Hereditary Spherocytosis Patients Research Articles

Analysis of Hereditary Elliptocytosis with Decreased Binding of Eosin-5-maleimide to Red Blood Cells.

Flow cytometric test for analyzing the eosin-5-maleimide (EMA) binding to red blood cells has been believed to be a specific method for diagnosing hereditary spherocytosis (HS). However, it has been reported that diseases other than HS, such as hereditary pyropoikilocytosis (HPP) and Southeast Asian ovalocytosis (SAO), which are forms in the category of hereditary elliptocytosis (HE), show decreased EMA binding to red blood cells. We analyzed EMA binding to red blood cells in 101 healthy control subjects and 42 HS patients and obtained a mean channel fluorescence (MCF) cut-off value of 36.4 (sensitivity 0.97, specificity 0.95). Using this method, we also analyzed 12 HE patients. Among them, four HE patients showed the MCF at or below the cut-off value. It indicates that some HE patients have decreased EMA binding to red blood cells. Two of these four HE patients were classified as common HE, and two were spherocytic HE with reduced spectrin. This study demonstrates that, in addition to patients with HPP or SAO, some HE patients have decreased EMA binding to red blood cells.

Comparison and evaluation of three screening tests of hereditary spherocytosis in Chinese patients.

The objective of this study is to compare and evaluate the diagnostic value of hereditary spherocytosis (HS) by three screening tests, comparing mean spherical corpuscular volume (MSCV) to mean corpuscular volume (MCV), mean corpuscular hemoglobin concentration (MCHC), and flow cytometric osmotic fragility test. Peripheral blood was collected from 237 participators diagnosed at the First Affiliated Hospital of Guangxi Medical University, including 56 hereditary spherocytosis patients, 86 thalassemia patients, and 95 healthy people. The samples were examined by three tests, and the three screening tests were evaluated by the sensitivity and specificity of tests. The sensitivity was only 41.07%, and specificity was 94.47% when using MCHC >355 g/L as diagnostic criteria. The sensitivity was 89.28%, and specificity was 96.14% when using MSCV < MCV as the optimum cutoff point. When using the residual red cell percentage <23.6% as the diagnostic threshold in flow cytometric osmotic fragility test, the sensitivity was 85.71% and the specificity was 97.24%. Flow cytometry osmotic fragility test or comparing MSCV to MCV combined with smear examination of peripheral red blood cells morphology can be a simple, practical, and accurate hereditary spherocytosis (HS) laboratory screening method.

No Association Was Found Between MTHFR and MTHFD1 SNPs and Vitamin Levels in People with Increased and Normal Erythropoiesis, after Compulsory Flour Fortification with Folic Acid

