O-12The use of control samples in the PAPANICOLAOU technical external quality assessment scheme

Technical external quality assessment (TEQA) in Wales is based on NHSCSP publication 19, which sets out policy and procedures for the scheme. The purpose of EQA is to sustain and improve the quality of patient care by promoting a high standard of performance. Following the introduction of liquid base cytology (LBC) technical limitations, particularly in assessing counterstaining, have been noted. LBC provides the means to address these limitations - As part of a development plan for TEQA in Wales, a control sample procedure was introduced to the scheme. A pooled control sample was composed, containing residual material from six 'matched' negative samples, re-suspended in collection fluid. Aliquots of this sample were distributed for processing and staining, to the 13 laboratories registered with the scheme. The slides produced were assessed at a scheduled TEQA assessment in accordance with the standard criteria. Initial overall scoring for these control samples produced acceptable levels of staining for 12 of the laboratories - one laboratory produced a marginal score. Repeat distributions have shown maintained or improved results. This method provides a prospective quality assessment tool, which counters the emphasis on slide selection and eliminates potential selection bias, whilst introducing consistency and improving comparability across participant laboratories. The method may also prove helpful in identifying technical inconsistencies such as equipment or handling errors that may occur during sample processing prior to or during staining. The control process, which is now used routinely in the Welsh TEQA scheme; is considered complimentary to, and not a replacement for, the selection process established in NHSCSP #19. However, we feel that this development could be considered as a new initiative in the National TEQA scheme. The control process is applicable to all LBC systems in current use.     

Cervical cytology EQA – the Northern experience

The experience of the Northern region using the 1995 draft National Gynaecological Cervical Cytology External Quality Assurance (EQA) Scheme is given. Over three rounds, 390 staff reporting Cervical Cytology took part, and using a cumulative non-numeric marking scheme, five participants were deemed "unacceptable" at the end of three rounds. A total of 3450 responses were given to the 40 test slides used, with an overall false-positive rate of 5.9% and a false-negative rate of 1.4%. Grading was assessed for pathologists, and many appeared to perform badly by accumulating discrepancies for non-clinically-significant grading differences. The problems of slide selection/staining and grading consistency/accuracy are highlighted. This EQA scheme serves its dual function of education and identifying poor performance. It must be seen as a viable EQA scheme, although other options must be considered, given advances in technology.     

External quality assessment schemes for inorganic elements in the clinical laboratory: Lessons from the OELM scheme

Measurements of inorganic elements in clinical laboratories produce results used for the diagnosis, the treatment and the monitoring of deficiencies or overloads. The main objective of External Quality Assessment Schemes is to verify, on a regular frequency, that clinical laboratory results correspond to the quality requirement for patient care. Therefore, External Quality Assessment Schemes represent an essential component of a laboratory’s quality management system. However, External Quality Assessment Schemes within the same analytical field remain heterogeneous for different reasons such as samples, determination of assigned value, acceptable limits, content of the reports. The aim of this review was to describe and illustrate some major critical aspects of External Quality Assessment Schemes based on Occupational and Environmental Laboratory Medicine external quality assessment scheme experience.     

Standardization of the Papanicolaou stain. I. A comparison of five nuclear stains.

The staining characteristics of five nuclear stains used in a Papanicolaou staining procedure were investigated. Alcohol-fixed cervical smears were stained with a modified Papanicolaou procedure using hematoxylin, alcoholic thionin bromide, alcoholic Victoria blue B, gallocyanin or the thionin Feulgen reagent (thionin-SO2) as the nuclear stain. The same anionic counterstain was used for all slides, and the optical densities of cell nuclei and cytoplasm were measured with the IBAS 2000 image analyzer. Alcoholic thionin gave the most intense nuclear stain, with a very high reproducibility of the staining pattern. Hematoxylin showed the highest coefficient of variation of the staining intensity. Both hematoxylin and gallocyanin gave some nonspecific cytoplasmic staining. Thionin-SO2 allowed a quantitative assessment of DNA, but gave a low staining intensity. Staining with the metal complex dyes interfered with subsequent staining with the pararosaniline Feulgen reagent. Alcoholic thioinin is thus recommended as a nuclear stain for cervical cytology in the Papanicolaou procedure, both for image analysis and for visual microscopy.     

