Irregular antibody screening by Groupamatic at the Paris (C.N.T.S.) and Toulouse (C.R.T.S.) blood transfusion centers

We report here the experience of 3 years of irregular antibody automated screening with Groupamatic, that is to say of 661,511 samples tested in Paris and 269, 162 samples tested in Toulouse. The positive reactions in Paris were 16,296 (2.46%) out of which 2,021 irregular antibodies were identified (0.30%). The positive reactions in Toulouse were 8,266 (3.07%) out of which 2,138 irregular antibodies were identified (0,79%). The difference between the number of screened positive reactions and the identified one is due to false positive reactions (half of the cases) and to autoantibodies whose number is roughly the same than the number of identified alloantibodies. During the years 1970, 1971 and 1972, the irregular antibodies were systematically screened in Toulouse on all blood donors with a manual technique on opaline plate using enzyme treated red cell tests (papain). 7,147 positive reactions were detected, out of 240,080 tests (2.98%). 1,954 (0,81%) were alloantibodies and 1,529 (0.64%) autoantibodies; 3.455 were false positive reactions and 209 were non identified antibodies. These figures are superimposed with those obtained with Groupamatic during the following years, thus pointing out the interest of this equipment.     

Automated Fluorescent Treponemal Antibody Test: Instrument and Evaluation

Two evaluations of the automated fluorescent treponemal antibody (AFTA) test for the serodiagnosis of syphilis are described. The results of AFTA and manually performed fluorescent treponemal antibody-absorption (FTA-ABS) tests were compared on serum samples from clinically defined donor groups, and the reproducibility of each procedure was studied. Significant improvement of AFTA test results was obtained in the most recent study after developmental modifications of the instrument and test technique. AFTA test agreement with both syphilis and nonsyphilis categories was considered good. With the increasing usage of the FTA-ABS test as an effective tool for the diagnosis of syphilis, successful automation of this procedure is particularly timely and significant.     

Quantitative measurement of red cell antigens with Groupamatic]

Groupamatic 360 can be adapted to the quantitative measurement of erythrocyte agglutination reactions. Here the authors give the necessary conditions to obtain the best sensitivity and the best reproductibility. They have considered globular suspensions, incubation of the erythrocyte antiserums mixtures, centrifugation, agitation, photometric measurement. As concerns the quantitative technique, we used the average peripheral measurement obtained in the twleve cuvettes of the same selected disc sector. The calibration is fixed to 50 mV for a non agglutinated red cell suspension and to 950 mV for albumin in a saline solution. In these conditions, the best discrimination is obtained for agglutination with measures between 400 and 800 mV. The maximum sensitivity is reached with reactions corresponding to 600 mV, that is to say in our selected conditions with 50% of free red cells. The choice of the zone between 500 and 600 mV enables to detect variations from 1.5 to 2% in an antibody solution concentration. This zone is particularly favorable for quantitative work. Although a part of these operations in this preliminary work were manually effected, the automatic centrifugation, agitation, photometric measurement and results print out make possible a large number of measurements with very satisfactory reproductibility conditions. Besides, these experiments pointed out some factors of improvement which will be helpful for the new 360 G Groupmatic equipments.     

Detection of HBs with latex tests]

Since 1971, several inert particles have been tested and proposed for HBs antigen screening, none of them being really used in routine. An evaluation is realized and presented, pointing out the main characteristics and the practical problems. We report our experience on two types of latex, simultaneously conducted through manual techniques and through Groupamatic 360 equipments. For one of them, TG Antigex, sensitivity and specificity are very satisfying and several batches are presently tested in order to quantify the reproducibility and stability features of the reagent.     

Systematic assignment of thermodynamic constraints in metabolic network models

Background  

The availability of genome sequences for many organisms enabled the reconstruction of several genome-scale metabolic network models. Currently, significant efforts are put into the automated reconstruction of such models. For this, several computational tools have been developed that particularly assist in identifying and compiling the organism-specific lists of metabolic reactions. In contrast, the last step of the model reconstruction process, which is the definition of the thermodynamic constraints in terms of reaction directionalities, still needs to be done manually. No computational method exists that allows for an automated and systematic assignment of reaction directions in genome-scale models.     