Background The methylenetetrahydrofolate reductase (MTHFR) and methylenetetrahydrofolate dehydrogenase (MTHFD1) enzymes play important roles in the metabolism of folate. MTHFR catalyses the reduction of 5,10-methylene-tetrahydrofolate (5,10-methylene-THF) to 5-methyl-THF and present a crucial role in the regulation of available folate to homocysteine remethylation, in a cobalamin (Cbl) dependent reaction. MTHFD1 has three distinct enzymatic activities in folate metabolism: the conversion of THF to 10-formyl-THF; the reversible conversion of 10-formyl-THF to 5,10-methenyl-THF; and the conversion of 5,10-methenyl-THF to 5,10-methylene-THF. The two enzymes were related with DNA synthesis and single nucleotides polymorphisms (SNPs) in their genes have been associated with lower folate levels. People who have increased cellular duplication could be affected by low folate levels. Hereditary spherocytosis (HS) patients had an increased erythropoiesis and need more amounts of acid folic. In Brazil, HS patients are usually treated with 5mg/day of folic acid, the only formulation available. The use of folic acid by β-thalassemia heterozygotes (β-TH) subjects is not common, and depends of the individual clinical feature. Furthermore, in Brazil, HS patients and β-TH people have been exposed to food fortification (wheat and corn flours) with 150 µg of FA since 2004. The influences of variants of MTHFR and MTHFD1 SNPs in people with increased erythropoiesis, especially in HS patients, are not known.Objective The aim of the study was to evaluate the interaction between MTHFR c. 677C>T and c. 1298A>C and MTHFD1 c. 1958G>A SNPs and the use or not of FA supplementation in HS patients. It was also our objective to compare the folate and Cbl levels according to genotypes for MTHFR and MTHFD1 SNPs in β-TH subjects and their controls.Methods Twenty-five patients with HS, 49 β-TH subjects and 98 healthy people exposed to mandatory fortification of wheat and corn flours with FA were included in this study. Serum folate and Cbl were determined by microbiologic assays. Genomic DNA was extracted from whole blood using commercial kit. The genotypes for MTHFR c. 1298A>C and MTHFD1 c.1958G>A SNPs were determined by Real Time PCR using TaqMan assays. PCR-RFLP was used for genotyping MTHFR c. 677C>T SNP. Haplotype frequencies (MTHFR c. 677C>T and c. 1298 A>G) and the standardized disequilibrium coefficient (D’) for pair-wise linkage disequilibrium were assessed by the Expectation-Maximization (EM) algorism using the Haploview Software. We considered linkage disequilibrium when D’ ≥0.50. The study of linkage disequilibrium was performed by calculating the coefficient D’ of Lewontin.Results The frequencies of genotypes for MTHFR (c. 677C>T and c. 1298A>C) and MTHFD1 c. 1958G>A SNPs were similar in HS, β-TH and control groups (p>0.05). When genotypes were grouped according to the presence of the mutated allele (CC vs. CT+TT for MTHFR c. 677C>T, AA vs. AC+CC for MTHFR c. 1298A>C and GG vs. GA+AA for MTHFD1 c. 1958G>A), no differences were also found (p>0.05). The MTHFR c.677C>T and c. 1298A>C SNPs are in complete linkage disequilibrium (D’=1.0). Regarding the correlation coefficient between the two loci (r2), the value found in this analysis was equal to 0.169 in HS versus control group and 0.205 in β-TH versus control group. The haplotype CA, which is formed by the ancestral allele of MTHFR c. 677C>T and c. 1298A>C SNPs respectively, was more frequent in controls (41.9%) than in the β-TH group (28.1%). However, this finding was not observed in HS group comared with control group. No association was found between genotypes for MTHFR c. 677C>T and c. 1298A>C and MTHFD1 c. 1958G>A and alterations in folate and Cbl levels in three groups. ANCOVA was used for evaluating the interaction between the use of FA supplementation and MTHFR c.677C>T SNP on folate levels in HS patients. Only FA supplementation was associated with increased serum folate, and no associations were observed with MTHFR SNP or interaction between both.Conclusion The SNPs were not associated with reduced serum folate levels in the three groups studied. This finding suggests that FA flour fortification in β-TH and controls, as well as, the supplementation plus fortification in HS group, could offset the effect of variants of SNPs, especially for MTHFR c.677C>T. DisclosuresNo relevant conflicts of interest to declare.

Elevated Serum Folic Acid Concentrations Were Associated with Higher mRNA Expression of DHFR Gene in Patients with Hereditary Spherocytosis

BackgroundFolic acid (FA) is the synthetic form of folate, a B complex vitamin which plays an important role in several reactions in the body. Folate deficiency produces several clinical complications including anemia and is associated with an increased risk of cancer, cardiovascular disease and neural tube defects. With the advent of FA and iron fortification of wheat and corn flour (150 µg of folic acid and 4.2 mg of iron/100 grams) in Brazil since 2004, the population was exposed to amounts of folate beyond that normally present in foods. The form present in fortified food is taken up by cells and reduced by the enzyme dihydrofolate reductase (DHFR) first to dihydrofolate (DHF) and then tetrahydrofolate (THF). DHF is the preferred natural substrate. Amounts of FA higher than the defined tolerable upper intake of 1 mg/day could impair the ability of DHFR to convert DHF to THF. Some patients with hemolytic anemia, such as hereditary spherocytosis (HS), need larger amounts of FA to compensate for their increase in erythropoiesis and have been receiving 5mg/day of supplemental FA, the only formulation available in Brazil, in addition to being exposed to compulsory food fortification with FA.ObjectiveThe aim of this study was to compare the effects of 5mg/day FA on serum folate levels, mRNA expression of DHFR, MTHFR, interferon-γ, TNF-α and interleukin-8 genes; and cytotoxicity of lymphocytes and NK cells in patients with HS and healthy individuals not receiving supplemental FA.Material and MethodsTwenty-five patients with HS exposed to mandatory fortification and in use or not of 5 mg/day of FA were included in this study. Forty-five healthy people were recruited as a control group, and matched with HS patients according to age, gender, body mass index and self-reported skin color. Blood count, including reticulocytes, C-reative protein and lactic dehydrogenase (LDH) were performed. Serum folate (SF) and vitamin B12 were determined by a microbiological method. The mRNA expression of DHFR, MTHFR, interferon-γ, TNF-α and interleukin-8 genes in mononuclear cells were performed in duplicate, using Real Time PCR. Cytotoxicity of lymphocytes and NK cells were also carried out using a flow cytometric assay.ResultsEight HS patients did not use FA 5 mg everyday (4 declared no use anytime and 4 reported intermittent use) and none of control group used FA supplementation. Reticulocytes and LDH were higher in HS group (P<0.05), however no difference was found between levels of C-reative protein (P=0.173) and vitamin B12 (P= 0.699) when compared with control group. The HS group had higher SF levels and elevated mRNA expression of DHFR, MTHFR, interferon-γ, TNF-α and interleukin-8 when compared with controls (P<0.05, Figure 1). It is not clear whether FA use or underlying disease was be responsible for increasing of mRNA expression of these genes. To verify the effect of SF levels on HS patients, this group was classified into two subgroups according to median SF (< 46.6 and ≥ 46.6 nmol/L). Interestingly, the subgroup with higher SF levels showed significantly elevated DHFR mRNA expression but no difference was found in the mRNA expression of the other genes studied. The two subgroups were similar according to WBC, RBC, hemoglobin, MCV, reticulocytes, LDH, C-reative protein, vitamin B12 and cytotoxic capacity of lymphocytes and NK cells.ConclusionsElevated SF concentrations were associated with higher mRNA expression of DHFR gene in HS patients, suggesting that the use of higher amounts of FA might influence the expression and activity of DHFR and thus affect folate metabolism in these patients. It is not known whether normal subjects receiving similar high doses of FA show the same effects.Financing: FAPESP 2012/12912-1 and CNPq 4826412012-6 [Display omitted] The line in each graphic is the median.The median and percentiles 25 and 75 (P25 – P75) for control and hereditary spherocytosis groups were, respectively: A- serum folate: 22.1 (14.5 – 36.6 nmolL) and 51.5 (19.5 – 95.2 nmolL), B- DHFR mRNA expression: 1.59 (1.00 – 2.48) and 35.8 (19.0 – 52.7). In Figure C, the median and percentiles 25 and 75 (P25 – P75) for DHFR mRNA expression, according to serum folate levels (SF <46.6 and SF ≥ 46.6 nmol/L), were, respectively: 19.6 (1.4 – 38.6) and 50.3 (23 – 66.8). DisclosuresNo relevant conflicts of interest to declare.

