Capture-S, a nontreponemal solid-phase erythrocyte adherence assay for serological detection of syphilis.

A solid-phase erythrocyte adherence assay has been developed for the serological detection of reagin antibodies in syphilis. Capture-S (Immucor, Inc., Norcross, Ga.) is a nontreponemal, qualitative screening test for the detection of immunoglobulin G (IgG) and IgM antilipid antibodies in serum or plasma samples from blood donors. The Capture-S assay utilizes a modified Venereal Disease Research Laboratory antigen bound to microtitration wells and anti-IgG- plus anti-IgM-coated indicator erythrocytes as the detection system. The Capture-S assay was evaluated at six separate sites on 10,942 specimens. For patient samples of clinically diagnosed syphilis categories (n = 366), the Capture-S assay yielded a sensitivity of 80.7% versus 80.3% for the rapid plasma reagin (RPR) card test (Becton Dickinson Microbiology Systems, Cockeysville, Md.). In comparative experiments on patient and donor samples (n = 10,222), the Capture-S assay demonstrated a sensitivity of 94% compared to 91.2% for the RPR card test. The Capture-S and RPR card tests produced essentially equivalent specificities of 99.2% and 99.3%, respectively, for this sample population. For five test sites, the Capture-S and RPR card test demonstrated a 98.3% agreement (10,085 of 10,264) of test results. These evaluations indicate that the Capture-S compares favorably to the RPR card test in assay sensitivity and specificity, with the added benefits of ease of use, accommodation of high-volume testing, and potential for automation.

Trimeresurus venom inhibition of anti-HPA-1a and anti-HPA-1b antibody binding to human platelets.

A solid-phase red cell adherence assay was used to demonstrate the specific inhibitory effect of seven species of Trimeresurus snake venom on the binding of HPA-1a- and HPA-1b-specific platelet antibodies. Trimeresurus venom did not inhibit the binding of HLA-, HPA-3a-, HPA-3b-, HPA-4a-, HPA-5a-, and HPA-5b-specific platelet antibodies. Venom from other genera of snakes, including representatives from Agkistrodon, Ancistrodon, Bitis, Bothrops, Bungarus, Causus, Crotalus, Dendroaspis, Ecis, Micrurus, Naja, Notechis, Ophiophagus, Pseudechis, Sepedon (Hemachatus), and Vipera, all failed to specifically inhibit anti-HPA-1a and HPA-1b binding. These results may indicate that the component in Trimeresurus snake venom previously reported to bind to the platelet GPIIb-IIIa complex, inhibiting fibrinogen binding, binds close to the HPA-1a and HPA-1b epitopes.

Detection of Lewis, P1, and some MNS blood group system antibodies by a solid phase assay.

Some solid phase red cell adherence (SPRCA) assays are designed to detect IgG antibodies to red blood cell (RBC) antigens. These assays use anti-IgG-coated red cells as the indicator. It is reported that most antibodies to Lea, Leb, P1, M, and N fail to react by solid phase (SP), presumably because they are IgM antibodies. Those detected are assumed to be IgG. In one year, during routine testing using SPRCA to screen patients for intended RBC transfusion, 28 of 59 such examples were found to react: anti-Lea(9), -Leb(1), -M(14), -N(1), and -P1(3). A study was undertaken to determine if reactivity was due to crosslinking by IgM antibodies of antigen-positive indicator RBCs to antigen-positive reagent RBC monolayers, or due to detection of IgG antibodies. Antibodies were tested according to standard SP protocols, except where IgG-neutralized indicator RBCs were substituted for anti-IgG-active indicator cells. The 59 samples were retested with antigen-positive and antigen-negative indicator RBCs. Only 5 of 59 reacted optimally when antigen-positive indicator cells were used: anti-Lea(2), -Leb(1), -M(1), and -N(1). The reactions of all antibodies were abolished when the anti-IgG component of the indicator was neutralized by soluble IgG. These findings show that detection of most Lewis, P1, M, and N antibodies by SPRCA is dependent on the presence of an IgG antibody in the serum.

Red cell antibody identification by solid phase red cell adherence utilizing dried RBC monolayers.

Recent technological advances in the immobilization and drying of red cell monolayers for use in solid phase red cell adherence (SPRCA) assays have resulted in the development of reagent red cells for antibody screening and identification that are stable at mom temperature. Panels consisting of twelve different RBC samples dried onto individual microplate wells were evaluated with 176 samples whose antibody specificities had previously been determined by conventional hemagglutination techniques. Identification tests performed with dried SPRCA panels proved to be more sensitive and less time consuming than hemagglutination tests. The red cell antigens of dried membranes were shown to be stable and reactive following storage for 120 days at mom temperature.

The evolution of pretransfusion testing: from agglutination to solid-phase red cell adherence tests.