Further Evaluation of the Automated Fluorescent Treponemal Antibody Test for Syphilis

Improvements in the equipment for the automated fluorescent treponemal antibody (AFTA) test for syphilis prompted this comparative study of the AFTA and its manual counterpart, the fluorescent treponemal antibody-absorption (FTA-ABS) test. The AFTA equipment operated satisfactorily, required only minimal monitoring, and afforded a three-to fourfold increase over the number of sera that could be tested manually by one serologist. The AFTA and FTA-ABS tests agreed well with only 2.1% of the sera yielding conflicting results. The AFTA was less precise than the FTA-ABS on sera retested because of original conflicting results and on sera retested within the same run to determine reproducibility. However, these differences were not large, and AFTA test performance was considered to be within the limits acceptable for a diagnostic serological procedure.     

Evaluation of the Qualitative and Automated Quantitative Microhemagglutination Assay for Antibodies to Treponema pallidum

Qualitative and quantitative microhemagglutination assays for antibodies to Treponema pallidum (MHA-TP) were performed on 314 syphilitic and 597 presumably nonsyphilitic sera, and the results were compared with those of the fluorescent treponemal antibody-absorbed (FTA-ABS), the Treponema pallidum immobilization (TPI), and the Veneral Disease Research Laboratory (VDRL) tests. MHA-TP sensitivity was similar to that of the other tests in all stages of syphilis except primary syphilis, in which MHA-TP reactivity was only 64% compared with 82% in the FTA-ABS test, 73% in the VDRL test, and 67% in the TPI test. MHA-TP specificity was satisfactory and comparable to that of the other treponemal tests. Quantitation of the MHA-TP test was automated by use of Autotiter II equipment. Titers tended to become elevated later in the course of syphilis and to remain elevated longer than did VDRL titers. Reproducibility of the quantitative MHA-TP test was satisfactory, with duplicate tests agreeing within one doubling dilution on 97.5% of 351 reactive sera. Poor reproducibility was obtained with sera giving minimal reactions in the qualitative test, and such sera should be routinely retested. The MHA-TP is less time-consuming and costly than the FTA-ABS test and could be used in conjunction with the VDRL or another reagin test for syphilis to eliminate a large number of the FTA-ABS tests now required.     

Tests for Syphilis—The Increasing Incidence of Biologic False Positive Reactions As Measured by Treponema Pallidum Immobilization

Analysis of data obtained in 25,787 Treponema pallidum immobilization tests in a ten-year period showed an increase in the incidence of biologic false positive (BFP) reactions for syphilis, and a decrease in the incidence of reactive TPI tests. The percentage of BFP tests increased from 54.2 per cent in 1953 to 70.7 per cent in 1962.Reaction to a standard serologic test for syphilis (STS) indicates only that the patient may have syphilis. A subsequent non-reactive TPI test remains the best procedure for ruling out a diagnosis of syphilis. Thus the clinician should be made more aware of the fact that a biologic false positive reaction strongly implies the existence of another disease, the cause of which should be investigated.     

Routine Serologic Testing for Syphilis in a Community Medical Practice

A retrospective review of 8,100 serologic tests for syphilis ordered during a 42-month period yielded positive rapid plasma reagin test results in 127 patients (1.6 percent) and a positive fluorescent treponemal antibody absorption reaction in 91 patients (1.1 percent). Of the 36 cases of biologic false-positive reactions, most were in prenatal patients. Forty-six cases of syphilis were previously undiagnosed but antibiotic therapy was given in only 26 of the patients. Some 24 percent of syphilitic patients were not treated because the positive serologic findings were overlooked. Cerebrospinal fluid determinations were analyzed and cost-effectiveness of finding a single case of previously undiagnosed syphilis was calculated. We found that routine serologic tests and cerebrospinal fluid studies for syphilis in asymptomatic patients had low rates of positivity in our community hospital and outpatient practice.     

