| Case Study: Immune Alloantibody A 42-year-old male received 6 units of RBCs during open heart surgery 6 months ago. His antibody screen was negative at that time. He has returned for a follow up surgery and his antibody screen is now positive with both screen cells at the AHG phase.Reactions are occurring at AHG phase, which indicates a possible clinically significant antibody, Jka showing dosage. Refer to Case Study 1 panel below to see reactions of antibody panel.IS = Immediate Spin; AHG = Antihuman Globulin Phase; CC = Check Cells; AC = Auto Control; ND= Not doneCase study 1 conclusion:Patient's previous transfusion 6 months ago exposed him to the Jka antigen, causing the formation of this antibody, which is known for showing dosage. | View Page |
| Initial Observations of Antibody Panel Look at the phase in which reactions are occurring. Reactions at immediate spin (IS) usually are not clinically significant. Reactions at AHG are clinically significant. Check for a match in the reactivity pattern by comparing sample reactions and individual antibody reactions Varying strengths of reactions could indicate dosage. Dosage means that there are two "doses" of the same antigen present on the red cells . Antibodies that exhibit dosage react more strongly with homozygous cells (e.g., Jkb Jkb ) than with heterozygous cells (e.g., Jka Jkb) . | View Page |
| Cold antibodies Most are IgM and not clinically significant May interfere with detection of clinically significant antibodies if they react at AHG phase. Screen cells and panel cells will have positive reactions in IS phase and strength will diminish or antibody will not be detected at AHG phase. Auto control will be positive if the cold antibody is an autoantibody. Binding of antibody to antigen occurs at room or colder temperatures and may start to disassociate from the red cell membrane at warmer temperatures. Reactions will appear weaker or be negative at warmer temperatures. (Example: 4+ at IS phase and W (weak)+ at AHG phase.) PrewarmingIf a non specific cold antibody or cold agglutinin is suspected, warm the sample and testing reagents, including saline, to 37° C. Only do reaction readings at AHG; bypassing the optimum reaction temperature prevents activation and binding of the cold antibody . | View Page |
| Effect of Enzymes and Dithiothreitol (DTT) Antibody Effect of Enzymes Effect of DTT Anti- D Ficin and Papain Enhanced DTT Resistant Anti-C Ficin and Papain Enhanced DTT Resistant Anti-c Ficin and Papain Enhanced DTT Resistant Anti-E Ficin and Papain Enhanced DTT Resistant Acid Resistant Anti-e Ficin and Papain Enhanced DTT Resistant Anti-Cw Ficin and Papain Enhanced DTT Resistant Anti-K Ficin and Papain Resistant DTT Sensitive Anti-k Ficin and Papain Resistant DTT Sensitive Anti-Kpa Ficin and Papain Resistant DTT Sensitive Anti-Jsa Ficin and Papain Resistant DTT Sensitive Anti-Fya Ficin and Papain Sensitive DTT Resistant Anti-Fyb Ficin and Papain Sensitive DTT Resistant Anti-Jka Ficin and Papain Enhanced DTT Resistant Anti-Jkb Ficin and Papain Enhanced DTT Resistant Anti-Lea Ficin and Papain Enhanced DTT Resistant Anti-Leb Ficin and Papain Enhanced DTT Resistant Anti-P1 Ficin and Papain Enhanced DTT Resistant Anti-M Ficin and Papain Sensitive DTT Resistant Anti-N Ficin and Papain Sensitive DTT Resistant Anti-S Usually Ficin and Papain Sensitive; Some Variable DTT Resistant Anti-s Usually Ficin and Papain Sensitive; Some Variable DTT Resistant | View Page |
| Case Study Two- Explanation Possible antibody is anti-C based on matching reaction pattern of sample at AHG. At least 3 positive reactions are present to rule in this antibody.Pink: negative reactions to use for rule-outsTurquoise: homozygous reactions used for rule-out (exceptions to homozygous rule are Rh group and Kk) Antibodies that can be ruled-out using "3 to rule out" rule: D, c, E, e, K, k, Fya, Fyb, Jka, Jkb, Lea, Leb, M, N, S, s, P, LubAntibodies that cannot be ruled out: Cw, Kpa, Jsa, LuaPoints to remember: The pattern of positives and negatives on an antibody panel cell indicates whether that particular antigen is present on the testing cells The phase in which the reactions are occurring will help determine if it is an IgG clinically significant antibody or IgM antibody (usually not considered clinically significant). Stronger reactions seen if antibody exhibiting dosage. Think multiple antibodies if reactions occurring at different reaction phases. | View Page |
| Panel 1 Example- Rule Outs Cells 4 and 9 may be used for rule outs due to negative sample reaction. Screen cell I may be used for rule outs due to negative sample reaction. Look at the antigens present on cells 4 and 9 that are in the homozygous state (highlighted in green). Remember the 3 to rule in and 3 to rule out procedure. Antibodies ruled out (with 3 reactions): e, k, Kpb, Jsb, Jka, Leb, P1, Lub. A selected panel should be set up to rule out (with 3 reactions) the remaining clinically significant antibodies (E, D, C,c, K, Fya, Fyb, Lea, M,N, S, and s). | View Page |
| Example of Dosage and/or Multiple Antibodies Influencing the Strength of Reactions Varying reaction strengths in the same phase could indicate antibody showing dosage, multiple antibodies, or both.Jka and S are the antibodies that are present. Weaker reactions can be seen when either of the target antigens is present alone and/or in the heterozygous state on the cell.4+ reaction in panel cell 1, 4 and 9: Both Jka and S are present4+ reaction in panel cell 7 and 10: S present (homozygous)3+ reaction in panel cell 6: Jka present (homozygous)3+ reaction in panel cells 2 and 8: S present (heterozygous)2+ reaction in panel cell 5: Jka present (heterozygous) | View Page |
| When to Use an Enzyme Panel - Results on a Regular Panel Rule-outs can be done using screen cell I and panel cells 4 and 8 (highlighted in green) Antibodies ruled out using these panel and screen cells: C, e, Kpb, Jsb, Jka, Leb, M, P1 and Lub Performing an enzyme panel could help further identify the suspected antibodies. Antibodies needing rule out: D, c, E,K, k, Fya, Fyb, Jkb, Lea, N, S, s If these antibodies are present, a stronger reaction will be seen on the enzyme panel: D, c, E, Jkb, Lea. If these antibodies are present, there will be no reaction on the enzyme panel, since the antigens are destroyed by enzymes: Fya, Fyb, N, S, s. | View Page |
| Reactions with an Enzyme Panel D, E, and Lea did not react with the enzyme panel cells (in green). If they had been present, the reactions would have been enhanced. Fya, Fyb, N, S, and s did not react with the enzyme panel cells (some are shown in green).Looking at the enzyme panel results, we can see the reaction pattern of c (in yellow) and the pattern of non-reaction for Fya (in pink). Suspected multiple antibodies are c and Fya. Fya will not react on the enzyme panel since the Duffy antigens are destroyed by enzymes. Enzymes will enhance the reaction of anti-c. | View Page |
| Multiple Antibodies: Example In this example the patient's plasma tests positive with both screening cells at a strength of 4+. In the panel below, reaction patterns show varying strengths, 2+ to 4+ (highlighted in green).4+ could indicate one strong antibody or a combination of several antibodies that increases the strength of the reaction.3+ could indicate the presence of just one strong antibody.2+ could indicate a weaker reaction of an antibody that commonly exhibits dosage if the panel cell is in the heterozygous state.Since Cw, Kpa, Jsa, Lua are not present on the testing cells, they are probably not causing these reactions. Perform rule outs using panel cells 5 and 7 (sample had no reaction in any phase with these panel cells)Antibodies that can probably be ruled out at this point because the corresponding antigens are present on cell 5 and/or 7: C, c, e, k, Kpb, Jsb, Fya, Jkb, Lea, M, N, s, P1, LubAntibodies that could not be ruled out with this panel: D,E, K, Fyb, Jka, Leb, SPredominant pattern of 4+ in panel cells 1,2,4,10 matches anti-D Varying strengths in reactions indicates a possible second antibody so selected cells should be picked to aid in identificationFind panel cells that do not contain D (antibody you suspect) and are homozygous positive for the antibodies you are trying to rule out. | View Page |
| Example- Choosing Selected Cells The selected cells should be antigen-negative for the antibody that you think is present and antigen-positive (homozygous) for what you are trying to rule out. You are designing a panel that addresses your testing needs. Example: JkbIf you suspect that your patient has an anti-Jkb and further rule out cells are needed, then those rule out cells should be negative for Jkb. In the table below, donor cells 1,2, 4, 6, 9 and 10 may be used when creating a select panel to test the patient and help rule out the remaining possible antibodies. The homozygous rule applies when choosing which cells to use for testing (antigens highlighted in light-yellow).Example: Picking cells to rule out CUse panel cell 1 and panel cell 2 (C is in the homozygous state). Explanation: Panel cells 1 and 2 do not contain the antigen Jkb (signified by "0" on cell panel). If these cells are tested with the patient's plasma and the reaction is negative, it can be assumed that the patient does not have an antibody to C. C is now ruled out because there would be a total of 3 negative patient reactions with C positive cells (These two reactions and screen cell I from the antibody screen, shown again below). This should be done for all clinically significant antibodies that you were unable to rule out on the first panel. | View Page |
| Case Study Three - Selected Cells List panel cells to test for ruling-in or ruling-out remaining antibodies in Case Study Three. These would be your selected cells. For rule-out, selected cells should be negative for the antigens that correspond to the antibodies you have possibly identified. In this case, the selected cells for rule-out should be antigen-negative for K and Fya. If you are trying to rule in a possible antibody like K, then the panel cell should be positive for that corresponding antigen so that reactions will occur if the antibody is present.Panel cells 1 and 7 could be used for rule-in of K.Panel cells 2, 4, 5, 6, and 9 can be used for rule-outsPanel cell 2: to rule out C, e, Fyb, Jka, N, s Panel cell 4: to rule out Jka, Lea, N, SPanel cell 5: to rule out C, e, Jkb,MPanel cell 6: to rule out E, Jkb, Lea, N, and sPanel cell 9: to rule out M, S | View Page |
| Case Study Three Rule-Outs Key Antibodies ruled out with 3 reactions: D, c, k, Kpb, Jsb, Leb, P1, and Lub (panel cells used for rule out are in green). Antibodies still needing selected cells for rule outs: C, Lea, E, M, Jka, Jkb, S, s (need 2 reactions)Fya, N, K (need 3 reactions)e, Fyb (needs 1 reaction) Jsa, Kpa, Cw, and Lua all need three reactions for rule-out but these are all low-frequency antigens. It is difficult to find panel cells with these antigens present to allow testing. They will fall in the "unable to rule out" category.Reactions are occurring in the AHG phase only and there is varying strengths of reactivity, which could indicate dosage and/or multiple antibodies.The pattern of reactivity closely matches Fya (cells 2,5,7,8,9 are positive). Of the remaining antibodies that have no rule-out reactions, anti-K is the possible second antibody (present on cell 2 and 10 and screen cell I). Explanation for the varying strengths in reactions: Panel cell 2: Fya (heterozygous) and K present so stronger reaction of 4+. Panel cell 5 and 8: Fya is heterozygous, so weaker reaction of 2+. Panel cell 7 and 9: Fya is homozygous, so stronger reaction of 3+. Panel cell 10: K is (homozygous, so stronger reaction of 3+. | View Page |
| Ruling Out Procedures, continued: Selecting Additional Rule-Out Cells Once an antibody hypothesis is generated, most laboratories will select additional cells to rule-out any other commonly encountered antibodies that could not be ruled-out with the initial antibody screen and panel. Cells should be selected that are negative for the antigen(s) that correspond to the hypothesized antibody and positive for the antigen (s) to commonly encountered antibodies that have not been ruled out. If not ruled-out most laboratories will select cells for at least the following: anti-D, anti-C, anti-c, anti-E, anti-e, anti-K, anti-k, anti-Fya, anti-Fyb, Anti-Jka, anti-Jkb, anti-Lea, anti-Leb, anti-P1, anti-M, anti-N, anti-S, and anti-s. Antibodies to antigens of very low incidence (for example, anti-Jsa) are generally not eliminated in initial testing, but in most settings it is not feasible to try and find rule-out cells. In these cases, it is important for the technologist to understand that these antibodies HAVE NOT been ruled-out due to limitations in the test system. | View Page |
| Selected Cell Panels Purpose: To design a set of panel cells that may help you to rule out additional antibodies and lead to the identification of the antibody that is present in the patient's plasma.Benefit of running selected cell panel: Decreases the use of reagents and specimen. How to choose selected panel cells: If you suspect that a specific antibody is present, the cells you choose for the select panel should be negative for that antigen and positive for the antigen you are trying to rule out (homozygous state). | View Page |
| Rule-Out Procedures Rule-out (also referred to as exclusion or cross-out) is a process by which antibodies are identified as being unlikely in a given sample because of the absence of an expected antigen-antibody reaction. In other words, the absence of a reaction is noted with a cell that is positive for the corresponding antigen. Rule-out, while very useful, can lead to error. Ruling out an antibody should be combined with other supporting data to increase confidence in the solution; the more data collected, the higher the probability that the final solution is correct.Non-reactive cells are selected for rule-out. To be classified as non-reactive, a cell must NOT have reacted in any phase of testing in a given panel or screen. In the case of cold antibodies: if reactions are only occurring at immediate spin and are negative in the AHG phase, then that panel cell can be used as a rule out cell for IgG reactive antibodies but not for antibodies that react at immediate spin (IgM).If there is no reaction with a panel cell then it is possible that antibodies to the antigens on that cell are not present in the sample being tested. | View Page |
| When to Suspect Dosage Suspect dosage if varying strengths in reactivity are seen and reactions are in the same phase. Weaker reactions will be seen if suspected antibody is reacting with antigens in the heterozygous state. Stronger reactions are seen if the antigen is present on the testing cells in the homozygous state. This allows more corresponding antibody to bind with the antigen. Remember the antibodies known for showing dosage are: Rh, Kidd, Duffy, MNSs, and Lutheran. Dosage may be seen if cells are R2R2 (DcE/DcE). These red cells have more D antigen sites so reaction with anti-D may be stronger.Refer to Example 5 on the following page. | View Page |
