Agglutination Patterns Research Articles

Agglutination of Helicobacter pylori coccoids by lectins.

AIM:To study the agglutination pattern of Helicobacter pylori coccoid andspiral forms.METHODS:Assays of agglutination and agglu-tination inhibition were applied using fifteen commercial lectins.RESULTS:Strong agglutination was observed with Mannose specific Concanavalin A(Con A), fucose specific Tetragonolobus purpureas(Lotus A) and N-acetyl glucosamine-specific Triticum vulgaris (WGA) lectins. Mannose and fucose specific lectins were reactive with all strains of H. pylori coccoids as compared to the spirals. Specific carbohydrates, glyco-proteins and mucin were shown to inhibit H. pylori lectin agglutination reactions.Pre-treatment of the bacterial cells with formalin and sulphuric acid did not alter the agglutination patterns with lectins. However, sodium periodate treatment of bacterial cells were shown to inhibit agglutination reaction with Con A, Lotus A and WGA lectins. On the contrary, enzymatic treatment of coccoids and spirals did not show marked inhibition of H. pylori-lectin agglutination. Interes-tingly, heating of H. pylori cells at 60° for 1 hour was shown to augment the agglutination with all of the lectins tested.CONCLUSION:The consi-derable differences in lectin agglutination patterns seen among the two different-tiated forms of H. pylori might be attributable to the structural changes during the events of morphological trans-formation, resulting in exposing or masking some of the sugar residues on the cell surface. Possibility of various sugar residues on the cell wall of the coccoids may allow them to bind to different carbohydrate receptors on gastric mucus and epithelial cells. The coccoids with adherence characteristics like the spirals could aid in the pathogenic process of Helicobacter infection. This may probably lead to different clinical outcome of H. pylori associated gastro-duodenal disease.

On the rôle of human salivary micelle-like globules in bacterial agglutination.

The hypothesis to be tested in this in vitro study was that the salivary micelle-like globules (SMGs) have a rôle in the agglutination of some oral bacteria. An attempt to determine the mechanisms for the interactions involved was also carried out. 4 laboratory and 4 native streptococci strains were tested. Human whole (HWS) and parotid (HPS) saliva was collected from 4 subjects, and SMGs were isolated from both salivas, and agglutination was recorded in the various bacterial suspensions over time. HPS, HWS and SMGs isolated from HPS and HWS caused typical agglutination patterns for the mutans strains. Salivary supernatants (without SMGs) caused a much delayed or no agglutination. Electron microscopy showed SMG-like structures on the surface of the agglutinated bacteria. Addition of pyrophosphate to HPS prevented agglutination, whereas guanidine HCl prevented normal agglutination of a sanguis strain, and urea had no obvious effect. Together, these results indicate that the SMGs are important in the agglutination of streptococci, and that both calcium-dependent, electrostatic and hydrophobic interactions may be involved.

Possible mechanisms for an irregular vessel coagulation when long laser pulses are used in the treatment of port-wine stains

The laser treatment of port-wine stains (PWS) has as a main aim the irreversible damage of ectatic vessels. Blood content of the subcutaneous venous plexus in PWS can be increased by a factor of seven or more, compared to that of the normal skin. The venous blood velocity ranges from 0.1–1 mm/s in capillaries to approximately 22 mm/s in larger vessels of about 300 μm in diameter. A PWS, selected for study, was irradiated with a multiline argon laser 488 515 nm wavelength, 1.5 W power, 200 ms pulse duration, 0.5 mm beam diameter and repetition frequency up to 5 Hz. Laser shots were placed adjacently in an area of 1 cm 2. Using these parameters, in the case of dilated PWS vessels with an optical penetration depth and thermal diffusion length less than the diameter of the vessel, together with a transit time of blood across the irradiated spot less than the pulse duration, and estimating that during a pulsed laser emission of 200 ms, the blood has travelled a distance of 3–4 mm, there is a strong indication that hemodynamics during irradiation may influence the pattern of coagulation and agglutination. Thrombosis should occur in the case of small vessels and, in larger vessels, the coagulated blood will only partly fill the lumen. The structure developed in the vessel interior may also change continuously with time, as the coagulated material is progressively replaced by fibrotic tissue and the irregular agglutination pattern may be due to inhomogeneity in the absorbed optical energy.

