OBJECTIVETo establish a new method for detection of red blood cell osmotic fragility by using flow cytometry.

METHODSThe hypotension salt solution of different concentrations (0.70 ml normal saline+0.3 ml deionized water, 0.60 ml normal saline+0.40 ml deionized water and 0.55 ml normal saline+0.45 ml deionized water) were prepared with normal saline and deionized water, in which the red blood cells were suspended, and the residual red blood cells were detected by flow cytometer.

RESULTSThere was no significant difference in percentage of residual red blood cells between different time points detected by flow cytometer in 3 different hypotonic salt solutions. The percentage of residual red blood cells in B+C+D+E+F+G detected time region was different among 3 NaCl dilution groups. The percentage of residual red blood cells in normal control was lower than that in hemoglobinopathy group. The percentage of residual red blood cells in hereditary spherocytosis (HS) group was obviously lower than that in hemoglobinopathy and normal control groups. The comparison of 3 different dilution concentrations found that the second concentration (0.60 ml normal saline+0.40 ml deionized water) is more suitable to screen HS by FC500 flow cytometer.

CONCLUSIONThe detection of red cell osmotic fragility by using flow cytometry is a simple, rapid, objective and economic way that can be an effective screening method for diagnose the HS.

BACKGROUND: Hereditary spherocytosis (HS) is a chronic hemolytic anemia characterized by microspherocytes in the peripheral blood and increased erythrocyte osmotic fragility (EOF). This study evaluated the cryohemolysis test (CHT); initial hemolysis (IH); immediate and incubated hemolysis percentage in 5.5 g/L NaCl (H5.5); mean corpuscular hemoglobin concentration (MCHC); red blood cell distribution width (RDW); and Hb/MCHC, Hb/RDW, and MCHC/RDW ratios for the diagnosis of HS. METHODS: Data from 13 patients with HS were evaluated at the Instituto de Bioquímica Aplicada and compared with data from 14 unaffected individuals and 11 patients with anemia due to another etiology. Total blood and reticulocyte counts, CHT, and immediate and incubated EOF were performed in all subjects; sensitivity, specificity, efficiency, and Youden index (YI) were calculated. RESULTS: Eight patients with HS had MCHC ≥345 g/L, 10 had RDW ≥14.5%, 12 had IH >5.0 g/L, 11 had immediate H5.5 ≥5%, and 13 had incubated H5.5 ≥50% (the cut-off value to consider HS). The efficiency and YI were: immediate H5.5 (0.94–0.85), incubated H5.5 (0.89–0.82), IH (0.89–0.78), MCHC (0.87–0.62), CHT (0.84–0.54), and Hb/MCHC (0.71–0.56), respectively. The calculated ratios could distinguish subjects with HS from unaffected individuals (P < 0.05), but not those with anemia of another etiology (P>0.05). CONCLUSION: Although the CHT and supplementary hematimetric indexes were useful to differentiate individuals with SH from healthy controls, they cannot distinguish from anemias of other etiology. CHT and MCHC, in addition to EOF, are recommended for diagnosing HS patients because of their low cost and efficiency.
Anemia ; Anemia, Hemolytic ; Diagnosis* ; Erythrocyte Indices ; Erythrocytes* ; Hemolysis ; Humans ; Osmotic Fragility* ; Reticulocyte Count ; Sensitivity and Specificity

We report a case of prolonged extreme reactive thrombocytosis in a post-splenectomy patient with hereditary spherocytosis. A 29-year-old female patient presented with gall stones detected incidentally by abdominal ultrasonography. Her laboratory findings showed hemolytic anemia with spherocytosis on the peripheral blood smear and increased osmotic fragility. She was diagnosed with hereditary spherocytosis and underwent a laparoscopic cholecystectomy and splenectomy. After undergoing surgery, the hemolytic anemia was resolved but thrombocytosis was newly detected. Nineteen months after the splenectomy, the thrombocytosis was still persistent and extremely high. To our knowledge, this is the first report of a prolonged extreme reactive thrombocytosis after a splenectomy in Korea.
Adult ; Anemia, Hemolytic ; Cholecystectomy, Laparoscopic ; Female ; Gallstones ; Humans ; Korea ; Osmotic Fragility ; Spherocytosis, Hereditary ; Splenectomy ; Thrombocytosis

