Anupam Mitra, MBBS, MD Pathology Resident, PGY2
Sarah Barnhard, MD Medical Director of Transfusion Services
The main purpose of testing prior to transfusion is to provide the most compatible blood to the patient in order to minimize the risk of hemolytic transfusion reactions. The type and screen are the first two tests required as pre-transfusion testing. As the name suggests, these are two tests: “type”- to detect the ABO and Rh type of the patient’s red blood cells and “screen” – to detect the presence of antibody(ies) against RBC antigen(s). Antibody/antigen complex formation is thermal range dependent. Antibodies against RBC antigens are optimally reactive at either warm (at or above body temperature) or cold (below body temperature) thermal amplitudes. Warm antibodies are usually acquired and of IgG type. They react at or above 37C. Cold antibodies are usually naturally occurring and of IgM type. They react below 37C.1
1. What is RBC phenotyping?
The phenotype of RBCs (RBC phenotyping) refers to determining the type of antigens present on the RBC. The ABO/Rh type in the ‘type and screen’ is performed on all patients requiring transfusions. However, an extended antigen phenotype may also be performed. This determines the antigen expression other than the A, B or D antigens. Red blood cell antigen extended phenotyping is almost always performed as a reflex test. That is, extended phenotyping usually supplements routine pre-transfusion testing in patients with clinically relevant alloantibody(ies) or in patients who are at risk for making clinically relevant alloantibody(ies). Four versions of RBC extended phenotyping panels are performed in the transfusion services laboratory - ‘cold phenotype’; ‘full warm phenotype’; ‘complete phenotype’ and ‘limited phenotype’.
As the name suggests, the ‘cold phenotype’ is a panel that determines the expression of all antigens with common corresponding cold-reacting antibodies (M, N, P, Lea and Leb). The ‘full warm phenotype’ is a panel that determines the expression of all the antigens with common clinically significant corresponding antibodies that are warm reacting (K, E, e, C, c, Fya, Fyb, Jka, Jkb, S and s). The ‘complete phenotype’ is a panel that determines the expression of all antigens with common corresponding antibodies, either warm reacting (K, E, e, C, c, Fya, Fyb, Jka, Jkb, S and s) or cold reacting (M, N, P, Lea and Leb). In some cases, a ‘limited phenotype’ panel is performed to detect one or a few specific antigens. RBC phenotyping is always performed from a pre-transfusion specimen to avoid interference from transfused red blood cells.2-3
1.1 Indication to perform a red blood cell antigen full warm phenotype
A full warm phenotype may be done in several different settings.2-3
1.1.1 To prevent RBC antibody formation- patients who are receiving chronic transfusions are exposed to multiple foreign RBC antigens repeatedly over a long period of time, which increases the possibility of developing new alloantibodies. Thus, performing a full warm phenotype prior to transfusions allows the transfusion services laboratory to provide fully or partially phenotype-matched units for these patients to prevent development of alloantibody(ies). The clinical indication for a full warm RBC phenotype prior to transfusions are as follows-
- Newly diagnosed sickle cell disease
- Patients with sickle cell disease who have not previously had a full warm phenotype performed
- Other hemoglobinopathies that are transfusion dependent
1.1.2 To prevent additional RBC antibody formation- patients with RBC alloantibody(ies) are at increased risk of developing other alloantibodies, especially if they are exposed to more immunogenic antigens. Performing RBC antigen phenotyping after identifying alloantibodies is critical to provide best matched transfusions and prevent additional antibodies from forming. This is performed either by a full warm phenotype or limited phenotype based on the reflex testing pathway.
1.1.3 To prepare for medications that interfere with all testing. In patients receiving anti-CD47 (such as Hu5F9), the medication is known to interfere with the red blood cell antibody screen. A full warm phenotype is performed prior to drug administration to allow phenotype matched transfusions during the period the medication is administered when new antibodies cannot be detected and thus allow safe transfusions.
1.2 Indication to perform a red blood cell antigen cold phenotype
Phenotyping the patient’s red blood cell antigens corresponding with common antibodies that are cold-reactive is typically performed when the patient has made a cold-reacting antibody. Common scenarios include anti-M (a naturally occurring antibody common in children) or anti-Lewis (a naturally occurring antibody common in pregnancy).
1.3 Indication to perform a red blood cell antigen complete phenotype
A complete phenotype is performed when the patient has multiple antibodies that are both cold-reacting and warm-reacting. This determines the patient’s antigen profile for K, E, e, C, c, Fya, Fyb, Jka, Jkb, S, s, M, N, Lea and Leb antigens.
1.4 Indication of performing a limited antigen phenotype
A limited or partial phenotype determines one or several specific RBC antigens instead of a full or complete phenotype. It is done in the following situations-
1.4.1 To help investigate the specificity of antibodies (patients develop antibodies against the antigens they lack). If an antibody panel identifies one or several alloantibodies of unclear specificity, performing a limited antigen phenotype may help determine the specificity of the antibody(ies).
1.4.2 In any patient that has made an antibody, the laboratory performs phenotyping for the corresponding antigen as well as any antigens which are more immunogenic.