| Case Study Four- Selected Cell Panel Cells 5 and 8 can be used for rule-out cells. Jkb, Lea, M, N, and s still need more rule-out cells. P1, C, E and Fya have no cells for rule-out.Running an enzyme panel would help to enhance Jkb, Lea,P1 C and E if these antibodies are present.If M,N,s and Fya are present, no reaction would be seen because these antigens are destroyed by enzymes. | View Page |
| Case Study Four- Selected Cell Panel 2 No reactions were seen with panel cell 1 and 2. This rules out M,N,and s.Notice that C, E, Fya, and P1 are not present on these cells so as not to interfere with ruling-in or ruling-out of the remaining antibodies.ConclusionThrough the processes of ruling-in and ruling-out and matching of reaction patterns, the antibodies that are identified are C,E, Fya, and P1.If the patient has not been transfused in the past 3 months, antigen typing for Fya may be done to further confirm the presence of Fya.Elution and autoadsorptions may also be necessary to confirm the presence of C,E, Fya and P1. | View Page |
| Naturally Occurring Antibodies Antibodies are immunoglobulin proteins secreted by B-lymphocytes after stimulation by a specific antigen. The antibody formed binds to the specific antigen in order to mark the antigen for destruction.The type of antigenic exposure occurring in the body determines if the antibody is a naturally occurring or immune antibody.Naturally occurring antibodies can be formed after exposure to environmental agents that are similar to red cell antigens, such as bacteria, dust or pollen. Sensitization through previous transfusions, pregnancy or injections is not necessary. These antibodies are usually IgM and react best at room temperature or lower. Most of these antibodies are not clinically significant with the exception of ABO antibodies. Examples of naturally occurring antibodies include anti-A, anti-B, anti-Cw, anti-M, and antibodies in the Lewis and P system. | View Page |
| Immune Antibodies Immune antibodies occur in the serum of individuals who become sensitized to foreign antigens through pregnancy or transfusion. IgM predominates in the primary response, IgG in the secondary response. Most react at 37°C and are considered clinically significant. Examples include antibodies in the Kell, Rh, Duffy, and Kidd systems. Immune antibodies can be classified as alloantibodies or autoantibodies.Alloantibodies Produced by exposure to foreign red cell antigens which are non-self antigens but are of the same species. They react only with allogenic cells. Exposure occurs through pregnancy or transfusion. Examples include anti-K and anti-E. Autoantibodies Produced in an autoimmune process and directed against one's own red cell antigens. React with patient's own cells and all cells tested. Can possibly mask the presence of other significant antibodies. It is very important to make sure that no underlying significant antibodies are present if an autoantibody is suspected. A positive direct antiglobulin test (DAT) or auto control could indicate the presence of an autoantibody. Examples include cold auto (P or I) or warm auto (Rh specificity). | View Page |
| Naturally occurring antibodies found in the ABO blood group system may be due to exposure to which of the following? | View Page |
| Products Used to Facilitate Antibody Identification Monospecific anti-human globulin (IgG) enables sensitized red cells to cross-link so that agglutination is visible.Enhancement media are sometimes used to further promote agglutination and reduce incubation time. Low ionic strength saline (LISS) is the most common enhancement media. LISS reduces the ionic strength in the testing sample and causes reduction of the zeta potential. It increases antibody uptake and decreases incubation time. Polyethylene glycol (PEG): brings red blood cells (RBCs) closer together and concentrates antibodies by removing water molecules from the testing sample. It is the most sensitive of the enhancement media; strengthening almost all clinically significant antibodies. However, it will also enhance some clinically insignificant antibodies as well. Centrifugation should be avoided when PEG is used. PEG can cause aggregates to form if the sample (red cell - serum mixture) with PEG added is centrifuged. Reaction readings should only be done at the AHG phase. 22% albumin: reduces zeta potential, bringing the RBCs closer together and enhancing agglutination. Albumin does not contribute much to antibody uptake. Longer incubation time is needed with this media than with the previously discussed media. Detection of some IgG antibodies can be enhanced with enzyme test methods. Proteolytic enzymes (papain and ficin) denature some RBC antigens and remove negative charges from the RBC membranes. This reduces the zeta potential, bringing the cells closer together. Enzyme techniques are particularly useful in the identification of Rh antibodies and antibodies in the Kidd, Lewis, P and I systems. However, enzymes destroy some antigens including Fya, Fyb, M, and N. The effect of proteolytic enzymes on the S and s antigens are variable. | View Page |
| Antibodies to Low- and High-Incidence Antigens Low-incidence antigens are antigens that occur in less than 1% of the population.Antibodies to low-incidence antigens Low-incidence antigens are not usually found on screen cell and antibody panels. Antibodies are hard to test for, but it is usually not difficult to find compatible blood. Suspect this antibody if an AHG crossmatch is incompatible and other causes have been ruled out, such as a positive donor DAT or ABO incompatibility. Examples of low-incidence antigens include: Cw, V, Kpa, Jsa. When going through the process of Ruling Out, antibodies like anti-V, anti-Cw, anti-Lua, anti-Kpa, and anti-Jsa usually fall into the "unable to rule out" category. High-incidence antigens are antigens that occur in greater than 99% of the population. Antibodies to high-incidence antigens Antibodies are rare and may be difficult to identify due to lack of negative panel cells for other high-incidence antigens (difficult to rule out). Reactions with screen and panel cells will all be positive (same strength and same phase). Auto control will be negative. Difficult to find antigen-negative compatible blood. Examples of antibodies to high-incidence antigens are: anti-k, anti-Kpb, anti-Jsb, and anti-Lub. If an antibody to either a high- or low-incidence antigen is present, it may be difficult to identify and may require further testing in a reference blood bank. | View Page |
| Examples of Antibodies to High-Incidence Antigens Suspect an antibody to a high-incidence antigen if: Reactions with all panel and screen cells are positive (same strength and same phase) Auto control is negative Antibodies to high-incidence antigens include: anti-k, anti-Kpb, anti-Jsb, anti-Lub (highlighted in turquoise) | View Page |
| Examples of Antibodies to Low-Incidence Antigens Antibodies to low-incidence antigens will be difficult to test for since most screen and panel cells do not have these antigens on the testing cells. Further testing may be needed at a reference laboratory where a larger selection of antibody panels are available to locate cells positive for these antigens.Suspect an antibody to a low-incidence antigen if: AHG crossmatch is incompatible and Other causes have been ruled out (positive donor DAT, ABO incompatibility) Examples of antibodies to low-incidence antigens are: anti-V, anti-Cw, anti-Kpa, anti-Jsa, and anti-Lua. | View Page |
| Fluorescent ANA Testing The most common method of ANA testing is indirect fluorescent assay (IFA) utilizing fluorescein isothiocyanate (FITC) as the marker on the secondary antibody.The fluorescent ANA test uses the indirect fluorescent antibody technique first described by Weller and Coons in 1954. Patient serum samples are incubated with antigen substrate to allow specific binding of autoantibodies to cell nuclei. If ANAs are present, a stable antigen-antibody complex is formed.After washing to remove non-specifically bound antibodies, the substrate is incubated with an anti-human antibody conjugated to fluorescein. When results are positive, a stable three-part complex forms, consisting of fluorescent antibody bound to human antinuclear antibody that is bound to nuclear antigen. This complex can be visualized with the aid of a fluorescent microscope. In positive samples, the cell nuclei will show a bright apple-green fluorescence with a staining pattern characteristic of the particular nuclear antigen distribution within the cells. If the sample is negative for ANA, the nucleus will show no clearly discernible pattern of nuclear fluorescence. The cytoplasm may demonstrate weak staining while the non-chromosome region of mitotic cells demonstrates brighter staining.The photo to the right demonstrates the 4 basic ANA patterns (clockwise from top left): Homogeneous, Speckled, Centromere, and Nucleolar. (Additional photos of these patterns will be seen in subsequent sections.) | View Page |
| Colorzyme® A similar procedure that is also widely used is called Colorzyme®.(Ref7) This system uses horseradish peroxidase rather than FITC as the marker on the secondary antibody. This technique offers the same advantages as the IFA procedure but also has the added benefits of being more photo-stable and not requiring a fluorescent microscope. The Colorzyme® ANA Test utilizes the indirect enzyme antibody technique. Patient serum samples are incubated with antigen substrate to allow specific binding of autoantibodies to cell nuclei. If ANA's are present, a stable antigen-antibody complex is formed. After washing to remove non-specifically bound antibodies, the substrate is incubated with an anti-human antibody reagent conjugated to horseradish peroxidase. When results are positive, there is the formation of a stable three-part complex consisting of enzyme antibody bound to human antinuclear antibody that is bound to nuclear antigen. This complex can be visualized by incubating the slide in an enzyme specific substrate. The reaction between the enzyme labeled antibody and enzyme specific substrate results in a color reaction on the slide visible by standard light microscopy. In positive samples, the cell nuclei will show a bright bluish purple staining with a pattern characteristic of the particular nuclear antigen distribution within the cells. If the sample is negative for ANA, the nucleus will show no clearly discernible pattern of nuclear staining. The cytoplasm may demonstrate weak staining while the non-chromosome region of the mitotic cells may demonstrate a darker staining. The photo to the right demonstrates the 4 basic ANA patterns (clockwise from top left): Homogeneous, Speckled, Centromere, and Nucleolar. (Additional photos of these patterns will be seen in subsequent sections.) | View Page |
| History of ANA Testing Slide-based ANA testing using a cell substrate started in the 1950s and continues to be the gold standard method. In the early days of ANA testing, rodent tissue (stomach, liver and/or kidney) was commonly used as the substrate. Rodent tissue however had several drawbacks such as small cell size, a lack of dividing cells (mitotics) and poor antigen expression that made interpretation of ANA patterns difficult. In the 1980s, cultured cell lines were examined for utility as an ANA substrate and the human epithelial- like cell line HEp-2 gained popularity. HEp-2's advantages over rodent tissue are: A large nucleus Better antigen expression Abundant mitotic cells that assist in interpretation of the ANA pattern (if grown properly).More recently a cell line called HEp-2000® has become popular for ANA detection. HEp-2000® is a HEp-2 cell line that has been transfected with the cDNA for overexpression of the SSA/Ro antigen. This results in a substrate with all of the original advantages of HEp-2 plus an added advantage of increased sensitivity for detection of antibodies directed to the SSA/Ro antigen and the ability to identify these clinically significant antibodies during the screening process.(Ref4)It has also been demonstrated that antibodies to SSA/Ro develop early in the disease process.(Ref5) Perhaps most importantly, if a woman has anti-SSA/Ro antibodies and becomes pregnant there is a risk of the antibodies crossing the placenta, resulting in the fetus developing neonatal lupus and congenital heart block in utero.The advantage of using these transfected cells is documented in the current Clinical and Laboratory Standards Institute (CLSI) guidelines for ANA testing. Here they note the "dramatically increased" sensitivity of transfected cells for the detection of SS-A/Ro and the unaltered effect of transfection on other ANA patterns.(Ref6) | View Page |
| Antigen Chart Pattern observed by indirect immuno fluorescence Antigen Disease(s) in which antibodies are seen Routine tests used to confirm specific antibody Homogeneous Double stranded DNA (dsDNA) Characteristic of SLE, lower levels in other rheumatic diseases IFA or CZ using Crithidia luciliae, RIA, ELISA, Addressable Laser Bead Assay (ALBIA) Nucleosome or Chromatin SLE, Drug-induced LE ELISA Histone Drug-induce LE, SLE ELISA, ALBIA Unusual Homogeneous Nuclear Membrane Lupoid hepatitis ELISA for gp-210 Speckled Sm (Smith) Marker antibody for SLE Immunodiffusion (ID), ELISA, ALBIA U1-RNP High levels in MCTD and SLE, low levels in other rheumatic diseases ID, ELISA, ALBIA Speckled (and/or SSA pattern if using HEp-2000®) Can also be ANA negative SS-A/Ro High prevalence in Sjögren syndrome sicca complex, lower prevalence in other rheumatic diseases With HEp-2000 characteristic ANA pattern is confirmatory, others confirm with ID, ELISA, ALBIA Fine speckled or ANA negative Ro52 Sjögren syndrome, myositis, Neonatal Lupus ELISA, ALBIA Fine speckled (sometimes with nucleolar staining as well) SS-B/La High prevalence in Sjögren syndrome sicca complex, lower prevalence in other rheumatic diseases ID, ELISA, ALBIA Fine speckled, Homogeneous, Nucleolar Scl-70 Marker antibody for Scleroderma ID, ELISA, ALBIA Cell Cycle Dependent Speckled PCNA Marker antibody for SLE ID, ELISA, ALBIA Coarse Speckled Nuclear Matrix Seen in some patients with evolving connective tissue disease NONE 3-20 dots NSp I, sp-100, MND, PBC 95 Associated with Primary Biliary Cirrhosis ELISA, ALBIA Cell Cycle Dependent Speckled with speckling in metaphase mitotics NSp II, CENP F Some association with malignancies NONE Staining in cleavage furrow between dividing cells Midbody Unknown Confirm by staining pattern Centromere CENP A, CENP B, CENP C Seen in 57-82% of patients with limited form (CREST) of scleroderma and Raynaud phenomenon Confirmed by staining pattern ELISA, ALBIA Nucleolar Fibrillarin (Clumpy nucleolar) Scleroderma ELISA, ALBIA RNA polymerase I, NOR-90, others? (Speckled nucleolar) Scleroderma and other connective tissue diseases ELISA, ALBIA PM-1 (PM/Scl), others?(Smooth nucleolar) Polymyositis/Scleroderma overlap ELISA, ALBIA | View Page |
