INTRODUCTION — Safe transfusion of red blood cells (RBC) is possible because donor RBC units can be selected for their compatibility with the recipient's blood type. Transfused RBC units do not need to be antigenically identical to the recipient's RBCs, but they do need to lack antigens that could provoke clinically significant hemolysis in the recipient (a blood group O donor unit transfused to a blood group A recipient is clinically acceptable, but a blood group A donor unit transfused to a blood group O recipient may lead to a fatal hemolytic transfusion reaction).
This topic discusses the practical aspects of compatibility testing and interpretation of potential results from this testing.
Separate topic reviews provide an overview of RBC antigens and their clinical significance, management of more complex compatibility testing, practical aspects of blood transfusion, and management of hemolytic transfusion reactions. (See "Red blood cell antigens and antibodies" and "Transfusion in individuals with complex serologies on pretransfusion testing" and "Practical aspects of red blood cell transfusion in adults: Storage, processing, modifications, and infusion", section on 'Specialized modifications and products' and "Hemolytic transfusion reactions".)
TERMINOLOGY — Terms that may be helpful in accurate communication regarding desired testing and interpretation of results from compatibility testing include the following:
●AABB – The Association for the Advancement of Blood & Biotherapies (AABB) is an international organization that provides technical input and guidance on standards and accreditation for transfusion practice and cellular therapies. AABB publishes a technical manual approximately every three years and standards for blood banks and transfusion services as well as cellular therapy every two years. The organization was formerly known as the American Association of Blood Banks; the name was changed in 2021 to reflect its expanded (international) scope.
●Antiglobulin (Coombs) testing – Testing that employs a secondary antibody directed against human immunoglobulins and complement proteins to detect antibodies in patient serum that bind RBCs (figure 1) [1]. This testing is important in pretransfusion testing for identifying antibodies that could potentially cause hemolysis in the recipient. This testing takes advantage of RBC agglutination (clumping of RBCs when an antibody binds to more than one RBC). Agglutination can be assessed visually or by an automated detection system.
•Direct antiglobulin testing (DAT, also called direct Coombs testing) is used to detect antibodies and/or complement on the surface of RBCs, demonstrating in vivo RBC coating. The DAT is performed by incubating RBCs in a tube with a secondary antibody directed against human immunoglobulins and/or complement. RBCs coated with immunoglobulins and/or complement will be agglutinated by the anti-human antibody. The DAT is positive when there is an RBC autoantibody (self antibody directed against self RBCs) or an alloantibody (antibody directed against foreign RBCs).
•Indirect antiglobulin testing (indirect Coombs testing) is used to detect alloantibodies or autoantibodies present in plasma/serum. The indirect antiglobulin test is performed by adding the secondary antibody to reagent RBCs following incubation with patient plasma/serum. If the patient's plasma/serum contains antibodies to antigens on the reagent RBCs, the RBCs will be agglutinated. This testing is used both to evaluate suspected immune mediated hemolysis, similar to the DAT, as well as in compatibility testing to determine whether patient plasma/serum contains antibodies that could react with transfused RBCs. (See 'Antibody screen' below.)
●Antibody screen – Testing that detects the presence of antibodies directed against RBC antigens that may require special attention should a patient require transfusion.
●Blood type – Blood type (also called blood group) refers to ABO and RhD antigens expressed on an individual's RBCs. Blood type from a donation card or military tag is never sufficient evidence for compatibility with a specific recipient. ABO and RhD typing are required every time a red cell transfusion is ordered. (See 'ABO and RhD type' below.)
●Clinically significant antibody – Clinically significant antibodies are antibodies that could decrease the survival of transfused RBCs. In practice, these are usually antibodies that react with RBC antigens and cause hemolysis at 37°C (98.6°F), rather than those that react at room temperature. Antibodies directed against a variety of red cell antigens are implicated; the vast majority of these antibodies are readily detected by the RBC antibody screen. (See 'Antibody screen' below.)
●Crossmatch (compatibility testing) – Crossmatch or compatibility testing refers to the testing of a specific donor unit of RBCs for transfusion to the recipient. Crossmatching is generally done when there is a reasonably high chance that transfusion will be required. The extent of testing required for a crossmatch depends on the recipient's antibody screen and prior transfusion history. (See 'Compatibility testing (crossmatch)' below and "Blood donor screening: Overview of recipient and donor protections", section on 'Protection of the recipient'.)
●Emergency release – Emergency release blood (generally blood group O) is available for immediate transfusion when there is insufficient time to perform pretransfusion testing (blood type, RBC antibody screen, and compatibility testing). This differs from "massive transfusion," in which a large number of units of RBCs (often with platelets and plasma) are required to treat major bleeding. In some cases of massive transfusion, emergency release blood is used (eg, treatment of massive trauma when pretransfusion testing is incomplete); in others, it is not needed (eg, scheduled liver transplantation). (See 'Emergency release blood for life-threatening anemia or bleeding' below.)
●Forward (front) type and reverse (back) type – Forward (front) type defines ABO antigens present on the surface of red cells. Reverse (back) determines the antibodies to A and B antigens in a patient’s plasma/serum. The forward (front) type and reverse (back) type must fit set criteria in order to assign an ABO type (see 'ABO and RhD type' below). If the criteria are not met, the ABO type is reported as unresolved.
●Mixed field agglutination – Descriptive term characterizing the agglutination pattern observed when there are two populations of RBCs, one of which expresses a particular antigen and one that does not. This may be observed post-transfusion.
●Panagglutinin – Descriptive term that refers to agglutination with all reagent red cells at the same strength of agglutination. This finding suggests the presence of an autoantibody and generally requires additional testing in the blood bank.
●Type and screen – Type and screen refers to testing only on the recipient sample, including ABO and RhD type and antibody screen. A compatible unit is not identified. Type and screen is appropriate for patients who are unlikely to require transfusion but would benefit from the information should transfusion become necessary (eg, in association with surgery).
EMERGENCY RELEASE BLOOD FOR LIFE-THREATENING ANEMIA OR BLEEDING — In some urgent or emergency situations, there may be insufficient time to complete all components of pretransfusion testing (eg, life-threatening anemia, brisk hemolysis, rapid bleeding). Decisions regarding transfusion in such settings depend on assessment of the risks and benefits of immediate transfusion versus completion of pretransfusion testing, including compatibility testing. These decisions are made by the clinician caring for the patient with involvement of transfusion medicine personnel. Importantly, "emergency release" blood is always available for immediate, lifesaving transfusion.
