Red cell osmotic fragility studies in hemoglobin C‐β thalassemia: osmotically resistant microspherocytes

Typically certain features of red cell morphology predict the results of osmotic fragility testing. Microspherocytes generally have increased and target cells decreased fragility. Blood smears in homozygous hemoglobin C disease show an interesting admixture of microspherocytes and target cells. Yet osmotic fragility studies generally show only reduced fragility and no population of fragile cells to correspond with the spherocytes. The present study demonstrates that the red cells of patients with hemoglobin C‐β thalassemia share many characteristics with hemoglobin C red cells, including the decreased osmotic fragility of all cells despite the presence of both spherocytes and target cells. These paradoxically osmotically resistant spherocytes probably arise because of cellular dehydration due to a K‐Cl transport system which may be activated by binding of hemoglobin C to the red cell membrane.     

联合试验在地中海贫血诊断中的应用

目的 :探讨红细胞平均体积 (MCV)、红细胞脆性及血红蛋白 (Hb)电泳联合试验在地中海贫血 (简称地贫 )中的诊断价值。方法 :用日本SysmexKX 2 1自动血细胞分析仪、中山医科大学红细胞脆性一管定量法、美国Helena公司KEP型全自动电泳分析系统 ,分别对本实验室经分子生物学技术确诊的 14 8例地贫及 81例非地贫样品进行MCV、红细胞脆性及Hb电泳测定 ,并对结果作相关统计学分析。结果 :MCV、红细胞脆性、Hb电泳单项试验对地中海贫血诊断的灵敏度及特异度分别为 91.9%、81.1%、83.8%及 79%、91.4 %、90 .1%。MCV与Hb电泳、红细胞脆性与Hb电泳二项试验平行联合的灵敏度及特异度分别为 98.6 %、96 .6 %及 71.6 %、82 .7% ;系列联合的灵敏度及特异度分别为 77%、6 8.2 %及 97.5 %、98.8%。MCV、红细胞脆性、Hb电泳三项试验平行联合的灵敏度、特异度为 10 0 %、6 5 .4 % ;系列联合的灵敏度及特异度为 6 2 .8%、10 0 %。经u检验 ,平行联合试验的灵敏度与各单项试验灵敏度之间、系列联合试验的特异度与各单项试验特异度之间差异有统计学意义 (P <0 .0 5 )。结论 :在地贫的诊断试验中 ,MCV、红细胞脆性、Hb电泳平行联合试验可提高灵敏度 ,系列联合试验可提高特异度 ,联合试验较单项试验有一定的优越性     

Comparison of acidified glycerol lysis test, Pink test and osmotic fragility test in hereditary spherocytosis: effect of incubation

In this study we compared the results of the acidified glycerol lysis test, the Pink test and the osmotic fragility test in 38 patients with hereditary spherocytosis and in healthy controls. The sensitivity of the acidified glycerol lysis test was 81.6% when performed within 3 h after blood collection. After incubating for 24 h, the sensitivity increased to 100% whereas the specificity remained maximal. Similar incubation did not improve the diagnostic utility of the Pink test. All patients, but none of the controls, showed a positive osmotic fragility test. It is concluded, because of sensitivity and specificity in this study, that the acidified glycerol lysis test after incubation and the osmotic fragility test are superior to the Pink test in detecting spherocytosis.     

Continuous flow method for determination of erythrocyte osmotic fragility

A simple and accurate micromethod for the determination of erythrocyte osmotic fragility is introduced. The method uses a laminar parabolic flow pattern, together with gravity, to retain cells in a long, small-diameter tube while a solution with decreasing osmolarity is passed through the tube. As the cells hemolyze, hemoglobin released from the cells is quickly removed by the axial flow pattern and monitored with a 547 nm optical detector for recording the hemolysis curve. Consequently, a continuous curve is obtained, with a peak occurring at the salt concentration that produces the maximum hemolysis rate. The advantages of this method are simplicity, accuracy, and small sample size (2 microliters of whole blood). The small sample size is of particular importance for infants. A comparison is made with the Parpart method using samples from 18 normal adults. Results are also given for a few abnormal adults and for a series of 26 normal newborns.     