The Italian External Quality Assessment scheme for fragile x syndrome: the results of a 5-year survey

The Italian External Quality Assessment scheme for fragile X syndrome started in 2001 as an activity funded by the National Health System and coordinated by the National Institute of Public Health. The aim of this work is to present the data of 5 years (2001--2004 and 2006) of survey. The External Quality Assessment scheme was designed to cover the following points: (a) genotyping and (b) interpretation and reporting of results. Overall, the scheme covered about 65% of all Italian public laboratories. The average reporting of results was 91.6%, with an overall success rate of 76%. The rate of diagnostic errors observed was on average 5%. Inaccuracy in sizing of CGG repeats of normal and premutated alleles was reported. During the survey the proportion of laboratories using a Southern blotting, polymerase chain reaction, and ABI sizing kit in combination rose from 36.8% to 70.6%. The reports from laboratories showed incompleteness and considerable variations in expected outcomes. For this reason, in 2004 a model for written reports was introduced. In conclusion, these data underscore the need to participate in External Quality Assessment schemes as an educational resource to ensure quality in molecular genetic testing.     

Reliability and accuracy of reporting cervical intraepithelial neoplasia (CIN) in 15 laboratories throughout Italy: phase 1 of a national programme of external quality control in cervical screening

This paper reports results of a first phase of a pilot study to assess and improve quality of diagnoses in cervical cytological laboratories located throughout Italy. It represents the first phase of an External Quality Assurance programme (EQA). In the first phase, two sets of cervical smears representing a range of diagnoses were circulated among participating laboratories. Responses were recorded on a standardized form. Participants were asked to assess the adequacy of the smear and formulate a diagnosis. They were also asked to recommend management of the patient on the basis of the smear report and judge the degree of diagnostic difficulty of each slide. Crude index of agreement, unweighted and weighted kappas, diagnostic specific kappas, sensitivity and specificity as well as clinical indices of variability were calculated. In the second phase, two additional sets of slides were circulated after discussion of the first phase. There was striking variability between laboratories, both in terms of diagnoses offered and recommendations for management on individual slides. Assessment of the degree of difficulty of each slide was also very variable. Discrimination between CINII and CINIII was poor, confirming the choice of merging these two categories in the Bethesda classification. However, discrimination between CINI and CINII was also unsatisfactory. The results were discussed in workshops and it was possible to reach a consensus diagnosis in 35 of 40 smears. This study confirms the need for external quality control programmes.     

细胞核内特征值的改变可引起DNA倍体的变化

目的通过测定不同DNA倍体细胞,研究细胞核内特征值的改变。方法用宫颈刷刷出宫颈细胞,经固定后,用涂片离心机制成二张玻片,一张行巴氏染色作TBS诊断,另一张行Feulgen染色做DNA定量测定。通过对宫颈细胞核图像内像素的统计,计算出细胞核内多种特征值,比较不同DNA倍体细胞内特征值的不同。结果 161873例妇女行宫颈细胞学检查,常规细胞学检查发现2454例低级别鳞状上皮内病变(low-grade squamous intraepithelial lesion,LSIL)和523例高级别鳞状上皮内病变(high-grade squamous intraepithelial lesion,HSIL);而DNA倍体分析发现3412例有3个以上>5c细胞。84%以上的LSIL和HSIL病例均可见倍体异常细胞。与2c细胞相比,4c、5c、7c及9c细胞核面积及核半径明显增大;7c、9c细胞核内平均光学密度和紧实度均值也有明显改变,而光密度方差和灰度熵无变化。结论宫颈细胞DNA倍体改变往往伴有细胞形态和DNA核内分布等特征值的改变。     

External quality assessment in gynaecological cytology: The Trent Region experience