Blood designated for emergency release is typically group O, RhD-negative. At many institutions, group O, RhD-positive RBC units may be used for males and females who are beyond childbearing age or when the usage is expected to be very high. The designated RBC units are generally stored separately from other RBC units to allow rapid access and to avoid potential mis-transfusion of non-group O blood. Specialized modifications may not be available (eg, there may not be irradiated or cytomegalovirus-safe RBC units).
Additional protocols to be followed during use of emergency release blood include written indication on the released unit stating that compatibility testing has not been completed, and written confirmation by the treating clinician that emergency release blood is required.
After the blood has been released, compatibility testing can be performed on a pretransfusion sample from the patient (if available). This is useful both for crossmatching subsequent units and for notification of the treating clinician of any unexpected results from the antibody screen and/or incompatibilities between the emergency release unit(s) and the recipient. This information is also recorded in the medical record.
The relatively low risk for hemolytic transfusion reactions in the setting of emergency release RBC transfusions (released in the setting of no or incomplete pretransfusion testing) was demonstrated in a retrospective review of 1002 emergency released RBC units transfused to 262 patients. In this study, emergency release of RBCs was associated with 0.1 percent risk of hemolytic transfusion reactions owing to pre-existing non-ABO RBC antibodies (anti-c and probable anti-Jka) [2].
SPECIMEN REQUIREMENTS
Specimen age/collection date — The blood sample used for testing must reflect the patient's blood type and the antibodies present in patient plasma at the time transfusion will be administered. These are usually constant, so there is no absolute requirement for the age/collection date of the sample in a patient who has not been exposed to foreign RBCs through recent transfusion or pregnancy.
In contrast, in patients who have been pregnant within the previous three months (or are currently pregnant); patients who have received a transfusion within the previous three months; or patients for whom the recent pregnancy and/or transfusion history is uncertain, samples for pretransfusion testing must be less than three days old. The rationale is that exposure to foreign antigens may have occurred and may have induced formation of an alloantibody. The collection day is counted as day 0, and the sample expires at midnight on day 3; so a sample collected on Monday would expire Thursday night at midnight.
Specimen collection tube — Compatibility testing can be performed on plasma, using blood drawn into a tube with EDTA; or serum, from a clotted sample. In general, plasma is preferred over serum for testing because it is anticoagulated, preventing the possibility of small clots that could interfere with the read-out. Specimen tubes with gel separator material (eg, red/black "tiger-top" tubes) should not be used for pretransfusion testing because contamination of the serum by the gel material may interfere with interpretation of agglutination [1].
In general, one 6 mL tube containing EDTA (eg, "pink top tube" or "purple top tube") is sufficient for all routine blood bank pre-transfusion testing in adults, including type and screen and compatibility testing. Smaller volumes (eg, 3 mL) are acceptable for infants. For patients with more complex serological issues (eg, multiple alloantibodies or warm autoantibodies), additional specimen tubes may be needed to complete transfusion testing. In these cases, the transfusion service will notify the clinician and request additional tubes.
Hemolysis or lipemia may interfere with interpretation of agglutination due to color and/or cloudiness of the plasma. The laboratory has specimen acceptance criteria to trigger a request for new samples for testing if this occurs.
Labeling — A properly labeled sample is critical to the safety of blood transfusion; a clerical error may lead to a fatal transfusion reaction.
●The person who draws the blood must identify the patient, preferably actively by asking the patient their name and date of birth. The tube must be labeled using at least two forms of unique patient-identifying information (eg, patient name, medical record number, date of birth); the date the blood was drawn; and identifying information for the individual who drew the blood sample.
●Labeling of the specimen must occur immediately after specimen collection.
●Information on the blood tube must match the information on the requisition for compatibility testing, and institution-specific specimen labeling policies should be followed.
●To mitigate risk of incompatible transfusion secondary to mislabeled specimens (referred to as "wrong blood in tube"), laboratories and transfusion services require two separate specimens for blood type verification [3]. Use of an electronic patient identification system is an alternative means of reducing mislabeling [4].
Mislabeled or incompletely labeled specimen tubes should be discarded and a new sample collected in order to avoid possible mis-transfusion [5]. If blood is needed urgently, emergency release blood should be requested. (See 'Emergency release blood for life-threatening anemia or bleeding' above.)
Other information — Other information important for the transfusion service includes the following:
●Indication for compatibility testing and relevant underlying conditions(s) that may need to be considered when selecting blood components for the patient, such as hematologic malignancy, hemolytic anemia, hemoglobinopathy (eg, sickle cell disease or thalassemia), pregnancy, or certain medications
●Prior transfusion history, including red cells, platelets, or plasma; hematopoietic cell transplantation, or solid organ transplantation with incompatible blood groups
●History of RBC antibodies (eg, from previous transfusion testing)
●History of transfusion reaction(s)
●For women of childbearing potential: either current pregnancy, including the approximate gestational age; prior pregnancy; or no future pregnancy possible (eg, patient has had a hysterectomy)
●Urgency of transfusion (eg, active bleeding, routine preoperative testing)
●Special modifications required such as irradiation, leukoreduction, cytomegalovirus-safe products, or other special transfusion requirements
PRETRANSFUSION TESTING — Tests that must be performed prior to release of an RBC component include ABO/RhD typing, antibody screen, and compatibility testing.
A patient sample can be tested for blood type and the presence of antibodies (ie, type and screen) without compatibility testing to a specific RBC unit for transfusion, or the type and screen can be combined with compatibility testing (ie, type and crossmatch).
●In patients who have a lower likelihood of requiring a transfusion, type and screen is a useful way to perform the initial pretransfusion testing. Once compatibility testing is performed, the donor RBC unit is reserved for a specific patient and removed from the general inventory.
●For patients with a high likelihood of requiring transfusion (anticipated surgery with high likelihood for significant blood loss) or who require immediate transfusion, compatibility testing is also performed so that a compatible RBC unit is immediately available. Any RBC unit released to the patient must be crossmatched, with the exception of emergency release blood.
