Lin Liao

Three cases of congenital dysfibrinogenemia in unrelated Chinese families: heterozygous missense mutation in fibrinogen alpha chain Argl6His.

AbstractCongenital dysfibrinogenemia (CD) is a qualitative fibrinogen disorder caused by an abnormal fibrinogen molecule structure, leading to dysfunctional blood coagulation. This study describes 3 cases of dysfibrinogenemia identified in the unrelated Chinese pedigrees.Routine coagulation screening tests were performed on the probands and their families. The antigens and functionality of fibrinogen was measured using an immunoturbidimetry assay and the Clauss method, respectively. To identify the genetic mutation responsible for these dysfibrinogens, genomic DNA extracted from the blood was analyzed using PCR amplification and direct sequencing. The presence of the mutant chains was determined using matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) mass spectroscopy. Purified plasma fibrinogen of 3 probands was analyzed using SDS–PAGE, fibrinogen clottability, fibrin polymerization, fibrinopeptide release, and scanning electron microscopy (SEM).The 3 probands had a long thrombin time. Levels of functional fibrinogen were found to be very low, while the fibrinogen antigen was within the normal range. DNA sequencing revealed a heterozygous Arg16His substitution in the fibrinogen A&agr; chain (FGA). The mutant chains were found to be expressed using MALDI-TOF mass spectroscopy. SDS–PAGE did not reveal any difference in the molecular weights of 3 polypeptide chains between normal and abnormal fibrinogens. Fibrinogen clottability showed a slower fibrin clot formation than the healthy control. Fibrin polymerization, after addition of thrombin, showed a prolonged lag phase and decreased final turbidity. The kinetics of fibrinopeptides release revealed a decreased amount of the released fibrinopeptide A. SEM of the patients fibrin clot was found to be abnormal.Results indicate that the 3 probands with dysfibrinogenemia were caused by mutations of A&agr; chain Arg16His. Mutation of this fibrinogen induced dysfunction of plasma fibrinogen.

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

Abstract Objectives To determine whether the values of mean cell volume (MCV) and mean sphered cell volume (MSCV) can distinguish hereditary spherocytosis (HS) from thalassemia. Methods The 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. Results The 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. Conclusion Measurements of the MCV higher than the MSCV can be considered an ideal index to distinguish rapidly HS from thalassemia.

Evaluation of a Flow-Cytometric Osmotic Fragility Test for Hereditary Spherocytosis in Chinese Patients.

Background: Osmotic fragility testing based on flow cytometry was recently introduced for the screening of hereditary spherocytosis (HS). This study was undertaken to evaluate the clinical diagnostic value of a flow-cytometric osmotic fragility test for HS. Methods: Peripheral blood was collected from 237 subjects at the First Affiliated Hospital of Guangxi Medical University, including 56 HS patients, 86 thalassemia patients and 95 healthy controls. The samples were examined by flow-cytometric osmotic fragility test and the percentage of residual red blood cells was used to determine HS. Peripheral blood smears were performed to examine the red blood cell morphology. Results: With clinical diagnosis of HS as the gold standard and the percentage of residual red blood cells <23.6% as the diagnostic threshold in the flow-cytometric osmotic fragility test, the sensitivity of the flow-cytometric osmotic fragility test for HS was 85.71% and the specificity was 97.24%. Conclusion: The flow-cytometric osmotic fragility test combined with a red blood cell morphology test by peripheral blood smear could be a simple, practical and accurate laboratory screening method for HS.

Misdiagnosis of two cases of hereditary spherocytosis in a family and review of published reports.

BACKGROUND Hereditary spherocytosis is usually diagnosed based on a combination of clinical and family histories, physical examination, and laboratory data. Milder or atypical cases can be difficult to diagnose. METHODS We report one case each of mild and moderate hereditary spherocytosis whose diagnoses were made by the First Affiliated Hospital, Guangxi Medical University in China. RESULTS In case 1, laboratory test results were as follows: hemoglobin 81.40 g/l, mean corpuscular hemoglobin concentration 339.00 g/l, mean corpuscular volume 82.88 fl, reticulocyte count 19%, mean sphered corpuscular volume 71.66 fl, total bilirubin 57.60 μmol/l, and blood smears showed increased spherocytosis. In case 2, laboratory test results were as follows: hemoglobin 133.10 g/l, mean corpuscular hemoglobin concentration 358.60 g/l, mean corpuscular volume 96.75 fl, reticulocyte count 17%, mean sphered corpuscular volume 77.78 fl, total bilirubin 62.50 μmol/l, and blood smears showed increased spherocytosis. CONCLUSIONS Mild and moderate hereditary spherocytosis can be easily misdiagnosed. Assessment of total, direct, and indirect serum bilirubin, erythrocyte morphology and red cell characteristics (particularly mean corpuscular volume and mean sphered corpuscular volume) clearly distinguishes hereditary spherocytosis from autoimmune hemolytic anemia, glucose-6-phosphate dehydrogenase deficiency, thalassemia, and autoimmune hepatitis.

