Cell Antibody Detection Research Articles

Nanomagnetic System for Rapid Diagnosis of Acute Infection.

Pathogen-activated antibody-secreting cells (ASCs) produce and secrete antigen-specific antibodies. ASCs are detectable in the peripheral blood as early as 3 days after antigen exposure, which makes ASCs a potential biomarker for early disease detection. Here, we present a magnetic capture and detection (MCD) assay for sensitive, on-site detection of ASCs. In this approach, ASCs are enriched through magnetic capture, and secreted antibodies are magnetically detected by a miniaturized nuclear magnetic resonance (μNMR) system. This approach is based entirely on magnetics, which supports high contrast against biological background and simplifies assay procedures. We advanced the MCD system by (i) synthesizing magnetic nanoparticles with high magnetic moments for both cell capture and antibody detection, (ii) developing a miniaturized magnetic device for high-yield cell capture, and (iii) optimizing the μNMR assay for antibody detection. Antibody responses targeting hemolysin E (HlyE) can accurately identify individuals with acute enteric fever. As a proof-of-concept, we applied MCD to detect antibodies produced by HlyE-specific hybridoma cells. The MCD achieved high sensitivity in detecting antibodies secreted from as few as 5 hybridoma cells (50 cells/mL). Importantly, the assay could be performed with whole blood with minimal sample processing.

Red cell autoantibodies: Selection of blood for transfusion

Introduction: Transfusion of serological safe blood is an essential requirement in transfusion medicine. Development of anti- red blood cell antibodies remains a major problem. Materials and Methods: A study was conducted on 24237 donors and 25155 patients between August 2013 and July 2016 to determine the prevalence of RBC autoantibodies and management of autoantibody screen positive cases. Results: 19 donors (0.078%) were identified with RBC autoantibodies. 16 of 19 donors showed warm autoantibodies (WAAs), one showed cold autoagglutinin and two showed autoantibodies; type not identified. Ten patients (0.039%) were identified with autoantibodies, of which nine showed warm autoantibodies and one showed cold autoagglutinin. Conclusion: Autoantibodies are mostly formed against the high incidence antigens and therefore react with red cells of random donors' blood, causing difficulty in interpretation of cell typing, compatibility testing, antibody detection and antibody identification. There are wide variations in testing and RBC selection practices in patients with WAAs, further studies are required to evaluate and compare different testing algorithms and transfusion strategies.

Red cell antibody detection by serology

Red cell antibody detection by serology

Unexpected red cell antibody detection by conditional combination of LISS/Coombs and NaCl/Enzyme gel tests at a tertiary care hospital in Korea: A 5-year study

BackgroundA combination of the LISS/Coombs and enzyme methods is recommended for identifying unexpected antibodies. However, many laboratories in which tests are to be performed within the limits of medical fees covered by insurance, use only the LISS/Coombs method because the permissible medical fee is low as compared to the price of reagents required for both methods. The NaCl/Enzyme gel is used as a secondary assay when the LISS/Coombs gel test yields inconclusive results. We compared the frequency of unexpected antibody identified by LISS/Coombs gel with that obtained by the conditional combination of LISS/Coombs and NaCl/Enzyme gels. We aimed at establishing evidence-based guidelines for antibody testing.MethodsFrom June 2007 to June 2012, antibody screening was performed for 69,986 samples; subsequently, antibodies were identified in samples showing positive screening results. These initial screenings and identifications were performed using the LISS/Coombs gel. We considered the results "inconclusive" when specific antibodies were not identified or reactions were too weak for accurate interpretation. For the inconclusive samples, we subsequently used NaCl/Enzyme gels.ResultsThe overall detection rate of unexpected antibodies was 1.23%. Among the samples analyzed using NaCl/Enzyme gels, 40.2% showed results different from those obtained using LISS/Coombs gels. Moreover, 41.9% of samples with nonspecific reactions in LISS/Coombs gels showed clinically significant Rh or Kidd antibodies with NaCl/Enzyme gels.ConclusionConsidering both patient safety and cost effectiveness, we recommend the use of conditional combination of LISS/Coombs and NaCl/Enzyme gels for antibody detection, especially in laboratories that must perform tests within an established budget.

