ORIGINAL ARTICLE Comparison and evaluation of three screening tests of hereditary spherocytosis in Chinese patients Yi-feng Tao & Zeng-fu Deng & Lin Liao & Yu-ling Qiu & Wen-qiang Chen & Fa-quan Lin Received: 28 July 2014 /Accepted: 27 November 2014 /Published online: 12 December 2014 # Springer-Verlag Berlin Heidelberg 2014 Abstract The objective of this study is to compare and eval- uate 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 collect- ed from 237 participators diagnosed at the First Affiliated Hospital of Guangxi Medical University, including 56 hered- itary 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 specific- ity 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 os- motic fragility test, the sensitivity was 85.71 % and the spec- ificity was 97.24 %. Flow cytometry osmotic fragility test or comparing MSCV to MCV combined with smear examina- tion of peripheral red blood cells morphology can be a simple, practical, and accurate hereditary spherocytosis (HS) labora- tory screening method. Keywords Hereditary spherocytosis . Mean corpuscular hemoglobin concentration . Mean sphered corpuscular volume . Flow cytometric . Osmotic fragility Introduction Hereditary spherocytosis (HS) is one of hereditary hemolytic diseases caused by red cell membrane disorders, characterized by anemia, jaundice, and splenomegaly [1]. Since the 1980s, HS cases had been reported from all over the country [2–6]. There was no exact incidence survey, but as reported, the incidence of HS was much higher than the clinical diagnosis rate [7]. It is not difficult to diagnose patients with HS if patients have classic symptoms of hemolysis, a family history of HS, and a positive laboratory test [1, 8, 9]. There are several ways to test for HS. Such as acidified glycerol lysis test (AGLT), eosin-5 ′ -maleimide (EMA)-binding test, cryohemolysis, and some red blood cell parameters. AGLT and EMA have been thoroughly tested. So far, none single test is perfect. In this study, flow cytometric osmotic fragility test was used to help diagnose HS quickly and accurately. Methods Subjects Fifty-six patients with HS were diagnosed based on their clinical symptoms, biochemistry, complete blood cell count, peripheral blood smear, osmotic fragility (OF) test, and other laboratory examinations at the First Affiliated Hospital of Guangxi Medical University, including 26 males and 30 The authors Yi-feng Tao and Zeng-fu Deng contributed equally to this work. Y.<f. Tao Affiliated Tumor Hospital of Guangxi Medical University, Nanning, Guangxi, China Z.<f. Deng : L. Liao : F.<q. Lin (*) Department of Clinical Laboratory, The First Affiliated Hospital of Guangxi Medical University, Nanning, Guangxi, China e-mail: [email protected] Y.<l. Qiu : W.<q. Chen Department of Pediatric Laboratory, The First Affiliated Hospital of Guangxi Medical University, Nanning, Guangxi, China Ann Hematol (2015) 94:747–751 DOI 10.1007/s00277-014-2270-2
Comparison and evaluation of three screening tests of hereditary spherocytosis in Chinese patients
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Comparison and evaluation of three screening tests of hereditary spherocytosis in Chinese patients
Yi-feng Tao & Zeng-fu Deng & Lin Liao & Yu-ling Qiu &
Wen-qiang Chen & Fa-quan Lin
Received: 28 July 2014 /Accepted: 27 November 2014 /Published online: 12 December 2014 # Springer-Verlag Berlin Heidelberg 2014
Abstract The objective of this study is to compare and eval- uate 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 collect- ed from 237 participators diagnosed at the First Affiliated Hospital of Guangxi Medical University, including 56 hered- itary 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 specific- ity 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 os- motic fragility test, the sensitivity was 85.71 % and the spec- ificity was 97.24 %. Flow cytometry osmotic fragility test or comparing MSCV to MCV combined with smear examina- tion of peripheral red blood cells morphology can be a simple,
practical, and accurate hereditary spherocytosis (HS) labora- tory screening method.
