Anti-d Information and Courses from MediaLab, Inc.

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.

Treatment guidelines recommend that patients receive treatment if they have any of the following: Significant bleeding risk <20 x 109/L platelets and moderate bleeding <10 x 10 9/L platelets with no bleeding symptomsCorticosteroids are effective treatments for 50 - 80% of individuals with either acute or chronic ITP. Even with a reduction or discontinuation of corticosteroid treatment, remission can be maintained.Anti-D immunoglobulin, administered intravenously, may be an effective treatment for Rh-positive children or adults diagnosed with acute or chronic ITP. Anti-D immunoglobulin forms red blood cell complexes that block the destruction of platelets. This treatment cannot be used for patients who are Rh-negative or who have undergone a splenectomy. When a patient is refractory to the above treatments, other treatment possibilities include thrombopoietic drugs to stimulate the megakaryoblast or Rituximab, a treatment that targets CD 20-positive B-cells.Splenectomy may be a last resort treatment for chronic ITP sufferers if their platelet counts are below 30 x 109/ L or if symptoms warrant it. In ITP, antibodies develop that coat the platelets. The spleen produces macrophages whose Fc receptors recognize and destroy these antibody-coated platelets. Removing the spleen would decrease platelet destruction, but it is a last resort since the immunologic function of the spleen would also be lost.

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?

Although HDFN can be life threatening, in the case of anti-D it is a disease that can be prevented. Regardless of causative antibody, HDFN's serious consequences can be lessened by early laboratory diagnosis and treatment. This course begins with an in-depth review of HDFN and later discusses its prevention in detail. In reviewing HDFN, key questions to be answered include: What are the typical signs and symptoms of severe HDFN? Which serologic tests does the transfusion service laboratory use to diagnose HDFN? How is severe HDFN treated? Which development dramatically changed the incidence of HDFN due to anti-D? Other than the causative antibodies, what are some of the main differences between ABO HDFN and HDFN due to anti-D and other antibodies?

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.

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).

Whenever possible, a hallmark of HDFN treatment is to induce labor as early as possible once lung maturity has been attained so that the newborn will be able to survive. Once the infant is born, the main treatment for severe HDFN due to anti-D (and other antibodies causing severe disease) is exchange transfusion. In exchange transfusions, up to 85–90% of the infant's blood can be exchanged with donor blood by a process of removing 5–20 mL of blood at a time, and injecting an equivalent amount until the exchange is complete. An exchange transfusion accomplishes the following: Removes bilirubin and thus helps prevent kernicterus; Removes sensitized red cells that have not been broken down yet; Removes circulating maternal antibody; Provides antigen-negative red cells that will not be destroyed by the maternal antibody, thus will survive and provide oxygen to the tissues.

Amniocentesis is a procedure used to obtain amniotic fluid for diagnostic tests.As applied to diagnosing severity of HDFN, amniocentesis can be done beginning at approximately 28 weeks and repeated to provide serial estimates of the amount of bile pigment (bilirubin) in amniotic fluid. The process measures the difference in optical density at a wavelength of 450 nm (DOD450) between the observed density and an extrapolated baseline (if no excess bilirubin was being produced).Serial DOD 450 values are plotted on charts (e.g., Queenan chart or the extended Liley* chart), which categorize HDFN into 3 zones of severity.See examples of Liley and Queenan charts (from e-Medicine/WebMD)

Before ABO HDFN is considered as a possible cause of jaundice and anemia in the newborn, other causes should be considered, for example, erythrocyte membrane defects or red cell enzyme deficiencies. The diagnosis of ABO HDFN in the laboratory differs from diagnosing Rh and other types of HDFN in which clinically significant antibodies must be identified. Diagnosis may be difficult, because the DAT on the newborn's red cells is unreliable. Indeed, many labs do not routinely do a DAT on infants born to Rh positive females, since many will be positive in the absence of clinically significant hemolysis. Cord blood is often retained (e.g., for 7 days) should the infant develop signs of HDFN and required testing.If ABO HDFN is possible, based on incompatible ABO blood groups and a positive DAT, and the mother's antibody screen is negative, many laboratories do not investigate the positive DAT as would be done for unexpected antibodies like anti-D or anti-K (the laboratory does not perform an elution on the newborn's red cells). Instead, the infant's plasma is tested against group A1 (or B cells) and group O screen cells using the indirect antiglobulin test (IAT). A positive reaction with A1 or B cells, but not group O cells, would suffice to report a case of possible ABO HDFN.

