Residency · Residency · Pathology
ABO/Rh Typing, Antibody Screening, and Crossmatch
Introduction
Pre-transfusion testing is the foundation of transfusion medicine safety. ABO and Rh typing, antibody screening, and crossmatch procedures ensure immunologic compatibility between donor and recipient, preventing potentially fatal hemolytic transfusion reactions.
ABO Blood Group System
Biochemistry and Genetics
ABO antigens are carbohydrate structures on glycoproteins and glycolipids of the red blood cell membrane. The H antigen is the precursor structure onto which A and B transferases add specific sugars. A transferase adds N-acetylgalactosamine to the H antigen, while B transferase adds D-galactose. The O phenotype encodes a non-functional transferase, leaving the H antigen unmodified. ABO genes are located on chromosome 9, and inheritance follows a codominant pattern.
ABO Typing: Forward and Reverse
Forward (cell) typing tests patient RBCs with known anti-A and anti-B reagents. Reverse (serum/plasma) typing tests patient serum with known A1 and B reagent red cells. Forward and reverse results must agree before a blood type is assigned, and any ABO discrepancy requires investigation before blood products are issued.
| Blood Type | Forward (Anti-A) | Forward (Anti-B) | Reverse (A1 cells) | Reverse (B cells) | Antibodies Present |
|---|---|---|---|---|---|
| O | 0 | 0 | + | + | Anti-A, Anti-B |
| A | + | 0 | 0 | + | Anti-B |
| B | 0 | + | + | 0 | Anti-A |
| AB | + | + | 0 | 0 | None |
ABO Discrepancies
Group I discrepancies involve weak or missing antibodies and are seen in neonates, elderly individuals, immunosuppressed patients, and those with hypogammaglobulinemia. Group II discrepancies feature unexpected antibodies, such as anti-A1 in A2 or A2B subgroups, cold autoantibodies, or passively acquired antibodies. Group III discrepancies involve weak or missing antigens and occur with subgroups of A (A2, A3, Ax, Aend), recent transfusion, bone marrow transplant, or leukemia. Group IV is a miscellaneous category encompassing polyagglutination, cold autoantibodies, and Wharton jelly contamination.
Rh Blood Group System
D Antigen and Rh Typing
The Rh system includes over 50 antigens, with D being the most immunogenic. Approximately 85% of the Caucasian population is RhD-positive (D antigen present), while RhD-negative individuals lack the D antigen and are at risk for anti-D immunization. Other clinically significant Rh antigens include C, c, E, and e. The Rh genes (RHD and RHCE) are located on chromosome 1.
Weak D and Partial D
Weak D (formerly Du) represents a reduced quantity of normal D antigen detected by the indirect antiglobulin test (IAT). Partial D is a qualitatively altered D antigen with missing epitopes, meaning the individual can form anti-D against the epitopes they lack. Current guidelines recommend RHD genotyping for weak D phenotypes to distinguish weak D types 1, 2, and 3 (who can safely receive RhD-positive blood) from partial D (who should receive RhD-negative blood).
Antibody Screening
Indirect Antiglobulin Test (IAT)
Patient plasma is incubated with reagent screening cells (group O RBCs with known antigen profiles), typically a two or three cell panel covering clinically significant antigens. After incubation, anti-human globulin (AHG/Coombs reagent) is added to detect IgG-coated cells. A positive screen indicates the presence of clinically significant alloantibodies.
Antibody Identification
Identification is performed using an extended panel of 10-16 reagent cells with known antigen profiles. The reaction pattern is matched against the antigen profile of each panel cell using a rule-out approach, where antigens present on non-reactive cells can be excluded. Multiple antibodies may coexist and require additional panels or selected cells. Common clinically significant antibodies include anti-D, anti-K (Kell), anti-E, anti-c, anti-Fya, and anti-Jka.
Techniques and Enhancement Media
The tube method is traditional and uses LISS (low ionic strength saline) or PEG (polyethylene glycol) enhancement. Gel/column agglutination (gel cards) provides standardized, stable reactions and is widely adopted. Solid-phase red cell adherence is used on automated platforms such as Immucor NEO and Ortho Vision. PEG enhances detection of weak IgG antibodies but can cause false-positive reactions.
Crossmatch
Types of Crossmatch
The immediate spin (IS) crossmatch detects ABO incompatibility by centrifuging patient plasma with donor RBCs immediately. The antiglobulin crossmatch (full crossmatch) includes 37 degrees C incubation and an AHG phase, and is required when the antibody screen is positive or the patient has a history of clinically significant antibodies. The electronic (computer) crossmatch involves no serologic testing; ABO/Rh is verified by a computer algorithm and is permitted when the antibody screen is negative and two concordant ABO/Rh typings are on file.
Requirements for Electronic Crossmatch
The electronic crossmatch requires a validated computer system with logic to prevent release of ABO-incompatible units, a current negative antibody screen, two concordant ABO/Rh determinations (one from the current sample), no history of clinically significant antibodies, and donor unit confirmation testing recorded in the system.
Special Situations
Emergency Release and Massive Transfusion
Uncrossmatched blood (group O RBCs, with O-negative for females of childbearing potential) is released in life-threatening emergencies. Type-specific blood is issued as soon as the ABO/Rh type is confirmed, typically within 5-10 minutes. Massive transfusion protocols may switch to type-specific blood after the initial O-negative units. All emergency release events must be documented for quality review.
Neonatal Transfusion
The mother's sample is used for the antibody screen; if negative, the infant can receive crossmatch-compatible or O-negative RBCs. ABO typing on cord or infant blood is performed, but reverse typing is not reliable due to maternal antibody transfer. RBCs for neonates must be irradiated, CMV-safe, and hemoglobin S-negative.
Clinical Pearls
Forward and reverse ABO typing must agree before a blood type is finalized, and any discrepancy must be resolved before transfusion. Weak D genotyping is recommended to distinguish patients who can safely receive RhD-positive products from those who cannot. Electronic crossmatch eliminates the need for serologic testing when the antibody screen is negative and system validation criteria are met. In emergencies, group O RhD-negative uncrossmatched RBCs should be issued immediately without waiting for compatibility testing.
References
- Fung MK, et al., eds. AABB Technical Manual. 20th ed. AABB Press; 2020.
- Sandler SG, et al. It's time to phase in RHD genotyping for patients with a serologic weak D phenotype. Transfusion. 2015;55(3):680-689.
- Tormey CA, Hendrickson JE. Transfusion-related red blood cell alloantibodies: induction and consequences. Blood. 2019;133(17):1821-1830.
- Delaney M, et al. Transfusion reactions: prevention, diagnosis, and treatment. Lancet. 2016;388(10061):2825-2836.