Laboratory Test of an Automated Cell Analysis System For Cervical Screening

An automated cell analysis system (Autoplan-MIAC) for the early detection of precancerous lesions of the cervix was tested under semi-routine conditions in a clinical cytology laboratory. A set of 1500 specimens, highly enriched with abnormal cases, was analysed. Cervical scrapings were collected in suspension and processed by cytocentrifugation for microscopy. Two slides were prepared from each sample: one for staining according to Papanicolaou for the visual reference diagnosis and one for Feulgen staining for automated analysis. the specimens were evaluated in two ways: the first one, which is referred to as the automated machine classification system (AMC), classifies the specimens according to the number and ratio of selected objects (alarms) and is a fully automated system. the second system classifies the specimens after visual evaluation of the stored alarms as they are displayed on a TV monitor, and is designated the interactive machine classification system (IMC). the AMC results showed a false positive rate of 16.5% when the cut-off threshold was selected so that all 117 positively diagnosed specimens were classified ‘positive’ by the system. In that case 87.4% of the CINI and 96.9% of the CINII cases were AMC-positive. the IMC results showed a false positive rate of 2.5%, when 86.3% of the CIN I cases, 96.9% of the CIN II cases and all CIN III and invasive carcinoma cases were positively classified.     

Image analysis combined with quantitative cytochemistry. Results and instrumental developments for cancer diagnosis

This paper describes the application of image analysis combined with a quantitative staining method for the analysis of cervical specimens. The image analysis is carried out with the Leyden Television Analysis System, LEYTAS, of which two versions are described. LEYTAS-1 as well as LEYTAS-2 have both been designed with a high degree of flexibility and interaction facilities. A much wider range of image analysis programs is however, possible with LEYTAS-2, enabling many applications. LEYTAS-1, the earlier version, consists of a Leitz microscope with automated functions, a TV camera, the Texture Analysis System (TAS, Leitz), a four-bit grey value memory and a minicomputer (PDP 11/23). Using this instrumentation 1,500 cervical smears prepared from cell suspensions and stained with acriflavin-Feulgen-Sits have been analysed in a completely automated procedure. Image transformations working in parallel on entire fields, have been used for cell selection and artefact rejection. Resulting alarms, consisting of selected single cells and non-rejected artefacts are stored in the grey value memory, which is displayed on a TV monitor. This option allows visual interaction after the machine diagnosis has been made. The machine diagnosis was correct in 320 out 321 specimens with a severe dysplasia or more serious lesion. The false positive rate in 561 morphologically negative specimens (normal and inflammation) was 16% (machine diagnosis). Visual interaction by subtracting the visually recognized false alarms from the total number of alarms reduces the false positive rate to 11%.(ABSTRACT TRUNCATED AT 250 WORDS)     

Image analysis combined with quantitative cytochemistry

Summary This paper describes the application of image analysis combined with a quantitative staining method for the analysis of cervical specimens. The image analysis is carried out with the Leyden Television Analysis System, LEYTAS, of which two versions are described. LEYTAS-1 as well as LEYTAS-2 have both been designed with a high degree of flexibility and interaction facilities. A much wider range of image analysis programs is however, possible with LEYTAS-2, enabling many applications. LEYTAS-1, the earlier version, consists of a Leitz microscope with automated functions, a TV camera, the Texture Analysis System (TAS, Leitz), a four-bit grey value memory and a minicomputer (PDP 11/23). Using this instrumentation 1,500 cervical smears prepared from cell suspensions and stained with acriflavin-Feulgen-Sits have been analysed in a completely automated procedure. Image transformations working in parallel on entire fields, have been used for cell selection and artefact rejection. Resulting alarms, consisting of selected single cells and non-rejected artefacts are stored in the grey value memory, which is displayed on a TV monitor. This option allows visual interaction after the machine diagnosis has been made. The machine diagnosis was correct in 320 out 321 specimens with a severe dysplasia or more serious lesion. The false positive rate in 561 morphologically negative specimens (normal and inflammation) was 16% (machine diagnosis). Visual interaction by subtracting the visually recognized false alarms from the total number of alarms reduces the false positive rate to 11%. In LEYTAS-2, which is based on LEYTAS-1 studies, the microscope is equipped with a new type of objective, enabling the analysis of microscope fields, which are four times as large as in LEYTAS-1. The image analysis part consists of the Modular Image Analysis Computer (MIAC, Leitz) and for alarm storage an eight-bit grey value processor is used. Comparison with LEYTAS-1 shows that cell selection capacities are similar and that the speed is four times higher.In honour of Prof. P. van Duijn     