A report of two cases of splenectomy in children younger than two years old with hereditary spherocytosis

Hereditary spherocytosis (HS) is a common pediatric hemolytic anemia and one of the most common types of familial hemolytic anemia caused by congenital red blood cell defects. HS often presents clinically as childhood anemia, jaundice, and progressive splenomegaly. It is generally accepted that splenectomy is the most effective treatment for HS, but development of overwhelming infections with symptoms such as chills, high fever, and septicemia may occur because of immune dysfunction after splenectomy. As a result, the generally proposed age for children to undergo splenectomy is after 5 years of age. Splenectomy in younger HS patients is rarely reported. Here, however, we report 2 cases of HS in children younger than 2 years of age (15 months and 18 months) who underwent splenectomy; we also report on their follow-up. • We report two cases of HS in children <2 yrs who underwent splenectomy. • Splenectomy in younger HS patients is rarely reported. • Splenectomy can be a solution for children <2 yrs with severe HS or hemolytic crisis.

Impairment of bone health in pediatric patients with hemolytic anemia.

IntroductionSickle cell anemia and thalassemia result in impaired bone health in both adults and youths. Children with other types of chronic hemolytic anemia may also display impaired bone health.Study DesignTo assess bone health in pediatric patients with chronic hemolytic anemia, a cross-sectional study was conducted involving 45 patients with different forms of hemolytic anemia (i.e., 17 homozygous sickle cell disease and 14 hereditary spherocytosis patients). Biochemical, radiographic and anamnestic parameters of bone health were assessed.ResultsVitamin D deficiency with 25 OH-vitamin D serum levels below 20 ng/ml was a common finding (80.5%) in this cohort. Bone pain was present in 31% of patients. Analysis of RANKL, osteoprotegerin (OPG) and osteocalcin levels indicated an alteration in bone modeling with significantly elevated RANKL/OPG ratios (control: 0.08+0.07; patients: 0.26+0.2, P = 0.0007). Osteocalcin levels were found to be lower in patients compared with healthy controls (68.5+39.0 ng/ml vs. 118.0+36.6 ng/ml, P = 0.0001). Multiple stepwise regression analysis revealed a significant (P<0.025) influence of LDH (partial r2 = 0.29), diagnosis of hemolytic anemia (partial r2 = 0.05) and age (partial r2 = 0.03) on osteocalcin levels. Patients with homozygous sickle cell anemia were more frequently and more severely affected by impaired bone health than patients with hereditary spherocytosis.ConclusionBone health is impaired in pediatric patients with hemolytic anemia. In addition to endocrine alterations, an imbalance in the RANKL/OPG system and low levels of osteocalcin may contribute to this impairment.