Hospital transfusion services and blood centers still use manual hemagglutination tests for most of their serological procedures. Automation of hemagglutination reactions has proven to be difficult, primarily because hemagglutination lacks an objective endpoint which can be easily interpreted by inexpensive instruments. Alternatively, solid-phase red cell adherence assays for ABO cell and serum grouping, Rh typing, red cell and platelet antibody screening, red cell and platelet crossmatching, IgA deficiency screening, hepatitis B surface antigen, and HIV antibody screening have been developed. The performance of these assays compares favorably with current hemagglutination and enzyme immunoassay methods. All of these tests share a common objective endpoint of adherence or nonadherence of indicator red cells. This uniformity allows easy interpretation of results visually, spectrophotometrically, or by image analysis. The latter technique has the potential to revolutionize the reading and interpretation of all agglutination tests. Solid-phase red cell adherence tests in microplates are ideal for batch processing large numbers of specimens. However, adherence tests are not restricted to this format. Therefore, blood grouping dipsticks have been produced, which permit testing of individual blood samples even outside of the laboratory.

Use of a solid phase red blood cell adherence method for pretransfusion platelet compatibility testing.

A solid phase red blood cell adherence method has been used for platelet antibody detection and crossmatching for refractory platelet recipients. Patient sera were first screened for HLA or platelet-specific antibodies, then crossmatched with potential apheresis platelet donors. The overall correlation of platelet crossmatch results with transfusion outcome was 97% in patients with no evidence of nonimmune platelet destruction. The solid phase red blood cell adherence method provided a feasible and effective alternative to HLA matching as a means of donor selection for refractory platelet recipients. The speed and simplicity of this method may allow most hospital laboratories to perform platelet antibody screening before routine platelet transfusions.

Solid phase red cell adherence immunoassay for anti-HIV 1: a simple, rapid, and accurate method for donor screening.

In technically developed countries in which acquired immunodeficiency syndrome is a risk to the recipients of blood or tissue, it is mandatory to screen the donor for evidence of HIV (human immunodeficiency virus) infection. Current tests, based on enzyme-linked immunoassay, are time-consuming and expensive and as such are unsuitable for developing countries. We describe a second generation test using anti-human IgG coupled to red cells as the indicator of antibody having reacted with test antigen (1). The test is complete within ten minutes, simple to perform and to read and has 100% sensitivity and 99% specificity compared with Western blot. It is ideal for the rapid screening of organ donors and for the screening of blood donors where cost is a major consideration.

The dot blot direct antiglobulin test.

The direct antiglobulin test (DAT) is the most widely used serologic method to determine whether antibody or complement has bound to red blood cells in vivo. A solid phase DAT, based on the dot immunobinding technic, has been developed (DOT DAT). The solid phase was prepared by attaching anti-human IgG to nitrocellulose membranes. Patients' red blood cells were washed in saline and layered on top of the membranes. After 5 minutes the membranes were washed and the results were read. A positive reaction had a red dot of adherent cells on the membrane, whereas a negative membrane remained white. Good correlation was observed between the DOT DAT and the hemagglutination DAT after testing of 131 patient samples. The primary advantages of the DOT DAT were its stability, simplicity, and objective end point.

Dipsticks for determining ABO blood groups.

Dipsticks for determining ABO blood groups were developed, based on the principles of dot immunobinding assays. Their sensitivity and specificity equalled those of conventional agglutination tests and they were simple, fast, stable, inexpensive, and easy to interpret. Since they used whole blood and did not require refrigeration or equipment, they should be useful for determining blood groups away from the hospital setting.

Solid-phase techniques in blood transfusion serology.

For nearly a century, erythrocyte agglutination has persisted as the most widely used method for the demonstration of antigen-antibody reaction in immunohematology. So far, no other system has been developed which can match its simplicity, versatility, and general reliability. The major disadvantage of agglutination reactions is the lack of an objective endpoint, which has severely hindered attempts to automate routine pretransfusion tests. To overcome this problem, we have designed a series of solid-phase assays for ABO and Rh grouping, antibody screening, compatibility, and hepatitis tests. Each of these solid-phase assays shares a common endpoint of red cell adherence, which is easily interpreted visually or spectrophotometrically. Computer interface permits the automatic interpretation and recording of results. We believe this solid-phase system should finally bring the blood bank laboratory into the age of automation.

A solid-phase antiglobulin test.

A solid-phase antiglobulin test was developed as an alternative to hemagglutination for compatibility testing. The solid-phase endpoint of red cell adherence allowed results to be read visually or spectrophotometrically. This method was easier to perform than a hemagglutination antiglobulin test and had increased sensitivity without loss of specificity.

Solid-phase ABO grouping and Rh typing.

A solid-phase adherence method (SPAM) for ABO grouping and Rh typing of red cells (RBCs) has been developed. Adherence reactions were read spectrophotometrically and interpreted by a computer. The SPAM had a 99.6 percent correlation with conventional microplate agglutination methods for ABO grouping and Rh typing. The increased sensitivity of the SPAM was demonstrated because it directly detected Du-positive RBCs and weak subgroups of A and B.

A solid phase antibody screen.

An automated solid phase antibody screen (SPAS) in microplates has been developed. Red blood cell (RBC) adherence was used as the end point instead of agglutination. Consequently, positive and negative reactions were readily distinguished by a microplate spectrophotometer. The SPAS performed as well as conventional antiglobulin methods for detecting IgG antibodies in donor sera and had increased sensitivity as determined by serial dilutions of antibodies.

The Rh antigen specificity of erythrocyte proteolipid.

A proteolipid was purified from erythrocytes by chloroform-methanol extraction and reconstituted into lipid micelles. This purified micellar proteolipid was used to demonstrate that different Rh antigens reside on the same protein molecule.