Can Flow Cytometry Reduce the Workload For Cervical Screening? the Results of A Series of 622 Specimens

A simple method permitting the flow cytometric examination of cervical specimens has been developed and an assessment made of the feasibility of relying on this method to screen women for cervical neoplasia. Examination of four flow cytometric parameters showed differences between morphologically normal and abnormal specimens and allowed identification of a proportion of the normal specimens. The system had a false negative rate of 8%. Our experience with cervical specimens has revealed a number of problems associated with their examination by flow cytometry and these are discussed.     

Identifying southern yellow pine cross sections from the southeastern United States using quadratic discriminant analysis on pith and second annual ring diameters

Southern yellow pine specimens collected from historical structures, stumps, and coarse woody debris in forests have been difficult to identify at the species level due to similar wood anatomy. This can be problematic for dendrochronologists when identifying the correct species used in the construction of historical structures, or reconstructing forest history on the landscape and using those specimens in the context of that history. We applied a quadratic discriminant analysis (QDA) to update a century-old method plotting pith diameters against second annual ring diameters to discern one species of southern yellow pine from others. Our analysis estimates error rates for false positive and false negative determinations when comparing longleaf pine (Pinus palustris Mill.) to shortleaf (Pinus echinata Mill.) and loblolly pine (Pinus taeda L.). The cross-validated false positive error rates for the smallest dataset (n = 46), was nearly twice (9.52%) that determined as a simple proportion by counting errant observations (4.76%). QDA of the largest dataset (n = 206) gave a flatter zero contour and false positive rate (3.13%) like the proportionally determined value (1.56%), despite one additional observation being falsely assigned to longleaf pine by QDA. An unknown, unearthed southern pine specimen from southeastern Virginia was radiocarbon dated up to 500 years prior and assigned as longleaf by our method (probability ≥ 0.9998). Thus, through a QDA, it is possible to greatly improve confidence in identifications of key unknown specimens that can provide evidence of discerning one species, longleaf pine, from other southern yellow pines.     

Direct and simultaneous identification of Mycobacterium tuberculosis complex (MTBC) and Mycobacterium tuberculosis (MTB) by rapid multiplex nested PCR-ICT assay

The Mycobacterium tuberculosis (MTB) shows different virulence and host infection range from other members of the M. tuberculosis complex (MTBC). Differential identification of MTB from MTBC is thus important in certain occasions. The currently commercially available molecular assays which use either IS6110 or 16S rDNA fragment as identification targets are mainly designed for identifying MTBC but not for MTB. Comparative genomic DNA analysis has provided valuable information on regions of difference (RD) present in MTB but not in other members of the MTBC. RD9 region is further suggested to be a potential target for differential identification of MTB from MTBC. In this study, using IS6110 and Rv3618 (belong to RD9) as the specific identification targets for MTBC and MTB, respectively, we developed and tested a multiplex nested PCR-ICT (immuno-chromatography test) assay for simultaneously and directly detecting not only MTBC but also MTB from 1500 clinical sputum specimens. The results were compared with traditional culture and biochemical identification results together with patients' clinical assessments. This assay showed a 95.5% sensitivity, 97.9% specificity, 2.1% false positive rate and 4.5% false negative rate towards detection of MTBC, and a 93.0% sensitivity, 99.8% specificity, 0.2% false positive rate and 7.0% false negative rate for detection of MTB. This detection system shows great potential in clinical application.     

Image cytometry in automated cervical screening

Severe restrictions with regard to false negative rates have played a major role in the development of the LEYden Television Analysis System (LEYTAS). The present paper describes a test with a continuous series of 1500 cervical samples illustrating the accuracy of LEYTAS in a fully automated screening procedure using cell selection transformations and artefact rejection procedures. Specimen classification with a cut-off at greater than 0.3% alarms (= percentage of automatically selected objects per epithelial cells) and greater than 10 alarms, results in a false negative rate (FNR) of 0.3% (1 case out of 321 cases with severe dysplasia or more serious lesions), a false positive rate (FPR) of 13% (663 negative cases) and a rejection rate of 2.7%. Besides a machine classification, LEYTAS offers a second, machine-interaction classification of those preparations which have been declared positive by the machine. Machine-interaction involves visual evaluation of the stored images of the detected objects (alarms) and reduces the FPR from 13 to 8%. Statistical tests further demonstrate the significance of the screening results. Presently the main drawback for routine use of automated screening with LEYTAS seems to be the time consuming preparation procedure, since instrumentation has now been updated to a new, fast and user-friendly version of LEYTAS.     

