Antibody Screening Procedures Research Articles

249 (PB029) - Identification of Nectin-4 Specific Monoclonal Antibodies Using a High-throughput Flow Cytometry Screening System

Background: Nectin-4 has recently emerged as a highly valuable therapeutic target. This is due to its overexpression in epithelial cancers, including ovarian, breast, colorectal, lung, and pancreatic, while exhibiting limited expression in normal adult tissue. This overexpression is functionally significant for tumor progression, making Nectin-4 an ideal target for potential cancer therapeutics. Using a high-throughput flow cytometry-based antibody screening procedure, wherein antibody binding to native cell-expressed Nectin-4 is assessed concomitantly with off-target binding, multiple antibodies were identified.

Red blood cell alloimmunisation and autoimmunisation in transfusion dependent beta thalassemics from Southern India

AbstractThe development of red blood cell (RBC) isoimmunization with alloantibodies and autoantibodies complicate transfusion therapy in multiply transfused thalassemia patients. We conducted a study to analyse the frequency in our population. Clinical and antibody profile from 55 multiply transfused thalassemic patients who were receiving transfusions were collected and analyzed prospectively. A commercially available 3 cell antigen panel was used for the antibody screening procedure. If antibody screening with the 3-cell antigen panel was positive, an extended 11-cell antigen panel was used for antibody identification in LISS (Low Ionic Strength Solution). All patients received blood matched for only ABO and Rh (D) antigens. A total of 55 transfusion dependent â thalassemics were included in this study out of which 30 (54.55%) were males and 25(45.45%) females with a male to female ratio of 1.2: 1. Frequency of red cell alloimmunization in this study was found to be 1.8%. None of the patients developed red cell autoimmunization. The alloantibody identified in the the patient who developed alloimmunisation was was anti-K. In conclusion, the transfusion of matched blood is essential for chronically transfused beta thalassemia patients in order to avoid alloimmunization.

HCV transmission in industrialized countries and resource-constrained areas

HCV is a blood-borne virus transmitted by percutaneous exposure to infected blood or blood-derived body fluids. The main routes of transmission are blood transfusions, medical procedures and injection drug use. In industrialized countries, HCV transmission through blood transfusions has been virtually eliminated and iatrogenic transmission occurs only sporadically during local breaches of infection control procedures. As most new cases originate from injection drug use, harm-reduction programmes (including opiate substitution, needle exchange and health education) can greatly reduce HCV transmission. Currently, the main approach to reduce the HCV disease burden is by increasing awareness of both the public and health-care providers to hepatitis C, enhancing screening opportunities and treatment of the infected population. In resource-limited countries, the priority is reducing transmission through blood transfusions and invasive medical procedures. This approach requires training of health-care providers and also structural changes and financial investments in countries where antibody screening, disposable materials and effective sterilization procedures are not routinely available. In these countries, reducing the HCV burden has been hampered by limited access to treatment, largely owing to the cost of drugs. Access to treatment is moving up on the agenda of international and non-governmental organizations in conjunction with the future availability of highly efficacious oral drug regimens.

Evaluation of the clinical utility of maternal alloantibody screening as a surrogate to antiglobulin crossmatch procedures in resource limited settings

