| 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 |
| 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 |
| 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 |
| 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 |
| 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 |
| 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- Antibody Panel Antibody panel resultsResults of the antibody panel show reactions at immediate spin and AHG with varying strengths. The pattern at IS matches P1. Remember that varied strengths can mean multiple antibodies, dosage or both.There are not enough rule-out cells to rule anything out with 3 negative reactions. You can use panel cells that reacted at IS and are negative at AHG for rule-out. Use cells 4 and 10 for rule-outs. Antibodies that have no rule-outs from this panel are: C, E, Cw, Kpa, Jsa, Fya, Lea, M,s, P1, and Lua. Cw, Kpa, Jsa, and Lua are usually not present on panels and fall under the "unable to rule out" catagory. C,E, Fya and s are clinically siginificant and should have further testing done to rule-out or rule-in these antibodies. Lea , P1 and M tend to react at IS, so if the pattern of reactivity is compared to the reaction pattern at IS, there is a match for P1.A selected cell panel was then performed. | View Page |
| Advance Organizer Before beginning the course take some time to review and think about what you already know about HDFN. For example, jot down brief notes to answer the following questions: Which antibody causes the most severe HDFN? Antibodies in which blood group system are the most common cause of positive direct antiglobulin tests (DATs) in newborns but rarely cause clinically significant hemolysis? Should DATs be performed on all newborns regardless of maternal ABO and Rh blood groups? What is Rh immune globulin (RhIg), its source, constituents, purpose, and mechanism of action? Which tests are used to determine postnatal RhIg dosage? Which type of D variant can produce anti-D? What follow-up tests are typically indicated if a pregnant female has a positive antibody screen when initially tested? Which laboratory findings would suggest that an infant may have ABO HDFN? How can the clinical status of fetuses at risk for HDFN be monitored? What are the characteristics of red cells suitable for intravenous transfusion to fetuses suffering from severe HDFN due to anti-D? | View Page |
| RhIg Dosage In North America, a standard dose of RhIg is considered to be 1500 IU (300 µg). Note: 1 µg of anti-D = 5 IU.300 µg of RhIg can suppress immunization to approximately 30 mL of D-positive whole blood (15 mL of packed rbc). If gestational age is known to be less than 12 weeks, a 600 IU (120 µg) dose may be sufficient.Depending on the gestation of the fetus, recommended dosages vary from country to country and within countries. Samples of recommendations that may change over time: USA: American Congress of Obstetricians and Gynecologists (1999, reaffirmed 2007): Antenatal RhIg dose of 300 µg (1500 IU) at 28 weeks and another 300 µg after delivery of a D-positive infant. Canada: Society of Obstetricians and Gynaecologists of Canada (2003): Antenatal RhIg dose of 300 µg (1500 IU)at 28 weeks (alternatively, 2 doses of 100–120 µg, one at 28 weeks and one at 34 weeks). After delivery of a D-positive infant, another 300 µg (alternatively, 120 µg IM or IV). UK: Royal College of Obstetricians and Gynaecologists (2002): Antenatal RhIg does of 100 µg (500 IU) at both 28 weeks and 34 weeks of gestation, and another 100 µg after delivery of a D-positive infant. All recommendations require testing to detect larger fetal bleeds, e.g., FMH larger than 30 mL of whole blood (for 300 µg doses) and FMH over 12 mL of rbc for 100 µg doses. | 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 'Failures' Numerous studies have shown that, if administered correctly, RhIg is effective at preventing D immunization. To work, RhIg must be given in sufficient dose, and it must be given before Rh immunization has begun.Unfortunately, despite RhIg's proven efficacy, some women continue to make anti-D in the perinatal period. Such 'failures' are mainly (but not totally) due to human error. Examples of how women may still produce anti-D some 40+ years after the implementation of RhIg