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Pretransfusion testing for red blood cell transfusion

Pretransfusion testing for red blood cell transfusion
Author:
Lynne Uhl, MD
Section Editor:
Aaron Tobian, MD, PhD
Deputy Editor:
Jennifer S Tirnauer, MD
Literature review current through: Sep 2023.
This topic last updated: Sep 14, 2023.

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. This topic discusses the practical aspects of pretransfusion testing and compatibility testing that facilitate transfusion.

Separate topic reviews discuss:

RBC antigens and their clinical significance – (See "Red blood cell antigens and antibodies".)

Management of more complex compatibility testing – (See "Red blood cell (RBC) transfusion in individuals with serologic complexity".)

Indications for transfusion

Children – (See "Red blood cell transfusion in infants and children: Indications".)

Adults – (See "Indications and hemoglobin thresholds for red blood cell transfusion in the adult".)

Practical aspects of blood transfusion

Children – (See "Red blood cell transfusion in infants and children: Administration and complications".)

Adults – (See "Practical aspects of red blood cell transfusion in adults: Storage, processing, modifications, and infusion".)

Evaluation and management of hemolytic transfusion reactions – (See "Hemolytic transfusion reactions".)

Blood donor screening and laboratory testing – (See "Blood donor screening: Medical history" and "Blood donor screening: Laboratory testing" and "Blood donor screening: Overview of recipient and donor protections".)

EMERGENCY RELEASE BLOOD FOR LIFE-THREATENING ANEMIA OR BLEEDING

Always available – Emergency release blood (generally blood group O, RhD-negative) is always available for immediate transfusion when needed for any reason, including insufficient time to determine blood type or perform an antibody screen or compatibility testing, or an incompatible crossmatch. Potentially life-saving transfusion should never be withheld from a patient with life-threatening anemia, brisk hemolysis, rapid bleeding, or other requirement for immediate transfusion.

Group O, RhD-positive – At many institutions, emergency release RBC units are group O, RhD-positive 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. Some institutions stock group O, RhD-negative units for females of childbearing potential. Specialized modifications may not be available (eg, there may not be irradiated or cytomegalovirus-safe emergency release RBC units).

Decision to use – The decision to use emergency release blood depends on clinical judgment of the treating clinician with input from transfusion medicine personnel; these individuals who are caring for the patient and performing the laboratory testing are best equipped to weigh the risks and benefits of immediate transfusion versus completion of the pretransfusion testing.

The relatively low risk for hemolytic transfusion reactions with emergency release RBC transfusions (released with no or incomplete pretransfusion testing) was demonstrated in a retrospective review of 1002 emergency released RBC units transfused to 262 patients, which found a 0.1 percent risk of hemolytic transfusion reactions owing to pre-existing non-ABO RBC antibodies (anti-c and probable anti-Jka) [1].

Protocols to follow – A pretransfusion sample can be obtained when possible to facilitate later testing for additional transfusions.

There should be a written indication on the released unit stating that compatibility testing has not been completed, as well as 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 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.

Massive transfusion – Emergency release differs from massive transfusion, in which a large number of RBC units (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 "Massive blood transfusion".)

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/serum 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. Some institutions may accept a specimen up to 14 or 30 days before surgery from a non-pregnant individual who has not been transfused within the prior three months for preoperative screening.

However, if information about pregnancy or prior transfusion within the prior three months is not documented on the order, a more current specimen (<3 days old) is required. The following individuals require a specimen <3 days old:

Pregnant within the previous three months (or are currently pregnant)

Received a transfusion within the previous three months

Uncertain/unknown history of recent pregnancy and/or transfusion history

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.

The rationale is that exposure to foreign antigens may have occurred and may have induced formation of an alloantibody.

Plasma or serum collection tube — Compatibility testing can be performed with plasma or serum.

Plasma – "Pink top" or "purple top" collection tube containing EDTA (ethylenediaminetetraacetic acid)

Serum – "Red top" tube for clotted sample

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 [2]. (See 'Definitions (front and back type, Coombs test, agglutination)' below.)

Plasma is generally preferred over serum for testing because it is anticoagulated, reducing the possibility of small clots that could interfere with the read-out of agglutination. (See 'Definitions (front and back type, Coombs test, agglutination)' below.)