Toward the harmonization of result presentation for the eosin-5′-maleimide binding test in the diagnosis of hereditary spherocytosis

The eosin-5'-maleimide (EMA) Binding test measures reduced mean channel fluorescence (MCF) reading of EMA-labeled red cells (EMA-RBCs) from patients with hereditary spherocytosis (HS). Reporting test results can be either in the actual MCF reading or as a ratio by normalization of the test MCF result to the mean MCF value of six normal controls. The latter format has potential for universal reporting. We analyzed three years' archival MCF data from HS and non-HS patient groups for establishment of reference ranges of ratios for normal adults and HS. A prospective study used FC500 and FACS Canto II cytometers to analyze contemporaneously EMA-RBCs from several patient groups and normal donors. Statistical analyses of the prospective data determined the cut-off values, and the sensitivity and specificity for HS respectively for the MCF and the ratio result presentations. The effect of using fewer than six normal controls for the ratio denominator was explored. The FC500 gave a mean ratio of 0.782 (SD=0.086) in HS patients with an optimal cut-off ratio of 0.918 (98.7% specificity, 95.6% sensitivity), and gray area ratio of 0.868-0.918. The Canto II gave a mean ratio of 0.774 (SD=0.085) with an optimal cut-off ratio of 0.925 (97.1% specificity and 100% sensitivity), and gray area ratio of 0.859-0.925. Harmonization of result presentation is feasible with no apparent constraint by instrument design. Interpretation of gray-area data requires an assessment of patient's clinical presentation and family history or performing a family study.

Towards the harmonization of result presentation for the eosin-5'- maleimide (EMA) binding test in the diagnosis of hereditary spherocytosis.

Background: The EMA Binding test measures reduced mean channel fluorescence (MCF) reading of EMA-labeled red cells (EMA-RBCs) from patients with hereditary spherocytosis (HS). Reporting test results can be either in the actual MCF reading or as a ratio by normalization of the test MCF result to the mean MCF value of 6 normal controls. The latter format has potential for universal reporting. Methods: We analyzed three years' archival MCF data from HS and non-HS patient groups for establishment of reference ranges of ratios for normal adults and HS. A prospective study used FC500 and FACS Canto II cytometers to analyze contemporaneously EMA-RBCs from several patient groups and normal donors. Statistical analyses of the prospective data determined the cut-off values, and the sensitivity and specificity for HS respectively for the MCF and the ratio result presentations. The effect of using fewer than six normal controls for the ratio denominator was explored. Results:. The FC500 gave a mean ratio of 0.782 (SD = 0.086) in HS patients with an optimal cut-off ratio of 0.918 (98.7% specificity, 95.6% sensitivity), and grey area ratio of 0.868 - 0.918. The Canto II gave a mean ratio of 0.774 (SD = 0.085) with an optimal cut-off ratio of 0.925 (97.1% specificity and 100% sensitivity), and grey area ratio of 0.859 - 0.925. Conclusion: Harmonization of result presentation is feasible with no apparent constraint by instrument design. Interpretation of grey-area data requires an assessment of patient's clinical presentation and family history or performing a family study. © 2014 Clinical Cytometry Society.

Screening and confirmation of hereditary spherocytosis in children using a CELL-DYN Sapphire haematology analyser.

Multidimensional optical scatter of sphered erythrocytes can identify and enumerate hyperchromic erythrocytes, which might be used for hereditary spherocytosis (HS) screening. The flow cytometric eosin-5'-maleimide test (EMA) is highly sensitive and specific for HS as a confirmatory method. The aims of this study were to assess the utility of hyperchromic erythrocytes in HS screening and to evaluate the EMA test performed on CELL-DYN Sapphire analyser compared with the reference method. Blood from 740 paediatric patients presenting at our institution was analysed in reticulocyte mode of the CELL-DYN Sapphire haematology analyser (Abbott Diagnostics) to obtain hyperchromic erythrocyte counts. The EMA test was performed using a flow cytometer as a reference, as well as CELL-DYN Sapphire as an investigational method. Hyperchromic erythrocytes were the highest in patients with HS (median 11.5%; range 5.1-29.2%). Patients with autoimmune haemolytic disease had significantly less hyperchromic erythrocytes (median 4.9%; range 0.0-18.3%). Hyperchromic erythrocytes showed a high area under the ROC curve: 0.972. At 4.9% cut-off, hyperchromic erythrocytes detected HS with 96.4% sensitivity and 99.1% specificity. The EMA test on CELL-DYN Sapphire correlated strongly with the reference test and had identical diagnostic power. Stability studies with blood from HS patients showed a significant decrease in hyperchromic erythrocytes after 6h storage. Measurement of hyperchromic erythrocytes is highly sensitive and specific for detecting HS and can be used for rapid and inexpensive screening. If required, the EMA test can be performed on CELL-DYN Sapphire or a standard flow cytometer for confirmation of HS.

Hereditary spherocytosis: evaluation of 68 children.

