Systematic analysis of direct antiglobulin test results in post-artesunate delayed haemolysis.

BACKGROUND: Post-artesunate delayed haemolysis (PADH) is common after severe malaria episodes. PADH is related to the "pitting" phenomenon and the synchronous delayed clearance of once-infected erythrocytes, initially spared during treatment. However, direct antiglobulin test (DAT) positivity has been reported in several PADH cases, suggesting a contribution of immune-mediated erythrocyte clearance. The aim of the present study was to compare clinical features of cases presenting a positive or negative DAT. METHODS: Articles reporting clinical data of patients diagnosed with PADH, for whom DAT had been performed, were collected from PubMed database. Data retrieved from single patients were extracted and univariate analysis was performed in order to identify features potentially related to DAT results and steroids use. RESULTS: Twenty-two studies reporting 39 PADH cases were included: median baseline parasitaemia was 20.8% (IQR: 11.2-30) and DAT was positive in 17 cases (45.5%). Compared to DAT-negative individuals, DAT-positive patients were older (49.5 vs 31; p = 0.01), had a higher baseline parasitaemia (27% vs 17%; p = 0.03) and were more commonly treated with systemic steroids (11 vs 3 patients, p = 0.002). Depth and kinetics of delayed anaemia were not associated with DAT positivity. CONCLUSIONS: In this case series, almost half of the patients affected by PADH had a positive DAT. An obvious difference between the clinical courses of patients presenting with a positive or negative DAT was lacking. This observation suggests that DAT result may not be indicative of a pathogenic role of anti-erythrocytes antibodies in patients affected by PADH, but it may be rather a marker of immune activation.

Survey of newborn direct antiglobulin testing practice in United States and Canadian transfusion services.

BACKGROUND: We hypothesized that variability in practice exists for newborn immunohematology testing due to lack of consensus guidelines. We report the results of a survey assessing that variability at hospitals in the United States and Canada. STUDY DESIGN AND METHODS: An AABB Pediatric Subsection working party developed and validated a survey of newborn immunohematology testing practice. The survey was sent electronically to transfusion service leadership at teaching institutions. RESULTS: The response rate was 67% (61/91); 56 surveys meeting inclusion criteria were analyzed. Approximately 90% (50/56) were from birth hospitals and 16.1% (9/56) were from pediatric hospitals. Newborn immunohematology testing is ordered as a panel by 66.0% (33/50) of birth hospitals. ABO group and DAT is mandated before discharge in 14/56 (25.0%) and 13/56 (23.2%), respectively. About 76.8% (43/56) selectively perform a DAT according to blood blank or clinical parameters. The most common DAT practices include anti-IgG only testing by 73.2% (41/56) and use of umbilical cord specimen type by 67.9% (38/56). A positive DAT is a critical value for 26.8% (15/56) and followed with eluate testing when a maternal antibody screen is positive for 48.2% (27/56). In the setting of a non-ABO maternal red cell antibody, 55.4% (31/56), phenotype neonatal red cells when the DAT is positive. Group O RBC are transfused irrespective of the DAT result for 82.1%, (46/56). CONCLUSION: There is variability in newborn immunohematology testing and transfusion practice and potential overutilization of the DAT. Evidence-based consensus guidelines should be developed to standardize practice and to improve safety.

Positive direct antiglobulin test in post-artesunate delayed haemolysis: more than a coincidence?

BACKGROUND: Delayed haemolysis is a frequent adverse event after treatment with artesunate (AS). Removing once-infected "pitted" erythrocytes by the spleen is the most accepted mechanism of haemolysis in these cases. However, an increasing number of cases with positive direct antiglobulin test (DAT) haemolysis after AS have been reported. METHODS: All malaria cases seen at Hospital Clinic of Barcelona between 2015 and 2017 were retrospectively reviewed. Clinical, parasitological and laboratory data from patients treated with intravenous artesunate-specifically looking for delayed haemolysis and DAT-was collected. RESULTS: Among the 36 severe malaria patients treated with artesunate at the hospital, 10 (27.8%) developed post-artesunate delayed haemolysis. Out of these, DAT was performed in six, being positive in four of them (at least 40%). DAT was positive only for complement-without IgG-suggesting drug-dependent immune-haemolytic anaemia of the immune-complex type. Three of the four patients were treated with corticosteroids and two also received blood transfusion, with a complete recovery. CONCLUSIONS: Drug-induced auto-immune phenomena in post-artesunate delayed haemolysis may be underreported and must be considered. The role of corticosteroids should be reassessed.

