Continuing Education Activity

Pretransfusion testing includes all activities from when the blood product is ordered by the physician to the final step when the blood product can be dispensed from the laboratory to the patient's bedside. This includes serological testing such as blood typing, antibody screening, and crossmatching in the laboratory. This activity emphasizes the importance of positive patient identification, compatibility testing, and appropriate product labeling to ensure that the right blood is given to the right patient at the right dose. An emphasis on the electronic crossmatch and red cell unit selection in situations where crossmatch compatible units are not available for transfusion. A summary of laboratory testing principles is also described. The role of attending physicians, nurses, laboratory physicians, and technical personnel involved in the entire transfusion chain is emphasized.

Objectives:

• Describe the flow of activities involved in pretransfusion testing.

• Explain the principles involved in various tests included in pretransfusion testing.

• Review critical elements in labeling samples and while ordering blood products.

• Identify the conditions that can interfere with pre-transfusion testing and the causes of positive pretransfusion testing.

Introduction

Pretransfusion testing is a multistep process that begins with the clinician's order for the right blood product and dose for the patient. It involves the following steps:

• Positive patient identification
• Collection of the blood samples of the patient for compatibility testing
• ABO and Rh blood grouping of the patient and the donor unit
• Red blood cell Antibody screening on serum/plasma sample of the patient
• Selection of appropriate blood component
• Performance of compatibility testing 
• Labeling of the unit with details of the product and the patient identification information
• Generation of compatibility report that is to be given along with the blood product

It is performed to ensure that the patient is positively identified with any special requirements, that transfusion records and history are reviewed and considered, that antibody detection and identification are performed, and that appropriate units are selected.[1]

Red blood cells have antigenic proteins and carbohydrates on the cell surface. These epitopes are classified into blood groups based on structure and similarities to a parent protein. Patients lacking certain epitopes may develop antibodies when exposed to these antigens through pregnancy, transfusion, or transplantation. Such red cell alloantibodies then lyse transfused red cells that possess the corresponding antigen.[2] 

Transfusion and pregnancy are the primary means of sensitization to red cell antigens. In a given population, 2% to 4% of the general population possess irregular red cell alloantibodies. These antibodies can cause hemolytic disease in the newborn or hemolysis of the donor's transfused red cells.[3][4] If time permits, pre-transfusion testing will identify the antibodies and provide antigen-negative units. 

In a "type and screen" procedure, the patient's red cells are typed for ABO and Rh (also known as D), and the patient's plasma is tested for clinically significant red cell antibodies.

A "type and crossmatch" involves selecting, matching, and reserving appropriate red cell components for the transfusion recipient. Many facilities have a Maximum Surgical Blood Ordering Schedule (MSBOS) that indicates when a type and screen are ordered and how many components should be reserved for each type of surgical procedure. Red cell antibodies of clinical significance are produced in response to pregnancy or transfusion; they can cause hemolysis or shortened survival of transfused red cells carrying the corresponding antigen (i.e., acute or delayed hemolytic transfusion reaction).

For this reason, the recipient's plasma is tested for the presence of these unexpected antibodies before red cell transfusion. If a patient has a clinically significant antibody, the transfusion service selects and reserves the appropriate red cell components that do not carry the corresponding antigen. Identifying a patient's red cell antibodies and crossmatching them with the appropriate red cell components can take hours or even days, depending on the antibody or antibodies found. This can be particularly problematic if the intended recipient has autoantibodies.[5] The various pretransfusion testing schemes are summarised in table 1.

Table 1. Pretransfusion testing schemes

Specimen Requirements and Procedure

Routine testing of blood components before transfusion includes receipt of an appropriately labeled patient sample by the transfusion service, testing for ABO and Rh D, the presence of unexpected antibodies, and crossmatching red cell components with the patient sample.