Aims: In resource limited settings, cross matching procedures are usually limited to the conventional antiglobulin technique. Pre-transfusion screening for red cell alloantibodies are not carried out routinely. The study was aimed at evaluating the usefulness of antibody screening as a surrogate to antiglobulin crossmatch procedure. Methods: A total 250 pregnant women attending the antenatal clinic of the Braithwaite Memorial Specialist Hospital (BMSH), Port Harcourt were screened for the presence of red cell alloantibodies using DiaMed screening and panel cells (DiaCell and DiaPanels). Results: Alloantibodies detected were anti-E (1.2%), anti-K (0.8%), anti-C (0.4%) and anti-Jsb (0.4%). The overall prevalence rate of red cell allo - antibodies was 4.8%. A blind crossmatch performed using the serum of the patients on donor's cells revealed the following results—incompatible 5 (2.0%) and 254 (98.0%) compatible. Taking incompatible results as positive and compatible as negative, the performance indices of the antibody screening procedure was obtained as follows: sensitivity (41.6%), specificity (100%), PPV (100%), NPV (97.1%), Efficiency (48.6%). Prevalence (4.8%) and percentage safety (41 .6%). The study did not show the type and screen to reach the expected safety level of 99.0%. Its usefulness was however shown through the detection of unexpected antibodies in 4.8% of the subjects. Conclusions: We concluded that with a high specificity obtained, the detection and identification of these antibodies would help select blood in advance for patients undergoing surgery in order to reduce the incidence of haemolytic transfusion reactions.

Delayed hemolytic transfusion reaction due to anti-S antibody in patient with anti-Jk a autoantibody and multiple alloantibodies

We describe the case of a 60-year-old woman with a delayed hemolytic transfusion reaction (DHTR). She had a history of an ulcerative colitis, blood transfusion because of rectal bleeding, and surgical removal of descendent and sigmoid colon. At admission, laboratory data showed Hb 6.3 g/dL, reticulocytes 120 x 10(9)/L, serum total bilirubin 1.2 mg/dL (direct bilirubin: 0.2 mg/dL). Pretransfusion antibody screening procedures were positive. A monospecific autoanti-Jk(a) and three alloantibodies (anti-c, -E, -K) were identified by immunohematologic studies. The patient received two units of crossmatch compatible concentrated red blood cells. Six days later biochemical serum values showed Hb 6.2 g/dL, LDH 975 I.U./L and total bilirubin 2.95 mg/dL (direct 0.35 mg/dL). Crossmatches with red cell suspension of transfused blood units and a post-transfusion serum were repeatedly positive. Laboratory tests showed the presence of anti-S alloantobody in the serum and eluate. Moreover, pre-transfusion serum of the patient was retrospectively retested: anti-S was not detected. These data suggested a DHTR. The present case is unusual and interesting because of the association of a rare autoanti-Jk(a), non responsible for anemia, and four alloantibodies of which anti-S involved in a DHTR.

Assessment of an instrument for automated reagent and specimen dispensing in blood banking

We have evaluated a semi-automated computer controlled dispensing device in conjunction with microtube technology for the performance of routine blood grouping and antibody screening procedures. A total of 787 specimens have been tested, 78 with unexpected antibodies previously identified by manual microtube methods. All were tested in duplicate in different order; there was complete agreement in blood group (ABO, RhD) determinations and the unexpected antibodies were found in the appropriate microtube location in all duplicate sets of results. We conclude that the device accurately and reproducibly dispenses cells, sera and reagents. The savings of technologists' time over traditional manual tube methods exceed 75%, excluding the time to perform elements common to both methods. The combination of automation and microtube technology offers the opportunity for substantial savings in technologist time with accuracy in reagent and specimen dispensing.

Assessment of an instrument for automated reagent and specimen dispensing in blood banking

We have evaluated a semi-automated computer controlled dispensing device in conjunction with microtube technology for the performance of routine blood grouping and antibody screening procedures. A total of 787 specimens have been tested, 78 with unexpected antibodies previously identified by manual microtube methods. All were tested in duplicate in different order; there was complete agreement in blood group (ABO, RhD) determinations and the unexpected antibodies were found in the appropriate microtube location in all duplicate sets of results. We conclude that the device accurately and reproducibly dispenses cells, sera and reagents. The savings of technologists' time over traditional manual tube methods exceed 75%, excluding the time to perform elements common to both methods. The combination of automation and microtube technology offers the opportunity for substantial savings in technologist time with accuracy in reagent and specimen dispensing.

Stereoselectivity of antibodies for the bioanalysis of chiral drugs.