Combined use of Clauss and prothrombin time-derived methods for determining fibrinogen concentrations: Screening for congenital dysfibrinogenemia

In this study, the significance of fibrinogen concentration assessed by a combination of Clauss and prothrombin time (PT)‐derived methods for screening for congenital dysfibrinogenemia were investigated, and the screening efficiency of fibrinogen PT‐derived/Clauss ratio on congenital dysfibrinogenemia was analyzed.

Mean reticulocyte volume: a specific parameter to screen for hereditary spherocytosis.

This study assessed the value of mean reticulocyte volume (MRV) for differential diagnosis of hereditary spherocytosis (HS) so as to develop conventional and new specific screen indexes. Subjects in this study were divided into three groups: 53 cases in HS group, 217 cases in hemolytic anemia control group (109 cases of thalassemia (THAL), 56 cases of glucose‐6‐phosphate dehydrogenase G6PD deficiency anemia, and 52 cases of autoimmune hemolytic anemia (AIHA)), and 100 cases in healthy control group. We analyzed erythrocyte and reticulocyte parameters including MRV, mean sphered corpuscular volume, mean corpuscular hemoglobin concentration, and immature reticulocyte fraction. Results demonstrated that MRV was significantly lower in the HS group but significantly higher in the AIHA and G6PD deficiency anemia groups than that in the healthy control group (P = 0.000). MRV was not significantly different between the AIHA and G6PD deficiency anemia groups (P = 0.977) and between the healthy control and THAL groups (P = 0.168). The area under the ROC curve of MRV for diagnosis of HS was 0.942, with a standard error of 0.019, 95% confidence interval of 0.905–0.979, and optimal critical diagnosis point of 95.77 fL. When the MRV was ≤95.77 fL, the sensitivity and specificity for diagnosis of HS were 86.80% and 91.20%, respectively. Therefore, MRV is a general and specific new index for screening HS and important for differential diagnosis of different types of hemolytic anemia.

Novel compound heterozygous SPTA1 mutations in a patient with hereditary elliptocytosis

Hereditaryelliptocytosis (HE) is a hereditary hemolytic disease, characterized by the presence of many elliptical erythrocytes in the peripheral blood that is caused by abnormal cytoskeletal proteins in the erythrocyte membrane. In the present study, a novel, causal HE mutation was reported. Routine blood examinations were performed on the proband and their family, and the fluorescence intensity of eosin-5-maleimide (EMA)-labeled erythrocytes was determined via flow cytometry. Subsequently, DNA was extracted from the peripheral blood of the proband and their family members, and amplified by quantitative polymerase chain reaction. The Sanger sequencing approach was used to determine and identify gene mutations, which were verified by matrix-assisted laser desorption-ionization time of flight (MALDI-TOF) mass spectrometry. To exclude genetic polymorphisms, newly identified mutations were subjected to large-scale gene screening using high-resolution melt analysis. Protein expression levels in the erythrocyte membrane of the proband were determined via SDS-PAGE, which demonstrated that, compared with healthy controls, the proband exhibited a reduction in EMA-labeled erythrocytes. In addition, DNA analysis demonstrated that the proband carried three mutations in the spectrin α chain erythrocytic 1 (SPTA1) gene: c.161A>C, c.5572C>G and 6531-12C>T. The corresponding mutant polypeptides were also analyzed by MALDI-TOF mass spectroscopy. SDS-PAGE analysis indicated that the proband exhibited normal levels of erythrocyte membrane proteins. In the present study, a novel HE case with a His54Pro mutation in the SPTA1 gene was reported. The results suggested that the His54Pro mutation influenced the role of erythrocyte membrane proteins without reducing its level of expression.

