Crossmatch and Antibody Screen

The antibody screen and crossmatch are the last laboratory safeguards before red cell transfusion.

The antibody screen uses the indirect antiglobulin technique to detect red cell alloantibodies in the recipient's plasma, with group O reagent cells of known antigen profile. The crossmatch tests the recipient's plasma against the red cells of the unit chosen, to confirm ABO compatibility and to exclude an antibody against an antigen carried by the donor cells.

Together with the ABO and RhD group, they decide which units are safe and how quickly they can be issued.

Clinically significant antibodies, those reactive at 37 °C or in the antiglobulin phase, are found in about 1 to 3% of hospital patients, and more often after multiple transfusions, pregnancies or transplantation. Most transfusion laboratories now issue blood electronically to patients with a negative screen and a verified group history, and keep serological crossmatching for the rest.

Indications. Any planned or likely red cell transfusion, pregnancy at booking and during gestation, surgery with a risk of major blood loss, regular transfusion in sickle cell disease or thalassaemia, transplantation, suspected transfusion reaction, and before and during anti-CD38 therapy.

Specimen. EDTA or clotted venous blood, labelled at the bedside with full patient identifiers. A sample is valid for 3 days when the patient has been transfused or pregnant in the previous 3 months (day of collection is day 0), and for up to 7 days otherwise, depending on local policy. A second independent sample, or a documented historical group, is needed before group specific red cells are issued.

Antibody screen. Two or three group O cells that together carry the clinically important antigens (D, C, c, E, e, K, k, Fya, Fyb, Jka, Jkb, S, s) are incubated with plasma, enhanced with low-ionic-strength solution or polyethylene glycol, washed, and read with antihuman globulin in tube, gel or solid phase systems.

Cells with double dose (homozygous) expression of Duffy, Kidd and MNS antigens are preferred, because antibodies against these systems react weakly with single dose cells. An autocontrol and direct antiglobulin test are run with a positive screen.

Electronic issue. No serological crossmatch is done. It is allowed only when the laboratory has a validated computer system that checks ABO and RhD compatibility at the point of issue, the patient has a group confirmed on two separate samples (or equivalent), the current screen is negative, and there is no history of a clinically significant antibody.

Immediate spin crossmatch. Donor red cells are mixed with recipient plasma, centrifuged and read for agglutination or haemolysis within about 5 minutes. It detects ABO incompatibility only, and is used when the screen is negative and electronic issue is not available.

Antiglobulin crossmatch. Donor cells and recipient plasma are incubated at 37 °C with enhancement, washed and read with antihuman globulin, taking 30 to 45 minutes. It is used when the screen is positive or an antibody has been recorded. Units are first typed and shown to lack the corresponding antigen, so that a compatible result is not the first line of defence.

Antigen negative selection. For each identified antibody, units lacking the antigen are chosen. With several antibodies, many units may need typing before enough compatible ones are found, which delays issue. In patients on regular transfusion, extended matching (Rh C, c, E, e and Kell at least) limits new antibody formation.

Usual timings. Group and screen take about 45 to 60 minutes. Electronic issue takes about 10 minutes after a valid group. Antibody identification and antigen negative selection can take several hours.

Positive screen. The antibody is identified, its clinical significance judged, and antigen negative blood selected. Clinically significant antibodies include anti-D, anti-C, anti-c, anti-E, anti-e, anti-K, anti-Fya, anti-Fyb, anti-Jka, anti-Jkb, anti-S and anti-s, and any antibody reactive at 37 °C. Cold reactive antibodies (anti-M, anti-N, anti-P1, anti-Lea, anti-Leb) that do not react at 37 °C are usually not significant.

Incompatible crossmatch with a negative screen. Causes are an antibody to a low frequency antigen on the donor cells, a positive direct antiglobulin test on the donor unit, an ABO error, rouleaux, and passively acquired antibody from plasma products or immunoglobulin.

Warm autoantibody. The screen is panreactive with a positive autocontrol. Adsorption with the patient's own cells, or with allogeneic cells, separates an underlying alloantibody, which matters because the patient is often transfused. Units matched for the Rh and Kell phenotype are selected when adsorption is not possible. Transfusion is not withheld in life threatening anaemia.

Anti-CD38 therapy (daratumumab and similar). Reagent cells carry CD38, so the screen is panreactive. Dithiothreitol treated reagent cells remove the reactivity but also destroy Kell antigens, so Kell negative units are selected by phenotype. A pre-treatment phenotype or genotype is needed.

Delayed haemolytic reaction. An antibody can fall to undetectable levels after sensitisation, then rise within 3 to 14 days of re-exposure. The haemoglobin falls, bilirubin and LDH rise, and the direct antiglobulin test turns positive. The antibody record is therefore kept for life, and antigen negative blood is issued even if the screen is now negative.

Emergency issue.

• Uncrossmatched group O RhD-negative red cells are issued when the group is unknown. Kell negative units are used in girls and women of childbearing potential.
• Group specific uncrossmatched units can be issued once a valid group is available, usually within 10 to 15 minutes.
• Antibody work continues after issue, and the clinician is told the units were released before testing was complete.
• Units with an incompatible crossmatch are released only on the documented request of a senior clinician who has accepted the risk, for example in life threatening haemorrhage with a panreactive screen.

Critical findings needing immediate action.

• A new clinically significant antibody in a patient who has just been transfused, or a rising titre in pregnancy.
• Fever, rigors, hypotension, loin pain, haemoglobinuria or bleeding during or after transfusion: stop the transfusion, recheck identity of the patient and unit, and contact the transfusion laboratory and a senior clinician immediately.
• Positive screen in a patient with massive haemorrhage: do not delay issue of emergency blood.

Pitfalls.

• Wrong blood in tube and labelling errors are the most dangerous failures, and the reason for the second group sample.
• Multiple antibodies mask one another, and each needs excluding separately.
• Antibodies to high frequency antigens give panreactivity with a negative autocontrol, and need a reference laboratory.
• Dosage effect gives weak or absent reactions with single dose cells.
• Reagent additives, rouleaux, and intravenous immunoglobulin (which carries passive ABO and other antibodies) can cause false positives.
• Antibody in a patient transfused in the last 3 months can be missed on an old sample.
• Massive transfusion dilutes the patient's plasma, and after a large exchange the screen reflects the donor plasma, so ABO-compatible units are given without further crossmatching.

Special groups.

• Infants under 4 months: they do not make red cell antibodies, so an initial screen on infant or maternal plasma is sufficient, and repeat crossmatching is not needed if the first is negative. Units must be compatible with any maternal antibody present.
• Pregnancy: screen at booking, at 28 weeks, and after sensitising events, with samples taken within 3 days of any transfusion.
• Sickle cell disease and thalassaemia: alloimmunisation is frequent, and extended phenotype matching is advised.
• Transplantation: the screen and crossmatch are part of immunological workup, together with HLA antibody testing.

Follow up tests. Antibody identification panel, direct antiglobulin test, red cell eluate, adsorption studies, antibody titration, extended red cell phenotype or genotype, haemolysis screen (reticulocytes, LDH, bilirubin, haptoglobin), repeat direct antiglobulin test after transfusion, anti-D quantification in pregnancy.