The Red Blood Cells (RBC) that are chosen for exchange transfusion must meet these criteria: ABO-compatible with mother and infant (usually group O) and lack antigens to any maternal IgG antibodies; If mother has anti-D, RBCs are group O Rh negative; No greater than 7 days old; Reconstituted with AB Fresh Frozen Plasma (FFP) to obtain a prescribed hematocrit, e.g., 45–60% (0.45–0.60); CMV negative (or equivalent, e.g., leukoreduced by filtration); Negative for hemoglobin S to prevent blood from hypoxia-induced sickling; Irradiated with a minimum dose of 25 Gray (Gy) to prevent graft-versus-host disease.RBC are normally crossmatched with maternal plasma, although the infant's plasma can be used if a maternal blood specimen is unavailable.

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.

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)

Policies for typing fathers vary widely and usually testing is not done unless the mother develops anti-D or another clinically significant antibody. However, for Rh negative women, some labs consider Rh typing the father if paternity is certain. For example: Tests on Father ABO and Rh type; Test for weak D if initial Rh typing appears to be D-negative. If father is Rh negative, the fetus will be Rh negative and antenatal RhIg is not needed. The purpose of DCEce typing Rh positive fathers is to determine if the father is homozygous or heterozygous for D in order to predict whether the fetus is Rh positive. The father's actual Rh genotype can be determined by molecular methods, if available.

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).

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.

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.

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.

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

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.

A scenario where anti-D is detected at delivery in a female who received RhIg during pregnancy raises the question, is the anti-D active or passive?Distinguishing between passive and immune anti-D is important clinically: If passive anti-D is misinterpreted as active, RhIg prophylaxis may be omitted, leading to D sensitization. If active anti-D is misinterpreted as passive, appropriate antibody investigation may be curtailed putting the fetus at risk of developing HDFN.When this occurs, two main serologic questions need to be answered: Are the reactions due to passive anti-D from RhIg or due to active anti-D? Are there other antibodies that need to be excluded?

Fetomaternal hemorrhage (FMH) greater than 30 mL of whole blood occurs in only about 0.3% of cases but must be detected to prevent the mother from producing anti-D. Once the mother has become immunized to D, RhIg is of no use.A typical test protocol is to first screen for a large FMH and then quantitate the bleed if the screen is positive. Some laboratories proceed directly to a test that can quantitate the size of the FMH.Once the size of the FMH is determined, a formula is used to determine how much RhIg is needed. Recall that A standard vial of RhIg contains 1500 IU (300 µg) of IgG anti-D; 300 µg of RhIg can suppress immunization to approximately 30 mL of D-positive whole blood.

The Kleihauer-Betke test is performed to quantitate the number of fetal cells present in the maternal circulation. Once the size of the feto-maternal hemorrhage (FMH) is determined, the appropriate RhIg dose can be calculated and administered to prevent the mother from making anti-D.The test is based on the principle that red cells containing fetal hemoglobin (HbF) are less susceptible to acid elution than cells containing adult hemoglobin (HbA).General descriptionA peripheral blood smear is made from the maternal postpartum sample and treated with acid. Fetal cells remain intact because of high concentrations of HbF, while HbA is eluted from the maternal cells.After acid treatment the slides are washed, stained, and examined microscopically.The number of fetal cells (which take up the stain) are counted per number of maternal cells (which appear as ghost cells) to give % fetal cells.The volume of fetal bleed is then calculated to determine how much additional RhIg is required.See Kleihauer-Betke graphic (Source: ENet Answers) The top image on the right illustrates a negative Kleihauer-Betke test. The blue arrows in the bottom image point to fetal cells that have taken up the stain. The red arrows indicate maternal cells, which appear as ghost cells.These images were provided courtesy of Mount Sinai Blood Transfusion Laboratory, Toronto, Ontario and can be found on Canada's Transfusion Safety Officer's Website. Available at: http://www.transfusionsafety.ca/library/kb-ros.html. Accessed September 26, 2011.Limitations: Despite its widespread use, the Kleihauer-Betke test has significant limitations, including Low sensitivityPoor reproducibility.