To determine the clinical and hematologic features of 68 children with hereditary spherocytosis (HS). In this retrospective study, we analyzed recorded information of 68 HS patients diagnosed between March 1997 and March 2007, including clinical manifestations at admission, gender, median age at diagnosis, family history, hematologic and biochemical data, patient management, complications, median age of splenectomy, and median follow-up time. Sixty-eight patients with HS (36 male and female) were investigated. The median age at diagnosis was 5.6years (range 3months to 18years). Twenty-seven (39.7%) had parents with consanguineous marriages, and 20 (29.4%) had parents with first-degree consanguinity. Predominant clinical manifestations at admission were anemia in 59 patients (86.76%), splenomegaly in 49 (72.05%), and jaundice in 33 (48.52%). Patients were classified as mild, moderate, or severe in 29.4, 61.7, and 8.8% of patients, respectively. Five patients (7.3%) underwent splenectomy. Major complications of HS were hemolytic, aplastic, and megaloblastic crises and cholelithiasis in 7 (10.2%), 1 (1.4%), 7 (10.2%), and 6 (8.8%) of patients, respectively. There were no deaths during follow-up. HS should be considered in evaluating possible diagnoses in patients with hemolytic anemia. In this study, the clinical course of patients with HS was relatively benign, with low proportions of patients having splenectomized and aplastic crises.

Synchronous splenectomy during cholecystectomy for hereditary spherocytosis: Is it really necessary?

Background/PurposeExpert guidelines recommend performing synchronous splenectomy in patients with mild hereditary spherocytosis (HS) and symptoms of gallstone disease. This recommendation has not been widely explored in the literature. The aim of this study is to determine if our data support expert opinion and if different practice patterns should exist. MethodsThis is an IRB-approved retrospective study. All HS patients under 18years of age who underwent cholecystectomy for symptomatic gallstones at a single institution between 1981 and 2009 were identified. Patients who underwent cholecystectomy without concurrent splenectomy were reviewed retrospectively for future need for splenectomy and evidence of recurrent gallstone disease. ResultsOf the 32 patients identified, 27 underwent synchronous splenectomy. The remaining 5 patients underwent cholecystectomy without splenectomy and had a mean age of 9.4years. One of the 5 patients eventually required splenectomy for left upper quadrant pain. None of the remaining 4 required hospitalization for symptoms related to hemolysis or hepatobiliary disease. Median follow-up is 15.6years. ConclusionThe need for splenectomy in patients with mild HS and symptomatic cholelithiasis should be assessed on a case by case basis. Our recommendation is to not perform synchronous splenectomy in conjunction with cholecystectomy for these patients if no indication for splenectomy exists.

Evaluation of red cell membrane cytoskeletal disorders using a flow cytometric method in South iran.

Objective: The diagnosis of hereditary red blood cell (RBC) membrane disorders, and in particular hereditary spherocytosis (HS) and Southeast Asian ovalocytosis (SAO), is based on clinical history, RBC morphology, and other conventional tests such as osmotic fragility. However, there are some milder cases of these disorders that are difficult to diagnose. The application of eosin-5’-maleimide (EMA) was evaluated for screening of RBC membrane defects along with some other anemias. We used EMA dye, which binds mostly to band 3 protein and to a lesser extent some other membrane proteins, for screening of some membrane defects such as HS. Materials and Methods: Fresh RBCs from hematologically normal controls and patients with HS, SAO, hereditary elliptocytosis, hereditary spherocytosis with pincered cells, severe iron deficiency, thalassemia minor, and autoimmune hemolytic anemia were stained with EMA dye and analyzed for mean fluorescent intensity (MFI) using a flow cytometer. Results: RBCs from patients with HS and iron deficiency showed a significant reduction in MFI compared to those from normal controls (p<0.0001 and p<0.001, respectively), while macrocytic RBCs showed a significant increase in MFI (p<0.01). A significant correlation was shown between mean corpuscular volume and MFI, with the exceptions of HS and thalassemia minor.Conclusion: Our results showed that the flow cytometric method could be a reliable diagnostic method for screening and confirmation, with higher sensitivity and specificity (95% and 93%, respectively) than conventional routine tests for HS patients prior to further specific membrane protein molecular tests.

Values of mean cell volume and mean sphered cell volume can differentiate hereditary spherocytosis and thalassemia

ObjectivesTo determine whether the values of mean cell volume (MCV) and mean sphered cell volume (MSCV) can distinguish hereditary spherocytosis (HS) from thalassemia.MethodsThe MCV, MSCV, and other erythrocyte indexes were measured in totally 263 people, 57 HS patients, 109 thalassemia patients, and 107 normal control subjects. All indexes were derived from measurements obtained by the Beckman–Coulter LH 750 Hematology Analyzer.ResultsThe MSCV was significantly lower in the HS group compared with the thalassemia group (P < 0.001), but the MCV was significantly higher in the HS group compared with the thalassemia group (P < 0.001). Among 57 patients with HS, the MCV was higher than the MSCV in 56 patients. The MCV was lower than the MSCV in one patient combined with β-thalassemia. In the control and thalassemia groups, the MCV was lower than the MSCV.ConclusionMeasurements of the MCV higher than the MSCV can be considered an ideal index to distinguish rapidly HS from thalassemia.

