Evaluating patients with autoimmune hemolytic anemia in the transfusion service and immunohematology reference laboratory: pretransfusion testing challenges and best transfusion-management strategies.

The serologic evaluation of autoimmune hemolytic anemia (AIHA) confirms the clinical diagnosis, helps distinguish the type of AIHA, and identifies whether any underlying alloantibodies are present that might complicate the selection of the safest blood for any needed transfusion. The spectrum of testing is generally dependent on the amount and class (immunoglobulin G or M) of autoantibody as well as the resources and methodologies where testing is performed. The approach may range from routine pretransfusion testing, including the direct antiglobulin test, to advanced techniques such as adsorptions, elution, and red cell genotyping. When transfusion is needed, the selection of the optimal unit of red blood cells is based on urgency and whether time allows for the completion of sophisticated serologic and molecular testing methods. From the start of when AIHA is suspected until the completion of testing, communication among the clinical team and medical laboratory scientists in the transfusion service and immunohematology reference laboratory is critical as testing can take several hours and the need for transfusion may be urgent. The frequent exchange of information including the patient's transfusion history and clinical status, the progress of testing, and any available results is invaluable for timely diagnosis, ongoing management of the patient, and the safety of transfusion if required before testing is complete.

Human leukocyte antigen (HLA)-incompatible mean fluorescence intensity-selected platelet products have corrected count increments similar to HLA antigen-matched platelets.

BACKGROUND: Donor specific antibody sum mean fluorescence intensity (MFI) values have been successfully used in transplant medicine to assess risk for organ rejection. However, little is known regarding whether MFI values could be similarly used to aid in platelet product selection. We have developed a novel protocol where MFI values are used to offer human leukocyte antigen (HLA)-incompatible platelet products when HLA antigen-matched products are not available. We aimed to evaluate the efficacy of this protocol. METHODS: We performed a 4-year retrospective chart review for all patients who received at least one MFI-selected platelet product. A corrected count increment (CCI) was calculated for each transfusion event. A mixed effects model was used to investigate the association between CCIs for MFI-selected, HLA antigen matched, and random donor platelet transfusions. A random effects expectation-maximization regression tree was used to define the extent to which other patient variables, such as age, sex, and diagnosis impacted the CCI for each platelet transfusion. RESULTS: Twenty highly HLA alloimmunized patients received a total of 591 platelets. MFI-selected platelet (low MFI) transfusions had a significantly higher median CCI 0-6 hour post-transfusion (13,559, interquartile range [IQR]: 8275-18,736) compared to random donor platelets (2121, IQR: 0-10,368, p < 0.0001). There was no significant difference in median CCI between HLA antigen matched and MFI selected platelet transfusions (p = 0.2). Mixed effects and regression modeling revealed that MFI-selected platelet products had a significantly higher CCI than non-matched platelets, even when accounting for other significant patient variables. CONCLUSION: MFI-selected HLA-incompatible platelet products could provide a comparable alternative to traditional HLA antigen-matched platelet products.

Chronic pain persists in adults with sickle cell disease despite regular red cell transfusions.

BACKGROUND: Pain affects over 50% of adults with sickle cell disease (SCD), and this pain is largely managed outside of the hospital. While chronic transfusion therapy is used to decrease the rate of acute pain events in patients with SCD, less is known about its impact on the day-to-day experience of pain. To address this knowledge gap, we provided pain diaries to patients with SCD receiving chronic transfusion. PATIENTS AND METHODS: A convenience sample of chronically-transfused adults with SCD successfully completed a diary over the course of at least 2 transfusion events. Patients receiving simple transfusions and red cell exchanges were included. Pain was rated on a scale of 0 to 10 each day, and patient laboratory values, co-morbidities, and hospital utilization were also obtained using the electronic medical record. The mean pain scores pre- and post-transfusion were evaluated using both a random effects-expectation maximization regression tree analysis and a generalized linear mixed regression model. RESULTS: Ten subjects (63%) in this cohort were defined as having chronic pain, while the remaining four (27%) subjects had episodic pain. Despite chronic transfusion and a suppressed HbS% (22.5% (16.5-25.9)), 10 patients (63%) continued to report nearly daily pain, and on almost 70% of diary days, the pain was significant (≥5/10). When the relationship between HbS% and reported pain intensity was examined, no association was found. DISCUSSION: These results suggest that, even with regular transfusions and a low HbS%, daily pain persists in many adults with SCD.