HTLV-I/II Indeterminate Serology and Natural Killer Cell Expansion

HTLV-I/II Indeterminate Serology and Natural Killer Cell Expansion

Serological analysis of monoclonal anti-E and -e with Japanese E/e variant red cells.

The agglutination patterns have been analyzed for the reaction between 24 monoclonal antibodies (MAB) with specificity for the Rh antigen E or e and red cells of E/e variant from Japanese blood donors. The MABs were tested for direct agglutination of papain treated cells at 20 degrees C. The reactions of a full titration series of each MAB with a E/e variant cell were compared to the reactions of that MAB with normal E + e + cells. Sixteen anti-E were divided into a minimum of 6 different agglutination patterns with 8 examples of E variant cells. Eight anti-e gave a minimum of 5 different agglutination patterns with 6 examples of e variant cells.

Reactivity of 62 Rh MAbs with weak and variant antigens.

We characterized serologically 5 anti-C (4 IgM and 1 IgG), 3 anti-c (2 IgM and 1 IgG), 4 anti-E (1 IgM and 3 IgG), 4 anti-e (3 IgM and 1 IgG) and 46 anti-D (16 IgM and 30 IgG) monoclonal antibodies, provided by the Rh Section of the Third International Workshop and Symposium on Monoclonal Antibodies against Human Red Blood Cells and Related Antigens (1996) for their ability to detect weak and variant antigens. The agglutination patterns were established using untreated and papain-treated red blood cells in a column agglutination technology system (BioVue, Ortho). Significant differences were found between the IgM and IgG antibodies. The papain treatment seemed to be important for IgM but not for IgG antibodies. Almost all of the IgM anti-D antibodies detected untreated DIV samples and almost all of the IgG anti-D antibodies detected untreated weak D samples. Both IgM and IgG anti-D antibodies showed the highest number of negative reactions with DVI and Rh 33 red blood cells. The CwCw sample was detected by only one of the 4 anti-C IgM MAbs using enzyme-treated red blood cells. All anti-c MAbs were able to detect treated Cx samples. Because of the small number of weakly expressed E and e samples, definitive conclusions cannot be drawn on the ability of these antibodies to detect these antigens.

Comparative pathogenicity study on antigenically variant strains of Enterococcus seriolicida

Abstract Enterococcus seriolicida strains were divided into two groups, agglutinating and nonagglutinating, by a slide agglutination test using antiserum against the YT‐3 strain. Intraperitoneal injection of agglutinating and nonagglutinating strains into yellowtail, Seriola quinqueradiata Temminck & Schlegel, revealed that nonagglutinating strains were more virulent than agglutinating strains. Two nonagglutinating and highly pathogenic strains SS91–014 and SS91–092 were subcultured 30 times in brain heart infusion broth, and the agglutination titres of 50 colonies from subcultures 1, 2, 3, 4, 5, 6, 11, 16, 21, 26 and 30 against anti‐YT‐3 serum were determined. Transformation from a nonagglutinating (1:<4) to an agglutinating (1: ≥4) pattern was first observed at the sixteenth subculture, and the ratio of agglutinating to nonagglutinating substrains rose until the thirtieth subculture. At this time, 70% of the SS91–014 population and 52% of the SS91–092 population were transformed to an agglutinating pattern. When the pathogenicity of four transformed substrains with different agglutination titres was tested in yellowtail, the nonagglutinating substrain showed higher pathogenicity than the agglutinating substrains, but no relationship between LD50 values and the agglutination titres of transformed substrains was observed. The pathogenicity of E. seriolicida appears to be related to the agglutination pattern, although it was not demonstrated that this property is solely responsible for pathogenicity.

Agglutination of "Streptococcus milleri" by lectins.