Screening For Hereditary Spherocytosis: EMA Binding Test and Flow Cytometric Osmotic Fragility Test Are Recommended, But Cryohemolysis Test Is Not Recommended

BackgroundThe hallmark of laboratory test for the diagnosis of hereditary spherocytosis (HS) has been osmotic fragility (OF) test. However, OF test gives false negative results in 10% - 20% of HS patients and false positive results in autoimmune hemolytic anemia (AIHA) patients. Currently, the eosin 5-maleimide (EMA) binding test and hypertonic cryohemolysis (HCH) test have been proposed as screening tests in recently published guidelines for the diagnosis of HS (Br J Haematol 2012;156:37-49). And the flow cytometric OF (FC OF) test is recently developed as an assay that can replace the classical OF test. In this study, we evaluated the performance of 3 screening tests (EMA binding test, FC OF test, and HCH test) for the diagnosis of HS. MethodsA total of 151 patients [32 for HS, 40 for AIHA, 40 for anemia of chronic disease (ACD), 39 for iron deficiency anemia (IDA)] and 140 normal controls (NCs) were enrolled in this study. EMA binding test, FC OF test, and HCH test were performed separately in each patient. Reference ranges (mean ± 1.96SD or 2.5th – 97.5th percentile) for each test were determined from 140 NCs. The cutoff value in EMA binding test (5th percentile) was estimated from 119 anemia patients (AIHA+ACD+IDA), and those in FC OF test (mean -1.65SD) and HCH test (95th percentile) were calculated from 140 NCs. Sensitivity, specificity, negative predictive values (NPV), positive predictive values (PPV), and accuracy of each test were calculated using these cutoff values and compared. Subsequently, receiver operating characteristics (ROC) analysis were performed to determine the best cutoff values of each test for the discrimination of HS from other anemia and area under curve (AUC) of three tests were compared. ResultsThe reference ranges of EMA binding test, FC OF test, and HCH test were determined as 86.9% - 118.68%, 6.6% - 71.6%, and 1.5% - 11.8%, respectively, and estimated cutoff values for the diagnosis of HS were calculated to be< 89.4% (EMA binding test),< 8.0% (FC OF test), and > 9.8% (HCH test). In the EMA binding test, HS patients showed significantly decreased EMA binding to RBC (median 71.3%) than AIHA (median 102.0%, P< 0.001), ACD (median 103.7%, P< 0.001), and IDA (median 100.5%, P< 0.001) patients. In the FC OF test, HS patients also showed significantly decreased residual RBC% (median 3.0%) than AIHA (median 44.6%, P< 0.001), ACD (36.0%, P< 0.001), IDA (median 59.4%, P< 0.001) patients and NCs (median 25.2%, P< 0.001). In the HCH test, HS patients showed significantly increased hemolysis (median 10.2%) than AIHA (median 3.6%, P< 0.001) and ACD (median 4.2%, P< 0.001) patients and NC (median 4.7%, P< 0.001), but IDA patients (median 16.4%) showed significantly increased hemolysis than HS patients (P = 0.027). The sensitivity/specificity/NPV/PPV/accuracy of three tests for the discrimination of HS from other anemia using estimated cutoff values mentioned above were calculated as 96.9%/95.0%/99.1%/83.8%/95.4% for EMA binding test, 84.4%/96.6%/95.8%/87.1%/94.0% for FC OF test, and 50.0%/73.1%/84.5%/33.3%/68.2% for HCH test. When the best cutoff values (≤ 85.0% for EMA binding test, ≤ 10.8% for FC OF test, > 7.1% for HCH test) obtained from ROC analysis are applied, both EMA binding test (AUC 0.996) and FC OF test (AUC 0.986) showed satisfactory performance for the discrimination of HS from other anemia. However, HCH test (AUC 0.703) showed significantly poor performance compared than EMA binding test (P< 0.001) and FC OF test (P< 0.001). ConclusionDespite the current guideline recommends EMA binding test and HCH test as screening tests for the diagnosis of HS, our present results demonstrated that both EMA binding test and FC OF test possess satisfactory performance for the diagnosis of HS but HCH test has relatively poor performance compared to the EMA binding test or FC OF test and failed to discriminate HS patients from IDA patients. These results partly contradict the recommendations from HS guideline and therefore, both EMA binding test and FC OF test is recommended as screening tests for the diagnosis of HS, but HCH test is not recommended. Disclosures:No relevant conflicts of interest to declare.