Gilbert syndrome is the most common inherited disorder of bilirubin glucuronidation. It is characterized by intermittent episodes of jaundice in the absence of hepatocellular disease or hemolysis. Hereditary spherocytosis is the most common inherited hemolytic anemia and is characterized by spherical, osmotically fragile erythrocytes that are selectively trapped by the spleen. The patients have variable degrees of anemia, jaundice, and splenomegaly. Hereditary spherocytosis usually leads to mild-to-moderate elevation of serum bilirubin levels. Severe hyperbilirubinemia compared with the degree of hemolysis should be lead to suspicion of additional clinical conditions such as Gilbert syndrome or thalassemia. We present the case of a 12-year-old boy with extreme jaundice and nausea. The diagnosis of hereditary spherocytosis was confirmed by osmotic fragility test results and that of Gilbert syndrome by genetic analysis findings.
Anemia ; Anemia, Hemolytic ; Bilirubin ; Child* ; Diagnosis ; Erythrocytes ; Gilbert Disease* ; Hemolysis ; Humans ; Hyperbilirubinemia ; Jaundice* ; Male ; Nausea ; Osmotic Fragility ; Spleen ; Splenomegaly ; Thalassemia

The effects of amphotericin B, an antifungal antibiotic, on erythrocyte volume and cation permeability were investigated by measuring the hematocrit, cell volume, cation content, fragility and osmotic behavior in rat erythrocytes, in vitro. 1. When erythrocytes were incubated in a Ringer solution containing amphotericin B (5-25 microgram/ml) the hematocrit and the cell volume increased, the effect being proportional to the concentration of the drug and the incubation time period. 2. Amphotericin B increased the Na content and decreased the K content of the erythrocyte. In normal Ringer solution (NaCl-Ringer)containing amphotericin B the magnitude of cellular Na gain was greater than that of K loss. Therefore, the total cellular cation content increased. On the other hand, when cells were incubated in the amphotericin B containing Ringer solution in which NaCl was replaced by Na2SO4 (Na2SO4-Ringer) the magnitude of cellular K loss exceeded that of cellular Na gain. Consequently, the total cellular cation content was reduced. 3. Amphotericin B increased cell volume (hematocrit) when erythrocytes were incubated in the Na2SO4-Ringer solution. 4. The fragility of erythrocytes increased when cells were preincubated in the amphotericin B containing normal Ringer solution, whereas it decreased in tile cells preincubated in the amphotericin B containing Na2SO4-Ringer solution. 5. The cell volume was linearly related to the reciprocal of medium osmolality(200 to 900 mOsm/kg H2O) in both NaCl-and Na2SO4-Ringer solutions, and the linearity was not altered by amphotericin B. The antibiotic did not change the slope of the correlation line (V vs. 1/OSM). It, however, increased the intercept of the line with the ordinate in normal Ringer solution and decreased that in the Na2SO4-Ringer solution. These results indicate that amphctericin B alters the cell volume by changing the permeability of Na and K across the membrane.
Amphotericin B/pharmacology* ; Animal ; Erythrocyte Volume/drug effects* ; Erythrocytes/analysis* ; In Vitro ; Osmotic Fragility/drug effects ; Potassium/blood* ; Rats ; Sodium/blood*

To observe the erythrocyte fragility and morphological changes of erythrocytes caused by roller pump. Ten tests were divided into two groups, Polystan pediatric pump group A (n = 5) and COBE pump group B (n = 5). Ten whole blood samples (each 400 ml) were circulated in the roller pump for 16 h. Erythrocyte fragility and free hemoglobin were measured before pumping and at every 2 hours during pumping. The possible morphological changes of erythrocytes caused by roller pump were observed by scanning electron microscope. The electron microscopic observation was made before pumping and at every 4 hours throughout pumping. Results showed that the erythrocyte fragility of two groups was not increased during a long period of pumping. The number of acanthocytes of two groups was 1.77/1.81% in the samples before pumping and 6.12/7.13, 9.18/8.73, 13.21/12.89, 16.53/17.21% at 4 h, 8 h, 12 h, and 16 h respectively. The free hemoglobin level of two groups was increased linearly during a long duration of pumping and the index of hemolysis of two groups was 0.296 mg/L/h and 0.3993 mg/L/h respectively. The result shows: 1. the erythrocyte fragility was not increased during a long period of pumping; 2. the erythrocyte membrane was injured or broken by roller pump directly; 3. the morphological changes of erythrocytes would be the basis of post operative hemolysis.
Erythrocyte Deformability ; Heart-Lung Machine ; adverse effects ; Hemoglobins ; analysis ; Hemolysis ; Humans ; In Vitro Techniques ; Osmotic Fragility ; Time Factors

