Analysis of alloantisera against bovine lymphocytes. Joint report of the 1st International Bovine Lymphocyte Antigen (BoLA) Workshop

The results and agreements of the 1st international BoLA workshop, held in Edinburgh, Scotland in August 1978, are reported. Most of these concern the results from a comparison test of 249 alloantisera to bovine lymphocytes, the antisera being contributed by 9 laboratories. These sera were compared directly in Edinburgh on a panel of lymphocytes from 130 cattle of 21 breeds. In the micro-lymphocytotoxicity test used 75% of the sera reacted. Sixty eight of these sera were grouped into clusters according to their reaction patterns against the lymphocyte panel. Eleven of these clusters were clearly defined and were given workshop BoLA designations. In addition 22 sera were assigned to subgroups of the agreed clusters. There was no evidence that the method of production of the sera had any effect on their specificity.
Although genetic data was not available, the phenotypes of the test panel of lymphocytes are consistent with the clusters detecting antigens controlled by multiple alleles at a single autosomal locus. It was agreed to name the genetic region where this putative locus is located BoLA (bovine lymphocyte antigen).     

Analysis of alloantisera against bovine lymphocytes. Joint report of the 1st International Bovine Lymphocyte Antigen (BoLA) workshop

The results and agreements of the 1 international BoLA workshop, held in Edinburgh, Scotland in August 1978, are reported. Most of these concern the results from a comparison test of 249 alloantisera to bovine lymphocytes, the antisera being contributed by 9 laboratories. These sera were compared directly in Edinburgh on a panel of lymphocytes from 130 cattle of 21 breeds. In the microlymphocytotoxicity test used 75% of the sera reacted. Sixty eight of these sera were grouped into clusters according to their reaction patterns against the lymphocyte panel. Eleven of these clusters were clearly defined and were given workshop BoLA designations. In addition 22 sera were assigned to subgroups of the agreed clusters. There was no evidence that the method of production of the sera had any effect on their specificity. Although genetic data was not available, the phenotypes of the test panel of lymphocytes are consistent with the clusters detecting antigens controlled by multiple alleles at a single autosomal locus. It was agreed to name the genetic region where this putative locus is located BoLA (bovine lymphocyte antigen).     

Joint report1 of the First International Workshop on Lymphocyte Alloantigens of the Horse held 24–29 October 1981

Six equine lymphocyte alloantigen (ELA) specificities were defined by an international antiserum comparison test and workshop held in 1981. Twelve laboratories from four countries submitted 195 antisera for analysis. The antisera were exchanged among the 12 laboratories and tested in a standard lymphocyte microcytoxicity assay against the isolated lymphocytes at 1009 horses of several breeds. The data was pooled and analysed by a single computer analysis. The calculated χ² values of all cells with all antisera provided comparisons between antisera. Fifteen antisera clusters were formed by this analysis, but only six of these clusters met the criteria established by the workshop for the identification of ELA antigens. No horses of the cell panel positively reacted with more than two of these six specificities. The consensus of the participants, although not substantiated in this workshop, was that these six clusters of antisera define alleles of a single genetic region, the ELA region, and it is likely that this genetic region is the major histocompatibility complex of the horse.     

Joint report of the First International Workshop on Lymphocyte Alloantigens of the Horse held 24-29 October 1981

Six equine lymphocyte alloantigen (ELA) specificities were defined by an international antiserum comparison test and workshop held in 1981. Twelve laboratories from four countries submitted 195 antisera for analysis. The antisera were exchanged among the 12 laboratories and tested in a standard lymphocyte microcytoxicity assay against the isolated lymphocytes at 1009 horses of several breeds. The data was pooled and analysed by a single computer analysis. The calculated chi 2 values of all cells with all antisera provided comparisons between antisera. Fifteen antisera clusters were formed by this analysis, but only six of these clusters met the criteria established by the workshop for the identification of ELA antigens. No horses of the cell panel positively reacted with more than two of these six specificities. The consensus of the participants, although not substantiated in this workshop, was that these six clusters of antisera define alleles of a single genetic region, the ELA region, and it is likely that this genetic region is the major histocompatibility complex of the horse.     

