Irradiation of red blood cells (RBCs) inactivates residual donor T lymphocytes to prevent transfu... more Irradiation of red blood cells (RBCs) inactivates residual donor T lymphocytes to prevent transfusion‐associated graft‐vs‐host disease (TA‐GVHD) but can have adverse effects on recipients and inventory management. Reported incidence of TA‐GVHD is lower when leukoreduced RBCs and older blood products are transfused; therefore, the impact of leukoreduction and storage was evaluated as an alternative prevention strategy.
A recent CMAJ Practice article1 on mod ern Rhesus (Rh) typing in transfusion and pregnancy and i... more A recent CMAJ Practice article1 on mod ern Rhesus (Rh) typing in transfusion and pregnancy and its associated cor respondence2 prompted a productive discussion on safe recommendations for pregnant patients with a weak D type 4.0 allele, originally described in 2000.3 The differing approaches1,2 represent the personal views of the respective authors. Based on ou r review of 20 years’ worth of literature on this spe cialized topic, we have agreed on the following 5 statements: 1. No published case reports have docu mented adverse clinical effects, such as hemolysis, among pregnant people with weak D type 4.0 caused by an allo or autoanti–D. 2. Similarly, no published case reports have documented adverse clinical effects, such as anemia or jaundice, among fetuses or newborns caused by such a mother’s allo or autoanti–D. 3. No published evidence has shown that Rh immunoglobulin (RhIg) is clinically effective in an individual with weak D type 4.0 (e.g., for preventing antiD for...
Topic: Safety/Quality Background: The clinical laboratory employs Standard Operating Procedures (... more Topic: Safety/Quality Background: The clinical laboratory employs Standard Operating Procedures (SOPs) extensively to describe work activities and to provide consistency in performing the steps or actions required within and between laboratories. The goal of a successful SOP is to ensure precise and accurate results through consistent performance of critical tasks. The SOP manual is regarded as the gold standard resource where all workers should be performing tasks according to these instructions. However, this does not always occur, leading to questions about the extent that current SOPs reflect local environments. Objectives: Firstly, to see if simulation can be used to discover barriers that prevent workers from following SOPs. Secondly, to determine if the use of simulation in conjunction with creating or editing SOPs could increase adherence levels. Description of the innovation: We have used simulation in several different areas to evaluate adherence to SOPs and observe proces...
Canadian family physician Medecin de famille canadien, 2020
OBJECTIVE To provide family physicians with an understanding of blood bank tests performed during... more OBJECTIVE To provide family physicians with an understanding of blood bank tests performed during pregnancy. The value of routine blood type and antibody tests, as well as the follow-up required when a patient develops a red blood cell antibody or experiences a fetal-maternal hemorrhage (FMH) will be reviewed. SOURCES OF INFORMATION The approach described is based on the authors' clinical expertise and peer-reviewed literature from 1967 to 2020. MAIN MESSAGE An ABO and RhD group and antibody screen test is performed on every pregnant patient during the first trimester. Although antibodies to red blood cell antigens occur infrequently, some can lead to substantial adverse fetal or neonatal consequences including hemolytic disease of the fetus and newborn. Early identification and quantification of important antibodies ensures that at-risk mothers are referred to and followed by obstetricians experienced with high-risk care. Another valuable and related test is the FMH test. For R...
Gwen Clarke, Melanie Bodnar, Miquel Lozano, Veera Sekaran Nadarajan, Christina Lee, David Baud, G... more Gwen Clarke, Melanie Bodnar, Miquel Lozano, Veera Sekaran Nadarajan, Christina Lee, David Baud, Giorgia Canellini, Tobias Gleich-Nagel, Oscar Walter Torres, Patricia L. Rey, Carolina Bonet Bub, Jos e Mauro Kutner, Lilian Castilho, Nabiha H. Saifee, Meghan Delaney, Theresa Nester, Agneta Wikman, Eleonor Tiblad, Luca Pierelli, Antonella Matteocci, Maddalena Maresca, Emeline Maisonneuve, Anne Cortey, Jean Marie Jouannic, Jordi Fornells, Arjan Albersen, Masja De Haas, Dick Oepkes & Lani Lieberman
The aims of the 19th International Society of Blood Transfusion Platelet Immunology Workshop were... more The aims of the 19th International Society of Blood Transfusion Platelet Immunology Workshop were to compare the sensitivity and specificity of in‐house and commercially available methods for the detection of alloantibodies against human platelet antigens. Survey regarding laboratory management of samples collected for the diagnosis of foetal neonatal alloimmune thrombocytopenia was also conducted.