prophylaxis: Immunization to D occurred before the administration of RhIg, e.g., before 28 weeks gestation*; Immunization to D occurred after the administration of RhIg at 28 weeks and before delivery because an antenatal fetomaternal hemorrhage (FMH) occurred that was too large for residual passive anti-D to give protection; Female was already immunized from a prior pregnancy but anti-D was too weak to be detected in antibody screen tests prior to RhIg administration; RhIg dosage was insufficient to clear a larger fetal bleed at delivery (e.g., FMH screen was not done or a false negative occurred); Incorrect calculation of RhIg dosage; RhIg administered too late , e.g., well after 72 hours of delivery; Antenatal RhIg not given, e.g., mother had no, or limited, access to prenatal care, or did not seek it, and a FMH occurred during pregnancy; Failure of physician to carry out prenatal blood testing; RhIg not given due to laboratory clerical or technical error in Rh typing the mother or child; RhIg not given in cases such as abortions, ectopic pregnancies, and trauma (e.g., car accidents).* Because anti-D production before 28 weeks is rare (the order of 0.24% to 0.31%), RhIg's use earlier in pregnancy is not recommended. It is not cost effective and would expose most women to an unneeded blood product. | View Page |
| Calculating RhIg Dosage Using the estimated volume of fetal bleed determined by the Kleihauer-Betke test or flow cytometry, the number of vials of RhIg (300 µg) to inject is calculated as follows: Number of vials of 300 µg RhIg = volume of fetal bleed/30 mLIn the interests of safety some American organizations recommend the following to deal with decimal points: If the number to the right of the decimal point is <5, round down and add 1 vial (e.g., 1.4 = 1 +1 = 2 vials) If the number to the right of the decimal point is greater than or equal to 5, round up and add 1 vial (e.g., 1.7 = 2 +1 = 3 vials) Sub-calculations: Volume of fetal bleed: % fetal cells x maternal blood volume Maternal blood volume: 70 mL/kg x weight (kg) (assume 5,000 mL if maternal information is unknown) Note: RhIg dose calculators are available (see Further Reading: Paxton A. Bringing new rigor to RhIG calculations). | View Page |
| The appropriate dosage of Rh immune globulin (RhIg) to administer post-delivery to an Rh-negative mother delivering an Rh-positive child is calculated based on the estimated volume of fetal bleed.What is the value of x in the formula given below that is used to calculate RhIg dosage?Number of vials of 300 µg RhIg = volume of fetal bleed/x mLEnter the number in the box below that is represented by x in the formula; do not spell out the number.(e.g., use "5" and not "five"). | View Page |
| RhIg Dosage In North America, a standard dose of RhIg is considered to be 1500 IU (300 µg). Note: 1 µg of anti-D = 5 IU.300 µg of RhIg can suppress immunization to approximately 30 mL of D-positive whole blood (15 mL of packed rbc). If gestational age is known to be less than 12 weeks, a 600 IU (120 µg) dose may be sufficient.Depending on the gestation of the fetus, recommended dosages vary from country to country and within countries. Samples of recommendations that may change over time: USA: American Congress of Obstetricians and Gynecologists (1999, reaffirmed 2007): Antenatal RhIg dose of 300 µg (1500 IU) at 28 weeks and another 300 µg after delivery of a D-positive infant. Canada: Society of Obstetricians and Gynaecologists of Canada (2003): Antenatal RhIg dose of 300 µg (1500 IU)at 28 weeks (alternatively, 2 doses of 100–120 µg, one at 28 weeks and one at 34 weeks). After delivery of a D-positive infant, another 300 µg (alternatively, 120 µg IM or IV). UK: Royal College of Obstetricians and Gynaecologists (2002): Antenatal RhIg does of 100 µg (500 IU) at both 28 weeks and 34 weeks of gestation, and another 100 µg after delivery of a D-positive infant. All recommendations require testing to detect larger fetal bleeds, e.g., FMH larger than 30 mL of whole blood (for 300 µg doses) and FMH over 12 mL of RBC for 100 µg doses. | View Page |