| Nucleolar This is an example of a Nucleolar ANA pattern.This pattern is characterized by staining in the nucleoli of the interphase cells (a). The nucleolar staining can display subtle variations in staining inside the nucleoli including smooth, speckled and clumpy. All are reported as ANA positive, Nucleolar. In this sample staining is present in the chromosomal area of the metaphase mitotic cells (b) along with some staining in the area outside of the chromosomal area. The staining of the mitotics can be different with different anti-nucleolar antibodies. Remember the ANA pattern is determined by staining in the interphase cells and the mitotics are used to assist in interpretation.Follow-up testing for anti-nucleolar antibodies is very limited and in most cases is not done. There are assays for identification of anti-RNP polymerase antibodies. Anti-nucleolar antibodies are primarily seen in patients with systemic sclerosis.This pattern is reported as ANA positive, Nucleolar; titering is necessary.Nucleolar:Interphase cells Staining only of the nucleoli Clumpy Smooth Speckled Metaphase mitotic cells Can demonstrate variable staining depending on the antigen involved | View Page |
| SSA/Ro Pattern SSA/Ro on HEp-2000®This is an example of the SSA/Ro ANA pattern using the HEp-2000® substrate. (This hyperexpressing pattern is not seen on standard HEp-2 substrates.)As stated earlier, the HEp-2000® substrate utilizes genetically engineered HEp-2 cells with increased expression of SS-A/Ro antigen in the cells.When the patient sample contains autoantibodies to the SS-A/Ro antigen, approximately 90% of the time the sample will produce a distinctive pattern with 10 - 15% of the hyperexpressing cells showing strong speckled staining, frequently with strong nucleolar staining (a). The metaphase mitotic cells are negative (b). The remaining cells demonstrate weak speckled and nucleolar staining (c). The presence of anti-SSA/Ro antibodies is confirmed by the unique staining pattern. Follow-up testing for antibodies to other extractable nuclear antigens (ENAs) is recommended. These antibodies are seen in patients with SLE, Sjögren's syndrome and low frequency in other diseases.This pattern is reported as ANA positive, SSA/Ro pattern, anti-SSA/Ro antibodies present. Most labs will titer these samples. However, because the significance is that the anti-SSA antibodies are present regardless of the titer endpoint, some labs do not titer the SSA/Ro pattern.SS-A/Ro (HEp-2000® only) Interphase:Seen with about 89% of samples containing anti-SS-A/Ro antibodiesHyperexpressing interphase cells 10-15% hyperexpress the SS-A/Ro antigen Stronger nucleolar staining Stronger speckled staining Non-hyperexpressing interphase cells May or may not stainSS-A/Ro (HEp-2000® only) Metaphase:Metaphase mitotic cells No staining in the chromosome region Region outside of the chromosome area will stain with variable intensitySome mitotics may also demonstrate brighter staining | View Page |
| Proliferating Cell Nuclear Antigen (PCNA) This is an example of a cell cycle dependent speckled ANA pattern called anti-proliferating cell nuclear antigen (PCNA).With this pattern the antigen that the antibodies are directed to is only expressed during a limited portion of the cell's growth cycle. During other parts of the growth cycle the antigen is not expressed. This creates a pattern where only 30-50% of the cells stain positive. The speckled staining within these positive cells varies between coarse speckled (a) and smooth speckled (b). Cells not expressing the antigen are negative (c).Follow-up testing to confirm antibodies to PCNA is recommended. Anti-PCNA antibodies are specific for SLE.This pattern is reported as ANA positive, Speckled, possible PCNA; titering is necessary.Proliferating Cell Nuclear Antigen (PCNA)Cell cycle dependent pattern:Antigen not expressed in all growth stagesInterphase cells 30-50% of interphase cells stain Coarse to fine speckling in the nucleus Nucleoli do not stain Metaphase mitotic cellsNo staining at this stage of the cell cycle | View Page |
| Other Mixed Patterns Mixed patterns are not limited just to the ones viewed previously. Virtually any combination is possible.Other Combinations: Centromere and SS-A/Ro (HEp-2000®) Homogeneous, speckled, and SS-A/Ro (HEp-2000®) Homogeneous and nucleolar Both patterns may be caused by antibodies to one antigen Remember: any combination is possible! | View Page |
| Scl-70 Antibodies to Scl-70, also known as anti-topoisomerase I antibodies, produce three ANA patterns: homogeneous, speckled and nucleolar. This is because the Scl-70 antigen is present in all three areas. In photographs it is difficult to capture the fine granular speckled staining (a), therefore, in the following photos the patterns are best described as Homogeneous (b) and Nucleolar (c).In cases of mixed homogeneous and speckled ANAs, follow-up testing for anti-dsDNA and anti-ENA antibodies is necessary.When mixed patterns are titered, the endpoint for each pattern is reported.This pattern is reported as ANA positive, Homogeneous, Nucleolar and Speckled; titering is necessary. (It should be noted that anti-Scl-70 cannot be identified simply by the ANA patterns. Follow-up ENA testing is necessary.)Scl-70:Antibodies target topoisomerase IDetection methodsANA: Three patterns present Homogeneous, Specked and Nucleolar Rarely seen with other marker antibodies Confirm by: ID, ELISA, WB, others Clinical significance Poor prognosis May precede onset of symptoms | View Page |
| Why would a unit of group O blood never be administered to a Bombay patient: | View Page |
| Anti-H: | View Page |
| Which of the following blood group antigens are most susceptible to destruction by the action of enzymes: | View Page |
| Which of the following group B antigens is generally associated with a mixed field reaction: | View Page |
| The term used to describe patients with absence of Rh antigens is: | View Page |
| The classification of Du refers to: | View Page |
| All of the following cellular antigens are important to an immunohematologist except: | View Page |
| Deglycerolized red cells are most effectively used to: | View Page |
| In HDN which of the following antigen-antibody reactions is occurring: | View Page |
| Which of the following antibodies is detected primarily in the antiglobulin phase of the crossmatch: | View Page |
| Which of the following blood group antigen-antibody reactions is enhanced by using enzymes: | View Page |
| Which of the following blood groups reacts least strongly with Anti-H: | View Page |
| If an Rh negative patient is administered a unit of R1R1 packed red cells, which one of the following antibodies would be most likely to develop: | View Page |
| Which of the following best describes the direct antiglobulin test principle: | View Page |
| An Rh positive individual is always positive for which of the following antigens: | View Page |
| Avidity is best described by which of the following statements: | View Page |
| Which of the following options gives in order from most to least important, the factors you would use to select blood for a transfusion: | View Page |
| Which of the following statements is not true about the Lewis blood group: | View Page |
| Which of the following is not a major Rh antigen: | View Page |
| Which of the following best describes the primary function of antibodies: | View Page |
| The most definite indication that a patient has been sensitized to a specific red cell antigen is: | View Page |
| DR antigens are found in which of the following systems: | View Page |
| Patients with antibody to the following antigen are immune to Hepatitis B: | View Page |
| Which of the following statements best describes Rh antibodies: | View Page |
| Which of the following Rh antigens is found the highest frequency in the Caucasian population: | View Page |
| Which of the following set of conditions would preclude hemolytic disease of the newborn as a result of ABO incompatibility: | View Page |
| Pre-transfusion testing should include all of the following except: | View Page |
| To detect the presence of blocking antibodies fixed on the red cells of a newborn infant: | View Page |
| Which of the following Rh antigens is found the highest frequency in the Caucasian population: | View Page |
| Unexpected positive reactions encountered during forward ABO typing may be due to: | View Page |
| The antigen marker most closely associated with transmission of HBV infections is: | View Page |
| HLA-A and HLA-B antigens can be detected using which of the following techniques? | View Page |
| Which of the following antigen groups is closely related to the ABO system: | View Page |
| Proteolytic enzyme techniques may be useful in identifying which of the following antigen groups: | View Page |
| Which of the following red blood cells contain the most H antigen: | View Page |
| A patient's serum reacts with all reagent red cell samples. The autocontrol is negative. An alloantibody to a high incidence antigen is suspected. Which of the following would be most likely to be a compatible donor: | View Page |
| The two or three reagent cells used for antibody screening will detect which of the following: | View Page |
| HLA antigen testing may be used for all except the following: | View Page |
| ABO blood groups were discovered by: | View Page |
| Immunization to D Antigen Since anti-D produces the most severe HDFN and was once relatively common, let's begin by reviewing how anti-D is produced.Immunization to D may occur when Rh-negative individuals are exposed to the D antigen, but developing anti-D varies greatly from person to person. Some individuals produce anti-D after being exposed to a small volume of D-positive red cells (e.g., 0.1 mL). For others, a relatively large volume of D-positive cells is required. Yet other persons will never produce anti-D, regardless of exposure. | View Page |
| Typical Case of Rh HDFN (Prior to RhIg) RhIg became widely available in 1968. Prior to that, HDFN due to anti-D typically developed as described below. Cases were much more prevalent in Caucasians due to the relative incidence of the D antigen in various populations, For example, approximate incidence of D+ individuals: Caucasians (European ancestry): ~85% African-Americans: ~93% Asians: ~99%In the first pregnancy, Rh positive fetal red cells enter the maternal circulation during the pregnancy and/or at delivery. The mother has a 1o immune response in which mainly IgM antibody is produced, with lower levels of IgG anti-D produced. Thus the first infant is rarely affected because: Larger fetal bleeds occur at delivery and these are more likely to cause antibody production than smaller antenatal bleeds. Antibody is produced slowly and is mostly IgM. In the second pregnancy, if the fetus is again D-positive, when fetal cells enter the mother, they cause a 2o immune response in which higher levels of IgG anti-D are produced. Depending on the antibody titer, the second child may suffer mild to severe HDN. If a third or fourth pregnancy results in D-positive infants, these infants (by also bleeding into the mother) cause the production of even higher titers of IgG anti-D and offspring will be more severely affected, perhaps dying in utero or soon after birth, if untreated. | View Page |
| Primary versus Secondary Response To understand the history of HDFN due to anti-D, it is useful to review the immune response. A primary (1°) immune response is the response that occurs following the first exposure to a foreign antigen. A secondary (2°)/anamnestic immune response occurs following subsequent exposures. The main differentiating features as related to producing anti-D during pregnancy are shown in the table and figure. 1o immune response 2o immune response 1. Following the first exposure to the D antigen, a lag phase occurs in which no anti-D is produced, but activated B cells differentiate into plasma cells. The lag phase can be as short as several days, but often is longer. 1. When exposure to D occurs in subsequent pregnancies, the lag phase is short (3–7 days) due to the presence of memory B cells that quickly differentiate into antibody-secreting plasma cells. 2. Depending on the antibody detection method, it often takes 5–15 weeks before anti-D is detectable in serologic tests. 2. An increase in anti-D is usually detectable within days. 3. The amount of anti-D produced is relatively low. 3. The amount of anti-D rises to a higher level. 4. Anti-D titers decline fairly rapidly and may become undetectable. 4. Anti-D titers tend to remain higher for longer but eventually decline. 5. The first anti-D produced is mainly IgM (although small amounts of IgG are usually also produced). 5. The main type of anti-D produced is IgG (although small amounts of IgM may be produced). | View Page |
| ABO HDFN - Expected Findings Diagnosis of ABO HDFN is supported by these findings: ABO incompatibility between mother and child, with mother typically group O; Maternal antibody screen negative; Cord DAT weakly positive or negative; Newborn hyperbilirubinemia with jaundice occurring in first 24 hours; Increased spherocytes and reticulocytosis in the newborn; Presence of IgG anti-A or anti-B in cord plasma / serum. | View Page |
| Factors That Affect Production of Anti-D Exposure to D+ red cells: Anti-D is red cell immune. The usual route of exposure to the D antigen is during pregnancy. Fetal bleeds into the mother occur more commonly at delivery but some may occur antenatally due to small lesions in the placenta or due to placenta previa, amniocentesis, abdominal trauma, abortion, ectopic pregnancy, etc. Transfusion is a relatively rare route of exposure since Rh-negative individuals normally receive only Rh-negative donor red cells. However, Rh-negative transfusion recipients may be exposed to small volumes of D-positive red cells in Rh-positive platelet concentrates. Also, there are rare reports of fresh frozen plasma, not normally matched for Rh(D), causing anti-D production.Volume of fetal bleed: In general, the larger the fetal bleed, the more likely the mother is to produce anti-D. Approximately 1 pregnancy in 400 result in a fetomaternal hemorrhage (FMH) of 30 mL or greater. ABO incompatibility between mother and fetus: If fetal red cells are ABO incompatible with the mother, maternal anti-A or anti-B will rapidly remove fetal cells from the circulation before anti-D can be produced. This protection decreases the chance of anti-D being produced but does not eliminate it entirely. | View Page |
| Choosing Donor RBC for IUT and IVT Donor RBC for IUTs and IVTs have these criteria: Group O Rh negative*; Crossmatched with maternal serum; Fresh: less than or equal to 7 days (or fresher); High hematocrit, e.g, 85–90% (0.85–0.90) to prevent volume overload; CMV seronegative (or equivalent, e.g., leukoreduced by filtration); Negative for hemoglobin S to prevent blood from hypoxia-induced sickling in the fetal circulation; Irradiated with a minimum dose of 25 Gray (Gy) to prevent graft-versus-host disease.* Some laboratories use red cells that are also K-negative since the K antigen is very immunogenic. This also applies to exchange transfusions. | View Page |