Detection and titration of anti-tetanus antibodies by passive hemagglutination]

Human red cells O Rh + are coated with purified tetanus toxoid, by means of chromic chloride as coupling agent. With this reagent and by passive haemagglutination technique, antitetanus antibodies can be automatically screened and titrated in blood donnors plasmas, on Groupamatic equipments. Comparative results of three techniques: counter immunoelectrophoresis; manual passive haemagglutination; automatic passive haemagglutination are given, 4, 92 percent among the 1.219 tested blood donnors plasmas have an antibody greater than or equal to 5 U.I., 16, 24 percent have a titre between 1 and 5 U.I.     

Automatic pre-transfusion serology]

This paper describes an automated apparatus combining Rosenfield's and Lalezari's antibody screening and identification basic technics. PVP bromelin and low ionic strength acid polybren channels are used; agglutinates are decanded; the remaining cells are hemolyzed and the optical density is then measured through a colorimeter and recorded on a chart; speed is of 40 samples an hour. This machine was also used for irregular antibody screening and identification. Sensitivity is shown to be equal to that of manual technics for ABO, Lewis, Lutheran as well as K, S, M, Kpb, Xga, U and Vel antibodies detection. Nevertheless, a much greater sensitivity is achieved (titers 3 to 10 times higher) than by manual technics for Rh, -k, S, Fya antibodies detection. Polybren channel is suitable for anti-Rh, Duffy, I and M (human detection; bromelin channel however, has a greater sensitivity for other specificities. Anti-M and anti-N sera from rabbits were shown to be non specific when using this machine. Over almost 15 000 sera tested, no antibody (detected by manual techniques) escaped the automated screening. This antibody detection machine was applied to compatibility tests prior to transfusion. (21 480 units were tested. aimed to be transfused to 5 611 patients). A third, PVP without bromelin, was set in parallel in order not to let escape any anti-M, even a weak one. The sera distributor was slaved to the cells distributor so that the whole procedure was automated. Furthermore, each serum was tested against red cells to be transfused, but also against the patient's own red cells to be transfused, but also against the patient's own red cells and against two selected red cells panels, so as to ensure irregular antibody detection at the same time. Using this machine, 3 to 4% of the cell samples were rejected, i.e. more than with usual techniques. All manually detected antibodies were identified, but also some others, which showed only weak reactions by classical techniques. Total results can be obtained within 20 to 30 minutes, which is quite rapid, compared to techniques using for example antiglobulin tests.     

PUBLIC HEALTH ASPECTS OF LYMPHOGRANULOMA VENEREUM

The clinical symptoms of lymphogranuloma venereum with the serious pathologic changes often occurring in the late stages of the disease warrant greater attention to the disease.The reported ratio of cases of lymphogranuloma venereum to cases of syphilis and gonorrhea is much higher in San Francisco than in other metropolitan ports of western United States, apparently because of greater use of diagnostic tests for the disease.Tests of persons likely to be exposed and other persons not likely to be exposed to venereal diseases indicate that a positive reaction to a Frei test implies past or present infection with lymphogranuloma venereum.Positive reactions to complement fixation tests are notably more frequent than positive response to Frei tests. The complement fixation test appears to be an unreliable diagnostic aid.The frequency of positive reactions associated with other venereal diseases, and their infrequency otherwise, suggests that lymphogranuloma venereum may exist, unrecognized, in many persons, who may be, potentially at least, carriers of the disease.     