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.

The composite image shown on the right illustrates the ABO typing reactions that were obtained for a patient. This particular case illustrates an ABO discrepancy. An ABO discrepancy occurs when the results of forward and reverse typing do not match. The reactions shown are described below in descending order:Patient red cells with reagent anti-A: negative reaction.Patient red cells with reagent anti-B: 4+ agglutination.Patient red cells with reagent anti-D: 4+ agglutination.Patient serum with reagent A1 red cells: negative reaction.Patient serum with reagent B red cells: negative reaction.This patient forward types as a group B, but reverse types as a group AB. (A group B patient should have anti-A. This patient demonstrates neither anti-A nor anti-B, similar to an AB patient). Further workup is necessary to determine the ABO type since the forward and back typing do not match. In this case, incubation at 40 C demonstrated the presence of weakened anti-A. The patient was therefore typed as group B. This case is an example of an ABO discrepancy which was due to a "missing" anti-A antibody. This could be due to old age, severe illness, or immunosuppression. Although evaluation of ABO discrepancies is beyond the scope of this course, it is important to note that all ABO discrepancies must be resolved before blood products can be released for transfusion.This patient is Rh (D) positive, as evidenced by the strong agglutination of his cells with reagent anti-D antibody.

This case concerns a common scenario in the transfusion service (TS) laboratory, the detection of anti-D at delivery in a female who has received Rh immune globulin (RhIg) during pregnancy.Distinguishing between passive and immune anti-D is important clinically: If passive anti-D is misinterpreted as immune, RhIg prophylaxis may be omitted leading to D sensitization. If immune anti-D is misinterpreted as passive, appropriate follow-up of the antibody may be curtailed putting the fetus at risk.Unfortunately, differentiating between immune and passive anti-D is often impossible. This case study presents an opportunity to review perinatal testing programs and the crucial role of RhIg in preventing hemolytic disease of the fetus and newborn (HDFN) due to anti-D. The case also examines practical aspects of routine serologic testing involving neonates and women who have received RhIg during pregnancy. The case is a companion to "Hemolytic Disease of the Fetus and Newborn" and complements its content.In brief, the case will: Guide participants through laboratory findings that need to be interpreted and resolved; Examine current best practices in perinatal testing programs; Review the characteristics of RhIg and its use in pregnancy; Review and investigate key issues associated with detection of anti-D in women who have received antenatal RhIg; Discuss crossmatch and LIS policies related to RhIg-derived passive anti-D.

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.

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).

Of the main D variants, a female with partial D (or partial weak D) may develop anti-D to the D epitopes that she lacks. Fortunately, partial D red cells adsorb little anti-D, thus leaving enough free RhIg to suppress immunization.However, most laboratories do not routinely differentiate between D variants and instead rely on routine tests and associated test protocols to determine a mother's Rh status for the purposes of RhIg administration. Policies differ between countries and even within countries. 1. Some labs do not test pregnant women for weak D and rely on routine D typing to determine Rh status. AABB Standards for Blood Banks and Transfusion Services, ed. 26 (2010) does not require weak D testing for Rh negative women.2. Some labs perform weak D tests on pregnant women who appear to be Rh negative and, if weak D, do NOT inject with RhIg.3. Some labs perform weak D tests on pregnant women who appear to be Rh negative and, if weak D, inject with RhIg based on the possibility that they may be partial D and capable of forming anti-D.

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.