ABO and RhD type — ABO and RhD typing is performed on all patient samples. It determines the presence of A, B, and/or RhD antigens on the patient's RBCs (ie, the front type) because individuals who lack any of these antigens can produce antibodies to them capable of causing severe hemolysis.
Antibodies to A and/or B are present in the plasma of the vast majority of individuals who lack these antigens (ie, the back type), even in the absence of prior transfusion, from exposure to gut bacteria that share similar epitopes (ie, molecular mimicry).
●Type O individuals lack both A and B antigens and make antibodies to both A and B. Type O individuals can receive only type O RBCs due to the presence of anti-A and anti-B in their plasma. However, they are considered universal blood donors because their RBCs lack the A and B antigens.
●Type AB individuals have both A and B antigens and do not make antibodies to A or B. They can receive RBCs of any ABO type (ie, "universal recipient").
●Type A individuals lack B antigens and make antibodies to B. They can receive type O or A RBCs.
●Type B individuals lack A antigens and make antibodies to A. They can receive type O or B RBCs.
The Rh blood group system includes several antigens, but only the RhD status is included in the blood type. The RhD blood type is often represented as the positive or negative after the ABO blood group (eg, O negative indicates the patient is blood group O and RhD negative). (See "Red blood cell antigens and antibodies", section on 'Rh blood group system'.)
●RhD-positive individuals express the RhD antigen and do not make antibodies to RhD. They can receive RhD-positive or RhD-negative RBCs.
●RhD-negative individuals do not express RhD and are easily induced to form anti-RhD antibodies through transfusion or pregnancy with an RhD positive fetus. (See "RhD alloimmunization in pregnancy: Overview".)
Females of childbearing potential are generally restricted to receive only RhD-negative red cells for transfusion to mitigate the risk for anti-RhD antibody formation and possible hemolytic disease of fetus and newborn (HDFN).
RhD-negative males and females who are beyond childbearing age may receive RhD-positive red cells in certain clinical situations (eg, massive transfusion) as long as there is no serologic evidence of an anti-RhD antibody. This serves to preserve the limited RhD-negative inventory for patients in whom alloimmunization can be clinically problematic, such as in prevention of HDFN [6].
Antibody screen
Overview of antibody screening — Antibody screening is performed on all patients who may require transfusion support. It is used to detect antibodies in patient plasma/serum that might react with antigens on transfused RBCs and cause hemolysis.
The antibody screen is performed by incubating the patient's plasma/serum at 37°C (98.6°F) with a two or three donor red cell panel of well-characterized, blood group O, "reagent" RBCs expressing combinations of commonly encountered, clinically significant RBC antigens. Antibody screening can be performed manually or on an automated system, in liquid or solid phase. Institution-specific guidelines for appropriate testing and quality controls should be used. In cases in which there is a need to deviate from standard protocols, involvement of the transfusion service is important to clarify any potential transfusion-related risks for the patient (eg, need for emergency release blood when antibody screen is incomplete).
●Alloantibodies that are always considered to be potentially clinically significant (associated with acute hemolytic transfusion reactions, delayed hemolytic transfusion reactions, or hemolytic disease of the fetus and newborn) include those directed against the following blood group systems (antigens within the system in parentheses): ABO (A, B), Rh (D, C, c, E, e), Duffy (Fya, Fyb), Kidd (Jka, Jkb), Kell (K, k), and SsU (S, s, U).
●Antibodies that are rarely or never considered to be clinically significant include those directed against: Lewis (Lea, Leb), MN, P1, Xga, Cartwright (Yta), Bg, Knops (Kna, McCa, Yka), Chido/Rodgers (Ch1/Rg1), Sda; as well as high titer low avidity (HTLA) antibodies.
Patients found to have clinically significant antibodies should be restricted to antigen-negative RBCs for all future transfusions, even if the antibodies are not apparent on subsequent routine testing. Many transfusion services send letters to patients and/or "wallet cards" alerting them to this requirement.
Screening methods — Several approaches are available for assaying the presence of an antibody; these all demonstrate the presence and degree of agglutination, but differ in the method of scoring the agglutination (figure 2). Most laboratories use a combination of methods (tube, solid phase red cell adherence [SPRC], and/or column agglutination) as the methods complement each other and all aid in the investigation of complex serologic test results.
Tube-method (liquid phase testing) — Tube (liquid phase) testing is carried out in a test tube to which an aliquot of reagent RBCs and patient's plasma/serum have been added in a specific ratio. Depending on institutional protocol, additive solutions are added to the mixture to potentiate interaction of RBC antibodies, if present in the patient's plasma/serum, with reagent RBCs. The mixture is incubated at 37°C for 15 to 30 minutes (depending on the additive solution used), and the cellular mixture is "washed" to remove additive. Following this, anti-human globulin (AHG) reagent is added to enhance detection of antibody coated RBCs. This mixture is centrifuged and assessed for RBC agglutination, which indicates the presence of antibody [1].
Various additive solutions may be used to enhance antibody detection. However, they may also enhance detection of antibodies that are not clinically significant (eg, non-pathologic cold autoantibodies). Additive solutions that may be used include the following:
●Albumin – Albumin is thought to reduce repulsive forces between cells (the Zeta potential), thereby enhancing antibody-antigen interactions.
●PEG (polyethylene glycol) – PEG is thought to promote exclusion of water molecules at the RBC surface, thereby promoting antibody-antigen interactions. PEG generally enhances sensitivity of antibody detection. However, it can enhance warm reactive autoantibodies and thus interfere with the detection of alloantibodies.
●LISS (low ionic strength saline) – LISS enhances antibody-antigen interactions by lowering the tonicity of the incubation mixture.
●Enzymes – The most commonly used enzymes include ficin and papain. These enzymes cleave sialic acid molecules from polysaccharides on the RBC surface, causing a reduction in RBC surface membrane charge, which in turn promotes antibody-antigen interactions. Enzymatic treatment must be used as an adjunct to the methods listed above because these enzymes can destroy some RBC antigens (eg, MNSs-system antigens, and Fy(a) and Fy(b) antigens) and could give misleading results if used in isolation [1].