一期梅毒实验室诊断差异性研究

目的通过梅毒螺旋体初筛试验、确认试验和鉴别诊断试验,探讨一期梅毒实验室诊断差异性,最大限度减少漏诊与误诊,为深入研发新型早期梅毒诊断试剂奠定基础。方法依据2000年中国卫生部防疫司颁布的性病诊断标准,临床筛选一期梅毒患者86例（研究组）和非梅毒患者100例（对照组）,对患者血清进行甲苯胺红不加热血清试验（TRUST）初筛和梅毒螺旋体明胶颗粒凝集试验（TPPA）确认。筛选临床体征、TRUST法和TPPA法三者结果有差异的患者进一步鉴别诊断,鉴别诊断主要应用荧光定量PCR（FQ-PCR）法、免疫PCR法与自身抗体检测等试验。结果初筛TRUST法灵敏度和特异性分别为62．8％、93．0％;确认TPPA法灵敏度与特异性分别为66．3％、100％。TRUST法和TPPA法两者结果差异占12．8％;临床体征诊断、TRUST法和TPPA法三者结果差异占41．9％。TPPA法与TRUST法两者均阴性的一期梅毒患者中,FQ-PCR阳性率达88．0％,免疫PCR阳性率占40．0％。TPPA法阳性、TRUST法阴性的一期梅毒患者免疫PCR法与TPPA法结果一致;TPPA法阴性、TRUST法阳性11例患者中结核抗体阳性2例,类风湿因子阳性3例与抗Sm抗体结果阳性6例。结论一期梅毒患者实验室诊断结果差异性较大,漏诊与误诊的比例较高,有待研发新型的诊断试剂和提高诊断水平。     

Microsequence analysis of peptides and proteins. IX. Manual gas-phase microsequencing of multiple samples

A novel apparatus for performing manual gas-phase Edman chemistry on protein and peptide samples is described. Edman chemistry is performed in 6 to 10 Teflon continuous flow reactors (CFR), previously described by J.E. Shively et al. (1987) Anal. Biochem. 163, 517-529). The CFRs are packed with 10-15 mg of Polybrene-coated spherical silica (Porasil B, Waters Associates). The gas-phase coupling reagent and cleavage reagent are 5% aqueous triethylamine and anhydrous trifluoroacetic acid, respectively, delivered by a stream of argon gas. The delivery of the gas-phase reagents is manually controlled with Hamilton 3-way valves and 2-way valves, and that of the solvents, ethyl acetate and butyl chloride, by syringe pipetting. The average cycle time is 15-20 min for 6 to 10 samples run simultaneously. Conversion of the anilinothiazolinone to phenylthiohydantoin (PTH) amino acid derivatives is accomplished manually with 25% aqueous trifluoroacetic acid. The PTH amino acids are analyzed by reversed-phase HPLC using an autosampler for handling multiple samples. Excellent results were obtained in the 100-200 pmol range. Protein samples can be sequenced from 15-20 cycles, and peptide samples usually to the COOH terminus. Initial yields ranged from 30 to 60% and repetitive yields ranged from 90 to 96%. The sample washout and size of background peaks are significantly reduced, compared to older methods of manual sequence analysis. The yields and background signal to noise are comparable to automated gas-phase Edman chemistry. The improved manual Edman described represents a low cost alternative to automated sequence analysis, and has the advantage being able to process multiple samples simultaneously.     

Automated measurement of MIB-1 positive area as an alternative to counting in follicular lymphoma

Manual counting of MIB-1 positive cells which has been suggested as an alternative to centroblast counting for the diagnostic grading of follicular lymphoma is a laborious task. In this study, the validity of automated measurement of the MIB-1 positive area is analyzed as an alternative approach. Archival MIB-1 stained tissue sections of 15 follicular lymphomas were assessed manually and automatically by three independent observers. Concordance correlation coefficients and coefficients of variation were calculated to study reproducibility and variability of both methods and to compare result from both methods. A good concordance was observed between the two methods. The reproducibility of the automated method was slightly better than the manual counting of positive nuclei. Measurement of MIB-1 positive surface area may be used as a simple and fast alternative to tedious manual counting of positive nuclei as a potential help in follicular lymphoma grading.