Coexistence of Southeast Asian ovalocytosis and beta-thalassemia: a molecular and hematological analysis

We describe hematological and molecular characterization of a Thai female who had Southeast Asian ovalocytosis (SAO) associated with beta+-thalassemia trait. The proband had mild microcytosis with Hb 12.9 g/dl, Hct 35.8%, MCV 74.4 fl, MCH 26.8 pg, MCHC 36.0 g/dl, and elevated Hb A2 (5.6%), characteristics of beta-thalassemia trait. Peripheral blood film examination revealed prominent ovalocytosis. However, a one-tube osmotic fragility (OF) test commonly used for thalassemia screening was negative and a normal OF curve was observed. Further polymerase chain reaction (PCR) analyses identified the beta(-28A-G) mutation in the beta-globin gene and a 27 bp deletion in erythrocyte band 3 protein gene, indicating a genetically compound heterozygote. Hematological data of the proband was comparatively presented with those of eight female and 15 male carriers of pure beta-thalassemia with the same mutation. The finding demonstrates that although the association of the SAO and beta-thalassemia does not produce a more severe clinical picture, this could lead to a mis-screening of beta-thalassemia using an OF test as a primary screening test. Additional blood film examination followed by PCR could help in the detection of this unusual genetic interaction in the region.     

Modified osmotic fragility test for the laboratory diagnosis of hereditary spherocytosis

A modified osmotic fragility test, based on measurement of hemolysis in four hypotonic NaCl solutions and logarithmic linearization of osmotic fragility curve is, like the "Pink test," a specific and sensitive test for the laboratory diagnosis of hereditary spherocytosis.     

Validation of osmotic fragility test and dichlorophenol indophenol precipitation test for screening of thalassemia and Hb E

The strategy for screening of thalassemia and Hb E by a combination of osmotic fragility (OF) test and dichlorophenol indophenol precipitation (DCIP) test was validated with 436 unrelated Thai subjects. Hemoglobin (Hb) typing, Hb A2 quantitation, PCR and DNA sequence analysis were used as confirmatory methods for diagnosis of thalassemia and hemoglobinopathy. The sensitivity and specificity of this strategy was 100% and 79.7%, respectively. The results assessed by two medical scientists were exactly the same with 93.3% accuracy in comparison with the confirmatory methods. A combination of OF and DCIP has been shown to be a reliable, rapid, simple and sensitive strategy for screening thalassemia and Hb E in the Thai population.     

Cryohemolysis for the detection of hereditary spherocytosis: Correlation studies with osmotic fragility and autohemolysis

Laboratory methods aimed to assess the presence of spheroidal cells such as osmotic fragility, autohemolysis, and glycerol lysis time are very elaborate, time consuming, and often give inconclusive results. We have developed a diagnostic test based on a unique sensitivity of HS cells to hypertonic cryohemolysis and analyzed blood samples of 55 HS patients. The patients were divided into two subgroups, clinically affected probands and their relatives. To get quantitative comparisons with the classic methods, the cryohemolysis results were compared to two parameters of the osmotic fragility test: the salt concentration that causes 50% hemolysis, and the percent lysis at a constant salt concentration. Autohemolysis results were also compared. To evaluate which of these tests has the best analytical power, we calculated the mean results and 2 SDs of each parameter in a control group, and then looked to see which of them was best in identifying the patients. The cryohemolysis test was the single parameter that identified all cases including asymptomatic carriers of the disease. The ability of this test to identify the less severe cases probably reflects the dependency of the cryohemolysis on factors that are more related to the primary membrane molecular defects and less by the surface area to volume ratio. Am. J. Hematol. 58:206–212, 1998. © 1998 Wiley-Liss, Inc.     

Prevalence of increased osmotic fragility of erythrocytes in German blood donors: Screening using a modified glycerol lysis test

Summary We screened for increased osmotic fragility of erythrocytes in 1464 healthy German blood donors. The osmotic fragility was determined by an acidified glycerol lysis test (AGLT) using glycerol-sodium phosphate-buffered NaCl solution. Since the original test described by Zanella et al. [23] showed only low specificity for hereditary spherocytosis, we used a modification with 0.0093M sodium phosphate-buffered glycerol-saline solution, pH 6.90, instead of the original 0.0053M sodium phosphate buffer, pH 6.85. Sixteen of the donors (1.1%) had a pathologic result, similar to that of 32 patients with hereditary spherocytosis: AGLT 50 <5 min (half-time of AGLT, defining normal and pathologic results). The osmotic fragility of the erythrocytes from 12 of these donors was further investigated using the conventional test with hypotonic NaCl solutions. With one exception, increased osmotic fragility was verified in all of them by both tests. Further hematologic data showed a mild reticulocytosis (2% and 2.6%) in two of the donors. One donor had a moderate reticulocytosis of 6.5%, probably due to a mild, previously undiagnosed spherocytosis; 99 of the donors had an intermediate result (AGLT 50: 5–30 min). Hypotonic lysis of their erythrocytes by the conventional method showed a normal result; there were no signs of increased hemolysis. Thus they are not definitely regarded as having increased osmotic fragility of their erythrocytes. Erythrocyte osmotic fragility shows a wide distribution range in the normal population and might be normally distributed. Thus the blood donors with pathologic AGLT (<5 min) probably represent only one end of a continuum of salt-dependent hemolysis, and not a separate entity. However, they did show additional minor signs of a functional defect of the erythrocyte membrane and therefore could be carriers of a spherocytosis trait. The frequency of carriers of an erythrocyte membrane defect (possible spherocytosis trait) could be as high as 1.1% in the general population and would distinctly exceed the prevalence of patients with apparent spherocytosis (0.02%).     

