基于社区水平的珠海市大人群地中海贫血的遗传筛查和产前诊断

目的 阐述在广东省珠海市开展基于社区的控制重型α和β地中海贫血(简称地贫)的预防模式.方法 构建由6家医院组成的二级地贫遗传服务网络,以珠海市婚前医学或产前检查人群作为筛查对象,采用常规杂合子筛查策略,以标准的血液学分析流程进行α和β地贫特征的筛查.对所有地贫疑诊对象进行随访和遗传咨询,并采用基于PCR的分子诊断技术对高风险夫妇进行确诊.在知情同意和选择的情况下,对高风险妊娠实施产前基因诊断并通过选择性引产淘汰受累的重型地贫胎儿.结果 从1998年1月至2005年12月,共筛查了85522例拟婚育龄青年和10439例孕妇,婚检地贫筛查覆盖率达到71.38%.在6563例地贫筛查阳性的病例中,α和β地贫分别为4312例(4.5%)和2251例(2.3%);总计发现148对有生育重型地贫儿可能的高风险夫妇(α地贫103对,β地贫45对),其中有142对(95.9%)高风险夫妇进行了产前诊断(α地贫98例,β地贫44例).本项目启动后共减少了41例重型地贫患儿的出生,其中包括Hb Barts水肿胎23例、Hb H病4例和重型β-地贫14例.结论 这是我国首次基于社区水平的、在拥有129万人口的珠海市实施的前瞻性α和β地贫预防监控计划.这一预防模式对我国其他地贫高发区和其他发展中国家开展地贫和其他血红蛋白病的预防有着重要的借鉴意义.     

A precision study of a photo-optical factor VIII assay technic.

Various methods of factor VIII analysis with a wide range of accuracies as confirmed by calculated coefficients of variation have been devised. The authors report a technic of factor VIII assay using a photo-optical clot-detection instrument with commercially available sources of factor VIII-deficient plasma and lyophilized normal control plasma, and compare its accuracy, ease of performance and reliability with those of existing methods. The accuracy of the technic was established by a survey of a normal control group, which revealed a mean value of 100% factor VIII activity, range 55-200%. The coefficients of variation of this unique technic, determined both by clotting time in sec (1.4%) and by the derived percentage activity (7.0%), reflect the greater reproducibility and reliability of the assay as compared with previously reported methods. We feel that this method of factor VIII assay should be utilized for specific diagnostic information regarding patients who have factor VIII disorders.     

A reliable screening protocol for thalassemia and hemoglobinopathies in pregnancy: an alternative approach to electronic blood cell counting

Primary screening for thalassemia and hemoglobinopathies usually involves an accurate blood count using an expensive electronic blood cell counter A cheaper alternative method was tested by using a modified osmotic fragility (OF) test and a modified dichlorophenolindophenol (DCIP) test. Altogether 423 pregnant Thai women participated in this project. Hemoglobin patterns and globin genotypes were determined using an automated high-performance liquid chromatography analyzer and polymerase chain reaction analysis of alpha- and beta-globin genes. Among the 423 subjects, 264 (62.4%) carried thalassemia genes. The combined OF and DCIP tests detected all pregnant carriers of the 3 clinically important thalassemias, ie, alpha0-thalassemia, beta-thalassemia, and hemoglobin E with a sensitivity of 100.0%, specificity of 87.1%, positive predictive value of 84.5%, and negative predictive value of 100.0%, which show more effectiveness than these values for the standard method based on RBC counts. A combination of modified OF and DCIP tests should prove useful and applicable to prenatal screening programs for thalassemia and hemoglobinopathies in communities with limited facilities and economic resources.     

State-based surveillance for selected hemoglobinopathies

PurposeThe lack of an ongoing surveillance system for hemoglobinopathies in the United States impedes the ability of public health organizations to identify individuals with these conditions, monitor their health-care utilization and clinical outcomes, and understand the effect these conditions have on the health-care system. This article describes the results of a pilot program that supported the development of the infrastructure and data collection methods for a state-based surveillance system for selected hemoglobinopathies.MethodsThe system was designed to identify and gather information on all people living with a hemoglobinopathy diagnosis (sickle cell diseases or thalassemias) in the participating states during 2004–2008. Novel, three-level case definitions were developed, and multiple data sets were used to collect information.ResultsIn total, 31,144 individuals who had a hemoglobinopathy diagnosis during the study period were identified in California; 39,633 in Florida; 20,815 in Georgia; 12,680 in Michigan; 34,853 in New York, and 8,696 in North Carolina.ConclusionThis approach provides a possible model for the development of state-based hemoglobinopathy surveillance systems.     