A Department of Health Executive Letter stated in 1998 that the principal function of external quality assessment (EQA) is educational. Subsequently, in England, it has no longer been acceptable to assess performance in gynaecological cytology by proficiency testing. This paper describes the EQA scheme in gynaecological cytology that has been run by the Trent Regional Gynaecological Pathology Quality Assurance Group for the NHS Cervical Screening Programme (NHSCSP) since 1998. It conforms as closely as possible to the recommendations published by the Department of Health Working Group on Histopathology EQA Accreditation, and replaced the national proficiency testing protocol. The educational value of the scheme is derived predominantly from a numerical score which provides confidential and quantitative feedback to all participants. Personal performance monitoring occurs as a secondary function. For primary screeners and checkers, this is based purely on the distinction between negative, inadequate and abnormal smears. For pathologists, personal performance monitoring also includes grading of abnormalities. The EQA has been designed so that all professional groups participate in a manner that closely mimics normal practice. Only slides that have achieved an 80% consensus amongst participants are used in the EQA. Substandard performance has been defined as those participants with scores falling below the 2.5%ile. The paper describes the EQA in detail and illustrates its use by means of the second round results. The EQA protocol developed within Trent and described in this paper has contributed to proposals contained in the current national EQA in gynaecological cytology for the NHSCSP. In particular this paper highlights the effectiveness of the scoring system contained within the Trent and National EQA protocols.     

Grading of laboratories on CD4+ T-lymphocyte evaluations based on acceptable data boundaries defined by the measurement error

BACKGROUND: We addressed the definition of limits of error of %CD4+ and CD4+ counts (AbsCD4+) typical of laboratories of excellence, as well as the grading of laboratories based on the decision to take these limits as boundaries of unacceptable data. METHODS: We studied the 99.9% confidence intervals of the means of 24 human immunodeficiency virus (HIV)+ and HIV- blood samples analyzed by 18 laboratories of the Liguria Region Quality Assessment Program (Liguria Region QALI). Regression equations of lower (L1) and upper (L2) confidence limits over the means of data cleared of unusual results were used to interpolate limits of error for mean values in the tested range. RESULTS: L1 and L2 were symmetric around the mean and a single absolute difference (Abs Res) between the limits and the mean was found. Abs Res significantly increased over mean values (P = 0.0005 for %CD4+, P < 0.0001 for AbsCD4+). Limits were compatible with errors shown with blind replicates. Unacceptable results, outside the limits, accounted for 25% and 30% of %CD4+ and for 18% and 35% AbsCD4+ in the Liguria Region QALI and in the Piemonte Region QA Program, respectively. Limits interpolated over the median showed a similar grading. A comparable fraction of unacceptable data was also found with the method used in the U.K. National External Quality Assessment Scheme (NEQAS) immune monitoring scheme. CONCLUSIONS: We propose the general use of these regression equations to determine bounds for unacceptable data in proficiency testing and to identify laboratories of excellence.     

Gram staining with an automatic machine

This study was undertaken to develop a new Gram-staining machine controlled by a micro-controller and to investigate the quality of slides that were stained in the machine. The machine was designed and produced by the authors. It uses standard 220 V AC. Staining, washing, and drying periods are controlled by a timer built in the micro-controller. A software was made that contains a certain algorithm and time intervals for the staining mode. One-hundred and forty smears were prepared fromEscherichia coli, Staphylococcus aureus, Neisseria sp., blood culture, trypticase soy broth, direct pus and sputum smears for comparison studies. Half of the slides in each group were stained with the machine, the other half by hand and then examined by four different microbiologists. Machine-stained slides had a higher clarity and less debris than the hand-stained slides (p<0.05). In hand-stained slides, some Gram-positive organisms showed poor Gram-positive staining features (p<0.05). In conclusion, we suggest that Gram staining with the automatic machine increases the staining quality and helps to decrease the work load in a busy diagnostic laboratory.     

Desmin detection in FNAB samples of rhabdomyosarcoma: an immunocytochemical study

We performed immunocytochemical (ICC) staining for desmin on 65 fine needle aspiration biopsies from 45 patients with rhabdomyosarcoma, using the avidin-biotin-peroxidase complex method (ABC), and two types of antibodies, D33 and DE-R-11. The material was fixed either in ether-alcohol or in Delaunay's solution. The ABC method was applied to Papanicolaou stained slides, without destaining. We compared the quality of staining on fresh, routinely prepared slides vs archival material and the quality of staining on smears vs cytospins. In 20 cases, D33 and DE-R-II were applied to a pair of slides from the same tumour sample in order to see if there was any difference in their ability to recognize desmin. Desmin was positive in all 18 cases in which ICC staining was performed at the same time diagnoses were given. Among the 27 cases where ICC staining was carried out on archival material, seven were negative. Slides from four of these cases were 20 or more years old and negative reaction could be attributed to heating of slides before coversliping and/or to uneven distribution of desmin immunoreactivity in tumours. The second reason was probably the cause of negative reactions in cases from 1985 and 87. The type of slide preparation had no influence on the quality of staining. However, results were easier to read on cytospins because cells were more evenly distributed. Finally, our results proved that there was no significant difference between D33 and DE-R-11 in their ability to recognize desmin.     