Recombinant blood group proteins for use in antibody screening and identification tests

The present review elucidates the potentials of recombinant blood group proteins (BGPs) for red blood cell (RBC) antibody detection and identification in pretransfusion testing and the achievements in this field so far. Many BGPs have been eukaryotically and prokaryotically expressed in sufficient quantity and quality for RBC antibody testing. Recombinant BGPs can be incorporated in soluble protein reagents or solid-phase assays such as ELISA, color-coded microsphere and protein microarray chip-based techniques. Because novel recombinant protein-based assays use single antigens, a positive reaction of a serum with the recombinant protein directly indicates the presence and specificity of the target antibody. Inversely, conventional RBC-based assays use panels of human RBCs carrying a huge number of blood group antigens at the same time and require negative reactions of samples with antigen-negative cells for indirect determination of antibody specificity. Because of their capacity for single-step, direct RBC antibody determination, recombinant protein-based assays may greatly facilitate and accelerate the identification of common and rare RBC antibodies.

Chlamydia Trachomatis: Milestones in clinical and microbiological diagnostics in the last hundred years

Chlamydia trachomatis an obligate intracellular, Gram-negative bacterium is the causative agent of several acute or chronic, local and systemic human diseases such as trachoma, oculogenital and neonatal infections. It was discovered in 1907 by Halberstaedter and von Prowazek who observed it in conjunctival scrapings from an experimentally infected orangutan. In the last hundred years the detection and study of the intracellular pathogens, including chlamydiae, passed through an enormous evolution. This memorial review is dedicated to these important research and diagnostic discoveries and to the scientists who significantly contributed to this evolution starting from the application of simple light microscopy through the cell culture technique, antibiotic susceptibility, antigen and antibody detection, serotyping, to the real-time nucleic acid amplification and restriction fragment lengths polymorphism analysis. Although the majority of these old and new excellent diagnostic methods have been introduced into the rutine practice, the trachoma has remained one of the leading causes of blindness, and oculogenital chlamydial infections still are the most frequent sexually transmitted bacterial diseases, furthermore lymphogranuloma venereum is a disease emerging in the developed countries at the beginning of the 21 st century.

Detection of red cell antibodies: comparison of two low ionic strength diluents

Various low ionic strength diluents are used routinely for red cell alloantibody detection in the antiglobulin test to increase the rate of antibody association to antigen, thereby allowing a reduction in the incubation time while achieving optimal agglutination. Two commercial low ionic strength diluents (DiaMed ID-CellStab and Inverclyde LISS) were assessed using the DiaMed-ID LISS Coombs’ microtube column system, to assess whether or not the choice of diluent influences red cell antibody detection. Effects of two low ionic strength diluents after 15-min incubation were assessed in 150 samples containing a wide range of typical red cell alloantibodies. Inverclyde LISS gave significantly higher reaction strengths in 25% of samples when compared with the same red cells suspended in ID-CellStab. Variation in reaction strengths ranged from 1+ to 2+, using Inverclyde LISS versus CellStab. Of 131 red cell alloantibodies directed against Rh, Kell, Kidd and Duffy antigens, Inverclyde LISS detected 90% after 15-min incubation, whereas 83% were detected with CellStab. This study suggests that Inverclyde LISS provides better red cell alloantibody detection than does ID-CellStab, and this may be due to the higher ionic strength of ID-CellStab.

Clinical associations of serum antiendothelial cell antibodies in patients with sudden sensorineural hearing loss.