Solid-phase red cell adherence method — The solid-phase red cell (SPRC) adherence method is similar to enzyme-linked immunosorbent assay (ELISA) methods, in that one component (antigen or antibody) is immobilized in a solid matrix affixed to a microplate well. For antibody detection, RBCs or RBC membranes with known antigens are affixed to the solid matrix. Patient plasma or serum is added to the wells and incubated, permitting antibody to interact with antigens. Following incubation and wash steps, indicator RBCs coated with anti-IgG are added. If antibodies to specific RBC antigens are present, the indicator cells will adhere and produce a disbursed pattern within the well. If no antibody is present, the indicator cells will settle to the bottom of the well as a pellet. A benefit of the SPRC adherence method is the ability to automate the antibody screening process [1].
Column agglutination — Column agglutination techniques employ glass beads or gel to separate agglutinated RBCs from non-agglutinated RBCs following incubation of patient plasma/serum with reagent RBCs. Advantages of this technique over tube-method include reduced sample size, ability to set up multiple specimens simultaneously on an automated centrifugation platform, improved objectivity of result interpretation, and stability of gel cards to allow review for up to 24 hours after testing, which is particularly helpful for questionable results [1].
RBC genotyping — An alternative approach to determining compatibility of donated blood is to perform a genotype for determinants of clinically important RBC antigens (ie, RBC genotyping). This may be especially useful in settings associated with a high rate of alloimmunization and/or confusing serologic results.
Examples include the following [7]:
●Sickle cell disease or thalassemia – These individuals often require frequent transfusions and are more likely to become alloimmunized to a number of RBC antigens [8]. (See "Red blood cell transfusion in sickle cell disease: Indications and transfusion techniques", section on 'Genetic RBC antigen typing' and "Management of thalassemia", section on 'Reduction of alloimmunization and other complications of transfusion'.)
●Complex results from antibody screen – These individuals may have a mixture of their own and donated RBCs and/or auto- and alloantibodies that make it difficult to determine the most compatible blood for transfusion. (See 'Patient with complex serologic testing precluding antibody identification' below.)
●Obstetrics – Genotyping could be helpful in resolving inconclusive or discrepant RhD typing (eg, due to a partial or weak D phenotype) or to determine fetal RBC genotype using cell-free fetal DNA in the maternal circulation. (See "RhD alloimmunization in pregnancy: Overview", section on 'D variants' and "RhD alloimmunization in pregnancy: Management", section on 'Cell-free DNA testing'.)
Generally, decisions regarding the need for genotyping to assist in transfusion management are made with the guidance of the transfusion service, with input from the clinical caregivers. This is particularly true in the case of discrepant RhD typing in women who are pregnant or of childbearing potential, in which prominent clinical societies in transfusion medicine (Association for the Advancement of Blood & Biotherapies [AABB]) and obstetrics (American College of Obstetricians and Gynecologists [ACOG]) advocate for molecular testing [9]. This approach has been shown to be cost-effective [10].
High-throughput RBC genotyping (ie, genotyping for multiple antigens using automated technology) is an evolving field. The first commercially available RBC genotyping platforms became available in Europe and the United States in 2014.
●The Precise Type HEA test uses polymerase chain reaction (PCR) to amplify DNA from nucleated blood cells followed by a bead capture array to identify genotypes for selected antigens in 11 blood groups (Rh, Kell, Kidd, Duffy, MNS, Diego, Dombrock, Colton, Lutheran, Landsteiner-Weiner, and Scianna) [11]. This test was approved by the US Food and Drug Administration (FDA) in 2014 [12].
●The ID CORE XT (BLOODChip) test uses PCR amplification of DNA from nucleated blood cells with hybridization-based assignment of single nucleotide polymorphisms that determine the genotype for selected antigens in 10 blood groups (Rh, Kell, Kidd, Duffy, MNS, Diego, Dombrock, Colton, Cartwright, and Lutheran) [13]. In a 2018 study that compared genotypes and phenotypes of 1000 samples (including 97 weak D donors), the genotype test was able to accurately predict all blood group antigens based on genotyping (100 percent sensitivity) and to accurately predict all negative results (100 percent specificity), including resolving 34 cases in which serologic testing gave a false-negative result [14]. There was one discrepancy in the identification of the "e" antigen that was resolved with bidirectional sequencing. Similar results were documented in an earlier study involving a smaller number of samples [15]. This platform was approved by the FDA in 2018 [16].
A number of other blood group genotyping tests including whole genome sequencing are under various stages of development or approval [7,17,18].
The use of genotyping may improve the availability of fully typed RBC units for patients with complex serologic issues, especially if the use of historical genotyping is incorporated (ie, using the genotyping from a previous donation by the same donor).
Individuals who use RBC genotyping for pretransfusion testing still require a crossmatch to ensure that the chosen unit is compatible. (See 'Compatibility testing (crossmatch)' below.)
Compatibility testing (crossmatch)
Overview of compatibility testing — Compatibility testing, also known as crossmatching, is required for all patients for whom a RBC unit is requested. Transfused RBC units do not need to be antigenically identical to the recipient's RBCs, but they do need to lack antigens that would provoke hemolysis by recipient alloantibodies and/or complement.
Crossmatching is performed on potentially compatible units of RBCs that have been selected based on the recipient's blood type and antibody screen. It can be done by computer matching or with serologic testing, depending on current and historical antibody results.
●For a patient with a negative antibody screen and no history of clinically significant antibodies, crossmatching typically is straightforward; a RBC unit often can be released to the recipient following an electronic or immediate spin crossmatch. (See 'Electronic crossmatch' below and 'Immediate spin crossmatch' below.)
●For a patient with a positive antibody screen or a history of RBC antibodies, additional testing that includes indirect antiglobulin testing (indirect Coombs testing) is used to ensure that RBCs in the selected unit do not express the antigen(s). (See 'IgG crossmatch (full crossmatch)' below.)
●For a patient whose antibody screen appears inconsistent with the results of the crossmatch, additional testing may be needed to clarify which units are safest for transfusion. (See 'Interpretation of pretransfusion testing results' below.)
The likelihood of finding a potentially compatible unit depends on the frequency at which the implicated antigen(s) are found in the donor population. It is often more challenging to find compatible blood for individuals with multiple alloantibodies. (See "Red blood cell antigens and antibodies".)