Report on a long-term trial of CYBEST Model 2 for prescreening for squamous cell carcinoma of the uterine cervix

The results of a 12 year field trial, on a large patient base, of the CYBEST Model 2, an automated screening system using an image analysis method, are summarized. The cell specimens were stained by a conventional Papanicolaou method. Individual cellular atypism was determined from the sum of the nuclear area, the N/C ratio, the mean nuclear optical density, and the nuclear roundness with an ambiguity function value of 0.4295. CYBEST's final assessment as 'suspicious' or 'normal' was statistically determined from a cumulative histogram of the cellular atypism of a maximum 300 detected cells for each cell population using the Kolmogrov-Smirnov test, and those of unsatisfactory samples were automatically classed as 'rejected', which occurred in 6% of the study. A total of 84 atypical preparations including 17 histologically proven carcinoma patients were encountered during the entire test period from 1977 to 1988, and the overall false negative rate was 1.19% (1/84). Among the results on a total of 42,988 slides during the test period of the last 9 years from 1980 to 1988, the false positives occurred at a rate of 30.7% (12,383/40,307 of non-dysplastic slides) and the false negatives at approximately 2% (1/55 of dysplastic slides). These results are compared with those of other important systems.     

A rapid immunochromatographic assay for the detection of Mycobacterium tuberculosis antigens in pulmonary samples from HIV seropositive patients and its comparison with conventional methods

As tuberculosis generates a highly heterogeneous antibody repertoire, its diagnosis requires tests based on cocktails of antigens. We describe a new, rapid method called rapid immunochromatographic assay (RICA) for cocktail-based diagnosis, which can detect Mycobacterial antigens in sputum specimens. Six antigenic fractions of pathogenic Mycobacterium tuberculosis were used in combination as the capture antigens in the control line of the flow-through assay. Antigen detection of 200 sputum samples from HIV seropositive patients by RICA assay gave a sensitivity of 97.9%, specificity of 99.0%, positive predictive value of 98.9%, negative predictive value of 98.0%, false positive rate of 0.9%, false negative rate of 2.0%, prevalence rate of 49%, likelihood ratio for positive results 97 and likelihood ratio for negative results 0.02. The combination of RICA and AFB staining gave a sensitivity of 100%, specificity of 100%, positive predictive value of 100%, negative predictive value of 100%, false positive rate of 0%, false negative rate of 0%, likelihood ratio for negative results 0. The assay was simple, rapid and economical for the detection of M. tuberculosis infection and suitable for large scale screening of samples in endemic areas without any sophisticated equipment. The results of the assay proved to be superior to conventional methods and combined with clinical data, could form the basis for starting an earlier course of treatment.     

Are seroepidemiological surveys for human immunodeficiency virus infection based on tests on pools of serum specimens accurate and cost-effective?

Serum specimens (n = 17668) from UK antenatal patients in the Thames Regions were tested by Wellcozyme HIV 1/2 EIA singly and in pools of 6, 12 and 24: 35 (0.2%, 1 in 505) were confirmed as anti-HIV positive. The pools of 12 were also tested for anti-HIV 1/2 by IAF Biochem, Behring and Diagnostics Pasteur EIAs. All 35 positive specimens were easily detectable after pooling in groups of 12. The false positive rate for Wellcozyme was nearly halved compared with individual testing (1 in 309 false positive compared with 1 in 174). For the other assays false positive rates on pools of 12 were: IAF Biochem 1 in 193, Behring 1 in 140, Diagnostics Pasteur 1 in 1547. Twenty-two known anti-HIV 2-positive sera were detected by all four EIAs when diluted as in pools of 6 and 12, but by only three EIAs in pools of 24 and 48. Pooling in groups of 6 did not seem to delay detection of HIV 1 seroconversion, but pooling in groups of 12, 24 and 48 might delay it by 1, 2 and 3 weeks respectively. For this study the effect of pooling in groups of 12 would have been a reagent saving of 87-91% and a labour saving of about 50%. Because of the low HIV incidence and rarity of specimens collected around seroconversion in UK, little, if any, loss of sensitivity would result from it. Pooling in groups of 12 has therefore been chosen for the screening of anonymous antenatal specimens in the UK.     

