Passive Hemagglutination Method Research Articles

Epidemiological and genetic analysis of human group C rotaviruses isolated from outbreaks of acute gastroenteritis in Yokohama, Japan, between 2006 and 2012

Group C rotavirus (GCRV) infection has been described in several parts of the world, predominantly as sporadic cases of acute gastroenteritis. Little is known about the yearly changes in the GCRV strains from diarrheal outbreaks. Stool samples collected from outbreaks of acute gastroenteritis in Yokohama, Japan, between 2006 and 2012 that were negative for norovirus, sapovirus, and group A rotavirus, were screened for GCRV using a reverse passive hemagglutination method. The GCRV strains were characterized by nucleotide sequence and phylogenetic analysis of their VP6, VP7, VP4, and NSP4 genes. Samples from nine of 735 outbreaks in Yokohama (1%) contained GCRV, and eight of these outbreaks occurred in primary schools. The nucleotide sequences of the strains detected in this study were more closely related to Asian strains than to those from other regions of the world. The nucleotide sequences of the VP7 gene in these nine strains differed, and yearly changes were observed in the amino acid sequences of the VP4 genes. Phylogenetic trees constructed using the nucleotide sequences of the VP6, VP7, VP4, and NSP4 genes showed that sublineage S1 has divided into S1-1 and S1-2 in the VP4 gene only. Our results confirm that the prevalent strains of GCRV change yearly in Yokohama. This is the first study to demonstrate GCRV-associated gastroenteritis outbreaks in Yokohama, Japan.

Detection of heat-stable antigens of Campylobacter jejuni and C. coli by direct agglutination and passive hemagglutination.

The two serotyping schemes for the detection of heat-stable antigens of Campylobacter jejuni and Campylobacter coli use the same strains for antiserum production but differ in the detection systems used for identifying agglutination. The Penner method uses passive hemagglutination (PHA) while the Laboratory of Enteric Pathogens method uses the same antisera but in a whole-bacterial-cell direct agglutination (DA) protocol. C. jejuni produces a polysaccharide capsule, which is antigenic, and is the main component detected by the PHA method. The DA method will detect both capsule antigens and lipopolysaccharide (LPS) or lipooligosaccharide (LOS) surface antigens. Comparison of both methods by using a selection of isolates from human infection has shown a range of variation in agglutination specificity, reflecting the differences in antigens detected by the two methods. While 27.4% of the 416 C. jejuni isolates reacted with the antisera raised against the same type strains by either method, the majority showed a range of more complex relationships. None of the 37 C. coli isolates reacted with the same antiserum by both methods. Together the two schemes gave a total of 102 distinct combined serogroups for C. jejuni and 16 for C. coli. Thus, while some clonally related isolates share the same capsule and LOS or LPS antigens, other strains appear to have a common capsule antigen but differ in their LPS or LOS structures or vice versa.

Significance of platelet-reactive antibody screening for patients facing frequent platelet transfusions

Abstract It is not clear whether platelet-reactive antibody screening is clinically significant for patients facing frequent platelet transfusions. On the basis of data from 96 patients who had been examined for platelet-reactive antibodies by the mixed passive hemagglutination method for a variety of reasons, we investigated the following three issues retrospectively: (1) the relationship between platelet-reactive antibodies and the occurrence of problems in platelet transfusions, such as refractoriness or nonhemolytic reactions;(2) the influence of a history of transfusion on the production of those antibodies; and (3) the effect of screening for those antibodies on the prompt administration of appropriate platelet components. More than half of the platelet transfusion-related problems were associated with platelet-reactive antibodies. For patients with a history of transfusion, the mean period before a clinical problem occurred with platelet transfusions was 9 days,compared with 66 days for those without such a history. Accordingly, during the period, patients with a history of transfusions received fewer units of platelets and had fewer donor exposures than did patients without such a history. On the other hand, most patients who had been screened in advance for those antibodies received appropriate platelet components without delay, whereas an average of 10 days was needed before those who had not been screened received compatible platelets. The patients who had not been screened were transfused with 68 units of random platelets on average during the period. When frequent platelet transfusions are anticipated, especially for patients with a history of transfusion, screening for platelet-reactive antibodies beforehand would be helpful for prompt administration of appropriate platelets, although problems, such as the cost of those platelets and the burden on donors, remain to be resolved. Immunohematology 2002;18:104–108

