk of transfusion-transmitted HTLV infection is 1 in 2.7 million units in the United States and 1 in 4.3 million in Canada [31,36-38].

Hepatitis C virus — Hepatitis C virus (HCV) is a cause of hepatitis with possible long-term sequelae from chronic infection. (See "Clinical manifestations and natural history of chronic hepatitis C virus infection".)

Screening for HCV antibody was implemented in 1990, and an improved multi-antigen second generation enzyme immunoassay (designated as EIA 2.0) was introduced in 1992 [39]. A third version of the assay (designated as EIA 3.0) was licensed by the FDA in 1996. HCV MP-NAT has also been performed since 1999 [27]. For many years, confirmation testing was performed with the FDA-licensed Recombinant ImmunoBlot Assay (RIBA) 3.0. Since there is no available FDA-licensed confirmatory serologic test, confirmation of antibody results is provided either by correlating with MP-NAT screening results or by performing a second manufacturer's enzyme or chemiluminescence-based immunoassay. HCV MP-NAT has been estimated to reduce the undetectable infectious window period to approximately 7.4 days compared with the 70-day window using HCV EIA 3.0 antibody testing [29,30,40,41]. (See "Screening and diagnosis of chronic hepatitis C virus infection", section on 'Introduction'.)

Estimates for the risk of transfusion-transmitted HCV infection range from 1 in 1 million to 1 in 2 million units in the United States, and 1 in 2.3 to 3 million in Canada; a marked decrease from the earlier estimate of 1 in 100,000 units prior to the use of MP-NAT [18,30,31,37,41-44].

Hepatitis B virus — Hepatitis B virus (HBV) is an uncommon cause of chronic hepatitis in adults. (See "Hepatitis B virus: Clinical manifestations and natural history".)

Screening for HBV surface antigen (HBsAg) was introduced in the early 1970s. Samples testing positive by EIA (or chemiluminescence enzyme immunoassay) are confirmed using a neutralization assay provided with the test kit. HBsAg may be positive in acute infection or as a result of a chronic carrier state. False positive neutralization results occasionally occur in the blood donor setting [45]. Neutralization-positive donors are deferred from future donation. Despite the improved sensitivity of the chemiluminescence enzyme immunoassay, the infectious window period prior to development of HBsAg positivity has been estimated to range from 18 to 27 days and possibly to last as long as 38 days [46,47].

Screening for antibody to HBV core antigen (anti-HBc) began in 1987 as a surrogate assay for carriers of non-A, non-B hepatitis [48,49]. In 1990, the anti-HBc assay was licensed by the FDA based upon its ability to decrease the risk of HBV infection by detecting some HBsAg-negative donors who are capable of transmitting HBV [49,50].

Anti-HBc develops early in the course of HBV infection and remains positive whether infection is resolved or becomes chronic. Approximately 1 percent of donors test anti-HBc positive [51]; however, some older FDA-approved assays had poor specificity and there are no confirmatory assays to determine with certainty which donation is potentially infectious [49,51]. (See "Epidemiology, transmission, and prevention of hepatitis B virus infection", section on 'Transfusion'.)

Deferral is not required by the FDA until two independent donations test anti-HBc positive; however, many blood collection centers will defer such donors after a single test for cost efficiency reasons. Upon referral to a physician, detection of antibody to HBV surface antigen (anti-HBs) often serves as a second independent assay to establish past HBV infection provided the donor has not received HBV vaccine.

The estimate for the risk of transfusion-transmitted HBV infection, prior to the introduction of HBV MP-NAT in the United States, ranged from 1 in 58,000 to 1 in 269,000 [31]. With HBV nucleic acid testing incorporated as part of routine MP-NAT screening in 2009, the risk of HBV transmission has decreased to 1 in 1 million to 1 in 1.5 million [17,18,44,52-54].

West Nile virus — West Nile virus (WNV) was shown to be transmitted by blood transfusion in the fall of 2002, with 23 cases eventually documented from that year [55,56]. (See "Epidemiology and pathogenesis of West Nile virus infection", section on 'Transmission'.)