A Fatal Case of Immune Hyperhemolysis with Bone Marrow Necrosis in a Patient with Sickle Cell Disease.

In patients with sickle cell disease, hyperhemolysis is a rare but life-threatening complication of transfusion. In this case report, we describe a 61 year-old woman with hemoglobin sickle cell (SC) disease and history of alloimmunization who developed hyperhemolysis associated with a transfusion. She was found to have a warm and a clinically-significant cold autoantibody. Severe anemia (Hb 2.7 g/dL) with reticulocytopenia and thrombocytopenia prompted a bone marrow biopsy, which demonstrated extensive bone marrow necrosis. Despite treatment, the bone marrow failure did not improve and the patient died on hospital day 38. This case illustrates the potential risks of transfusion in a patient with sickle cell disease, especially one with previous hemolytic reactions. While uncommon, hyperhemolysis can cause death, in this case by extensive bone marrow necrosis. In patients with sickle cell disease, judicious use of red cell transfusions with phenotypically-matched units can diminish, but never completely abrogate, the risks associated with transfusion.

Patient factors associated with unidentified reactivity in solid-phase and polyethylene glycol antibody detection methods.

BACKGROUND: Several publications have reported an increase in nonspecific reactions when automated technologies such as solid phase are used for the detection of red blood cell alloantibodies. However, there is little known about patient-specific factors associated with these reactions and the clinical importance of these nonspecific reactions. STUDY DESIGN AND METHODS: We performed a 6-year retrospective review of our blood bank records and all newly reported unidentified (UID) reactivity using a test tube polyethylene glycol (t-PEG) and solid-phase method for the detection and identification of alloantibodies was recorded. Patient factors, such as underlying diagnosis, age, sex, ABO, Rh type, ethnicity, and subsequent antibody formation were recorded in each case. RESULTS: We determined that there was a significant increase in new UID reactions recorded in solid phase (20 per 10,000 tests) when compared to the t-PEG (1.8 per 10,000 tests) method for the detection of antibodies (p ≤ 0.0001). Solid-phase UID reactions were significantly associated with female sex (p = 0.04) and certain diagnoses, such as chronic or autoimmune disease, cancer, pregnancy, surgery, and trauma. Approximately 16% of patients developed a new auto- or alloantibody subsequent to their detected UID using solid phase. CONCLUSIONS: When solid phase is used for antibody identification, there is greater sensitivity toward nonspecific reactivity when compared to the t-PEG method. Patient sex and underlying diagnosis may explain the increased incidence of new UID reactivity in the solid-phase technology. Finally, UID reactivity should not be overlooked due to a notable percentage of subsequent clinically significant antibodies after UID detection.

Detection and identification of red cell alloantibodies in multiply transfused thalassemia major patients: a prospective study.

Life long red blood transfusion remains the main treatment for ß thalassemia major patients. The development of alloantibodies complicates transfusion therapy in thalassemia patients. Alloimmunization to red cell antigens is one of the most important immunological transfusion reaction and causes delayed type of transfusion reaction. A prospective study was conducted from January 2007 to January 2010. This was a cohorts of 115 patients were selected from regular transfusion group and they were followed for two and half year. They were followed up for the effect of transfusion during study period. There was a decline in patient number from 115 to 96 due to mortality and transfer of patient. A total of 96 multiply transfused thalassemia patients were prospectively included in this study and three consecutive samples collected after every 6 months and investigated for the development of alloantibody to red cell antigens. Tests for antibody screening and identification were performed on preserved sample to investigate prevalence and development of red cell alloimmunization by standardized laboratory techniques by same person at Prathama Blood Centre. A total of 96 patients were included in the study. 63 patients were males and 33 females. A total of five single alloantibodies were formed in five patients out of them four (80 %) belonged to Kell blood group system and one (20 %) from Rh system. It was observed that two (1.92 %) of new thalassemia patients developed red cell alloantibodies during study period. Red cell alloimmunization should be kept in mind in the patients receiving multiple transfusions. In present study, alloimmunization rate was 5.21 %. Mean transfusion duration in these patients was 23.90 days, probably due to presence of alloantibody. RBC alloantibody detection on regular interval and corresponding antigen negative blood transfusion is strongly recommended in transfusion dependent thalassemia patients.