The role of antiendothelial cell antibodies in systemic vasculitis has been reported. The aim of the study was to define the clinical associations of serum antiendothelial cell antibodies in patients with sudden sensorineural hearing loss. A prospective study in patients with sudden sensorineural hearing loss. Serum samples were taken from 59 consecutive patients with sudden sensorineural hearing loss at time of presentation and from 28 normal control subjects. Indirect immunofluorescence assay was used to detect antiendothelial cell antibodies. The prevalence of antiendothelial cell antibody detection was 54% (32 of 59 patients), with a statistically significant difference between patients and control subjects (P =.0004). Antiendothelial cell antibody positivity was significantly associated with absent recovery of hearing loss (P =.0020). The cytotoxicity to endothelial cells of the inner ear by antiendothelial cell antibody-positive sera might play a role in causing the stria vascularis damage in immune-mediated sudden sensorineural deafness. The appearance of antiendothelial cell antibody is related to the poor outcome of hearing loss, and its detection could be helpful in the selection of particular patients with sensorineural hearing loss for specific immunosuppressive treatments.

Detection of Red Blood Cell Alloantibodies by Gel Centrifugation Test

Background: Gel centrifugation techniques and solid-phase antiglobulin tests have increased sensitivity and specificity for red blood cell antibody (rbc-ab) detection compared to classical tube techniques. In this report we present results of rbc-ab screening and differentiation of patient and donor sera using two variants of gel centrifugation test. Material and Methods: 95,138 sera of 76,693 patients and blood donors of Rostock University Medical School (samples of a 7.5-year period) were screened for rbc-abs by gel centrifugation test (DiaMed ID microtyping system, papain 37 °C, and indirect antiglobulin test with LISS). Positive sera were further differentiated in the same test system. Results: The incidence of alloimmunization was 2.68%. Differentiation of 6,407 positively screened sera resulted in 3,160 unspecific reactions preferably in enzyme test, 532 negative sera, 484 sera containing anti-D following Rhesus prophylaxis, 73 rbc autoantibodies, 103 cases of dubious antibody specificity, and 2,055 cases of rbc alloantibodies with defined single or multiple specificity. Anti-E was most frequently found (27.3%) followed by anti-D (18.1%), anti-Le(a) (12.5%), anti-Cw (9.7%), anti-C (8.7%), anti-K (7.9%), anti-c (3.9%), anti-Fy(a) (2.8%), anti-Jk(a) (1.6%), and further specificities in lower frequency. Especially for detection of antibodies directed against antigens of Rhesus system, the enzyme test was more sensitive than the antiglobulin test. Conclusion: Data of this investigation indicate that differentiation of rbc-abs by enzyme test plays an important role for early detection of Rhesus antibodies. Unfortunately, the enzyme test yields more unspecific reactions than the antiglobulin test. Therefore, unclear test results obtained with enzyme-treated rbcs in gel centrifugation test require critical interpretation.

Gastric parietal cell antibody detection by kidney/stomach IFA and H+/K+ ATPase ELISA procedures

Objective: Evaluate the performance of a new ELISA kit for the detection of anti-gastric parietal cell antibodies in normal and clinically-defined specimens. Compare ELISA results obtained with conventional testing using indirect immunofluorescence (IFA) on rodent kidney/stomach-slides.Methods: Specimens from patients with pernicious anemia, various autoimmune or infectious disease conditions, healthy individuals, and specimens submitted to a reference laboratory for gastric parietal cell antibody testing were collected. Specimens were tested by the INOVA QUANTA Lite™ Gastric parietal antibody (GPA) ELISA, which utilizes purified H+/K+ ATPase antigen as the substrate, and by IFA on rodent kidney/stomach-slides.Results: Testing of 210 specimens from normal, healthy individuals with ages ranging from 17 to 77 years old, resulted in a specificity of 96.6% (199/206). Of the 7 GPA positive specimens, the 2 specimens with the highest values were found to be GPA IFA positive.Antibodies to gastric parietal cells have been reported in up to 90% of patients with pernicious anemia. Testing a panel of blinded specimens from 20 patients with pernicious anemia resulted in 15 of the 20 interpreted as positive by the GPA ELISA. IFA test results performed at INOVA were in complete agreement with the GPA ELISA results, while results at the Italian site interpreted 18 specimens 1+ or 2+ and 2 specimens as weakly reactive by IFA. Several of the GPA ELISA negative pernicious anemia specimens were positive for intrinsic factor antibodies by a prototype intrinsic factor ELISA kit and suggest the value of testing for both GPA and intrinsic factor antibodies. Additional testing of a panel of specimens submitted to various laboratories for GPA testing found a 92% (45/49) agreement between the GPA ELISA and IFA assays.Sera from 49 patients with autoimmune, infectious, or other clinical conditions were tested on the GPA ELISA. Three specimens were positive for GPA antibodies and subsequently found to be positive for GPA by IFA. While the results of IFA testing generally agreed between laboratories, a few samples were consistently interpreted differently in different laboratories.Conclusion: The QUANTA Lite™ GPA ELISA allows the sensitive and specific detection of gastric parietal antibodies in human sera using a defined target antigen, H+/K+ ATPase. In contrast to the conventional GPA IFA procedure, the assay is not dependent on subjective interpretation and allows more objective, reproducible, and automatable testing for GPA.