WNV generally causes a self-limited febrile illness, although neurologic disease with meningitis, encephalitis, and/or paralysis can occur. Infection is seasonal. (See "Clinical manifestations and diagnosis of West Nile virus infection", section on 'Clinical manifestations'.)

Minipool NAT — WNV transfusion-transmission occurs solely from acutely infected asymptomatic donors rather than from donors with chronic infection. Therefore, WNV blood donor screening is performed using only a NAT system; serological screening is not used in conjunction with NAT [57].

WNV NAT was implemented nationwide in the United States under FDA approval in July 2003, using MPs of 6 or 16 different donation samples, depending on the manufacturer's format. However, in late 2003, data indicated that despite MP-NAT screening, a small number of breakthrough transfusion-transmitted cases of WNV occurred; these were due to donors who were very early in infection and whose viral loads were below the threshold of detection by MP-NAT [57-63]. This led to the strategy of targeted individual donation nucleic acid testing (ID-NAT).

Targeted individual donation testing — Unlike other transfusion-transmitted viruses, WNV is seasonal in its occurrence and has shown significant geographic and temporal variation throughout the United States [58,59,64]. The targeted ID-NAT testing strategy was designed to balance the residual risk of transfusion transmission from units screened with MP-NAT against the limitations in testing capacity due to lack of sufficient automation for performing ID-NAT. It involves real-time tracking of MP-NAT results in defined geographic regions, based on a predetermined initiation trigger, at which point all units will be tested by ID-NAT rather than MP-NAT.

The initiation trigger for ID-NAT at most blood centers is detection of a single MP-NAT-positive donation. The criterion for discontinuing ID-NAT and reverting to MP-NAT is the absence of ID-NAT-reactive donations for a defined time frame (usually a minimum of 14 consecutive days, although it may be longer in some programs in WNV epidemic areas) [11,65-67].

All WNV transfusion-transmissions, with one possible exception, have been from viremic donors who were IgM negative, leading to the conclusion that the presence of IgM antibody is protective against transmission via transfusion [57].

It has been estimated that if ID-NAT testing is implemented early in the course of a region-specific epidemic, 5 to 10 percent of the units detected would be MP-NAT-negative, ID-NAT-positive, IgM- and IgG-negative, and therefore potentially infectious [58,59].

Identification of viremic donors — In 2003, approximately 1000 WNV viremic donors were identified in the United States, either by MP-NAT or ID-NAT, resulting in approximately 1500 potentially infectious blood components being discarded prior to transfusion [58,59,64].

In the ensuing years, testing has proven highly effective with 300 to 1000 WNV RNA-positive donations interdicted each year [68]. From year to year, there continues to be geographic and temporal variation in the number of detected donors, but no positive donors have been identified from mid-December through early April [11].

The average time viremia is detectable by MP-NAT is approximately seven days [69].

Based on a few cases in which low level viremia in the presence of IgM has persisted for several months, the FDA requires that donors found positive by WNV NAT screening be temporarily deferred from donation for 120 days [70].

Cytomegalovirus — Cytomegalovirus (CMV) infection can cause severe disease in immunocompromised patients, such as hematopoietic cell transplant (HCT) and organ transplant recipients and those infected with HIV. CMV-safe blood is required only for these selected patient populations. In the past, CMV-safe blood needed to be supplied from donors who had no evidence of current or past CMV infection; such donors were identified by testing negative on a CMV antibody assay.

Unlike the previous screening assays, CMV antibody testing is not performed on each unit of donated blood prior to transfusion, but only on enough units to establish a sufficient inventory of CMV-negative units to supply patient needs. The presence or absence of CMV antibody does not affect a donor's eligibility.