Flow cytometric osmotic fragility testing does reflect the clinical severity of hereditary spherocytosis

Osmotic fragility (OF) testing based on flow cytometry (FCM) was recently introduced for the screening of hereditary spherocytosis (HS). This study was undertaken to compare the test efficiencies of the FCM OF test and the conventional OF test, and to investigate the correlation between FCM OF results and the clinical severity of HS. The test efficiency of FCM OF test was retrospectively compared with one of conventional OF test. FCM OF test results are expressed in two ways, that is, as percentages of residual red cells (%RRC) in hypotonic saline (%RRC values) and by expressing as a healthy individual versus patient ratio of %RRC (%RRC ratio). Cutoff values were defined using 47 subjects including 22 HS patients. HS severity scores were determined using a scoring system devised to quantify the clinical severity of HS. Using cutoff values of 1.68 for %RRC ratio and 61.9 for %RRC value, the test efficiency of %RRC ratio (93.6%) was higher than that of %RRC value (89.4%). However, their difference was not significant (P = 0.357). The FCM OF test (93.6%) achieved a higher test efficiency than the conventional OF test (68.9%) in 115 subjects, which included 75 HS patients (P = 0.002). %RRC ratios and %RRC values were significantly correlated with HS severity scores (P < 0.001 and P = 0.004, respectively). The FCM OF test was found to have greater test efficiency than the conventional OF test. Furthermore, FCM OF test results quantitatively reflected the clinical severity of HS. Using %RRC ratio is recommended to minimize false positivity although its superiority over %RRC value could not be verified statistically. The FCM OF test could be the method of choice for routine diagnostic use to screen HS and assess its clinical severity.

Is activity restriction appropriate for patients with hereditary spherocytosis? A population-based analysis

Patients with hereditary spherocytosis (HS) are often thought to have an increased risk of blunt splenic injury (BSI) from trauma due to splenomegaly. We aim to quantify this risk. Using a population-based database consisting of all injury-related admissions in Canada from 2001 to 2010, we identified patients with BSI and HS based on the discharge diagnoses. Intercensal population estimates were used to derive rates of BSI. The HS population at risk for BSI was estimated based on population rates of HS obtained from the literature. Rates of BSI in the HS population were estimated and the relative rates of BSI were calculated to compare the populations with and without HS. There were 10,106 patients with BSI over 202,405,788 person-years of observation, yielding an overall rate of BSI in the general population of 5.0 BSI per 100,000 person-years. Of these BSI patients, only two had a history of HS. Population rates of HS in the literature range from 1 in 2,000 to 5,000, corresponding to a low estimate of 2.0 and a high estimate of 4.9 BSI per 100,000 person-years in the HS population. The relative rate of BSI in the population with HS compared to the population without HS ranged from a low of 0.4 (95 % CI 0.1-1.4) to a high of 1.0 (0.1-3.6). The rate of BSI in the HS patient population appears not to differ significantly from those in the general population.

Reply to "Testing for hereditary spherocytosis: a French experience". Haematologica. 2012;97(12):e48-9

The data presented by Mayeur-Rousse et al. [1][1] in this issue of the journal are very interesting. We stratified the hereditary spherocytosis (HS) patients of our series[2][2] in the 4 categories adopted by the authors finding a perfect correspondence of the results among classes in which a 15-21

Testing for hereditary spherocytosis: a French experience

In a recent article, Bianchi et al. have compared several laboratory tests for diagnosis of hereditary spherocytosis (HS) by studying 150 HS patients.[1][1] They concluded that the association of the eosin-5-maleimide (EMA) binding test and the acidified glycerol lysis test (AGLT) could identify all

Flow Cytometric Osmotic Fragility Test; A Sensitive and Quantitative Diagnostic Approach for Hereditary Spherocytosis