Post-Baccalaureate Laboratory Specialist Certifications and Master's Degrees in Laboratory Medicine.

Opportunities to advance one's knowledge and position are available within the clinical laboratory arena. By obtaining a specialist credential in chemistry, hematology or microbiology, a laboratorian has demonstrated advance knowledge and ability in their respective discipline. These specialist certifications open doors within and outside the laboratory profession and may lead to promotion. The specialist in blood banking credential is unique in that accredited training programs are available, some of which are affiliated with universities and graduate credit is granted for program completion. Other avenues available include pathologist assistants programs, diplomats in laboratory management and Master of Science degrees in clinical laboratory science. There are a number of choices available to achieve your professional goal.

A prospective study for prevalence and/or development of transfusion-transmitted infections in multiply transfused thalassemia major patients.

OBJECTIVE: To evaluate the rate of seropositivity to hepatitis B and C and Human Immunodeficiency Virus (HIV) infections among children with ß-thalassemia major receiving multiple transfusions in Ahmedabad, India, compared with healthy controls. MATERIALS AND METHODS: The study was performed during January 2007 to January 2009 on multi-transfused children suffering with ß-thalassemia major registered in the Prathama Blood Centre, Ahmedabad; Jeevandeep hospital, Ahmedabad; and Red Cross Blood Centre, Ahmedabad, and investigated for the prevalence and development of transfusion-transmitted infections. Hepatitis B surface Antigen (HBsAg), anti-Hepatitis C Virus (HCV) Antibodies (Ab), and HIV Ab were checked using a fourth-generation Enzyme-Linked Immunosorbent Assay (ELISA). Positive tests were confirmed by western blots. Healthy blood donors were used for the control group. RESULTS: Hepatitis B surface antigen, anti-HCV Ab, and HIV Ab were positive in one of 96 (1.04%; 95% Confidence Interval (CI) = 0.17-1.3), 24 of 96 (25%; 95% CI = 11.4-14.2), and one of 96 (1.04%; 95% CI = 0.12-1.3), respectively. The rate of anti-HCV Ab was significantly higher in multi-transfused children suffering with ß-thalassemia major. In thalassemia patients, the rate of positive anti-HCV Ab was significantly higher than that for positive HBsAg (P<0.001) and HIV Ab (P<0.001). CONCLUSION: It is concluded that HCV is the current major problem in multi-transfused children with thalassemia major and more careful pretransfusion screening of blood for anti-HCV must be introduced in blood centers.

Mass-scale red cell genotyping of blood donors.

Blood centers are able to recruit and process large numbers of blood donations to meet the demand for antigen-matched blood. However, there are limitations with the use of hemagglutination that can be circumvented with blood group genotyping. Antisera do not exist for several clinically important blood group antigens and many methods have been developed (direct hemagglutination, indirect antiglobulin-dependent, solid phase, or gel column). There is increasing interest to apply mass-scale red cell genotyping of blood donors to find rare (predicted) phenotypes, rare combinations of antigens and locus haplotypes, and to have access to information on the common clinically relevant blood group antigens. This review outlines technological advances, emerging algorithms, and the future of mass-scale red cell genotyping of blood donors.

Molecular determination of RHD zygosity: predicting risk of hemolytic disease of the fetus and newborn related to anti-D.