Volume – One 6 mL tube is typically 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 or individuals who are severely anemic. For patients with more complex serological issues (multiple alloantibodies or warm autoantibodies), additional specimen tubes may be needed to complete pretransfusion testing. In these cases, the transfusion service will notify the clinician and request additional tubes. (See "Red blood cell (RBC) transfusion in individuals with serologic complexity".)

Sources of interference – 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; clerical error may lead to a fatal transfusion reaction. (See "Approach to the patient with a suspected acute transfusion reaction", section on 'Acute hemolytic transfusion reaction (AHTR)'.)

Phlebotomist – The person who draws the blood must identify the patient, preferably 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.

Label – 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.

Quality control – To mitigate risk of incompatible transfusion secondary to mislabeled specimens (referred to as "wrong blood in tube"), many 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 accidental transfusion of the wrong unit to the wrong patient [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, which is generally available from the medical record, the treating clinician, or a blood bank to blood bank communication (for patients who are transferred):

Indication and urgency – Reason 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. This may affect urgency and extent of testing as well as specific modifications. (See "Practical aspects of red blood cell transfusion in adults: Storage, processing, modifications, and infusion", section on 'Specialized modifications and products'.)

Prior transfusion history – Includes RBC, platelet, or plasma transfusion, as well as hematopoietic stem cell transplantation or solid organ transplantation with incompatible blood groups. This helps identify risks of alloantibodies. History of prior transfusion reactions is also important. If transfusion occurred at another institution, it is helpful to provide the name of the institution so that they can be contacted. Some countries are working on maintaining a national database of this information.

History of RBC alloantibodies – Obtained from previous transfusion testing. This will be available in the local institution, but results from other institutions may not be available, especially in the United States. Many hospital blood banks will call the other hospital laboratories or reference laboratories where the patient's samples have been previously evaluated to ensure all alloantibodies are known.

Reproductive status – Includes whether the person has ever been, is, or may become pregnant. For current pregnancy, includes the approximate gestational age. This information is relevant to the risk of hemolytic disease of the fetus and newborn.

Special modifications required – Information about the need for irradiation is critical to avoid transfusion-associated graft-versus-host disease (TA-GVHD), which is almost uniformly fatal; need for cytomegalovirus-safe products should also be specified. (See "Transfusion-associated graft-versus-host disease", section on 'Risk factors'.)

The need for leukoreduction may be specified, but most institutions use universal leukoreduction. (See "Practical aspects of red blood cell transfusion in adults: Storage, processing, modifications, and infusion", section on 'Pre-storage leukoreduction'.)

Other special transfusion requirements such as volume reduction may apply. (See "Practical aspects of red blood cell transfusion in adults: Storage, processing, modifications, and infusion", section on 'Specialized modifications and products'.)

How is it regulated — Regulations can be complex and can differ by country or jurisdiction.

CMS – Centers for Medicare & Medicaid Services (CMS) regulates all clinical laboratory testing performed on humans in the United States through the Clinical Laboratory Improvement Amendments (CLIA) [6].

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 and then simply "AABB"; the name was changed in 2021 to reflect its expanded (international) scope. Sites around the world can become AABB-accredited such that they meet standards set by the AABB.

CAP – The College of American Pathologists (CAP) is a membership organization that fosters and advocates for best practices in pathology and laboratory medicine.

TJC – The Joint Commission (TJC) assesses and accredits health care organizations.

On behalf of CMS in the United States, the AABB, CAP, and TJC can inspect transfusion services and cell therapies. Institutions can choose which of these organizations participate in these inspections.

US FDA – In the United States, review, approval, and regulatory oversight of instrumentation and laboratory information systems used in donor and patient testing are under the purview of the US Food and Drug Administration (FDA).

SEROLOGIC TESTING (TYPE AND SCREEN)

Definitions (front and back type, Coombs test, agglutination)

Type and screen – Type and screen refers to testing on a patient sample in preparation for transfusion.

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).

Blood type – Blood type (also called blood group) refers to ABO and RhD antigens expressed on an individual's RBCs. This includes forward and back type for ABO and RhD antigens. Typing is required every time a red cell transfusion is ordered and is performed on all patient samples. Blood type from a donation card or military tag is never sufficient evidence for compatibility with a specific recipient. (See 'Blood type (ABO and RhD type)' below.)