Prevalence of positive direct antiglobulin test and clinical outcomes in Surinamese newborns from D-negative women.

BACKGROUND: In low-resource countries, screening for D antibodies to detect pregnancies at risk for hemolytic disease of the newborn is not routine practice. Retrospective data showed that 5.5% of Surinamese newborns of D-negative women had a positive direct antiglobulin test (DAT), indicating the presence of maternal antibodies against fetal antigens. Here, the frequency and clinical relevance of DAT positivity is evaluated. STUDY DESIGN AND METHODS: Between April 2015 and June 2016, an observational, multicenter cohort study was undertaken among Surinamese newborns born to D-negative women. In newborns, the DAT was performed, and clinical outcomes between DAT-negative and DAT-positive newborns were compared. RESULTS: Of the 232 evaluable newborns, 19 (8.2%) had a positive DAT, of which 11 of 15 antibody-tested newborns had D antibodies. DAT-positive newborns had lower hemoglobin levels (p = 0.02) and a trend toward higher bilirubin concentrations (p = 0.09) in the first days of life compared with DAT-negative newborns. DAT-positive newborns were admitted more frequently (p = 0.02), needed phototherapy treatment almost four times as often as DAT-negative newborns (26% vs. 7%; p = 0.008), and therapy took 2 days longer (p = 0.01). Exchange transfusions were performed in two newborns with D antibodies, both complicated with sepsis. The hospital stay was 2.5 days longer for DAT-positive newborns (p = 0.007). Overall, the prevalence of hemolytic disease of the newborn requiring treatment was 2.2% among the whole cohort of newborns. CONCLUSION: We found a high prevalence of DAT positivity with substantial need for hyperbilirubinemia treatment in newborns in Suriname. These results stress the necessity for better management procedures in D-negative women.

Comparison of a Novel Test (ODAK Brucella Coombs Gel Test) with Commonly Used Serological Tests in Human Brucellosis.

BACKGROUND: Since Brucellosis is difficult to diagnose based on clinical symptoms, the diagnosis mostly relies on the results of serological testing. ODAK Brucella Coombs Gel Test is a novel and rapid gel microcolumn agglutination test which is performed in microcolumns containing gel matrix and Coombs antibodies. In this study, we aimed to compare ODAK Brucella Coombs Gel Test with other commonly used serological tests. METHODS: 150 blood samples of patients, preliminarily diagnosed as Brucellosis, were included in this study. Rose Bengal (RB), ODAK Brucella Coombs Gel Test (CGT), Brucellacapt (BCAP), and Standard Agglutination Test (SAT) were performed for all samples. Also, Coombs Agglutination Test (CAT) was performed for all SAT negative samples. 1/160 and above titers were accepted as positive result except RB which is a qualitative test. RESULTS: 100 (67%) out of 150 samples were found positive by RB. All of the 50 RB negative samples were also found negative by SAT and CAT test. However, 2 (4%) and 7 (14%) of them were positive by CGT and BCAP tests, respectively. Additionally, among 100 RB positive samples, only 68, 77, and 87 were positive by SAT+CAT combination, CGT, and BCAP tests, respectively. CONCLUSIONS: Currently, CGT is the only rapid (< 1 hour) serological test in which Coombs antibodies are used. Our results showed that negative results of RB, as a screening test, are not reliable enough as compared to CGT. However, positive RBT results confirmed with SAT were almost always, in most of the cases with higher titers, positive with CGT and BCAP. On the other hand, even if SAT is found negative with RB positivity, samples still must be investigated with CAT, CGT or BCAP. Consequently, CGT may be used as a rapid screening test instead of RB and it furthermore has similar sensitivity with the other confirmation tests in which Coombs antibodies are used. Therefore, ODAK Brucella Coombs Gel Test seems to be a very useful diagnostic tool for Brucellosis.