Laboratories use different protocols to confirm anti-D from RhIg administration and simultaneously exclude other antibodies in pregnant females at delivery. The following are sample protocols (not all inclusive) with comments. All protocols assume that the patient has received RhIg (this needs to be confirmed); that the antibody screen is positive (2+ or less); reacts only in the IAT phase; reacts only with D+ screen cells.In other words, the following protocols assume that the antibody looks like a relatively weak IgG anti-D consistent with RhIg administration.1. Mini-panelProbably the most common protocol is to perform a mini-panel to exclude other antibodies and report "probable passive anti-D due to RhIg administration"; "passive anti-D consistent with recent RhIg administration" or similar. Some commercial panels indicate which panel cells are useful to rule out other antibodies in the presence of anti-D. A mini-panel of 5-7 carefully selected red cells is typically all that is needed.2. Full panel: Some labs do a full antibody identification panel to confirm ant-D and exclude other antibodies. This protocol is acceptable, but may be 'overkill' given that the same results can be achieved with fewer red cells.

Red cell reaction strengths at delivery from an antenatal RhIg injection at 26–30 weeks (usually 28 weeks) are typically 2+ or less, although stronger reactions are possible depending on the detection method, time since injection, and other factors. Multiple variables can affect the reaction strength of passive anti-D seen post-RhIg injection: Amount of RhIg injected (the greater the number of IU of anti-D administered, the stronger reactions will be); Titers of anti-D in the plasma pool used to manufacture RhIg (occasionally a donor with an exceptionally strong anti-D may be in the pool); Maternal physical size and related blood volume (a larger volume of maternal plasma will dilute RhIg more); Time between RhIg administration and testing (passive antibody will decrease in strength over time); Sensitivity of antibody detection method (e.g., gel-IAT and PEG-IAT may give stronger reactions than LISS-IAT); Volume of FMH (amount of D-positive fetal RBC available in the mother to adsorb anti-D); Route of RhIg administration: Some RhIg products can be administered IM only, whereas others can be given both IM and IV (see later). Peak levels of RhIg are reached faster with IV compared to IM administration (within hours with IV administration compared to days with IM administration). Also, with IV administration, higher levels of IgG anti-D are achieved. Operator variability (technologist techniques vary in removing cell buttons when reading IATs). Because of these variables, many laboratories consider 2+ or less reaction strengths to be consistent with passive anti-D.

As noted, policies for further testing to confirm anti-D, exclude other antibodies, and assess whether the anti-D is passive or immune vary among TS laboratories. Even though patient A.D. had a negative antibody screen at 28 weeks and her positive antibody screen appears to be anti-D from RhIg administration at 28 weeks, some TS laboratories may set up a full antibody identification panel to confirm the presence of anti-D. Others would proceed straight to a mini-panel of red cells specifically selected to exclude other clinically significant antibodies in the presence of anti-D.In this case the laboratory's protocol was to set up a mini-panel of six selected red cells (rr, r'r, and r'r cells), along with a positive Ror control and an autocontrol.

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.

As noted earlier, in this case study the laboratory's protocol is to set up a mini-panel, providing these criteria are met: Mother is Rh-negative and has been tested on two separate occasions; Laboratory has confirmed administration of RhIg prophylaxis; Result of current antibody screen is positive and typical of anti-D due to RhIg; There is no record or history of an unexpected antibody. All criteria were met and a selected mini-panel was set up to confirm the presence of anti-D and exclude possible co-existing maternal antibodies. Other clinically significant antibodies have implications for possible HDFN and for transfusion to both the mother and newborn, thus must be excluded.

Fetomaternal hemorrhage (FMH) greater than 30 mL of whole blood occurs in only about 0.3% of cases but must be detected to prevent the mother from producing anti-D. Once the mother has become immunized, it cannot be undone and RhIg is of no use.A typical test protocol is first to screen for a large FMH and then quantitate the bleed if the screen is positive. Some laboratories proceed directly to a test that can quantitate the size of the FMH.Once the size of the FMH is determined, a formula is used to determine how much RhIg is needed. Recall that: A standard vial of RhIg contains 1500 IU (300 µg) of IgG anti-D; 300 µg of RhIg can suppress immunization to approximately 30 mL of D-positive whole blood. Several methods are available to detect FMHs that require additional RhIg.Acceptable screening tests for FMH include Rosette method; Commercial fetal bleed screening tests; Gel agglutination fetal cell screening technique.Note: The weak D (microscopic Du) test is not a reliable screening test for FMH. The rosette method will be briefly reviewed.