Cytologic Diagnosis of Gastric Cancer

Follow-up of cytologic examinations of specimens obtained from 187 patients by gastric lavage with saline solution, without the use of enzymes or abrasive devices, showed a false positive rate of 0.6 per cent and a false negative rate of 17 per cent.To achieve satisfactory results with such examinations, great attention to detail in obtaining, preparing and examining the specimens is essential. Cytologic examination should be done in any case in which gastric cancer is suspected clinically or roentgenographic or gastroscopic findings arouse suspicion.     

Accuracy of liquid-based Pap tests: comparison of concurrent liquid-based tests and cervical biopsies on 782 women with previously abnormal Pap smears

OBJECTIVE: To evaluate the diagnostic performance of a liquid-based Pap test, the ThinPrep Pap test (TP) (Cytyc Corp., Boxborough, Massachusetts, U.S.A.), by comparing concurrent TP and cervical biopsy results on 782 patients who were referred for colposcopy because of previously abnormal conventional Pap smears (CPs). STUDY DESIGN: The ability of TP diagnoses of atypical cells of undetermined significance (ASC-US) and squamous intraepithelial lesions (SILs) to predict biopsy diagnoses of cervical intraepithelial neoplasia (CIN) was analyzed using chi2 and McNemar tests. RESULTS: The rate of agreement between diagnoses of SIL by TP and CIN by biopsy was 74.7%. ASC-US accounted for 16.0% of TP diagnoses. ASC-US had biopsy diagnoses of CIN 1 in 60% and CIN 2/3 in 12.8% of cases. For TP diagnosis of low grade SIL, biopsy diagnoses of CIN 2/3 were found in 13.5% of cases. For TP diagnoses of ASC-US and higher, the proportions of TP and cervical biopsies in comparable diagnostic categories were statistically significant (p < 0.001), with TP having sensitivity of 89.4% and positive predictive value of 89.7% for the detection of CIN. The false positive rate for TP was 8.1%, but rescreening confirmed the presence of abnormal cells in 51 of 63 (81.0%) cases of ASC-US or higher having negative biopsies. TP had a false negative rate of 8.3% and negative predictive value of 61.3%. Rescreening showed that most (77.6%) of the false negative TP specimens failed to have abnormal cells on the slides. CONCLUSION: For patients having previously detected cervical abnormalities by CP, concurrent TP demonstrated the following: (1) that it has high diagnostic accuracy for SIL, (2) that ASC-US was diagnostically equivalent to LSIL, and (3) that false negative TP for SIL can be attributed primarily to sampling rather than cytotechnologists' screening errors.     

Les anticorps fluorescents dans le diagnostic bactériologique des Escherichia coli entéro-pathogènes: Comparaison avec l'isolement de ces germes par culture classique

Enteropathogenic E. coli were sought routinely by the fluorescent antibody technique, using monovalent and polyvalent conjugates (rhodamine sulfonyl· chloride, fluorescein and rhodamine isothiocyanate).In 2061 stool specimens examined with monovalent antibody to E. coli 0127:B8, there were 61 false positives, 14 of which were from previously known cases of E. coli 0127:B8 infection, and 33 specimens that were negative by fluorescence but positive on culture. In 457 stool specimens examined with polyvalent antiserum, there were 15 false positives, five of which came from cases previously infected by the corresponding serotypes, and there were 20 specimens negative by fluorescence but positive on culture. The disagreement amounted, therefore, to 4.6% in the former instance and 7.6% in the latter. This fluorescent technique permits rapid sufficiently precise detection of enteropathogenic E. coli in stools.     