Radioimmunoassay for Glutamic Acid Decarboxylase (GAD65) Autoantibodies as a Diagnostic Aid for Stiff-Man Syndrome and a Correlate of Susceptibility to Type 1 Diabetes Mellitus

To establish and validate a double-antibody radioimmunoassay (RIA) for detecting serum auto-antibodies against glutamic acid decarboxylase (GAD65). This enzyme catalyzes synthesis of the neurotransmitter gamma-aminobutyric acid in neurons and pancreatic islet cells. We compared the frequency of GAD65 and other "thyrogastric" autoantibodies in adult patients with stiff-man (Moersch-Woltman) syndrome, type 1 diabetes, or polyendocrine disorders and in healthy subjects. The frequency of pancreatic islet cell antibody (ICA) detection was also assessed. The GAD65 RIA was validated by testing blinded samples, by confirming the specificity of low-titered positive results by "cold" antigen inhibition, and by comparing the RIA results with results of a kit assay incorporating staphylococcal protein A as immunoprecipitant. Recombinant GAD65 protein labeled with 125I was used as antigen, and a combination of anti-human IgG and IgM was used as immunoprecipitant. Seropositivity was determined for ICA and gastric parietal cell antibodies by indirect immunofluorescence assays and for thyroid peroxidase (microsome) and thyroglobulin antibodies by agglutination assays. We detected GAD65-specific antibodies in all but 1 of 46 local patients with stiff-man syndrome (98%); 16 had evidence of diabetes. Positive values exceeded 20 nmol/L in 96%, and 89% were ICA-positive; 76% had additional thyrogastric antibodies. Of 41 patients with type 1 diabetes (17 local and 24 workshop serum specimens), 33 were GAD65 antibody-positive (80%); 85% of these positive values were 20 nmol/L or lower. Only 18% of sera from patients with type 1 diabetes were ICA-positive, but 59% had other thyrogastric autoantibodies. Of 20 patients with autoimmune endocrinopathies without diabetes or stiff-man syndrome, 35% were GAD65 antibody-positive, 5% were ICA-positive, and 90% were thyrogastric antibody-positive. Of 117 healthy control subjects, 8% were GAD65 antibody-positive, and a third of those had other thyrogastric antibodies (14% overall); none was ICA-positive. Seropositivity in the double-antibody RIA for GAD65 autoantibody is a sensitive and specific marker of predisposition to type 1 diabetes and related organ-specific autoimmune disorders. As such, this RIA is complemented by assays for thyroid and gastric parietal cell autoantibodies.

The use of pooled red cells and column techniques for routine red cell antibody detection.