Proceedings of the Second International Bovine Lymphocyte Antigen (BoLA) Workshop

The results of the second International BoLA Workshop, held in Wageningen, Netherlands in July 1980, are reported. These results arise from a comparison of 362 alloantisera to bovine lymphocytes, originating from 9 laboratories. The an-tisera were tested against a selected panel of 144 lymphocyte samples, originating from 7 laboratories. Some of the antisera and lymphocytes had also been tested during the First International BoLA Workshop in 1978.
Ten of the eleven specificities defined at the first workshop were confirmed, and an additional six new specificities were designated. Two of the additional specificities are subgroups of the w6 specificity. The data from this workshop are consistent with the hypothesis that BoLA antigens are controlled by a series of codominant alleles at a single autosomal locus.     

Evaluation of the system for controlling the quality of the HBsAg detection in the system of screening laboratories

The two-stage control system, ensuring the high quality of serological investigations in the network of screening laboratories in Moscow was developed and introduced into practice. At the first stage the entry control of the quality of the test system for the detection of HbsAg, coming to the screening laboratories, is made in the reference laboratory with the use of specially developed "representative" panels, as well as the test systems for comparison ("reference" test systems). Then "minipanels" are formed from specimens included into the "representative" panels, which are used for the evaluation of the quality of laboratory investigations made in the screening laboratories. The high quality of such system for controlling the quality of the detection of HBsAg in the screening laboratories of Moscow is shown.     

In vitro micronucleus assay with Chinese hamster V79 cells - results of a collaborative study with in situ exposure to 26 chemical substances

A collaborative study with 10 participating laboratories was conducted to evaluate a test protocol for the performance of the in vitro micronucleus (MN) test using the V79 cell line with one treatment and one sampling time only. A total of 26 coded substances were tested in this study for MN-inducing properties. Three substances were tested by all 10 laboratories and 23 substances were tested by three or four laboratories in parallel. Six aneugenic, 7 clastogenic and 6 non-genotoxic chemicals were uniformly recognised as such by all laboratories. Three chemicals were tested uniformly negative by three laboratories although also clastogenic properties have been reported for these substances. Another set of three clastogenic substances showed inconsistent results and one non-clastogenic substance was found to be positive by one out of three laboratories. Within the study, the applicability of the determination of a proliferation index (PI) as an internal cytotoxicity parameter in comparison with the determination of the mitotic index (MI) was also evaluated. Both parameters were found to be useful for the interpretation of the MN test result with regard to the control of cell cycle kinetics and the mode of action for MN induction. The MN test in vitro was found to be easy to perform and its results were mainly in accordance with results from chromosomal aberration tests in vitro.     

A comparison of lymphocyte antigen specificities in Norwegian and Swiss goats

In all, 363 alloantireagents were tested in Berne, Switzerland and in Oslo, Norway against lymphocytes from 1679 goats of different breeds. The same lymphocytotoxicity test was used at both laboratories. The test data were pooled, and correlation coefficients for pairs of sera were used to group the sera in clusters. Twelve clusters were accepted as defining lymphocyte antigen specificities believed to be coded by genes within the major histocompatibility complex (MHC). The specificities defined by these clusters were designated Eu1-Eu12. Two clusters defined specificities which were not coded from loci within the MHC. These were designated GLY-1.1 and GLY-2.1, and the loci GLY-1 and GLY-2, respectively. GLY-1.1 was also located on erythrocytes.     

A comparison of lymphocyte antigen specificities in Norwegian and Swiss goats

Summary. In all, 363 alloantireagents were tested in Berne, Switzerland and in Oslo, Norway against lymphocytes from 1679 goats of different breeds. The same lymphocytotoxicity test was used at both laboratories. The test data were pooled, and correlation coefficients for pairs of sera were used to group the sera in clusters. Twelve clusters were accepted as defining lymphocyte antigen specificities believed to be coded by genes within the major histocompatibility complex (MHC). The specificities defined by these clusters were designated Eu 1-Eu12. Two clusters defined specificities which were not coded from loci within the MHC. These were designated GLY-1.1 and GLY-2.1, and the loci GLY-1 and GLY-2, respectively. GLY-1.1 was also located on erythrocytes.     