| Follow-up Investigative Tests (Mother) If a pregnant woman is found to have an unexpected clinically significant antibody, routine antenatal serologic tests on the mother include Antibody identification to detect clinically significant antibodies. Antigen typing: Once the antibody is identified, the mother is tested for the corresponding antigen, which she should lack. Antibody titration: Laboratories have different protocols. Depending on the antibody titer, titration may be performed at 2 or 4 week intervals after 18 weeks gestation.Notes (titration): Maternal antibody titer is an unreliable indicator of fetal disease and is mainly done to determine if clinical fetal monitoring is warranted, e.g., Doppler ultrasonography of fetal cerebral blood flow or, more rarely, invasive monitoring such as amniocentesis. Careful quality control is needed for titrations. QC includes using red cells from donors with the same phenotype or likely genotype (e.g., R2r or R2R2) and titrating the new sample in parallel with the prior sample. A two-tube rise or more in a doubling dilution is considered a significant rise in titer. In the case of anti-D, a predetermined critical titer (often 16 or 32 for anti-D depending on the method) indicates the need for clinical fetal monitoring. | View Page |
| Follow-up Investigative Tests (Fetus) If a mother has a clinically significant antibody, fetal blood for phenotyping can be obtained by several procedures, depending on gestational age and the antigen involved. These include Amniotic fluid sampling* Chorionic villus sampling* Cell-free fetal DNA in maternal plasma* Percutaneous umbilical cord blood sampling (PUBS) / cordocentesis** * molecular genotyping / ** serologic testsAs noted, typing the fetus is warranted when the father's blood type is unknown or the father tests as heterozygous positive. | View Page |
| Follow-up Investigative Tests (Father) Investigative tests on the father depend on which maternal antibodies are present.1. Mother has anti-D ABO and Rh typing with anti-D, -C, -E, -c,-e to determine probable Rh genotype* to predict the chance the fetus has of being Rh positive and affected by HDFN; Test for weak D if initial Rh typing appears to be D-negative. * For D+ fathers, the probable Rh genotype can be determined using serologic tests, i.e., DCEce typing to determine if the father is probably homozygous or heterozygous for D.2. Other maternal clinically significant antibodies Phenotype father for the corresponding antigen and its antithetical antigen (e.g., K and k) | View Page |
| Molecular Genotyping - Introduction The application of DNA analysis to typing blood group antigens started in the early 1990s but is not yet widely available. Molecular methods exist for typing Rh (RHD and RHCE), Kell (K & k), Duffy (Fya & Fyb), and Kidd (Jka &Jkb) loci.In perinatal testing programs, molecular typing can determine the Rh type of the mother, father, and fetus and may be done if the mother has anti-D or another antibody known to cause HDFN. More specifically, if available, DNA methods are typically used in these circumstances: For women who type as weak D in serologic tests, to determine the Rh genotype of the mother to identify if she is partial D or weak D; For women who have made anti-D, to determine the Rh genotype of the father to see if fetal monitoring is needed; For women who have made anti-D, to determine the Rh type of the fetus if the father is heterozygous for RhD or unavailable for testing. Fetal blood typing can be done using fetal DNA from cells obtained by amniocentesis or by testing cell-free, fetal-derived DNA present in maternal plasma at 5 weeks gestation and later. Like all diagnostic methods, DNA typing has limitations and is not 100% sensitive and specific. For example: The blood group's molecular basis may be unknown; Not all alleles in ethnic populations are known; Rare mutations in the RHD and other genes may not be detected; Silencing changes (switching off of a gene) may affect antigen expression; Fetal typing using amniotic fluid may give false-negative results because of maternal cell contamination. | View Page |
| Molecular Genotyping - Mother Mother's Rh Type - Weak D or Partial DRecall that some individuals have a variant RHD gene that encodes a reduced concentration of D antigen (weak D) or a D antigen with missing D epitopes (partial D). Various anti-D reagents react differently with these red cells and interpreting Rh(D) type can vary with the method used, e.g., tubes, solid phase, gel. Differentiating between weak D and partial D is important in pregnant women. Those with partial D, but not usually weak D, may make anti-D and should be considered D negative for transfusion and as RhIg candidates. Currently, serologic reagents cannot distinguish the two D variants, but RHD genotyping can. | View Page |
| Molecular Genotyping - Father and Fetus Rh Genotype (Father and Fetus)As noted, usually molecular typing of the father is done only if the mother has anti-D or an antibody to another antigen for which molecular methods exist. In the case of a mother with anti-D and a father who is D+ using serologic methods, molecular typing can determine the father's RHD heterozygosity or homozygosity*. If the father is homozygous for the RHD allele, all of his offspring will be Rh positive, negating the need for fetal D testing, but indicating that the fetus should be monitored for HDFN. If the father is heterozygous for RHD, the Rh type of the fetus should be determined to see if HDFN is possible. * For D+ fathers, the probable Rh genotype can be determined using serologic tests, i.e., DCEce typing to determine if the father is probably homozygous or heterozygous for D (see later). | View Page |
| Immunogenicity Immunogenicity is the ability of an antigen to provoke an immune response in an antigen-negative recipient. Why some antigens are more immunogenic than others is unknown. Not considering antigens in the ABO system, Rh(D) is the most immunogenic red cell antigen, followed by K in the Kell blood group system. Other immunogenic antigens include c and E in the Rh system. In routine blood banking, assessments of an antigen's immunogenicity are typically based on the prevalence of the corresponding antibody and do not take into account the frequency of the antigen in the general population. For example, k in the Kell system may be very immunogenic but anti-k is rare since 99.8% of Caucasians are k+ and cannot make anti-k. | View Page |
| Use in Pregnancy As applied to pregnancy, RhIg's purpose is to prevent immunization to the D antigen in the perinatal period and thus prevent HDFN due to anti-D. If the mother has already produced anti-D, RhIg is of no use in moderating the immune response.Accordingly, RhIg is routinely administered to Rh negative women not previously sensitized to the D antigen under the following circumstances:1, Antenatal. Antepartum prophylaxis of 300 µg (1500 IU) at about 28 weeks gestation in the USA and Canada, which could be weeks later, depending on how appointments are scheduled. To illustrate variation in antenatal international practice, in the UK, smaller doses of RhIg (e.g., 500 IU) may be given at 28 weeks and 34 weeks, although many UK facilities issue a 1500 IU dose at 28–30 weeks. With antenatal administration, the Rh of the fetus is usually unknown. Some transfusion services recommend a further antenatal dose if the infant is undelivered after 40 weeks.2. Postnatal. Prophylaxis of 300 µg (1500 IU) at delivery of an Rh positive or weak D infant within 72 hours of delivery whenever possible. If RhIg administration is delayed beyond 72 hours, laboratory policies differ as to when it would no longer be administered. The longer the delay, the more likely RhIg may fail to suppress production of anti-D, but it is still worth trying. Note: Because RhIg contains IgG anti-D, when given antenatally, it can cross the placenta and sensitize fetal D-positive red cells. Occasionally the fetus may be born with a weakly positive DAT, but significant hemolysis does not occur. For this reason some guidelines recommend that labs do NOT routinely perform DATs on infants whose mothers have received antenatal RhIg. | View Page |
| RhIg and Rh Complexity Policies for administering RhIg when the mother's Rh type is weak D vary among countries and within some countries. To understand the issues involved, we need to review the genetics of the Rh D antigen and the types of weak D (formerly Du). The Rh system is complex and only a basic overview will be given.In brief, Rh system inheritance is determined by two sets of genes: RHD codes for the proteins carrying D expression. In most people, the presence or absence of the RHD gene results in being Rh positive or Rh negative, respectively. RHCE codes for different combinations of the proteins carrying CcEe expression and Rh-associated glycoprotein (RhAG). RhAG is needed for the expression of Rh antigens. The RHCE locus is adjacent to RHD on chromosome #1. See a model for the Rh locus (NCBI)D variantsFor a small percentage of people, the inheritance of the RHD gene and the expression of the D antigen may be altered leading to several variants of D encoded by more than 100 RHD alleles | View Page |
| Mechanism of Action When first developed in the 1960s, RhIg was believed to work by a simple clearance mechanism, i.e., by coating D-positive fetal red cells with IgG anti-D, which resulted in clearance of the sensitized cells in the spleen by macrophages with receptors for IgG.Current research shows that a simple model of antigen clearance by antibody-sensitized D-positive RBCs is not the mechanism of anti-D suppression by RhIg. More is involved at the molecular level, possibly involving a down-regulation of antigen-specific B cells and related mechanisms.Regardless, if given soon enough following exposure to D+ red cells, and in a suitable dosage, RhIg has the ability to prevent immunization to D. | View Page |
| RhIg & Variants of D As noted, policies for administering RhIg to mothers with a variant of D vary among countries and within some countries. An Rh(D) red blood cell phenotype with a weak or variant expression of the D antigen occurs in 0.2% to 1% of whites and is slightly more common in African Americans. The phenotype is routinely called weak D, although several variants exist. A simple model includes these D variants: 1. Weak DMultiple weak D variants exist. Red cells have fewer D antigens/red cell (quantitative difference) and only minor variations in D antigen proteins. Some, but not all, weak D phenotypes are detected by today's Rh typing sera and may be classified as Rh positive or Rh negative by routine testing but will be positive when a weak D test (IAT with anti-D) is done. An extreme form of weak D is the Del phenotype, in which the D antigen is so weakly expressed that it may be demonstrated only by adsorption and elution of anti-D. Weak D individuals do NOT produce anti-D and can be considered to be Rh positive for transfusion and RhIg purposes.2. Partial DPartial D variants have altered Rh(D) proteins that differ sufficiently from normal D antigens (qualitative difference) to allow anti-D production. Partial D red cells may react with some but not all anti-D typing reagents. There are many categories of partial D antigens (e.g., DIIIa, DVI, DAR), each with a unique genetic basis.Some persons with partial D have weakly expressed D epitopes and are designated "partial weak D."In practice, partial D and weak partial D can be considered similarly, i.e., ideally they should be transfused with Rh negative RBC and are candidates to receive perinatal Rh immune globulin depending on the policy in their location. | View Page |
| Clinical Relevance of D Phenotypes Clinically relevant information on D phenotypes can be summarized as follows: D phenotype D antigen expression Rh(D) typing Produce anti-D RBC to transfuse RhIg recommended** D+ normal direct agglutination no D+ or D– no Weak D normal but weak IAT no D+ or D– no Partial D altered direct agglutination* & IAT yes D– yes Partial weak D altered & variable direct agglutination* & IAT yes D– yes D– none IAT yes D– yes * Depending on anti-D reagent used ** USA, UK and parts of Canada | View Page |
| RhIg prevents anti-D production mainly by clearing antibody-sensitized D-positive rbc from maternal circulation. | View Page |
| Literature and Online Resources The following published literature and online resources, while useful, should not be used as a substitute for technical and clinical judgment. Medical and technical information becomes obsolete quickly and current sources relevant to the user's location should always be consulted.References indicated by * provide a broad overview of HDFN and are highly recommended.LITERATUREAvent ND, Reid ME. The Rh blood group system: a review. Blood 2000 Jan 15;95 (2):375-87.Bowman J. Thirty-five years of Rh prophylaxis. Transfusion 2003 Dec;43(12):1661-6.* Eder AF. Update on HDFN: new information on long-standing controversies. Immunohematology 2006;22(4):188–195. (scroll to article)Eder, AF, Manno, C.S. Alloimmune hemolytic disease of the fetus and newborn. In Wintrobe's Clinical Hematology, 11th ed. (Greer JP, Foerster J, Lukens JN, Rodgers GM, Paraskevas F, Glader BE, (eds). Philadelphia, PA: Lippincott, Williams & Wilkins, 2004.Flegel WA. Molecular genetics of RH and its clinical application. Transfus Clin Biol. 2006 Mar-Apr;13(1-2):4-12. Kennedy MS, McNanie J, Waheed A. Detection of anti-D following antepartum injections of Rh immune globulin. Immunohematology 1998;14(4):138-40.Koelewijn JM, de Haas M, Vrijkotte TG, van der Schoot CE, Bonsel GJ. Risk factors for RhD immunisation despite antenatal and postnatal anti-D prophylaxis. BJOG. 2009 Sep;116 (10): 1307-14. Epub 2009 Jun 17.* Kumar S, Regan F. Management of pregnancies with RhD alloimmunisation. BMJ. 2005 May 28;330(7502):1255-8. (UK perspective but much valuable information relevant to all)* Murray NA, Roberts IAG. Haemolytic disease of the newborn. Arch Dis Child Fetal Neonatal Ed 2007 Mar; 92(2): F83–F88. Oepkes D, Seaward PG, Vandenbussche FP, Windrim R, Kingdom J, Beyene J, Kanhai HH, Ohlsson A, Ryan G; DIAMOND Study Group. Doppler ultrasonography versus amniocentesis to predict fetal anemia. N Engl J Med. 2006 Jul 13;355(2):156-64.Ramsey G. Inaccurate doses of Rh immune globulin after Rh-incompatible fetomaternal hemorrhage: survey of laboratory practice. Arch Pathol Lab Med 2009 Mar; 133(3):465-9. Reid ME. The Rh antigen D: a review for clinicians. Blood Bulletin 2008 Apr; 10(1).Sandler SG. Effectiveness of the RhIg dose calculator. Arch Pathol Lab Med 2010 Jul;134(7): 967-8.Shulman IA, Calderon C, Nelson JM, Nakayama R. The routine use of Rh-negative reagent red cells for the identification of anti-D and the detection of non-D red cell antibodies. Transfusion 1994 Aug;34(8):666-70.Tamul KR. Determining fetal-maternal hemorrhage with flow cytometry. Advance 2000. Posted online June 5, 2000.Westhoff CM, Sloan SR. Molecular genotyping in transfusion medicine. Clin Chem 2008;54(12): 1948-50.ONLINE RESOURCESPaxton A. Bringing new rigor to RhIg calculations. CAP TODAY. May 2008. Accessed January 18, 2011.*Wagle S, Deshpande PG. Hemolytic disease of the newborn. eMedicine / WebMD. Updated Apr. 9, 2010. Accessed January 18, 2011. | View Page |