During the 2019-2020 coronavirus disease (COVID-19) pandemic, the AABB issued tips for extending the blood supply that include aspects of crossmatching to make more units available to more individuals [19]. (See "Indications and hemoglobin thresholds for red blood cell transfusion in the adult", section on 'Extending the blood supply'.)
Electronic crossmatch — In some institutions, the electronic (computer) crossmatch (EXM) is performed instead of an immediate spin (IS) XM; this can only be performed if the patient has been ABO-typed on two separate occasions and has no clinically significant antibodies on the current antibody screen and no history of a clinically significant antibody, and institutional informatics and quality controls are available to support accurate electronic matching. In EXM, a computer algorithm compares the recipient's previous and current transfusion testing. If no discrepancies are found, the computer system allows release of an ABO and RhD compatible RBC unit. This may be completed in only a few minutes.
Quality controls imbedded in the computer-driven selection algorithm must include checking for ABO, RhD typing of at least two patient samples, warnings of ABO-type discrepancies, and antibody screen results (both current and historical) [20]. The criteria for using EXM are outlined in the guidance from the US Food and Drug Administration (FDA) and the AABB [21]. The electronic crossmatching algorithm must also allow specification of special attributes of the selected unit (eg, CMV negative, leukoreduction). (See "Practical aspects of red blood cell transfusion in adults: Storage, processing, modifications, and infusion", section on 'Specialized modifications and products'.)
Advantages of EXM are savings in cost, faster turnaround time, and reduced labor for laboratory personnel once familiarity with the system has been established. However, if an EXM system has not been properly validated or laboratory personnel have not been adequately trained, the risks of releasing incompatible RBC units may be increased. The EXM is standard practice at large hospitals throughout the United States.
Immediate spin crossmatch — The immediate spin crossmatch (IS XM) is performed using patient plasma/serum and RBCs from a potentially compatible RBC unit. This test is done at room temperature and takes only a few minutes. Patient plasma/serum and donor RBCs are mixed in a tube, gently centrifuged, resuspended, and analyzed visually for hemolysis (red serum) and RBC agglutination (failure of RBCs to re-disperse upon gentle shaking). Hemolysis or agglutination during the IS XM suggests the presence of an IgM antibody that can fix complement or bind multiple RBCs, which is concerning for ABO mismatch.
●For patients with a negative antibody screen, the IS XM is often sufficient to verify ABO compatibility. If the IS XM is negative, the selected RBC unit can be released.
●For patients with a positive antibody screen, the IS XM is followed by an IgG crossmatch (see below) to ensure that the donor RBCs lack the antigen to which recipient antibodies are directed.
Benefits of using IS XM without further testing in appropriate patients include savings in turnaround time, cost, and labor. Potential risks include the possibility that the recipient has IgG antibodies to a rare (low frequency) RBC antigen in the donor unit, which could cause an acute or delayed hemolytic transfusion reaction. The likelihood of this possibility is less than 1 in 10,000 to 1 in 100,000 [22].
IgG crossmatch (full crossmatch) — An IgG crossmatch must be performed in the following situations:
●Positive antibody screen for clinically significant antibody (see 'Antibody screen' above)
●History of a clinically significant antibody (either allo or autoantibody)
●Positive immediate spin crossmatch (see 'Immediate spin crossmatch' above)
The IgG crossmatch can use one of several variations of an indirect antiglobulin test (indirect Coombs test), in which patient plasma/serum is added to donor RBCs, warmed to body temperature (37°C [98.6°F]), incubated for 15 to 30 minutes, washed, treated with an antibody to human IgG, and inspected for hemolysis or agglutination.
Modifications may include the use of low ionic strength sodium (LISS), polyethylene glycol (PEG), or other methods to increase the sensitivity of antibody detection. (See 'Tube-method (liquid phase testing)' above.)
The IgG crossmatch may take two or more hours to complete; the amount of time depends on the complexity of the patient's serologic test results.
INTERPRETATION OF PRETRANSFUSION TESTING RESULTS — Interpretation of blood type, antibody screen, and crossmatching is straightforward and relies on macroscopic detection of antibody-antigen interactions. In the case of tube-method and column agglutination, antibody-antigen interactions lead to RBC agglutination, which appears as a non-dispersible precipitate in tube testing and impedance in the passage of light through gel/beads in column agglutination (figure 2). For solid-phase testing, positive results appear as disbursement of RBCs within a microwell.
However, there are situations in which results of any of the blood bank tests may be more challenging to interpret [23].
ABO-type discrepancies — ABO type discrepancies occur when the RBC "front-type" is inconsistent with the plasma "back-type." This can be a consequence of massive transfusion (eg, type A recipient who receives more than five units of type O RBCs during trauma resuscitation); waning antibody titers in the setting of an underlying medical condition that impairs antibody production (eg, type O patient with hematologic malignancy with undetectable anti-A and/or anti-B titers) [1]; or ABO-incompatible hematopoietic cell transplant (eg, type O patient who receives a stem cell product from a type A donor). In situations of ABO-type discrepancies, type O RBCs and type AB plasma should be selected for transfusion.
Antibody screen — A "positive auto-control" is a potentially confounding finding in the antibody screen that results when the patient's plasma/serum causes the patient's own saline-washed RBCs to agglutinate in the absence of any foreign RBCs or plasma. This may be seen in the following settings:
●Patient is experiencing a hemolytic transfusion reaction (see "Hemolytic transfusion reactions")
●Patient was transfused with plasma or platelets containing an antibody that reacts with patient RBCs (see 'Massive transfusion' below)
●Patient has a RBC autoantibody (eg, autoimmune hemolytic anemia) (see 'Autoimmune hemolytic anemia' below)
●Patient has a condition that causes rouleaux formation (eg, multiple myeloma), which is mistaken for agglutination in the reaction tube (see "Multiple myeloma: Clinical features, laboratory manifestations, and diagnosis", section on 'Peripheral smear')
Incompatible crossmatch — A negative antibody screen followed by an incompatible crossmatch with selected RBC units may occur in the following settings:
●Unit is ABO matched, but differs at certain within-class ABO antigens (eg, A2 individual produces anti-A1)
●Unit is compatible, but the recipient or the donor has an RBC autoantibody (eg, autoimmune hemolytic anemia) (see 'Autoimmune hemolytic anemia' below)
●Unit is incompatible because recipient has an alloantibody to an antigen on donor RBCs that was missed during antibody testing (eg, reagent RBCs were heterozygous for the antigen but donor unit is homozygous for the antigen)
●Unit is incompatible because the recipient has an antibody to an extremely rare (low prevalence) antigen that was not present on the reagent RBCs used for the antibody screen
Evaluation and interpretation of compatibility testing in these settings depends on the patient's underlying condition, and management depends on the likely explanation(s) for the unexpected result. These issues and our approach to their management are discussed in more detail separately. (See "Transfusion in individuals with complex serologies on pretransfusion testing".)
