Clinicopathologic Spectrum of Lysozyme-Associated Nephropathy.

Introduction: Lysozyme-associated nephropathy (LyN), a rare cause of kidney injury in patients with chronic myelomonocytic leukemia (CMML), has not been well described to date. We report the clinicopathologic spectrum of LyN from a multi-institutional series. Method: We identified 37 native kidney biopsies with LyN and retrospectively obtained clinicopathologic data. Results: Thirty-seven patients had a median age of 74 years and included 78% males. Their most common presentation was acute kidney injury (AKI) or AKI on chronic kidney disease (CKD) (66%) with median estimated glomerular filtration rate (eGFR) of 21.7 ml/min per 1.73 m2, and proteinuria of 1.7 g. A minority (15%) had partial Fanconi syndrome. Serum lysozyme levels were elevated in all tested. Hematologic disorder (n = 28, 76%) was the most common etiology, including CMML (n = 15), acute myeloid leukemia (n = 5), and myelodysplastic syndrome (MDS) (n = 5). Nonhematologic causes (n = 5, 14%), included metastatic neuroendocrine carcinoma (n = 3), sarcoidosis, and leprosy. Etiology was unknown in 4 (11%). Pathology showed proximal tubulopathy with abundant hypereosinophilic intracytoplasmic inclusions, with characteristic staining pattern by lysozyme immunostain. Mortality was high (8/30). However, among the 22 alive, including 85% treated, 7 had improved kidney function, including 1 who discontinued dialysis and 6 with increase in eGFR >15 ml/min per 1.73 m2 compared with eGFR at the time of biopsy. Conclusion: Increased awareness of the full clinicopathologic spectrum of LyN may lead to prompt diagnosis, earlier treatment, and potentially improved outcome of this rare entity.

Circulating SARS-CoV-2+ megakaryocytes are associated with severe viral infection in COVID-19.

Several independent lines of evidence suggest that megakaryocytes are dysfunctional in severe COVID-19. Herein, we characterized peripheral circulating megakaryocytes in a large cohort of inpatients with COVID-19 and correlated the subpopulation frequencies with clinical outcomes. Using peripheral blood, we show that megakaryocytes are increased in the systemic circulation in COVID-19, and we identify and validate S100A8/A9 as a defining marker of megakaryocyte dysfunction. We further reveal a subpopulation of S100A8/A9+ megakaryocytes that contain severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) protein and RNA. Using flow cytometry of peripheral blood and in vitro studies on SARS-CoV-2-infected primary human megakaryocytes, we demonstrate that megakaryocytes can transfer viral antigens to emerging platelets. Mechanistically, we show that SARS-CoV-2-containing megakaryocytes are nuclear factor κB (NF-κB)-activated, via p65 and p52; express the NF-κB-mediated cytokines interleukin-6 (IL-6) and IL-1ß; and display high surface expression of Toll-like receptor 2 (TLR2) and TLR4, canonical drivers of NF-κB. In a cohort of 218 inpatients with COVID-19, we correlate frequencies of megakaryocyte subpopulations with clinical outcomes and show that SARS-CoV-2-containing megakaryocytes are a strong risk factor for mortality and multiorgan injury, including respiratory failure, mechanical ventilation, acute kidney injury, thrombotic events, and intensive care unit admission. Furthermore, we show that SARS-CoV-2+ megakaryocytes are present in lung and brain autopsy tissues from deceased donors who had COVID-19. To our knowledge, this study offers the first evidence implicating SARS-CoV-2+ peripheral megakaryocytes in severe disease and suggests that circulating megakaryocytes warrant investigation in inflammatory disorders beyond COVID-19.

A Novel Approach to Estimating M-Protein Concentration: Capillary Electrophoresis Quantitative Immunosubtraction.

INTRODUCTION: M-protein quantification is routinely performed by demarcating serum protein electrophoresis (SPE) regions. However, quantification of ß-migrating M-protein is hindered by overlapping nonimmunoglobulin protein. Immunosubtraction (ISUB) on capillary electrophoresis is a method currently used qualitatively to subtract out (and therefore highlight) immunoglobulin isotypes in serum, thus reducing the masking effect of normal serum proteins. This study expands on traditional ISUB by developing a quantitative immunosubtraction (qIS) methodology. METHODS: qIS is achieved by estimating the monoclonal class-specific immunoglobulin contribution to the SPE region containing the M-protein. We conducted a recovery study by use of serial dilutions from 3 patients with ß-region M-proteins (n = 22), performing SPE and ISUB on each dilution. We visualized the difference between the ISUB electrophoresis trace and the involved ISUB isotype-subtracted trace to distinguish M-protein and background polyclonal immunoglobulins, which was demarcated independently by 3 pathologists. The M-protein contribution to the ß-region was calculated and applied to the ß-region protein concentration producing the quantitative M-protein concentration, while minimizing contamination by nonimmunoglobulin or polyclonal immunoglobulin proteins. RESULTS: Using a quality target of 25% error, we determined that our analytical measurable range spanned the maximum concentration tested (0.81 g/dL) to 0.05 g/dL. Passing-Bablok regression between qIS and the expected M-protein produced a slope of 1.04 (95% CI, 0.94-1.09), r = 0.99. Total CV was 4.8% and intraclass correlation between pathologists was 0.998. DISCUSSION: qIS promises quantification of ß-migrating M-proteins at concentrations an order of magnitude lower than traditional SPE methodology, allowing earlier detection of increasing or decreasing M-protein.

Technical feasibility of isolated Bowman layer graft preparation by femtosecond laser: a pilot study.

PURPOSE: To evaluate the technical feasibility of isolated Bowman layer (BL) graft preparation by femtosecond laser (FSL) and to compare the ultrastructural morphology to manually dissected grafts. METHODS: Five whole globes were placed in custom-made eye holders and debrided of epithelium. After programming a dissection depth of 20 µm, the FSL was docked into position and 5 isolated BL grafts were created. From 5 additional globes, corneoscleral buttons were procured, mounted in artificial anterior chambers, and stripped of BL via the previously described manual technique. Three specimens from both series were randomly selected and assigned to transmission electron microscopy for ultrastructural evaluation and thickness measurements. RESULTS: All dissections were uneventful and 10 total grafts were produced: 5 by FSL and 5 by manual dissection. Mean graft thickness was 37 (±8.6) µm (n = 3) for the FSL group and 11.7 (±1.6) µm (n = 3) for the manually dissected group. Transmission electron microscopy revealed a thick but relatively smooth posterior cut edge in the FSL group, versus a virtually isolated BL with irregular rests of dispersed stroma in the manually dissected group. CONCLUSIONS: Femtosecond laser may have potential for harvesting intact BL and with a smooth posterior surface, but accompanied by variable amounts of anterior stroma owing to technical limitations.