Red Cell Alloimmunization

Genetics

Initial concepts on the genetics of the Rh antigens proposed the presence of three distinct genes. Newer DNA techniques have allowed for the localization of the Rh locus to the short arm of chromosome 1. Only two genes have been identified: an RHD gene and an RHCE gene. Each gene is 10 exons in length with 96% homology. These genes, presumably, represent a duplication of a common ancestral gene. Production of two distinct proteins from the RHCE gene probably occurs as a result of alternative splicing of messenger RNA. One nucleotide difference, cytosine to thymine, in exon 2 of the RHCE gene results in a single amino acid change of a serine to proline. This causes the expression of the C antigen as opposed to the c antigen. A single cytosine-to-guanine change in exon 5 of the RHCE gene, produces a single amino acid change of a proline to alanine, which results in formation of the e antigen instead of the E antigen.

The gene frequency found in different ethnic groups can be traced to the Spanish colonization in the fifteenth and sixteenth centuries. Populations native to certain land masses have a less than 1% incidence of RhD negativity—Eskimos, Native Americans, Japanese, and Chinese individuals. The Basque tribe in Spain is noted to have a 30% incidence of Rh negativity. This may well be the origin of the RHD gene deletion that is the most common genetic basis of the RhD-negative state in whites ( Fig. 40.2 ). Whites of European descent exhibit a 15% incidence of RhD negativity, whereas an 8% incidence is found in blacks and Hispanics of Mexico and Central America. This latter incidence probably reflects ethnic diversity secondary to Spanish colonization of the New World.

Further study of the RHD gene has revealed significant heterogeneity. Several of these genetic modifications result in a lack of expression of the RhD phenotype. Although these individuals may have an aberrant RhD gene present, serologic methods do not detect the RhD antigen on the surface of the red cells. One such example is the RHD pseudogene, which has been found in 69% of South African blacks and 24% of American blacks (see Fig. 40.2 ). In this situation, all 10 exons of the RHD gene are present. However, translation of the gene into a messenger RNA (mRNA) product does not occur, owing to the presence of a stop codon in the intron between exons 3 and 4. Thus, no RhD protein is synthesized, and the patient is serologically RhD negative. Similarly, the RHCcdes gene has been detected in 22% of American blacks. It appears to contain exons 1, 2, 9, and 10 as well as a portion of exon 3 of the original RHD gene, with other exons being duplicated from the RHCE gene. In the Taiwanese population of RhD-negative individuals, five different exons of the RHD gene were evaluated. Seventeen percent of individuals had all five exons detected, and an additional 135 demonstrated the presence of at least one of the five exons tested.

History

The history of rhesus prophylaxis can be traced to three unique individuals. Vincent Freda was an obstetric resident who developed an interest in HDFN. He was allowed to spend part of the fourth year of his residency at Columbia Presbyterian Medical Center in the laboratory of Alexander Weiner, one of the first investigators to identify the “Rh factor.” When Freda returned to Columbia, he went on to establish a serology laboratory and later organized the Rh Antepartum Clinic in 1960. A seat on the hospital transfusion committee became vacant and, in an unprecedented move, based on his interest, the chairman of obstetrics and gynecology, Howard C. Taylor Jr, appointed Freda to this position, even though he had not completed his residency. The chairman of pathology responded with the appointment of John Gorman to the committee, a resident in pathology with an interest in blood banking. It is here that these two individuals met and developed the collaboration that would one day end in the introduction of RhIG. In 1906, Theobald Smith found that guinea pigs given excess passive antibody failed to become immunized to diphtheria toxin. Freda and Gorman proposed that anti-D could be used in a similar fashion to prevent alloimmunization after delivery. They enlisted the aid of William Pollack, a senior protein chemist at Ortho Diagnostics, who developed an IgG globulin fraction from high-titered donor plasma. An initial grant application to the National Institutes of Health was rejected; however, funding was secured from the New York City Health Research Council on a second attempt. This was followed by a year-long negotiation with lawyers in the state capital to allow the investigators to perform their clinical trials at the Sing Sing prison in New York beginning in 1961 (John Gorman; personal communication, 2009). Nine RhD-negative male volunteers were injected monthly with RhD-positive cells for 5 successive months. Four of the men were immunized with intramuscular RhIG 24 hours before the injection of the red cells. Four of the five controls became alloimmunized to RhD, whereas none of the treated individuals developed anti-RhD antibodies. Their second experiment involved 27 inmates at Sing Sing, 13 controls and 14 treated. Red cells were given intravenously. However, the warden of Sing Sing would not allow the investigators to return on any fixed schedule that would enable the prisoners to know the time and day of their revisit. He was concerned that this exact foreknowledge could involve the prisoners in an escape plan. The investigators gladly accepted this limitation, as they reasoned that pregnant women who delivered over a weekend would probably not receive RhIG until Monday, up to 72 hours after delivery, owing to the closure of blood banks on weekends, as was commonly practiced at the time. None of the men who received RhIG were alloimmunized, whereas 8 of 13 controls developed anti-RhD antibodies. After two additional experiments at Sing Sing in this second group of individuals, Freda and Gorman went on to conduct a clinical trial in postpartum women at Columbia Presbyterian Medical Center starting in March of 1964. Of the 100 patients that received RhIG, none became sensitized, as compared with a rate of 12% sensitization to RhD in the control group. In a follow-up study in these patients, in their next pregnancy, none of the treated patients developed antibodies; 5 of 10 controls were alloimmunized and delivered infants affected by HDFN.