Potential confounders
Autoimmune hemolytic anemia — Patients with autoimmune hemolytic anemia (AIHA) have circulating autoantibodies directed against their own RBCs. While the primary treatment involves reducing or eliminating the autoantibody (eg, using immunosuppression with a glucocorticoid), patients may have anemia severe enough to require transfusion before therapy becomes effective. (See "Warm autoimmune hemolytic anemia (AIHA) in adults", section on 'Initial management'.)
The presence of RBC autoantibodies may make interpretation of antibody screen and compatibility testing results more challenging because the antibodies are often detectable in the test system and may mask the presence of an alloantibody. Approaches to circumvent these confounding antibodies include use of techniques that remove the autoantibody (autoadsorption or heterologous adsorption), or alternative additive solutions that are less likely to detect the autoantibody (eg, LISS or saline). In either case, testing for clinically significant alloantibodies in a patient with autoantibodies requires more time than routine testing. In some cases, it may be necessary to refer testing to an outside reference laboratory, and the evaluation may take up to many hours to a few days. Some institutions will also only transfuse phenotypically matched RBCs to all clinically significant antigens to avoid missing potential alloantibodies.
Massive transfusion — Massive transfusion involves transfusion of multiple RBC units over a short period of time, with or without plasma and platelet transfusions. Various specific definitions have been developed, as discussed separately. (See "Massive blood transfusion".)
Massive transfusion replaces a significant portion of the patient's blood volume with donor blood. In this setting, a patient sample obtained prior to initiating transfusion will accurately reflect the patient's blood type at the start of the massive transfusion, but subsequent samples may not reflect blood type or alloantibodies accurately, depending on the ABO type of the RBCs and plasma selected for emergency resuscitation. For example, if a trauma protocol initiates transfusion with type O RBCs and AB plasma, a patient who is type A may no longer front- or back-type as a type A due to dilution of circulating RBCs by transfused type O red cells, and the endogenous anti-B by transfusion of AB plasma (devoid of anti-A and anti-B). If a pretransfusion specimen was not drawn prior to initiation of resuscitation, the transfusion service is obligated to continued release of type O red cells and type AB plasma, which can pose a significant drain on precious resources. In order to avoid this situation, many institutional trauma resuscitation policies mandate immediate collection of patient specimen for ABO and RhD typing so that type-specific blood can be released for resuscitation.
Comprehensive management of patients undergoing massive transfusion is discussed in detail separately. (See "Massive blood transfusion".)
Out-of-group platelet transfusion — Non-type O patients requiring platelet transfusion frequently receive ABO incompatible platelets. Since platelet products may contain up to 500 mL of plasma, such transfusions, particularly in the setting of multiple out-of-group transfusions, may lead to coating of endogenous RBCs with passively acquired anti-A or anti-B. In some cases, the amount of circulating anti-A or B may interfere with crossmatching. This can be circumvented by restricting patients to group O red cells for transfusion. In rare cases, passively acquired antibodies have led to hemolytic transfusion reactions [24]. In response to these reports, many transfusion services have initiated the practice of antibody titration of donor platelet components; if components are found to contain high titer antibodies to RBC antigens, the products are restricted to group O recipients.
Other transfusion services have initiated the practice of limiting the quantity of out-of-group plasma transfusion to only 300 to 600 mL every 24 hours.
Drugs and therapeutic antibodies — Certain drugs or therapeutic antibodies may confound interpretation of the antibody screen, the direct antiglobulin test, or both. In some cases, these findings are of clinical significance (ie, associated with hemolysis); in others they are not.
Drug-induced antibodies — Several antibiotics including penicillin, ampicillin, and many cephalosporins, as well as several chemotherapeutic agents have been associated with drug-induced RBC antibody formation, through a hapten-mediated or other mechanism (figure 3). Examples of drugs associated with drug-antibody immune complexes that bind non-covalently to the RBC member include quinidine and ceftriaxone [25,26]. A list of implicated drugs is provided in the table (table 1). Patients may present with evidence of hemolysis and a positive direct antiglobulin test (positive for IgG, C3, or both). Detection of the serum antibodies requires testing with drug-treated reagent RBCs or inclusion of the drug in the antibody screening step. Hemolysis generally abates upon discontinuation of the offending drug; future use of the offending medication should be avoided [27]. (See "Drug-induced hemolytic anemia", section on 'Immune-mediated'.)
Anti-D immune globulin — Anti-D immune globulin (also called Rho[D] immune globulin) is used for immune prophylaxis in RhD-negative females during pregnancy to prevent sensitization. It may also be used therapeutically to treat immune thrombocytopenic purpura (ITP) in patients who are RhD-positive. As a consequence of passive immunization, the presence of circulating anti-D may be detected on routine antibody screening for up to 12 weeks following administration of the anti-D immune globulin. For both patient populations it is important to seek information regarding recent administration of anti-D immune globulin to allow for correct interpretation of serologic test findings [1]. (See "RhD alloimmunization in pregnancy: Overview".)
IVIG — Intravenous immune globulin (IVIG) products may contain sufficiently high enough titers of antibodies to ABO antigens (anti-A and anti-B) to lead to a positive direct antiglobulin test in blood type A or B individuals, respectively. There are numerous case reports in the literature reporting on clinically significant hemolysis as a consequence of passive sensitization by these antibodies. At-risk clinical scenarios include administration of high dose IVIG (eg, 1 to 2 g/kg over two to seven days) to blood type A or B patients [28,29].(See "Intravenous immune globulin: Adverse effects", section on 'Hemolysis'.)