LOVD–DASH: A comprehensive LOVD database coupled with diagnosis and an at‐risk assessment system for hemoglobinopathies

Hemoglobinopathies are the most common monogenic disorders worldwide. Substantial effort has been made to establish databases to record complete mutation spectra causing or modifying this group of diseases. We present a variant database which couples an online auxiliary diagnosis and at‐risk assessment system for hemoglobinopathies (DASH). The database was integrated into the Leiden Open Variation Database (LOVD), in which we included all reported variants focusing on a Chinese population by literature peer review‐curation and existing databases, such as HbVar and IthaGenes. In addition, comprehensive mutation data generated by high‐throughput sequencing of 2,087 hemoglobinopathy patients and 20,222 general individuals from southern China were also incorporated into the database. These sequencing data enabled us to observe disease‐causing and modifier variants responsible for hemoglobinopathies in bulk. Currently, 371 unique variants have been recorded; 265 of 371 were described as disease‐causing variants, whereas 106 were defined as modifier variants, including 34 functional variants identified by a quantitative trait association study of this high‐throughput sequencing data. Due to the availability of a comprehensive phenotype‐genotype data set, DASH has been established to automatically provide accurate suggestions on diagnosis and genetic counseling of hemoglobinopathies. LOVD‐DASH will inspire us to deal with clinical genotyping and molecular screening for other Mendelian disorders.     

Immunophenotypic characterization of acute leukemia by immunocytology

The potential for specific immunophenotypic characterization of the acute leukemias has been enhanced greatly by the development of monoclonal antibodies. Currently, this immunologic data is obtained most commonly by flow cytometric analysis or cellular cytotoxicity assays. The former is an expensive technic that lacks morphologic evaluation unless cell sorting is performed. The latter precludes morphologic assessment by the nature of the assay. The authors have developed an immunostaining procedure utilizing cytospin preparations and immunoperoxidase methods that are relatively inexpensive and allow simultaneous assessment of the immunologic markers and cellular morphology. Although a comparison of flow cytometry and immunocytology revealed quantitative differences for individual cell surface markers, the "qualitative" immunologic phenotype of the leukemic population was virtually identical by the two technics.     

Intraoperative diagnosis of breast lesions. Comparison of cytologic with frozen section technics

A retrospective double-blind examination of the cytologic smears and frozen section tissue slides from 140 lesions of the breast was performed in order to assess the diagnostic accuracy of each method. The cases with diagnostic errors were reevaluated to define the pitfalls encountered in the intraoperative diagnosis of breast lesions by both methods. Results with the cytologic technics are more variable than with frozen sections, but when read by experienced observers the smears are as accurate as frozen sections and have the additional advantages of rapidity, sparing of tissue for other studies, better correlation with fine-needle aspiration cytologic results, and more complete sampling of large or multiple specimens. The authors recommend that intraoperative cytologic examination be employed routinely in breast diagnosis, initially always in conjunction with frozen section examination but eventually in many cases as an alternative technic.     

Hemoglobinopathy prevention in primary care: a reflection of underdetection and difficulties with accessibility of medical care,a quantitative study

As in most Northern European countries, the prevalence of hemoglobinopathies in The Netherlands is increasing due to migration. Although hemoglobinopathies are severe chronic diseases with few treatment options, timely detection of carriers allows at-risk couples to make informed reproductive choices such as pre-implantation diagnosis, prenatal diagnosis or termination of affected pregnancies. Using a quantitative design, we evaluated the prevalence of hemoglobinopathies in The Hague region, The Netherlands. Patient and carrier registries from hospital, laboratory and general practitioners allowed this quantitative analysis. The highest prevalence of hemoglobinopathies was seen in immigrant neighborhoods, and a large gap was noted between estimated carrier prevalence and the actual registration of carriers in electronic patient records. Carrier prevalence was estimated to be 13,704; however, the ELAN database contains only 1542 cases with ICPC codes for sickle cell disease or thalassemia. Although more research is needed to define the requirements of the healthcare system to address this challenge, this study clearly shows the gap between estimated carrier prevalence and registration and thereby the pressing need for action.Subject terms: Population screening, Anaemia     