The agglutination of 218 clinical isolates and three ATCC type strains of "Streptococcus milleri" was tested with 25 different lectins from plants and fungi. An agglutination reaction with one or more lectins was observed with 42 isolates when the cells were untreated. After trypsinisation of the bacteria, 109 strains yielded a positive reaction and after boiling the bacterial cells at pH2, 218 isolates were agglutinated. As an overall result of our experiments with untreated, trypsinised and boiled cells, 17, 37 and 45 different agglutination patterns, respectively, were obtained. The lectins from Datura stramonium, Robinia pseudoacacia and Dolichos biflorus agglutinated isolates belonging only to Lancefield group C, being non-reactive with other isolates. These lectins were also found to be specific for "large colony type" streptococci of group C. The use of lectin agglutination in epidemiological and ecological studies of "S. milleri" is discussed.

Wild-type and cultured Ehrlich ascites tumour cells differ in tumorigenicity, lectin binding patterns and binding to basement membranes.

Three different variants of the Ehrlich ascites tumour (EAT) cell were derived and the lectin surface reactivities, as well as the malignant characteristics of each variant, were studied. Wild-type cells (EAT-wt) were selected for growth on basement membranes and tissue culture plastic to give EAT-c cells. The EAT-c were passaged in mice by i.p. injection, giving rise to a third variant (EAT-c/m). Each of these three cell variants was characterized for: (i) specific lectin agglutinability patterns; (ii) the ability to produce ascites tumours in mice; (iii) the ability to produce solid tumours; and (iv) the attachment to and growth on basement membranes and purified extracellular matrix molecules. Analysis of the total protein and carbohydrate content of each cell line showed that there was an increase in the glycosylation of the EAT-c cells compared to EAT-wt cells. After repeated passage of the EAT-c/m cells in mice, the glycosylation level of the EAT-c/m cells returned to that of the EAT-wt cell line. In addition, the EAT-c cells displayed an increase in the number of terminal non-reducing sugars which could indicate either an increase degree of branching or the presence of additional N- and/or O-linked oligosaccharide chains of the cellular glycoproteins. This phenotype was retained by the EAT-c/m cells which had been passaged repeatedly in mice. The most significant increase was in the content of sialic acid-containing glycoproteins found in the EAT-c cells. The sialic acid-binding lectin Maackia amurensis leukoagglutinin (MAL) agglutinated all three EAT cell variants, while the sialic acid-binding Sambucus nigra (elderberry bark) lectin (SNA) agglutinated only the EAT-c and early-passage EAT-c/m cells. These findings indicate the presence of alpha 2,3-linked sialic acid on all three variants, but only the cultured cells and early-passage EAT-c/m cells possess the Neu5Ac alpha 2,6 linkage. The EAT-c cells attached avidly to wells coated with either laminin or fibronectin, as well as extracellular matrix produced by cultured bovine endothelial cells, but the EAT-wt and EAT-c/m cells did not. Paradoxically, the EAT-c cells were incapable of producing solid tumours when injected into a basement membrane-rich skeletal muscle bed, whereas the EAT-wt and EAT-c/m cells produced rapidly growing tumours when injected into the same environment. Lectin agglutination patterns established that ascitic tumour cells within the peritoneal cavity were derived from injected EAT-c cells.

The agglutination of beta-haemolytic streptococci by lectins.

The ability of 25 lectins, isolated from different plants and fungi, to agglutinate 95 clinical isolates of beta-haemolytic streptococci was examined. Cell suspensions were untreated, trypsin-treated or boiled at pH 2.0. None of the 95 untreated cell suspensions gave a visible reaction with any of the lectins. When the cells were trypsinised, 42 strains were agglutinated with one or more lectin and after boiling at pH 2, all the strains were agglutinated. After treatment with trypsin, 20 different agglutination patterns were observed, and after boiling, 19 patterns, four of which were similar. A correlation was found between Lancefield group C and some of these patterns. Some lectins reacted specifically with group C streptococci; DBA and WFA, both specific for D-GalNAc, DSA, a GlcNAc-specific lectin, and RPA, which showed a complex specificity, reacted only with group C strains. Furthermore, the lectin of Maackia amurensis reacted with 50% of group B streptococci only. Agglutination assays with lectins were reproducible, easy to perform, relatively inexpensive and, therefore, applicable to studies of cell-wall structure and epidemiology of beta-haemolytic streptococci.