RhD DEL variants may show complete or partial expression of RhD epitopes. There have been only ra... more RhD DEL variants may show complete or partial expression of RhD epitopes. There have been only rare reports of anti‐D causing hemolytic disease of the fetus and newborn (HDFN) in this context. We report a case of severe HDFN associated with a recently described DEL variant.
Many countries maintain rare blood programs to provide access to blood for patients with complex ... more Many countries maintain rare blood programs to provide access to blood for patients with complex serologies. These include a process to screen donors and a registry to record information about rare donors; blood agencies may also freeze some units. However, frozen blood is much more expensive than liquid blood. A two-phase approach to analysis was used to evaluate how rare a blood type must be before a frozen inventory is necessary and what screening rates are required to support a rare blood program. A simulation model was employed to evaluate the impact of inventory on patient access. Results suggested that, for 27 of 29 phenotypes managed by Canadian Blood Services, insufficient donors had been identified to ensure a stable inventory. Analytic results showed the screening rate necessary to ensure a stable inventory and the time frame to build a rare donor base. Twenty-nine simulation scenarios were executed to evaluate patient access to rare blood against inventory levels. Results show that some amount of frozen inventory is necessary for phenotypes rarer than 1 in 3000. However, holding more than two units apiece of O-, O+, A-, and A+ did not improve patient access. While some level of frozen blood is needed for rare blood, large inventories do not improve access. Modest amounts of frozen inventory, combined with increased door screening, provides the greatest chance of maximizing patient access.
Rh is a complex blood group system with diverse genotypes that may encode weak and partial D vari... more Rh is a complex blood group system with diverse genotypes that may encode weak and partial D variants. Standard serologic analysis may identify clinically significant D variants as D+; nevertheless, individuals with these D variants should be managed as D- patients to prevent antibody formation to absent D epitopes. Variant identification is necessary during pregnancy to allow for timely and appropriate Rh immune globulin (RhIG) prophylaxis for hemolytic disease of the fetus and newborn (HDFN) as D alloimmunization can occur with some D variants. Here, we describe two cases of the RHD*DAU5 allele associated with maternal alloanti-D in patients of African ancestry. Two obstetric patients were initially serologically classified as D+ with negative antibody detection tests on routine prenatal testing. Repeat testing at delivery identified anti-D in both patients with no history of RhIG administration or transfusion. DNA sequencing revealed that both patients possessed the RHD*DAU5 alle...
Elucidation of the molecular basis of blood group expression has led to the development of highth... more Elucidation of the molecular basis of blood group expression has led to the development of highthroughput molecular methods for predicting blood group antigens. Red blood cell (RBC) antigen alloimmunization of some multiply transfused patients poses a challenge to safe transfusion and RBC antigen typing by molecular methods offers a new approach that may help mitigate the risk of alloimmunization. The commonly used single-nucleotide polymorphism arrays require nucleic acid isolation, which is typically achieved by extracting genomic DNA from whole blood. This method requires venipuncture and may not be an ideal approach for severely anemic patients, for children managed in outpatient sickle cell anemia clinics, or potential donors that are unable to provide a sample of whole blood due to their remote location. DNA extracted from buccal swab samples offers a noninvasive and cost-effective alternative to venipuncture and it would provide a safe and efficient means of transporting nonbiohazardous samples from remote locations to reference laboratories for extended blood type prediction. Canadian Blood Services has performed large-scale DNA extraction and HLA genotyping for the OneMatch Stem Cell and Marrow Registry using buccal swabs since 2007; buccal swabs are also used by other unrelated donor stem cell registries, such as the US National Marrow Donor Program. We sought to assess the accuracy and reliability of using DNA extracted from buccal swabs in predicting blood group antigen expression. We performed parallel RBC genotyping on an automated typing platform, the Progenika/Grifols ID CoreXT assay (Progenika BiopharmaGrifols) using DNA extracted from blood and buccal tissue from volunteers. For antigen systems with available serologic reagents, we also compared results with serologic typing. We evaluated three different methods of DNA extraction and performed testing regardless of DNA yield or purity. Two buccal swabs (Puritan Medical Products) were used for each test. Swabs were stored at room temperature, and DNA extraction was performed within 6 days of collection. In the initial phase of the study, buccal swab samples (n 5 15) were processed with the automated Biorobot M48 Robot using the MagAttract DNA Mini M48 extraction method (Qiagen), a high-throughput instrument that has been optimized for DNA extraction from buccal swabs. Extracted DNA had a mean concentration and purity of 38.1 ng/mL and 1.83, respectively. In the second phase of the study (n 5 39), DNA extractions from buccal swabs were performed using methods available in our national RBC immunohematology reference laboratory: the QIAamp DNA mini kit, using either manual or an automated QiaCube robotic