Lyse or not to lyse: Clinical significance of red blood cell autoantibodies.

Autoimmune hemolytic anemia (AIHA) is a rare autoimmune disease characterized by a hemolytic anemia caused by autoantibodies against red blood cells (RBCs). These autoantibodies are routinely detected via the direct antiglobulin test (DAT). As expected, the DAT score correlates with the presence of clinical symptoms, but this correlation is far from perfect. Regularly, strongly positive DAT scores are encountered with no sign of hemolysis, while severe hemolysis can be seen even in patients with a negative DAT score. Apparently, the mere amount of antibody is not the sole predictor of disease. In this paper, we review the current literature on aspects of both the autoantibodies and the patients' clearance system that together determine the clinical significance of an anti-RBC autoantibody, ranging from antibody isotype to antibody Fc-glycosylation to alternative clearance mechanisms. From this, the ensemble of tests is inferred that in our view best assesses the main determinants for pathogenicity of autoantibodies.

Unexplained extreme hyperbilirubinemia among neonates in a multihospital healthcare system.

We report a series of neonates who developed a total serum bilirubin (TSB) >20mg/dL during a recent ten-year period in a multihospital healthcare system. The incidence of a TSB >20mg/dL fell after instituting a pre-hospital discharge bilirubin screening program in 2003/2004 (91.3 cases/10,000 births before vs. 72.4/10,000 after), but the incidence has subsequently remained unchanged. No specific cause for the hyperbilirubinemia was identified in 66% of (n=32) cases with a TSB >30 mg/dL or in 76% of (n=112) cases with a TSB 25.0-29.9 mg/dL. We hypothesized that hemolysis was a common contributing mechanism, but our review of hospital records indicated that in most instances these infants were not evaluated sufficiently to test this hypothesis. Records review showed maternal and neonatal blood types and direct antiglobulin testing were performed in >95% cases, but rarely were other tests for hemolysis obtained. In the ten-year period reviewed there were zero instances where erythrocyte morphology from a blood film examination or Heinz body evaluation by a pediatric hematologist or pathologist were performed. In 3% of cases pyruvate kinase was tested, 3% were evaluated by hemoglobin electrophoresis, 3% had a haptoglobin measurement, and 16% were tested for G6PD deficiency. Thus, determining the cause for hyperbilirubinemia in neonates remains a problem at Intermountain Healthcare and, we submit, elsewhere. As a result, the majority of infants with a TSB >25mg/dL have no specific causation identified. We speculate that most of these cases involve hemolysis and that the etiology could be identified if searched for more systematically. With this in mind, we propose a "consistent approach" to evaluating the cause(s) of hyperbilirubinemia among neonates with a TSB >25mg/dL.

Outcome and cost analysis of implementing selective Coombs testing in the newborn nursery.

OBJECTIVE: (1) To determine whether infants born to O+ mothers who had selective cord-blood testing would have higher rates of clinically significant hyperbilirubinemia compared with those newborns who had routine cord-blood testing. (2) To determine the amount of cost savings by implementing a policy of selective cord-blood testing in newborns born to O+ mothers. STUDY DESIGN: We conducted a retrospective pre/post intervention chart review on all infants in the normal newborn nursery at Loyola, born to blood type O+ women between 1 April 2008 and 1 April 2009. The pre-intervention group (routine testing) included infants born within 6 months before implementation of a new policy. The post-intervention group (selective testing) included infants born within 6 months following the implementation of a new policy. Data were collected for each of these groups regarding clinically significant hyperbilirubinemia. RESULT: All 250 of the infants in the routine testing group had a cord-blood type and Coombs done, whereas 42 of 164 (25%) infants in the selective group had testing done. By the end of the 6 months following the policy change, only 8% of infants were undergoing cord testing. When comparing routine vs selective testing, there was no statistically significant difference in the 24-h serum bilirubin, rate of phototherapy during the birth hospitalization, rate of readmission for hyperbilirubinemia or peak serum bilirubin level at readmission. The 92% reduction of cord-blood typing and Coombs testing would lead to a cost saving of $4100 per year to our hospital and $18 900 per year to our patients, and 95 h per year of technician time to perform these tests. When extrapolated to Illinois births in 2008, this would lead to an annual cost saving of almost $800 000 to Illinois hospitals and about $3.6 million to patients. CONCLUSION: Selective newborn cord testing of infants born to O+ mothers can decrease the use of resources and costs without increasing the risk of clinically significant hyperbilirubinemia.