Positive patient identification: To ensure safe blood transfusion, it is critical that a properly labeled blood sample for pretransfusion testing is collected from the correct patient. The person collecting the sample must identify the patient using the wristband, which contains two unique patient identifiers (usually the patient's full name and the hospital's unique registration number) and remains on the patient throughout the hospital stay and blood transfusion. The information on the requisition form must be compared with that on the wristband; blood samples should not be collected if there is a discrepancy.[6]

The request form: This, either in paper or electronic form, for a transfusion must contain the following information:

• The patient's full name, including first and last (family and given) and gender
• A unique identifier like date of birth (DOB) or a hospital or health card number
• The recipient's address
• The required blood component/product with appropriate dose/volume
• The indication for transfusion
• History of previous transfusion or transfusion reactions
• Date and time of the order
• Identity of the qualified medical person ordering the blood products
• Date and time of the intended transfusion
• If any special requirements, like CMVnegative unit, leukoreduced, irradiated, washed, or reduced volume

Blood Sample: Pretransfusion testing requires a clotted sample from the recipient for serum and an EDTA sample for red cells and plasma. Hemolyzed or lipemic samples are unacceptable because it is difficult to visualize agglutination, the endpoint of pretransfusion testing. Institutional policies determine how far in advance a sample can be collected. For patients with negative antibody screening and no history of transfusion or pregnancy in the previous three months, samples can be collected up to one month before surgery. However, if the patient has been transfused or pregnant in the prior three months, or if this history is uncertain, a pre-transfusion sample is valid for only three days. For most hospitalized patients, a fresh sample must be taken every three days.[7]

It is advisable to collect a clotted sample from the mother for pretransfusion testing in infants under four months of age.

Labeling the sample: It should contain the following information:  

1. Patient's full first and last names
2. Patient's health care record number  
3. Date and time of specimen collection
4. Initials (if collected by laboratory personnel) or signature (if collected by nonlaboratory personnel) of a phlebotomist,
5. Possibly a unique blood bank number (found on a special blood bank identification band)

Samples not complying with any of the above information should be rejected except for minor inconsistencies like spelling mistakes of last name or short form of a full name like Jon for Jonathan. Strict adherence to the specimen-labeling policy decreases the error rate in reporting the patient's blood group. Accurate labeling of specimens is more critical in transfusion medicine, where specimen misidentification may result in the administration of incompatible blood components, leading to fatal acute hemolytic transfusion reactions. Specimens are to be labeled immediately after being drawn at the patient's bedside.[8] 

Table 2: Compatibility chart for choosing the blood products

Diagnostic Tests

The various tests included under pretransfusion testing are mostly serological, with some assistance from molecular methods in rare instances. The methods available for serological testing are based on test tube methods, column agglutination (CAT), or solid phase. The principle involved is the detection of antigen and antibody reactions by observation of agglutination or hemolysis in vitro.

The AHG (antihuman globulin) phase detects the red cell antibodies, usually IgG in nature, that do not produce direct agglutination by bridging red cells to complete the microscopically detectable lattice. These AHG reagents are usually IgM antibodies directed against the Fc portion of those IgG molecules.[7]

Testing Procedures

ABO and Rh Typing

The patient's ABO grouping is performed by ascertaining concordance with both cell typing (forward grouping) and serum testing (reverse typing). Discrepancies noted, if any, should be resolved before proceeding with further pretransfusion testing. Blood requirement if urgent O group unit should be selected for crossmatching. Testing two samples collected at different times is required before finalizing the ABO typing report.

Rh D typing is performed by testing the recipient's red cells with anti-D sera. Weak D testing is not required in the RhD-negative samples unless the recipient is a neonate born to RhD negative mother.[6]

Antibody Screening

These are serological tests designed to detect clinically significant antibodies to blood group antigens using an Indirect antiglobulin test (IAT). The recipient serum or plasma is incubated with a panel of red cells; usually, 2,3 or 4 (unpooled) cells with a known blood group antigen profile are used for antibody screening. 

Antibody identification

If the screening is positive, the next step is to identify the specificity of the antibody and for which an extended panel of unpooled reagent red blood cells is used (11-20 cell panel). The method includes testing samples against a sufficient number of reagent red cells that lack or express a particular blood group antigen. The specificity of the antibody is determined based on the reactivity pattern of the antibody against the cell panel. Then probability calculation is performed, allowing a minimum requirement of the p-value of 0.05 to zero down on the antibody/antibodies. Additional testing strategies may be required, namely the use of enhancement media (albumin, polyethylene glycol, low ionic strength solution, or chemical/enzyme treatment of the panel cells to aid identification.