workstation (Qiagen). Results are shown in Table 1. The manufacturer’s recommended analytical range for DNA concentration was 20 to 80 ng/mL and the recommended purity was an absorbance ratio of 1.63 to 2.1 (A260/280) for use of the ID CoreXT platform. DNA extraction using two buccal swab samples did not meet these specifications in several cases and is a limitation to using this method (Table 1). However, in most cases, a lower concentration of DNA was adequate for prediction of phenotype and would be a sensitive means to identify variant alleles (Fig. 1). Repeat analysis on the remaining two buccal swab samples after 2 weeks of room temperature storage showed the same results. The Dombrock system was the most susceptible to failure of interpretation in the samples with a low DNA concentration, with “nocall” results reported. There was 100% concordance in genotyping results when source DNA was extracted from whole blood or buccal tissue; there was also 100% concordance between predicted phenotype and serologic testing results. This study supports the use of genomic DNA extracted from buccal tissue on the ID CoreXT for predicting RBC phenotype with high accuracy. Extraction methods may require optimization to achieve DNA yields within the recommended analytical range of the assay.
Despite the existence of long-standing, well-organized programs for Rh immune globulin (RhIG) pro... more Despite the existence of long-standing, well-organized programs for Rh immune globulin (RhIG) prophylaxis, immune anti-D continues to be detected in the D– perinatal population. Between 2006 and 2008, 91 prenatal patients, found to have a previously unidentified anti-D, were followed up with a survey to their treating physician and with additional serologic testing where possible. The physician survey requested pregnancy and RhIG history information, including recent or distant potential alloimmunizing events, and the physicians were asked their opinion on the likely cause for the anti-D. Based on survey responses, updated RhIG information, and results of follow-up serology, anti-D was determined to be attributable to previously unreported RhIG in 44 of 91 (48.3%) cases and to active immunization (immune anti-D) in 36 of 91 cases (39.6%). A probable cause for alloimmunization was reported in 14 of 52 (26.9%) returned surveys. Anti-D alloimmunization continues to occur in our prenata...
Appropriate management of non‐infectious adverse transfusion reactions begins with recognition th... more Appropriate management of non‐infectious adverse transfusion reactions begins with recognition that a change in clinical status during or following a transfusion may represent an adverse event. Appropriate monitoring of patients during transfusion and explicit training of staff to recognize the signs and symptoms of adverse transfusion reactions is the key to diagnosis and to management. As some reactions may occur in the hours following transfusion, patient education and instruction on reporting relevant symptoms are also important. The typical symptoms that herald the onset of a transfusion‐related adverse event include fever, rash, shock and respiratory distress. Haemoglobinuria may also be a presenting feature. Early signs or symptoms may reflect more than one type of reaction. All transfusionists must be aware of the steps in acute management of a suspected adverse transfusion reaction. For those events that occur while the transfusion is ongoing, stopping the infusion and maintaining the intravenous access are the important first step. Rapid evaluation of the patient's vital signs, a bedside check of the unit and patient identification, as well as assessment of the appearance of the blood component, are early steps. Supportive care based on the patient's signs and symptoms must occur while additional laboratory and clinical investigations are initiated. In most cases of transfusion‐related adverse events, a ‘posttransfusion’ blood sample should be evaluated for the possibility of serological incompatibility. In addition, most moderate and severe reactions would be investigated with a blood count, renal and liver function tests and assessment of urine for haemoglobin. Other specific investigations depend on the presenting features and initial serologic findings. Based on the clinical, laboratory and/or imaging studies, most transfusion‐related adverse events can be classified into one of the categories of acute transfusion reactions. These include acute haemolytic transfusion reactions, febrile non‐haemolytic, allergic, anaphylactoid, septic, circulatory overload, hypotension and transfusion‐related acute lung injury. Transfusion‐associated graft‐versus‐host disease and posttransfusion purpura can be considered in some circumstances and delayed haemolytic transfusion reactions may be seen in the days to week following a transfusion. This diagnostic classification is important in optimizing acute management and may also contribute to decisions about component selection for subsequent transfusion. Reporting of adverse transfusion events is also an important part of management. Reporting to the hospital blood bank assists with diagnosis and decisions regarding future blood component therapy. The hospital transfusion committee may monitor transfusion reaction rates and trends as a quality indicator that can be used to change practices. The blood supplier must be notified of all reactions which may be attributable to a particular donor or donor unit, especially if recall or quarantine of associated blood components may be necessary. Regional or national haemovigilance programmes may require notification and can contribute to changes in standard practices to address common or serious adverse transfusion events and in early recognition of uncommon complications.