Because the presence of CMV antibody is a nonspecific assay for CMV infectivity and because 30 to 70 percent of blood donors (depending upon geographic region) test CMV antibody positive, much effort has gone into finding another method of providing CMV-safe blood [71]. Leukoreduction with depletion of leukocytes (white blood cells) to <5 million leukocytes per unit is an acceptable alternative [72,73]. Nevertheless, CMV antibody screening is used in some parts of the United States to provide CMV-safe blood. (See "Practical aspects of red blood cell transfusion in adults: Storage, processing, modifications, and infusion", section on 'Pre-storage leukoreduction' and "Platelet transfusion: Indications, ordering, and associated risks", section on 'Leukoreduction'.)

Zika virus — Zika virus is a mosquito-borne flavivirus (the same virus family that includes WNV and dengue virus). Infection causes mild disease or no symptoms in most individuals but carries a risk for fetal loss, microcephaly, and other adverse fetal and newborn outcomes if infection occurs during pregnancy. (See "Zika virus infection: An overview" and "Zika virus infection: Evaluation and management of pregnant patients".)

Policies with regard to Zika virus testing have evolved over time. In May 2021, the FDA removed guidance from 2016 and 2018 that had required testing of donated blood for Zika virus [74,75].

Prior to test licensure, universal ID-NAT testing was instituted in the United States and its territories in mid to late 2016 using investigational tests [76]; subsequently, two blood donor NAT screening assays that detect Zika virus RNA were licensed by the FDA. Blood donor deferral criteria used prior to this testing had included additional donor history questions [77,78]. (See "Blood donor screening: Medical history".)

In mid-2018, the FDA changed to recommending minipool testing using either of the licensed tests based on the changed epidemiology of Zika virus infection in the Western Hemisphere (very few cases occurred in 2017 and 2018) [79]. Similar to the approach to WNV testing, the testing algorithm approved by the FDA specified a transition to ID-NAT in a given geographic area if minipool testing identified a Zika-reactive donation.

In May 2021, FDA stated that there had been no local, mosquito-borne Zika virus transmission in the United States since 2017 and only four travel-related cases in 2020, and the Centers for Disease Control and Prevention (CDC) indicated that there are no countries with active Zika outbreaks [74]. The last confirmed Zika virus-positive blood donation in the United States occurred in March 2018, and there have been no reported cases of transfusion-transmitted Zika virus in the United States [80]. Based on these data, the FDA removed the requirement for testing donated blood for Zika virus as of May 2021 [74,75].

Other transfusion-transmitted infections

Syphilis — Serologic testing for syphilis (STS) has been performed on donated blood for many decades. Algorithms for testing are described in a December 2020 FDA guidance document [81].

In the past, nontreponemal screening assays (eg, rapid plasma reagin [RPR] or Venereal Disease Research Laboratory [VDRL] tests) were used, despite their poor specificity. In the 1990s, automated high throughput instrumentation using T. pallidum specific antigens replaced these manual methods. This resulted in an increased detection of donors with remote infection, sometimes acquired many decades previously [82].

When a positive treponemal test is found, the donated blood is not transfused and the donor is deferred indefinitely. Additional testing with a nontreponemal test can be performed to assist with donor notification and counseling. If screening is performed with a nontreponemal test, additional specific treponemal tests can be performed; under some instances as described in the Guidance document, such as having been treated for syphilis the donor can be reinstated for future donation [81]. (See "Syphilis: Screening and diagnostic testing".)

Syphilis testing presents no value as a surrogate marker for incident HCV, HBV, HTLV, or HIV infection in blood donors [83].

Bacteria — Transfusion-transmitted bacterial infection (TTBI) has been associated with a spectrum of bacterial organisms. This is a rare complication. Platelets have been most frequently implicated due to their storage at room temperature, but other blood components (RBCs or plasma) can also transmit bacterial infection. (See "Transfusion-transmitted bacterial infection", section on 'Epidemiology' and "Platelet transfusion: Indications, ordering, and associated risks", section on 'Room temperature storage'.)