Interaction of coagulase negative staphylococci with lectins.

To investigate the reaction of 118 blood culture isolates of coagulase negative staphylococci with a panel of seven lectins. The interactions between the bacterial suspensions and lectins from Arachnis hypogaea (peanut agglutinin), Bauhina purpurea, Solanum tuberosum (potato starch), Triticum vulgaris (wheat germ agglutinin), Wisteria floribunda, Concanavalin ensiformis and Limulus polphemus (horse-shoe crab agglutinin) were assayed in microtitre plates incubated for 1 hour at room temperature then left overnight at 4 degrees C. Agglutinating activity was detected by examining the pattern of cell settlement compared with that of the controls. Lectins from Solanum tuberosum, Triticum vulgaris, Concanavalin ensiformis and Limulus polyphemus agglutinated 90% of the strains and displayed 11 agglutination patterns which were unrelated to species, clinical relevance, or antibiotic resistance. Fifty three per cent of the isolates fell into three reaction patterns and the other patterns were represented by nine or fewer strains. Replicate cultures investigated simultaneously gave consistent results, but some strains exhibited variation in agglutination patterns on repeat testing. Based on these observations lectin agglutination patterns seem to offer a method with potential for strain differentiation among coagulase negative staphylococci. Reproducibility may be improved by the use of biotinylated lectins and growing the coagulase negative staphylococci on defined media. Discrimination can be increased by the inclusion of other reactive lectins.

Direct Inoculation of Food as the Cause of an Outbreak of Group A Streptococcal Pharyngitis

An investigation was conducted of a food-related outbreak of group A streptococcal pharyngitis following an elementary school banquet. Of 166 surveyed banquet attendees, 71 (43%) reported outbreak-associated pharyngitis, and 21 (88%) of 24 tested attendees had evidence of group A streptococcus (GAS) in the throat. Attendees who ate macaroni and cheese were three times more likely to develop pharyngitis than those who did not (66/132 [50%] vs. 5/30 [17%], P = .002). None of the food handlers had GAS recovered by throat culture. However, the cook who prepared the macaroni and cheese had a hand wound; a wound culture grew GAS with the same T agglutination pattern and M- and/or opacity factor type as that of all available GAS strains from ill attendees. Under laboratory conditions, macaroni and cheese supported rapid growth of the outbreak-associated strain of GAS. To the authors' knowledge, this is the first documented foodborne outbreak of GAS pharyngitis in which the only apparent source of contamination was a food handler's skin lesion.

An analysis of lectin agglutination as a means of sub-dividing gonococcal serovars.

Sixteen lectins were examined for their ability to agglutinate 298 strains of Neisseria gonorrhoeae. Seven lectins failed to agglutinate any of the strains; the remaining nine lectins gave 22 different agglutination patterns. The 298 strains were divided into 14 serovars with a single panel of monoclonal antibody typing reagents; lectin agglutination subdivided these into 57 serovar/lectin patterns. A combination of two monoclonal antibody serotyping panels divided the strains into 32 serovar combinations; lectin agglutination further subdivided these into 79 serovar/lectin patterns. There was no correlation between lectin pattern and serovar. Lectin agglutination is a simple supplementary typing method and could be particularly useful in micro-epidemiological studies.

The isolation and identification of Leptospira interrogans serovar bratislava from a pig in Germany.