Molecular genetic confirmatory testing from newborn screening samples for the common African-American, Asian Indian, Southeast Asian, and Chinese beta-thalassemia mutations

beta-Thalassemia is a serious health problem in the United States, especially in California, due to increased Asian immigration. Neonatal screening by using high-performance liquid chromatography (HPLC) or isoelectric focusing (IEF) may lead to confusion due to interactions of various hemoglobinopathies with beta-thalassemia. Our purpose was to develop single-tube multiplexed PCR assays using original neonatal screening specimens to identify the mutations responsible for beta-thalassemia in order to expedite diagnostic confirmation. Primers were designed for two to six common ethnic-specific mutations using the amplification refractory mutation system (ARMS). This multiplex ARMS approach was standardized using DNA samples with known mutations for beta-thalassemia in those of Asian (Southeast Asian, Chinese, and Asian Indian) and African-American descent. Specimens from African-American neonates were tested for two mutations (-88 and -29); Asian Indians for five mutations (IVSI-1, IVSI-5, codons (Cd) 41/42, Cd 8/9, and 619-bp deletion); Chinese, Taiwanese, and Southeast Asians for seven mutations (Cd 41/42, Cd 17, -28, IVSII-654, Cd 71/72, IVSI-5, and IVSI-1). We identified each of these beta-thalassemia mutations in multiplexed ARMS from positive control samples. We tested 25 anonymized dried blood specimens from neonates who had been diagnosed with beta-thalassemia and who also belonged to these ethnic groups. We detected a mutation specific to the neonate's ethnic group using the ARMS approach in nearly all specimens, and the results were confirmed by sequencing. Multiplexed ARMS for ethnic-specific beta-thalassemia mutations from the original newborn screening dried blood specimens is a rapid and efficient approach for diagnostic confirmation.     

Bias-corrected diagnostic performance of the naked-eye single-tube red-cell osmotic fragility test (NESTROFT): an effective screening tool for beta-thalassemia

It is being increasingly recognized that a majority of the countries in the thalassemia-belt need a cost-effective screening program as the first step towards control of thalassemia. Although the naked eye single tube red cell osmotic fragility test (NESTROFT) has been considered to be a very effective screening tool for beta-thalassemia trait, assessment of its diagnostic performance has been affected with the reference test- and verification-bias. Here, we set out to provide estimates of sensitivity and specificity of NESTROFT corrected for these potential biases. We conducted a cross-sectional diagnostic test evaluation study using data from 1563 subjects from Central India with a high prevalence of beta-thalassemia. We used latent class modelling after ensuring its validity to account for the reference test bias and global sensitivity analysis to control the verification bias. We also compared the results of latent class modelling with those of five discriminant indexes. We observed that across a range of cut-offs for the mean corpuscular volume (MCV) and the hemoglobin A2 (HbA2) concentration the average sensitivity and specificity of NESTROFT obtained from latent class modelling was 99.8 and 83.7%, respectively. These estimates were comparable to those characterizing the diagnostic performance of HbA2, which is considered by many as the reference test to detect beta-thalassemia. After correction for the verification bias these estimates were 93.4 and 97.2%, respectively. Combined with the inexpensive and quick disposition of NESTROFT, these results strongly support its candidature as a screening tool-especially in the resource-poor and high-prevalence settings.     

HIV抗体筛查试验阳性-确证试验阴性标本的分析

目的了解HIV抗体筛查试验假阳性现象的特点及引起假阳性反应的原因。方法对2004～2009年本实验室常规监测检测中筛查试验阳性-确证试验阴性标本的相关资料进行分析。结果394例筛查试验阳性-确证试验阴性标本主要来源于没有或甚少高危行为的各类普通人群;采用"明胶颗粒凝集试剂(PA)+ELISA"或双ELISA试剂组合检测,83%以上为一阴一阳结果,近70%的标本ELISAS/CO值在1～4之间;采用"吉比爱ELISA+第3代梅里埃ELISA"或"吉比爱ELISA+第4代梅里埃ELISA"组合检测,S/CO值同处于1～6范围的标本分别占78.95%和64.0%。结论HIV抗体筛查检测假阳性存在主、客观原因;实验室应采取应对措施最大限度地保证结果的准确以及对检测结果进行正确的解释。     