白血球除去フィルター使用輸血における同種免疫感作の頻度と危険因子 プロスペクティブ研究

Seventy-eight patients multitransfused using leukocyte-reduction filters at bedside were prospectively studied to determine the rate of post-transfusion alloimmunization and its risk to platelet transfusion. HLA- and HPA-antibodies were detected by standard lymphocytotoxicity test (LCT), antihuman globulin-lymphocytotoxicity test (AHG-LCT) and mixed passive haemagglutination method (MPHA).Of these 78 patients, all of whom had a negative antibody screening on admission, 11 (14%) developed HLA antibodies but only one (1%) developed an HPA antibody. HLA antibodies were detected in 9 (24%) of 38 patients with previous transfusion, pregnancy or both but in only 2 (5%) of 40 patients with no presensitization (p<0.02). Among children (<20 years), one (3%) of 35 patients developed HLA antibodies whereas 10 (23%) of 43 adult patients (≥20 years) developed HLA antibodies (p<0.01). An HPA antibody (anti-HPA-2b) was detected with multireactive HLA antibodies in a woman with previous pregnancy.We conclude that WBC-reduced transfusions decrease primary immunization for HLA and HPA antigens. In presensitized adults, however, these transfusions may provide sufficient antigenic stimulation to boost or sustain a secondary alloimmune response. Women with previous pregnancy were identified as a high-responder group with regard to alloimmunization.

Anti‐Granulocyte Antibody Screening with Extracted Granulocyte Antigens by a Micro‐Mixed Passive Hemagglutination Method

Abstract Background and Objectives: Serologic tests for granulocyte antibodies, i.e., the granulocyte agglutination test and the granulocyte immunofluorescence test, require panels of typed granulocytes that cannot be preserved for more than a few hours. We have developed a new method in which granulocyte antigens, extrcated into saline containing 3% sucrose, are coated onto U‐type Terasaki plates. With this new method, we evaluated the micro‐mixed passive hemagglutination test (EG‐MPHA) for screening for granulocyte antibodies. Materials and Methods: We tested the ability of the EG‐MPHA to detect granulocyte antigens using 5 human antibodies specific for NA1, NA2, NB1, 5b, and Sara, and 8 different monoclonal antibodies for NA1, CD11a, CD11b, CD13, CD16, CD18 and HLA class I. Sera from 94 alloimmunized patients were screened by the chloroquine‐treated EG‐MPHA method. Results: Na1, NA2, NB1, 5b, Sara, CD11a, CD11b, CD13, CD16, CD18 and HLA class I antigens were present in the extracted granulocyte antigen preparation. CD11b and HLA class I antigens were removed when the extracted granulocyte antigens were treated with chloroquine. Granulocyte antibody screening of sera from alloimmunized patients showed that approximately 30% of the anti‐HLA‐positive for granulocyte antibody by the chloroquine‐treated EG‐MPHA. The extracted granulocyte antigen panels could be stored frozen for at least 1 year at −80°C. Conclusion: This new method is preferable for screening for granulocyte antibodies. In addition, it has the advantage of requiring only 5 μl of serum for each test.