Evaluation of radiation-induced chromosomal aberrations in human peripheral blood lymphocytes in vitro results of an IAEA-coordinated programme

The results of an IAEA coordinated programme on radiation induced chromosomal aberrations in human peripheral blood lymphocytes in vitro are presented. In a master experiment, a whole blood sample from one donor was irradiated with 200 R of X-rays. Different fixation times from 46 to 82 h were used. The progression of cells into mitosis was monitored by BrdUrd incorporation. 14 investigators took part in the scoring of chromosomal aberrations. The main conclusions of this study are: (1) The mean frequencies of aberrations changed with fixation time. (2) The number of cells scored as aberrant by different laboratories was very similar, but there was variability in the number of aberrations scored per aberrant cell. (3) The differences in the frequencies of aberrations between laboratories were minimal when the scoring was restricted to the first major peak of mitotic activity and sufficient cells were scored.

It is concluded that using controlled experimental conditions, human peripheral blood lymphocytes can effectively be used as a reliable biological dosimeter for absorbed radiation dose.     

Inter-laboratory variability in in vitro spinal segment flexibility testing

In vitro spine flexibility testing has been performed using a variety of laboratory-specific loading apparatuses and conditions, making test results across laboratories difficult to compare. The application of pure moments has been well established for spine flexibility testing, but to our knowledge there have been no attempts to quantify differences in range of motion (ROM) resulting from laboratory-specific loading apparatuses. Seven fresh-frozen lumbar cadaveric motion segments were tested intact at four independent laboratories. Unconstrained pure moments of 7.5 Nm were applied in each anatomic plane without an axial preload. At laboratories A and B, pure moments were applied using hydraulically actuated spinal loading fixtures with either a passive (A) or controlled (B) XY table. At laboratories C and D, pure moments were applied using a sliding (C) or fixed ring (D) cable–pulley system with a servohydraulic test frame. Three sinusoidal load-unload cycles were applied at laboratories A and B while a single quasistatic cycle was applied in 1.5 Nm increments at laboratories C and D. Non-contact motion measurement systems were used to quantify ROM. In all test directions, the ROM variability among donors was greater than single-donor ROM variability among laboratories. The maximum difference in average ROM between any two laboratories was 1.5° in flexion-extension, 1.3° in lateral bending and 1.1° in axial torsion. This was the first study to quantify ROM in a single group of spinal motion segments at four independent laboratories with varying pure moment systems. These data support our hypothesis that given a well-described test method, independent laboratories can produce similar biomechanical outcomes.     

Rate of Pyrite Bioleaching by Thiobacillus ferrooxidans: Results of an Interlaboratory Comparison

Ten laboratories participated in an interlaboratory comparison of determination of bioleaching rates of a pyrite reference material. A standardized procedure and a single strain of Thiobacillus ferrooxidans were used in this study. The mean rate of bioleaching of the pyrite reference material was 12.4 mg of Fe per liter per h, with a coefficient of variation (percent relative standard deviation) of 32% as determined by eight laboratories. These results show the precision among laboratories of the determination of rates of pyrite bioleaching when a standard test procedure and reference material are used.     

Proficiency testing in cytology laboratories in Ontario, Canada: a decade of experience. I. Introduction and description of the testing model

There are few formally documented proficiency testing programs for cytology laboratories, and those that have been documented are not entirely comparable in format. The first of three papers documenting a mandatory universal proficiency testing program for cytology laboratories in the Province of Ontario, Canada, presents data on the structure and function of the participating laboratories (including a comparison of the data for 1974 and 1980) and on the organization of the testing model (including selection of terminology, construction and use of the survey and assessment of responses). In 1980, of the 463 medical laboratories in Ontario, 91 of 222 hospital laboratories and 65 of 216 nonhospital laboratories were licensed in cytology. In that year, the 156 cytology laboratories processed 1.48 million cytology specimens, 92% of which were gynecologic. Hospital laboratories processed 87.5% of the nongynecologic cytology specimens and 30% of the gynecologic cytology specimens. These proportions have been virtually constant for several years. Between 1974 and 1980, there was a trend in Ontario to fewer laboratories processing less than 5,000 cytology specimens per annum. Subsequent papers in this series describe the results of the initial surveys in this program and a precision study to evaluate the consistency of reporting by individual laboratories.     