During a study on the improvement of selective media for the isolation of leptospires from clinical material, leptospires were isolated from the kidneys of a pig. Test material was cultured in EMJH-medium containing 0.4% rabbit serum and 3 inhibitory substances (Rifampicin 10 micrograms/ml, Amphotericin B 2 micrograms/ml, 5-Fluorouracil 100 micrograms/ml). The cultured leptospira-strain (Berlin 406) was identified to serogroup level using the cross agglutination method and it was further typed to serovar level by MAB's, factor analysis and restriction endonuclease analysis (REA). MAB typing of Berlin 406 gave an agglutination pattern profile typical of serovar bratislava. Factor serum identification clearly identified this strain as serovar bratislava. On REA examination, Berlin 406 gave a profile identical to that of the serovar bratislava type strain Jez Bratislava i.e. genotype B1. This is the first report of a confirmed isolate of Leptospira interrogans serovar bratislava from domestic animals in Germany. There exist a number of possibly important implications as this agent is being incriminated in the cause of clinical disease not only in pigs, but also in horses and dogs in other countries.

False-Positive Titers of Thyroid Autoantibodies in Patients Undergoing Hemodialysis?

Abstract Concentrations of thyroid-related hormones in serum of patients with chronic renal failure are known to be abnormal (1, 2). In our study on thyroid-function testing of patients undergoing hemodialysis, we determined, in addition to concentrations of free and total triiodothyronine, free and total thyroxin, “reverse” triiodothyronine, and thyroxin-binding globulin, the titers of thyroglobulin and microsomal autoantibodies (TGA and TMA, respectively). The study was provoked by the appearance of an uncommon agglutination pattern in the control wells of some samples from patients with chronic renal failure during the standard procedures of detecting TGA and TMA with hemagglutination methods (Thymune-T and Thymune-M assays from Welcome, London, U.K.). For these samples we were not certain whether positive titers for TGA and (or) TMA represented false-positive or true-positive values. Therefore, we assayed the absorbed serum samples and samples after addition of excess nonspecific immunoglobulin. Furthermore, we wanted to determine the difference in TGA and TMA titers of serum samples before and after hemodialysis.

Lewis Blood Group Determination in Bloodstains by Planimetric Measurement of Eluted Monoclonal Antibodies

Planimetric measurements were employed for reading the results of an elution test to determine Lewis blood groups in dry human bloodstains. In the absorption-elution test, two varieties of indicators were used to detect eluted Lewis antibodies. First, 64 blood-stains aged between 2 to 8 months were tested with glutaraldehyde (GLA)-treated erythrocytes (planimetric hemagglutination assay, PMHA). This method demonstrated that dry stains weighing approximately 0.4 mg (equivalent to 3 microliters of whole blood) were sufficient for detection of Lea or Leb antigen. Results were obtained within 1 h. Then, 37 of these stains were tested with Lewis substance-coated latex particles (planimetric latex agglutination assay, PMLA). The presence of Lea and Leb antigen were detected from dry stains weighing 0.1 mg (equivalent to 1 microL of whole blood) within 3 h. Both these assays are faster and simpler with accuracy than the enzyme-linked immunosorbent assay (ELISA). Latex particles coated with Lewis substance are, in particular, strongly agglutinated and show agglutination patterns more clearly than erythrocytes. The blind tests using these two methods properly classified 7 Le(a + b-) and 23 Le(a-b + ) bloodstains; whereas, 5 Le(a-b-) stains were undetermined by the criteria for these tests. These results indicate the usefulness of the PMHA and PMLA for typing Lewis blood groups from small bloodstains.

Erythrocyte binding factors in the sera of the adult lamprey Geotria australis

Adults of the Southern Hemisphere lamprey Geotria australis possess three major factors in their sera which bind to erythrocytes in vitro; in addition, an induced factor can also be demonstrated in the sera of animals immunized against human erythrocytes. These factors are a hemagglutinin, a hemolysin and a further binding factor together with a probable antibody. Furthermore, differences in agglutination patterns following immunization were noted between animals of different age groups. Old adults possessed a unique dilution pattern, containing a negative zone which occurred shortly after the initial dilutions, followed by a return to the normal agglutination pattern in the higher dilutions to a titer of 2 18. While the greatest agglutinating activity took place in sera taken from immunized animals, all animals possessed weak, low titer nonspecific direct agglutination together with a strong nonspecific binding activity which was revealed only by the use of an antiserum to lamprey plasma proteins. It is suggested that the interaction of these factors may account, in some measure, for the confusion concerning the nature and magnitude of the immune response in this phylogenetically important group of animals.