Anti-CD38 mAbs (daratumumab and isatuximab) — CD38 is a membrane-bound enzyme that degrades nicotinamide adenine dinucleotide (NAD), which serves as a cofactor (an electron donor) in redox reactions and is involved in calcium signalling; it is also highly expressed on the surface of multiple myeloma cells [30]. Daratumumab (DARA) and isatuximab are human monoclonal antibodies (mAbs) directed against CD38 that may be administered for the treatment of multiple myeloma. (See "Multiple myeloma: Treatment of first or second relapse" and "Multiple myeloma: Treatment of first or second relapse", section on 'Daratumumab, bortezomib, dexamethasone (DVd)'.)
Because all RBCs express CD38, the presence of these antibodies in patient plasma can result in pan-reactivity against reagent RBCs used in antibody screening (panagglutination), which can mask a clinically significant alloantibody. (See 'Terminology' above.)
The generally accepted approach to patients who have received (or will receive) an anti-CD38 mAb and require (or are likely to require) transfusions includes the following:
●Obtain a sample for the blood bank to perform a type and antibody screen prior to administration of the anti-CD38 therapy, and inform the blood bank that anti-CD38 treatment is planned. Many institutions have a reminder system (eg, pharmacy alert) to make sure this happens the first time the anti-CD38 agent is ordered for a patient.
The pre-treatment sample can be used to determine whether any allo- or autoantibodies are present and will facilitate matching for ABO, Rh, and Kell for any future transfusions.
●If an RBC transfusion is needed, notify the blood bank that the individual has received an anti-CD38 mAb. They will review the results of the baseline (pre-anti-CD38) sample and the current pretransfusion sample to determine whether anti-CD38 is interfering with the antibody screen.
•A negative antibody screen suggests there is no interference by the anti-CD38 mAb. Any previously identified, clinically significant alloantibodies should be respected, as noted above. (See 'Antibody screen' above.)
•A panagglutinin suggests the anti-CD38 mAb is interfering with the antibody screen.
If a panagglutinin is present, the interference can be circumvented through various methods [31,32]:
●Use of dithiothreitol (DTT) or low ionic strength saline (LISS) to eliminate interference by anti-CD38.
•DTT is a reducing agent that breaks disulfide bonds and destroys the CD38 antigen present on red blood cells [33].
DTT also destroys the Kell antigen; if DTT-treated reagent red cells are used in antibody identification, it is not possible to exclude the presence of anti-K1. Thus, K1-negative units should be provided (unless the individual is known from previous testing to express Kell on their own RBCs) [34].
•LISS decreases the tonicity of the antibody screen solution and in some cases eliminates the interference by anti-CD38.
●For daratumumab, add daratumumab F(ab) fragments to the antibody screen [35]. These fragments bind to CD38 and block binding by free daratumumab in patient serum. When tested in a gel-based antibody detection system using several samples with known clinically relevant antibodies to RBC antigens, this reagent prevented daratumumab interference and allowed detection of the clinically important anti-RBC antibodies. This approach to mitigating interference by therapeutic mAbs is promising, and similar reagents could be produced for other anti-CD38 mAbs; however, broad application awaits commercial availability of the relevant F(ab) fragments and the validation with other RBC antibody detection systems.
The choice among these methods is determined by the local blood bank or testing laboratory. Samples can be sent to a reference laboratory if these methods are unavailable.
The likelihood of a panagglutinin in an individual treated with anti-CD38 therapy may be high (91 of 91 patients in one report) [31]. Among 65 who also had a pre-anti-CD38 treatment sample obtained, 5 (8 percent) had a preexisting alloantibody, perhaps due to the high likelihood of prior transfusion in this population.
The effect of anti-CD38 antibodies can potentially last as long as six months following administration, although there is no well-established typical range.
Anti-CD47 (Hu5F9-G4) — Hu5F9 is a humanized mAb directed against CD47, a cell surface molecule on hematopoietic stem cells and some tumor cells that protects them from phagocytosis (ie, a "don't eat me" signal). This therapy is under investigation for treatment of refractory non-Hodgkin lymphoma and hematopoietic cell transplantation. (See "Diffuse large B cell lymphoma (DLBCL): Suspected first relapse or refractory disease in medically-fit patients" and "Hematopoietic cell transplantation for severe combined immunodeficiencies", section on 'Nonchemotherapy-based myeloablation'.)
CD47 is an integral glycoprotein associated with the Rh protein complex, and is highly expressed on RBCs [36,37]. Consequently, its presence in patient plasma can interfere with both ABO typing of non-group O patients (ie, interferes with reverse typing) (see 'Terminology' above) and antibody screening (panagglutination).
Unlike the interference caused by daratumumab, mitigation of interference by Hu5F9-G4 is not readily resolvable. Thus, it is imperative for transfusion services to develop robust algorithms for serologic evaluation of this patient population including baseline testing and consideration for extended red cell phenotyping and/or genotyping.
SPECIAL POPULATIONS
Infant <4 months — Infants under four months of age have limited antibody production and thus are less likely to develop alloantibodies to RBC antigens. However, maternal antibodies introduced into the fetal circulation prior to birth may still be present. Approaches to testing and identifying compatible units are presented separately but generally include transfusing only group O, RhD-negative RBCs. (See "Red blood cell transfusion in infants and children: Selection of blood products", section on 'Compatibility testing (crossmatching)'.)
Pregnancy — Challenges in the management of pregnant women include the risk(s) of severe bleeding due to placental or postpartum hemorrhage; the possibility of alloimmunization during the current or previous pregnancy; and the desire to avoid alloimmunization to prevent hemolytic disease of the fetus and newborn. These issues and associated management recommendations are discussed in detail separately. (See "RhD alloimmunization in pregnancy: Overview" and "RhD alloimmunization: Prevention in pregnant and postpartum patients" and "RhD alloimmunization in pregnancy: Management" and "Management of non-RhD red blood cell alloantibodies during pregnancy".)
Sickle cell disease — Individuals with sickle cell disease may require frequent transfusions, and are more likely to develop alloantibodies than in the general population [8]. The use of more extensive crossmatching for this population is discussed in detail separately. (See "Red blood cell transfusion in sickle cell disease: Indications and transfusion techniques", section on 'Transfusion techniques'.)