Only platelet products are subject to specific testing and/or risk mitigation procedures. Details are presented separately. (See "Transfusion-transmitted bacterial infection", section on 'Reducing risk of bacteria in the product' and "Platelet transfusion: Indications, ordering, and associated risks", section on 'Strategies for reducing bacteria and other pathogens'.)

Chagas disease — Chagas disease is caused by infection with the protozoan parasite, T. cruzi that causes nonspecific symptoms (fever, malaise); rarely, severe neurologic or cardiac manifestations may occur. (See "Chagas disease: Acute and congenital Trypanosoma cruzi infection", section on 'Acute T. cruzi infection'.)

In the 1990s and early 2000s, data on Chagas disease indicated that the risk to transfusion recipients in the United States appeared to be growing based upon the changing demographics of the United States population. Historically, transfusion transmission occurred at high rates in endemic countries (eg, Mexico and other Central and South American countries) in the absence of donor laboratory testing. Blood stream parasites are detectable and potentially transmissible decades after immigration from an endemic area [84]. Transfusion transmitted cases in the United States have occurred almost exclusively with platelet components [85]. (See "Blood donor screening: Medical history", section on 'Chagas disease' and "Chagas disease: Epidemiology, screening, and prevention", section on 'Blood transfusion'.)

Two EIA blood donor screening tests for T. cruzi antibody have been approved by the FDA (in 2006 and 2010). The majority of United States blood centers implemented universal screening for evidence of past Chagas infection in 2007 [86]. Subsequently, the FDA also approved a recombinant antigen-based immunoblot as a confirmatory test [87]. Prior to that date, most blood collection centers used the radioimmune precipitation assay (RIPA).

Since 2010, many blood centers have adopted a selective testing screening approach in which donors are screened with the T. cruzi antibody test on their initial donation, but the testing is not repeated on subsequent donations if the initial testing was negative [88]. While infection with T. cruzi could have been acquired in the past (and hence requires one-time screening and deferral from future donation), there is very little ongoing transmission of the T. cruzi parasite to blood donors currently residing in the United States [89]. The FDA issued formal guidance for blood component screening and appropriate use of the tests in December 2010 and again in 2017, including confirmation of continued use of selective testing [90].

Babesia microti — Babesia are obligate intraerythrocytic protozoan parasites that can cause babesiosis, a potentially life-threatening malaria-like illness. In the United States, B. microti is the primary agent of babesiosis. Naturally occurring B. microti infection acquired from infected tick bites is regionally distributed within the United States, with high-risk areas located in the Northeast and upper Midwest [91]. (See "Babesiosis: Clinical manifestations and diagnosis" and "Babesiosis: Microbiology, epidemiology, and pathogenesis".)

From 1979 to 2009, more than 160 cases of transfusion-transmitted babesiosis were recognized in the United States, contributing to at least 28 associated deaths; almost all of these cases were caused by B. microti [91]. Subsequently, an additional manufacturer's NAT assay was approved, which detects not only B. microti DNA but also the DNA of several other Babesia species; this assay can be used to test individual samples or minipools of 16 samples. It is performed on a lysed whole blood sample (as opposed to plasma for other NAT assays). A second manufacturer has an approved assay for MP testing in pools of six samples. Due to the high sensitivity of the NAT assay, there is no requirement for Babesia antibody testing; however, some blood collectors still perform this test to gather further information and/or for counseling purposes. [92,93].

NAT assays for babesiosis have been used for several years to test a substantial portion of the blood supply in high-risk (B. microti endemic) regions. In 2019, the FDA issued a guidance document that requires blood collectors in 14 endemic or at-risk states (Connecticut, Delaware, Maine, Maryland, Massachusetts, Minnesota, New Hampshire, New Jersey, New York, Pennsylvania, Rhode Island, Vermont, Virginia, Wisconsin) plus the District of Columbia (Washington, DC) to perform B. microti NAT on all units that they collect. One allowable exception is that if an FDA-approved pathogen-reduction technology is used to treat the collected blood unit or blood component (such as has been approved and is being done for some apheresis platelet units), then NAT is not required [94].