Prevalence of hepatitis G virus and characterization of viral genome in Ghana

The prevalence of hepatitis G virus (HGV) infection was investigated in 85 human immunodeficiency virus (HIV)-infected and 30 uninfected individuals' sera obtained from Ghanaians. HGV RNA in serum was identified by a nested reverse transcription polymerase chain reaction (RT-PCR) using primers derived from the 5′-noncoding region. We also tested for hepatitis C virus by nested RT-PCR and for hepatitis B surface antigen (HBsAg) by passive hemagglutination method. HGV RNA was detected in 17 of 85 (20%) HIV sero-positive and three of 30 (10%) sero-negative Ghanaians, respectively. The prevalence of HGV infection was much greater than hepatitis C (0.9%) and hepatitis B virus (7.8%) infections in the present study. Ninety four percent of HGV infected patients were seronegative for hepatitis B and C virus infections. The nine different Ghanaian isolates in the 5′-untranslated region of the HGV genome had one nucleotide deletion at the same position when compared with other HGV isolates. Phylogenetic analysis showed that all Ghanaian isolates belonged to type 1 (West Africa type) of the HGV genotypes. Moreover, we determined nearly full-length nucleotide sequence of the HGV genome (denoted HGV-GA128) recovered from a Ghanaian infected with HIV. The HGV-GA128 was composed of 9231 nucleotides and had a single open reading frame, encoding 2843 amino acid residues. This isolate differed from previously reported HGV/GBV-C isolates by 10–15% of the nucleotide sequence and 2–5% of the amino acid sequence. Our data indicate a high prevalence of HGV, especially genotype 1, in Ghana.

好中球特異抗原ＮＡ１に対するモノクローナル抗体の作製

The neutrophil-specific antigen NA is biallelic, Composed of NA1 and NA2.NA antigens are often targets of antibodies which cause immune neutropenia. The phenotype of the NA system has been determined by the reaction of specific antibodies. We developed a new NA1-specific monoclonal antibody, TAG1. The reactivity of TAG1 to neutrophils was identical to that of MG38, which has been previously used to detect NA1 antigen. TAG1 did not react with any other blood cells or with cell lines derived from hematological malignancies, suggesting specificity for mature neutrophils. Preincubation of NA1/NA1 neutrophils with MG38 remarkably abolished the binding of TAG1. Further, the reactivity of TAG1 to solubilized antigen from NA1 neutrophils was significantly lower than that of MG38 by the mixed passive hemagglutination method. These results suggest that TAG1 specificically recognizes the NA1 antigen through different epitopes to those detected by MG38.

Prognostic Value of Suppressed Thyrotropin Level and Positive Thyrotropin-Receptor Antibody Activity in Graves’ Disease with Long-Lasting Clinical Remission

To determine the prognostic value of suppressed thyrotropin (TSH) level and positive TSH-receptor antibodies (TSH-R Ab) in patients with Graves' disease who have long-lasting clinical remission. We retrospectively studied patients with Graves' disease who underwent follow-up for a mean of 55 months after the withdrawal of antithyroid drug treatment. Study patients were 84 consecutive subjects in clinical remission, with normal serum free thyroxine (FT(4)) and free triiodothyronine (FT(3)) levels, regardless of serum TSH levels, a mean of 35 months (range, 6 to 135) after discontinuation of carbimazole therapy. Eighty-seven euthyroid subjects were used as control study participants. All subjects had serum determinations of FT(4) and FT(3) (radioimmunoassay), TSH (highly sensitive immunoradiometric method), TSH-R Ab (radioreceptor assay), and microsomal antibodies (M Ab, passive hemagglutination method). In the study patients, serum TSH was suppressed (</=0.10 mU/L) in 13 cases (15%), TSH-R Ab were positive (>/=15%) in 11 cases (13%), and M Ab were positive (>/=1:100) in 54 cases (64%). Simultaneous suppressed TSH and positive TSH-R Ab levels were present in six patients. During the follow-up, 11 patients had a relapse, demonstrated by above-normal values for serum FT(4) and FT(3) in association with clinical symptoms of hyperthyroidism. Five of them had a previously suppressed TSH level, three had a positive TSH-R Ab level, and six had a positive M Ab titer. Relapse was significantly more likely in patients with a previously suppressed TSH level (P<0.02) but not in patients with a previously positive TSH-R Ab level or positive M Ab titer. Patients with Graves' disease and long-lasting clinical remission after discontinuation of carbimazole therapy may have a suppressed TSH level, a positive TSH-R Ab level, or a positive M Ab titer (or some combination of these findings). Although positive TSH-R Ab and M Ab have no significant prognostic value, a suppressed TSH level is indicative of subclinical hyperthyroidism and higher risk of relapse.