Antigen phenotyping: Whenever an antibody is identified in a patient, confirmation can be done by phenotyping the recipient for the corresponding antigen to confirm he is negative for that antigen. Antigen phenotyping also needs to be performed on the donor units to select negative units for the antibody identified in the patient or to give phenotype-matched blood as in thalassemia or pregnancy. The method used for phenotyping is similar to forward typing by using the particular antisera. In patients who have recently transfused red cells in the circulation that are interfering with phenotyping or the antigens for which commercial antisera are not available, as, in Dombrock, molecular genotyping may be utilized for ascertaining the phenotype.[9]

Compatibility Testing

Immediate spin method: The recipient serum/plasma is mixed with saline-suspended donor cells (major crossmatch) and vice-versa (minor crossmatch) at room temperature. Immediate spin can be the only crossmatch method when the recipient does not have current or previously detected clinically significant antibodies.[6]

Antiglobulin crossmatch: In the liquid phase, i.e., tube method, 2 to 5% saline-suspended red cells of the donor unit are mixed with recipient serum and incubated at 37 C for about 45 minutes and washed to remove unbound antibodies. The AHG serum is added to the remaining cell button after washing, centrifuged, and read for agglutination. This can also be done using CAT or solid-phase systems.

Computer / electronic crossmatch: In this method, the patient is screened for antibodies and entered into the electronic system, which contains all the donor units in the inventory with their phenotype reports. The system picks the compatible units for the particular patient and displays them from the inventory; compatibility may be verified by computer. Electronic crossmatch can be used as the sole method only after ensuring that the recipient has no clinically significant antibodies present historically or currently.

 However, the validation of the system is to be done on-site. The system should recognize and correlate anti­ body detection results, compare previous records, concor­dant ABO on the recipient from at least two determinations, donor component and unit number, and ABO/Rh retype results, as well as have logic to alert the user to discrepancies between donor unit labeling and confirmatory test interpretation, and ABO incompatibilities between the recipient and the donor unit.[10]

Interfering Factors

Several factors influence the results of pretransfusion testing giving rise to false positive or negative results.[11] These include:

Some of the causes leading to false results are mentioned below;

 False Positive Result

• Rouleaux formation
• Passively transferred antibodies due to transfusion of platelets or administration of IVIG
• Antibodies to the preservatives present in the reagents used

False Negative Result

• Technical error
• The antibodies showing dosage effect may not react if the donor cells have a heterozygous expression of a particular antigen
• Loss of potency of the reagents used
• Failure to add reagents
• Equipment failure

 Massive transfusion replaces a portion of the patient's blood volume with donor blood. A  patient sample after transfusion may not accurately reflect blood type or alloantibodies, depending on the ABO type of the RBCs and plasma selected for emergency resuscitation. Hence it is imperative to continue the blood products based on the samples collected before the transfusion.

Results, Reporting, and Critical Findings

 Table 3:

Interpretation:

If the result is positive (antibody detected), antibody identification is required to determine the specificity and clinical significance of the detected antibody. If alloantibodies are detected in pregnant patients, a titer test is performed.

Negative results mean that no antibodies were detected.

A positive antibody screen with incompatible crossmatch with negative auto-control indicates the presence of alloantibody(s). If the auto-control test is positive, the cause could be nonspecific antibodies directed against the enhancement medium or rouleau formation.

Crossmatching by indirect antiglobulin testing method: A negative test (crossmatch compatible) indicates that the serum or plasma contains no detectable antibodies that are reactive with the red cells being transfused.