A parallel track of investigation was being undertaken by a group of British researchers in Liverpool. This group reasoned that the natural protective effect of ABO incompatibility between a mother and her fetus in preventing the formation of anti-D antibody could be used as a preventative strategy. A preparation of plasma that contained anti-D immunoglobulin M (IgM) was formulated and was administered intravenously to male volunteers. Although initial short-term antibody studies were promising, eventually 8 of 13 treated men became immunized to RhD, compared with only 1 of 11 controls. After the publication of the initial work of Freda and colleagues describing the use of a gamma globulin fraction of the plasma, the British group visited the New York investigators and obtained a sample of their gamma globulin preparation. The Liverpool group began their clinical trial in postpartum women with evidence of FMH by Kleihauer-Betke (KB) stain in April 1964, and they were subsequently credited for the first publication of a successful clinical trial in women.

An observational trial in Canada was initiated and determined that the baseline rate of antenatal sensitization to RhD was 1.8%. Between 1968 and 1974, a trial of antenatal prophylaxis using injections of 300 µg of RhIG at 28 and 34 weeks’ gestation followed. As compared with the previous observational study, none of the women demonstrated the development of anti-D antibodies. In a subsequent investigation that involved RhIG administered only at 28 weeks’ gestation, only 0.18% of women became sensitized.

In 1968, RhIG was approved by the Division of Biologics Standards of the National Institutes of Health for general clinical use in the United States as RhoGAM (Ortho-Clinical Diagnostics, Inc.). Recommendations for the use of RhoGAM during the immediate postpartum period were set forth by the American College of Obstetricians and Gynecologists (ACOG) in 1970, while the Food and Drug Administration (FDA) approved the use of antenatal RhIG in 1981. Routine antenatal prophylaxis at 28 to 29 weeks’ gestation was subsequently proposed by ACOG later that year.

Preparations

Four polyclonal products derived from human plasma are currently available in the United States for the prevention of RhD alloimmunization. Two of the products, RhoGAM (Kedrion Biopharma) and HyperRho S/D (Grifols USA), can only be given intramuscularly, because they are derived from human plasma through Cohn cold ethanol fractionation, a process that results in contamination with IgA and other plasma proteins. The remaining two products, WinRho-SDF (Cangene Corporation) and Rhophlac (CSL Behring), are prepared through sepharose column and ion-exchange chromatography, respectively. At present, all available products are subject to solvent detergent treatment to inactivate enveloped viruses; many manufacturers also use an additional micropore filtration step to further reduce the chance for viral contamination. Additionally, thimerosal, a mercury-based preservative used to prevent bacterial and fungal contamination, has been removed from all RhIG products used in the United States.

The dwindling resource of plasma donors for RhIG manufacture has led to the search for a synthetic product. One synthetic monoclonal anti-D antibody (roledumab) is currently being developed in human clinical trials. In the future, this antibody may replace the current polyclonal products derived from human plasma.

Indications

All pregnant patients should undergo blood type determination and an antibody screen at the first prenatal visit. In the past, all Rh-negative patients underwent additional testing to see if they were Du positive . This terminology was later changed to classify these patients as weak Rh positive individuals. In one series of 500 pregnant patients, this occurred in 1% of whites, 2.6% of blacks, and 2.7% of Hispanics. The recommendation in the past was that these individuals should be considered Rh positive, and RhIG was not indicated. Subsequent research found that the weak D individuals can belong to one of two groups; some of these patients have intact D antigens that are expressed in reduced numbers on the surface of the red cells ( Fig. 40.3 ). These individuals are not at risk for rhesus alloimmunization. In others with a weak D phenotype, the individual has inherited a gene that results in a variant expression of the D antigen. In these cases, one or more of the D antigen epitopes are missing, and the patient can become alloimmunized to these missing portions of the D antigen. Severe cases of HDFN have been reported in instances when a maternal antibody develops to the missing epitope. Although clinical trials have not been undertaken, the current recommendation is that these patients should receive RhIG.