The use of immunoassay techniques represents an alternative approach to the more common chromatographic methods for the determination of the concentrations of chiral drugs. In the development of the former technique, the inherent stereoselectivity of the antibodies used appears to be an important parameter to be studied. The structural features of the drug, including the environment around the asymmetric center, the flexibility of the molecule and the ability of the molecule to undergo racemization, contribute to the molecule's ability to be recognized by stereoselective antibodies. These parameters are intrinsic and can not be influenced by the investigator. On the other hand, other parameters can be modified to favor the raising of highly stereoselective antibodies. These include the synthesis of an appropriate hapten; the selection of an optimal spacer arm between the hapten and the carrier protein used for the immunization procedure; and the choice of appropriate immunization and antibody-screening procedures. The purification of antibodies using affinity chromatography may also facilitate the selection of stereoselective antibodies.

A MONOCLONAL ANTIBODY TO HUMAN PLACENTAL LACTOGEN HORMONE FACILITATES ISOLATION OF FETAL CELLS FROM MATERNAL BLOOD IN A MODEL SYSTEM

A cocktail of trophoblast-reactive monoclonal antibodies (MAbs) is required for efficient isolation of trophoblasts from maternal blood. A modified antibody screening procedure was used to identify a clone, in a COS cell placental cDNA expression library, that expressed the gene product recognized by MAb FDO202N. The antigen recognised by MAb FDO202N was identified as human placental lactogen (hPL) hormone. hPL hormone is secreted into the maternal blood by trophoblasts at high levels during pregnancy. Immunohistochemical localization of hPL hormone was consistent with expression in the syncytiotrophoblast and extravillous cytotrophoblast. A model system was used where known numbers of syncytiotrophoblast sprouts were seeded into saline or maternal blood, bound by trophoblast-specific MAbs, recovered magnetically, and then counted. MAb FDO202N was shown to facilitate the efficient recovery of trophoblast sprouts from saline and maternal blood.

Evaluation of 4,4′‐diisothiocyanatostilbene‐2,2′‐disulfonic acid in the inhibition of rouleaux formation

DIDS (4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid) inhibits the formation of serum-or plasma-induced rouleaux through its ability to bind to band 3 on red cell membranes. This property of DIDS was evaluated in the serologic testing of specimens exhibiting rouleaux. Optimal test conditions for DIDS treatment of reagent red cells were determined by varying the volume and concentration of DIDS solution and the incubation temperature and duration and comparing the results of antibody screening procedures using specimens that exhibit macroscopically visible rouleaux. Blind titration studies compared untreated and DIDS-treated red cells to evaluate the maintenance of antigen integrity. The use of DIDS-treated red cells for antibody detection and identification was evaluated by comparing the results in donor specimens containing antibodies with those in untreated and DIDS-treated selected panel cells. In addition, 4-percent (wt/vol) dextran in serum was used to induce rouleaux formation as a way of determining the capability of DIDS to resolve ABO serum grouping discrepancies. Complete inhibition of rouleaux formation occurred when reagent red cells were treated by incubation at 37 degrees C for 10 minutes with 150 microliter (approx. 5 drops) of 0.05 mg per mL of DIDS in 0.9-percent NaCl. There were no significant differences in titration scores of untreated and DIDS-treated red cells tested with the 19 antisera used to assess antigen integrity. Antibody identification studies showed that DIDS-treated reagent red cells reacted similarly to untreated reagent red cells. In addition, DIDS resolved dextran-induced ABO serum grouping discrepancies. DIDS effectively resolved the serologic problems associated with the presence of rouleaux, without affecting the results of the test system itself. Implementation of this method to inhibit the rouleaux-forming properties of serum and plasma specimens can be useful in serologic testing.