Interaction of lectins withYersinia pestis strains

The ability of lectins to interact with Yersinia pestis strains isolated from rodent fleas and human biological fluids, obtained from different geographic areas, was examined. Lectins of Canavalia ensiformis, Ulex europaeus, Phaseolus vulgaris, and Triticum vulgaris, as well as a new autochthonous lectin of Swartzia pickellii of undefined specificity, were used. Most of the Y. pestis strains did not agglutinate with U. europaeus or C. ensiformis lectin. However, P. vulgaris lectin agglutinated suspensions of all the bacillus strains used. Fifteen of the 19 strains tested positive for assays using S. pickellii lectin. It is believed this is the first report of Y. pestis strain agglutination by lectins. A similar agglutination pattern was obtained for lectins with specificity for oligosaccharides containing N-acetylglucosamine and S. pickellii lectin, which did bind to the affinity matrix chitin, a polysaccharide of N-acetylglucosamine. The use of bacterial strains and commercial lectins of defined specificity may be an approach to providing evidence about the lectin binding sites of undefined monosaccharide specificity.

Sensitivity and Interobserver Variability of the Recombigen®-HIV-1 LA Test

The Recombigen-HIV-1 LA Test (Cambridge BioScience Corporation, Worcester, MA) uses recombinant peptides derived from the env gene product in a latex agglutination assay for the detection of antibodies to human immunodeficiency virus type 1 (HIV-1) and was recently approved by the Food and Drug Administration for marketing in the United States. It is intended for use as a screening test in physicians' offices, emergency rooms, and other settings where enzyme immunoassays are not practical or available. Concern has been raised over the sensitivity, specificity, and difficulty in interpretation of the agglutination pattern. The authors report on the sensitivity and interobserver variability of the assay as performed in a blinded fashion in a hospital laboratory by technologists experienced with other latex agglutination assays. In the first study, sera from 50 patients positive by enzyme immunoassay (EIA) (Abbott HIV EIA) and western blot (WB), performed with EPITOPE HIV western blot strips were assayed by one technologist using the latex agglutination technique. Forty-six samples were positive and four were negative, yielding a sensitivity of 92%. In the second study, 30 samples consisting of 10 negative by EIA and WB, 10 borderline by EIA and/or indeterminate by WB, and 10 positive by EIA and WB were evaluated by three technologists with the latex agglutination technique. There was agreement among all three technologists in 24 of 30 samples (80%). There was disagreement over one sample from the negative group (one technologist obtained a single false positive result), three from the borderline/indeterminate group, and two from the positive group (three technologists obtained false negative results on two samples). In summary, the authors report interobserver variation in interpreting 20% of tests, reflecting difficulty in assessing weak agglutination. Sensitivity of 92% is below that achievable with the EIA or WB techniques and limits the usefulness of the latex agglutination assay as a screening test.

Demonstration of Seven Epitopes on the Rh Antigen D Using Human Monoclonal Anti‐D Antibodies and Red Cells from D Categories

The agglutination patterns have been established for the reaction between 29 monoclonal antibodies with specificity for the Rh antigen D and red cells of D categories IIIa, IIIc, IVa, IVb, Va, Vc, VI and VII, which are known to lack certain epitopes on the D polypeptide. Six different agglutination patterns were recognized and interpreted to indicate the recognition of seven different epitopes. These epitopes are termed epD1 through to epD7. The separate existence of epD6 and epD7 is deduced from previous observations in inhibition studies using purified 125I-labelled antibodies; they cannot yet be distinguished in agglutination tests. The number of epitopes lacking from cells of each category varied between two and five. As all the antibodies agglutinated cells of categories IIIa, IIIc and VII and cells of categories II, IIIb and Vb were not available, it is probable that there are epitopes other than the seven presently recognized. Eighteen out of the 29 antibodies which were examined recognized epitopes epD6/7 and it is suggested either that antibodies recognizing these epitopes predominate in polyclonal anti-D sera, or that the lymphocytes producing these antibodies are preferentially selected during establishment of cell lines.