Keywords Hereditary spherocytosis .Mean corpuscular hemoglobin concentration .Mean sphered corpuscular volume . Flow cytometric . Osmotic fragility
Introduction
Hereditary spherocytosis (HS) is one of hereditary hemolytic diseases caused by red cell membrane disorders, characterized by anemia, jaundice, and splenomegaly [1]. Since the 1980s, HS cases had been reported from all over the country [2–6]. There was no exact incidence survey, but as reported, the incidence of HS was much higher than the clinical diagnosis rate [7]. It is not difficult to diagnose patients with HS if patients have classic symptoms of hemolysis, a family history of HS, and a positive laboratory test [1, 8, 9]. There are several ways to test for HS. Such as acidified glycerol lysis test (AGLT), eosin-5 ′-maleimide (EMA)-binding test, cryohemolysis, and some red blood cell parameters. AGLT and EMA have been thoroughly tested. So far, none single test is perfect. In this study, flow cytometric osmotic fragility test was used to help diagnose HS quickly and accurately.
Methods
Subjects
Fifty-six patients with HS were diagnosed based on their clinical symptoms, biochemistry, complete blood cell count, peripheral blood smear, osmotic fragility (OF) test, and other laboratory examinations at the First Affiliated Hospital of Guangxi Medical University, including 26 males and 30
The authors Yi-feng Tao and Zeng-fu Deng contributed equally to this work.
Y.<f. Tao Affiliated Tumor Hospital of Guangxi Medical University, Nanning, Guangxi, China
Z.<f. Deng : L. Liao : F.<q. Lin (*) Department of Clinical Laboratory, The First Affiliated Hospital of Guangxi Medical University, Nanning, Guangxi, China e-mail: [email protected]
Y.<l. Qiu :W.<q. Chen Department of Pediatric Laboratory, The First Affiliated Hospital of Guangxi Medical University, Nanning, Guangxi, China
Ann Hematol (2015) 94:747–751 DOI 10.1007/s00277-014-2270-2
females, aged 2∼38 years. Eighty-six patients with thalasse- mia were diagnosed by hemoglobin (Hb) electrophoresis and genetic test at the Thalassemia Laboratory of Medical Science Experimental Center, Guangxi Medical University, including 41 males and 45 females, aged 4∼37 years. The healthy control group was composed by 95 volunteers (52 males and 43 females, aged 3∼36 years) who received physical exami- nations at the First Affiliated Hospital of Guangxi Medical University. The subjects of this study had given their informed consent, and the study was approved by the Ethics Committee of Guangxi Medical University.
Tests of HS
Red blood cell (RBC), Hb, mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular he- moglobin concentration (MCHC), and mean spherical corpus- cular volume (MSCV) were determined using the Beckman Coulter GEN·S (Beckmann Coulter Inc., Fullerton, CA) ac- cording to manufacturer’s instructions.
Flow cytometric osmotic fragility test was performed ac- cording to the method described by Won and Suh [10]. The red cell suspension was prepared firstly. The volume of anticoagulated venous blood mixed with 1 mL isotonic saline was calculated by the following formula: blood volume (μl)= 130/red blood cells (1012/L) [11]. For example, if the red blood cell count was 4×1012/L, 32.5 μl (130 divided by 4) blood would be mixed with 1 mL normal saline. And then 10 μl diluted blood was thoroughly mixed with 1.1 mL nor- mal saline to make the final red cell suspension ready for the flow cytometric acquisition (about 3.7×104 cells). The final red cell suspension was installed at the sample injection port. After the first region elapsed during acquisition (R1), the tube was removed without ending acquisition, and 0.9 mL deion- ized water was added (about 0.45 % NaCl). The thoroughly mixed tube was then reinstalled at the port, and acquisition was again continued up to the eighth region. One region took about 11 s. The last region (R9) was not used to calculate the residual red cell percentage.
Only regions R7 and R8 were used in the calculation. Forward scatter characteristics (FSCs) and side scatter char- acteristics (SSCs) were acquired on the FACSCalibur flow cytometer (Becton Dickinson, USA). The degree of osmotic hemolysis was expressed as the percentage of residual red
cells divided by the number of red cells initially present and was calculated based on the following formula:
Residual red cell percentage ¼ h Meanevent countof last tworegions=
Eventcountof first region 1:1=2:0ð Þ i 100%
The multiplying factor for correction is 1.1/2.0 as it is assumed that the first region is also in a diluted state for comparison with the remaining regions which are diluted by spiking with 0.9 mL deionized water.