The forward (front) type defines ABO antigens present on the recipient's RBCs. The reverse (back) type reacts patient plasma/serum against reagent group A and B RBCs to determine the patient's antibodies to A and B antigens. The front and back type must fit set criteria in order to assign an ABO type. If the criteria are not met, the ABO type is reported as unresolved. Reasons for discrepancies are discussed below. (See 'ABO-type discrepancies' below.)

Antibody screen – The screen tests the patient's plasma/serum for antibodies against RBC antigens including anti-RhD. (See 'Antibody screen' below.)

Antiglobulin (Coombs) testing – Coombs testing is a serologic (antibody-based) assay (figure 1) [7].

Direct – Direct Coombs (also called direct antiglobulin test [DAT]) detects antibodies and/or complement that are already present 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 (figure 1). 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 – Indirect Coombs (also called indirect antiglobulin test [IAT]) is performed by incubating reagent RBCs with patient plasma/serum and then adding the secondary antibody. If the patient's plasma/serum contains antibodies to antigens on the reagent RBCs, the RBCs will be agglutinated. (See 'Antibody screen' below.)

Coombs testing is also used for non-transfusion indications to evaluate hemolysis or hemolytic anemia and determine if it is immune or non-immune. (See "Diagnosis of hemolytic anemia in adults", section on 'Cause not obvious - start with Coombs test'.)

Agglutination – Refers to clumping of RBCs in the test tube. Occurs when an antibody against human IgG binds to multiple antibody-coated RBCs. Occurs without a secondary antibody when the anti-RBC antibody is an IgM, which is pentameric. This can occur in cold agglutinin disease. (See "Cold agglutinin disease", section on 'Cold agglutinins'.)

Agglutination can be assessed visually or by an automated detection system.

Mixed field agglutination – 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 in a patient who has been transfused in the prior three months. (See "Hemolytic transfusion reactions", section on 'Delayed hemolytic transfusion reactions and delayed serologic transfusion reactions'.)

Panagglutinin – Agglutination with all reagent RBCs at the same strength of agglutination. This finding has several possible causes and generally requires additional testing in the blood bank. (See "Red blood cell (RBC) transfusion in individuals with serologic complexity", section on 'Panagglutination'.)

What testing is required? — Tests that must be performed prior to release of an RBC component include ABO and RhD type, antibody screen, and compatibility testing (crossmatching).

A patient sample can be tested for blood type and the presence of antibodies (ie, type and screen) without compatibility testing on a specific RBC unit, 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 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. (See 'Emergency release blood for life-threatening anemia or bleeding' above.)

Blood type (ABO and RhD type)

Clinical relevance – Individuals who lack any A or B antigens can produce antibodies to them capable of causing severe hemolysis. (See "Hemolytic transfusion reactions", section on 'Acute hemolytic transfusion reactions'.)

An RhD-negative individual who becomes pregnant with an RhD-positive fetus can have hemolytic disease of the fetus and newborn (HDFN). (See "RhD alloimmunization in pregnancy: Overview" and "RhD alloimmunization: Prevention in pregnant and postpartum patients".)

Which blood can they receive?

ABO type Antibodies to A and/or B are present in the plasma of the vast majority of individuals who lack these antigens, even in the absence of prior transfusion, due to exposure to gut bacteria that share similar epitopes (molecular mimicry).

-Type O individuals can only receive type O RBCs. Type O individuals lack A and B antigens and make antibodies to A and B. Type O blood is the "universal donor" for RBCs.

-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 ( "universal recipient").

-Type A individuals lack B antigens and make antibodies to B. They can receive type O or type A RBCs.

-Type B individuals lack A antigens and make antibodies to A. They can receive type O or type B RBCs.

Plasma compatibility is different from RBC compatibility. As an example, type O individuals have anti-A and anti-B in their plasma, so their plasma can only be given to type O individuals. Type AB plasma is the "universal donor" plasma, and type O individuals are the "universal recipient" for plasma.

RhD type – The Rh blood group system includes several antigens, but only the RhD status is included in the blood type. The RhD type is often represented as positive or negative after the ABO blood group (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, in general, they do not make antibodies to RhD. They can receive RhD-positive or RhD-negative RBCs. An exception is individuals who are "partial D" and type as RhD-positive but can make anti-RhD antibodies following exposure (eg, transfusion, pregnancy) due to immune response to the portion of the RhD antigen they are missing.

-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 HDFN.