Anti-HLA antibody screening with extracted platelet HLA antigens by the mixed passive hemagglutination method.

For the detection of anti-HLA antibody in the serum of patients receiving frequent platelet transfusions, we developed a new method in which platelet antigens are extracted into physiological saline containing 3% sucrose and coated as a layer on a U-type Terasaki plate. In the present study, screening of anti-HLA antibodies was conducted with this plate by the mixed passive hemagglutination test although the plate contained both HLA and HPA. The reactivity determined by this method correlated well with antihuman immunoglobulin-lymphocyte cytotoxicity test (AHG-LCT) results (r = 0.963). The plate can be preserved for at least 2 years at -80 degrees C and is easier to handle than the frozen lymphocyte panels used in the LCT test in which lymphocytes must be kept alive. This new method is an alternative way to screen HLA antibodies. In addition, it is expected that this method will be used to screen anti-HPA antibodies.

Anti-HLA Antibody Screening with Extracted Platelet HLA Antigens by the Mixed Passive Hemagglutination Method

For the detection of anti-HLA antibody in the serum of patients receiving frequent platelet transfusions, we developed a new method in which platelet antigens are extracted into physiological saline containing 3% sucrose and coated as a layer on a U-type Terasaki plate. In the present study, screening of anti-HLA antibodies was conducted with this plate by the mixed passive hemagglutination test although the plate contained both HLA and HPA. The reactivity determined by this method correlated well with antihuman immunoglobulin-lymphocyte cytotoxicity test (AHG-LCT) results (r = 0.963). The plate can be preserved for at least 2 years at -80 °C and is easier to handle than the frozen lymphocyte panels used in the LCT test in which lymphocytes must be kept alive. This new method is an alternative way to screen HLA antibodies. In addition, it is expected that this method will be used to screen anti-HPA antibodies.

Prevalence of anti-thyroid peroxidase antibodies in autoimmune and nonautoimmune thyroid disorders in a relatively low-iodine environment

We evaluated the prevalence of antithyroid peroxidase antibodies (anti-TP0 Ab) in 402 patients with thyroid disease and 30 healthy controls by a commercial radioimmunoassay (RIA) and compared the results with the passive hemagglutination (HA) method. The patients in the study had autoimmune thyroid disorders (AITD) such as Graves' disease and Hashimoto's disease or had nonautoimmune thyroid diseases (NAITD) such as thyroid cancer, congenital goiter, endemic goiter, and nodular goiter. Subjects were recruited from a population with a mild iodine deficiency (Sao Paulo, Brazil). The effect of specific therapy (for either thyrotoxicosis or chronic thyroiditis) on the circulating anti-TPO levels was also investigated. Positive anti-TPO Ab was detected in 89.9% of the patients with AITD as compared with a prevalence of positive tests of only 4.8% in patients with NAITD. Positive microsomal antibody (M Ab) was found in 68.4% of the patients with AITD and in 6.4% of the patients with NAITD. A positive and significant correlation was obtained between M Ab and anti-TPO Ab. A positive anti-TPO test with negative anti-M was found in 14.1% of the patients with AITD but in only 4.3% of the patients with NAITD and normal controls. These results suggest that anti-TPO Ab by RIA is more sensitive and specific than M Ab by HA. In patients with AITD, anti-TPO Ab levels usually decreased after treatment, suggesting that this parameter could be used in the follow-up of these thyroid disorders.

Antithyroid antibodies and thyroid dysfunction in Saudi children with Down syndrome.