OBJECTIVE: Development of an accurate molecular method for paternal RHD zygosity to predict risk to a fetus for hemolytic disease of the fetus and newborn (HDFN) related to anti-D. METHODS: Quantitative fluorescence polymerase chain reaction (QF-PCR) was used to detect RHD exons 5 and 7, using RHCE exon 7 as an internal control. The genotype and zygosity were determined from the peak area ratios of RHD exon 5 or 7 to RHCE exon 7. We tested 25 Caucasian and 25 African American (AA) samples whose zygosity was predicted from the Rh phenotype and an alternate molecular method. In addition, we tested 71 paternal samples from prenatal cases where fetal testing was performed. RESULTS: RHD/RHCE ratios clearly distinguished the RHD/D and RHD/d genotypes. RHD variants were recognized when RHD exon 5 copy number was discordant with exon 7. The molecular assay identified eight cases where the phenotype incorrectly assigned zygosity and we observed three false-negatives in the hybrid Rhesus box assay. The prenatal results were consistent with the zygosity determined for the paternal samples in our study. CONCLUSIONS: This QF-PCR method accurately determines RHD zygosity in Caucasians and AAs and will help predict the risk that a fetus will inherit RHD.

Trimethoprim-induced immune hemolytic anemia in a pediatric oncology patient presenting as an acute hemolytic transfusion reaction.

A 10-year-old male with acute leukemia presented with post-chemotherapy anemia. During red cell transfusion, he developed hemoglobinuria. Transfusion reaction workup was negative. Drug-induced immune hemolytic anemia was suspected because of positive direct antiglobulin test, negative eluate, and microspherocytes on smear pre- and post-transfusion. Drug studies using the indirect antiglobulin test were strongly positive with trimethoprim and trimethoprim-sulfamethoxazole but negative with sulfamethoxazole. The patient recovered after discontinuing the drug, with no recurrence in 2 years. Other causes of anemia should be considered in patients with worse-than-expected anemia after chemotherapy. Furthermore, hemolysis during transfusion is not always a transfusion reaction.

High-throughput red blood cell antigen genotyping using a nanofluidic real-time polymerase chain reaction platform.

BACKGROUND: Serologic testing of donors to obtain antigen-negative blood for transfusion is limited by availability and quality of reagents. Where sequence variant information is available, molecular typing platforms can be used to determine the presence of a variant allele and offer a high-throughput format correlated to the blood group antigen. We have investigated a flexible high-throughput platform to screen blood donors for antigen genotypes in the African American population. STUDY DESIGN AND METHODS: Genomic DNA from 427 African American donors was analyzed for single-nucleotide polymorphisms responsible for red blood cell (RBC) antigens E/e, Fy(a)/Fy(b), Fy gene promoter, Jk(a)/Jk(b), Lu(a)/Lu(b), K/k, Js(a)/Js(b), Do(a)/Do(b), Jo(a), and Hy using primer/probe sets (Taqman, Applied Biosystems) on a high-throughput genotyping platform (OpenArray, BioTrove). Where available, the phenotype obtained by serologic testing was compared to genotype data. RESULTS: Serologic antigen types were available for 2037 of the 4270 genotypes generated. There were five discordant results. Three resolved with repeat serologic typing, one resolved after repeat genotyping, and one discordance was clarified by confirmation of the BioTrove genotype by Sanger sequencing. Triplicate determinations were made for each sample genotype and the results were identical more than 99% of the time. CONCLUSIONS: The nanofluidic genotyping platform described here provides an accurate method for predicting blood group phenotypes. The user-specified array layout provides flexibility of target selection and number of replicate determinations and is suitable for screening antigen types.

Erythroid urea transporter deficiency due to novel JKnull alleles.