Females who are beyond childbearing age and RhD-negative males may receive RhD-positive units in certain clinical situations (eg, emergency release, 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 [8].

Antibody screen

Auto-control — The auto-control screens the recipient's own plasma/serum for antibodies to the recipient's own RBCs.

Most individuals will have a negative auto-control, meaning that any antibodies identified on the antibody screen will be alloantibodies rather than autoantibodies.

A "positive auto-control" is a potentially confounding finding in the antibody screen that results when the recipient's plasma/serum causes the recipient's own saline-washed RBCs to agglutinate in the absence of any foreign RBCs or plasma. Causes include:

Patient has an RBC autoantibody (eg, autoimmune hemolytic anemia). (See 'Autoimmune or drug-induced hemolytic anemia' below.)

Patient is experiencing a hemolytic transfusion reaction. (See "Hemolytic transfusion reactions".)

Patient was transfused with plasma or platelets containing an antibody that reacts with their own RBCs. (See 'ABO-type discrepancies' below.)

Patient has a condition that causes rouleaux formation, such as in multiple myeloma, which is mistaken for agglutination in the reaction tube. (See "Multiple myeloma: Clinical features, laboratory manifestations, and diagnosis", section on 'Peripheral smear'.)

ABO-type discrepancies — ABO type discrepancies occur when the RBC "front-type" is inconsistent with the plasma/serum "back-type." (See 'Definitions (front and back type, Coombs test, agglutination)' above.)

Many of these can be addressed by evaluating a pre-transfusion (or pre-transplant) specimen. Causes include [7]:

Massive transfusion – Massive transfusion involves transfusion of multiple RBC units over a short period of time, with or without plasma and platelet transfusions. (See "Massive blood transfusion".)

Massive transfusion replaces a significant portion of the patient's blood volume with donor blood. A type A recipient who receives >5 units of type O RBCs may acquire enough anti-A from the type O units to cause front-back type discrepancies. Because massive transfusion can pose a significant drain on precious resources, many institutional trauma resuscitation policies mandate immediate collection of a patient specimen for ABO and RhD typing so that type-specific blood products can be released.

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 with multiple 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. In rare cases, passively acquired antibodies have led to hemolytic transfusion reactions [9]. (See "Platelet transfusion: Indications, ordering, and associated risks", section on 'ABO, Rh, and HLA matching'.)

This underlying cause of ABO type discrepancy can be circumvented by restricting patients to group O RBCs for transfusion, although the laboratory findings will still be present. 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. Alternatively, some 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.

ABO-incompatible hematopoietic stem cell transplant – Type discrepancies depend on the residual recipient B and T cell function and the degree of donor chimerism. (See "Red blood cell (RBC) transfusion in individuals with serologic complexity", section on 'Allogeneic hematopoietic stem cell transplantation recipients'.)

Impaired humoral immunity – Waning antibody titers due to an underlying medical condition that impairs antibody production can result in loss of anti-A or anti-B. As an example, a type O patient with hematologic malignancy may develop undetectable anti-A and/or anti-B titers.

With ABO-type discrepancies, universal donor products (type O RBCs and type AB plasma) should be selected for transfusion. (See "Clinical use of plasma components", section on 'ABO matching'.)

Clinically significant alloantibodies — Antibody screening is performed on all patients who may require transfusion. It identifies clinically significant alloantibodies in patient plasma/serum that might react with antigens on transfused RBCs and cause hemolysis (hemolytic transfusion reaction), decreasing the survival of the transfused RBCs and possibly causing symptoms or complications. Clinically relevant hemolysis usually occurs at 37°C (98.6°F), rather than room temperature.

Always clinically significant – 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). (See "Red blood cell antigens and antibodies", section on 'Clinically significant (common)' and "Red blood cell antigens and antibodies", section on 'Clinically significant (rare)'.)

Rarely clinically significant – 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. (See "Red blood cell antigens and antibodies", section on 'Limited or no clinical significance'.)

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. (See 'Other information' above.)

Some alloantibodies may not be identified by the antibody screen. This includes alloantibodies from a remote transfusion that are below the level of detection in plasma/serum but might increase upon re-exposure due to an anamnestic response. This is why information about previous transfusions and previous alloantibodies is important to convey to the transfusion service.

Screening methods — The antibody screen is performed by incubating the patient's plasma/serum at 37°C (98.6°F) with two or three donor RBC panels 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. If 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).