Novel developments in rapid mycotoxin detection

Rapid antibody-based mycotoxin screening techniques are designed to be used outside a laboratory environment, at the place of sampling. Results are expected immediately, so that commodities can be further processed without delay. Because they are used for mycotoxin analysis, very low levels (ppb and ppt range) should be detected. A further requirement is that the obtained results are accurate with a false negative rate of <5% at the level of interest. At first, plastic microtiter plates were used as solid phase materials for immobilizing antibodies (enzyme-linked immunosorbent assays). However, to increase speed and user-friendliness, plastics were replaced by microporous membranes. As an example a flow-through enzyme immunoassay for the detection of fumonisins in cornflakes with a cut-off value of 275 μg/kg is described. No false negative results were observed and the false positive rate was 18%. However, enzyme labels, used to enable visual evaluation of results, did not seem to be completely satisfactory in terms of stability and repeatability of the generated signal. Therefore microparticle labels such as colloidal gold particles are used more and more,e.g. in a lateral flow dipstick immunoassay. When applied to the detection of aflatoxin B₁ in pig feed a cut-off value of 5 μg/kg could be reached with no false negative results and a false positive rate of only 10%. Sample pretreatment for screening techniques should be rapid and simple. Preferably a simple solvent extraction is used, followed by a filtration and dilution step. However, for strongly coloured or complex food matrices, this did not seem to work. The combination of clean-up and detection in one single test device is a new approach. When using this clean-up tandem assay column for the detection of ochratoxin A in roasted coffee, a cut-off value of 6 μg/kg was reached. No false positive results were obtained, however, the false negative rate was 8%. Presented at the EU-USA Bilateral Workshop on Toxigenic Fungi & Mycotoxins, New Orleans, USA, July 5–7, 2005 Financial support: Belgian Federal Science Policy Office (SPSDII-CPAA 15 project), IWT-Flanders (research project 020448/Toxi-Test) and Bijzonder Onderzoeksfonds Ghent University (01 1D02803)     

Evaluation of the Cost Effectiveness of Establishing A Fine Needle Aspiration Cytology Clinic In A Hospital Out-Patient Department

In an attempt to reduce the number of inadequate smears processed by our laboratory and the false negative rate of fine needle aspiration cytodiagnosis, we have introduced a fine needle aspiration cytology service where aspirates are taken by the cytopathologist in a clinic. In the 12 month period since the introduction of this service, the number of inadequate smears fell sharply. Nine per cent of the specimens were inadequate compared with 43% of specimens from other sources. The establishment of the clinic resulted in a threefold reduction in the cost of diagnosing breast lesions.     

Multi-centre testing and validation of current protocols for the identification of Gyrodactylus salaris (Monogenea)

Despite routine screening requirements for the notifiable fish pathogen Gyrodactylus salaris, no standard operating procedure exists for its rapid identification and discrimination from other species of Gyrodactylus. This study assessed screening and identification efficiencies under real-world conditions for the most commonly employed identification methodologies: visual, morphometric and molecular analyses. Obtained data were used to design a best-practice processing and decision-making protocol allowing rapid specimen throughput and maximal classification accuracy. True specimen identities were established using a consensus from all three identification methods, coupled with the use of host and location information. The most experienced salmonid gyrodactylid expert correctly identified 95.1% of G. salaris specimens. Statistical methods of classification identified 66.7% of the G. salaris, demonstrating the need for much wider training. Molecular techniques (internal transcribed spacer region-restriction fragment length polymorphism (ITS-RFLP)/cytochrome c oxidase I (COI) sequencing) conducted in the diagnostic laboratory most experienced in the analysis of gyrodactylid material, identified 100% of the true G. salaris specimens. Taking into account causes of potential specimen loss, the probabilities of a specimen being accurately identified were 95%, 87% and 92% for visual, morphometric and molecular techniques, respectively, and the probabilities of correctly identifying a specimen of G. salaris by each method were 81%, 58% and 92%. Inter-analyst agreement for 189 gyrodactylids assessed by all three methods using Fleiss’ Kappa suggested substantial agreement in identification between the methods. During routine surveillance periods when low numbers of specimens are analysed, we recommend that specimens be analysed using the ITS-RFLP approach followed by sequencing of specimens with a “G. salaris-like” (i.e. G. salaris, Gyrodactylus thymalli) banding pattern. During periods of suspected outbreaks, where a high volume of specimens is expected, we recommended that specimens be identified using visual identification, as the fastest processing method, to select “G. salaris-like” specimens, which are subsequently identified by molecular-based techniques.     