Statistical analysis
SPSS 13.0 was used for statistical analysis. All data were presented as mean±SD. Normally distributed variables were compared pairwise by one-way ANOVA. The sensitivity and specificity of the three tests for HS were calculated; the receiver operating characteristic (ROC) curves were drawn, and the areas under the curve (AUC) were calculated.
Results
Red cell parameters in healthy control group, thalassemia group, and HS group
Compared within groups, MSCVof healthy control group and thalassemia group was greater than MCV (P<0.05); however MSCVof HS group was less than MCV (P<0.05). Compared with healthy control group, all the five parameters of thalas- semia group were less (P<0.05). Red blood cell (RBC), hemoglobin (Hb), and MSCV of HS group were less (P<0.05) than those of healthy control group, while there were no significant differences in MCHC and MCV (P>0.05). MCV and MCHC of HS group were greater than those of thalassemia group (P<0.05), and MSCV was less (P<0.05) (Table 1).
Table 1 Five parameters of erythrocyte of healthy control group, thalassemia group, and HS group. The numbers with the symbol asterisk present P<0.05, vs healthy control group, and the number sign presents P<0.05, vs thalassemia group
Group Healthy control group (n=95) Thalassemia group (n=86) HS group (n=56)
RBC (1012) 4.62±0.41 3.70±1.06* 3.10±0.37*
Hb (g/L) 135.00±11.53 79.54±22.81* 94.96±26.53*#
MCV (fL) 89.36±4.37 71.85±10.73* 81.90±6.12#
MSCV (fL) 93.70±5.46 80.73±11.69* 68.50±7.49*#
MCHC (g/L) 334.02±9.38 304.57±18.60* 348.06±28.12#
748 Ann Hematol (2015) 94:747–751
Evaluation of HS diagnosis method of comparing reticulocyte MSCV to MCV
MSCV was greater than MCV in all subjects of healthy control group. In HS group, MSCV was less than MCV in 50 patients, while in the other 6 patients, MSCV was greater. In thalassemia group, MSCV was greater than MCV in 7 patients. When using clinical diagnosis of HS as the gold standard, MSCV<MCV as the optimum cutoff point, com- paring reticulocyte MSCV to MCV had a sensitivity of 89.28% and a specificity of 96.14% to diagnoseHS (Table 2). Figure 1 shows the ROC curve of comparing MSCV to MCV to diagnose HS, and the AUC is 0.931.
Evaluation of HS diagnosis method of red cell MCHC
MCHC was more than 335 g/L in five subjects of healthy control group. In HS group, MCHCwas more than 335 g/L in nine patients. MCHC was less than 335 g/L in all patients of thalassemia group.When using clinical diagnosis of HS as the gold standard, MCHC >355 g/L as diagnostic criteria, the sensitivity was only 41.07 % and the specificity was 94.47 % to diagnose HS (Table 3). The ROC curve of diag- nosing HS using MCHC is shown in Fig. 2, and the AUC is 0.768.
Evaluation of HS diagnosis method by flow cytometric osmotic fragility test
The percentage of residual red cells in HS group (20.9± 3.65 %) was significantly less than that in healthy control group (48.37±7.62 %) (P<0.05) and thalassemia group (55.12±6.90 %) (P<0.05). The residual red cell percentage of thalassemia group was higher than that of healthy control group (P<0.05). There were 90 cases with the residual red cell percentage of flow cytometric osmotic fragility test above 23.6 % and only 5 cases with that under 23.6 % in healthy control group. In HS group, there were 48 cases with the percentage of residual red cells less than 23.6 % and 8 cases above 23.6 %. In thalassemia group, the residual red cell percentages of all the patients were above 23.6 %. For the clinical diagnosis of HS as the gold standard and the residual
red cell percentage under 23.6 % as the diagnostic threshold, the sensitivity of flow cytometric osmotic fragility test for HS was 85.71 % and the specificity was 97.24 % (Table 4). The ROC curve of flow cytometric osmotic fragility test for HS is shown in Fig. 3, and the AUC is 0.921.