Quality assurance in cervical cancer screening

Objectives. to examine the effectiveness of introducing External Quality Assessment (EQA) into all laboratories which undertake gynaecological cytopathology. to assess pathologists and cytotechnologists regularly for their competence to screen cervical smears, regardless of their standing in the laboratory hierarchy or their experience of gynaecological cytopathology.
Methods. Each participant was asked to screen and report on 10 slides during a 2 h period. the assessment was carried out by a facilitator under the direction of a specially appointed EQA Committee. A maximum score of 20 points was awarded for a completely correct set of answers. A minus score was awarded for a missed abnormal smear. Seventeen pathology laboratories in North West Thames Regional Health Authority participated; 146 cytologists were assessed.
Results. A pilot and four rounds of EQA have been completed and a total 5350 smears examined. Out of 2568 dyskaryotic (abnormal) smears screened, 0.7% were not identified correctly. of the 146 cytologists taking part in the assessment, 95% achieved a score of 17 or more. Three participants were identified who did not reach an acceptable level of competence and appropriate remedial action was taken.
Conclusion. the EQA scheme detected unacceptable levels of performance which can be quickly rectified. Participation of 100% has been maintained on a voluntary basis, and 4 years experience of the scheme confirms that a very high standard of screening prevails in the Region. the study illustrates that voluntary self-regulation is acceptable in the NHS, and the introduction of similar EQA schemes on a national scale will go a long way to establishing confidence in the cervical cancer screening programme.     

An audit of the positive predictive value of high-grade dyskaryosis in cervical smears: 2001–2002

The positive predictive value (PPV) of high-grade dyskaryosis for cervical intraepithelial neoplasia grade 2 (CIN2) or worse on histology is published annually for the laboratories in the UK National Health Service Cervical Screening Programme (NHSCSP). The PPV fell in 2001 compared with 2000 for four of the five consultants reporting cervical smears in our laboratory, the greatest fall being from 91.6% to 77.9%. Investigation of the possible reasons for the fall suggested the main cause lay outside the laboratory in the type of biopsy taken at colposcopy. We conclude that biopsy type affects accuracy of PPV calculations. There is variation in collection and submission of KC61 data including PPV across laboratories. This factor needs to be taken into account when publishing and comparing laboratory data for the NHSCSP.     

Monitoring standards for molecular genetic testing in the United Kingdom, the Netherlands, and Ireland

Molecular genetic techniques have entered many areas of clinical practice. Public expectations from this technology are understandably high. To maintain confidence in this technology, laboratories must implement the highest standards of quality assurance (QA). External quality assessment (EQA) is recognized as an essential component of QA. The United Kingdom National External Quality Assessment Service (UKNEQAS) for Molecular Genetics, first set up in 1991, is currently the longest provider of EQA to molecular genetic testing laboratories in the UK, The Netherlands, and Ireland. Errors in the scheme are sporadic events. However, evidence from this and other EQA schemes suggests that a residual error rate persists, which should be taken into account in clinical practice. This EQA scheme has evolved from the respective scientific bodies of the constituent countries and retains a strong emphasis on collective peer review. It is essential that the steps taken to ensure quality in this rapidly expanding field are clear and transparent to participants and public alike. We describe the procedures developed and the governance imposed to monitor and improve analytical and reporting standards in participant laboratories and we compare our experiences with those of equivalent EQA services in the United States.     

REQUIREMENT OF AN INTERNAL STANDARD FOR MICROSPECTROPHOTOMETRIC MEASUREMENTS OF DNA

Cytophotometric measurements of DNA quantity per cell from 45 different Pinus rigida trees obtained from 135 slides showed that an internal standard was necessary in order to avoid serious errors. Chicken red blood cells were incorporated on the same slide as the pine cells, permitting the calculation of the actual mass units of DNA per pine nucleus. The use of these units makes the results of different investigations and investigators directly comparable. Staining variation between slides and staining batches was shown to be the reason for the requirement of an internal standard. DNA quantity in Feulgen units varied as much as 2.9 fold between different slides of red blood cells although the actual quantity of DNA is constant at 2.8 picograms per nucleus.     