The object of antibody screening is to detect all clinically relevant antibodies. In order to do this effectively red cells are selected with an appropriate antigen profile. The introduction of column techniques for antibody screening by indirect antiglobulin testing (IAT) and two-stage enzyme testing (ETC) is perceived to lead to an increased sensitivity and an ability to detect red cell antibodies more easily than by traditional tube techniques because reactions in columns are more easily read and are stable. We evaluated the use of a column technology with pooled red cells for routine antenatal screening. The pooled cells used contained at least one cell with homozygous antigen expression for the majority of clinically significant antibodies known to be present, except for Kell. Pooled cell results were not as easy to read in gel columns when compared with single cell results due to weaker reactions which were often diffused throughout the gel in the column. We concluded that the use of pooled cells led to a decreased sensitivity which proved problematic for the interpretation of results. We used a two-cell and a three-cell pool and found that detection of known antibodies was reduced in IAT and ETC methods.

Is it safe to omit the 37 degrees C reading from pretransfusion red blood cell antibody detection testing?

The routine pretransfusion red blood cell antibody detection test (PADT), performed at the authors' institution, consists of a three-phase, saline-antiglobulin technique (immediate spin, 30-minute incubation at 37 degrees C, IgG indirect antihuman globulin test [IAT]), and each phase is examined for hemolysis and agglutination. To determine if it would be safe to omit reading the 37 degrees C phase of the PADT, a 6-year retrospective review of records (February 1986 to February 1992) was undertaken. Of approximately 280,000 sera tested for unexpected red cell alloantibodies, 1480 (.53%) were reactive at only 37 degrees C. Of 1480 sera, 1313 contained alloantibodies of no or questionable significance (eg, anti-Le(a), anti-Leb, and anti-P1), 71 sera contained antibodies of undetermined specificity, and 10 sera were reactive because of rouleaux. Eighty-six serum samples from 53 different patients contained alloantibodies of potential significance (anti-K, anti-E, etc). These 86 sera represented approximately 2.2% of all reactive sera that contained potentially significant alloantibodies. Although most antibodies (approximately 94%) detected only at 37 degrees C were of no or questionable significance, the other 6% could have increased the risk of missing a potentially significant antibody from approximately 1 in every 4700 sera tested to 1 in every 1875 sera tested, had they not been detected. The authors suggest that the increased risk of an acute or delayed hemolytic transfusion reaction would be too high to justify a procedural change. Based on these data, the authors continue to read the 37 degrees C phase of the PADT for hemolysis and agglutination.

Non-human pancreas as substrate for cytoplasmic islet cell antibodies

To investigate qualitative and quantitative differences in islet cell antibody (ICA) detection within and between species we evaluated 8 pancreatic substrates (2 human, 2 baboon, 2 macaque, 1 pig and 1 rat). All substrates were able to detect strongly positive sera (with ICA greater than 160 JDF units). Non-human primate pancreata showed a larger size and greater abundance of islets than human, pig or rat. Significant differences in end point titer were observed both within and between species. In comparing the two best pancreata (1 human and 1 macaque) in masked screening of 50 normal controls and 108 nondiabetic first degree relatives of type 1 diabetics, very similar results were obtained. We concluded that pancreatic substrates from a variety of species may be useful for detection of ICA. This study, however, underscores the need for between and within assay standardization of ICA such as those currently underway in the International Immunology of Diabetes Workshops and Proficiency Tests.

Islet cell antibody detection standardisation

Islet cell antibody detection standardisation

A prozone phenomenon interferes in islet cell antibody detection: Direct comparison of two methods in subjects at risk of diabetes and in insulin dependent diabetics at onset