| Match the blood types from the drop-down boxes with the appropriate descriptions to the right of the boxes. | View Page |
| An individual with type AB blood will demonstrate the complete absence of which of the following antigen sites? | View Page |
| Reverse typing is done using known antisera to detect ABO antigens present on the patient's red blood cells. | View Page |
| The History of the ABO System In 1900, a German scientist, Karl Landsteiner, discovered that blood groups differ from one individual to another. He took blood samples from five associates and himself, allowed them to clot, and then separated the serum from the cells. Landsteiner found that when he mixed the serum and red cells from different individuals, some samples clumped and some didn’t. Our present day classification of the ABO system is based on Landsteiner’s realization that agglutination occurred because of highly reactive antigens present on the red blood cell which corresponded to antibodies present in the serum. Landsteiner isolated and named the red cell antigens “A” and “B” and the corresponding antibodies “Anti-A” and “Anti-B.” If the red cells contained neither antigen, he called these cells “O,” representing zero antigens present. The fourth type of red cells, “AB” was discovered in 1902 by Von Decastello and Sturli, associates of Landsteiner. “AB” cells contained both A and B antigens on their surface. | View Page |
| The History of the ABO System, continued Landsteiner, knowing that none of his subjects had been immunized, realized that “natural” antibodies must develop which are directed against antigens not present on the red cells. Individuals with “A” antigens on their red cells had sera containing “Anti-B” antibody. Individuals with “B” antigens had sera containing “Anti-A.” “AB” individuals had sera with no ABO antibodies present and “O” individuals’ sera contained “Anti-A” and “Anti-B.” Sera from group O individuals may contain a separate antibody, “Anti-A,B.” Anti-A,B possesses serologic activity not found in mixtures of Anti-A and Anti-B. Anti-A,B sera will agglutinate A, B, and AB cells. It is particularly useful in detecting weak A and B antigens. See the table on the next page. | View Page |
| Table 1: ABO Blood Group System Antigen on Red Cells Antibodies in Serum ABO Blood Group A Anti-B A B Anti-A B Neither A nor B Anti-A, Anti-B, Anti-A,B O A and B Neither Anti-A nor Anti-B AB | View Page |
| Table 2: Testing the Patient Red Cells with Known Antisera (Forward Typing) In routine practice, specially prepared blood grouping sera containing anti-A, anti-B, (and optionally anti-A,B) are used to identify the four types of red cells. These sera will agglutinate cells with the corresponding antigen. This is called forward typing.ABO Blood GroupPatient Red Cells Tested with Known AntiseraAnti-AAnti-BAnti-A,BA3-4+03-4+B03-4+3-4+O000AB3-4+3-4+3-4++ = agglutination (graded 1+ to 4+)0 = no agglutination | View Page |
| Table 3: Testing the Serum with Known Red Cells (Reverse Typing) Antibodies occur predictably in the sera of all normal adults in association with the ABO antigens. Demonstration of these antibodies is necessary for definitive classification of an individual's ABO cell type. The individual's serum is therefore tested against reagent red cells containing known antigens. Patient ABO Blood GroupPatient Serum Tested with Known Reagent CellsA CellsB CellsA0 3-4+ B3-4+ 0 O3-4+ 3-4+ AB0 0 + = agglutination (graded 1+ to 4+)0 = no agglutination or hemolysis | View Page |
| Importance of Understanding the ABO System The predictability of ABO antibodies appearing in serum lacking the corresponding antigens makes ABO typing a simple process in most cases. However, the importance of getting it right cannot be stressed enough when a patient will be transfused with blood from a donor. If a patient receives donor cells containing A or B antigens and the transfused patient's serum contains the corresponding antibody, the donor cells will be destroyed almost immediately, causing a severe (hemolytic) and sometimes fatal reaction. Therefore, it is of utmost importance to thoroughly understand the ABO blood group system. In addition to red cells, ABO antigenic determinants (epitopes) are found in many tissues, body fluids, and other cells, including endothelial cells and platelets. Because ABO antigens are so widely expressed, ABO antigens are also a major consideration in solid organ and bone marrow transplants. | View Page |
| Epitopes In addition to red cells, ABO antigenic determinants (epitopes) are found in many tissues, body fluids, and other cells including endothelial cells and platelets. Because ABO antigens are so widely expressed, ABO antigens are also a major consideration in solid organ and bone marrow transplants. | View Page |
| Why does agglutination (clumping) sometimes occur when red blood cells (RBCs) from one individual are mixed with serum from another? | View Page |
| Match the blood types in the drop down boxes with the characteristics on the right. | View Page |
| Galactose and ABO Antigen Precursor Substance Specific sugars, attached to the red cell membrane in unvarying linkage conformations, determine ABO antigenic activity. Galactose resides at the end of this specific sugar chain. This configuration constitutes the ABO antigen precursor substance. | View Page |
| Fucose Another sugar, fucose, must be attached to the galactose in a specific configuration for further antigen development to take place. This "galactose-plus-fucose" configuration has antigenic activity called "H". | View Page |
| "A" Antigenic Activity Without H substance (also known as H antigen or substance H), there is no way for additional sugar attachment to take place. Additional sugar attachment is necessary for the development of A and B antigens. Therefore, without substance H there is no development of A and B antigens. Once substance H is developed, the addition of the sugar N-acetylgalactosamine to the terminal position of the chain gives the molecule "A" antigenic activity. | View Page |
| The H gene Three separate loci (ABO, Hh, and Se) contain the genes that control the location and occurrence of the A and B antigens. Hh and Se genes are closely linked on chromosome 19. The precursor substance is acted upon by the H gene and is converted to H substance. The product of the H gene is an enzyme fucosyltransferase, responsible for attaching fucose to the terminal galactose of the precursor substance on the RBC membrane and thus forming H substance. There are only two recognized alleles at this locus: the active form, H, and an amorph, h. The H gene is a high-incidence gene. People who inherit hh are extremely rare. Since the h gene is amorphic, it does not act on the precursor substance. | View Page |
| A, B, and O Genes The ABO locus is on chromosome number 9. There are three major allelic genes and numerous rare genes. The three principle genes are A, B, and O. The A gene determines the product N-acetylgalactosaminyltranferase activity. The B gene determines galactosyltransferase activity. The O gene does not produce a functional enzyme. The enzyme products of the A and/or B genes act on H substance to convert it to A and/or B antigens. Not all H substance is converted; thus, all cells normally contain some H substance along with the A and/or B antigens. If both the A and B genes are present, some H antigen sites are converted to A antigen and other H antigen sites are converted to B antigen. (A single antigen site does not have both A and B antigens.) The O gene is an amorph and doesn't act on H substance, therefore group O cells contain only H substance. See the diagram on the next page. | View Page |
| Bombay Blood Group Genes As mentioned previously, the A and B genes cannot act directly on the precursor substance. Thus, since individuals with the Bombay phenotype have only the precursor substance and no H antigen, they cannot have A or B antigens, even if they have the A and/or B gene. | View Page |
| Inherited Genes The A, B, and H antigens, like many other blood group antigens, are the expression of genes inherited from the previous generation. If the antigen is demonstrated, the gene controlling it must have been inherited from one or both of the parents. As previously mentioned, the genes A, B, and O are allelic genes. Assuming the production of H substance, these three genes, in various possible combinations of two, account for the four recognized ABO groups: A, B, AB, and O. Each individual inherits two ABO genes, one from each parent, and these genes determine which ABO antigen will be present on that individual’s red cells. These genes exhibit co-dominance, meaning that if both A and B genes are present, both will be expressed. | View Page |
| Deducing the Gene The presence of A and/or B antigen on the red cells can be recognized by serological tests with the appropriate antisera so that the presence of the gene that controls its production can be deduced in the absence of both A and B genes (when no A or B antigen is present on the red cells). | View Page |
| Genotyping Those who type as group O must have two O genes present (since both the A and B genes would have produced recognizable antigens, neither of which is present on group O cells). Therefore, in the case of an AB individual or an O individual, we can tell exactly which genes are present, or a genotype. Group A or group B typing reveals only one gene product and thus only a phenotype can be determined. Persons of phenotype A can be genotype AA or AO , while those of phenotype B can be genotypically BB or BO. Family studies may be done to determine the genotype of an A or B individual. For example, if the mating of one A and one O parent produced a group O child, the second gene present in the A parent must have been O since the child has inherited one O gene from each parent. | View Page |
| How many gene loci regulate red cell ABO antigen development? | View Page |
| Which of the following is true of the classic Bombay phenotype? | View Page |
| ABO Antibodies In most other blood group systems, antibody may be formed after an individual has been immunized by an antigen that is missing from his or her red cells; perhaps as the result of pregnancy or transfusion. In the ABO system, when the antigen is missing from the cells, the corresponding antibody will predictably be found in the serum and must be found before determining the ABO type. There are few exceptions to this rule and any exception must be explained before the true ABO blood type can be determined. | View Page |
| Anti-A and Anti-B Development It is possible that since anti-A and anti-B develop so predictably, without a recognizable immunizing event, that they are naturally occurring. However, some exposure to antigen must take place for antibodies to be produced. ABO antibodies are generally not present in the serum of newborns. It is postulated that production of the antibodies occurs as a result of exposure to environmental and internal (digestive tract) bacteria, which have been shown to contain carbohydrate structures (antigens). Antibody production occurs after exposure to these carbohydrate antigens, in accordance with the person's genetically predetermined ABO group. | View Page |
| Immunoglobulin The predominant immunoglobulin class for the B antibodies produced by individuals with group A phenotype and the A antibodies produced by individuals with group B phenotype is IgM. Small quantities of IgG and IgA may also be present.The ABO antibodies found in the serum of group O individuals include anti-A and anti-B. An antibody designated anti-A,B is also present. Anti-A,B in group O individuals tends to be predominantly IgG, although IgM and IgA components are also present.Infants of group O mothers are at higher risk for hemolytic disease of the fetus and newborn (HDFN) than those born to mothers with group A or B because IgG immunoglobulins readily cross the placenta. IgM molecules do not cross the placenta because of their larger size. However, the HDFN that results is usually mild and often subclinical. Infants generally survive with little or no intervention.It is important to note that immune antibodies are usually IgG. Both naturally occurring and immune ABO antibodies are critically important in transfusion since both sensitize, and usually hemolyze, red cells with the corresponding antigen. | View Page |
| Anti-A and anti-B may be stimulated by bacteria which have been shown to contain substances that are chemically similar to human A and B antigens. | View Page |
| Strength of the A Antigen The strength of the A antigen can vary considerably, and although most A cells react strongly with anti-A and anti-A1B, some cells have been found that are very weakly reactive. The blood group has been divided into subgroups and is classified not only by the strength of the A antigen but also by certain other serologic characteristics. | View Page |
| A1 and A2 Subgroups The most common subgroups of group A phenotype are A1 and A2. These account for over 99% of individuals who are classified as Group A. Of this 99%, A1 comprises approximately 80%. Commercial anti-A typing serum does not differentiate between A1 and A2 cells. A1 cells contain "A" antigen and "A1" antigen. A2 is not really a unique antigen. It is thought to be simply "A" antigen with no "A1" antigen. Several preparations are available that will react with A1 cells, but not other subgroups of A. The most commonly used reagent is Anti-A1 lectin, an extract of the seeds of the plant, Dolichos biflorus, which has specific anti-A1 activity.Approximately 4% of individuals who are subgroup A2 have naturally occurring anti-A1 in their serum. | View Page |
| Inherited Antigens A subgroup antigens are inherited, as are other ABO antigens with A1 being dominant over A2. Individuals who are phenotypically A1 may be genotypically A1O, A1A1, or A1A2. A phenotypically A2 individual may be genotypically A2A3. These alleles are passed to offspring in the same manner as other ABO antigens. Weak variant forms of the B antigen (B3, Bx, and Bel) also exist, but are very rare. | View Page |
| Why Knowledge of A Subgroups Is Important For Laboratorians For the most part, subgroups are merely of academic interest, but occasionally they present clinical problems. The antigen may be so weak that it is not detected and the red cells are mistyped as group O. This is especially dangerous if the cells are those of a donor. Problems may arise because the serum of an A2 or A2B, A3, or Ax individual might contain anti-A1. This antibody may be detected in serum typing and cause confusion. You would not expect to find a person with A antigen on his/her red cells and anti-A in the serum. Anti-A1 is produced by about 4% of group A2 individuals and about 25% of group A2B individuals. Subgroups may be determined by reactions with antisera as seen in the table on the next page. | View Page |
| Given the results below, what is the most probable ABO type for this individual?Forward (Cell) Grouping Reverse (Serum) Grouping Anti-AAnti-BAnti-A,BA1 CellsB Cells4+04+2+4+ | View Page |
