Flow Cytometric Osmotic Fragility Testing Does Reflect the Clinical Severity of Hereditary Spherocytosis

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1 Cytometry Part B (Clinical Cytometry) 86B: (2014) Original Article Flow Cytometric Osmotic Fragility Testing Does Reflect the Clinical Severity of Hereditary Spherocytosis Ye Jee Shim 1 and Dong II Won 2 * 1 Hanyoung Children s Hospital and Department of Pediatrics, Kyungpook National University School of Medicine, Daegu, Republic of Korea 2 Department of Clinical Pathology, Kyungpook National University School of Medicine, Daegu, Republic of Korea Background: 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. Methods: 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. Results: 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 ). 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 ). %RRC ratios and %RRC values were significantly correlated with HS severity scores (P < and P , respectively). Conclusions: 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. VC 2013 International Clinical Cytometry Society Key terms: hereditary spherocytosis; flow cytometry; osmotic fragility How to cite this article: Shim YJ and Won DI. Flow Cytometric Osmotic Fragility Testing Does Reflect the Clinical Severity of Hereditary Spherocytosis. Cytometry Part B 2014; 86B: Hereditary spherocytosis (HS) is one of the major causes of inherited chronic hemolysis, and presents with diverse clinical features and laboratory results (1,2). Because of defects in proteins that comprise the red cell membrane (ankyrin, band 3, spectrin, and protein 4.2), red cells acquire a spherocytic shape and are destroyed in the spleen. The clinical severity of HS is reflected in laboratory results associated with the degree of hemolysis, namely, hemoglobin and serum bilirubin levels, as well as reticulocyte and spherocyte counts (3 5). It is not difficult to diagnose patients with HS if they have classic symptoms of hemolysis, a family history of Disclaimer: The authors declare that they have no proprietary, commercial, or financial interests that could be construed to have inappropriately influenced this study. *Correspondence to: Dong II Won, MD, PhD, Department of Clinical Pathology, Kyungpook National University Hospital, 50 Samduk-Dong, 2-Ga, Jung-Gu, Daegu , Republic of Korea. wondi@knu.ac.kr Grant sponsor: BioMedical Research Institute grant, Kyungpook National University Hospital (2013). Received 7 June 2013; Revised 3 October 2013; Accepted 23 October 2013 Published online 30 October 2013 in Wiley Online Library (wileyonlinelibrary.com). DOI: /cyto.b VC 2013 International Clinical Cytometry Society

2 FCM OF TEST REFLECTS THE SEVERITY OF HS 437 HS, and a positive laboratory test (2,4,5). However, for patients with atypical symptoms, the eosin-5 0 -maleimide (EMA) binding test, the cryohemolysis test, or sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS- PAGE) have been recommended (4,5). However, there are practical problems applying these methods because they are not yet available in many laboratories. The conventional osmotic fragility (OF) test, which uses a series of hypotonic solutions, is the most widely used diagnostic method. However, this test has a low test efficiency and is no longer recommended for routine testing in HS (2,4,5). Recently, the flow cytometry based OF test flow cytometry (FCM OF test) was introduced. It has many advantages over the other tests: (1) it is quantitative and objective; (2) it saves time and labor; (3) it does not require preincubation of blood samples; (4) is costeffective; and (5) it has high test efficiency (6 8). Our aim in this study was (1) to confirm the higher test efficiency of the FCM OF test versus the conventional OF test. The comparison was performed retrospectively; (2) to define cutoff values for the two FCM OF indices, that is, the percentage of residual red cells (%RRC) in hypotonic saline (%RRC values), and a healthy individual versus patient ratio of %RRC (%RRC ratio) in a HS group and a non-hs group (disease control); (3) to compare the diagnostic power between two FCM OF indices; and (4) to investigate the correlations between FCM OF indices and the clinical severity of HS. METHODS Subjects In