S. Gerald Sandler

It's time to phase in RHD genotyping for patients with a serologic weak D phenotype

In 2014, the College of American Pathologists (CAP) Transfusion Medicine Resource Committee (TMRC) reported the results of a survey of more than 3100 laboratories concerning their policies and procedures for testing serological weak D phenotypes and administration of Rh immune globulin (RhIG).1 Among the findings of this survey is the observation that there is a lack of standard practice in the United States for interpreting the RhD type when a serological weak D phenotype is detected. In some laboratories, an individual with a serological weak D phenotype, especially if a blood donor, is interpreted to be RhD-positive. In the same or other laboratories, especially if a serological weak D phenotype is detected in a female of child-bearing potential, the individual is likely to be managed as RhD-negative for transfusions and, if pregnant, considered a candidate for RhIG. Also, the performance characteristics of serological typing methods for RhD vary. For patients, including pregnant women, the majority of laboratories have policies and procedures that do not use the indirect antiglobulin (weak D) test, thereby avoiding detection of a serological weak D phenotype so that the RhD type will be interpreted to be RhD-negative. Other laboratories typically perform a weak D test for the same category of patients. For blood donors and newborns, it is standard practice for laboratories to have policies and procedures for RhD typing to ensure that serological weak D phenotypes are detected and interpreted as RhD-positive.1 n nThe goal of these RhD typing practices is to protect RhD-negative persons from inadvertent alloimmunization to the D antigen by exposure to RhD-positive RBCs, including RBCs expressing a serological weak D phenotype. Although there has not been a recent prospective study in the United States, it is estimated that current RhD typing practice, together with contemporary obstetrical practices for administration of antepartum and postpartum RhIG, is 98.4 to 99 percent successful in preventing RhD alloimmunization and RhD hemolytic disease of the fetus/newborn.2 However, there are unwarranted consequences associated with the practice of not determining the RHD genotype of persons with a serological weak D phenotype, including unnecessary injections of RhIG and transfusion of RhD-negative RBCs -- always in short supply -- when RhD-positive RBCs could be transfused safely. n nCAP’s TMRC reviewed the current status of RHD genotyping and proposed that selective integration of RHD genotyping in laboratory practices could improve the accuracy of RhD typing results, reduce unnecessary administration of RhIG in women with a serological weak D phenotype, and decrease unnecessary transfusion of RhD-negative RBCs to recipients with a serological weak D phenotype.1 In response to the findings of the CAP TMRC survey, AABB and CAP convened a Work Group on RHD Genotyping and charged it with developing recommendations to clarify clinical issues related to RhD typing in persons with a serological weak D phenotype. As an initial step for formulating recommendations, the Work Group reviewed the current state of molecular science of RHD, including more than 140 publications covering background;1-12 D variants with anti-D;13-29 molecular basis of serological weak D phenotypes;30-92 reviews, editorials and commentaries;93-129 technical resources;130-142 and standards and guidelines.143-149 This Commentary summarizes the proceedings and recommendations of the Work Group.

Financial implications of RHD genotyping of pregnant women with a serologic weak D phenotype

Hemolytic disease of the fetus and newborn, classically caused by maternal–fetal incompatibility of the Rh blood group D antigen, can be prevented by RhIG prophylaxis. While prophylactic practices for pregnant women with serologic weak D phenotypes vary widely, RHD genotyping could provide clear guidance for management. This analysis evaluated the financial implications of using RHD genotyping to guide RhIG prophylaxis among pregnant females.

Bar Code and Radio-Frequency Technologies Can Increase Safety and Efficiency of Blood Transfusions

Bar-coded labels on wristbands, blood sample tubes, and blood components can facilitate matching the “right” blood unit and the “right” patient. Nurses preferred to verify their patientblood unit identification matches by scanning bar code labels rather than seeking out a second nurse for a conventional visual “double check” verification. Occasional scans of barcoded wristbands failed because of crinkles, food spills, and blurring due to bathing. Also, inadvertently scanning the wrong bar code on a blood component signaled the program that a mismatch was occurring and “shut down” the system, according to the requirements of our software program. Radio-frequency identification (RFID) labels on wristbands and blood components were easier to scan, but bar coded labels may be adequate for personal identification cards and labels on blood sample tubes for budget-conscious transfusion safety systems. We envision significant advantages to combining transfusion safety and medication dispensing systems, using common hardware and complementary software programs.

The entity of immunoglobulin A-related anaphylactic transfusion reactions is not evidence based.