Gene diagnosis of hemoglobinopathies--the determination of breakpoints for a large deletion

A large number of mutations leading to hemoglobinopathies(abnormal hemoglobins, thalassemias) have been discovered. Gene diagnosis for a point mutation or a deletion/insertion of a few nucleotides is now readily performed. For a large deletion, once the precise breakpoints are unraveled, the same type of deletion is promptly diagnosed by gap PCR. However, a number of new types of large deletions remain unexamined. They need meticulous Southern blot analysis and/or cloning. Here we present a new technique to determine their precise breakpoints. One of the two breakpoints needs to be first assigned within the 5-kb portion estimated by gene dosage by quantitative PCR. The other breakpoint is left unexamined. The genome DNA is digested with one of eight kinds of endonucleases and subjected to recombination with pUC18 cloning vector digested with the same endonuclease. The gap PCR is subsequently performed between the common primers of pUC18 and five arbitrary primers within the aforementioned 5-kb portion. An abnormal gap PCR product, if detected by electrophoresis, discloses precise 5' and 3' breakpoints after direct sequencing. This method successfully disclosed the breakpoints for two epsilon gamma delta beta-thalassemias, one delta beta-thalassemia and one beta-thalassmeia in a relatively short period. All are new mutations. This method uses neither cloning procedures nor Southern blot, but employs gene dosage estimation and PCR. Thus, it is relatively simple. The progress of the genome project facilitated analysis of any large deletions.     

Parathyroid hormone (PTH) assay of parathyroid cysts examined by fine-needle aspiration biopsy

The authors report three cases of parathyroid cysts examined by the fine-needle aspiration biopsy technic. A presumptive diagnosis of parathyroid cyst was made when characteristic water-clear fluid was aspirated. The diagnosis was then confirmed by parathyroid hormone (PTH) assay. The authors believe that the C-terminal/midmolecule determination should be the assay of choice, because the N-terminal-specific assay gave normal or slightly elevated results in all the cases studied. If only an N-terminal-specific PTH assay is obtained, potential for a false negative diagnosis exists. With a correct PTH assay, a specific diagnosis of parathyroid cyst can be rendered, which enables appropriate treatment of total fluid aspiration, which thereby eliminates the need for thyroid hormone treatment or surgery in most cases. A discussion of PTH assays is presented along with speculations concerning the secretion of PTH by the parathyroid gland. The previous literature detailing cytologic findings and the PTH assays of parathyroid cysts diagnosed by the fine-needle aspiration biopsy are reviewed.     

An enzyme-linked immunosorbent assay for the detection of complement components on red blood cells

A new technic using the principle of enzyme-linked immunoassay (ELISA) has been developed for the detection of complement components on red blood cells sensitized in vivo or in vitro. Using a double-antibody technic, anticomplement antisera (anti-C3c or anti-C3c/C3d) produced in rabbits was incubated with the red blood cells, followed by incubation with antirabbit alkaline phosphatase conjugated antiglobulin. The amount of the enzyme fixed was measured spectrophotometrically by the enzymatic hydrolysis of the substrate PNPP. A calibration curve was made from red blood cells on which complement was deposited by the method of Fruitstone . The technic showed a greater sensitivity than the standard antiglobulin tests and allowed simultaneous qualitative and semiquantitative estimates. The technic can be performed in any laboratory equipped with the standard equipment found in a blood bank, including a spectrophotometer. The authors made a modification of Alsever 's solution, which allowed the safe and stable preservation of complement coated red blood cells for 15 days. Significant positive results were obtained clinically using this technic, while negative or weakly positive reactions were obtained by the conventional antiglobulin tests.     