Compatibility testing in infants (< 4 months old): Crossmatch compatibility testing is not required if the antibody screen is negative, the transfused red cells are either ABO identical, compatible, or Group O and RhD negative or identical. Compatibility testing is to be performed with maternal serum or plasma as well.[12]

Evidence supports the use of fresher red cells (less than seven days after collection) when the transfusion volume in neonates is greater than 25 ml/kg.[13]

Emergency release: Uncrossmatched ABO identical group or group O red cells or group O whole blood (without hemolysins) may be issued if the recipient's group is unknown. However, compatibility testing should be continued and completed, and if incompatibility is detected, the recipient's physician should be notified immediately.[14]

Clinical Significance

In emergencies and before crossmatch compatible blood components are available, group O red cells and group AB plasma (some facilities use group A plasma) can be transfused appropriately. A properly labeled blood sample from the intended recipient before transfusion is critical to safe blood transfusion. Most hemolytic transfusion reactions are caused by misidentification of the patient or labeling errors of blood samples.

Selection of appropriate red cell units: Recipients should receive ABO and Rh D identical or compatible units unless warranted. In situations where Rh D negative units are unavailable, Rh D positive units may be given to men and women over reproductive age after they have been determined to lack anti-D antibodies. Fresh red cell units (less than seven days old) should be selected for large-volume transfusions (> 25 ml/kg) in neonates.[13]

Selection of other blood products: All plasma-containing components should be compatible with the recipient's red cells. All products containing more than 2 ml of red cells must be ABO compatible with the recipient's plasma. When RhD-positive products are given to RhD-negative recipients, RhIg should be administered.

Quality Control and Lab Safety

The critical steps of pretransfusion testing must be identified, and checkpoints implemented to prevent errors. All reagents used in pretransfusion testing, such as AHG reagent, must meet all quality criteria established by regulatory agencies or certification and accreditation bodies. Laboratory SafetyUniversal precautions must be observed when handling blood specimens in the laboratory. When disposing of the materials and blood samples, the guidelines for biomedical waste disposal must be followed. The basic elements of laboratory safety include the following:

• Regular training of the lab personnel
• Use of appropriate personal protective equipment
• Following safe work practices
• Reporting of accidents and injuries
• The protocol should be in place to manage blood spill
• Hepatitis B prophylaxis to all the technical staff

Enhancing Healthcare Team Outcomes

Ensuring that the correct blood is delivered to the right patient requires a team effort and the involvement of clinicians, nurses, and laboratory personnel. Transfusion medicine physicians play a critical role in establishing an efficient transfusion chain in a hospital. However, from the attending physician's decision to transfuse to the bedside administration of blood products, the medical staff involved, including the phlebotomist who collects the sample, the laboratory staff who performs the tests, and the staff who collects and transports the blood products to the transfusion site, which may be a ward or operating room, as well as the nurses who administer and monitor the transfusion, are an indispensable link in the transfusion chain.Transfusion medicine staff work with physicians to implement patient blood management and the rational use of blood products. They oversee blood donation, pre-transfusion testing for compatibility and blood-related antigens and antibodies, and the selection of blood for patients undergoing transplantation. Therapeutically, they handle transfusion reactions, plasmapheresis, exchange transfusions, and peripheral stem cell collections. Transfusion physicians manage and support clinical transfusion therapy, which requires modified blood products such as intrauterine transfusions and exchange blood transfusions. Transfusion laboratories apply key quality assurance systems to provide safe and high-quality blood products. Transfusion medicine is highly complex, with many opportunities for redundancy. Designing transfusion medicine processes with human factors in mind will reduce the possibility of errors. Creating causal trees provides a realistic view of how a system works and facilitates the development of effective and durable solutions. Because of the potential impact of blood administration, transfusion medicine is at the forefront of safety innovation in healthcare. The reduction and elimination of numerous clinical hazards have been demonstrated over many years. Although there is still a heavy reliance on procedural methods for the essentially manual steps that make up the phases of the transfusion chain, recognizing this continuing vulnerability has led to increased attention to the transfusion process itself. An encouraging example of this current phase of transfusion safety improvement is the creation of the new Transfusion Safety Officer role in hospitals to support efforts and progress in monitoring, identifying, and eliminating conditions that may compromise safety. Key Lessons: Safety in transfusion medicine has evolved from an exclusive focus on disease transmission and clinical outcomes to attention to error-prone processes. Knowledge of the key steps in pre-transfusion testing and the critical points in the process flow will help strengthen the transfusion chain.