Fifty children (ages seven months to nine years) with Down syndrome compared with age and sex matched controls were tested for antithyroid antibodies. Seven (14%) of the Down group were found to be seropositive; six (12%) for antimicrosomal antibodies; three for both antimicrosomal and antithyroglobulin; none was positive for antithyroglobulin alone. All the control group were seronegative and had no clinical evidence of thyroid disease. Three (6%) of the Down group had hypothyroidism, two due to autoimmune thyroiditis and one had thyroid dysgenesis; one of the previous two had insulin dependent diabetes mellitus. One child had Graves disease. Thyroid dysfunction was not previously suspected in the two symptomatic children. In view of our findings, recommendations are made to regularly screen these children for autoantibodies and evidence of thyroid dysfunction.

Rapid determination of hepatitis B surface antigen (HBsAg) by latex agglutination using an integrating sphere turbidimetric assay

A method for determining levels of serum HBs antigen has been developed, applying the principles of the integrating sphere turbidimetric assay (ISTA). Using this method, the minimum detectable level of HBs antigen is 15 ng/ml, i.e., it is three times more sensitive than the reversed passive hemagglutination (RPHA) method. Reproducibility and specificity are also excellent with ISTA. With a cut-off level of 20 ng/ml, the highest reading possible with this method is 1000 ng/ml. Serum HBs antigen can readily be measured by this method if a fully automated EL-1000 analyzer is used. This rapid and simple method of measurement should be clinically useful.

Quick assay of serum HBs antibody levels using latex agglutination‐integrating sphere turbidimetric assay (ISTA)

A method for determining serum HBs antibody applying the principle of integrating sphere turbidimetric assay (ISTA) by latex agglutination was developed. The minimum detectable level of HBs antibody by this method is 12.5 IU/L, indicating that this method is 3 times or more sensitive with better reproducibility and specificity than the passive hemagglutination (PHA) method. With a cut-off level of 25 IU/L, the possible highest simultaneous reading by this method was 1,000 IU/L. Serum HBs antibody can be readily measured in 10 or so minutes by this method if a fully automated EL-1000 analyzer is used. This rapid and simple method for determining serum HBs antibody will be useful not only clinically, but also in preventive medicine.

Immunization in children with HIV seropositivity at birth

To evaluate the humoral response to routine childhood immunization of HIV-infected children. Response rate, antibody titres and persistence after polio and tetanus vaccination were compared in 72 children with HIV seropositivity at birth and divided according to HIV infection status as determined by clinical and laboratory tests. Polio antibodies were titred in a microneutralization test (positive titres, > or = 1:4), and antibody to tetanus toxoid with a passive haemagglutination method (protective titres, > or = 1:1024). The response rates to polio and tetanus vaccination (> 80 and > 75%) were similar in the HIV-infected and non-infected children, as were antibody levels. In the subgroup with sera obtained some months after the last dose of vaccine, polio antibody levels decreased in all four HIV-infected and in three of the seven non-infected children; protective tetanus antitoxin levels were detected in three of the six infected and in all three non-infected children. This study demonstrates the ability of children with HIV infection to respond adequately to the two vaccines considered, although tetanus antitoxin levels were inferior, compared with those in the seroreverted children. The unsatisfactory antibody levels observed in the admittedly few HIV-positive children studied some months after the last vaccination could be the result of a lower initial protective level and not necessarily an expression of severely impaired immunocompetence. The administration of booster doses in addition to the traditional immunization schedule could be useful in children with HIV infection.

The use of dacron plates for DOT enzyme-linked immunosorbent assay (DOT-ELISA)

Dacron (polyethylenetherephthalate) is proposed as a matrix for dot-ELISA procedures, as an alternative to nitrocellulose. Plates of dacron were partially hydrazinolyzed and hydrazide groups introduced were converted to azide groups. The derivative dacron-antigen was covalently linked on to the plates through these azide groups. The derivative dacron-antigen was exaustively washed according to CROOK and antigen was still fixed onto the plates. Protein F1A purified from Yersinia pestis was used as a model. Titration of sera from immunized and non immunized rabbits against this protein was carried out by employing the dot-ELISA method. No significant difference was observed using dacron-antigen and nitrocellulose-antigen preparations. However, both procedures showed to have a significant better performance in comparison with the passive hemagglutination method. The specificity and reproductibility of the dot-ELISA assay using both preparations showed a similar behaviour. Nitrocellulose preparation was stable at 4 degrees C, 28 degrees C and -20 degrees C for 90 days, whereas the dacron-antigen derivative was stable only when stored at 4 degrees C. Dacron-antigen derivative could be re-used when the spot developing was proceeded using 4-chloro-1-naphtol as substrate.