AbstractAbstract 5151 Background & Aim:Hereditary spherocytosis (HS) is the most common cause of congenital inherited hemolytic anemia. Traditional osmotic fragility test (OF) using a series of hypotonic solutions of NaCl, the most widely used diagnostic approach, has relatively low sensitivity. Further the ‘positive' result cannot quantify the disease severity. We have performed OF using flow cytometric method (FCM OF) to make a diagnosis of HS in Kyungpook National University Hospital (Daegu, South Korea) from September 2008 until now. In this new test, deionized water (a hemolysis-inducing agent) is spiked to a red cell suspension during acquisition and the count of red cell is measured sequentially in real-time FCM. The healthy/patient ratio of %residual red cell over 3. 0 is considered ‘positive'. The aim of this study is to investigate the usefulness of FCM OF (from September 2008 to July 2012) in comparison with the traditional one (January 2002 to August 2008). Methods:The HS patients’ laboratory results were divided into two groups based on the diagnostic methods (FCM OF vs. traditional one). The values were described as ‘mean ± standard deviation'. Statistical analyses were conducted using SPSS ver. 19. 0 (Chicago, IL, USA). The independent T-test was performed to compare inter-group differences for the disease severity variables at the time of test - hemoglobin, hematocrit, reticulocyte, corrected reticulocyte (c-reticulocyte), spherocyte percentage in peripheral blood (PB), and total bilirubin. To determine the factors which influence the healthy/patient ratio of %residual red cell, Pearson or Spearman correlation were performed according to the aforementioned severity variables in the subjects who underwent FCM OF. Absolute values of rho > 0. 3 and p < 0. 05 were considered statistically significant. Results:Nineteen HS patients (male: female = 8: 11) underwent a total of 23 times of OF (FCM OF: traditional one = 11: 12). Their mean age at the time of diagnosis was 8. 8 years (range, 0–72). The hemoglobin and hematocrit were higher in FCM OF group than in traditional one (both, p = 0. 038). The mean value of severity variables and respective p-value are summarized in table 1. And sixteen subjects (male: female = 8: 8) underwent a total of 19 times of FCM OF (positive: negative = 11: 8). A negative correlation was observed between the healthy/patient ratio of %residual red cell and hemoglobin (p = 0. 039). We also observed a positive correlation between The healthy/patient ratio of %residual red cell and the reticulocyte/c-reticulocyte/spherocyte percentage in PB (respectively, p = 0. 040, 0. 014, and 0. 018). The rho and p-value are described in table 2. Conclusions:Considering the higher level of hemoglobin and hematocrit at the time of diagnosis in FCM OF group than those in the case of traditional one, we supposed that less severe cases could be diagnosed as HS by using this new test. Furthermore, the value of healthy/patient ratio of %residual red cell correlated with the severity of the disease. Thus FCM OF could be an useful first line screening test for HS due to its sensitivity and quantitative advantage.Table 1Inter-group differences of the severity variables at the time of diagnosis.FCM OF (n = 11)Traditional OF (n = 12)p-valueHemoglobin (g/dL)10.2 ± 1.78.6 ± 1.70.038*Hematocrit (%)27.5 ± 4.323.7 ± 4.00.038*Reticulocyte (%)14.0 ± 11.114.7 ± 6.30.853C-reticulocyte (%)9.2 ± 6.59.0 ± 3.60.941Spherocyte (%)21.7 ± 20.526.9 ± 18.70.531Total bilirubin (mg/dL)4.7 ± 2.63.6 ± 1.20.214Table 2The correlation between healthy/patient ratio of %residual red cell in FCM OF and severity variables.The healthy/patient ratio of %residual red cellrhop-valueHemoglobin (g/dL)−0.476*0.039*Hematocrit (%)−0.3250.175Reticulocyte (%)0.474*0.040*C-reticulocyte (%)0.556*0.014*Spherocyte (%)0.538*0.018*Total bilirubin (mg/dL)0.2710.261*The value of statistical significance Disclosures:No relevant conflicts of interest to declare.

Delay in the measurement of eosin-5′-maleimide (EMA) binding does not affect the test result for the diagnosis of hereditary spherocytosis

The eosin-5′-maleimide (EMA) binding test is a flow cytometric test widely used to detect hereditary spherocytosis (HS). EMA binds to plasma membrane proteins of red blood cells (RBCs), mainly to band 3 protein. The mean fluorescence of EMA-stained RBCs in HS patients is lower when compared with control RBCs due to the decreased amount of target proteins. EMA dye in aqueous solution is sensitive to light and high temperature. Its fluorescence can decrease when exposed to light or ambient temperatures higher than 4°C. The aim of the study was to evaluate the stability of fluorescence readings of EMA-labeled RBCs over a period of 24 h. The EMA test was performed in peripheral blood from 35 patients with microcytic anemia (five with HS, and 30 without HS). Peripheral blood samples were stained immediately after blood collection and analyzed using a flow cytometer at three time points: 0, after 1 and 24 h of storage at 4°C in the darkness. The results are presented as the percentage of normal control RBCs fluorescence. Flow cytometric studies were performed with Cytomics FC500 (Beckman Coulter, USA). In HS patients the mean result of the test reached 66.72%±9.26% of normal controls, and in non-HS patients the EMA result was 99.48%±5.03% of normal control cells. The results of patients with HS were 66.72%±9.26%, 66.90%±10.24% and 67.86%±11.31% at 0 h, and after 1 and 24 h of storage, respectively. The results obtained from non-HS patients at time 0, after 1 and 24 h of storage reached 99.48%±5.03%, 99.49%±5.34% and 99.78%±6.13%, respectively. There was no difference between the results from each time point in samples from patients with or without HS. Results of the EMA binding test do not depend on storage time of stained samples when stored at 4°C up to 24 h after staining.