this retrospective study, we reviewed the medical records of 115 subjects including 75 HS patients that underwent the FCM or conventional OF test between February 1992 and December The test method was changed from the conventional to the FCM OF test on September 2008, we refer to the period before and after this date as phase I and II; details of diagnoses are presented in Table 1. No significant difference was found between phase I and II with respect to the clinical parameters of HS patients at initial diagnosis. At our institute a diagnosis of HS is based on: (1) clinical manifestations; (2) the exclusion of other causes of hemolytic anemia or secondary spherocytosis; and (3) elevated conventional OF test result (2,9). Even if the results of an OF test are negative, other laboratory findings, such as, spherocytosis, an increased mean corpuscular hemoglobin concentration (MCHC), and reticulocytosis together with a family history could be used to confirm a diagnosis of HS (4,5). Patients with atypical clinical findings and a negative OF test result undergo further evaluations to ensure accurate diagnosis. SDS-PAGE was performed in 8 patients and the EMA binding test in 1 patient. FCM OF Test FCM OF testing was performed according to the method of Won and Suh (6), our previous study. Briefly, for flow cytometric acquisition, the appropriately diluted red cell suspension was installed at the sample injection port of FACSCalibur Flow Cytometer (BD Biosciences, San Jose, CA) after thorough mixing. After the first region on the time vs. forward scatter plot elapsed during acquisition, the tube was removed without ending acquisition and 0.9 ml deionized water was added and acquisition was again continued up to the last region. The first and the last two regions were used to calculate % of %RRC. The two FCM OF indices were calculated as follows: 1. %RRC value 5 (RRC count after DW spiking)/(initial red cell count) 3 2.0/ (%). 2. %RRC ratio 5 (%RRC of a normal control)/(%rrc of a patient). Thus, a low %RRC value and a high %RRC ratio indicated increased OF. Table 1 Number of Involved Subjects in Each Phase According to the OF Test Type Performed Total Phase I (conventional OF test) Phase II (FCM OF test) Period Feb 1992 Dec 2012 Feb 1992 Aug 2008 Sep 2008 Dec 2012 HS group Non-HS group Immune hemolytic anemia Iron deficiency anemia HE a 1 1 Myelodysplastic syndrome Other hematologic diseases b Non-hematologic diseases c Healthy 6 6 Total a One patient with hereditary eliptocytosis (HE) was included in the hereditary spherocytosis (HS) group when data were analyzed to obtain the test efficiency of the test. b Other hematologic diseases included 2 cases of congenital dyserythropoietic anemia in phase I and 1 case each of leukemia and pernicious anemia in phase II. c Nonhematologic diseases included 3 cases of cholelithiasis, 1 case each of Gilbert syndrome and alcoholic fatty liver in the phase I, 2 cases of Gilbert syndrome, 1 case each of Crigler-Najjar syndrome, cholelithiasis, meningoencephalitis, and gastritis in the phase II.

3 438 SHIM AND WON Conventional OF Test Freshly drawn red cells were suspended in a series of tubes containing hypotonic NaCl solution (from 0.9% to 0.0%), incubated at room temperature for 30 min, and centrifuged. Supernatant absorbance was measured at 541 nm using a spectrophotometer. Percentage of hemolysis was plotted for each NaCl concentration. When the OF of freshly drawn blood appeared normal, the blood was incubated at 37 C for 24 h before performing the second OF test. The System Used to Score the Clinical Severity of HS A scoring system was used to quantify the clinical severity of HS (Table 2). We adopted several severity parameters from two known classification systems to subdivide the clinical severity of HS further. The hemoglobin, reticulocytes, and total bilirubin were adopted from both classification systems from Bolton-Maggs et al. (4,5) and Perrotta et al. (2). For more accurate assessment, corrected reticulocyte (c-reticulocyte) were calculated. Spherocytosis in peripheral blood smears, transfusion history, and heredity were also used according to the recent classification system devised by Perrotta et al (2). One expert graded spherocytosis in peripheral