The prevalence of B. microti in the donor pool was illustrated by a 2016 analysis of almost 90,000 blood donation samples from four states in high-risk regions [95]. Nucleic acid testing for B. microti DNA and serologic testing for anti-babesia antibodies identified 335 (0.38 percent) to be positive on one or both of the screening assays. Furthermore, 20 percent of the positives contained detectable B. microti DNA, and in an animal model, such samples had an infectivity rate of approximately 50 percent. Follow-up testing of positive donors at one year demonstrated that 86 percent of PCR-positive donors had clearance of parasite DNA, but only 8 percent of antibody-positive donors showed loss of antibody reactivity. Based on these data, the FDA has recommended a two-year deferral following a reactive NAT result.

The clinical effectiveness of donor testing was demonstrated in the same 2016 study by comparing transfusion-transmitted clinical cases from screened and unscreened units in two high-risk states in New England [95]. There were no reported cases of transfusion-transmitted babesiosis from 75,000 screened donations, whereas there were 14 cases from approximately 250,000 unscreened donations (transmission rate, 1 case per 18,074 unscreened units).

EMERGING INFECTIOUS DISEASE AGENTS

SARS-CoV-2/COVID-19 blood safety policies — Based on recommendations from the Association for the Advancement of Blood & Biotherapies (AABB) and the US Food and Drug Administration (FDA), donated blood is not screened for the presence of SARS-CoV-2 (the virus that causes COVID-19). At the outset of the pandemic, this policy was established based on prior observations that viruses transmitted primarily by the respiratory route have not been documented to be transmitted by transfusion [96-98].

In the absence of laboratory testing, blood collection centers have adopted donor deferral policies related to recent infection with SARS-CoV-2. Furthermore, blood centers inform donors that they should contact the blood center if they develop symptoms compatible with COVID-19 or are diagnosed with COVID-19 in a defined period after donation (ranging from 48 hours to 14 days depending on the blood center). Units from these individuals are removed from inventory. These policies are discussed separately. (See "Blood donor screening: Medical history", section on 'Active or prior COVID-19'.)

Data have accumulated with respect to SARS-CoV-2 RNA-emia in plasma and on donations by asymptomatic persons who later develop symptoms of SARS-CoV-2 or a positive SARS-CoV-2 diagnostic RNA test.

●The largest study evaluating SARS-CoV-2 in donated blood is a 2020 multicenter study that used a highly sensitive nucleic acid amplification test to screen blood donations collected over a six-month interval in six United States metropolitan areas [99]. Out of 257,809 screened donations tested in minipools (MP) of 16, three RNA-positive samples were detected, for an estimated prevalence of 1.16 in 100,000 (95% CI 0.40-3.42). The RNA-reactive samples were non-reactive for antibody, and the estimated viral loads of the (presumed single) positive donations within each MP were low, ranging from <1000 to <4000 copies/mL. Because samples were tested retrospectively in a deidentified manner, there are no data on the recipients who received these units.

●A French study of 268 blood donors who reported COVID-19 symptoms after donation identified only three SARS-CoV-2 RNA-reactive donations given by these donors (rate of 1.1 percent); none of these RNA-reactive plasma samples were infectious in cell culture [100].

●A United States study of 2250 donors who reported symptoms following donation revealed a higher rate of RNA-emia (8.7 percent) but found that RNA was present at very low concentration (median, 6 genome equivalents per mL) [101]. Plasma units with the highest RNA levels were not infectious in a sensitive in-vitro cell culture system nor in an enhanced sensitivity mouse model system.

●A case report from the United States documented a positive SARS-CoV-2 RNA by RT-PCR in a blood sample from a donor who had recovered from COVID-19-like symptoms 40 days earlier; this unit was not transfused [102].

●Additional studies reported failure to detect infectious virus in culture despite RNA-emia [100,103].