In 1968, Vyas and colleagues described three immunoglobulin (Ig)A-deficient patients with “alarming transfusion reactions” after administration of whole blood, plasma, or gamma globulin. Sera from these patients agglutinated eight IgA multiple myeloma–coated reagent red blood cells (RBCs), fulfilling the authors’ criterion for an anaphylactic reaction. Three additional IgA-deficient patients experienced urticaria and/or anaphylactoid symptoms. Their sera agglutinated some, but not all, of the eight IgA myeloma-coated RBCs. The authors described these patients as having IgA-related anaphylactoid reactions. Since this original publication in 1968, more than 40 additional case reports of IgA-related anaphylactic transfusion reactions have been published in peer-reviewed journals. Nearly all of these reports define a transfusion reaction to be anti-IgA related if the patient’s plasma agglutinated IgA myeloma-coated reagent RBCs in a direct hemagglutination assay. During the 46 years since publication of this initial report, clinicians have accepted IgA-related anaphylactic transfusion reaction as a valid diagnosis and tested patients for IgA and anti-IgA if they experienced a serious allergic transfusion reaction. Transfusing physicians have requested IgAdeficient blood components for patients with severe allergic reactions and IgA deficiency or patients that they consider to be at higher risk of an anaphylactic reaction due to IgA deficiency with or without anti-IgA. Despite the limitations of the laboratory tests and the inability to convincingly establish a diagnosis or confirm the pathogenicity of the detected anti-IgA, this approach often invokes misguided transfusion restrictions and unnecessary reliance on rare IgA-deficient blood components. Nevertheless, blood services worldwide have established registries of IgA-deficient donors to meet the demand for IgA-deficient blood components. Given the rarity of transfusion-related anaphylactic reactions and the specialized testing needed to demonstrate the presence of anti-IgA, the entity of IgA-related anaphylactic transfusion reaction has been difficult to study and validate. With the advent of large-scale hemovigilance systems and the implementation of donor screening programs by several blood services, more data are available on the association of IgA deficiency and anaphylactic transfusion reactions, as well as the frequency of anti-IgA in the healthy donor population. The authors of this commentary evaluated the evidence for the entity of IgA-related anaphylactic transfusion reactions by reviewing published cases and unpublished data from diagnostic laboratories, blood services, and hemovigilance systems. We conclude that the analogy of the emperor’s clothes is fitting for most, if not all, cases of suspected IgA-related anaphylactic transfusion reactions. The entity of “IgA-related anaphylactic transfusion reaction” has not been established by evidence-based medicine.

Serological weak D phenotypes: a review and guidance for interpreting the RhD blood type using the RHD genotype

Approximately 0·2–1% of routine RhD blood typings result in a “serological weak D phenotype.” For more than 50 years, serological weak D phenotypes have been managed by policies to protect RhD‐negative women of child‐bearing potential from exposure to weak D antigens. Typically, blood donors with a serological weak D phenotype have been managed as RhD‐positive, in contrast to transfusion recipients and pregnant women, who have been managed as RhD‐negative. Most serological weak D phenotypes in Caucasians express molecularly defined weak D types 1, 2 or 3 and can be managed safely as RhD‐positive, eliminating unnecessary injections of Rh immune globulin and conserving limited supplies of RhD‐negative RBCs. If laboratories in the UK, Ireland and other European countries validated the use of potent anti‐D reagents to result in weak D types 1, 2 and 3 typing initially as RhD‐positive, such laboratory results would not require further testing. When serological weak D phenotypes are detected, laboratories should complete RhD testing by determining RHD genotypes (internally or by referral). Individuals with a serological weak D phenotype should be managed as RhD‐positive or RhD‐negative, according to their RHD genotype.

Blood group genotyping: faster and more reliable identification of rare blood for transfusion