1.4.3 To evaluate risk of hemolytic disease of the newborn. Paternal or cord blood RBC antigen typing determines the antigen expression that corresponds to the maternal antibody.
1.4.4 Rarely performed after a transfusion to determine if the RBC recovery is as expected. Determining the patient’s own RBC phenotype in the post-transfusion circulating RBCs and compared it to pre-transfusion antigen typing can aid in determining the RBC recovery.
1.5 Sample collection
Blood is collected in EDTA tube (lavender top). EDTA chelates calcium and thus acts as an anticoagulant. The plasma in the sample is used for antibody testing while the red blood cells are used for antigen phenotyping.
1.6 Assay time and technique
Usually, the phenotype panels listed require testing time of 1.5-2 hrs. Determining the phenotype of some antigens requires an extended incubation time while others are determined rapidly. The assays are performed using known monoclonal or polyclonal antisera incubated with the patient’s red blood cells. Coombs reagent is required when using monoclonal IgG based antisera to induce agglutination and determine if the antigen is present.
1.7 What are the clinical implications?
In patients who require extended antigen phenotype matching, the antigen profile of any donor units that are transfused match the NEGATIVE antigens in the patient’s antigen profile in addition to any antibodies. If the patient is not chronically transfused and one or several antibodies are detected for the first time, transfusion services will perform extended antigen typing for highly immunogenic antigens and provide packed red blood cells from a donor who lacks the pertinent antigen(s). This will avoid a hemolytic transfusion reaction and formation of new antibodies. If the patient’s care will require lifelong chronic transfusions, a full warm RBC phenotype is performed prior to all transfusions. At times, the negative antigen profile is difficult to match and transfusion services pathologists need to risk stratify the antigens by immunogenicity to find the best available match. By performing this reflex testing and obtaining donor red blood cell units that are extended phenotype matched, we prevent hemolysis from current antibodies and prevent new antibodies from forming.
1.8 Limitations to RBC Phenotyping
Red blood cell antigen phenotyping cannot be performed in certain situations:
1.8.1 The Coombs test is positive (DAT+). In autoimmune hemolytic anemia (AIHA), the patient’s red blood cells are coated in IgG or IgM, an autoimmune phenomenon. The presence of the IgG/IgM causes false positive phenotype testing, and thus the antigen profile cannot be reliably determined.
1.8.2 The patient has been recently transfused. The RBC antigen phenotype is performed on a sample of the patient’s own RBCs. In the setting of recent transfusion, circulating RBCs consist of a mixture of the patient’s RBCs and the donor’s RBCs. Thus, phenotyping will not represent the patient’s native RBCs. The clinical history of transfusion is very important in this setting.
1.8.3 RBCs are coated with drugs and cause interference (anti-CD47). If the patient’s RBCs are coated with medications, then the antigen profile cannot be reliably determined.
2. What is RBC genotyping?
As the name suggests, red blood cell antigen expression may also be determined using genetic testing. Current testing allows the patient’s genomic DNA to be isolated and red cell antigen genotyping performed to predict the red cell antigen phenotype for select antigens. This is a send-out test, currently performed at Versiti laboratories. There are two panels available - Red cell genotyping panel (44 antigens) and a STAT panel (24 antigens).4
2.1 Indication of doing RBC antigen genotype -
- Patient’s red blood cell antigens cannot be reliable phenotyped due to recent history of receiving blood transfusion
- Prior to significantly interfering drugs (such as anti-CD47)
- To evaluate for genetic changes such as partial antigens that may explain alloantibodies but positive phenotyping
- In rare cases when uncommon antibodies form and antigen profiling beyond the common antigens mentioned is required.
2.2 Sample collection
EDTA (lavender top) blood- 5 ml
2.3 Methodology and turn-around time
For the 44-panel assay, 72 PCR-hybridization probes are used in 36 polymerase chain reactions to identify the alleles associated with 44 blood group antigens. For the STAT Panel, 32 PCR-hybridization probes are used in 16 polymerase chain reactions to identify the alleles associated with 24 blood group antigens.4 The 44-antigen panel turn-around time is 2-5 days, whereas the STAT (24-antigen panel) panel turn-around time is 24-48 hours.
The RBC genotype is performed using specific probes against RBC antigen alleles. Thus, any mutations outside of the targeted region will not be detected. Novel mutations leading to altered or partial antigen expression and null phenotypes may not be detected. Results from hematopoietic stem cell transplant recipients may not match the genotype obtained from other tissues.4
- Harmening, Denise. Modern Blood Banking and Transfusion Practices 5th Edition. Philadelphia PA, FA Davis Company, 2005.
- Fung MK, Grossman BJ, Hillyer CD, Westhoff CM Editors. AABB Technical Manual 18th Edition. Bethesda MD, AABB Press, 2014.
- Reid ME, Lomas-Francis C, Olsson ML. The Blood Group Antigen Facts Book 3rd Edition. London UK, Elsevier. 2012.
- Red Blood Cell Genotyping Panels. Versiti Laboratories, Blood Center of Wisconsin. Information online at: https://www.versiti.org/Custom/Files/Versiti/68/682cf64b-4508-4714-ae29-7f1a414b7a1e.pdf