An international collaborative study of an anti-infectious bursal disease virus reference serum

Fourteen laboratories participated in a collaborative study of a freeze-dried preparation of anti-infectious bursal disease virus serum to assess the suitability of the serum as a standard for use in the infectious bursal disease virus neutralization test. Ten laboratories carried out micro-virus neutralization tests and six carried out plaque reduction tests, two laboratories carrying out both tests. When titres were expressed as a proportion of that obtained for a reference preparation there was a marked reduction in variation between results from different laboratories. The use of a reference preparation was therefore of value when comparing results from different laboratories. It is proposed that the reference preparation used in this study be used as a standard to facilitate the comparison of results from different laboratories. The proposed standard contains by definition 10,000 UK units.     

Isolation procedures for blood lymphocytes produce artifacts. Detection by changes of electrophoretic mobilities of lymphocytes.

The changes induced in the distribution of the electrophoretic mobility (EPM) of human peripheral blood lymphocytes (HPBL), by various methods used to prepare the lymphocyte suspensions and eliminate platelets from them, were investigated on blood samples collected from healthy individuals and thrombopenic patients. Data showed that the distribution of the lymphocyte EPMs, i.e., the "lymphocyte electrophoregram," was dependent on the method chosen to enrich the suspension in the cell type of interest. The relative percentages of the low and high mobility cells, the two main subpopulations defined by lymphocyte electrophoresis, were different. The most striking artifactual differences in the lymphocyte electrophoregram were induced by the method of elimination of platelets; the distribution was unimodal and asymmetric when thrombin was used and bimodal when the blood sample, or the lymphocyte suspension, was placed on ice for 30 min (as is the practice in some laboratories). The "split" of the lymphocyte electrophoregram was found to be reversible within 90 min. Similar changes were observed on lymphocyte suspensions and blood samples of thrombopenic patients when the step for the elimination of platelets was not involved.     

Isolation procedures for blood lymphocytes produce artifacts

The changes induced in the distribution of the electrophoretic mobility (EPM) of human peripheral blood lymphocytes (HPBL), by various methods used to prepare the lymphocyte suspensions and eliminate platelets from them, were investigated on blood samples collected from healthy individuals and thrombopenic patients. Data showed that the distribution of the lymphocyte EPMs, i.e., the “lymphocyte electrophoregram,” was dependent on the method chosen to enrich the suspension in the cell type of interest. The relative percentages of the low and high mobility cells, the two main subpopulations defined by lymphocyte electrophoresis, were different. The most striking artifactual differences in the lymphocyte electrophoregram were induced by the method of elimination of platelets; the distribution was unimodal and asymmertric when thrombin was used and bimodal when the blood sample, or the lymphocyte suspension, was placed on ice for 30 min (as is the practice in some laboratories). The “split” of the lymphocyte electrophoregram was found to be reversible within 90 min. Similar changes were observed on lymphocyte suspensions and blood samples of thrombopenic patients when the step for the elimination of platelets was not involved.

     

Clinical application of four and five-color flow cytometry lymphocyte subset immunophenotyping