RhIG prophylaxis for D- pregnant women prevents hemolytic disease of the newborn and typically de... more RhIG prophylaxis for D- pregnant women prevents hemolytic disease of the newborn and typically depends on results of serologic D typing. Interpretation and follow-up of weak D serology is variable. Recent recommendations promote genotyping for RHD status determination in those with weak D serology. Canadian Blood Services performs comprehensive serologic prenatal testing in four provinces. Genotyping is used to determine D typing in patients with weak D. A serologic algorithm identified which patients require genotyping for RHD determination. Genotyping was performed on one of two commercially available platforms. Only 0.4% of D- patients met criteria for genotyping. Sixty-one percent were weak D Type 1, 2, or 3. Thirty percent had a partial or weak D other than Type 1, 2, or 3. Eleven had variants which remained unresolved. Seventeen were D+ and four were D-. Genotyping of patients with weak D serology led to an identified genotype in most patients. RhIG administration was avoided in 66% who were weak D Type 1, 2, or 3 or were D+. The use of a serologic algorithm to select patients for RHD genotyping identifies a majority of patients with weak D types not at risk for alloimmunization. This approach limits the number of genotyping investigations and the cost of providing classification for weak D types.
We read with interest the article by Chapuy and colleagues proposing the pretreatment of reagent ... more We read with interest the article by Chapuy and colleagues proposing the pretreatment of reagent screening cells to exclude clinically significant alloantibodies (CSAs) in patients receiving treatment with daratumumab (DARA). DARA is an IgG1 human monoclonal antibody directed against a unique epitope of CD38, which is strongly expressed on malignant plasma cells. DARA is being used successfully in the management of advanced plasma cell myeloma. CD38 is weakly expressed on normal human red blood cells (RBCs) and consequently DARA can bind to human reagent RBCs interfering with detection of CSAs. Chapuy and colleagues demonstrate that dithiothreitol (DTT) abolishes binding of daratumumab to RBCs. Our laboratory serves as a cross-match and serologic reference laboratory for a regional cancer program that has recently joined clinical trials assessing DARA effectiveness in patients with plasma cell myeloma. In February and March 2014, six male patients aged 32 to 84 years were entered into a clinical trial protocol at DARA doses of 8 or 16 mg/kg. The patients were group O, D1 (2); group A, D1 (2); group A, D– (1); and group B, D– (1). Antibody screens and direct antiglobulin tests (DATs) were negative on all patients before initiation of therapy. Extended phenotyping (C, c, E, e, K, Fy, Fy, Jk, Jk, S, s) and/or genotyping (Immucor BioArray HEA BeadChip) were performed at the time our laboratory was notified by the clinical service that DARA may interfere with indirect antiglobulin testing (IAT). The patients were followed serologically throughout their course of treatment. No abnormalities were noted in the ABO or D grouping. One patient developed a positive DATon Day 14 of treatment. Panreactivity developed in the antibody screen on Day 1 (one patient) and Day 13 (two patients), with strength of reactivity by solid-phase assay of 1 to 41 and by polyethylene glycol IAT of 11. Five patients required transfusion for hemoglobin (Hb) levels as low as 45 g/L. Blood was selected on the basis of extended phenotype and/or the phenotype predicted on the basis of genotyping. All patients obtained the expected Hb increment with the exception of the patient with a positive DAT. The range of RBC units transfused was 1 to 20, and no adverse transfusion events were reported. Panreactivity persisted throughout the course of treatment. It was not possible to follow the patients after therapy to see if the serologic findings persisted. It is becoming increasingly common to use the results of phenotyping and genotyping to support transfusion decisions in complex clinical situations, particularly sickle cell anemia. Our patients were successfully managed in the same manner as all other patients presenting to our laboratory with panreactivity in the antibody screen or cross-match. Although the use of DTT may be effective and of academic interest, this approach does not lend itself well to a routine transfusion laboratory where a large volume of patient samples must be tested on a daily basis because it is time-consuming and represents a deviation from normal laboratory procedures. Conversely, antigen typing is a simple procedure that allows for selection of antigen-matched units in patients with a panreactive antibody, including those in whom DARA treatment may be responsible for the serologic incompatibility.