Discussion
It is necessary to determine the red cell parameters for the diagnosis, classification, and treatment of anemia, since ane- mia patients with different pathogeneses have diverse param- eters. Because unique hemorheology characteristics can help to diagnose anemia, red cell parameters were used in the diagnosis and differential diagnosis of anemia since the 1990s in the southern Iran and Japan [12, 13]. Reticulocytes reflect the hematopoiesis of the bonemarrow, so it can be used to classify the anemia. With the hematology analyzer widely used in hospitals, clinicians can analyze reticulocyte parame- ters automatically. In 2010, Broseus et al. [14] observed that the delta (MCV minus MSCV) values >9.6 fL showed sensi- tivity of 100 % and specificity of 90.57 % in discriminating HS from other subjects. In our study, when using MSCV< MCV as the optimum cutoff point, the sensitivity was 89.28 %, specificity was 96.14 %, and the ROC was 0.931. It is basically the same as the first report in China [15]. MCHC is usually used to diagnose anemia combined with MCH and MCV. The age of subjects, the degree of anemia, and other complications might affect the sensitivity. MCHC has been
Table 2 the diagnosis of HS by comparing MSCV to MCV
The diagnosis by comparing MSCV to MCV
Gold standard Total number
Positive results 50 6 56
Negative results 7 174 181
Total number 56 181 237
Fig. 1 The ROC curve of comparing MSCV to MCV to diagnose HS
Ann Hematol (2015) 94:747–751 749
reported as a valuable index in the diagnosis of iron deficiency anemia (IDA) and megaloblastic anemia (MA), but its value for the diagnosis of hemolytic anemia remains to the further studies.
Flow cytometer can rapidly measure the number of corre- lated properties on a cell-by-cell basis. So we used flow cytometer to count the number of red blood cells in the isotonic solution before and after spiking with deionized wa- ter. The residual red cell percentagewas then calculated for the diagnosis of HS. Similar to the study of Prashant Warang et al. [11], the sensitivity of this diagnosis method could reach 85.71 %, and the specificity was 97.24 %. There are several disadvantages in conventional hemolytic anemia tests for the diagnosis of HS. Coombs test is not much specific because patients with autoimmune hemolytic anemia (AIHA), cold agglutinin syndrome, neonatal alloimmune hemolysis, and many other diseases all have positive test results [16]. AGLT50
reduces in HS, AIHA, renal failure, gestation, and others [17–20], so acidified glycerol lysis test (AGLT) is not specific, too. The relatively high sensitivity of the eosin-5′-maleimide (EMA)-binding test for detecting abnormal red blood cells of
varying sizes can be attributed to the use of flow cytometry as a detection system, which analyzes single red cells in a sample [21]. Flow cytometric osmotic fragility test is simple, rapid, and practical means of diagnosing HS than the tradition OF. Red cell membrane protein analysis using sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS- PAGE) is a useful diagnosis method in patients whose diag- nosis was not definite or if the treating physician chose to include this analysis in the diagnostic workup [22]. Neverthe- less, it is not sensitive for mild HS or asymptomatic carriers. A meticulously microscopic examination of the blood smear examination is the first important step for the diagnosis of HS and all the red cell disorders [23]. Spherocyte percentage greater than 10 % or concentrated red cell percentage above 4 % in a peripheral blood smear could be a diagnosis criterion of HS. However, red cells can change into sphere in some autoimmune hemolysis and parasitic protozoa infection he- molytic patients.
There was a case in our study with symptoms of Hb 88.0 g/ L, RET 6.8 %, MSCV<MCV, 2 % spherocytes, and MCHC
Table 3 The diagnosis of HS by MCHC
The diagnosis by MCHC Gold standard Total number
HS group Non-HS group
Fig. 2 The ROC curve of diagnosing HS using MCHC
Table 4 The diagnosis of HS by flow cytometric osmotic fragility test
The diagnosis by flow cytometric osmotic fragility test
Gold standard Total number
Positive results 48 5 53
Negative results 8 176 184
Total number 56 181 237
Fig. 3 The ROC curve of flow cytometric osmotic fragility test for HS
750 Ann Hematol (2015) 94:747–751
increasing indicating HS. The child’s parents also took exam- inations for HS, and the mother had positive results. In the examination of red cell morphology for the child, there were only 2 % spherocytes. It is considered to be an error caused by inspection technology. And there were 19 % spherocytes in the repeat test. It is suggested that it was possible to have been misdiagnosed by red cell morphology test, so the quality of the test must be appreciated. The red cell morphology exam- ination is necessary for the first screening of hemolysis and combined with blood routine examination can prevent the misdiagnosis or missed diagnosis. In conclusion, there has not been a laboratory test that can be used to diagnose HS alone. The diagnosis of HS must be reached through the screening tests combined with clinical symptoms and positive family history.