| Forward Typing Forward typing is done using known antisera to detect ABO antigens present on the patient’s red cells. In the tube test, known antisera and patient cells are placed in labeled test tubes, centrifuged, and observed for agglutination. Each manufacturer has specific instructions for its own antisera, detailing the percent of cell suspension, number of drops of cell suspension versus number of drops of antisera, and the rate and length of centrifugation. Though the details differ, the theory behind the tests is the same. | View Page |
| Testing the Red Cells With Known Antisera Patient Red Cells Tested With Known AntiseraABO Antigens Present on Red CellAnti-AAnti-BAnti-A,B3-4+03-4+A03-4+3-4+B000Neither A nor B3-4+3-4+3-4+A and B+ = agglutination (graded 1+ to 4+) 0 = no agglutination or hemolysis | View Page |
| Which of the following statements best describes forward typing? | View Page |
| Which of the following best describes reverse typing? | View Page |
| Prior to 1985 Once relegated to the domain of research laboratories, molecular methods for the diagnosis of infectious disease had little, if any place, in a clinical diagnostic laboratory prior to 1985. Procedurally, molecular methods were very complex and required specialized instrumentation and dedicated laboratory space. They were also susceptible, initially, to the influence of variation of technique. Although they represented valuable research tools, and were helpful as esoteric testing for unique clinical situations, their performance characteristics simply did not fit well into most clinical laboratories.Certain pathogens were logical targets for development. Organisms that were of concern for significant patient populations, were difficult to sustain in transport, and/or were difficult to cultivate and detect by traditional methods represented some of the first targets of commercially offered molecular based assays.Sexually transmitted diseases, affecting significant numbers of people, with key pathogens affected by lability in transport or poor sensitivity with traditional cultivation or antigen detection methods, were among the first targets for development. | View Page |
| Why were Chlamydia and Neisseria logical targets for the development of a commercial molecular assay? (Choose all that apply.) | View Page |
| The Key Benefits: Improved Sensitivity of Detection There are three key benefits that molecular methods can offer, in contrast to traditional culture methods:Improved sensitivity of detectionImproved specificity of identificationReduced turnaround timeImproved sensitivity of detectionSuccessful cultivation or detection of an organism depends on many factors, including the:Ability of the organism to survive transport Fastidious nature of the organism/its ability to grow in available culture media/systems Number of organisms present in a specimen Ability of a staining/culture system to visualize/recover low numbers of organisms Sensitivity of non-culture (antigen detection) methodsOrganisms that have been shown to be very labile are difficult to cultivate even when they are present in significant numbers. The immediacy of transport, plating, and incubation are critical factors that frequently cannot be controlled in a positive direction.Even under the ideal transport conditions, some organisms may require culture media and conditions that are not routinely available in every laboratory, which reduces the likelihood of successful cultivation. | View Page |
| The Key Benefits: Improved Sensitivity of Detection, continued Some organisms are present in infections in very low numbers, which may be undetectable with direct staining methods. These organisms may also prove to be difficult to recover with currently available culture methods.Although non-culture antigen methods have been developed to address some of these difficulties (examples include direct fluorescent antibody (DFA) and enzyme immunoassay (EIA) methods), the sensitivity of these methods has not always been desirable.Molecular methods offer the prospect of:Detecting nonviable organisms that did not survive transport Detecting organisms difficult/impossible to cultivate Detecting organisms present in low numbers Providing better detection capability than other non-culture methods | View Page |
| In traditional culture or antigen detection methods, the sensitivity of detection is adversely affected by which of the following? (Choose all that apply.) | View Page |
| Challenges for Implementation: Cost Implementing molecular methods may involve purchasing an equipment platform that represents a significant capital investment. Reagents for the assays are frequently more expensive, on a cost per test basis, than either culture or antigen detection methods. Reimbursement issues, although improving, can be more complex. Realistically, implementations of molecular methods are likely to represent increased costs that do need to be weighed against the potential benefits that can be realized.When considering the implementation of a molecular method, the following question should be asked:Will the methods significantly impact/improve clinical management and patient outcomes, reduce antimicrobial costs and lengths of stay, and/or facilitate infection control, epidemiology, or antibiotic stewardship programs?The answer may not be "yes" for every single agent of infectious disease for which molecular methods are now available. | View Page |
| Prior Traditional Methods and the Need for Change Rapid detection of influenza was a key focus for method development for many years. Traditional viral culture methods require special transport mediums, appropriate cell culture lines, and staff well versed in the manipulation of these cultures. Although the introduction of shell vial cultures and monoclonal fluorescent staining provided some improvement, the availability of results did not always meet the clinical need.Direct fluorescent antibody (DFA) staining of specimen smears can provide more immediately available results; however the availability of trained staff to interpret these smears is an obstacle for many laboratories. Antigen detection kits employing enzyme immunoassay (EIA) or immunochromatographic membrane principles did provide easily performed alternatives that fit well in most laboratory settings and provided more immediate results. Despite the fact that published studies demonstrated less than desirable sensitivity, these assays had found a niche and remained in place, even as molecular methods began to target these viruses. | View Page |
| Previous Methodologies: Antigenic Detection of Toxin and Glutamate Dehydrogenase (GDH) Toxin assaysThe most common laboratory tests for the detection of C. difficile are enzyme immunoassays (EIA) for the detection of C. difficile toxin A and toxin B. The immunoassays are simple to perform, provide rapid results, and are easily incorporated into the workflow of most laboratories. Sensitivities of these tests do NOT compare favorably to culture, cell cytotoxicity neutralization assay (CCNA), or molecular methods. There are many test kits commercially available for detection of C. difficile toxins. Results are available in 15 minutes to 2 hours, depending on the assay. Initially, toxin A was thought to be the toxin responsible for the majority of the effects of C. difficile disease, so most early kits only detected toxin A. With the realization that there are strains that produce aberrant or no toxin A (A-) that are known to produce infection, and more recently toxin B negative (B-) strains, it is now recommended to use kits detecting BOTH toxins.Glutamate Dehydrogenase (GDH) assaysPublished studies have indicated that toxin immunoassays, by themselves, may not provide adequate sensitivity of detection. GDH assays initially attracted attention as a possible means to provide a rapid but more sensitive means for screening for C. difficile.GDH is an enzyme produced by C. difficile. EIAs negative for the GDH antigen have been associated with high negative predictive values. However, positive results are not necessarily associated with a toxin producing strain. A second assay on GDH positive samples is required to confirm the presence of a toxigenic strain. Initially, CCNA assays were recommended as the confirmatory method of choice; molecular methods (PCR for the toxin gene) were subsequently explored for this purpose. | View Page |
| Several methods of detection are available for the detection of Clostridium difficile in clinical samples. Which methods have the capability for detection in less than 48 hours? (Choose all that apply.) | View Page |
| What statements are TRUE about the glutamate dehydrogenase (GDH) assay for Clostridium difficile? (Choose all that apply.) | View Page |
| Use in Pregnancy As applied to pregnancy, RhIg's purpose is to prevent immunization to the D antigen in the perinatal period and thus prevent HDFN due to anti-D. If the mother has already produced anti-D, RhIg is of no use.Accordingly, RhIg is routinely administered to Rh negative women* not previously sensitized to the D antigen under the following circumstances:1, Antenatal. Antepartum prophylaxis of 300 µg (1500 IU) at about 28 weeks gestation in the USA and Canada, which could be weeks later, depending on how physician appointments are scheduled. To illustrate variation in antenatal international practice, in the UK smaller doses of RhIg (e.g., 500 IU) may be given at 28 weeks and 34 weeks, although many UK facilities issue a 1500 IU dose at 28–30 weeks. With antenatal administration, the Rh of the fetus is usually unknown. Some transfusion services recommend a further antenatal dose if the infant is undelivered after 40 weeks.2. Postnatal. Prophylaxis of 300 µg (1500 IU) at delivery of an Rh positive or weak D infant, preferably within 72 hours of delivery but can be given up to 28 days later if administration is delayed. If RhIg administration is delayed beyond 72 hours, laboratory policies differ as to when it would no longer be administered.* Policies related to women who are weak D (formerly Du) are discussed later.Note: Because RhIg contains IgG anti-D, when given antenatally, it can cross the placenta and sensitize fetal D-positive red cells. Occasionally the fetus may be born with a weakly positive DAT, but significant hemolysis does not occur. | View Page |
| RhIg and Rh Complexity Policies for administering RhIg when the mother's Rh type is weak D vary among countries and within some countries. To understand the issues involved, we need to review the genetics of the Rh D antigen and the types of weak D. The Rh system is complex and only a basic overview will be given.In brief, Rh system inheritance is determined by two sets of genes: RHD codes for the proteins carrying D expression. In most people, the presence or absence of the RHD gene results in being Rh positive or Rh negative, respectively. RHCE codes for different combinations of the proteins carrying CcEe expression and Rh-Associated Glycoprotein (RhAG). RhAG is needed for the expression of Rh antigens. The RHCE locus is adjacent to RHD on chromosome #1. See a model for the Rh locus (NCBI)D variantsFor a small percentage of people, the inheritance of the RHD gene and the expression of the D antigen may be altered leading to several variants of D encoded by more than 100 RHD alleles | View Page |
| Mechanism of Action When first developed in the 1960s, RhIg was believed to work by a simple clearance mechanism, i.e., by coating D-positive fetal red cells with IgG anti-D, which resulted in clearance of the sensitized cells in the spleen by macrophages with receptors for IgG.Current research shows that a simple model of antigen clearance by antibody-sensitized D-positive RBC is not the mechanism of anti-D suppression by RhIg. More is involved at the molecular level, possibly involving a down-regulation of antigen-specific B cells and related mechanisms (see Further Reading). | View Page |
| RhIg & Variants of D As noted, policies for administering RhIg to mothers with a variant of D vary among countries and within some countries. An Rh(D) red blood cell phenotype with a weak or variant expression of the D antigen occurs in 0.2% to 1% of whites and is slightly more common in African Americans. The phenotype is routinely called weak D, although several variants exist. A simple model includes these D variants: 1. Weak DMultiple weak D variants exist. Red cells have fewer D antigens/red cell (quantitative difference) and only minor variations in D antigen proteins. Some, but not all, weak D phenotypes are detected by today's Rh typing sera and may be classified as Rh positive or Rh negative by routine testing but will be positive when a weak D test (IAT with anti-D) is done. An extreme form of weak D is the Del phenotype, in which the D antigen is so weakly expressed that it may be demonstrated only by adsorption and elution of anti-D. Weak D individuals do NOT produce anti-D and can be considered to be Rh positive for transfusion and RhIg purposes.2. Partial DPartial D variants have altered Rh(D) proteins that differ sufficiently from normal D antigens (qualitative difference) to allow anti-D production. Partial D red cells may react with some but not all anti-D typing reagents. There are many categories of partial D antigens (e.g., DIIIa, DVI, DAR), each with a unique genetic basis.Some persons with partial D have weakly expressed D epitopes and are designated "partial weak D."In practice, partial D and weak partial D can be considered similarly, i.e., ideally they should be transfused with Rh negative RBC and are candidates to receive perinatal Rh immune globulin depending on the policy in their location. | View Page |
| Clinical Relevance of D Phenotypes Clinically relevant information on D phenotypes can be summarized as follows: D phenotype D antigen expression Rh(D) typing Produce anti-D RBC to transfuse RhIg recommended** D+ normal direct agglutination no D+ or D– no Weak D normal but weak IAT no D+ or D– no Partial D altered direct agglutination* & IAT yes D– yes Partial weak D altered & variable direct agglutination* & IAT yes D– yes D– none IAT yes D– yes * Depending on anti-D reagent used ** USA, UK and parts of Canada | View Page |
| RhIg prevents anti-D production mainly by clearing antibody-sensitized D-positive RBCs from maternal circulation. | View Page |
| Which D variant has a qualitative difference in the D antigen that allows individuals with the D variant to produce anti-D? Select all that apply. | View Page |
| Using the initial screen cell antigram below, which antibodies have not been eliminated? Include all antibodies even if they are unlikely to cause HDFN.Screen CellRhRhesusKellDuffyKiddMNSsPLewisResultsCellCDEceKkFyaFybJkaJkbMNSsP1LeaLebGelIAT1R1R1++00+0+++0++00++002+12R2R20+++0++0++++++++0+3+23rr000++0++00+0++0+S+003Auto0Auto | View Page |