Several assay platforms are available; these all demonstrate agglutination but differ in the scoring method (figure 2); they can be complementary. In the tube-method and column agglutination, RBC agglutination appears as a non-dispersible precipitate in tube testing and impedance in the passage of light through gel/beads in column agglutination. For solid-phase testing, positive results appear as disbursement of RBCs within a microwell. Most laboratories use a combination of methods.

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 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 [7].

Additive solutions enhance antibody detection, but 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 since 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 [7].

Solid-phase red cell (SPRC) adherence method — The 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/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 [7].

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 the 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 [7].

RBC GENOTYPING — Genotyping for determinants of clinically important RBC antigens is an evolving field; genotyping is an alternative approach to determining blood type and risk for alloantibody formation.

Indications for genotyping — Genotyping may be especially useful in populations with a high rate of alloimmunization and/or confusing serologic results. Benefits include ability to evaluate multiple antigens using automated technology, ability to check rare antigens, and ability to avoid interference from autoantibodies and transfused RBCs. (See 'ABO-type discrepancies' above and 'Special populations and potential confounders and special populations' below.)

Generally, decisions regarding the need for genotyping to assist in transfusion management are made by the transfusion service, with input from the clinical caregivers.

Examples of individuals who may benefit include [10]:

Sickle cell disease or thalassemia – These individuals often require frequent transfusions and are more likely to become alloimmunized to a number of RBC antigens [11]. (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'.)

In 2023, the National Health Service (NHS) in England announced plans to include blood group genotyping for patients with sickle cell disease or thalassemia, along with creation of a national donor database to facilitate matching RBC units to patients [12].

Complex results from antibody screen – These individuals may have a mixture of their own and donated RBCs and/or auto- and alloantibodies or other conditions that make it difficult to determine the most compatible blood for transfusion. 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 (using the genotype obtained at the time of a previous donation by the same donor). (See "Red blood cell (RBC) transfusion in individuals with serologic complexity".)

Obstetrics – Genotyping could be helpful in resolving inconclusive or discrepant RhD typing (eg, due to a partial or weak D phenotype), particularly in females who are pregnant or of childbearing potential. Prominent clinical societies that advocate for molecular testing include the Association for the Advancement of Blood & Biotherapies (AABB) and the American College of Obstetricians and Gynecologists (ACOG) [13]. This approach has been shown to be cost-effective [14]. (See "RhD alloimmunization in pregnancy: Overview", section on 'D variants' and "RhD alloimmunization in pregnancy: Management", section on 'Cell-free DNA testing'.)

Fetal RBC genotype can be determined using cell-free fetal DNA in the maternal circulation. (See "Cell-free DNA screening for fetal conditions other than the common aneuploidies" and "RhD alloimmunization: Prevention in pregnant and postpartum patients", section on 'Fetal D-negative status known based on evaluation of cell-free DNA'.)

Individuals who use RBC genotyping for pretransfusion testing and require transfusion still must have a crossmatch to ensure that the chosen unit is compatible. (See 'Compatibility testing (crossmatch)' below.)

Genotyping technology — Genotyping can only be useful in identifying units for compatibility testing when there is sufficient time to perform the testing. Unlike serologic testing, which can be done within minutes to hours in the blood bank, genotyping can take hours to days and is often a send-out test that may not be returned for 7 to 14 days.

The first commercially available RBC genotyping platforms became available in Europe and the United States in 2014. Available tests include:

Precise Type HEA – 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) [15]. This test was approved by the US Food and Drug Administration (FDA) in 2014 [16].

ID CORE XT (BLOODChip) – 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) [17]. 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 [18]. 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 [19]. This platform was approved by the FDA in 2018 [20].

A number of other blood group genotyping tests including whole genome sequencing are under various stages of development or approval [10,21,22].

COMPATIBILITY TESTING (CROSSMATCH)

Overview of compatibility testing

Definition and indications – Compatibility testing (also called crossmatching) refers to the testing of a specific donor unit of RBCs for transfusion to the recipient. It is performed using patient plasma/serum and one or more potentially compatible RBC units selected based on the recipient's blood type and antibody screen. It is generally done when there is a reasonably high chance that transfusion will be used and is required for all patients for whom an 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 could provoke hemolysis in the recipient due to recipient alloantibodies and/or complement. As an example, 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.