In most studies, the potentially infectious units were interdicted prior to transfusion, so there are limited data to directly evaluate transfusion-transmission. However, a few studies have evaluated recipients for the acquisition of transfusion-transmitted infection. In a Korean study, six blood donors were found to have confirmed COVID-19 within two weeks post donation. Nine patients received units from these donations, none developed COVID-19 symptoms, and the three recipients who underwent SARS-CoV-2 RNA testing in nasopharyngeal samples were negative [104]. Several other case reports of donors who reported post-donation COVID also have failed to identify COVID symptoms in recipients [105-107]. In addition, almost three years into the pandemic, no cases of transfusion-transmitted COVID-19 have been reported in the United States or internationally [101].

One explanation for these findings is that the presence of SARS-CoV-2 RNA in blood may not represent fully competent infectious virus but instead could be due to incomplete RNA fragments that are not infectious; this hypothesis is supported by the low concentration of SARS-CoV-2 RNA in PCR-positive units and by the negative infectivity results in cell culture and mouse models [99,101].

Taken together, these studies indicate a low prevalence of SARS-CoV-2 RNA-emia in donated blood and document that viral RNA is at low concentration when detected, and they show no ability to transmit infection in limited investigations in humans. Although it cannot be ruled out that transfusion transmission may occur rarely, all studies to date support the conclusion that SARS-CoV-2 is not transmissible by transfusion.

Data on the epidemiology of COVID-19 are presented separately. (See "COVID-19: Epidemiology, virology, and prevention", section on 'Epidemiology'.)

Other emerging pathogens — There have been no reports of mpox (monkeypox) transmission from blood transfusion, and the FDA is not recommending that donated blood be tested for mpox [108].

A comprehensive review of 68 emerging infectious disease (EID) organisms was compiled in 2009 by the members of the Transfusion Transmitted Diseases Committee of the Association for the Advancement of Blood & Biotherapies (AABB) [109]. The intent of this review was to provide a set of tools for identifying, describing, and prioritizing those EID organisms that have an actual or potential risk of transmission by transfusion and for which there is no currently implemented effective intervention. The authors prioritized the organisms according to the consensus opinion about their anticipated impact on blood safety by considering the following factors:

●Biology and epidemiology

●Subjective assessment of public and regulatory concerns

●Availability of sensitive and specific donor screening approaches

●Geographic range, including potential shifts in the near future

●Frequency of potential risk of prospective donor exposure

Subsequent to the initial AABB publication, revised information about some of these organisms and new information about additional EID organisms has been added to the AABB website section on emerging infectious diseases. As an example of a new concern, several research studies have shown a high prevalence of hepatitis E virus (HEV) RNA in donors in several European countries (approximately 1 in 2000 to 1 in 3000), prompting routine donor screening for HEV RNA by nucleic acid testing (NAT) to be implemented in the United Kingdom and the Netherlands [110]. However, the prevalence in United States blood donors is far lower (approximately 1 in 10,000), and no screening is needed [111].

There continues to be a need for ongoing surveillance for new organisms and for determining the criteria that should be used to make rational and balanced decisions about whether additional laboratory tests should be added to blood donor screening [5].

NON-INFECTIOUS DISEASE LABORATORY TESTING

Routine RBC antigen and antibody testing — Red blood cell (RBC) compatibility is essential to avoid causing hemolytic transfusion reactions. (See "Hemolytic transfusion reactions".)

All donor units are tested to determine the ABO blood group and the Rh type.

●ABO group – The ABO group is determined using appropriate commercially supplied anti-A and anti-B antibodies to detect the A and/or B antigens on donor RBCs (forward type) and using reagent RBCs to detect the reciprocal antibody in the donor's serum (reverse type) [112]. Testing of donor units is conducted with automated high throughput equipment, and any discrepancies between forward and reverse type are flagged for further investigation. (See "Pretransfusion testing for red blood cell transfusion", section on 'Blood type (ABO and RhD type)' and "Pretransfusion testing for red blood cell transfusion", section on 'Antibody screen'.)