| Antibody Titration Some TS laboratories try to determine if anti-D is passive or immune by performing titrations to determine the titer of the anti-D. Such a protocol usually assumes that an anti-D titer greater than 4 likely represents active immunization. Unfortunately, a titer of 4 or 8 could be active or passive, although a high titer (e.g., 64 or more) almost certainly means the anti-D is immune.Titration results can be affected by several variables: Red cell phenotype; Donor antigen variability (even if the same phenotype); Method used; Operator variability.Because lower titers could be due to both passive and immune anti-D, in the absence of test results that suggest immune anti-D, routine antibody titration is not a good use of time compared to assuming that anti-D is passive. Most transfusion medicine best practice guidelines do NOT recommend routine titration for women known to be injected with RhIg and exhibiting a 2+ or less reaction with D+ red cells, i.e., test results consistent with RhIg-derived passive anti-D. | View Page |
| Crossmatch Implications of RhIg-associated Passive Anti-D Once again, policies vary from laboratory to laboratory since the issue is not directly addressed by blood safety standards. For example, AABB and other standards require a version of the following: When clinically significant red cell antibodies are detected or the recipient has a history of such antibodies, RBC components shall be prepared for transfusion that lack the corresponding antigen and are serologically crossmatch-compatible, where serologically is taken to be an IAT at 37oC. If no clinically significant antibodies were detected in antibody screen tests and the patient has no record of such antibodies, detection of ABO incompatibility is required, which can be accomplished by immediate spin crossmatch or an electronic crossmatch. The key issues are whether detectable passive anti-D from RhIg or a record of passive anti-D from RhIg should be considered clinically significant for crossmatch purposes. Because standards do not directly address these issues, TS laboratories are left to interpret what is required to meet the standards. Practices may be further complicated because of the transfusion service's laboratory information system (LIS). | View Page |
| Immunogenicity Immunogenicity is the ability of an antigen to provoke an immune response in an antigen-negative recipient. Why some antigens are more immunogenic than others is unknown. Not considering antigens in the ABO system, Rh(D) is the most immunogenic red cell antigen, followed by K in the Kell blood group system. Other immunogenic antigens include c and E in the Rh system. In routine blood banking assessments of an antigen's immunogenicity are typically based on the prevalence of the corresponding antibody and do not take into account the frequency of the antigen in the general population. For example, k in the Kell system may be very immunogenic but anti-k is rare since 99.8% of Caucasians are k+ and cannot make anti-k. | View Page |
| Literature and Online Resources The following published literature and online resources, while useful, should not be used as a substitute for technical and clinical judgment. Medical and technical information becomes obsolete quickly and current sources relevant to the user's location should always be consulted.References indicated by * provide a broad overview of HDFN and are highly recommended.LITERATUREAvent ND, Reid ME. The Rh blood group system: a review. Blood. 2000 Jan 15;95 (2):375-87.Bowman J. Thirty-five years of Rh prophylaxis. Transfusion 2003 Dec;43(12):1661-6.* Eder AF. Update on HDFN: new information on long-standing controversies. Immunohematology. 2006;22(4):188–195. (scroll to article).Eder, AF, Manno, C.S. Alloimmune hemolytic disease of the fetus and newborn. In Wintrobe's Clinical Hematology, 11th ed. (Greer JP, Foerster J, Lukens JN, Rodgers GM, Paraskevas F, Glader BE, (eds). Philadelphia, PA: Lippincott, Williams & Wilkins, 2004.Flegel WA. Molecular genetics of RH and its clinical application. Transfus Clin Biol. 2006 Mar-Apr;13(1-2):4-12. Kennedy MS, McNanie J, Waheed A. Detection of anti-D following antepartum injections of Rh immune globulin. Immunohematology 1998;14(4):138-40.Koelewijn JM, de Haas M, Vrijkotte TG, van der Schoot CE, Bonsel GJ. Risk factors for RhD immunisation despite antenatal and postnatal anti-D prophylaxis.BJOG. 2009 Sep;116 (10): 1307-14. Epub 2009 Jun 17.* Kumar S, Regan F. Management of pregnancies with RhD alloimmunisation. BMJ. 2005 May 28;330(7502):1255-8. (UK perspective but much valuable information relevant to all)* Murray NA, Roberts IAG. Haemolytic disease of the newborn. Arch Dis Child Fetal Neonatal Ed 2007 Mar; 92(2): F83–F88. Oepkes D, Seaward PG, Vandenbussche FP, Windrim R, Kingdom J, Beyene J, Kanhai HH, Ohlsson A, Ryan G; DIAMOND Study Group. Doppler ultrasonography versus amniocentesis to predict fetal anemia. N Engl J Med. 2006 Jul 13;355(2):156-64.Ramsey G. Inaccurate doses of Rh immune globulin after Rh-incompatible fetomaternal hemorrhage: survey of laboratory practice.Arch Pathol Lab Med 2009 Mar; 133(3):465-9. Reid ME. The Rh antigen D: a review for clinicians. Blood Bulletin 2008 Apr; 10(1).Sandler SG. Effectiveness of the RhIg dose calculator. Arch Pathol Lab Med 2010 Jul;134(7): 967-8.Shulman IA, Calderon C, Nelson JM, Nakayama R. The routine use of Rh-negative reagent red cells for the identification of anti-D and the detection of non-D red cell antibodies. Transfusion 1994 Aug;34(8):666-70.Tamul KR. Determining fetal-maternal hemorrhage with flow cytometry. Advance 2000. Posted online June 5, 2000.Westhoff CM, Sloan SR. Molecular genotyping in transfusion medicine. Clin Chem 2008;54(12): 1948-50.ONLINE RESOURCESPaxton A. Bringing new rigor to RhIg calculations. CAP Today May 2008. *Wagle S, Deshpande PG. Hemolytic disease of the newborn. eMedicine / WebMD. Updated Apr. 9, 2010. | View Page |
| Why might screen cell #2 be reacting stronger than screen cell #1? | View Page |
| Antigram to explain prior question The antigram below explains possible reasons for cell #2 reacting stronger: The patient may have anti-D and another antibody whose corresponding antigen is on cell # 2 (e.g., anti-E or anti-K). The patient has an antibody other than anti-D (e.g., anti-Jka) and cell #2 has a double dose of the antigen but cell #1 has only single dose. Screen Cell Rh Rhesus Kell Duffy Kidd MNSs P Lewis Lu Results Cell C D E c e Cw K k Kpa Fya Fyb Jka Jkb M N S s P1 Lea Leb Lua Gel IAT 1 R1R1 + + 0 0 + 0 0 + 0 + + + + 0 + 0 + + + 0 0 2+ 1 2 R2R2 0 + + + 0 0 + + 0 0 + + 0 + + + + + 0 + 0 3+ 2 3 rr 0 0 0 + + 0 0 + 0 + 0 0 + + 0 + 0 +S 0 + 0 0 3 Auto 0 Auto | View Page |
| Evaluating inconsistencies Once an antibody has been identified and other clinically significant antibodies have been excluded, the case must be looked at as a whole to confirm the logical consistency of all results and data.This process includes assessing any inconsistencies.For example:1. Is the patient negative for the corresponding antigen? Yes: The patient is Jk(a-).2. Is the antibody specificity consistent with the typical phase(s) of reactivity for the antibody? Yes: Kidd antibodies are IgG and react in the antiglobulin phase. | View Page |
| Unexpected anomaly 3. Do the results of the initial antibody screen support the presence of the identified antibody?No: All 3 screen cells reacted in the initial screen. Upon review, however, only Screen Cells 1 and 3 were Jk(a+); Screen Cell 2 reacted but was Jk(a-).This anomalous result was investigated by a reference laboratory. It was discovered that the patient had anti-Rd, an antibody to the low frequency antigen Radin (Rd). By chance, Screen Cell 2 was Rd-positive. Radin has a frequency of less than 0.5% in several populations tested. The screen cell manufacturer was notified. They would likely confirm that the cell was Rd-positive, make their clients aware of it, and document it in future antigrams. | View Page |
| Antibody identification checklist To improve the quality of conclusions when identifying antibodies, a checklist is a simple quality control tool to increase transfusion safety. If a specific antibody pattern cannot be identified with acceptable confidence, or if significant serologic or non-serologic data are inconsistent and cannot be rationalized, further testing will be required.Before concluding that the investigation is complete, unless not applicable, mentally reply to each question in the checklist. If any answer is no, has it been resolved? Antibody Identification Checklist Yes/No/NA 1. For a single antibody, does the reaction pattern fit only one antibody specificity? 2. Is antibody specificity consistent with the results of the initial antibody screen? 3. Are reaction phases consistent with antibody specificity? 4. If multiple antibodies are present, can all reactions be explained by the antibody combination? 5. If the autocontrol is negative, are patient red cells negative for the corresponding antigen(s)? 6. Have additional possible antibodies been excluded by selected red cells? 7. Can all variable reaction strengths be explained? 8. If tested, are antigen-negative donor cells compatible by antiglobulin crossmatch? 9. If there are data that do not fit antibody specificity or if there are results that are improbable, are they explainable? 10. Have all results and conclusions been systematically evaluated for consistency? | View Page |
| Understanding the "rule of three" In immunohematology textbooks, the "rule of three" is sometimes presented as follows:1. If a patient plasma or serum gives positive results with a minimum of three antigen-positive cells and negative results with a minimum of three antigen-negative cells, concluding that the serum contains an antibody directed against the antigen has a p value of 0.05.2. Therefore, a p value of 0.05 requires at least three positives and three negatives.The first statement is correct but second statement is a misinterpretation of the p value.Three positives and three negatives are required to identify an antibody with a p value of 0.05 ONLY if you have only a 6-cell panel. It does not mean that you always need three positive cells and three negative cells to get p=0.05.For example: A 10-cell panel with eight Jk(a+) cells and two Jk(a-) cells gives a probability of 0.02 if all the positive cells and none of the negative cells react. A 10-cell panel with eight K- cells and two K+ cells gives a probability of 0.02 if all the positive cells and none of the negative cells react. Learning point: You do not need three positive cells and three negative cells to get an acceptable p value of 0.05. | View Page |
| The patient's red cell eluate initially was unidentifiable, reacting weakly with only two panel cells that did not fit a pattern. Once anti-Jka was identified, a check of the eluate panel results showed that both reactive cells were Jk(a+b-) but two other JkaJka panel cells did not react.Consider the question below, then click on the answer. | View Page |
| Summary This case study presents a scenario in which a patient had an unexpected antibody that disappeared after he was transfused with 2 units of unmatched group O Rh negative RBC. The patient developed a positive DAT with MFA but an antibody identification using the post-transfusion red cell eluate was inconclusive, making the antibody unidentifiable. Fortunately, the patient improved and further transfusion was not required. Ultimately, the patient's antibody was identified as anti-Jka, with a second antibody to a low frequency antigen (Radin) also unexpectedly present.The case illustrates the risks involved in using unmatched blood. | View Page |
| Risks of transfusing unmatched RBC We often "get away" with transfusing unmatched RBC because the incidence of unexpected antibodies in patients experiencing medical emergencies is thought to be relatively low ( ~3-5% is sometimes cited, but with little solid evidence).Antibody incidence may vary according to several factors: Genetic disposition Patient's underlying disease Number of prior transfusions Gender (females may get exposed to foreign antigens via fetomaternal bleeds as well as transfusion) Concordance of antigen phenotypes of patients vs blood donors in a given locale.In general, antibody incidence increases with the number of transfusions that are given, although most antibody producers will respond within the first 3 - 4 transfusions. Antibody incidence in transfusion-dependent patients, such as those with sickle cell anemia or thalassemia, is very high. Regardless of likelihood, transfusing uncrossmatched blood to a patient with unexpected antibodies can result in a serious hemolytic transfusion reaction. | View Page |
| Balancing the risks Life-Threatening HemorrhageDespite potential risk, sometimes immediate transfusion is necessary, even for patients with red cell antibodies. In such cases transfusion service staff should alert the medical director, who can discuss options with clinical staff.The medical director will generally talk to the staff attending the patient and indicate that, if possible, they should hold off transfusion. But if it is a case of massive bleeding where exsanguinating hemorrhage is likely, it is better to give some blood and monitor for a delayed hemolytic transfusion reaction than to let the patient bleed to death.Transfusing when bleeding is brisk will result in much of the autologous and incompatible blood bleeding out, with the possibility of a delayed hemolytic reaction once the patient's antibody rebounds and destroys still present antigen-positive donor red cells.Some transfusion services also try to minimize the risk of unmatched blood by typing their emergency supply of O Rh negative RBCs for the K antigen, since anti-K is a relatively common clinically significant antibody. See Resources for two papers that discuss the risks of transfusing un-crossmatched emergency blood. | View Page |
| Literature and online resources LiteratureDutton RP, Shih D, Edelman BB, Hess J, Scalea TM. [abstract]. Available at: Safety of uncrossmatched type-O red cells for resuscitation from hemorrhagic shock.J Trauma. 2005 Dec;59(6):1445-9. Accessed November 5, 2012.Johnson ST, Rudmann SV,Wilson, SM. Serologic problem solving strategies:a systematic approach. Bethesda, MD: AABB, 1996.Online resourcesThe following are online examples of good practice. The information should not be used as a substitute for technical and clinical judgment. Medical and technical information becomes obsolete quickly and current sources relevant to the user's location should always be consulted.Transfusion reactions: Transfusion complications (Canadian Blood Services)Education website for CBS's hospital customersREACT (Sunnybrook HSC, Toronto, ON, Canada) Pocket reference card for nurseson signs and symptoms of transfusion reactionsQuick cals (online calculator of p values for Fisher's exact test) Use a one-tailed test (since we would expect an antibody to react with red cells that are positive for the corresponding antigen) | View Page |
| The patient is Rh positive, but what is the patient's ABO group? | View Page |
| The antibody screen is positive but the transfusion of the O Rh-negative RBCs is already in progress. What are the transfusion service (TS) laboratory's priorities in this case?Place the following procedures that will be followed by the TS in the appropriate order of priority. | View Page |
| Which of the following statements about mixed-field agglutination (MFA) are true? Select all that are correct. | View Page |