Abnormal fibrinogen with an Aα 16Arg → Cys substitution is associated with multiple cerebral infarctions

We found a heterozygous dysfibrinogenemia caused by a substitution of AαArg16Cys. The proband suffered multiple cerebral infarctions. Routine coagulation tests revealed a prolonged thrombin time. The fibrinogen levels in the functional assays were considerably lower than the levels in the immunological assays. The polymerization of the purified fibrinogen was strongly impaired in the presence of calcium. As previously observed in other heterozygous Aα R16C variants, the release rate and amount of fibrinopeptide A (FPA) were lower in the proband than those in normal controls. Additionally, the release of fibrinopeptide B (FpB) was delayed. The immunoblotting analysis using antibodies against human serum albumin indicated that albumin is bound to Aα R16C. The mass spectrometry analysis showed that the Aα R16C fibrinogen chains appeared in the patient’s circulation. The clot structure analysis using scanning electron microscopy (SEM) revealed that the fibrin network was dense and consisted of thin and highly branched fibres. Using overlaid fibrinolytic enzymes in a clot lysis experiment, clot degradation was observed to be delayed. These results indicated that the thrombotic tendency may be ascribed to a fibrinolytic resistance caused by an abnormal clot structure with thin fibres and fibrinogen–albumin complexes.

Application of albumin/globulin ratio in elderly patients with acute exacerbation of chronic obstructive pulmonary disease

Background Acute exacerbation of chronic obstructive pulmonary disease (AECOPD) has become an important disease of hospitalized elderly patients, which lack simple and inexpensive indicators for evaluating the condition and prognosis. This study was performed to investigate the clinical significance of the serum albumin/globulin ratio (AGR) in elderly patients with AECOPD. Methods The data of 252 hospitalized elderly patients with AECOPD, 89 stable COPD patients and 115 elderly healthy individuals were analyzed and compared. The differences in the AGR, logarithm of the serum C-reactive protein (LogCRP) level, prealbumin (PA) level, and immunoglobulin G (IgG) level were compared. AECOPD patients were grouped using the optimal cutoff values of each index to compare the difference in the combined infection rate. The correlation between hospital stays and AGR was analyzed. Results The AGR, LogCRP, PA level, and IgG level were different among the AECOPD group, stable COPD group and healthy control groups (P<0.05). The AGR, LogCRP, and PA level were different (P<0.05) among the Global Initiative for Chronic Obstructive Lung Disease (GOLD) I, II, II, and IV groups. Age, AGR, LogCRP, and PA level were different (P<0.05) between the infection and non-infection groups. After grouping according to the optimal cutoff values, the combined infection rate was different (P<0.05). The AGR was negatively correlated with the hospital stay (r=-0.583, P<0.001). The hospital stay was longer in patients with an AGR of <1.37 than ≥1.37 (P<0.001). Conclusions The AGR can be regarded as a reference index for evaluating the condition of elderly patients with AECOPD, determining the presence of combined infection, and predicting the prognosis.

Single and combined use of red cell distribution width, mean platelet volume, and cancer antigen 125 for differential diagnosis of ovarian cancer and benign ovarian tumors

BackgroundCancer is widely believed to result from chronic inflammation, and red cell distribution width (RDW) and mean platelet volume (MPV) are considered as inflammatory markers for cancer. We investigated the values of RDW, MPV, and cancer antigen 125 (CA125), alone or in combination, for distinguishing between ovarian cancer and benign ovarian tumors.MethodsThe study included 326 patients with ovarian cancer, 290 patients with benign ovarian tumors, and 162 control subjects. Hematologic tests were performed at initial diagnosis.ResultsRDW was increased and MPV was decreased in the ovarian cancer group compared with the control and benign ovarian tumor groups. RDW was positively correlated and MPV was negatively correlated with cancer stage. Area under the curve (AUC) analysis for ovarian cancer versus benign ovarian tumors revealed that the specificity and sensitivity were increased for the combination of MPV and CA125 compared with either marker alone, and the specificity was increased for the combination of RDW and CA125, compared with either alone. The AUCs for RDW plus CA125 and MPV plus CA125 were significantly larger than for any of the markers alone.ConclusionsIn conclusion, combinations of the markers RDW, MPV, and CA125 may improve the differential diagnosis of ovarian cancer and benign ovarian tumors.