blood smears based on the mean ranges of spherocytes seen on 10 oil emersion fields: absent (0); slight (1 5); moderate (6 15); marked ( 16). We conducted a family study on all patients that underwent the FCM OF test to determine inheritance patterns. Routine laboratory workups for hemolysis and detailed history taking were performed on all available parents, siblings, grandparents, and relatives of patients. Three HS patients had HS siblings and healthy parents, showing autosomal recessive inheritance. Six HS patients belonged to three or two consecutive generations of HS (parent/grandparent or parent/sibling), showing autosomal dominant inheritance. The remaining 13 HS patients (10 patients with no family history of HS and three patients of unclear inheritance) were classified into de novo mutations or as having an unclear inheritance. Individual parameters were scored as 0 4. The sum of all parameter scores was defined as the patient s HS severity score. Our system was based on the clinical severity of HS on the day of FCM OF testing. Statistical Analysis Fisher s exact test or v 2 test were used to compare the test efficiency of FCM OF and conventional OF tests. Receiver-operator characteristic (ROC) curves were used to define cutoff of FCM OF test. Pearson s correlation analysis (for parametric and normally distributed data) or Spearman s correlation analysis (for nonparametric or non-normally distributed data) were used to define the correlation between FCM OF indices and clinical severity of HS. Statistical analyses were performed using SPSS version 20.0 software (Knowledge Dynamics, Canyon Lake, TX). Dependent comparison of area under the curve (AUC) was used to compare the diagnostic powers between two FCM indices using SAS (SAS Institute, Cary, NC). Test efficiency was defined as the fraction of cases correctly classified, that is, [(true positives 1 true negatives)/total cases]. Data are reported as means 6 SDs. Absolute correlation coefficient values (r) of > 0.3 and P values of < 0.05 were taken to indicate statistical significance. Ethics Statement This study was approved by the institutional review board of Kyungpook National University Hospital (Approval No., ), which waived the requirement for informed consent. RESULTS Cutoff Definitions and the Test Efficiency of the FCM OF Test Measured %RRC ratios and %RRC values of the patients in phase II are presented in Figure 1 according to their disease group. One patient with hereditary elliptocytosis (HE) was included in the HS group because HE may show increased OF (HS group [N 5 23] and non- HS group [N 5 24]). Using the non-hs group as disease controls, optimal cutoff values for %RRC ratio and %RRC value are presented in Table 3 (1.68 for %RRC ratio and 61.9 for %RRC value). When these cutoff values were applied, the test efficiency of %RRC ratio (93.6%) was Table 2 Scoring System Used to Assess the Clinical Severity of HS Parameter Hemoglobin (g/dl) >6.0 C-reticulocytes (%) < Total bilirubin (mg/dl) < Spherocytosis in peripheral Absent Slight Moderate Marked Marked spherocytosis blood smear a and poikilocytosis Transfusion >2 Regular Heredity AD de novo mutation or unclear AR a Spherocytosis in peripheral blood smears was graded based on the mean ranges of spherocytes seen on 10 oil emersion fields: absent (0); slight (1 5); moderate (6 15); marked (16). Abbreviations: AD, autosomal dominant; AR, autosomal recessive; C-reticulocytes, corrected reticulocytes. Score

4 FCM OF TEST REFLECTS THE SEVERITY OF HS 439 FIG. 1. Measured two indices of FCM OF test (%RRC ratio and %RRC value) by disease group in the phase II (N 5 47). Two HS patients showed false negativities by both FCM OF indices, and two false positive non-hs cases by %RRC value were true negativities by %RRC ratio. NHDs included two cases of Gilbert syndrome, 1 case each of Crigler-Najjar syndrome, cholelithiasis, meningoencephalitis, and gastritis. Others include one case each of myelodysplastic syndrome, leukemia, and pernicious anemia. Abbreviations: FCM, flow cytometry; OF, osmotic fragility; %RRC, percentage of residual red cells; HS, hereditary spherocytosis; NHD, nonhematologic disease; IHA, immune hemolytic anemia; IDA, iron deficiency anemia. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.] found to be higher than that of %RRC value (89.4%). However, the dependant comparison of AUC revealed no statistical difference between them (0.938 and 0.904, respectively, P ). Comparison of the Test Efficiencies of the FCM OF and Conventional OF Tests The test efficiency of the conventional OF test (using fresh and incubated blood) in phase I is presented in Table 4. The comparison was performed retrospectively. To compare the test efficiencies of the FCM OF and conventional OF tests, optimal determination criteria were used for each test: the FCM OF test is positive when both %RRC ratio and %RRC value are positive according to the aforementioned cutoff definitions, and the conventional OF test is positive when both fresh and incubated blood are positive or when only positive fresh blood is enough for the diagnosis of HS without incubation. Comparisons using the v2 test or Fisher s exact test disclosed that the FCM OF test achieved a significantly higher diagnostic sensitivity (P ) and test efficiency (P ) than the conventional OF test. No significant difference toward higher diagnostic specificity (P ) was found for the FCM OF test versus the conventional test. Correlations Between the Clinical Severity of HS and FCM OF Indices Regarding correlations between individual severity parameters and %RRC ratio, a positive correlation was found between spherocytosis grade in peripheral blood smear and %RRC ratio (P ). In the case of hemoglobin, c-reticulocytes, and total bilirubin, no significant differences were found (P , P , and P , respectively). The correlations between individual severity parameters and %RRC values are described in Figure 2. A positive correlation was found between hemoglobin and %RRC values (P ). Furthermore, a negative correlation was found between c-reticulocytes or spherocytes and %RRC values (P and P < 0.001, respectively). In the case of total bilirubin, no significant difference was found (P ). Table 3 Cutoff Definitions and the Test Efficiencies of %RRC Ratio and %RRC Value Diagnostic Cutoff Sensitivity Specificity Efficiency AUC %RRC ratio % 95.8% 93.6% a %RRC value 61.9% 91.3% 87.5% 89.4% a a There was no significant difference between the AUCs of two indices (P ). Abbreviations: AUC, area under the curve; %RRC, percentage of residual red cells.

5 440 SHIM AND WON Table 4 Comparison of the Test Efficiencies of the FCM OF Test During Phase II and the Conventional OF Test During Phase I HS group Non-HS group Test efficiency (%) TP FN FP TN Sensitivity Specificity Efficiency FCM OF test (N 5 47) %RRC ratio %RRC value If 1ve both indices, 1ve Conventional OF test (N 5 68) Fresh blood Incubated blood (N 5 58) If 1ve both samples, 1ve a a This category includes the three cases without incubation because the positive results of fresh samples were enough to make a diagnosis of HS. Abbreviations: %RRC, percentage of residual red cells; 1ve, positive; FN, false negative; FP, false positive; HS, hereditary spherocytosis; TN, true negative; TP, true positive. The correlations between HS severity scores and FCM OF indices are presented in Figure 3. A significant positive correlation was found between HS severity scores and %RRC ratios (P < 0.001), and a negative correlation was found between HS severity scores and %RRC values (P ). Furthermore, both FCM OF indices were graded into three categories as follows: (1) trait or mild (%RRC ratio < 2.0); (2) moderate (2.0 %RRC ratio < 4.0); and (3) severe (%RRC ratio 4.0). This classification concurred with that of Bolton-Maggs with a concordance rate of 71.4% (15/21). These border %RRC ratios (2.0 and FIG. 2. Correlations between individual severity parameters and %RRC values of HS patients in phase II. A positive correlation was found between hemoglobin and %RRC values (A) and negative correlations between c-reticulocytes or spherocytosis in peripheral blood smear and %RRC values (B, C). No significant difference was found between total bilirubin and %RRC values (D). One HS female, whose laboratory records were incomplete, was included in the hemoglobin 2 %RRC plot only (A). Abbreviations: %RRC, percentage of residual red cells; HS, hereditary spherocytosis; c- reticulocytes, corrected reticulocytes.