A RAPID FLOW CYTOMETRY ASSAY FOR HLA ANTIBODY DETECTION USING A POOLED CELL PANEL COVERING 14 SEROLOGICAL CROSSREACTING GROUPS1

Many studies have demonstrated the usefulness of flow cytometry crossmatching (FC-XM) for selection of regraft recipients, and more recently this assay has been shown to correlate with allograft survival in primary cadaveric transplant patients. The need now exists for a practical antibody screening procedure which uses the same methodology. We describe here a simple and sensitive method to screen for HLA antibodies by FC using a pool of 6 cells selected to cover the 14 serological crossreacting groups defined by Rodey. Screenings of 367 sera (255 primary transplant sera, 112 regraft sera) received for monthly antibody testing were performed by both pooled cell FC and complement-dependent cytotoxicity (CDC) assays. Forty of these sera were also FC-screened using a panel of 16 individual cells for comparison with the pooled cell FC screenings. Analysis indicated a strong correlation between the pooled FC-PRA and the individual cell panel FC-PRA (P = .0001) with mean values of 60% and 73%, respectively. Only 2 of the 40 sera screened by both FC methods resulted in PRAs that differed by > 40%. The majority (82%) of the primary patients did not exhibit HLA antibodies by CDC--however, 22% of the CDC negative patients were positive by flow cytometry. Females were more likely to be positive by FC (35%) than males (16%) (P = .0001). Similarly, black patients were more likely to have FC-demonstrable antibodies (28%) than white candidates (14%) (P = .014). The regraft patients who tested positive by either or all methods had a mean PRA for CDC, pooled FC-PRA, and individual cell FC-PRA of 40, 75, and 85, respectively. FC-PRA proved to be a more sensitive technique in both primary and regraft patients.

Isolation and expression analysis of tyro3, a murine growth factor receptor tyrosine kinase preferentially expressed in adult brain.

Growth factors and their receptors function in the nervous system to induce proliferation and differentiation of neuronal precursor cells and to support survival of mature neurons. We have isolated a murine growth factor receptor tyrosine kinase using an anti-phosphotyrosine antibody screening procedure and studied the pattern of expression. The deduced amino acid sequence of the kinase has all the characteristics of a growth factor receptor and consists of a putative extracellular domain, a transmembrane domain, and a tyrosine kinase domain. Sequence comparison with known receptor tyrosine kinases indicated that the murine kinase is a mouse homolog of tyro3. tyro3 belongs to the Axl/Ufo growth factor receptor family. In the putative extracellular domain, there are two Ig-like domains and two fibronectin type III repeats which are conserved in other members of the Axl/Ufo family receptors. Northern blot hybridization analysis showed that tyro3 is expressed at high levels in the brain of adult mice, although considerable expression was also observed in the testis. In situ hybridization analysis revealed that high levels of tyro3 are expressed in the cerebral cortex, the lateral septum, the hippocampus, the olfactory bulb, and in the cerebellum. The highest levels of tyro3 expression in the brain are associated with neurons. The preferential expression of tyro3 in specific regions of the adult mouse brain suggests that tyro3 may function as a novel neurotrophic factor receptor.

Detection of anti‐Fya, anti‐S and anti‐Jka in relation to the genotypes of the panel red cells: report of a U.K. NEQAS survey

Three antibody-containing samples (anti-Fya, anti-S and anti-Jka), each at two dilutions, were distributed to U.K. hospitals and transfusion centres together with an antibody screening panel comprising red cells homozygous for the corresponding antigens. Participants in the study subjected the samples to their routine antibody screening procedures using both their own antibody screening panels and the screening panel provided. A within-group comparison of those participants using their own screening panels having a heterozygous expression of the antigens, with the same participants when using the screening panel provided, showed for five of the samples a greater detection rate in routine antibody screening procedures when using the panel provided, having homozygous expression of the corresponding antigens. The sixth sample, the most potent, was detected equally using both panels. The difference in overall detection rate is statistically significant (chi-square test, 2P < 0.001). The study shows that the use of red cells presumptively homozygous for Fya, S and Jka improved the detection of the corresponding antibodies.