Streptococci from primary isolation plates grouped by reverse passive haemagglutination.

Reverse passive haemagglutination, a novel microtitre based assay, was compared with the Streptex (Wellcome UK) latex slide agglutination kit for streptococcal grouping in a diagnostic microbiology laboratory. Three hundred and fifty two extracts from 349 consecutive primary isolation plates were assayed by both methods. Reverse passive haemagglutination gave identical grouping results for 98.0% of the 345 streptococci identified by Streptex, and the kappa coefficient of agreement between the methods for all 352 extracts tested was 0.973. Cross reactions with Listeria spp seen with Streptex were not found by reverse passive haemagglutination. In the reverse passive haemagglutination method 11 streptococci could be grouped on each 96-well plate and most reactions were stable for at least 30 minutes. Reverse passive haemagglutination is more rapid to perform than latex slide agglutination when many organisms are to be grouped, and the patterns of haemagglutination are easily recognised. If the method was taken into routine use in a diagnostic laboratory, the persistence of reverse passive haemagglutination reactions would enable grouping results to be checked for quality control purposes.

Seroepidemiology of Herpes Simplex Virus and Restriction Endonuclease Cleavage Analysis of Herpes Simplex Virus Type 2 in Okinawa

A seroepidemiologic study of herpes simplex virus (HSV) in Okinawa was performed. A total of 423 serum samples were collected from all over Okinawa, and the positivity rate of antibody against HSV was measured using a passive hemagglutination method. The sero-positive rate for HSV in age groups of over 40 years was 100%. Seven HSV type 2 (HSV 2) isolates were obtained in Okinawa, and DNA preparations from Vero cells infected with the isolates were analyzed using five restriction endonucleases: Bam HI, Hind III, Kpn I, Bgl II and Eco RI. Variations in the genomic region were demonstrated in five of the isolates. Such variations have not been reported previously in HSV 2 in mainland Japan. This is the first report of a seroepidemiologic study of HSV and restriction endonuclease cleavage analysis of HSV 2 in Okinawa, is a subtropical island where HSV is endemic.

A reconstructed cohort study on the hepatitis B virus infection as a risk factor of liver cancer in Korea

A stable cohort (n = 369,725) was reconstructed to test the association of hepatitis B virus (HBV) infection with subsequent occurrence of liver cancer in a healthy Korean population. The cohort consisted of male beneficiaries of the Korea Medical Insurance Corporation over 30 years of age, living nationwide. The HBV infection was tested by the reversed passive hemagglutination method for the HBsAg and by the passive hemagglutination method for the anti-HBs at the time of recruitment in 1984. Admissions due to liver diseases were identified through a computerized system for reimbursements on medical insurance claims from January 1, 1985 to December 31, 1987. For a more valid estimate of incidence, a sample survey was carried out, obtaining correction coefficients against misclassification of the diagnosis, as well as those for duplicate claims in a year. The incidence rate of liver cancer steadily increased with age (kappa 2 TREND = 51.1, df = 1, p = 0.00).(ABSTRACT TRUNCATED AT 250 WORDS)

Seroepidemiology of Herpes Simplex Virus Type 1 in Yanji, Jilin, China

Sera from 158 individuals in Yanji, Jilin, China, were tested for antibodies to herpes simplex virus type 1 (HSV-1) by the passive hemagglutination method. Age-specific incidence rates for antibodies to HSV-1 were calculated. For sera from persons in the age group 10 years or less, the positive rate was 54% but in the age group higher than 10 years, it was more than 91% (P less than 0.01). In the part of China surveyed, primary HSV-1 infection occurred in early generation before about age 10. In children, the positive rate in the Han race was significantly higher than that in the Korean race (P less than 0.05).