In our study, we suggest that comparing MSCV to MCV and flow cytometric osmotic fragility test were simple, prac- tical, and accurate laboratory screening methods.
Acknowledgments This study was supported by the National Natural Science Foundation of China (No. 81360263) and the Provincial Natural Science Foundation of Guangxi (2011GXNSFA018290).
Conflict of interest The authors declare that they have no conflict of interest.
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15. Lin FQTJ, Ju Y, Qiu YL (2010) The value of mean spherical corpus- cular volume in diagnosis of hereditary spherocytosis. J Pract Med 26(14):2537–2539
16. Zarandona JM, Yazer MH (2006) The role of the Coombs test in evaluating hemolysis in adults. CMAJ Can Med Assoc J J Assoc Med Can 174(3):305–307
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Ann Hematol (2015) 94:747–751 751
Comparison and evaluation of three screening tests of hereditary spherocytosis in Chinese patients
Abstract
Introduction
Methods
Subjects
Results
Red cell parameters in healthy control group, thalassemia group, and HS group
Evaluation of HS diagnosis method of comparing reticulocyte MSCV to MCV
Evaluation of HS diagnosis method of red cell MCHC
Evaluation of HS diagnosis method by flow cytometric osmotic fragility test
Discussion
References
Yi-feng Tao & Zeng-fu Deng & Lin Liao & Yu-ling Qiu &
Wen-qiang Chen & Fa-quan Lin
Received: 28 July 2014 /Accepted: 27 November 2014 /Published online: 12 December 2014 # Springer-Verlag Berlin Heidelberg 2014
Abstract The objective of this study is to compare and eval- uate 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 collect- ed from 237 participators diagnosed at the First Affiliated Hospital of Guangxi Medical University, including 56 hered- itary 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 specific- ity 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 os- motic fragility test, the sensitivity was 85.71 % and the spec- ificity was 97.24 %. Flow cytometry osmotic fragility test or comparing MSCV to MCV combined with smear examina- tion of peripheral red blood cells morphology can be a simple,
practical, and accurate hereditary spherocytosis (HS) labora- tory screening method.
Keywords Hereditary spherocytosis .Mean corpuscular hemoglobin concentration .Mean sphered corpuscular volume . Flow cytometric . Osmotic fragility
Introduction
Hereditary spherocytosis (HS) is one of hereditary hemolytic diseases caused by red cell membrane disorders, characterized by anemia, jaundice, and splenomegaly [1]. Since the 1980s, HS cases had been reported from all over the country [2–6]. There was no exact incidence survey, but as reported, the incidence of HS was much higher than the clinical diagnosis rate [7]. It is not difficult to diagnose patients with HS if patients have classic symptoms of hemolysis, a family history of HS, and a positive laboratory test [1, 8, 9]. There are several ways to test for HS. Such as acidified glycerol lysis test (AGLT), eosin-5 ′-maleimide (EMA)-binding test, cryohemolysis, and some red blood cell parameters. AGLT and EMA have been thoroughly tested. So far, none single test is perfect. In this study, flow cytometric osmotic fragility test was used to help diagnose HS quickly and accurately.
Methods
Subjects
Fifty-six patients with HS were diagnosed based on their clinical symptoms, biochemistry, complete blood cell count, peripheral blood smear, osmotic fragility (OF) test, and other laboratory examinations at the First Affiliated Hospital of Guangxi Medical University, including 26 males and 30
The authors Yi-feng Tao and Zeng-fu Deng contributed equally to this work.