Estudio clínico serológico de Brucelosis en nuestro medio / Clinical and serological study of Brucella in our means

OBJETIVO.Conocer la seroprevalencia de brucelosis, los factores deriesgo y las patologías asociadas a la presencia de anticuerposespecíficos frente a Brucela sp en pacientes del área dependientedel Consorcio Hospital General de Valencia, destacandolos pertenecientes a poblaciones próximas a zonas endémicasde esta patología.MÉTODOS.En un año, se analizaron 580 muestras de suero de pacientesde los distintos servicios del Hospital General de Valencia yArea de Salud correspondiente. Todos presentaban síndromefebril a estudio y/o manifestaciones clínicas compatibles conbrucelosis. Se determinó la reactividad frente a Brucela spmediante las pruebas; Rosa de Bengala (RB), Seroaglutinación(SAT) y Test de Coombs. Obtuvimos resultados positivos en163 muestras (28,1 %) de 122 pacientes. Sólo tuvimos accesoa la historia clínica de 46, siendo las variables revisadas;sexo, edad, procedencia, manifestaciones clínicas y diagnóstico.RESULTADOS.La prueba Rosa de Bengala fue positiva en 35 muestras de8 pacientes con clínica de brucelosis. Títulos significativos deAglutininas y Ac bloqueantes se encontraron en 14 pacientes y a títulos bajos, en 32. Clinica y serológicamente se diagnosticaron,8 pacientes de brucelosis aguda, 3 de brucelosis crónicay 3 de complicaciones de brucelosis aguda no tratada.CONCLUSIONES.Nuestra seroprevalencia (28,1 %), fue superior a la descritapor otros autores. Encontramos discrepancia entre las pruebasRB, SAT y Test de Coombs, lo mismo que otros autores ypensamos que seria necesario, ante una sospecha clínica debrucelosis, realizar más determinaciones para ver seroconversiónevitando así problemas de diagnóstico, tratamiento y controlde esta patología

OBJECTIVE.Knowing the seroprevalencia of brucellosis, the risk factorsand the pathologies associated to the presence of specificantibodies opposite Brucella sp in patients of the area contingenton the Consortium Hospital General from Valencia, highlightingthe proximate populations to this pathology’s endemiczones.METHODS.In a year, we examined 580 patients’s serum from differentservices of the Hospital General from Valencia and correspondentHealt’s Area. They all presented feverish syndrome tostudy and or clinical compatible manifestations with brucellosis.The intervening spdetermined the reactivity in front of Brucellaitselftests; Bengali rose ( RB ), Seroaglutination ( SAT ) andCoombs Test. We obtained positive results in 163 sera sample(28.1 %) of 122 patients. Only we had access to clinical historyof 46, being the revised variables; Sex, age, procedence,clinical manifestations and diagnosis.RESULTS.The RB test was positive in 35 samples of 8 patients withclinic of brucellosis. Significant levels of agglutinins and blockingantibodies, met in 14 patients and to low titles, in 32. Theclinical and serological diagnosed themselves, 8 patients ofacute brucellosis, 3 of chronic brucellosis and 3 with complicationsof acute brucellosis without treatment. CONCLUSIONS.Our seroprevalence ( 28.1 % ), was higher to the describedfor another authors. We found discrepancy among tests RB,SAT and Coombs Test, the same as another authors and wethought than it will be necessary, in front of a clinical suspicionof brucellosis, accomplishing more determinations to see seroconvertionavoiding thus problems of diagnosis, treatment andcontrol of this pathology

The Coombs' test in veterinary medicine: past, present, future.