LENTICULE discs provide a homogenous format for external quality assessment samples: a comparison with freeze-dried samples for shellfish microbiology

AIMS: The aim was to compare the variability in Escherichia coli enumeration data and detection of Salmonella spp. between four samples of LENTICULE discs and freeze-dried samples for the Health Protection Agency's External Quality Assessment (EQA) scheme for shellfish microbiology. METHODS AND RESULTS: Four samples of known but undisclosed microbiological content were dispatched in both freeze-dried and LENTICULE disc formats to 57 participating laboratories in 20 countries. Participants examined samples using their routine methods for the most probable number (MPN) of E. coli per 100 g and the presence/absence of Salmonella spp. There was no significant difference between the Food and Environmental Proficiency Testing Unit and participating laboratories for E. coli and Salmonella spp. results. There were significantly less outlying results using the LENTICULE discs than freeze-dried sample format and equivalent or less variance for the former for E. coli MPN. There was no significant difference between LENTICULE discs and freeze-dried samples for the presence/absence of Salmonella spp. CONCLUSIONS: Overall the results indicated that there was equivalent or less variance in results for the LENTICULE discs than for freeze-dried samples, therefore LENTICULE discs are a homogenous and stable matrix for EQA samples. SIGNIFICANCE AND IMPACT OF THE STUDY: This study provides validation data for the replacement of freeze-dried samples by LENTICULE discs for the Health Protection Agency Shellfish EQA Scheme.     

Liquid-based cytology can improve efficiency of cervical smear readers: evidence from timing surveys in two NHS cytology laboratories

OBJECTIVE: Cervical screening programmes in England and Wales were advised by the National Institute for Clinical Excellence in 2003 to adopt liquid-based cytology (LBC) in place of conventional Papanicolaou (Pap) cytology to facilitate laboratory efficiency. Pilot evaluations in England and Scotland monitored daily or weekly workloads of smear readers and concluded that LBC could increase hourly throughput rates. This study, instead, used timing surveys to determine screening rates. METHODS: Two National Health Service cytology laboratories in Manchester and Stockport were partially converted to the LBC ThinPrep process for a cervical screening trial. Three 1-week timing surveys were conducted over 7 months. The surveys covered all LBC-trained staff. The first survey in Manchester also covered staff undertaking conventional Pap screening. The smear readers used timers to record time taken for examining and reporting each slide. RESULTS: In Manchester, in the first survey, nearly 1 minute per slide was saved by the LBC method during primary microscopy. In both laboratories, the mean microscopy time for primary screening of LBC slides was reduced by almost 1 minute between the first and second surveys. There was no difference between the second and third surveys. Microscopy by cytopathologists was also 1 minute per slide quicker with LBC than conventional Pap. The LBC inadequate rates for both laboratories were <2.0%. Organizational factors impacted on the hourly LBC primary screening rates in the laboratories, the rate for Stockport being higher than the rates in the pilot evaluations. CONCLUSIONS: The timing surveys confirm that the LBC ThinPrep technology can improve laboratory efficiency. However, decision-makers should also consider the overall costs and benefits of introducing the technology in screening programmes, including the capital investment and workforce implications.     