Y.<f. Tao Affiliated Tumor Hospital of Guangxi Medical University, Nanning, Guangxi, China
Z.<f. Deng : L. Liao : F.<q. Lin (*) Department of Clinical Laboratory, The First Affiliated Hospital of Guangxi Medical University, Nanning, Guangxi, China e-mail: [email protected]
Y.<l. Qiu :W.<q. Chen Department of Pediatric Laboratory, The First Affiliated Hospital of Guangxi Medical University, Nanning, Guangxi, China
Ann Hematol (2015) 94:747–751 DOI 10.1007/s00277-014-2270-2
females, aged 2∼38 years. Eighty-six patients with thalasse- mia were diagnosed by hemoglobin (Hb) electrophoresis and genetic test at the Thalassemia Laboratory of Medical Science Experimental Center, Guangxi Medical University, including 41 males and 45 females, aged 4∼37 years. The healthy control group was composed by 95 volunteers (52 males and 43 females, aged 3∼36 years) who received physical exami- nations at the First Affiliated Hospital of Guangxi Medical University. The subjects of this study had given their informed consent, and the study was approved by the Ethics Committee of Guangxi Medical University.
Tests of HS
Red blood cell (RBC), Hb, mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular he- moglobin concentration (MCHC), and mean spherical corpus- cular volume (MSCV) were determined using the Beckman Coulter GEN·S (Beckmann Coulter Inc., Fullerton, CA) ac- cording to manufacturer’s instructions.
Flow cytometric osmotic fragility test was performed ac- cording to the method described by Won and Suh [10]. The red cell suspension was prepared firstly. The volume of anticoagulated venous blood mixed with 1 mL isotonic saline was calculated by the following formula: blood volume (μl)= 130/red blood cells (1012/L) [11]. For example, if the red blood cell count was 4×1012/L, 32.5 μl (130 divided by 4) blood would be mixed with 1 mL normal saline. And then 10 μl diluted blood was thoroughly mixed with 1.1 mL nor- mal saline to make the final red cell suspension ready for the flow cytometric acquisition (about 3.7×104 cells). The final red cell suspension was installed at the sample injection port. After the first region elapsed during acquisition (R1), the tube was removed without ending acquisition, and 0.9 mL deion- ized water was added (about 0.45 % NaCl). The thoroughly mixed tube was then reinstalled at the port, and acquisition was again continued up to the eighth region. One region took about 11 s. The last region (R9) was not used to calculate the residual red cell percentage.
Only regions R7 and R8 were used in the calculation. Forward scatter characteristics (FSCs) and side scatter char- acteristics (SSCs) were acquired on the FACSCalibur flow cytometer (Becton Dickinson, USA). The degree of osmotic hemolysis was expressed as the percentage of residual red
cells divided by the number of red cells initially present and was calculated based on the following formula:
Residual red cell percentage ¼ h Meanevent countof last tworegions=
Eventcountof first region 1:1=2:0ð Þ i 100%
The multiplying factor for correction is 1.1/2.0 as it is assumed that the first region is also in a diluted state for comparison with the remaining regions which are diluted by spiking with 0.9 mL deionized water.
Statistical analysis
SPSS 13.0 was used for statistical analysis. All data were presented as mean±SD. Normally distributed variables were compared pairwise by one-way ANOVA. The sensitivity and specificity of the three tests for HS were calculated; the receiver operating characteristic (ROC) curves were drawn, and the areas under the curve (AUC) were calculated.
Results
Red cell parameters in healthy control group, thalassemia group, and HS group
Compared within groups, MSCVof healthy control group and thalassemia group was greater than MCV (P<0.05); however MSCVof HS group was less than MCV (P<0.05). Compared with healthy control group, all the five parameters of thalas- semia group were less (P<0.05). Red blood cell (RBC), hemoglobin (Hb), and MSCV of HS group were less (P<0.05) than those of healthy control group, while there were no significant differences in MCHC and MCV (P>0.05). MCV and MCHC of HS group were greater than those of thalassemia group (P<0.05), and MSCV was less (P<0.05) (Table 1).