The glycohaemoglobin assay as a screening test for diabetes mellitus: the Islington Diabetes Survey

Blood glucose 2 h after an oral glucose load (2hBG) and glycohaemoglobin (GHb) (Corning agar-gel electrophoresis) levels were used as screening tests in a general practice diabetic screening programme. The diagnosis of diabetes (DM) was based on a separate oral glucose tolerance test (OGTT) in 223 of 1040 screened subjects, selected as a stratified sample biased towards higher levels of 2hBG and GHb. The GHb assay was also repeated at the recall examination and urine was tested for glycosuria before and after glucose administration. At a cut-off level of 8.1%, the screening GHb assay correctly identified 90% of all probable diabetics with a specificity of 85.3% (95% Cl 83.3-87.3%) and a positive predictive value of 14.0% (9.0-19.0%). The specificity of the screening GHb assay as a screening test for true DM was 45.8% (39.0-52.4%) at 90% sensitivity, and that of the recall GHb assay was 64.5% (57.9-71.1%). The screening 2hBG was 93.3% (88.9-97.7%) specific at 90% sensitivity as a screening test for true DM diagnosed by OGTT at recall. The test characteristics for fasting glycosuria were: sensitivity 16.7% (0-37.8%) and specificity 98.0% (96.0-100.0%). Equivalent values for the post-glucose test for glycosuria were: 72.7% (46.4-99.0%) and 77.4% (70.1-84.7%), respectively. While GHb assay is a poorer screening test for DM than the 2hBG at the single cut-off level quoted, comparison of the accuracy of the two tests shows that the GHb assay is only marginally less accurate. It is superior to testing for glycosuria as a screening test for DM and can be performed on any random blood sample, facilitating its use in population screening.     

Clinical usefulness of carbohydrate antigen 19-9 as a screening test for pancreatic cancer in an asymptomatic population

BACKGROUND AND AIM: Although the prognosis for pancreatic cancer is generally poor, it is well known that the survival rate for resected pancreatic cancer is much higher than that for more conservative treatment. The importance of early detection is emphasized for resection of pancreatic cancer. Measurement of serum carbohydrate antigen (CA) 19-9 has shown satisfactory sensitivity and predictive value in symptomatic patients, but no available data has been found on healthy asymptomatic subjects. Thus, the authors aimed to determine the clinical usefulness of CA 19-9 as a screening tool for pancreatic cancer in asymptomatic subjects. METHODS: From December 1994 to November 2000, 70 940 asymptomatic persons visiting the Health Promotion Center at the Samsung Medical Center, Seoul, Korea, participated. All subjects underwent abdominal ultrasonography and serum CA 19-9 measurement. The authors analyzed the sensitivity, specificity, and predictive values of CA 19-9 for detecting pancreatic cancer. Also, those subjects who had a serum CA 19-9 level above the cut-off value were followed up using a serial check of CA 19-9, computed tomography, or endoscopic retrograde cholangiopancreatography. RESULTS: The number of subjects with a level of CA 19-9 above the cutoff of 37 U/mL was 1063 (1.5%), including four cases diagnosed with pancreatic cancer. The prevalence of pancreatic cancer over the age of 30 years is 13.66 per 100 000 population in Korea. Therefore, the sensitivity is 100% and the specificity 98.5%. However, the positive predictive value of CA 19-9 for detecting pancreatic cancer is only 0.9% in the asymptomatic population. CONCLUSION: Mass screening for pancreatic cancer using CA 19-9 levels in asymptomatic subjects is ineffective because of a very low positive predictive value, despite its high sensitivity and specificity.     

A novel test tube method of screening for hemoglobin E

Introduction: Hemoglobin (Hb) E is a β‐structural variant common worldwide. This Hb disorder can form a compound heterozygous state with the β‐thalassemia gene, leading to life‐threatening hereditary hemolytic anemia, HbE/β‐thalassemia. Screening of HbE has proven to be a challenging practice in prevention and control of the HbE/β‐thalassemia. Methods: A novel test tube method for HbE screening using diethyl aminoethyl (DEAE)‐cellulose resin was described. With the developed system, HbE/A₂ did not bind to the resin and remained dissolved in the supernatant, whereas other Hbs completely bound to the resin. The red color of the supernatant observed in the test tube indicated the presence of HbE. Colorless or markedly pale color of the supernatant indicates the absence of HbE. Results: Accuracy and efficiency of the established method in detecting HbE was comparable with the standard cellulose acetate electrophoresis method. The developed method is cheap and simple with no requirement of sophisticated equipment. The reagent could be stored at 4 °C for up to 5 months. Hemolysate samples aged up to 5 months were still suitable for this test. Conclusion: The described novel test tube method could be an alternative method of mass population screening for HbE, particularly in small health care facilities.