Prozone rates in the solid-phase platelet crossmatch assay and correlation with class I HLA antibody levels.

BACKGROUND: Solid-phase platelet crossmatch (PXM) testing is used to help manage patients with platelet transfusion-refractoriness. Recently, we published the first report of false-negative PXM results from prozone effect that was mitigated using sample dilution. This study aimed to describe the prevalence of PXM prozone effect and the levels of class I HLA antibodies (HLA-Abs) associated with positive PXM results and with false-negative PXM results from prozone effect. STUDY DESIGN AND METHODS: A cross-sectional study of patients undergoing PXM testing from July 2019 through December 2020 was performed. All PXM tests were run simultaneously using undiluted and 1:4 diluted patient plasma. Prozone effect was defined as a negative PXM result using undiluted patient plasma but a positive PXM result using 1:4 diluted patient plasma. RESULTS: Among 59 patients, 830 individual ABO-compatible PXM results yielded an overall positivity rate of 25.8% (214/830) and a false-negative rate from prozone effect of 4.7% (10/214). Among the 28 patients with class I HLA-Ab testing and no other anti-platelet antibodies, maximum HLA-Ab mean fluorescence intensity (MFI) was significantly associated with a positive PXM result (p < .0001; AUC approx. 0.9) and categorized into negative (<3700), indeterminate (3700-10300), and positive (>10300) maximum HLA-Ab MFI zones. Maximum HLA-Ab MFI, however, was not associated with prozone effect (p = .17; AUC approx. 0.6). DISCUSSION: While there is a strong predictive association between class I HLA-Ab levels and positive PXM results, PXM prozone effect is a common occurrence not associated with class I HLA-Ab levels, so additional testing with diluted samples should be considered.

False-negative solid-phase platelet crossmatch results due to prozone phenomenon.

Prozone is a known phenomenon affecting immunoassays causing falsely low or negative results when excess target is present in the test system. For assays used to evaluate immune-mediated platelet (PLT) transfusion refractoriness, prozone-like phenomenon has been described in solid-phase human leukocyte antigen (HLA) antibody testing and can be mitigated by diluting samples or pretreating samples with ethylenediaminetetraacetic acid (EDTA) or dithiothreitol. Prozone phenomenon has not yet been described in solid-phase red blood cell (RBC) adherence PLT crossmatch assays. CASE REPORT: A 40-year-old female with myeloid sarcoma and PLT transfusion refractoriness underwent repeated solid-phase PLT crossmatches; however, crossmatch-compatible PLTs units did not yield adequate PLT count responses. Class I HLA antibody testing with neat, diluted, and EDTA-pretreated serum demonstrated significant prozone-like effect and the presence of numerous high strength HLA antibodies. Based on this HLA antibody profile, HLA antigen-negative PLTs gave an adequate PLT count response. It was noted that the HLA types of her crossmatch-compatible PLTs were incompatible with her HLA antibody profile (eg, HLA-A2). With ABO-identical, HLA-A2-positive PLT units, a solid-phase PLT crossmatch was repeated using undiluted and diluted EDTA plasma. Undiluted EDTA plasma demonstrated negative or weakly positive PLT crossmatches while the diluted EDTA plasma demonstrated strongly positive PLT crossmatches. CONCLUSION: The prozone phenomenon can cause false-negative results in solid-phase RBC adherence PLT crossmatch assays, which can be mitigated with sample dilution. In immune-mediated PLT transfusion-refractory patients with high-strength HLA antibodies, sample dilution should be considered to correctly identify compatible PLT inventory.

HLA-B*45:22: a novel allele that differs from HLA-B*45:01:01 at several locations.

Discovery of a novel HLA-B*45:22 allele in a Middle Eastern heart and lung transplant candidate.

Steroidogenic acute regulatory protein in white sturgeon (Acipenser transmontanus): cDNA cloning, sites of expression and transcript abundance in corticosteroidogenic tissue after an acute stressor.

