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Biomedical subjects

F V Plapp

Publications and source records attributed to F V Plapp.

At least 19 recordsLinked to original sources

Automation in blood banking. Machines for clumping, sticking, and gelling.

Hospital transfusion services and blood donor centers have continued to rely on manual hemagglutination methods for pretransfusion compatibility testing. Automation with continuous flow and batch analyzers has been practical only for the largest donor centers. During the last 20 years, other methods of streamlining compatibility testing have evolved. Two of the most successful approaches have used microplates to perform liquid agglutination tests or solid-phase, red cell adherence tests. More recently, a gel test has been developed. On the basis of these technologies, increasing numbers of semiautomated systems for compatibility testing have become commercially available. However, these systems primarily address the needs of large donor centers. New technologies are needed to automate the transfusion services of hospitals and other small laboratories.

Automation

A rapid screening test for detection of IgA deficiency.

A solid-phase red cell adherence (SPRCA) assay has been developed to screen blood donors for IgA deficiency, and 6117 donor sera have been screened by this method. Eighteen sera were found to be IgA deficient, which represents a frequency of 1 in 340. Seventeen of these sera were retested by passive hemagglutination inhibition, which has a sensitivity of approximately 0.1 mg per dL. Eight sera were confirmed as IgA deficient, and nine were found to contain low levels of IgA (less than 1 mg/dL). The approximate sensitivity of the SPRCA assay is 1 mg per dL. The speed, simplicity, and sensitivity of this assay make it a good alternative to conventional methods of screening.

Blood Donors

Hyperkalemia after irradiation of packed red blood cells: possible effects with intravascular fetal transfusion.

Plasma potassium, calcium, and albumin concentrations in irradiated blood, and in fetal blood before and after transfusion, were measured. Dangerously high plasma potassium levels were observed in some units of irradiated packed red blood cells (range, 13.9 to 66.5 mEq/L; mean, 44.7 mEq/L) and could be one possible explanation for the high incidence of fetal arrhythmia associated with fetal intravascular transfusion. There are many factors operative in the preparation of irradiated packed red blood cells that may predispose to high potassium levels: the age of the red blood cells, the number of procedures used to concentrate the blood, the duration of time elapsed from concentration, the duration of time elapsed from irradiation, and the hematocrit. Use of fresh blood, avoidance of multiple packing procedures, limiting the hematocrit in the donor unit to less than or equal to 80%, and minimizing the time between concentration, irradiation and transfusion may minimize the potassium levels, and therefore making an additional washing procedure unnecessary.

Arrhythmias, Cardiac

Bedside blood grouping.

Early attempts to provide bedside ABO grouping were abandoned in favour of the convenience of centralised laboratory testing. However, transport of samples involves the inherent risk of clerical error, which may result in a haemolytic transfusion reaction. One way to minimise these errors would be to verify the recipient's ABO group at the bedside immediately prior to transfusion. Card or slide haemagglutination methods may be utilised for this purpose, but a recently described dipstick technique is ideally suited for bedside ABO grouping, as well as a variety of other near-patient testing applications.

ABO Blood-Group System

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.

Blood Grouping and Crossmatching

New techniques for compatibility testing.

Hospital transfusion services and blood centers have continued to use manual hemagglutination methods for most of their pretransfusion compatibility testing. Automation with continuous-flow and batch analyzers has been practical only for the largest blood-processing centers. During the past 20 years, other methods of streamlining compatibility testing have evolved. Two of the most successful approaches have employed microplates to perform either liquid agglutination tests or solid-phase red blood cell adherence tests. Increasing numbers of automated systems for compatibility testing, based on these technologies, have become commercially available. These systems were optimally designed for batch processing. More recently, dipsticks for ABO grouping and direct antiglobulin testing have been developed, which were ideally suited for individual testing.

ABO Blood-Group System

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.

Antibodies

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.

Antibodies

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.

ABO Blood-Group System

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.

ABO Blood-Group System

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.

Autoanalysis

Detection of an antigen on the inner surface of Rh negative erythrocytes which binds anti-D IgG.

Previous investigation have demonstrated the presence of the Rho(D) antigen in Rh negative erythrocytes. The intact Rh negative cell, however, does not bind anti-D IgG. Presently we have shown that an anti-D binding antigen resides on the cytoplasmic surface of Rh negative erythrocyte membranes. Unsealed Rh negative membranes, in which both the inner and outer surface are exposed, bind anti-D IgG. Dicyclohexylcarbodiimide specifically blocked the binding of anti-D IgG to these membranes. Sealed Rh negative membranes which expose only their external surface, failed to bind anti-D antiserum. These results were confirmed by proteolytic digestion of membrane preparations and subsequent Rho(D) antigen purification. Only when protease had access to the inner surface of Rh negative erythrocyte membranes did degradation of this 'D' antigen occur. Thus, intact Rh negative erythrocytes contain an antigen which binds anti-D antibody but is located on the inner surface of the membrane. In contrast, Rh positive erythrocytes expose Rho(D) antigen on the external surface of the membrane.

Antigens, Surface

The Rhesus D antigen. A dicyclohexylcarbodiimide-binding proteolipid.

Previous studies on the human Rhesus D antigen revealed several similarities between the D antigen and proteolipids. Proteolipids are a family of low-molecular-weight, hydrophobic proteins that are soluble in chloroform/methanol. In addition, many proteolipids bind dicyclohexylcarbodiimide (DCCD), an ATPase inhibitor. For determination of whether the D antigen was a proteolipid, the chloroform/methanol solubility and DCCD binding of the antigen were investigated. DCCD specifically inhibited the binding of anti-D IgG to Rh-positive red blood cells and to partially purified D antigen as determined by enzyme-linked immunoassays. The antigen was not only soluble in chloroform/methanol but was purified to apparent homogeneity by extraction with these solvents and subsequent discontinuous sucrose gradient centrifugation. The antigen's chloroform/methanol solubility, DCCD binding, low molecular weight, and previously reported phospholipid dependence allow classification of the D antigen as a proteolipid. The discovery that the D antigen is a proteolipid provides further clues to the antigen's cellular function.

Antigen-Antibody Reactions