Rhodamine B fluorescence as a stain for amniotic fluid squames in maternal pulmonary embolism and fetal lungs

Rhodamine B fluorescence is presented as a simple, rapid, highly sensitive, permanent method for the histologic detection of squames in maternal amniotic fluid emboli and fetal lungs in cases of intrauterine asphyxia. The method may be used on alcohol fixed smears or formalin fixed paraffin sections. The application of this procedure allows for identification of sparsely distributed fetal squames which otherwise may be overlooked by less sensitive tinctorial methods which may also be more tedious in technical preparation and screening.     

Rapid recovery of mycobacteria from clinical specimens using automated radiometric technic

Automated radiometric technic (BACTEC Johnston Laboratories, Towson, MD) was compared with conventional mycobacterial culture procedure (Lowenstein-Jensen plus Gruft modification of Lowenstein-Jensen) in this study of 1,000 clinical specimens. In addition, 8-azaguanine inhibition was tested by radiometric technic as a rapid procedure for the differentiation of Mycobacterium tuberculosis from other mycobacterial species. A total of 59 mycobacteria was recovered. Of 28 clinically significant isolates (M. tuberculosis, M. kansasii, M. avium, M. fortuitum), the BACTEC system detected 26 (93%). Conventional methods recovered 23 (82%). The BACTEC system required an average of seven days to recover M. tuberculosis from smear-positive specimens compared with 18 days required by Lowenstein-Jensen or Gruft slants. From smear-negative specimens, the BACTEC detected M. tuberculosis in an average of 20 days versus 28 days by conventional procedure. All 20 isolates of M. tuberculosis were inhibited by 8-azaguanine, whereas 39 isolates of mycobacteria other than M. tuberculosis were not inhibited. The BACTEC system accomplishes more rapid recovery of mycobacteria and provides a higher yield than conventional methods.     

Quality control of electrolyte analyzers. Evaluation of the anion gap average

The authors have shown previously that averaging at least eight patient anion gaps provides a sensitive technic for the detection of systematic error in electrolyte analysis (Am J Clin Pathol 79:688-696, 1983). They conducted a retrospective and prospective evaluation of this technic on the ASTRA 4. One month of patient and control data were studied retrospectively and showed that 17/71 abnormally low patient anion gap averages were associated with violations in a multi-rule procedure, and 41/71 low averages were associated with violations in cusum, a more sensitive procedure. In the prospective study, a total of 36 runs of eight patient specimens with low anion gap averages (less than 7.5 mmol/L) were reanalyzed after appropriate recalibration and/or maintenance. Thirty-one of the 36 groups had significant changes in either Na (nine groups, delta Na = +1.5 mmol/L), Cl (14 groups; delta Cl = -1.8 mmol/L), or in both Na and Cl (eight groups; delta Na = +1.2 mmol/L; delta Cl = -0.9 mmol/L). Because the average error detected was small, the authors recommend that the average of anion gaps be used as an early indicator of drift. It must be used, however, in conjunction with standard quality control procedures such as the multi-rule approach.     

The reliability and rapidity of the coagglutination technic and its comparison with precipitin technic in the grouping of streptococci.

Three methods of the grouping of 170 strains of beta-hemolytic streptococci belonging to Groups A, B, C, F, and G by capillary and agar-gel precipitation using Lancefield extract and by coagglutination of antibody-coated protein A-containing staphylococci were compared. Capillary and agar-gel precipitin technics correctly grouped all 170 strains, with no cross-reaction. One hundred sixty-nine out of the 170 strains were also correctly grouped by the coagglutination technic, and the one strain with a cross-reaction was correctly grouped after blood--agar subculture. Although 20 of the 170 strains showed minor cross-reactions by the coagglutination technic, the specific groups were easily and unquestionably detected, and the minor cross-reactions were easily overcome. A rapid method of grouping by coagglutination technic using 4--6-hour broth culture, done on 75 strains, showed that 73 strains could be correctly grouped by the rapid method and two after overnight incubation. Thus, the coagglutination technic of grouping was found to be easy, reliable, and economical, and could be adopted in any routine diagnostic laboratory as a rapid grouping procedure.     