A recent international workshop documented marked interlaboratory variation in end point titers of standard islet cell antibody (ICA) positive sera. End titers were lower using a modified assay which utilizes fluorescein labeled protein A (ICA-pA) rather than fluoresceinated anti-IgG (ICA-IgG) to detect antibody binding to islets. In this study we sought to compare directly two ICA assays with respect to future development of IDDM. Sera were obtained from 26 prospectively evaluated high risk subjects identified by family screening or history of transient hyperglycemia and 12 normal controls. As expected, end point titers for ICA positive sera were 10 times greater using the ICA-IgG assay than with the ICA-pA assay. However, despite higher end point titers, the ICA-IgG assay failed to detect more ‘prediabetics’ and showed a prozone effect. Fourteen subjects were positive at a 1:2 dilution using the ICA-pA assay. Only 10 of these 14 were positive at a 1:2 dilution using the ICA-IgG assay but all became positive at greater sera dilutions. No normal controls were positive using either assay. A similar prozone was observed with anti-islet monoclonal antibodies A2B5 and 4F2. Sera from 14 long-standing IDDM patients (where titers of ICA may have decreased relative to time of onset of diabetes) which were negative using ICA-pA were also assayed using ICA-IgG. Five sera positive for ICA-IgG but negative for ICA-pA were identified. In addition two sera in which a prozone effect was seen with ICA-pA were identified. Our studies suggest that both diluted and undiluted sera should be assayed in order to avoid false negatives using either assay although an interfering prozone occurs more often with the ICA-IgG assay.

Red cell alloantibodies in thalassemia major. Results of an Italian cooperative study.

Clinical and serological data on 1435 Italian thalassemia major patients were collected during a cooperative study involving 19 centers in 10 regions. The main findings were as follows: 18 percent of the patients were under 6 years of age, 63 percent between 6 and 15, and 19 percent over 15. Forty-one percent had undergone splenectomy. Sixty-two percent of the patients were maintained at pretransfusion hemoglobin levels higher than 10 g per dl, 36 percent between 8 and 10 g per dl, and 2 percent below 8 g per dl. Overall, 5.2 percent of the patients had clinically significant red cell alloantibodies (136 alloantibodies in 74 patients). One-half of the immunized patients had more than one and one-fourth had more than two alloantibodies. The specificities of the 136 alloantibodies were almost exclusively confined to the common antigens of the Rh, Kell, Kidd, and Duffy systems, in that decreasing order of frequency. The antibody screening procedure, using a low-ionic-strength solution antiglobulin test against a three-red-cell panel and the patient's own red cells (autocontrol) with a serum to cell ratio of 100 to 1 was shown to be an adequate technique for red cell antibody detection.

Automated red cell antibody detection. An improved method.

A single-channel AutoAnalyzer was used to screen potential blood-grouping sera to exclude the presence of unwanted antibodies. In order to detect the widest possible range of antibodies, a low-ionic strength Polybrene method was employed, but difficulty was encountered because the aggregated cells adhered to the glass surfaces of the reaction coils, resulting in instability of the system. Modifications were made to the apparatus, and the improvements in the demonstration of antibodies that resulted are described.

Automated Red Cell Antibody Detection

Automated Red Cell Antibody Detection

Automated red cell antibody analysis. A parallel study. II. Identification of serological specificity.

Abstract. The AutoAnalyzer method of red blood cell antibody detection can be adapted to identify the serological specificity of these same antibodies. In the present study, 1,152 AA‐positive sera obtained during AA screening were investigated. In 217 (19%) of these sera blood group antibodies were identified by manual techniques. Since the AA techniques are generally more sensitive than manual methods an attempt was made to identify antibodies by the AA methods in 823 sera which were negative manually. The low‐ionic strength‐Polybrene (LISP) method was found to be suitable for antibody identification because of its sensitivity in the detection of most red cell antibodies and the small technical deviations of OD from the base‐line. The bromeline‐methyl cellulose (BMC) technique is less sensitive and often more difficult to interpret in antibody identification. In some cases, however, this method was the only one that gave positive reactions. In 214 instances antibodies were identified by the AA techniques, about half of which were anti‐D's, the others being RBC antibodies of more or less rare varieties. The relative frequency of these rare antibodies was higher in the group of manual‐negative sera, 47 versus 21%, normally found. Positive identification but no clear‐cut blood type antibody was found in 155 cases (19%). Indications were obtained that some of the reactions were due to HL‐A antibodies. Antibodies to HL‐A7 and HL‐A5 were found to react with red cells. In addition the AA techniques seemed to be more sensitive to detect RBC autoantibodies than standard manual methods. However, the usefulness of the AA techniques for routine antibody identification is diminished by the relative slowness of the machine.