Preparation and Testing

Before any grouping or compatibility work starts, the technologist must be satisfied that the tube on the bench came from the patient named on the request. That means comparing the label with the requisition and the patient's identification band, confirming the collector's initials and the date and time of collection, and refusing anything unlabelled, mislabelled or altered at the bedside. Most Canadian laboratories also require a second, independently collected sample before group-specific red cells go out to a patient with no historical ABO record. Mistakes at this step are the commonest route to an ABO-incompatible transfusion.

Serological results are only as good as the materials behind them. This area covers preparing and handling antisera, reagent red cells, enhancement media and control materials: checking lot numbers and expiry dates, inspecting vials for haemolysis, turbidity or contamination, letting reagents reach the temperature the product insert specifies, and making cell suspensions at the concentration the method requires. Positive and negative controls are run before patient samples each day and whenever a new lot is opened. Every lot, expiry date and control outcome is recorded, and a reagent that fails its control is quarantined rather than used.

Every serological method has a blind spot, and knowing where it lies is part of the job. Immediate-spin testing will not find warm IgG antibodies; gel and tube methods differ in sensitivity; enhancement media change what is detected. A recent transfusion produces mixed cell populations that make serological phenotyping unreliable, a positive direct antiglobulin test can mask an alloantibody, and screening cells may lack low-incidence antigens altogether. The technologist has to recognise when a negative result cannot rule an antibody out, and when to move on to an extended panel, adsorption, enzyme treatment or molecular typing.

Slide work in the transfusion laboratory has two main uses. The first is resolving a doubtful tube reading: the cell button is transferred gently to a slide and examined so that true agglutination can be told apart from rouleaux, fibrin strands or debris. The second is the acid elution film used to estimate a foetomaternal haemorrhage, where a thin, even smear, correct fixation and staining times, and a control slide containing known foetal cells are all needed before any count is credible. Poor spreading or over-fixation ruins the count long before the microscope is switched on.

A sample that is not fit for purpose cannot give a safe answer. The technologist checks that the correct tube type was drawn, that the volume reaches the fill line so the anticoagulant ratio is right, that an EDTA sample is free of clots, and that the draw is recent enough for the testing being asked for. Where a patient has been transfused or pregnant within the previous three months, the sample must usually be no more than 96 hours old, because a new antibody may have appeared since. Inadequate samples are rejected and recollected, never salvaged.

Phenotyping asks which antigens a set of red cells actually carries. The technologist follows the antiserum insert exactly, since some reagents work at immediate spin while others need thirty-seven degrees or an antiglobulin phase, and each run carries its own positive and negative controls plus a diluent control where the method demands one. Results are read against the patient's transfusion history, because cells transfused within the last three months make a serological type unreliable. Phenotypes confirm that a suspected antibody is an alloantibody and guide the choice of antigen-negative units for future transfusion.

This is the core bench work: ABO forward and reverse grouping, Rh(D) typing with weak D testing where policy requires it, antibody screening by indirect antiglobulin technique, and identification on a panel when the screen is positive. Each phase detects something different, since immediate spin picks up cold-reacting IgM while thirty-seven degrees and the antiglobulin phase find the clinically significant IgG antibodies that destroy transfused cells. Washing quality, incubation time and the addition of check cells to every negative antiglobulin tube decide whether a negative result can be trusted at all.

Compatibility testing joins a specific unit to a specific patient. When the antibody screen is negative, two concordant ABO determinations are on file and there is no antibody history, an immediate-spin or validated computer crossmatch is sufficient. A clinically significant antibody, present now or recorded in the past, changes the rules: antigen-negative units must be selected and a full antiglobulin crossmatch performed. An incompatible crossmatch is investigated rather than overridden, and emergency issue of uncrossmatched group O units follows a written protocol with the ordering physician's authorisation documented.

Attention also has to fall on the products. Donor red cell units are confirmed for ABO and Rh where policy requires it, and every unit is inspected before issue for clots, haemolysis, unusual colour, leaks and a damaged segment line, with expiry date and storage temperature checked at the same time. Some patients need modified components: irradiated units to prevent transfusion-associated graft versus host disease, washed cells for severe reactions to plasma proteins, or small aliquots for neonates. Each modification, inspection and release decision is recorded against the unit number.

Appearance is data. Red plasma may mean in vivo destruction or simply a traumatic draw, and either way free haemoglobin can hide the in vitro haemolysis that signals a complement-binding antibody, so a fresh sample is normally requested. Milky, lipaemic plasma makes a cell button hard to read and is easily mistaken for a weak reaction, while deep icterus discolours the supernatant and interferes in much the same way. The technologist grades the appearance, decides whether testing can proceed or the sample must be recollected, and documents the finding with the result.