Irradiation of red blood cells (RBCs) inactivates residual donor T lymphocytes to prevent transfu... more Irradiation of red blood cells (RBCs) inactivates residual donor T lymphocytes to prevent transfusion‐associated graft‐vs‐host disease (TA‐GVHD) but can have adverse effects on recipients and inventory management. Reported incidence of TA‐GVHD is lower when leukoreduced RBCs and older blood products are transfused; therefore, the impact of leukoreduction and storage was evaluated as an alternative prevention strategy.
A recent CMAJ Practice article1 on mod ern Rhesus (Rh) typing in transfusion and pregnancy and i... more A recent CMAJ Practice article1 on mod ern Rhesus (Rh) typing in transfusion and pregnancy and its associated cor respondence2 prompted a productive discussion on safe recommendations for pregnant patients with a weak D type 4.0 allele, originally described in 2000.3 The differing approaches1,2 represent the personal views of the respective authors. Based on ou r review of 20 years’ worth of literature on this spe cialized topic, we have agreed on the following 5 statements: 1. No published case reports have docu mented adverse clinical effects, such as hemolysis, among pregnant people with weak D type 4.0 caused by an allo or autoanti–D. 2. Similarly, no published case reports have documented adverse clinical effects, such as anemia or jaundice, among fetuses or newborns caused by such a mother’s allo or autoanti–D. 3. No published evidence has shown that Rh immunoglobulin (RhIg) is clinically effective in an individual with weak D type 4.0 (e.g., for preventing antiD for...
Topic: Safety/Quality Background: The clinical laboratory employs Standard Operating Procedures (... more Topic: Safety/Quality Background: The clinical laboratory employs Standard Operating Procedures (SOPs) extensively to describe work activities and to provide consistency in performing the steps or actions required within and between laboratories. The goal of a successful SOP is to ensure precise and accurate results through consistent performance of critical tasks. The SOP manual is regarded as the gold standard resource where all workers should be performing tasks according to these instructions. However, this does not always occur, leading to questions about the extent that current SOPs reflect local environments. Objectives: Firstly, to see if simulation can be used to discover barriers that prevent workers from following SOPs. Secondly, to determine if the use of simulation in conjunction with creating or editing SOPs could increase adherence levels. Description of the innovation: We have used simulation in several different areas to evaluate adherence to SOPs and observe proces...
Canadian family physician Medecin de famille canadien, 2020
OBJECTIVE To provide family physicians with an understanding of blood bank tests performed during... more OBJECTIVE To provide family physicians with an understanding of blood bank tests performed during pregnancy. The value of routine blood type and antibody tests, as well as the follow-up required when a patient develops a red blood cell antibody or experiences a fetal-maternal hemorrhage (FMH) will be reviewed. SOURCES OF INFORMATION The approach described is based on the authors' clinical expertise and peer-reviewed literature from 1967 to 2020. MAIN MESSAGE An ABO and RhD group and antibody screen test is performed on every pregnant patient during the first trimester. Although antibodies to red blood cell antigens occur infrequently, some can lead to substantial adverse fetal or neonatal consequences including hemolytic disease of the fetus and newborn. Early identification and quantification of important antibodies ensures that at-risk mothers are referred to and followed by obstetricians experienced with high-risk care. Another valuable and related test is the FMH test. For R...
Gwen Clarke, Melanie Bodnar, Miquel Lozano, Veera Sekaran Nadarajan, Christina Lee, David Baud, G... more Gwen Clarke, Melanie Bodnar, Miquel Lozano, Veera Sekaran Nadarajan, Christina Lee, David Baud, Giorgia Canellini, Tobias Gleich-Nagel, Oscar Walter Torres, Patricia L. Rey, Carolina Bonet Bub, Jos e Mauro Kutner, Lilian Castilho, Nabiha H. Saifee, Meghan Delaney, Theresa Nester, Agneta Wikman, Eleonor Tiblad, Luca Pierelli, Antonella Matteocci, Maddalena Maresca, Emeline Maisonneuve, Anne Cortey, Jean Marie Jouannic, Jordi Fornells, Arjan Albersen, Masja De Haas, Dick Oepkes & Lani Lieberman
The aims of the 19th International Society of Blood Transfusion Platelet Immunology Workshop were... more The aims of the 19th International Society of Blood Transfusion Platelet Immunology Workshop were to compare the sensitivity and specificity of in‐house and commercially available methods for the detection of alloantibodies against human platelet antigens. Survey regarding laboratory management of samples collected for the diagnosis of foetal neonatal alloimmune thrombocytopenia was also conducted.