Table 1 Five parameters of erythrocyte of healthy control group, thalassemia group, and HS group. The numbers with the symbol asterisk present P<0.05, vs healthy control group, and the number sign presents P<0.05, vs thalassemia group
Group Healthy control group (n=95) Thalassemia group (n=86) HS group (n=56)
RBC (1012) 4.62±0.41 3.70±1.06* 3.10±0.37*
Hb (g/L) 135.00±11.53 79.54±22.81* 94.96±26.53*#
MCV (fL) 89.36±4.37 71.85±10.73* 81.90±6.12#
MSCV (fL) 93.70±5.46 80.73±11.69* 68.50±7.49*#
MCHC (g/L) 334.02±9.38 304.57±18.60* 348.06±28.12#
748 Ann Hematol (2015) 94:747–751
Evaluation of HS diagnosis method of comparing reticulocyte MSCV to MCV
MSCV was greater than MCV in all subjects of healthy control group. In HS group, MSCV was less than MCV in 50 patients, while in the other 6 patients, MSCV was greater. In thalassemia group, MSCV was greater than MCV in 7 patients. When using clinical diagnosis of HS as the gold standard, MSCV<MCV as the optimum cutoff point, com- paring reticulocyte MSCV to MCV had a sensitivity of 89.28% and a specificity of 96.14% to diagnoseHS (Table 2). Figure 1 shows the ROC curve of comparing MSCV to MCV to diagnose HS, and the AUC is 0.931.
Evaluation of HS diagnosis method of red cell MCHC
MCHC was more than 335 g/L in five subjects of healthy control group. In HS group, MCHCwas more than 335 g/L in nine patients. MCHC was less than 335 g/L in all patients of thalassemia group.When using clinical diagnosis of HS as the gold standard, MCHC >355 g/L as diagnostic criteria, the sensitivity was only 41.07 % and the specificity was 94.47 % to diagnose HS (Table 3). The ROC curve of diag- nosing HS using MCHC is shown in Fig. 2, and the AUC is 0.768.
Evaluation of HS diagnosis method by flow cytometric osmotic fragility test
The percentage of residual red cells in HS group (20.9± 3.65 %) was significantly less than that in healthy control group (48.37±7.62 %) (P<0.05) and thalassemia group (55.12±6.90 %) (P<0.05). The residual red cell percentage of thalassemia group was higher than that of healthy control group (P<0.05). There were 90 cases with the residual red cell percentage of flow cytometric osmotic fragility test above 23.6 % and only 5 cases with that under 23.6 % in healthy control group. In HS group, there were 48 cases with the percentage of residual red cells less than 23.6 % and 8 cases above 23.6 %. In thalassemia group, the residual red cell percentages of all the patients were above 23.6 %. For the clinical diagnosis of HS as the gold standard and the residual
red cell percentage under 23.6 % as the diagnostic threshold, the sensitivity of flow cytometric osmotic fragility test for HS was 85.71 % and the specificity was 97.24 % (Table 4). The ROC curve of flow cytometric osmotic fragility test for HS is shown in Fig. 3, and the AUC is 0.921.
Discussion
It is necessary to determine the red cell parameters for the diagnosis, classification, and treatment of anemia, since ane- mia patients with different pathogeneses have diverse param- eters. Because unique hemorheology characteristics can help to diagnose anemia, red cell parameters were used in the diagnosis and differential diagnosis of anemia since the 1990s in the southern Iran and Japan [12, 13]. Reticulocytes reflect the hematopoiesis of the bonemarrow, so it can be used to classify the anemia. With the hematology analyzer widely used in hospitals, clinicians can analyze reticulocyte parame- ters automatically. In 2010, Broseus et al. [14] observed that the delta (MCV minus MSCV) values >9.6 fL showed sensi- tivity of 100 % and specificity of 90.57 % in discriminating HS from other subjects. In our study, when using MSCV< MCV as the optimum cutoff point, the sensitivity was 89.28 %, specificity was 96.14 %, and the ROC was 0.931. It is basically the same as the first report in China [15]. MCHC is usually used to diagnose anemia combined with MCH and MCV. The age of subjects, the degree of anemia, and other complications might affect the sensitivity. MCHC has been
Table 2 the diagnosis of HS by comparing MSCV to MCV
The diagnosis by comparing MSCV to MCV
Gold standard Total number
Positive results 50 6 56
Negative results 7 174 181
Total number 56 181 237
Fig. 1 The ROC curve of comparing MSCV to MCV to diagnose HS
Ann Hematol (2015) 94:747–751 749
reported as a valuable index in the diagnosis of iron deficiency anemia (IDA) and megaloblastic anemia (MA), but its value for the diagnosis of hemolytic anemia remains to the further studies.