Evaluation of the ID-gel test for antibody screening and identification.

A gel technique for the detection of red blood cell (rbc) antigen-antibody reactions was evaluated for use in antenatal antibody screening and identification procedures. The evaluation was undertaken on 3,900 random antenatal samples. Results obtained in the gel test system were compared with those obtained from parallel testing using conventional serological methods. The ID gel system detected 148 (3.7%) red cell antibodies, compared with 95 (2.4%) using traditional techniques. The number of non-specific antibodies and false-positive screens were reduced using the gel test system. Antibody titres performed using the gel system were more sensitive than with our tube IAT method. The gel system was easy to use and gave reliable, reproducible results. Antibody detection rates were enhanced compared with our existing routine techniques.

Blood transfusion and caesarean section.

A clinical audit of 1,000 consecutive Caesarean sections at the Royal Women's Hospital indicated that 9.4% of patients required blood transfusion. Urgent blood transfusion was required in 53% of the 33 patients requiring Caesarean section because of placenta praevia, 43% of the 23 with accidental haemorrhage, 11% of the 36 with severe preeclampsia, but in only 2.3% of the remaining 908 patients. Blood was cross-matched preoperatively in 47.7% of patients and 90% of this blood was either not administered or given as a delayed nonurgent procedure. It is suggested that routine preoperative cross-matching of blood could be replaced by a blood type and antibody screening procedure for Caesarean sections being performed for those indications associated with low risk of blood transfusion requirement.

Use of a urease-antibody conjugate in an enzyme immunoassay for the detection of blastomycosis.

A urease-antibody conjugate was used in an enzyme-linked immunoassay (ELISA) for the detection of antibodies in blastomycosis. A Blastomyces dermatitidis immunodiffusion antigen was used as the reference antigen in a solid phase indirect ELISA procedure and the endpoints were determined visually. Urease ELISA results on serum specimens from patients with blastomycosis compared favorably (90%) with results obtained spectrophotometrically by the alkaline phosphatase enzyme system. Specificity was evaluated with assays on sera from patients with histoplasmosis (20% cross reactivity), coccidioidomycosis (0% cross reactivity) and on Histoplasma capsulatum skin-test positive individuals (0% cross reactivity). The ease of performance of the urease ELISA combined with no requirements for specialized spectrophometric equipment are factors that favor the continued development of the test as an alternative serodiagnostic method. The assay may prove to be useful and a valuable adjunct to fungal antibody screening procedures.

The efficacy of performing red cell elution studies in the pretransfusion testing of patients with positive direct antiglobulin tests.

In order to evaluate the efficacy of performing red cell elutions in pretransfusion testing, the serologic records of 638 patients with positive direct antiglobulin tests (DAT) were reviewed. These patients were identified by routine antibody screening procedures that included an autologous control. DAT results on the red cells of these patients showed 279 with IgG and C3d sensitization, 319 with IgG alone, and 40 with C3d sensitization alone. Of 638 patients' red cell eluates, 401 demonstrated no reactivity, 154 demonstrated panagglutination, and 60 demonstrated passively acquired anti-A,B. Only 23 of 638 patients had alloantibody sensitization of their red cells. Of the 23, 19 had serum antibody corresponding to the specificity of antibody detected in the eluate. Thus, only four of 638 (0.6%) eluates gave results unavailable by serum testing alone. This study indicates that routine eluate investigation provides little useful information in assuring compatibility. Serum antibody testing and careful review of the clinical and transfusion history constitute appropriate pretransfusion testing in patients with positive direct antiglobulin tests. Eluate testing should be restricted to cases in which immune hemolysis is suspected clinically.