Confusion can arise depending on when and where the patient undergoes red cell typing. Standards from the American Association of Blood Banks (AABB) recommend that reagents that detect weak D should not be used for prenatal typing. This guideline results in all weak D patients being called Rh negative and subsequently being considered candidates for RhIG. Newer monoclonal reagents used in the indirect antiglobulin test that can detect weak D are used at blood donor centers. These reagents are used to be sure that weak D blood is not administered to an Rh-negative recipient, resulting in a potential for alloimmunization. This will result in the same individual, now a blood donor, being called RhD positive —a very confusing scenario for the RhD negative patient and the clinician.

More recently, a work group of the AABB and the College of American Pathologists has suggested that weak D types 1, 2, and 3 can be managed as if they are RhD positive with no need for RhIG. RHD genotyping would be required in all pregnant patients to identify this subgroup with weak D. This proposal has not yet been adopted by ACOG. Thus all patients typed as weak D should receive antenatal and appropriate postpartum RhIG.

If there is no evidence of anti-D alloimmunization in the RhD-negative woman, the patient should receive 300 µg of RhIG at 28 weeks’ gestation. The 2% background incidence of RhD alloimmunization in the antenatal period can be expected to decline to 0.1%. In the United Kingdom, an antenatal protocol of administering 100 µg (500 IU) of RhIG at 28 and 34 weeks is used in primigravidae. Limited resources have not allowed for extension of this protocol to all subsequent pregnancies. Although the incidence of RhD alloimmunization prior to 28 weeks is rare, the current recommendation is to obtain a repeat antibody screen at the time of the antenatal RhIG. The maternal blood sample can be drawn at the same office visit as the RhIG injection. Although the administration of the exogenous anti-D will eventually result in a weakly positive titer, this will not occur in the short interval of several hours due to the slow absorption from the intramuscular site.

A new paradigm is developing in antenatal prophylaxis. Early studies in pregnant women carrying a male fetus indicated that 3% of the free DNA in the maternal circulation in the first trimester is fetal in origin; this increases to 6% by the third trimester. The source of this DNA appears to be apoptosis of placental villi. Fetal DNA is rapidly cleared from the maternal circulation with a mean half-life of 16 minutes after cesarean delivery; after vaginal delivery, fetal free DNA is cleared by 100 hours. The presence of fetal RHD DNA sequences in the maternal circulation was first reported by Lo and colleagues. Clinical assays for the determination of the fetal RhD status were subsequently developed. Approximately 40% of Rh-negative pregnant women will carry an Rh-negative fetus; thus RhIG would not be indicated in the antepartum period if this can be accurately determined.

Screening of RhD negative pregnant patients to determine whether antepartum RhIG should be undertaken is now routinely practiced in Denmark and the Netherlands, as well as in regions of Sweden, and France. The National Institute for Health and Care Excellence in England has recommended the nationwide institution of this practice. Canada is planning to implement a program in the near future. In some of these situations, ccffDNA screening was implemented as part of a new antepartum prophylaxis program, because of the limited availability of RhIG. However, in the United States, plasma collected from sensitized male volunteer donors is used to manufacture RhIG. Therefore, the availability of RhIG is unrestricted. Others have argued that the potential for infection with prions and other viruses supports an ethical approach to limiting antenatal RhIG to only those patients who need it. A cost-neutral strategy would appear to be the optimal approach to the implementation of ccffDNA. Several studies have determined that the break-even costs of ccffDNA testing would range from $29 to $119 for the saved doses of RhIG to offset the costs of evaluating all RhD-negative pregnant women. In the U.S., each year, over 500,000 RhD negative women give birth to over 350,000 RhD positive infants. As compared to the universal use of RhIG in these patients, incorporation of ccffDNA to triage the use of antenatal RhIG would result in between 1 and 57 new cases of Rh alloimmunization annually. This has led to concerns regarding increased alloimmunization if this practice was to be adopted in the U.S. Currently, the use of ccffDNA to guide antenatal RhIG use is not a guideline from any major U.S. organization.

