Donated blood is subjected to a rigorous series of tests before it is deemed safe for transfusion. The list of tests performed by the Red Cross seems ever expanding, and this has resulted in an overall safer blood supply and fewer transfusion-associated risks. Blood testing consists of screening for infectious disease as well as ABO screening, Rh screening, and testing for various red-cell antibodies.

Infectious disease screening is accomplished two ways. An initial level of screening is by self-report of previous diseases and living or travel situations. Since March 2005, all blood donors in the United States must complete a 54-question survey called the Universal Donor Survey. For example, donors are asked if they have had oral surgery in the last 3 days (to eliminate the possibility of transient bacteremia of mouth pathogens) or have ever had certain blood-borne infections such as malaria or babesiosis (both intracellular erythrocyte parasites). They are also queried about residence in Europe during the interval that may increase the epidemiologic risk for bovine spongiform encephalopathy. Affirmative answers on the test can disqualify the donor temporarily or permanently.

Serum tests for infectious disease entities include HIV-1 and -2, hepatitis C (HCV), hepatitis B (HBV), human T-lymphocyte virus (HTLV)-I and-II, West Nile virus, and Treponema palladium (syphilis). Alanine aminotransferase is also tested as a laboratory marker of infectious disease. All viruses are initially tested for by enzyme-linked immunoassay (ELISA). An initial positive result is then rechecked in two separate ELISA tests, and if one of those two subsequent tests is also positive, the blood is discarded. Only if both are negative is the blood taken out of special quarantine and subjected to further testing.

A new method of testing blood was started in 1999. Nucleic acid testing or NAT is used to screen blood for the presence of HIV, HCV, and, as of 2003, West Nile virus. Nucleic acid testing is done by polymerase chain reaction (PCR) or transcription-mediated amplification (TMA). As both of these are fairly expensive tests, most commonly nucleic acid testing is done on a minipool sampling. This involves pooling multiple donations, and if an infection is present, then all of the individual donations in the pool are tested. This supersensitive assay is responsible for detecting HIV an average
of 11 days earlier than other forms of testing, and in concert with ELISA and Western blot tests for anti-HIV antibodies, has decreased the risk of transfusion-related infection to 1 in 2 million in the United States, 1 in 5 million in Germany, and 1 in 10 million in Canada. HCV infection has been dramatically reduced using nucleic acid testing, from 1 in 100,000 in 1996 to 1 in 2 million in the United States in 2005. Nucleic acid testing results in a decrease from an average of a 70-day window for undetectable infection using ELISA screening for HCV antibodies to an 8- to 10-day window of undetectable infection.

Blood infected with HBV is screened for by hepatitis B surface antigen and core antigen testing. The latency period of about 60 days results in a higher amount of undetected virus in the donor pool and thus HBV infection risk remains the highest among the diseases tested for—estimates vary from 1 in 60,000 to 1 in 270,000. HTLV-I and -II are screened for by ELISA and confirmed by Western blot or PCR. The resulting infection rate is estimated at 1 in 2 million for HTLV. Syphilis is also screened for by a number of serologic assays varying from lab to lab using highly sensitive specific antigens for T. palladium. Finally, cytomegalovirus (CMV) is screened for only in a select number of cases, because of the need for CMV-negative blood for transplant recipients and the immunosuppressed population, especially HIV-infected individuals.