Extent of testing – The extent of testing required for a crossmatch depends on the recipient's current and historical screen results and prior transfusion history.

Computer or immediate spin crossmatch – For a patient with a negative antibody screen and no history of clinically significant antibodies, crossmatching typically is straightforward; one or more RBC units often can be released to the recipient following an electronic or immediate spin crossmatch. (See 'Electronic crossmatch' below and 'Immediate spin crossmatch' below.)

IgG/AHG (anti-human globulin) crossmatch (full crossmatch) – 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 and 'Definitions (front and back type, Coombs test, agglutination)' above.)

Additional testing – For a patient whose crossmatch results appear to be inconsistent with their antibody screen, additional testing may be needed to clarify which units are safest for transfusion. (See 'Special populations and potential confounders and special populations' below and "Red blood cell (RBC) transfusion in individuals with serologic complexity".)

Likelihood of compatible unit(s) – The likelihood of finding a potentially compatible unit depends on the frequency of the implicated antigen(s) in the donor population. Rare alloantibodies are listed separately. (See "Red blood cell antigens and antibodies", section on 'Clinically significant (rare)'.)

It is often more challenging to find compatible blood for individuals 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, since only 2 percent of ABO compatible RBC units would be fully antigen compatible.

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. (See "Red blood cell (RBC) transfusion in individuals with serologic complexity", section on 'Multiple alloantibodies'.)

During the coronavirus disease 2019 (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 [23]. (See "Indications and hemoglobin thresholds for red blood cell transfusion in the adult", section on 'Extending the blood supply'.)

Electronic crossmatch — The electronic (computer) crossmatch (EXM) is done using a computer algorithm rather than serologic testing. The 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.

In some institutions, EXM is performed instead of an immediate spin (IS) crossmatch; this can only be done if the following conditions are met:

The patient has been ABO-typed on two separate occasions.

The patient has no history of a clinically significant antibody.

Institutional informatics and quality controls are available to support accurate electronic matching, including checking for ABO and RhD typing of at least two patient samples, warnings of ABO-type discrepancies, and antibody screen results (both current and historical) [24].

The criteria for using EXM are outlined in the guidance from the US Food and Drug Administration (FDA) [25]. 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 recipient has an IgM antibody that can fix complement or bind multiple RBCs, which is concerning for ABO mismatch (the unlikely event that an ABO incompatible unit was selected, or the ABO typing of patient was incorrect).

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 to ensure that the donor RBCs lack the antigen to which recipient antibodies are directed. (See 'IgG crossmatch (full crossmatch)' below.)

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 [26].

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. (See 'Definitions (front and back type, Coombs test, agglutination)' above.)

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.

SPECIAL POPULATIONS AND POTENTIAL CONFOUNDERS AND SPECIAL POPULATIONS

Infant <4 months — Infants <4 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 or thalassemia — Individuals with sickle cell disease or thalassemia often require frequent transfusions and are more likely to develop alloantibodies than individuals in the general population [11]. The use of RBC genotyping and more extensive crossmatching for this population is discussed separately. (See "Red blood cell transfusion in sickle cell disease: Indications and transfusion techniques", section on 'Transfusion techniques'.)

Autoimmune or drug-induced hemolytic anemia

AIHA – Patients with autoimmune hemolytic anemia (AIHA) have circulating autoantibodies directed against their own RBCs. While the primary treatment involves reducing or eliminating the autoantibody (typically 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'.)

RBC autoantibodies may make interpretation of antibody screen and compatibility testing results more challenging because the antibodies may mask a clinically significant 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, low ionic strength sodium [LISS] or saline). Even though the crossmatch is incompatible in patients with RBC autoantibodies, it is incredibly important to ensure transfusions are not withheld from individuals who require transfusion due to severe anemia [27]. (See 'Emergency release blood for life-threatening anemia or bleeding' above and "Red blood cell (RBC) transfusion in individuals with serologic complexity", section on 'Autoantibodies'.)

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). A list of implicated drugs is provided in the table (table 1) and details of the mechanisms and management are presented separately. (See "Drug-induced hemolytic anemia", section on 'Immune-mediated'.)