This study was purposed to evaluate the effect of trehalose-loading on physiological and biochemistry properties of red blood cell (RBC) membrane. The samples were divided into the control group (RBC without trehalose loading) and the test group (RBC with trehalose loading). Osmotic fragility reaction was used to determine the osmotic fragility change of loaded RBC membrane in NaCl solution of different osmotic concentration. Flow cytometry and deformeter were used to assay the integrality and deformability of the RBC, respectively. The results showed that the NaCl solution osmotic concentrations were 160 mOsm and 121.4 mOsm, respectively when the haemolysis rate was 50% of the control group and the test group. Flow cytometry data demonstrated that incubation of RBC in a hypertonic trehalose solution resulted in a fraction of cells with different complexity that attached to little Annexin V-FITC, and that it could be removed by washing and resuspending the RBC in an iso-osmotic (300 mOsm PBS) medium. The deformability of the loaded RBC descend, the statistical difference was significant between control and test groups (P < 0.01). It is concluded that the membrane physiological and biochemistry stability and membrane integrality of RBC in a hyper osmotic pressure can be retained after trehalose loading.
Blood Preservation ; methods ; Cryopreservation ; methods ; Erythrocyte Membrane ; drug effects ; Erythrocytes ; drug effects ; Humans ; Osmotic Fragility ; drug effects ; Trehalose ; pharmacology

In patients with hemolytic anemia associated with spherocytosis, differential diagnosis has to be made whether the hemolysis is immune-mediated or of non-immune origin. We report a case of hereditary spherocytosis in a 12-yr-old male child, in whom flow-assisted diagnosis was made. In this case, diagnosis was not determined because routine laboratory workups for hereditary spherocytosis yielded discrepant RESULTS: positive osmotic fragility test, positive direct antiglobulin test, and normal result in the red cell membrane protein sodium dodecyl succinimide polyacrylamide gel electrophoresis. However, all flow cytometry-based tests, such as osmotic fragility, direct antiglobulin, and eosin 5-maleimide binding test, yielded results compatible with hereditary spherocytosis. Additionally, in family study, the results of eosin 5-maleimide binding test suggested his disease being hereditary. In cases with diagnostic difficulties, flow cytometry may be used as an alternative tool, which can provide additional information in the differential diagnosis of hemolytic anemia with spherocytosis.
Anemia, Hemolytic/complications/*diagnosis ; Child ; Coombs' Test ; Diagnosis, Differential ; Eosine Yellowish-(YS)/analogs & derivatives/chemistry ; Erythrocytes/immunology/metabolism ; Flow Cytometry ; Humans ; Male ; Osmotic Fragility ; Spherocytosis, Hereditary/complications/*diagnosis