Restricted Expression of a New Acute Myelogenous Leukemia-Associated Antigen (NHL-30.5) on Normal Hemopoietic Progenitor Cells

Abstract We have previously reported the isolation of a monoclonal antibody (NHL- 30.5) that reacts with an antigen expressed on a substantial proportion of marrow and blood cells of most patients with newly diagnosed or relapsing acute myeloid leukemia. This antigen is also found on several cell lines derived from myeloid malignancies of human origin. It is not present on mature hemopoietic cells or on the majority of differentiating bone marrow cells. In order to determine whether the NHL-30.5 antigen may, nevertheless, be expressed on low-frequency primitive normal hemopoietic cells, not detected in standard antibody screening procedures, its expression was studied on clonogenic erythropoietic and granulopoietic cells. Light-density (less than 1.077 g/mL) suspensions of normal or chronic myelogenous leukemia bone marrow and peripheral blood cells were stained with NHL-30.5 and fluorescein isothiocyanate labeled second antibody and then sorted into two fractions using the fluorescence-activated cell sorter. The first contained the top 5% of cells with the highest fluorescence intensity. The remainder were collected in the second fraction. Colony assays of both fractions showed the first to be enriched in CFU-E, BFU-E, and CFU- C content (fourfold to 17-fold). The second fraction was correspondingly depleted of these progenitors. These findings reveal NHL-30.5 antigen expression to be a transient event during normal hemopoiesis that characterizes primitive hemopoietic cells on several pathways. Subsequent experiments showed that the presence of up to 10 micrograms/mL of purified NHL-30.5 antibody in colony assay cultures neither inhibited nor stimulated colony formation. Marrow fibroblasts (subcultured marrow adherent cells) were NHL-30.5 negative. Immunoprecipitation studies showed that the antigen detected by NHL- 30.5 is clearly distinct from that identified by My-10, another monoclonal antibody that has previously shown some similarities to NHL- 30.5. It thus appears that the NHL-30.5 antibody reacts with a new myeloid differentiation antigen of as yet unidentified function that is normally restricted in its expression to early stages of hemopoiesis.

Restricted expression of a new acute myelogenous leukemia-associated antigen (NHL-30.5) on normal hemopoietic progenitor cells

We have previously reported the isolation of a monoclonal antibody (NHL-30.5) that reacts with an antigen expressed on a substantial proportion of marrow and blood cells of most patients with newly diagnosed or relapsing acute myeloid leukemia. This antigen is also found on several cell lines derived from myeloid malignancies of human origin. It is not present on mature hemopoietic cells or on the majority of differentiating bone marrow cells. In order to determine whether the NHL-30.5 antigen may, nevertheless, be expressed on low-frequency primitive normal hemopoietic cells, not detected in standard antibody screening procedures, its expression was studied on clonogenic erythropoietic and granulopoietic cells. Light-density (less than 1.077 g/mL) suspensions of normal or chronic myelogenous leukemia bone marrow and peripheral blood cells were stained with NHL-30.5 and fluorescein isothiocyanate labeled second antibody and then sorted into two fractions using the fluorescence-activated cell sorter. The first contained the top 5% of cells with the highest fluorescence intensity. The remainder were collected in the second fraction. Colony assays of both fractions showed the first to be enriched in CFU-E, BFU-E, and CFU-C content (fourfold to 17-fold). The second fraction was correspondingly depleted of these progenitors. These findings reveal NHL-30.5 antigen expression to be a transient event during normal hemopoiesis that characterizes primitive hemopoietic cells on several pathways. Subsequent experiments showed that the presence of up to 10 micrograms/mL of purified NHL-30.5 antibody in colony assay cultures neither inhibited nor stimulated colony formation. Marrow fibroblasts (subcultured marrow adherent cells) were NHL-30.5 negative. Immunoprecipitation studies showed that the antigen detected by NHL-30.5 is clearly distinct from that identified by My-10, another monoclonal antibody that has previously shown some similarities to NHL-30.5. It thus appears that the NHL-30.5 antibody reacts with a new myeloid differentiation antigen of as yet unidentified function that is normally restricted in its expression to early stages of hemopoiesis.

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.