Diagnostic Immunohistochemistry: What Can Go Wrong and How to Prevent It

Context.—There are a number of critical factors that can lead to incorrect results if the diagnostic pathologist performing immunohistochemistry is unaware of, or not vigilant about, their influence. Objective.—To highlight 3 arenas in which errors may be introduced. Data Sources.—For choosing the correct primary antibody, selection of the most appropriate antibodies for a given clinical application can be aided by obtaining information from the vendor; however, this can yield incomplete information. There are a number of online databases that have comparisons of antibodies from different vendors, particularly with respect to their use and properties. Reading the published literature can assist in this process, particularly with respect to determining antibody sensitivity and specificity, but it is a daunting task to keep up with all of the immunohistochemistry-related papers published. Finally, Web sites of a number of quality assurance organizations are accessible and can provide a wealth of information comparing the “real world” performance characteristics of different antibodies to the same target protein. False-positive signals can result from a number of factors, including the use of inappropriately high antibody concentration, and “pseudospecific” signal that is in the wrong compartment of the cell. False-negative signal can result from factors such as use of a nonoptimized epitope retrieval method. It is critical that epitope retrieval methods be optimized for each antibody employed in the laboratory. Conclusions.—By paying attention to these potential problems, the “black box” of diagnostic immunohistochemistry can be made more transparent.

Contributions of Y- and W-cell pathways to response properties of cat superior colliculus neurons: comparison of antibody- and deprivation-induced alterations

The cat's superior colliculus (SC) receives direct inputs from retinal W-cells (a W-D input) and Y-cells (Y-D input) and an indirect Y-cell input via the lateral geniculate nucleus and visual cortex (Y-I input). In previous studies we have shown that intraocular injection of antibodies raised against large retinal ganglion cells produces a dose-dependent reduction in the Y retinogeniculate pathway. Furthermore, when a sufficiently high antibody concentration is used, there is a substantial loss of the Y pathway and no apparent loss of the W pathway. In the present study, we used the antibodies to investigate the contributions of the Y and W pathways to functional organization within the SC. Binocular injections of low (330 micrograms/100 microliters) or high (1,000 micrograms/100 microliters) antibody concentrations were made. The antibody-mediated effects on SC cells' response properties were compared directly with effects of early binocular deprivation, which have been attributed to a loss of Y-I input. Extracellular single-cell recordings were made from the SC, and cells were classified as receiving Y-D, Y-I, or W-D inputs on the basis of their response latencies to electrical stimulation of the optic chiasm and optic tract. Injections of the low antibody concentration produced no significant effects on inputs to the SC. However, injections of the high antibody concentration resulted in a 70% reduction in SC cells with a Y-D input and an 82% reduction in SC cells with a Y-I input. There was no effect on the percentage of cells with a W-D input. Binocular deprivation produced a 76% reduction in the percentage of cells with Y-I input. Visual response properties of SC cells also were assessed. Injections of the high antibody concentration produced a 55% reduction in cells that respond with a directional preference and a 51% reduction in cells that respond to high-velocity stimuli. Binocular deprivation produced a 78% reduction in the proportion of directional cells and a 25% reduction in cells that respond to the ipsilateral eye. Taken together, the results of this and previous studies using cortical lesions, visual deprivation, and immunoablation suggest that Y-D input is the primary basis for responses to high stimulus velocity, Y-I input is an important basis for directional responses and response through the ipsilateral eye, and W-D input is important for responses to low stimulus velocity.(ABSTRACT TRUNCATED AT 400 WORDS)

Fluid-Phase Immunoradiometric Assay of F. VIII/WF Demonstrating Abnormal Antigenic Reactivity in Variants of Von Willebrand’s Disease

Antigenic reactivity of F. VIII/MF was studied in cryo-supernatant (n = 3) and in plasma from variants of vWd (n = 4) by fluid phase IRMA. Specific anti-human F. VIII/WF rabbit or goat 125-I-Fab fraqments were used as antibody. The slopes of the dose-response curves of cryo-supernatant and plasma from variants were significantly lower (p<0.001) than those of normal (n = 7), classical vWd (n = 2) or hemophilia A (n = 2) plasma and normal cryoprecipitate (n = 5). The ratio of the slopes of variant to control plasma was 0.60, 0.76, 0.74 and 0.86 using rabbit Fab fragments and 0.43, 0.51, 0.73 and 0.80 using goat Fab fragments (normal plasma : 0.98 ± 0.01 and 1.01 ± 0.04 respectively). Antigen-antibody binding was studied according to Scatchard by reacting a constant amount of VIIIR:Ag with serial dilutions of antibody. In control plasma and cryoprecipitate, at least two groups of binding sites were demonstrated, of high (K1 = 13.2) and low (K2 = 1.7) affinity. In variants of vWd and cryo-supernatant, weak binding sites observed at high antibody concentration were normal. At low antibody concentration, the strong binding sites differed from normal, the curves demonstrating a positive cooperativity. This abnormal antigenic reactivity is associated in cryo-supernatant and variants of vWd with a lack of VIIIR:RCo and of the large, slow-moving, forms of VIIIR:Ag by electrophoresis. Such data suggest an abnormality of aggregation of F. VIII/WF in variants of vWd.