| In this case, which red blood cells (RBCs) do you think are agglutinating in the DAT and why? | View Page |
| Consulting the patient's physician If the physician had decided to continue transfusing the patient at this stage, the following information should be communicated: Although all donors appear to be compatible in the post-transfusion crossmatch, they are not. The results are false negatives - the patient's antibody has been "mopped up" by adsorbing to the incompatible transfused O Rh-negative RBC. Given that 6 donors were positive using the pretransfusion plasma, the antigen is a higher frequency antigen and most donors would likely be antigen-positive and incompatible. The patient's physician should consult the TS medical director before any decision to transfuse is made. Transfusing RBC before tests are complete requires physicians to sign an emergency release form in which they assume full responsibility. | View Page |
| Cause of Delayed HTR Delayed HTR result from a secondary (anamnestic) immune response causing a weak, undetectable antibody to become stronger.Upon re-stimulation by donor RBC positive for the antigen corresponding to the patient's antibody:* Patient's memory B cells differentiate into antibody-producing plasma cells.* As new IgG antibody is produced, it sensitizes antigen-positive transfused donor red blood cells.* The IgG-sensitized donor red blood cells are then removed by extravascular hemolysis (EVH) mainly in the spleen. | View Page |
| Investigating weak antibodies In this case the patient's antibody has disappeared from the plasma by adsorbing to transfused donor red cells. It is detectable but unidentifiable in the post-transfusion red cell eluate. Several trial and error procedures exist to enhance weak antibodies. Which methods will enhance the reactivity of a given antibody depend on its characteristics. Methods to investigate weak antibodies include: Use a higher plasma to red cell ratio (add more antibody-containing plasma or eluate) Increase incubation time (if consistent with manufacturer instructions, if applicable) Use enzyme-treated panel red cells (enzymes enhance IgG antibodies in Rh and Kidd blood systems but denature some antigens, e.g., Fya, Fyb, S) Try alternative antibody detection methods, e.g., if using LISS routinely, try polyethylene glycol (PEG) or column agglutination methods such as gel, providing they have been validated for use in the TS laboratory. | View Page |
| Antigen phenotyping A standard follow-up to antibody identification is to antigen phenotype: Patient's red cells (expecting them to lack the corresponding antigen) Donor red cells (in this case, those transfused before an antibody was identified, or, more typically, to find suitable antigen-negative donors to crossmatch prior to transfusion).If you had wanted to type the patient for any antigens at this point in the investigation (2-weeks post-transfusion), which specimen would you have used? Think about any antigen typing problems and how to overcome them before proceeding to the next page. | View Page |
| Antigen phenotyping issues There are two potential problems in typing a recently transfused patient who develops a positive DAT: There will be two cell populations, patient and donor red blood cells. If the typing sera reacts by IAT, the positive DAT will cause false positives. In the case presented, the DAT has become negative. This also suggests that most (if not all) transfused donor red cells have been removed from the patient's circulation.Regardless, to be on the safe side, the patient's initial pretransfusion specimen, which was DAT negative and consisted of only the patient's red blood cells, should be used for antigen phenotyping. | View Page |
| Antigen phenotyping results The patient's pretransfusion red cells and all donor red cells involved in the case (two group O Rh-negative RBC and four group O Rh-positive red cells initially crossmatched) were phenotyped for Jka.As expected, the patient typed as Jk(a-). The six donor RBC that were incompatible in the initial crossmatch were Jk(a+).The frequency of the Jka gene in Caucasians is ~77%, with most Caucasian red cells (50%) typing as Jk(a+b+). | View Page |
| Which of the following statements about antigen phenotyping are true? (Select all that apply) | View Page |
| Categories of Transfusion Reactions Adverse complications of transfusions can be classified into several categories: Immune-mediated transfusion reactions are those that trigger a response from the patient's immune system. Many transfusion reactions are mediated by the recipient's immune system. These reactions occur as a result of antigen-antibody interactions. Antibodies involved include those with specificity towards antigens on red cells, white cells, or platelets. In general, the immune responses occur in three stages: the immune system detects foreign material (antigen) the immune system processes the antigen the immune system mounts a response to remove the antigen from the body Non-immune mediated hemolytic transfusion reactions are caused by the physical or chemical destruction of transfused RBCs, bacterial contamination, circulatory overload, or citrate toxicity. Acute reactions are those that occur during or within 24 hours after the transfusion. There is usually a rapid onset of symptoms and these reactions may be fatal. Delayed reactions occur weeks or months after the transfusion of blood or blood components. | View Page |
| Additional Testing If preliminary testing suggests hemolysis or if the results are misleading, additional testing may be required. If human error has been ruled out during the clerical check, repeat ABO/Rh testing should be performed on the unit of blood or its segment and the pre-transfusion sample to detect any sample mix ups and clerical errors. Antibody detection studies should be performed on the pre- and post-transfusion samples to look for any unidentified antibodies. If an antibody is identified, the donor cells should be tested for the corresponding antigen. The crossmatch should be repeated with pre-and post-tranfusion specimens using the indirect antiglobulin test (IAT). An incompatible crossmatch with the pre-transfusion sample indicates an original error, either clerical or technical. Incompatibility with only the post-transfusion sample indicates a possible anamnestic response, as in a delayed hemolytic transfusion reaction (DHTR), or sample misidentification. The patient's first voided urine specimen should be examined for the presence of free hemoglobin. The patient's bilirubin levels may also be evaluated. A change from normal pale yellow serum to a post-transfusion bright or deep yellow serum should prompt an investigation for hemolysis. The maximum concentration of bilirubin following hemolysis is not usually detectable until 3 to 6 hours after transfusion. The hemoglobin and hematocrit can be tested to detect a drop in hemoglobin or failure of the hemoglobin to rise after transfusion. Important information about physical or chemical hemolysis may be gained from examining the returned unit bag. If hemolysis is present in the bag or tubing, a process which affected the blood, such as inappropriate warming or faulty infusion pump, should be suspected. If bacterial contamination is suspected, the unit can be cultured. A positive culture indicates a reaction due to bacterial contamination. | View Page |
| An acute hemolytic reaction may be caused by which of the following? (Choose all that apply) | View Page |
| Pathophysiology The exact mechanism of lung injury in transfusion-related acute lung injury (TRALI) has not be identified. It is believed that the mechanism may vary from patient to patient. The most common finding is leukocyte antibodies in donor or patient plasma. Anitbodies to human leukocyte antigen (HLA) have been associated with TRALI. These anti-HLA antibodies can be formed in response to exposure to foreign antigens from transfusion or pregnancy. The source of the antibody is usually the donor not the patient. Transfused antibodies react with the recipient which results in leukocyte emboli aggregating in the lung capillary bed. Capillary damage triggers interstitial edema and fluid in the alveolar spaces, causing decreased air exchange and hypoxia. | View Page |
| Evaluation of Donors Associated with Transfusion-Related Acute Lung Injury (TRALI) The AABB published an interim standard in 2005 that states, "Donors implicated in TRALI or associated with multiple events of TRALI shall be evaluated regarding their continued eligibility to donate." A donor is associated with TRALI when one of his/her donor units is transfused 6 hours before the clinical presentation of TRALI in a patient. A donor is implicated in TRALI if he/she is found to have an antibody to an HLA class I or II antigen and the antibody is specific for an antigen on the recipient's leukocytes or a positive crossmatch is obtained.*It is suggested that donors at greatest risk of developing HLA antibodies be tested, such as multiparous women. It has also been suggested that donors that present with demonstrable antibodies and have been implicated in TRALI be permanently deferred from donating. Studies have shown that donors implicated in TRALI reactions may present a future danger to transfusion recipients. Although, there are some instances where donors with HLA antibodies have not caused TRALI reactions. Another option would be to wash all red cell products from these donors in special circumstances such as rare donors. Reference: Association bulletin #05-09. AABB; August 2005. Available at: http://www.aabb.org/resources/publications/bulletins/Pages/ab05-09.aspx. Accessed November 12, 2010. | View Page |
| Which type of antibodies are known to cause transfusion-related acute lung injury (TRALI) reactions? | View Page |
| Definition and Incidence Delayed hemolytic transfusion reactions (DHTR) are reactions that occurs 3 to 10 days after the transfusion. Usually, the blood appears serologically compatible at initial testing. Delayed reactions are common in patients who have been immunized to a foreign antigen from a previous transfusion or pregnancy, but the antibody titers decrease over time and the antibody is not detectable during pre-transfusion testing. The transfusion leads to a secondary (anamnestic) response, causing increased antibody production that sensitizes antigen-positive donor red cells. Hemolysis is extravascular. Sensitized cells are removed from circulation by the reticuloendothelial system, also called the monocyte-macrophage system. Because there is a delay in the presentation of symptoms, DHTR is not usually considered as a cause of the clinical presentation. The transfusion service usually initiates investigation of a DHTR because of serologic findings in a post-transfusion specimen. DHTRs occur more frequently than acute hemolytic reactions. Approximately 1:2500 transfusions result in a DHTR. | View Page |
| Diagnosis The symptom most commonly associated with a delayed hemolytic transfusion reaction (DHTR) is unexplained decrease in hemoglobin and hematocrit. Patients may also present with fever and jaundice. Hemolysis occurs slowly and is primarily extravascular. Unlike an acute hemolytic transfusion reaction (AHTR), hemoglobinuria, acute renal failure, and disseminated intravascular coagulation (DIC) are not generally seen. On some occasions, patient's may not present with any symptoms. Serologic findings include a positive direct antiglobulin test (DAT) and/or a positive antibody screen in post-transfusion testing. In many cases, the physician will send a request for an additional transfusion because of the decreased hemoglobin levels, and not suspect a DHTR. The positive antibody screen will trigger an investigation including antibody identification. The DAT may have a mixed field appearance because of the antibody-sensitized transfused red cells and the non-sensitized patient red cells. Segments from the donor unit can be tested for the offending antigen once the antibody has been identified.Antibodies that are most often reported as the cause of DHTR are anti-Jka and anti- Jkb. Other antibodies that are also commonly implicated in a DHTR include Kell, Rh, and Duffy system antibodies.The patient's physician should be notified so that additional clinical and laboratory evidence can be evaluated. | View Page |
| Definition/Manifestation/Prevalence Post-transfusion purpura (PTP) is a very rare complication of blood transfusion. It has been most commonly associated with the transfusion of red blood cells (RBCs) and whole blood, but has also been seen in platelet and plasma transfusions. It is characterized by a rapid onset of thrombocytopenia, or decreased platelet count, which results from the product of a platelet alloantibody. Platelet counts are usually less than 10,000/uL. Reactions occur around 7 to 14 days post-transfusion. Patients present with purpura, bleeding from the mucous membranes, gastrointesinal ,and/or urinary tract bleeding. Melena and vaginal bleeding have also been reported. The thrombocytopenia is usually self-limiting. Platelet counts and coagulation studies aid in the diagnosis. Patients can also be tested for platelet specific antibodies, human leukocyte antigen (HLA) antibodies and lymphocytotoxic antibodies. The differential diagnosis includes other causes of thrombocytopenia. | View Page |
| Pathophysiology, Treatment and Prevention Post-transfusion purpura (PTP) is caused by platelet-specific antibodies in a patient who has been previously exposed to platelet antigens through pregnancy or transfusion. The most frequently identified antibody is Anti-PLA1 which reacts with platelet antigen HPA-1a. The platelet antibody binds to the platelet surface which allows for extravascular removal through the liver or the spleen. The patient's own platelets are destroyed as well, thus aggravating the thrombocytopenia. Three theories are suggested regarding the destruction of autologous platelets. One suggests that immune complexes bind to the platelets through the Fc receptor and cause destruction. The second theory proposes that the patient's platelets absorb a soluable platelet antigen from the donor plasma. The third hypothesis, which has the most support, states that the platelet alloantibody has autoreactivity that develops when the patient is exposed to the foreign platelet antigen. Platelet transfusion is NOT a treatment option. Steroids, whole blood exchange, and plasma exchange are accepted options for treatment. According to the AABB, intravenous IgG (IVIG) is the treatment of choice (AABB Technical Manual, p. 744). Most patients will respond to treatment within several hours to four days. PTP does not usually re-occur but it is recommended that patient's with a previous reaction be transfused with antigen-matched components. Autologous donations or directed donations from antigen matched family members may be the best sources of blood. PTP has been known to occurr even after the transfusion of deglycerolized rejuvenated or washed red cells, so these processes do not prevent a reaction. | View Page |