BACKGROUND: Hereditary spherocytosis(HS) is a clinically and biochemically very heterogeneous disorder The purpose of this study is to detect erythrocyte membrane protein abnormalities by SDS-PAGE and to investigate the frequency of erythrocyte membrane protein defects in hereditary spherocytosis and correlation between some of the hereditary spherocytosis biochemical subsets and the selected clinical phenotype. METHODS: We evaluated the clinical and laboratory characteristics of 14 normal healthy persons and 23 hereditary spherocytosis patients and 8 their family members. The patients were divided into three groups based on clinical and hematological severity(mild, typical, severe). In addition to routine hematologic determlnatlons, osmotic fragility and autohemolysis, RBC membrane protein analysis were performed in all patients by densitometric tracing of SDS-PAGE(sodium dodecyl sulphate polyacrylamide gel electrophoresis) stained by Coomassle blue utilizing both the discontinuous buffer system of Laemmli with acrylamide linear gradient from 4% to 12% and the continuous buffer system of Fairbank with exponential gradient of acrylamide from 3.5% to 17%. RESULTS: 1) The patients could be seperated into three classes of different clinical severity as mild(3 cases), moderate(16 cases) and severe(4 cases) on the clinical feature. 2) Eighteen patients(82.6%) among 23 hereditary spherocytosis revealed abnormal erythrocyte membrane protein and we detected six patients(26.1%) with spectrin deficiency combined with ankyrin reduction, 4 patients(17.4%) with ankyrin deficiency, 4 patients(17.4%) with isolated spectrin deficiency and 3 patients(13.0%) with band 3 deficiency. Five HS patients(21.7%) showed normal RBC membrane protein. 3) Eight HS and their family members showed same RBC membrane protein deficiency. 4) The type and degree of RBC membrane protein reduction were variale with spectrin at 66~94%, with ankyrin at 48~82% of normal levels. These showed that each patient had different clinical severities according to different RBC membrane protein levels and type. CONCLUSION: RBC membrane protein abnormalities were observed in 82.6% of HS patients. The combined spectrin and ankyrin deficiency is the most common molecular defect in HS. The clinical severity and biochemical expression is heterogeneous. SDS-PAGE analysis of RBC membrane protein was provided the diagnosis of RBC membrane defects and basic molecular studies. We believed that the early identification of the biochemical defect responsible for HS is important because it is helpful starling point for the identification of the primary molecular defect, and it could help to anticipate the clinical outcome of the disease. For these reasons, we consider the SDS-PAGE of the red cell membrane to be of crucial importance for a complete evaluation of children with HS. Further studies with more cases would be to clarify the correlation between clinical and biochemical phenotypes.
Acrylamide ; Ankyrins ; Cell Membrane ; Child ; Diagnosis ; Electrophoresis, Polyacrylamide Gel* ; Erythrocyte Membrane* ; Erythrocytes* ; Erythrocytes, Abnormal ; Humans ; Membrane Proteins ; Membranes ; Osmotic Fragility ; Phenotype ; Population Characteristics* ; Spectrin ; Starlings

Among the erythrocyte membrane defects, hereditary spherocytosis is the most common. The erythrocyte membrane defect results from a deficiency of spectrin, the most important structural protein in red cell. Hereditary spherocytosis often presents with hemolytic anemia, jaundice, moderate splenomegaly. Diagnosis is established by the presence of spherocytes in the peripheral blood, reticulocytosis, an increased osmotic fragility, and a negative Coombs test. In children, splenectomy is usually performed after age 6 years but can be done at a younger age if warranted by the severity of the anemia and the need for frequent transfusions. In the period December 1987 to Agust 1993, 9 patients with hereditrary spherocytosis underwent splenectomy and the following results were obtained. 1. Nine patients were comprised of five males and four females. 2. Five patients(55.6%) had been admitted to our hospital during age 6-10 years. 3. Four of the nine patients had autosomal dominant inheritance with variable expression. The other five patients had no known inheritance. 4. The diagnosis of the spherocytosis was based on the increased osmotic fragility and increased autohemolysis of the erythrocytes, as well as on the appearance of spherocytes in the peripheral blood smear. 5. In all cases splenectomy was performed. Two patients had concomitant gall stones and choledocholithiasis, respectively. One patient with concomitant gall stones underwent simultaneous cholecystectomy and splenectomy. The other patients associated with choledocholithiasis underwent splenectomy, cholecystectomy, choledocholithotomy, and T-tube drainage. 6. Complete hematologic recovery was obtained by the splenectomy in all cases. 7. Postoperative complication was not occurred.
Anemia ; Anemia, Hemolytic ; Child ; Cholecystectomy ; Choledocholithiasis ; Coombs Test ; Diagnosis ; Drainage ; Erythrocyte Membrane ; Erythrocytes ; Female ; Gallstones ; Humans ; Jaundice ; Male ; Osmotic Fragility ; Postoperative Complications ; Reticulocytosis ; Spectrin ; Spherocytes ; Splenectomy* ; Splenomegaly ; Wills