BACKGROUND: The Kidd blood group antigens Jka and Jkb are encoded by the red blood cell (RBC) urea transporter gene. Homozygosity for silent JK alleles results in the rare Jk(a-b-) phenotype. To date, seven JKnull alleles have been identified, and of these, two are more frequent in the Polynesians and Finns. This study reports the identification of other JKnull alleles in Jk(a-b-) individuals of different ethnic or geographic origins. STUDY DESIGN AND METHODS: Nine Jk(a-b-) samples and a sample from a Jk(a-b+) mother of a Jk(a+b-) baby were investigated. Polymerase chain reaction amplification and sequence analysis of the JK gene was performed. Western blotting and urea lysis were used to confirm Jk(a-b-) RBCs. RESULTS: Four novel alleles were identified: two different nonsense mutations, 202C>T (Gln68Stop) and 723delA (Ile262Stop) were identified on otherwise consensus JK*1 and JK*2 alleles, respectively. A missense mutation, 956C>T (Thr319Met), was identified in a JK*1 allele from an African-American and a JK*2 allele in two people of subcontinental Indian descent. Immunoblotting and urea lysis confirmed absence of JK glycoprotein in RBC membranes from a sample carrying the 956C>T mutation. Other previously described JKnull mutations were found in samples of origins other than in which they were first identified. CONCLUSION: The molecular bases of the Jk(a-b-) phenotype are diverse and this is the first report of JKnull alleles in individuals of African and subcontinental Indian descent. Although rare, these alleles should be taken into consideration when planning genotyping strategies for blood donors and patients.

One center's experience: the serology and drugs associated with drug-induced immune hemolytic anemia--a new paradigm.

BACKGROUND: Drug-induced immune hemolytic anemia (DIIHA) is an uncommon finding characterized by a sudden decrease in hemoglobin after treatment with the putative drug. The full range of drugs causing DIIHA and the initial serologic presentation are not fully appreciated. This work identifies additional drugs associated with DIIHA and offers additional insights about diagnosis. STUDY DESIGN AND METHODS: A 20-year retrospective review of testing in one laboratory was performed. Patient sex, age, medication history, initial direct antiglobulin test (DAT) and indirect antiglobulin test, method of drug-dependent antibody (DDA) detection, and specificity were reviewed. RESULTS: Seventy-one patients with 73 DDAs to 23 different drugs were identified. The following DDA specificities were identified: cephalosporins (37), penicillin and/or penicillin derivatives (12), nonsteroidal anti-inflammatory drugs (NSAIDs) (11), quinine and/or quinidine (7), and others (6). Fifty-two percent (37) were due to cephalosporins with 27 cefotetan-dependent antibodies detected. Four NSAIDs required urinary metabolite for detection. DAT was strongly positive, at least 2+, in 75 percent (51/68) of patients with a positive DAT. Initial eluate was negative in 52 patients, weak positive (<2+) in 14 patients, and strong positive (>or=2+) in 2 patients. Serologic results showed characteristics of warm autoimmune hemolytic anemia (AIHA) in 22 or 31 percent of all cases and cold-reactive AIHA in 2 cases. CONCLUSIONS: It is important to consider DIIHA when a patient serologically presents as either warm- or cold-type AIHA to avoid erroneous diagnosis. Based on these findings, the strength of the initial DAT is much stronger than previously reported for all types of drug-induced immune hemolysis. This report is also unique in the number of NSAIDs reported. A new classification of categorizing DDA is proposed.

An immunohematological 'Wet' workshop.

A practical workshop on 'Immunohematology' was conducted in conjunction with the Indian Society of Blood Transfusion and Immunohaematology annual scientific program. The participants, from many parts of India, were able to obtain valuable practice in key areas of blood group serology and by the end of the workshop were able to carry out 'tube' techniques for antibody detection and identification. Column agglutination methods were also demonstrated. A preliminary questionnaire was completed by participants. Results showed a wide variety in types of pretransfusion (serologic) testing being performed. Less than half of the participants had encountered hemolytic transfusion reactions. The program was rated as excellent by most participants in response to a postworkshop evaluation questionnaire, with requests for longer and more frequent workshops. Safety of blood for transfusion depends on maintenance of high standards of both microbiological and immunohematological performance by the blood bank staff.