GRAMA: genetic mapping analysis of temperature gradient capillary electrophoresis data

Temperature gradient capillary electrophoresis (TGCE) is a high-throughput method to detect segregating single nucleotide polymorphisms and InDel polymorphisms in genetic mapping populations. Existing software that analyzes TGCE data was, however, designed for mutation analysis rather than genetic mapping. Genetic recombinant analysis and mapping assistant (GRAMA) is a new tool that automates TGCE data analysis for the purpose of genetic mapping. Data from multiple TGCE runs are analyzed, integrated, and displayed in an intuitive visual format. GRAMA includes an algorithm to detect peaks in electropherograms and can automatically compare its peak calls with those produced by another software package. Consequently, GRAMA provides highly accurate results with a low false positive rate of 5.9% and an even lower false negative rate of 1.3%. Because of its accuracy and intuitive interface, GRAMA boosts user productivity more than twofold relative to previous manual methods of scoring TGCE data. GRAMA is written in Java and is freely available at .     

多仪器联合分析尿液的应用价值

目的:探讨干化学分析、UF-100尿有形成分分析与DIASYS沉渣镜检分析联合检测尿液的应用价值。方法:用尿干化学分析仪MiditronM、UF-100型尿液分析仪、DIASYS R/S 2003沉渣分析仪分别对390例随机尿液标本进行检测。结果:以DIASYSR/S 2003沉渣分析结果为标准,干化学法与DIASYS镜检法相比,测定红细胞阳性符合率为92.68%,假阳性为13.96%,假阴性为7.32%;测定白细胞阳性符合率为58.91%,假阳性为9.58%,假阴性为41.09%;UF-100与DIASYS相比,检测红细胞阳性符合率为82.93%,假阳性为7.47%,假阴性为17.07%;测定白细胞阳性符合率为81.40%,假阳性为12.26%,假阴性为18.60%;检测上皮细胞、小园上皮细胞、管型、结晶和类酵母菌阳性符合率较低,分别为69.09%、60.53%、68.18%、78.95%、57.14%。390例尿标本中干化学法白细胞、红细胞、蛋白及亚硝酸盐均阴性的176例标本中,UF-100检测红细胞阳性14例,白细胞阳性19例,DIASYS检出红细胞阳性5例,白细胞阳性11例。结论:UF-100型尿沉...     

Flow cytometric prescreening of cervical smears.

One-parameter (nuclear DNA) and two-parameter (nuclear DNA and protein or cellular light scatter) measurements of cervical smears were performed using an ICP 11 and a cytofluorograf 4800 respectively. A total of about 1000 cases was analyzed. For the estimation of nuclear DNA alone two fluorochromes were tested (ethidium bromide (EB) and mithramycin (MMC)) combined with three different methods of cell preparation. For the two-parameter measurements cells were double stained with EB and fluorescein isothiocyanate (FITC). Red fluorescence (EB) versus green fluorescence (FITC) or red fluorescence versus scatter were recorded. A computer analysis of the one-parameter histograms was performed using discriminant analysis and the results were compared with the cytodiagnosis of microscopic specimens stained with the Papanicolaou technique. The error rates of the flow cytometric (FCM) data were as follows: (a) standard EB staining, 11% false negative, 26% false positive, 6% unsatisfactory results; (b) pepsination of vital cells and EB staining, 12% false negative, 14% false positive and 4% unsatisfactory results; (c) MMC staining, 10% false negative, 65% false positive and 5% unsatisfactory results. Our two-parameter measurements prove that, as confirmed by cell sorting, red fluorescence versus scatter allows separation of at least three subpopulations in most analyzed samples: (a) anucleated cells; (b) leukocytes; and (c) intermediate and superficial cells.     

A comparison of HPLC and TLC in the assessment of amniotic fluid lecithin/sphingomyelin ratio

One hundred amniotic fluid (AF) specimens of women at 28 to 37 weeks gestation were obtained during labor for lecithin/sphingomyelin ratio (L/S ratio) study by using high performance liquid chromatography (HPLC) and thin layer chromatography (TLC). The results are divided into two groups: one with L/S ratio at or more than 2, and the other with L/S ratio of less than 2. Of the 80 measured by HPLC with the ratio of above 2 or 1.3%, one suffered respiratory distress syndrome (RDS); of the 65 measured by TLC also with the ratio of above 2 or 3.1%, two developed RDS. The result bears no great significance here. However, in the groups with L/S ratio of less than two, the results were very significant: 17 of 20 estimated by HPLC happened to have RDS for an incidence of false positive rate of 15% compared with a 54.3% in 16 of the 35 women evaluated by TLC.