6 FCM OF TEST REFLECTS THE SEVERITY OF HS 441 FIG. 3. Correlations between HS severity scores and FCM OF indies, %RRC ratios (A), and %RRC values (B), in HS patients (N 5 21) in phase II. Both FCM OF indices categorized as trait or mild, moderate, or severe showed reasonable agreement with the Bolton-Maggs classification, which is indicated by different dot colors. The categories of the two FCM OF indices are shown by the horizontal dotted lines inside the plots, and their names are labeled beside Y-axis. Abbreviations: %RRC, percentage of residual red cells; HS, hereditary spherocytosis. 4.0) corresponded to %RRC values of 50 and 25%, respectively. DISCUSSION A diagnosis of HS is usually based on a combination of the following: (1) clinical manifestations; (2) hereditable factors; and (3) laboratory findings (2,4,5). In addition, conventional OF tests have been widely used as diagnostic tools in HS (2,9). Physicians also need to examine the results of direct antiglobulin tests (DAT), bone marrow studies, and iron status to exclude other potential causes of hemolytic anemia, such as autoimmune or congenital dyserythropoiesis (2). The diagnosis of a milder form of HS can be complicated, and some HS patients have no apparent symptoms (10). In this study, the conventional OF test, which was based on fresh blood showed a sensitivity of 73.6%. This is similar to previously reported data (9,11,12). The use of incubated blood, however, did not improve the diagnostic sensitivity of the conventional OF test. We believe that this is because the conventional OF test using incubated blood was not performed for 10 patients with positive conventional OF test results using fresh blood. The test efficiency of the FCM OF test was much superior to that of conventional OF test. Crisp et al. also recently reported that a FCM OF test using capillary blood had a sensitivity of 94% (8). This is similar to the sensitivity of the EMA binding test and cryohemolysis. In previous studies, the cutoff values for %RRC ratio and %RRC value were set at 3.0 and %, respectively, with sensitivities of % and specificities of 96 98% (6 8). These cutoff values were obtained by averaging the results of healthy individuals and subtracting two standard deviations. Warang et al. also reported a cutoff for %RRC of 24% using the same statistical method (7). In this study, the cutoff values of %RRC ratio and %RRC value were set at 1.68 and 61.9%, respectively. These cutoff values are different from those of other and our previous studies (6 8). This might be (1) because this study obtained cutoff values from ROC curve analysis for both HS and non-hs groups (disease control); and (2) because we included HS patients with a milder phenotype. Had the cutoff values of Crisp et al. been applied in this study, one infant with HS, whose %RRC value was 56.9%, would have also fallen in the diagnostic range of the FCM OF test (8). Therefore, we expect that the newly revised cutoff values might have increase the discriminatory power of the FCM OF test in a practical clinical setting. Traditionally, the clinical severity of HS has been classified into trait, mild, moderate, and severe based on laboratory parameters of hemolysis. This grading system includes hemoglobin, reticulocytes, and total bilirubin and helps physicians decide an appropriate timing for splenectomy (4,5). Recently, the new grade moderately severe was defined for patients with a lower hemoglobin level, more frequent reticulocytes (>15%), and more prominent hyperbilirubinemia than moderate patients by Perrotta et al. (2). Other clinical parameters, that is, spherocytosis in peripheral blood smear, a transfusion history, and heredity were included in this new classification system. In this study, we adopted several severity parameters from two classification systems to subdivide clinical severity further. In phase II of our study, two FCM OF indices, %RRC ratio and %RRC value, were found to be correlated even with the raw values of individual severity parameters. The HS severity scores were also significantly correlated with both FCM OF indices, which suggest that the two FCM OF indices would quantitatively reflect the clinical severity of HS. Furthermore, %RRC ratio was categorized as trait or mild (<2.0), moderate (2.0 and <4.0), or severe (4.0). Our classification agreed reasonably with that of Bolton-Maggs, which also suggests that %RRC ratio is an independent parameter that could be used to classify the clinical severity of HS.