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).

Before discussing crossmatch policies for women with passive anti-D likely due to RhIg, LIS-related issues will be outlined. A transfusion service's LIS and how it is configured determines under which circumstances an electronic crossmatch is possible.Regardless of crossmatch policy, almost all laboratories use a special designation or code in their LIS for anti-D likely due to RhIg. Often this designation is entered in the patient history comment field and not the antibody field, thus eliminating the need to remove the passive anti-D from the antibody field when the antibody disappears. Using this policy, once the passive antibody no longer reacts, the patient becomes eligible for an electronic crossmatch without the need to remove the antibody history.In essence, using a special designation for passive anti-D allows the lab to bypass the LIS's normal requirements for patients with clinically significant antibodies, i.e., allows them to omit doing an IAT crossmatch. Examples of how RhIg-derived anti-D is designated in lab information systems: Passive anti-D (eg., code 'PD', 'DPAS', etc.); Probably passive anti-D; Anti-D consistent with RhIg; Anti-D due to RhIg.Depending on the LIS, other variations are possible.

The Kleihauer-Betke test is performed to quantitate the number of fetal cells present in the maternal circulation. Once the size of the FMH is determined, the appropriate RhIg dose can be calculated and administered to prevent the mother from making anti-D.The test is based on the principle that red cells containing fetal hemoglobin (HbF) are less susceptible to acid elution than cells containing adult hemoglobin (HbA).General description A peripheral blood smear is made from the maternal postpartum sample and treated with acid. Fetal cells remain intact because of high concentrations of HbF, while HbA is eluted from the maternal cells. After acid treatment the slides are washed, stained, and examined microscopically. The number of fetal cells (which take up the stain) are counted per number of maternal cells (which appear as ghost cells) to give % fetal cells. The volume of fetal bleed is then calculated to determine how much additional RhIg is required. See Kleihauer-Betke graphic (Source: ENet Answers) Limitations: Despite its widespread use, the Kleihauer-Betke test has significant limitations, including Low sensitivity; Poor reproducibility.

Tests done routinely as part of perinatal testing programs vary from country to country and within countries. Below is one example of serologic tests typically done when pregnant females lack clinically significant antibodies. Other test protocols exist.Mother ABO, Rh, and antibody screen at first prenatal visit; Optional (not mandated by blood safety standards): Test for weak D, if initial Rh typing appears to be D-negative; D-negative females: Tested again (ABO, Rh, and antibody screen) at ~ 28 weeks weeks gestation prior to administration of RhIg (depending on the country) and again at delivery. Note: The application of DNA analysis to typing blood group antigens started in the early 1990s but is not yet widely available. When available, the mother can be typed for D using molecular methods, but this is usually not done unless she is weak D. The purpose is to determine using molecular methods which D variant the mother has, weak D or partial D, since the latter can produce anti-D. (see Further Reading) Molecular typing is reviewed more fully in Refresher on Hemolytic Disease of the Fetus and Newborn and Its Prevention, a companion course that complements this one.

A group O Rh negative female who had received RhIg at 28 weeks gestation had a weak anti-D when a type and screen was done prior to performance of a Cesarean section (C-section). A mini-panel of selected red cells confirmed the presence of anti-D and excluded other antibodies. The laboratory decided that the anti-D was likely passive and consistent with RhIG administration. A group O Rh positive child was delivered by C-section. The newborn had a weakly positive DAT but was healthy and required no treatment. A rosette test to screen for FMH was negative and A.D. was injected with 1500 IU (300 µg) of RhIG within 72 hours of delivery.

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.

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

These ABO, Rh, and antibody screen results were obtained by the TS using the blood specimen that was collected prior to starting the emergency transfusion with O Rh-negative RBCs. ABO and Rh typing ABO Forward Group ABO Reverse Group Rh anti-A anti-B A1 cells B cells anti-D 0 0 4+ 4+ 3+ Antibody screen Cells Gel IAT* Screen Cell I 3+ Screen Cell II 2+ Screen Cell III 2+ * IAT = indirect antiglobulin test