For repeat donors, the ABO group of the current donation is verified against historical computer records for accuracy. Upon receipt at the hospital, the ABO group of each unit must be verified by repeat forward typing.

●Rh type – Although RBCs have many different antigens in multiple blood group systems, the RhD antigen is the only non-ABO antigen for which all units of donated blood are routinely screened. There are many antigens of the Rh system other than D that are present on every individual's blood cells; nevertheless, the presence of the D antigen designates a unit as Rh-positive, whereas its absence is designated as Rh-negative. (See "Red blood cell antigens and antibodies".)

Other than ABO mismatches, RhD is the most immunogenic of the common RBC antigens, with approximately 40 to 80 percent of RhD-negative individuals forming an anti-D upon exposure to an RhD-positive RBC unit. Historically, anti-D is the primary cause of hemolytic disease of the fetus and newborn (HDFN), although routine screening and administration of anti-D immune globulin has reduced rates of HDFN due to anti-D. (See "RhD alloimmunization in pregnancy: Overview".)

There are other important antigens of clinical significance; these are less immunogenic than RhD, with alloantibodies developing in a minority of recipients negative for the antigen who are exposed to an antigen-positive unit. These include antigens in the Kell system (with approximately 10 percent of K-negative individuals forming anti-K upon exposure to a K-positive RBC unit), followed by lesser antibody formation rates from other Rh system antigens (C, E) as well as for certain antigens in the Duffy, Kidd, and other blood group systems [113,114]. (See "Red blood cell antigens and antibodies", section on 'Clinically significant (common)'.)

Blood centers maintain an inventory of special RBC units that have been determined to be negative for common antigens in the Rh and other common blood group systems (Kell, Duffy, Kidd). These antigen-negative units are identified by more extensive RBC phenotyping or genotyping of selected donors. The units can be made available to patients who have developed antibodies against specific RBC antigens and, if transfusion is needed, who require RBCs lacking those specific antigens. Examples of individuals more likely to have developed alloantibodies include multiply-transfused patients with sickle cell disease, thalassemia, aplastic anemia, or a myelodysplastic syndrome (MDS). (See "Red blood cell transfusion in sickle cell disease: Indications and transfusion techniques", section on 'RBC antigen matching'.)

Donor serum is also tested for unexpected RBC antibodies, which may have been formed through prior transfusion or pregnancy in the donor. For a recipient, these antibodies in the donor serum could cause a transfusion reaction if the recipient's RBCs carry the relevant antigen [112].

The antibody screen is performed using one of several different methods (solid phase, gel card, tube testing), all of which use indicator RBCs that have the common significant RBC antigen specificities. Observational data compiled from over a 4.5 year timeframe from four United States blood centers indicated that 0.5 percent of donations were antibody screen-positive and that the likelihood of a positive antibody screen increased with increasing donor age, female sex, RhD-negative status, and history of transfusion or pregnancy [115]. Components from units with a positive antibody screen will usually not be transfused, although hospital transfusion services have the option to decide whether to do so.

Sickle cell trait — Sickle cell trait is generally an asymptomatic carrier condition due to heterozygosity for the sickle cell variant in the beta globin locus. Despite routine newborn screening in the United States, many individuals are not informed of results that indicate sickle cell trait, and as a consequence many individuals with sickle cell trait are unaware that they carry the sickle cell variant. (See "Sickle cell trait", section on 'Newborn screening'.)

Most blood collectors in the United States screen some blood donors for sickle cell trait. However, there are no uniform requirements for such testing, nor is there a single standardized testing method, and policies vary across institutions. More information can be obtained by contacting individual blood collection agencies. The main reason for testing is to be able to provide recipients with sickle cell disease with blood that does not contain sickle hemoglobin. (See "Red blood cell transfusion in sickle cell disease: Indications and transfusion techniques", section on 'Overview of transfusion techniques'.)