The Coombs' test, also known as the antiglobulin test, is used most frequently in veterinary medicine as an aid in the diagnosis of immune-mediated hemolytic anemia. The test also is used widely in human medicine to screen for red blood cell alloantibodies. Polyspecific reagents for veterinary use typically contain anti-IgG, anti-IgM, and anti-C3. Monospecific reagents also are available. False-positive and false-negative test results can be obtained. Inadequate sensitivity of the standard test in human and veterinary medicine has necessitated development of alternate, more sensitive technologies.

Isoimmunization is unlikely to be the cause of hemolysis in ABO-incompatible but direct antiglobulin test-negative neonates.

OBJECTIVE: It is stated that the direct antiglobulin (Coombs') test (DAT) may be negative in ABO hemolytic disease of the newborn. Thus, significant jaundice in neonates who are A-B incompatible with their mothers but DAT test negative is often attributed to isoimmunization and another diagnosis is not sought. We wished to determine the rate of bilirubin production, as an objective measure of hemolysis, in 2 groups of DAT-negative neonates--ABO-compatible and ABO-incompatible--and in DAT-positive ABO-incompatible neonates. METHODS: In consecutive, term, healthy newborns who were admitted to the general care nursery, we measured the level in parts per million (ppm) of end-tidal breath carbon monoxide (CO), corrected for inspired CO (ETCOc), an index of the rate of bilirubin production. We compared the levels in DAT-negative ABO-incompatible neonates with those in ABO-compatible neonates and with the levels in DAT-positive ABO-incompatible neonates. Statistical analysis was performed using 2-sample t and chi(2) tests. RESULTS: There was no significant difference between the mean 12-hour ETCOc levels in DAT-negative ABO-incompatible neonates (n = 60, 2.2 +/- 0.6 ppm) versus DAT-negative ABO-compatible neonates (n = 171, 2.1 +/- 0.6 ppm), although there was a difference between the mean levels in DAT-positive ABO-incompatible neonates (n = 14, 3.4 +/- 1.8 ppm) and the DAT-negative groups. Four DAT-negative ABO-incompatible neonates had elevated ETCOc levels; in 2, we diagnosed a specific hematologic abnormality, namely, glucose-6-phosphate dehydrogenase deficiency in 1 and elliptocytosis in the other. CONCLUSION: In DAT-negative newborns with significant jaundice or increased bilirubin production, even if ABO-incompatible, a cause other than isoimmunization should be sought.

The intra- and inter-assay variation of the indirect mixed antiglobulin reaction test: is a quality control suitable?

The test most commonly used to detect sperm antibodies is the mixed antiglobulin reaction (MAR), standardized by the World Health Organization. The indirect MAR test detects soluble sperm antibodies in seminal plasma by using healthy donor spermatozoa as antigen. In this study we systematically investigated the influence of donor spermatozoa and the source of sperm antibodies upon the results of the indirect MAR test, and calculated the intra- and inter-assay variations. Using one individual seminal plasma and the same donor semen, results of the indirect MAR test are highly reproducible (low intra-assay variation). Two dimensions of inter-assay variation must be considered: (i) serial ejaculates of an individual donor may be used at different times; (ii) different donors may be applied to identical antibody sources. Donor spermatozoa strongly influenced the results of the indirect MAR test. Using multivariate statistical tests, highly significant main effects between the different donors (P < 0.001) and specific reciprocal effects between donor spermatozoa and seminal plasma samples (P < 0.001) were observed. The high inter-assay variation of the indirect MAR test will lead to incorrect results. There is urgent need of a reliable and reproducible test for sperm antibody detection to improve quality control of the methods.