Flow cytometer can rapidly measure the number of corre- lated properties on a cell-by-cell basis. So we used flow cytometer to count the number of red blood cells in the isotonic solution before and after spiking with deionized wa- ter. The residual red cell percentagewas then calculated for the diagnosis of HS. Similar to the study of Prashant Warang et al. [11], the sensitivity of this diagnosis method could reach 85.71 %, and the specificity was 97.24 %. There are several disadvantages in conventional hemolytic anemia tests for the diagnosis of HS. Coombs test is not much specific because patients with autoimmune hemolytic anemia (AIHA), cold agglutinin syndrome, neonatal alloimmune hemolysis, and many other diseases all have positive test results [16]. AGLT50
reduces in HS, AIHA, renal failure, gestation, and others [17–20], so acidified glycerol lysis test (AGLT) is not specific, too. The relatively high sensitivity of the eosin-5′-maleimide (EMA)-binding test for detecting abnormal red blood cells of
varying sizes can be attributed to the use of flow cytometry as a detection system, which analyzes single red cells in a sample [21]. Flow cytometric osmotic fragility test is simple, rapid, and practical means of diagnosing HS than the tradition OF. Red cell membrane protein analysis using sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS- PAGE) is a useful diagnosis method in patients whose diag- nosis was not definite or if the treating physician chose to include this analysis in the diagnostic workup [22]. Neverthe- less, it is not sensitive for mild HS or asymptomatic carriers. A meticulously microscopic examination of the blood smear examination is the first important step for the diagnosis of HS and all the red cell disorders [23]. Spherocyte percentage greater than 10 % or concentrated red cell percentage above 4 % in a peripheral blood smear could be a diagnosis criterion of HS. However, red cells can change into sphere in some autoimmune hemolysis and parasitic protozoa infection he- molytic patients.
There was a case in our study with symptoms of Hb 88.0 g/ L, RET 6.8 %, MSCV<MCV, 2 % spherocytes, and MCHC
Table 3 The diagnosis of HS by MCHC
The diagnosis by MCHC Gold standard Total number
HS group Non-HS group
Fig. 2 The ROC curve of diagnosing HS using MCHC
Table 4 The diagnosis of HS by flow cytometric osmotic fragility test
The diagnosis by flow cytometric osmotic fragility test
Gold standard Total number
Positive results 48 5 53
Negative results 8 176 184
Total number 56 181 237
Fig. 3 The ROC curve of flow cytometric osmotic fragility test for HS
750 Ann Hematol (2015) 94:747–751
increasing indicating HS. The child’s parents also took exam- inations for HS, and the mother had positive results. In the examination of red cell morphology for the child, there were only 2 % spherocytes. It is considered to be an error caused by inspection technology. And there were 19 % spherocytes in the repeat test. It is suggested that it was possible to have been misdiagnosed by red cell morphology test, so the quality of the test must be appreciated. The red cell morphology exam- ination is necessary for the first screening of hemolysis and combined with blood routine examination can prevent the misdiagnosis or missed diagnosis. In conclusion, there has not been a laboratory test that can be used to diagnose HS alone. The diagnosis of HS must be reached through the screening tests combined with clinical symptoms and positive family history.
In our study, we suggest that comparing MSCV to MCV and flow cytometric osmotic fragility test were simple, prac- tical, and accurate laboratory screening methods.
Acknowledgments This study was supported by the National Natural Science Foundation of China (No. 81360263) and the Provincial Natural Science Foundation of Guangxi (2011GXNSFA018290).
Conflict of interest The authors declare that they have no conflict of interest.
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Ann Hematol (2015) 94:747–751 751
Comparison and evaluation of three screening tests of hereditary spherocytosis in Chinese patients
Abstract
Introduction
Methods
Subjects
Results
Red cell parameters in healthy control group, thalassemia group, and HS group
Evaluation of HS diagnosis method of comparing reticulocyte MSCV to MCV
Evaluation of HS diagnosis method of red cell MCHC
Evaluation of HS diagnosis method by flow cytometric osmotic fragility test
Discussion
References
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