Some blood centers use self-reported donor race to select units donated by African Americans or multiracial persons for sickle cell trait testing. Policies with regard to notification of positive test results and future donation eligibility vary across blood collectors; these include deferral from whole blood or RBC donation and restriction to platelet and/or plasma donation only. As one example, the American Red Cross screens for sickle cell trait in donors who self-identify as African American or multiracial [116]. Additional confirmatory testing is not performed, but the donor is notified of their screening results.

Another reason for sickle cell trait testing is when the leukoreduction filter becomes clogged or fails to pass quality control requirements due to inadequate removal of leukocytes; these complications are known to occur at higher rates in RBC units from donors with sickle cell trait.

SOCIETY GUIDELINE LINKS — Links to society and government-sponsored guidelines from selected countries and regions around the world are provided separately. (See "Society guideline links: Transfusion and patient blood management".)

INFORMATION FOR PATIENTS — UpToDate offers two types of patient education materials, "The Basics" and "Beyond the Basics." The Basics patient education pieces are written in plain language, at the 5^th to 6^th grade reading level, and they answer the four or five key questions a patient might have about a given condition. These articles are best for patients who want a general overview and who prefer short, easy-to-read materials. Beyond the Basics patient education pieces are longer, more sophisticated, and more detailed. These articles are written at the 10^th to 12^th grade reading level and are best for patients who want in-depth information and are comfortable with some medical jargon.

Here are the patient education articles that are relevant to this topic. We encourage you to print or e-mail these topics to your patients. (You can also locate patient education articles on a variety of subjects by searching on "patient info" and the keyword(s) of interest.)

●Basics topic (see "Patient education: Blood donation (giving blood) (The Basics)")

●Beyond the Basics topic (see "Patient education: Blood donation and transfusion (Beyond the Basics)")

SUMMARY

●Risks of specific infections – The risk of transfusion-transmitted HIV is approximately 1 in 1.6 to 1 in 1.23 million. This and other risks are summarized in the table (table 2).

●List of tests – Donated blood in the United States is tested for ABO blood group and RhD blood type, screened for the presence of red blood cell (RBC) antibodies, and screened for the following infectious agents (table 1):

•HIV-1 and HIV-2 (see 'HIV-1 and HIV-2' above)

•Human T-lymphotropic virus (HTLV)-I and HTLV-II (see 'HTLV-I and HTLV-II' above)

•Hepatitis C virus (HCV) (see 'Hepatitis C virus' above)

•Hepatitis B virus (HBV) (see 'Hepatitis B virus' above)

•West Nile virus (WNV) (see 'West Nile virus' above)

•Treponema pallidum (syphilis) (see 'Syphilis' above)

•Trypanosoma cruzi (Chagas disease) (see 'Chagas disease' above)

●CMV – Cytomegalovirus (CMV)-safe blood can be provided by screening units with antibody testing and identifying CMV-negative units, or by leukodepletion that reduces donor leukocytes to <5 million per unit of blood. (See 'Cytomegalovirus' above.)

●Babesia – An assay for Babesia microti is used in high-risk regions of the United States. (See 'Babesia microti' above.)

●COVID-19 and mpox – There is no evidence for transfusion-transmission of SARS-CoV-2, the virus that causes COVID-19, or for mpox. Research, surveillance, and careful monitoring of transfusion-transmission for emerging infectious disease agents of concern is ongoing in the United States and internationally. (See 'Emerging infectious disease agents' above.)

●ABO and RhD type – Blood grouping and typing consists of ABO and RhD testing (forward type) and antibody screening (reverse type). An inventory of special RBC units is made available for patients who have antibodies against specific RBC antigens. (See 'Routine RBC antigen and antibody testing' above and "Pretransfusion testing for red blood cell transfusion".)

●Sickle cell trait – Most blood collectors in the United States screen some blood donors for sickle cell trait. However, there are no uniform requirements for such testing, nor is there a single standardized testing method, and policies vary across institutions. More information can be obtained by contacting individual blood collection agencies. (See 'Sickle cell trait' above.)