The white sturgeon, Acipenser transmontanus, is a primitive bony fish that is recognized as an important emerging species for aquaculture. However, many aspects of its stress and reproductive physiology remain unclear. These processes are controlled by various steroid hormones. In order to investigate the regulation of steroidogenesis associated with acute stress in sturgeon, a cDNA-encoding steroidogenic acute regulatory protein (StAR) was isolated from white sturgeon. The putative amino acid sequence of sturgeon StAR shares high homology (over 60%) with other vertebrates. Phylogenetic analysis grouped sturgeon StAR within Actinopterygii, but it was clearly segregated from teleost StARs. RT-PCR analysis revealed that transcripts were most abundant in yellow corpuscles found throughout the kidney and weaker signals were detected in gonad and kidney. Very weak signals were also detected in brain and spleen by quantitative real-time PCR. In situ hybridization revealed that StAR is expressed in the cells of yellow corpuscles. No significant changes in StAR gene expression were detected in response to an acute handling stress. These results suggest that StAR is highly conserved throughout vertebrates, but the expression of the functional protein during the stress response may be partially regulated post-transcriptionally.

Calcium-induced quiescence of sperm motility in the bluegill (Lepomis macrochirus).

Before dilution in hypoosmotic media sperm of freshwater fish are maintained quiescent by a range of factors including osmolality, K+ and pH, and the onset of motility is generally associated with an increase in cytoplasmic Ca2+. In contrast, Ca2+ in conjunction with osmolality was found to inhibit motility of intact bluegill sperm. Consistent with seminal plasma composition, 0.16 mmol/L Ca2+ and greater, in conjunction with an osmotic concentration of 290 mOsm/kg, inhibited the onset of bluegill sperm motility; sperm diluted in saline at 290 mOsm/kg without Ca2+ became motile. Cations Mn2+ and Sr2+, in conjunction with osmolality, had an inhibitory effect on initiation of sperm motility similar to that of Ca2+. Sperm motility was inhibited by Ca2+ channel blockers nimodipine and nifedipine, the mitochondrial Ca2+ uniporter inhibitor ruthenium red and the calmodulin inhibitors W-7 and trifluoperazine dihydrochloride. These results provide evidence that elevated cytoplasmic Ca2+ inhibits sperm motility and yet low levels permit or promote motility. This study demonstrates a unique inhibitory action of Ca2+ on the motility of intact fish sperm at physiologically relevant levels.

Exhaustive exercise, animal stress, and environmental hypercapnia on motility of sperm of steelhead trout (Oncorhynchus mykiss).

Motility of salmonid sperm is inhibited by the presence of carbon dioxide (CO2) in vitro; however, whether this occurs in response to challenges to the adult in vivo is not known. To determine whether CO2 negatively impacts sperm function in vivo, mature males were exposed to exhaustive exercise as well as to acute stress, chronic stress, tricaine anesthesia and environmental hypercapnia and sperm motility and semen CO2 tensions and pH values assessed. Semen CO2 rose and pH decreased significantly only in response to exhaustive exercise and environmental hypercapnia (13 kPa CO2). These changes in semen CO2 and pH were associated with reductions in numbers of sperm becoming motile upon water activation. Chronic and acute stress and tricaine anesthesia were without effect on sperm motility or on semen CO2 or pH. The time course of CO2 inhibition and recovery was evaluated in vitro. At least 50 min was required to note 50% of the inhibitory effect of low CO2 tensions on motility when sperm were exposed to 1.6-3.1 kPa CO2. At higher CO2 levels sperm motility displayed 50% of the inhibitory effect of these tensions within about 30 min. Sperm recovered maximal motility within 1 h of being placed in a nominally CO2-free environment. This study demonstrates sperm vulnerability to not only in vitro CO2 exposure but also in vivo exposure during exhaustive exercise and as result of environmental hypercapnia.