Therapeutic antibodies (IVIG, anti-D, monoclonals)

IVIG – Intravenous immune globulin (IVIG) products may contain sufficiently high enough titers of antibodies to ABO antigens (anti-A and anti-B) to cause a positive direct antiglobulin test in blood type A or B individuals, respectively. Less commonly, other alloantibodies may be involved. There are numerous case reports of clinically significant hemolysis as a consequence of passive sensitization by these antibodies, especially with high dose IVIG (1 to 2 g/kg over two to seven days) administered to blood type A or B patients [28,29]. (See "Intravenous immune globulin: Adverse effects", section on 'Hemolysis'.)

Anti-DAnti-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 [7]. (See "RhD alloimmunization in pregnancy: Overview".)

Monoclonal antibodies – Therapeutic monoclonal antibodies that bind RBCs can interfere with the antibody screen and crossmatching. Details are presented separately. (See "Red blood cell (RBC) transfusion in individuals with serologic complexity", section on 'Monoclonal antibodies that affect the antibody screen'.)

Transplant recipients

Solid organ transplantation – Solid organ transplantation generally uses ABO-identical donor organs. However, "minor ABO-incompatible" solid organ transplantation can be performed (eg, group O donor organ transplanted to non-group O recipient) and may cause transient RBC hemolysis due to 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 [30]. Hemolysis due to passenger lymphocytes is transient, generally abating within a month after transplantation and clearance of donor lymphocytes. (See "Warm autoimmune hemolytic anemia (AIHA) in adults", section on 'Associated conditions'.)

Allogeneic hematopoietic stem cell transplantation – (See "Red blood cell (RBC) transfusion in individuals with serologic complexity", section on 'Allogeneic hematopoietic stem cell transplantation recipients'.)

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

Emergency release blood (no time for testing) – For patients with life-threatening anemia or bleeding in whom compatible blood is not readily available, we recommend emergency release blood rather than delaying transfusion (Grade 1B). Transfusion should never be withheld if needed while awaiting compatibility testing for a patient with life-threatening anemia, brisk hemolysis, or rapid bleeding. Emergency release red blood cell (RBC) units are typically type O, RhD-negative or RhD-positive. (See 'Emergency release blood for life-threatening anemia or bleeding' above.)

Recipient blood specimen – Quality controls are in place to ensure that the patient specimen is from the correct patient and reflects the patient's blood type and alloantibodies at the time transfusion will be administered. A properly labeled tube containing patient plasma or serum 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 medical history, prior transfusions, transfusion reactions, allogeneic hematopoietic cell or solid organ transplantation, pregnancy history, and special modifications required can help prevent hemolytic transfusion reactions and other serious complications. (See 'Specimen requirements' above.)

Type and screen – 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 that may be especially useful in individuals with a high rate of alloimmunization and/or confusing serologic results. (See 'Blood type (ABO and RhD type)' above and 'Antibody screen' above and 'RBC genotyping' above.)

Crossmatch – Crossmatching is performed when requested or when release of an RBC unit is requested. (See 'Compatibility testing (crossmatch)' above.)

Negative antibody screen and no history of clinically significant antibodies – One or more RBC units often can be released to the recipient following a negative electronic or immediate spin crossmatch. (See 'Electronic crossmatch' above and 'Immediate spin crossmatch' above.)

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.)

Additional testing – Some situations may be more challenging and/or may require additional testing:

-Early infancy and pregnancy – (See 'Infant <4 months' above and 'Pregnancy' above.)

-Heavily transfused individuals – (See 'Sickle cell disease or thalassemia' above and "Red blood cell (RBC) transfusion in individuals with serologic complexity", section on 'Multiple alloantibodies'.)

-Massive transfusion or out-of-group platelet transfusion – (See 'ABO-type discrepancies' above.)

-Autoimmune or drug-induced hemolytic anemia – (See 'Autoimmune or drug-induced hemolytic anemia' above and "Red blood cell (RBC) transfusion in individuals with serologic complexity", section on 'Autoantibodies'.)

-Antibody-containing products – (See 'Therapeutic antibodies (IVIG, anti-D, monoclonals)' above and "Red blood cell (RBC) transfusion in individuals with serologic complexity", section on 'Monoclonal antibodies that affect the antibody screen'.)

-Recipients of solid organ or hematopoietic stem cell transplantation – (See 'Transplant recipients' above and "Red blood cell (RBC) transfusion in individuals with serologic complexity", section on 'Allogeneic hematopoietic stem cell transplantation recipients'.)

ACKNOWLEDGMENTS — 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.

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Topic 7953 Version 40.0

References

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