RhD DEL variants may show complete or partial expression of RhD epitopes. There have been only ra... more RhD DEL variants may show complete or partial expression of RhD epitopes. There have been only rare reports of anti‐D causing hemolytic disease of the fetus and newborn (HDFN) in this context. We report a case of severe HDFN associated with a recently described DEL variant.
Many countries maintain rare blood programs to provide access to blood for patients with complex ... more Many countries maintain rare blood programs to provide access to blood for patients with complex serologies. These include a process to screen donors and a registry to record information about rare donors; blood agencies may also freeze some units. However, frozen blood is much more expensive than liquid blood. A two-phase approach to analysis was used to evaluate how rare a blood type must be before a frozen inventory is necessary and what screening rates are required to support a rare blood program. A simulation model was employed to evaluate the impact of inventory on patient access. Results suggested that, for 27 of 29 phenotypes managed by Canadian Blood Services, insufficient donors had been identified to ensure a stable inventory. Analytic results showed the screening rate necessary to ensure a stable inventory and the time frame to build a rare donor base. Twenty-nine simulation scenarios were executed to evaluate patient access to rare blood against inventory levels. Results show that some amount of frozen inventory is necessary for phenotypes rarer than 1 in 3000. However, holding more than two units apiece of O-, O+, A-, and A+ did not improve patient access. While some level of frozen blood is needed for rare blood, large inventories do not improve access. Modest amounts of frozen inventory, combined with increased door screening, provides the greatest chance of maximizing patient access.
Rh is a complex blood group system with diverse genotypes that may encode weak and partial D vari... more Rh is a complex blood group system with diverse genotypes that may encode weak and partial D variants. Standard serologic analysis may identify clinically significant D variants as D+; nevertheless, individuals with these D variants should be managed as D- patients to prevent antibody formation to absent D epitopes. Variant identification is necessary during pregnancy to allow for timely and appropriate Rh immune globulin (RhIG) prophylaxis for hemolytic disease of the fetus and newborn (HDFN) as D alloimmunization can occur with some D variants. Here, we describe two cases of the RHD*DAU5 allele associated with maternal alloanti-D in patients of African ancestry. Two obstetric patients were initially serologically classified as D+ with negative antibody detection tests on routine prenatal testing. Repeat testing at delivery identified anti-D in both patients with no history of RhIG administration or transfusion. DNA sequencing revealed that both patients possessed the RHD*DAU5 alle...
Elucidation of the molecular basis of blood group expression has led to the development of highth... more Elucidation of the molecular basis of blood group expression has led to the development of highthroughput molecular methods for predicting blood group antigens. Red blood cell (RBC) antigen alloimmunization of some multiply transfused patients poses a challenge to safe transfusion and RBC antigen typing by molecular methods offers a new approach that may help mitigate the risk of alloimmunization. The commonly used single-nucleotide polymorphism arrays require nucleic acid isolation, which is typically achieved by extracting genomic DNA from whole blood. This method requires venipuncture and may not be an ideal approach for severely anemic patients, for children managed in outpatient sickle cell anemia clinics, or potential donors that are unable to provide a sample of whole blood due to their remote location. DNA extracted from buccal swab samples offers a noninvasive and cost-effective alternative to venipuncture and it would provide a safe and efficient means of transporting nonbiohazardous samples from remote locations to reference laboratories for extended blood type prediction. Canadian Blood Services has performed large-scale DNA extraction and HLA genotyping for the OneMatch Stem Cell and Marrow Registry using buccal swabs since 2007; buccal swabs are also used by other unrelated donor stem cell registries, such as the US National Marrow Donor Program. We sought to assess the accuracy and reliability of using DNA extracted from buccal swabs in predicting blood group antigen expression. We performed parallel RBC genotyping on an automated typing platform, the Progenika/Grifols ID CoreXT assay (Progenika BiopharmaGrifols) using DNA extracted from blood and buccal tissue from volunteers. For antigen systems with available serologic reagents, we also compared results with serologic typing. We evaluated three different methods of DNA extraction and performed testing regardless of DNA yield or purity. Two buccal swabs (Puritan Medical Products) were used for each test. Swabs were stored at room temperature, and DNA extraction was performed within 6 days of collection. In the initial phase of the study, buccal swab samples (n 5 15) were processed with the automated Biorobot M48 Robot using the MagAttract DNA Mini M48 extraction method (Qiagen), a high-throughput instrument that has been optimized for DNA extraction from buccal swabs. Extracted DNA had a mean concentration and purity of 38.1 ng/mL and 1.83, respectively. In the second phase of the study (n 5 39), DNA extractions from buccal swabs were performed using methods available in our national RBC immunohematology reference laboratory: the QIAamp DNA mini kit, using either manual or an automated QiaCube robotic