| RhIg 'Failures' Numerous studies have shown that, if administered correctly, RhIg is effective at preventing D immunization. To work, RhIg must be given in sufficient dose, and it must be given before Rh immunization has begun.Unfortunately, despite RhIg's proven efficacy, some women still make anti-D in the perinatal period. Such 'failures' are mainly (but not totally) due to human error. Examples of how women may still produce anti-D some 40+ years after the implementation of RhIg prophylaxis: Immunization to D occurred before RhIg was administered, e.g., before 28 weeks gestation*; Immunization to D occurred after the administration of RhIg at 28 weeks and before delivery because an antenatal FMH occurred that was too large for residual passive anti-D to give protection; Female was already immunized from a prior pregnancy but anti-D was too weak to be detected in antibody screen tests prior to RhIg administration; RhIg dosage was insufficient to clear a larger fetal bleed at delivery (e.g., FMH screen or quantification was not done or a false negative occurred); Incorrect calculation of RhIg dosage; RhIg administered too late , e.g., well after 72 hours of delivery; Antenatal RhIg not given, e.g., mother had no or limited access to prenatal care, or did not seek it, and a FMH occurred during pregnancy; Failure of physician to carry out prenatal blood testing; RhIg not given due to laboratory clerical or technical error in Rh typing the mother or child; RhIg not given in cases such as abortions, ectopic pregnancies, and trauma (e.g., car accidents). * Because anti-D production before 28 weeks is rare (the order of 0.24% to 0.31%), RhIg's use earlier in pregnancy is not recommended. It is not cost effective and would expose most women to an unneeded blood product. | View Page |
| Antibody Exclusion Protocol (General) Transfusion service (TS) laboratories use different protocols to exclude antibodies. For example:For antibodies whose corresponding antigens exhibit dosage, some laboratories exclude them based on a negative reaction with one homozygous cell.* Other laboratories require negatives with two homozygous cells to increase the confidence that the antibody is not present.If a homozygous cell is not available, some laboratories exclude such antibodies based on a negative reaction with two heterozygous cells.* Other laboratories require negatives with three heterozygous cells to increase confidence that the antibody is not present. * Homozygous and heterozygous do not refer to the red cells per se but to the red cell donors who are homozygous or heterozygous for the genes that determine the red cell phenotypes. See the general antibody exclusion protocol to be used in this case. | View Page |
| Assessing FMH and RhIg Dosage The remaining issue in this case is to determine if one vial of RhIG is sufficient or if there has been a FMH >30 mL of whole blood, requiring more than one vial of RhIg (300 µg). Recall that the incidence of FMH greater than 30 mL at delivery is rare and estimated to be about 1 in 400 deliveries (~0.3%). The laboratory used the rosette test to screen for FMH and it was negative. Accordingly, quantitation using the Kleihauer-Betke test or flow cytometry was not needed.RhIg dosageBased on the negative rosette test, the mother was injected with one vial of RhIg (300 µg). She was later discharged along with her healthy infant. | View Page |
| Calculating RhIg Dosage Using the estimated volume of fetal bleed determined by the Kleihauer-Betke test or flow cytometry, the number of vials of RhIg (300 µg) to inject is calculated as follows: Number of vials of 300 µg (1500 IU) RhIg = volume of fetal bleed/30 mLIn the interests of safety some organizations recommend the following to deal with decimal points: If the number to the right of the decimal point is <5, round down and add 1 vial (e.g., 1.4 = 1 +1 = 2 vials) If the number to the right of the decimal point is greater than or equal to 5, round up and add 1 vial (e.g., 1.7 = 2 +1 = 3 vials) Sub-calculations: Volume of fetal bleed: % fetal cells x maternal blood volume Maternal blood volume: 70 mL/kg x weight (kg) (assume 5,000 mL if maternal information is unknown) Note: RhIg dose calculators are available (see Further Reading: "Bringing new rigor to RhIG calculations"). | View Page |
| For those facilities that in the interest of safety use a special calculation for RhIg dosage, regardless if they round up or round down, they always add one vial. | View Page |