Patient with multiple alloantibodies — On occasion, it may be challenging to meet transfusion needs of a patient with multiple alloantibodies. As an example, if a patient who has anti-c, anti-E, anti-Jka, anti-Fya, anti-S, and anti-K presents with a gastrointestinal bleed, it may not be possible to find a sufficient number of units to meet transfusion requirements (in this case only 2 percent of ABO compatible red cells are antigen compatible given the alloantibodies present). In such cases, one would need to consider the hemolytic potential of each antibody and the current antibody titers. Any decisions regarding selection of antigen positive RBCs in the setting of detectable antibody should be made with the guidance of the blood bank medical director following discussion with the medical team caring for the patient.
Patient with complex serologic testing precluding antibody identification — For some patients found to have positive antibody screens, determining antibody specificities can be difficult if not impossible. In such cases, genotyping may provide an alternative method for identifying patient RBC antigens and determining unequivocally which antigens are not present. (See 'RBC genotyping' above.)
Hematopoietic cell transplant recipient — Allogeneic hematopoietic cell transplantation (HCT) can be carried out using donor hematopoietic cells of a different blood type from the recipient. Compatibility is categorized as follows:
●Major ABO-incompatible transplant – Recipient has antibody directed against donor red blood cells (RBCs)
●Minor ABO-incompatible transplant – Donor has antibody directed against recipient RBCs
●Bidirectional incompatibility – Combination of major and minor incompatibility
In some cases, the recipient may develop transient RBC chimerism, resulting in mixed field agglutination with ABO-typing (reflecting circulation of donor derived RBCs and residual recipient RBCs, as well as transfused type O RBCs). Eventually, the recipient will convert to the donor RBC type following stem cell engraftment. The immediate post-transplant period raises complex issues for compatibility testing and selection of RBCs and sometimes other components. The goal of component selection in this setting is to minimize risks of RBC hemolysis and to optimize hematopoiesis from donor cells [1]. Recommendations for selection of RBCs, platelets, and plasma are listed in the table (table 2).
Solid organ transplant recipient — In general, solid organ transplant is carried out using ABO identical donor organs. However, similar to hematopoietic cell transplantation "minor ABO-incompatible" solid organ transplants can be performed (eg, group O donor organ transplanted to non-group O recipient). In some cases of minor ABO-incompatible solid organ transplant, transient RBC hemolysis may be observed. This is caused by the production of anti-A or anti-B by donor lymphocytes (passenger lymphocytes) that accompany the transplanted organ. If hemolysis due to anti-A or anti-B is brisk, necessitating RBC transfusion, donor-type RBCs (eg, group O RBCs) may be used [38]. Hemolysis due to passenger lymphocytes is transient, generally abating within a month following transplant following clearance of donor lymphocytes.
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 5th to 6th 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 10th to 12th 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 transfusion (The Basics)")
●Beyond the Basics topic (see "Patient education: Blood donation and transfusion (Beyond the Basics)")
SUMMARY AND RECOMMENDATIONS
●Definitions – Terminology used in compatibility testing is presented above. (See 'Terminology' above.)
●Emergency release blood (no time for testing) – In urgent/emergency situations, time may be insufficient to perform full compatibility testing (eg, life-threatening anemia, brisk hemolysis, rapid bleeding). Decisions regarding transfusion in such settings depend on assessment of the risks and benefits of immediate transfusion versus full compatibility testing. Importantly, "emergency release" blood is always available for immediate lifesaving transfusion. Blood designated for emergency release is typically group O. (See 'Emergency release blood for life-threatening anemia or bleeding' above.)
●Recipient blood specimen – It is critically important to ensure the specimen received for compatibility testing is from the correct patient and reflects the patient's blood type and the antibodies present in patient plasma at the time transfusion will be administered. A properly labeled tube containing patient serum or plasma obtained within three days is appropriate for most patients; those without exposure to foreign RBCs can use an older specimen in accordance with blood bank policy. Additional information regarding prior transfusions, transfusion reactions, allogeneic hematopoietic cell or solid organ transplantation, special modifications required, and pregnancy history can be very helpful to the transfusion service and may prevent hemolytic transfusion reactions. (See 'Specimen requirements' above.)
●Type and screen for low risk patients – Patients with a lower likelihood of requiring a transfusion can have a blood type and antibody screen performed without a crossmatch. This includes ABO and RhD typing as well as a screen for antibodies capable of causing hemolysis (figure 1 and figure 2), including those of the ABO, Rh, Duffy, Kidd, Kell, SsU, and Lutheran blood group systems. RBC genotyping is an alternative approach to determining compatibility of donated blood that may be especially useful in settings associated with a high rate of alloimmunization and/or confusing serologic results. (See 'ABO and RhD type' above and 'Antibody screen' above and 'RBC genotyping' above.)
●Crossmatching – Crossmatching is performed when a crossmatch or release of an RBC unit is requested. (See 'Compatibility testing (crossmatch)' above.)
•In a patient with a negative antibody screen and no history of clinically significant antibodies, an RBC unit often can be released to the recipient following a negative electronic or immediate spin crossmatch. (See 'Electronic crossmatch' above and 'Immediate spin crossmatch' above.)
•In a patient with a positive antibody screen or a history of RBC antibodies, additional testing that includes indirect antiglobulin testing (indirect Coombs testing) is used to ensure that RBCs in the selected unit do not express the antigen(s). (See 'IgG crossmatch (full crossmatch)' above.)
●Interpretation – For some patients, interpretation of the results of the antibody screen and/or crossmatching may be more challenging, and additional testing may be needed. Potential confounding conditions include autoimmune or drug-induced hemolytic anemia, hemolytic transfusion reaction, recent transfusion of ABO incompatible plasma or platelets, or massive transfusion. (See 'Interpretation of pretransfusion testing results' above.)
●When is additional testing needed – Additional aspects of pretransfusion testing and crossmatching may apply to certain patients, including infants less than four months of age, pregnant individuals, individuals with sickle cell disease, those treated with certain drugs and therapeutic antibodies, and those undergoing hematopoietic cell transplantation (table 2). (See 'Special populations' above.)
ACKNOWLEDGMENT — The UpToDate editorial staff acknowledges extensive contributions of Arthur J Silvergleid, MD to earlier versions of this and many other topic reviews.
UpToDate staff would also like to acknowledge David W Cohen, MA, MT(ASCP)SBB, who also contributed to earlier versions.