workstation (Qiagen). Results are shown in Table 1. The manufacturer’s recommended analytical range for DNA concentration was 20 to 80 ng/mL and the recommended purity was an absorbance ratio of 1.63 to 2.1 (A260/280) for use of the ID CoreXT platform. DNA extraction using two buccal swab samples did not meet these specifications in several cases and is a limitation to using this method (Table 1). However, in most cases, a lower concentration of DNA was adequate for prediction of phenotype and would be a sensitive means to identify variant alleles (Fig. 1). Repeat analysis on the remaining two buccal swab samples after 2 weeks of room temperature storage showed the same results. The Dombrock system was the most susceptible to failure of interpretation in the samples with a low DNA concentration, with “nocall” results reported. There was 100% concordance in genotyping results when source DNA was extracted from whole blood or buccal tissue; there was also 100% concordance between predicted phenotype and serologic testing results. This study supports the use of genomic DNA extracted from buccal tissue on the ID CoreXT for predicting RBC phenotype with high accuracy. Extraction methods may require optimization to achieve DNA yields within the recommended analytical range of the assay.
Despite the existence of long-standing, well-organized programs for Rh immune globulin (RhIG) pro... more Despite the existence of long-standing, well-organized programs for Rh immune globulin (RhIG) prophylaxis, immune anti-D continues to be detected in the D– perinatal population. Between 2006 and 2008, 91 prenatal patients, found to have a previously unidentified anti-D, were followed up with a survey to their treating physician and with additional serologic testing where possible. The physician survey requested pregnancy and RhIG history information, including recent or distant potential alloimmunizing events, and the physicians were asked their opinion on the likely cause for the anti-D. Based on survey responses, updated RhIG information, and results of follow-up serology, anti-D was determined to be attributable to previously unreported RhIG in 44 of 91 (48.3%) cases and to active immunization (immune anti-D) in 36 of 91 cases (39.6%). A probable cause for alloimmunization was reported in 14 of 52 (26.9%) returned surveys. Anti-D alloimmunization continues to occur in our prenata...
Appropriate management of non‐infectious adverse transfusion reactions begins with recognition th... more Appropriate management of non‐infectious adverse transfusion reactions begins with recognition that a change in clinical status during or following a transfusion may represent an adverse event. Appropriate monitoring of patients during transfusion and explicit training of staff to recognize the signs and symptoms of adverse transfusion reactions is the key to diagnosis and to management. As some reactions may occur in the hours following transfusion, patient education and instruction on reporting relevant symptoms are also important. The typical symptoms that herald the onset of a transfusion‐related adverse event include fever, rash, shock and respiratory distress. Haemoglobinuria may also be a presenting feature. Early signs or symptoms may reflect more than one type of reaction. All transfusionists must be aware of the steps in acute management of a suspected adverse transfusion reaction. For those events that occur while the transfusion is ongoing, stopping the infusion and maintaining the intravenous access are the important first step. Rapid evaluation of the patient's vital signs, a bedside check of the unit and patient identification, as well as assessment of the appearance of the blood component, are early steps. Supportive care based on the patient's signs and symptoms must occur while additional laboratory and clinical investigations are initiated. In most cases of transfusion‐related adverse events, a ‘posttransfusion’ blood sample should be evaluated for the possibility of serological incompatibility. In addition, most moderate and severe reactions would be investigated with a blood count, renal and liver function tests and assessment of urine for haemoglobin. Other specific investigations depend on the presenting features and initial serologic findings. Based on the clinical, laboratory and/or imaging studies, most transfusion‐related adverse events can be classified into one of the categories of acute transfusion reactions. These include acute haemolytic transfusion reactions, febrile non‐haemolytic, allergic, anaphylactoid, septic, circulatory overload, hypotension and transfusion‐related acute lung injury. Transfusion‐associated graft‐versus‐host disease and posttransfusion purpura can be considered in some circumstances and delayed haemolytic transfusion reactions may be seen in the days to week following a transfusion. This diagnostic classification is important in optimizing acute management and may also contribute to decisions about component selection for subsequent transfusion. Reporting of adverse transfusion events is also an important part of management. Reporting to the hospital blood bank assists with diagnosis and decisions regarding future blood component therapy. The hospital transfusion committee may monitor transfusion reaction rates and trends as a quality indicator that can be used to change practices. The blood supplier must be notified of all reactions which may be attributable to a particular donor or donor unit, especially if recall or quarantine of associated blood components may be necessary. Regional or national haemovigilance programmes may require notification and can contribute to changes in standard practices to address common or serious adverse transfusion events and in early recognition of uncommon complications.