Application of Ultrafast Papanicolaou stain to body fluid cytology

OBJECTIVE: To investigate the applicability of the Ultrafast Papanicolaou stain to the cytology of fluids and to compare it with other methods. STUDY DESIGN: Over a 30-month period, 528 unfixed fluids (462 serous effusions, 48 pelvic washings, 16 cyst fluids and 2 bile duct drain fluids) were mixed thoroughly and centrifuged. Two Swedish-style air-dried smears were made and stained with Diff-Quik (Mercedes Medical, Inc., Sarasota, Florida, U.S.A.) and Ultrafast Papanicolaou stain (Richard Allan Scientific, Kalamazoo, Michigan, U.S.A.), and the remaining sediment was fixed in CytoRich Red (TriPath Imaging, Inc., Burlington, North Carolina, U.S.A.), centrifuged onto a 17.5-mm circle with a Hettich cytocentrifuge and stained by the Papanicolaou method. RESULTS: For the 115 malignant fluids, Ultrafast Papanicolaou stain was the preferred method in the 94 non-hematopoietic malignant fluids, Diff-Quik was the preferred method in the 9 hematopoietic malignancies, and CytoRich Red was the preferred preparation in 8 bloody effusions containing rare cancer cells and 4 malignant pelvic washings. The diagnostic turnaround time of smears stained by Ultrafast Papanicolaou stain was < 15 minutes, fast enough for intraoperative consultations. CONCLUSION: It seems that Ultrafast Papanicolaou stain improves the resolution of cytoplasmic and nuclear details of nonhematopoietic cells in body fluids. However, to detect cancer in all types of fluids, Diff-Quik and CytoRich preparations are also required. We now examine three slides per fluid sample, one slide by each of the three techniques.     

Modified Papanicolaou staining protocol with minimum alcohol use: a cost‐cutting measure for resource‐limited settings

S. Gupta, K. L. Chachra, P. Bhadola and P. Sodhani
Modified Papanicolaou staining protocol with minimum alcohol use: a cost‐cutting measure for resource‐limited settings Objective: To devise a simple, cost‐effective protocol for Papanicolaou (Pap) staining of cervicovaginal smears. Methods: Five hundred coded paired cervical smears were collected from women as part of routine cervical cancer screening. One set of smears was stained by conventional Pap staining protocol (CP) and the other by a modified protocol (MP) in which alcohol was replaced by 1% acetic acid in all the steps except during fixation and prior to mounting; in addition, one alcohol‐based counterstain, OG, was omitted. The smears were examined blindly by the pathologists and then decoded. Each pair of smears was compared and the two protocols were analysed for staining quality and diagnoses by McNemar and chi‐square tests. Results: The staining quality in the MP was satisfactory. The nuclear and cytoplasmic features were comparable to the CP. Cytoplasmic transparency was maintained in the MP and the differential staining of blue/green and pink was acceptable to the pathologists and technicians. The diagnoses agreed in all cases and there was no compromise in interpreting the smears. With MP it took only 3–4 minutes to stain a batch of 50 slides. in contrast to the 20 minutes taken by CP. The MP used almost one‐seventh of the amount of alcohol compared with CP, which translated into a significant cost reduction per smear. Conclusions: The improvised Pap staining protocol with minimum alcohol use is a simple, cost‐effective and technician‐friendly procedure that can be easily adopted in high‐volume, resource‐limited laboratories for mass cervical cancer screening.     

A Versatile Stain for Pollen Fungi, Yeast and Bacteria

A versatile stain has been developed for demonstrating pollen, fungal hyphae and spores, bacteria and yeasts. The mixture is made by compounding in the following order: ethanol, 20 ml; 1% malachite green in 95% ethanol, 2 ml; distilled water, 50 ml; glycerol, 40 ml; acid fuchsin 1% in distilled water, 10 ml; phenol, 5 g and lactic acid, 1-6 ml. A solution has also been formulated to destain overstained pollen mounts. Ideally, aborted pollen grains are stained green and nonaborted ones crimson red. Fungal hyphae and spores take a bluish purple color and host tissues green. Fungi, bacteria and yeasts are stained purple to red. The concentration of lactic acid in the stain mixture plays an important role in the differential staining of pollen. For staining fungi, bacteria and yeasts, the stain has to be acidic, but its concentration is not critical except for bacteria. In the case of pollen, staining can be done in a drop of stain on a slide or in a few drops of stain in a vial. Pollen stained in the vial can be used immediately or stored for later use. Staining is hastened by lightly flaming the slides or by storing at 55±2 C for 24 hr. Bacteria and yeasts are fixed on the slide in the usual manner and then stained. The stock solution is durable, the staining mixture is very stable and the color of the mounted specimens does not fade on prolonged storage. Slides are semipermanent and it is not necessary to ring the coverslip provided 1-2 drops of stain are added if air bubbles appear below the coverslip. The use of differentially stained pollen mounts in image analyzers for automatic counting and recording of aborted and nonaborted pollen is also discussed.