A 1995 survey of clinical flow cytometry laboratories in the United States determined that 63% of clinical laboratories used one or two-color, 33% used three-color, 4% used four-color, and none used five-color panels. We show the feasibility and advantages of acquiring routine clinical four-color, six-parameter and five-color, seven-parameter analysis on blood samples derived from a pediatric population. The panels were evaluated by comparing the following cell characteristics: size, internal structure, and up to five distinct fluorochrome-conjugated antibodies to cell surface antigens: CD3, CD16+CD56, CD19, CD8, and CD4. These samples were processed on a commercially available instrument without any special modifications. A comparison of two-color and four-color as well as four-color and five-color panel analysis showed no statistical difference between the groups. We propose that the five-color, single-tube panel will (1) eliminate the need for isotype controls; (2) the relative proportions of lymphocyte subpopulations may be used to validate the operator-defined window, replacing CD45; (3) eliminate the need to run a common factor, in order to establish and maintain a reproducible lymphocyte window between tubes; and (4) create a more complete clinical picture by generating 32 unique, mutually exclusive phenotypes (permutations). Our results show that it is feasible to acquire and integrate seven-parameter data. This may be a powerful tool for immunophenotyping cells in a modern clinical diagnostic cytometry laboratory.     

Joint report of the Third International Workshop on Lymphocyte Alloantigens of the Horse, Kennett Square, Pennsylvania, 25-27 April 1984

The Third International Workshop on Lymphocyte Alloantigens of the Horse was held on 25-27 April 1984 in Kennett Square, Pennsylvania. Twelve laboratories from five countries participated. The principal purpose of this Workshop was to determine the phenotypic and gene frequencies of the 10 equine lymphocyte antigens (ELA) and a non-ELA lymphocyte antigen, ELY-2.1, in several breeds of horse. A total of 86 alloantisera characterized in previous workshops were tested against lymphocytes from 1179 horses. In addition, several experimental antisera were also tested against the same panel of lymphocytes. As a result of analysis of these data, the Workshop recognized two new equine lymphocyte alloantigens: W11 of the ELA system, and ELY-1.1, an antigen not linked to the ELA system.     

Joint Report of the Third International Workshop on Lymphocyte Alloantigens of the Horse, Kennett Square, Pennsylvania, 25–27 April 1984

Summary. The Third International Workshop on Lymphocyte Alloantigens of the Horse was held on 25–27 April 1984 in Kennett Square, Pennsylvania. Twelve laboratories from five countries participated. The principal purpose of this Workshop was to determine the phenotypic and gene frequencies of the 10 equine lymphocyte antigens (ELA) and a non-ELA lymphocyte antigen, ELY-2.1, in several breeds of horse. A total of 86 alloantisera characterized in previous workshops were tested against lymphocytes from 1179 horses. In addition, several experimental antisera were also tested against the same panel of lymphocytes. As a result of analysis of these data, the Workshop recognized two new equine lymphocyte alloantigens: W11 of the ELA system, and ELY-1.1, an antigen not linked to the ELA system.     

Round robin investigation of methods for the recovery of poliovirus from drinking water.

Six laboratories actively involved in water virology research participated in a methods evaluation study, conducted under the auspices of the American Society for Testing and Materials Committee on Viruses in the Aquatic Environment, Task Force on Drinking Water. Each participant was asked to examine the Viradel (virus adsorption-elution) method with cartridge-type Filterite filters for virus adsorption and organic flocculation and aluminum hydroxide-hydroextraction for reconcentration. Virus was adsorbed to filter media at pH 3.5 and eluted with either glycine buffer (pH 10.5) or beef extract-glycine (pHG 9.0). Considerable variation was noted in the quantity of virus recovered from four 100-liter samples of dechlorinated tapwater seeded with low (350 to 860 PFU) and high (1,837 to 4,689 PFU) doses of poliovirus type 1. To have a more uniform standard of comparison, all the test samples were reassayed in one laboratory, where titers were also determined for the virus seed. Test results of the Viradel-organic flocculation method indicated that the average percentage of virus recovery for low-input experiments was 66%, with a range of 8 to 20% in two laboratories, 49 to 63% in three laboratories, and 198% in one laboratory. For the high-input experiments, two laboratories reported recoveries of 6 to 12%, and four laboratories reported recoveries of 26 to 46%. For the Viradel aluminum hydroxide-hydroextraction procedure, two laboratories recovered 9 to 11%, whereas four obtained 17 to 34% for low-input experiments. For the high-input tests, two laboratories reported a recovery of 3 to 5%, and four recovered 11 to 18% of the seeded virus.(ABSTRACT TRUNCATED AT 250 WORDS)