RhIG prophylaxis for D- pregnant women prevents hemolytic disease of the newborn and typically de... more RhIG prophylaxis for D- pregnant women prevents hemolytic disease of the newborn and typically depends on results of serologic D typing. Interpretation and follow-up of weak D serology is variable. Recent recommendations promote genotyping for RHD status determination in those with weak D serology. Canadian Blood Services performs comprehensive serologic prenatal testing in four provinces. Genotyping is used to determine D typing in patients with weak D. A serologic algorithm identified which patients require genotyping for RHD determination. Genotyping was performed on one of two commercially available platforms. Only 0.4% of D- patients met criteria for genotyping. Sixty-one percent were weak D Type 1, 2, or 3. Thirty percent had a partial or weak D other than Type 1, 2, or 3. Eleven had variants which remained unresolved. Seventeen were D+ and four were D-. Genotyping of patients with weak D serology led to an identified genotype in most patients. RhIG administration was avoided in 66% who were weak D Type 1, 2, or 3 or were D+. The use of a serologic algorithm to select patients for RHD genotyping identifies a majority of patients with weak D types not at risk for alloimmunization. This approach limits the number of genotyping investigations and the cost of providing classification for weak D types.
We read with interest the article by Chapuy and colleagues proposing the pretreatment of reagent ... more We read with interest the article by Chapuy and colleagues proposing the pretreatment of reagent screening cells to exclude clinically significant alloantibodies (CSAs) in patients receiving treatment with daratumumab (DARA). DARA is an IgG1 human monoclonal antibody directed against a unique epitope of CD38, which is strongly expressed on malignant plasma cells. DARA is being used successfully in the management of advanced plasma cell myeloma. CD38 is weakly expressed on normal human red blood cells (RBCs) and consequently DARA can bind to human reagent RBCs interfering with detection of CSAs. Chapuy and colleagues demonstrate that dithiothreitol (DTT) abolishes binding of daratumumab to RBCs. Our laboratory serves as a cross-match and serologic reference laboratory for a regional cancer program that has recently joined clinical trials assessing DARA effectiveness in patients with plasma cell myeloma. In February and March 2014, six male patients aged 32 to 84 years were entered into a clinical trial protocol at DARA doses of 8 or 16 mg/kg. The patients were group O, D1 (2); group A, D1 (2); group A, D– (1); and group B, D– (1). Antibody screens and direct antiglobulin tests (DATs) were negative on all patients before initiation of therapy. Extended phenotyping (C, c, E, e, K, Fy, Fy, Jk, Jk, S, s) and/or genotyping (Immucor BioArray HEA BeadChip) were performed at the time our laboratory was notified by the clinical service that DARA may interfere with indirect antiglobulin testing (IAT). The patients were followed serologically throughout their course of treatment. No abnormalities were noted in the ABO or D grouping. One patient developed a positive DATon Day 14 of treatment. Panreactivity developed in the antibody screen on Day 1 (one patient) and Day 13 (two patients), with strength of reactivity by solid-phase assay of 1 to 41 and by polyethylene glycol IAT of 11. Five patients required transfusion for hemoglobin (Hb) levels as low as 45 g/L. Blood was selected on the basis of extended phenotype and/or the phenotype predicted on the basis of genotyping. All patients obtained the expected Hb increment with the exception of the patient with a positive DAT. The range of RBC units transfused was 1 to 20, and no adverse transfusion events were reported. Panreactivity persisted throughout the course of treatment. It was not possible to follow the patients after therapy to see if the serologic findings persisted. It is becoming increasingly common to use the results of phenotyping and genotyping to support transfusion decisions in complex clinical situations, particularly sickle cell anemia. Our patients were successfully managed in the same manner as all other patients presenting to our laboratory with panreactivity in the antibody screen or cross-match. Although the use of DTT may be effective and of academic interest, this approach does not lend itself well to a routine transfusion laboratory where a large volume of patient samples must be tested on a daily basis because it is time-consuming and represents a deviation from normal laboratory procedures. Conversely, antigen typing is a simple procedure that allows for selection of antigen-matched units in patients with a panreactive antibody, including those in whom DARA treatment may be responsible for the serologic incompatibility.
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