Although not well studied, level A scientific evidence has been cited by ACOG to address additional indications for the antepartum administration of RhIG. These include: spontaneous miscarriage, elective abortion, ectopic pregnancy, genetic amniocentesis, chorionic villus sampling, and cordocentesis ( Table 40.1 ). A dose of 50 µg of RhIG is effective until 13 weeks’ gestation owing to the small volume of red cells in the fetoplacental circulation. However, most hospitals and offices do not stock this dose of RhIG, because the cost is equivalent to that of the standard 300 µg dose.

The use of RhIG in other scenarios that involve the possibility of FMH are lacking. However, most experts agree that such events as hydatidiform mole, threatened miscarriage, fetal death in the second or third trimester, blunt trauma to the abdomen, and external cephalic version warrant strong consideration for the use of RhIG.

The practice of evaluating a persistent maternal anti-D titer as an indication that additional RhIG is not required after an antenatal event is to be discouraged. Although the precise mechanism for the protective effect of RhIG is unknown, an excess amount of exogenous antibody in relation to the volume of RhD-positive red cells in the maternal circulation is essential for effective prophylaxis. Both animal and human studies have demonstrated that a low level of RhIG can actually enhance the chance for alloimmunization. In the words of Vincent Freda, “The rule of thumb should be to administer Rh immune globulin when in doubt, rather than to withhold it.”

Because the half-life of RhIG is approximately 16 days, 15% to 20% of patients receiving it at 28 weeks’ gestation have a very low anti-D titer (usually 2 or 4) at the time of admission for labor at term. In North America, the current recommendation is to administer 300 µg of RhIG within 72 hours of delivery if umbilical cord blood typing reveals an RhD-positive infant. This is sufficient for protection from sensitization due to an FMH of 30 mL of fetal whole blood. In the United Kingdom, 100 µg is given at delivery. Approximately 1 in 1000 deliveries will be associated with an excessive FMH; risk factors identify only 50% of these cases. ACOG and AABB recommend routine screening of all women at delivery for excessive FMH. A qualitative yet sensitive test for FMH, the rosette test, is performed first. Results return as positive or negative; a negative result warrants administration of a standard 300 µg dose of RhIG. If the rosette is positive, a KB stain or fetal cell stain using flow cytometry is undertaken to quantify the amount of the FMH. The AABB then recommends that the percentage of fetal blood cells be multiplied by a factor of 50 (to account for an estimated maternal blood volume of 5000 mL) to calculate the volume of the FMH. This volume is divided by 30 to determine the number of vials of RhIG to be administered. A decimal point is rounded up or down for values greater than 0.5 or less than 0.5, respectively. Because this calculation includes an inaccurate estimation of the maternal blood volume, one additional vial of RhIG is added to the calculation. As an example, a 3% KB stain is calculated to indicate a 150-mL FMH. Dividing this number by 30 yields five vials of RhIG with one additional vial added; therefore, the blood bank would prescribe 6 vials of RhIG (a total of 1800 µg) for this patient. However, a survey by the American College of Pathologists of its member blood banks noted that even following these guidelines, an inadequate dose of RhIG was recommended in 9% of cases and an excessive dose was recommended in 12% of cases.

No more than 5 mL RhIG should be administered by the intramuscular route in one 24-hour period. Should a large dose of RhIG be necessary, an alternative method would be to give the calculated dose using one of the currently available intravenous (IV) preparations of RhIG. Doses of up to 600 µg (3000 IU) can be administered every 8 hours until the total dose has been achieved. Should RhIG be inadvertently omitted after delivery, some protection has been proven with administration within 13 days; recommendations have been made to administer it as late as 28 days after delivery. If delivery is planned within 48 hours of amniocentesis for fetal lung maturity, RhIG can be deferred until after delivery. If delivery occurs less than 3 weeks from the administration of RhIG used for antenatal indications, such as external cephalic version, a repeat dose is unnecessary unless a large FMH is detected at the time of delivery.

Failed prophylaxis after the appropriate dose of RhIG is administered is rare. However, once postpartum administration is undertaken, the anti-D antibody screen may remain positive for up to 6 months. Anti-D that persists after this time is likely to be the result of sensitization.

The administration of RhIG after a postpartum tubal ligation is controversial. The possibility of a new partner, in conjunction with the availability of in vitro fertilization, would seem to make the use of RhIG, in these situations, prudent. In some cases, RhD-negative red cells may be in short supply if the patient presents after major trauma such as a motor vehicle accident with the need for massive transfusion. In these cases, RhD-positive blood could not be used as a life-saving alternative if the patient is alloimmunized to RhD through her previous delivery. RhIG is not effective once alloimmunization to the RhD antigen has occurred. At present, prophylactic immune globulin preparations, to prevent other forms of red cell alloimmunization, such as anti-K1, do not exist.

Diagnostic Methods