Rapid diagnosis of thalassemias and other hemoglobinopathies by capillary electrophoresis system

Basic diagnosis of hemoglobinopathies can be performed by analysis of erythrocyte indices as well as by the separation and quantification of the common hemoglobin (Hb) fractions Hb A(2), Hb S, Hb C, Hb D, Hb E, and Hb F. This study used an automatic capillary zone electrophoresis system to diagnose various types of hemoglobinopathies common in the Thai population. A total of 459 adults were recruited, which consisted of normal, various types of thalassemia carriers, and thalassemia patients with different genotypes. Hb types and quantification of all Hb components were determined by an automated capillary zone electrophoresis. The automatic capillary electrophoresis system can separate and quantitate Hbs A, F, E, A(2), Constant Spring (CS), H, and Bart's in a way that is comparable with other Hb analysis methods. Moreover, the Hb A(2) peak can be distinguished clearly from the Hb E peak in individuals who carry Hb E. The slightly increased levels of Hb A(2), 3.5% +/- 0.4%, which is shown in the carriers of Hb E, confirm that Hb E is the silent phenotype of beta(+)-thalassemia.     

Analysis and comparison of two methods to detect hepatitis B antigen. Counterelectrophoresis (CEP) and reversed passive latex-agglutination (RPLA) technic in relationship to radioimmunoassay.

The authors evaluated two methods for detection of hepatitis B surface antigen, reversed passive latex agglutination and counterelectrophoresis, using radioimmunoassay as a control. The RPLA test (Antigen I, Pfizer Inc., Lot no. 403-94301-1) was compared with radioimmunoassay (RIA, Austria) and counterelectrophoresis (CEP) using serum specimens obtained at the Mount Sinai Medical Center, Miami Beach, Florida. Of the 100 selected sera tested as unknowns, 64 were positive by RIA and 36 were negative. The RPLA test yielded results comparable to those of the RIA technic in that the same 64 positives and 36 negatives identified by RIA were obtained with the RPLA test. Of the 64 positive samples obtained with the RIA technic, five yielded negative results with the CEP method. The RPLA test proved to be more sensitive than CEP in this survey.     

Recombinant DNA technology and laboratory medicine

Recombinant DNA technology has already had a major impact on our understanding of microbiology, cell biology, and genetic diseases and it will certainly have extensive applications in laboratory medicine. The techniques of restriction endonuclease analysis of DNA, nucleic acid hybridization after electrophoretic separation of nucleic acid fragments, and molecular cloning of bacterial, viral, and human genes are already being used in epidemiologic studies and the prenatal diagnosis of certain genetic diseases, such as sickle cell anemia and the thalassemias. New insights into genes that may be involved in human cancer are being developed and may lead to improved methods for diagnosis and classification of tumors.     

Hemoglobin comparisons between African American and European American males with hemoglobin values in the normal range

There are conflicting reports about the nature and magnitude of hemoglobin (Hb) differences between African Americans (AA) and European Americans (EA). Several reports suggest that AA have lower mean Hb values and that differences between AA and EA are large and are due to “racial” differences. Some have even called for separate Hb standards. In this study, Hb comparisons were made between AA and EA males in the Second National Health and Nutrition Examination Survey, 1976–1980. Comparison of AA and EA males with normal Hb values (i.e., 13.0 g/dl or greater) indicated that observed mean Hb differences exist primarily at the lower end of the Hb distribution (i.e., the distribution is skewed to the left). Several factors are likely contributors to lower hemoglobin values at the lower end of the distribution, including hemoglobinopathies, thalassemias, and/or nutritional deficiencies. If racial differences exist between these groups, controlled prospective studies are necessary to demonstrate this possibility. This study supports a single Hb standard for AA and EA males. © 1992 Wiley-Liss, Inc.     

Sichelzellenanämien in der perinatalen Plazentadiagnostik

Hereditary hemoglobinopathies should be considered as differential diagnosis when examining placental specimens for fetal growth retardation and spontaneous abortion. They can cause various macroscopic and microscopic changes in the placenta that are relevant for routine pathology examination. The importance of interdisciplinary co-operation between obstetrics and pathology to achieve optimum diagnostics and therapy planning is demonstrated using the case of a pregnant woman with heterozygous genotype and her child with homozygous genotype. Within this context, the influence of hemoglobinopathies on placental pathology and fetal development are summarized and exemplified.