In The Lancet Haematology, Willy Flegel and colleagues describe how they established a database of 43 066 blood donors whose extended blood group phenotypes had been identifi ed with an automated process that genotyped for 42 clinically relevant antigens. The investigators used this database to locate compatible—ie, blood group antigen-negative— red blood cell units to respond to 5672 requests from hospitals for red blood cell units that included requirements for specifi c blood group antigen-negative phenotypes. During the 3-year study period, the investigators’ blood centre (BloodCenter of Wisconsin, Milwaukee, WI, USA) was able to fi ll 5339 (94·1%) of requests using the genotyped donor database. The remaining 333 requests were managed by conventional serological laboratory methods and searches of the blood centre’s inventory. The successful outcome of this study resulted in the authors integrating the programme into the blood centre’s routine procedures for identifi cation of compatible antigen-negative red blood cell units. The results of this study represent a substantial advance for the management of patients who need red blood cell units with specifi c antigen-negative blood group phenotypes for their transfusions. Put simply, these are patients who need so-called rare blood as a consequence of one or more previous transfusions that resulted in the formation of blood group alloantibodies and a requirement that all future transfusions must be matched with red blood cells that lack the corresponding antigens. Two categories of rare red blood cell units exist that make special searches necessary. One category consists of patients who need red blood cells with an absence of a single antigen that is prevalent in the donor population. Conventionally, these red blood cells are defi ned as expressing a phenotype that is negative for a highfrequency antigen, and are present in fewer than one in 1000 random donors—eg, Kp(b–), Lu(b–), or Yt(a–). The second category of rare blood is for patients who need red blood cells with an absence of several blood group antigens, which would not be problematic individually, but becomes a search for rare blood when a combination is needed—eg, C–, E–, K–, S–, Jk(b–). The investigators’ use of an in-house-developed, highthroughput genotyping process to create an inventory database of 42-blood group antigen profi les for large blood donor cohorts replaced the previous lengthy, labour-intensive serological searches for compatible units. The novel routine blood group genotyping process is more effi cient than existing processes. Most importantly, the availability of a database of genotyped antigen-negative donors or red blood cell units can potentially improve the speed and reliability of delivery of compatible red blood cell units to patients, increasing patient safety by saving time and saving the cost of shipping red blood cell units from other blood centres. For more than 100 years since Karl Landsteiner’s fi rst report of blood group antigens on red blood cell membranes, hospital transfusion services have matched patients with compatible red blood cell units for transfusion by serological methods. Typically, these serological laboratory methods are subjective and manual, although serological methods have improved in accuracy and effi ciency through the development of monoclonal antibodies and automated blood typing analysers. Nevertheless, many technological limitations to serological compatibility testing exist, including the absence of a reliable supply of patientderived antibodies for all pertinent blood group antigens and the fact that there are no routine tests for some cell-mediated mechanisms of haemolysis. The development of automated genotyping instruments for the prediction of blood group phenotypes is a substantial advance that makes large-volume testing for all clinically relevant blood group antigens possible, as shown in this pilot study. Manual genotyping methods for prediction of an individual’s blood group phenotype have been available for two decades. However, the use of genotyping to identify blood group phenotypes has mostly been restricted to research and resolution of complex alloantibody problems for individual patients. Flegel and colleagues’ study shows that genotyping can be productively applied for blood donors and it enables blood centres to increase effi ciency and shorten time needed to locate compatible red blood cell units. Published Online June 3, 2015 http://dx.doi.org/10.1016/ S2352-3026(15)00093-9

Nonhemolytic passenger lymphocyte syndrome

An 8‐month‐old recipient of a liver segment transplant had anti‐D detected for the first time in her Day 5 posttransplant plasma and anti‐C detected for the first time in her Day 55 posttransplant plasma. The donors plasma contained anti‐C and anti‐D. Clinical and laboratory findings established a diagnosis of passenger lymphocyte syndrome (PLS). Hemolysis did not occur, because the recipients blood group phenotype was, by chance, D– C–.

Delayed hemolytic transfusion reaction captured by a cell phone camera

A 43-year-old male with AIDS was transferred to our hospital after transfusion of 5 units of crossmatch-compatible red blood cells (RBCs). Ten days after the transfusions, the patient developed weakness, anemia (Hb 7.8 g/dL), and a positive DAT result (anti-IgG, 21; anti-C3d, negative). Anti-Jk was identified in his plasma and in an eluate of his peripheral RBCs. A peripheral blood smear revealed multiple microspherocytes and an absence of schistocytes, confirming the serologic diagnosis of a delayed hemolytic transfusion reaction (see figure, left, illustrating dilute RBCs and abundant [donor’s] microspherocytes [arrows]). The Kidd phenotype of the transfused RBCs was not available, but given the prevalence of Jk(a1) RBCs among random blood donors, we estimate that 4 of the transfused RBC units were likely to be Jk(a1). Following the method of Morrison and Gardner, the admitting house officer used her cell phone camera (iPhone 6, Apple, Cupertino, CA) to photograph the morphologic findings directly through the 103 eyepiece and 1003 oil objective of an Olympus BH-2 microscope (Center Valley, NJ). A few hours later, she projected the images during her presentation at morning report. One month later the patient returned with an essentially normal peripheral blood smear (see figure, right, recovery from anemia, repopulation of [patient’s] corpuscular RBCs, and absence of microspherocytes). These images, while not as crisp as they would be if taken using a camera-fitted microscope, illustrate that images obtained using a cell phone camera are adequate for demonstrating abnormal morphologic findings on a peripheral blood smear. Cell phone photography may also facilitate timely communication to achieve consensus in complex or

Rh Immunoprophylaxis for Women With a Serologic Weak D Phenotype

It is standard practice for pregnant RhD-negative women who have not already formed anti-D to receive antepartum Rh immunoprophylaxis and, if they deliver an RhD-positive neonate, to receive postpartum Rh immunoprophylaxis. An estimated 0.6% to 1.0% of white women have red blood cells that express a serologic weak D phenotype. Of these women, approximately 80% will have a weak D type 1, 2, or 3 that could be managed safely as RhD-positive. Surveys of laboratory practice reveal a lack of standards for interpreting the RhD type for women with a serologic weak D and for determining their need for Rh immunoprophylaxis. RhD genotyping is recommended to determine the molecular basis of serologic weak D phenotypes in pregnant women as a basis for determining their candidacy for Rh immunoprophylaxis.

Immunosuppressive medication and alloimmunization to red blood cell antigens

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