7 442 SHIM AND WON The %RRC ratio was found to be better correlated with HS severity scores than %RRC value and showed a higher AUC value by ROC curve analysis, although this was not statistically significant. Whereas, the %RRC value was better correlated with individual severity parameters of HS. Thus, in this study, we were not able to decide which index was the better for determining the clinical severity of HS. As shown in our previous study, delays between preparation of the final red cell suspension and injection into the port of the flow cytometer cause significant reductions in the percentages of RRCs (6). Hence, it is important to proceed to the next step immediately after blood is diluted. We can prevent aforementioned error by using %RRC ratio because it is corrected by %RRC value of a healthy individual tested in the same batch. This is the reason why we recommend %RRC ratio rather than %RRC value although its superiority over %RRC value could not be verified statistically. In the present study, two false positives by %RRC value were changed to true negatives by %RRC ratio. Two patients (1 with HE and 1 with IHA) had a positive FCM OF test result, but did not have HS. HE is a rarer type of hemolytic anemia than HS. In HE, red cell membrane defects (due to a spectrin or protein 4.1 deficiency) result in unstable elliptical red cells in peripheral blood (1,13). In these patients, OF test results could be normal or elevated depending on disease severity. The finding that one HE patient in our study was positive by the FCM OF test (%RRC ratio of 3.30) suggests that this test could be used to screen HE, as was reported previously by Warang et al. (7). Regarding the five patients with IHA in phase II of our study, these patients were positive for DAT. One of them had a positive FCM OF test result. Spherocytes may also appear in peripheral blood secondary to IHA, and OF can be increased by spherocytes in the peripheral blood irrespective of their pathophysiological origin (14). It is, therefore, essential to verify the DAT results in conjunction with the OF test findings in patients with peripheral spherocytes. Two patients were diagnosed as HS although FCM OF test results were negative. One patient fulfilled the laboratory diagnostic criteria for HS (reticulocytes 5.9%; MCHC 38.8 g/dl; and spherocytes 5.5%). In addition, this patient had a family history of HS his father was splenectomized because of HS. The main symptom of HS in this patient was jaundice (total bilirubin 12.8 mg/ dl; direct bilirubin 0.9 mg/dl) rather than anemia (hemoglobin 14.2 g/dl). These laboratory findings indicate compensated hemolysis, which is quite different from the hemolysis of his father (hemoglobin 8.4; reticulocytes 30.6%; spherocytes 13.0%; total bilirubin 2.5 mg/ dl). These findings suggest that heterozygosity can result in subclinical or carrier HS. We presumed that the patient s father was homozygous and that he had an autosomal recessive trait. Interestingly, this patient s follow-up laboratory tests results were almost normal (hemoglobin 14.5 g/dl; no spherocytes) except for hyperbilirubinemia (total bilirubin 6.8 mg/dl; direct bilirubin 0.6 mg/dl). We also considered that this patient might have a hepatobiliary condition, such as, Gilbert syndrome (15,16). Another HS patient with a negative FCM OF test had been previously reported by us (17). He underwent two FCM OF tests and different results were obtained: one was positive and the other was negative. He was finally diagnosed as having HS by the EMA binding test. This case indicates that the FCM OF test reflects the presence of illness throughout entire disease course. The limitation of our study is that the FCM OF and conventional OF tests were not performed simultaneously in the enrolled subjects. Such a study could probably be undertaken as a part of a large multi-centered prospective study involving several HS diagnostic methods. Nevertheless, our results are important because, to our knowledge, this is the first attempt to compare these two OF tests. In addition, we included a wide range of patients and considered the presence of subclinical HS or carriers even among healthy donors (10). In conclusion, the FCM OF test showed much better test efficiency than the conventional OF test. Furthermore, two FCM OF indices, %RRC ratio and %RRC value, reflected the clinical severity of HS quantitatively. The %RRC ratio is recommended to minimize the false positivity. We are of the opinion that the FCM OF test could become the method of choice for routine diagnostic use to screen for HS and to assess disease severity. LITERATURE CITED 1. 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