Karen Quillen

The COVID-19 pandemic has significantly impacted work, economy, and way of life. The SARS-CoV-2 virus displays unique features including widely varying symptoms and outcomes between infected individuals. Sensitive measurement of SARS-CoV-2 specific antibodies would provide new insight into virus transmission dynamics, pre-existing cross-reactive immunity, and the nuances of SARS-CoV-2 pathogenesis. To date, existing SARS-CoV-2 serology tests have limited utility due to insufficient detection of antibody levels lower than what is typically present after several days of symptoms. To measure lower quantities of SARS-CoV-2 IgM, IgG, and IgA with higher resolution than existing assays, we developed a new ELISA protocol with a distinct plate washing procedure and timed plate development via use of a standard curve. This ‘BU ELISA’ method exhibits very low signal from plasma or serum samples added to uncoated wells at as low as a 1:5 dilution. Use of this method revealed circulating SARS-CoV-2 receptor binding domain (RBD) and nucleocapsid protein (NP) reactive antibodies from blood samples drawn prior to May 2019. Of our prepandemic cohort, no SARS-CoV-2 RBD-reactive IgG antibodies were detected in subjects over 70 years of age, and SARS-CoV-2 NP-reactive antibodies were present at similar levels to infected subjects in some individuals and very low in others. Also, samples drawn in May 2020 from two individuals with no symptoms or no known virus exposure contained SARS-CoV-2 RBD-reactive antibodies at intermediate amounts compared with other subject groups (higher than pre-pandemic and lower than confirmed SARS-CoV-2 infected). The one asymptomatic SARS-CoV-2 convalescent subject in our study possessed comparable amounts of SARS-CoV-2 NP-specific IgM and IgG but drastically lower IgA than the symptomatic counterparts. Also, our assay detected positive signal from samples that gave negative results in a commercially available Lateral Flow Device (LFD) and the EUA approved Abbott IgG chemiluminescent microparticle immunoassay for SARS-CoV-2 antibody detection. We propose that this improved ELISA protocol, which is straightforward to perform, low cost, and uses readily available commercial reagents, is a useful tool to elucidate new information about SARS-CoV-2 infection and has promising implications for improved detection of all analytes measurable by this platform.

Background: A subset of COVID-19 patients exhibit clinical features of cytokine storm. However, clinicopathologic features diagnostic of hemophagocytic lymphohistiocytosis (HLH) have not been reported. Pathologic studies to date have largely focused on the pulmonary finding of diffuse alveolar damage (DAD). To this aim, we study the reticuloendothelial organs of four consecutive patients dying of COVID-19 and correlate with clinical and laboratory parameters to detect HLH. Methods: Autopsies restricted to chest and abdomen were performed on four patients who succumbed to COVID-19. Spleen, liver, and multiple pulmonary hilar/mediastinal lymph nodes were sampled in all cases. Bone marrow was obtained by rib squeeze in a subset of cases. Routine H&E staining as well as immunohistochemical staining for CD163 was performed to detect hemophagocytosis. Clinical and laboratory results from pre-mortem blood samples were used to calculate H-scores. Findings: All four cases demonstrated DAD within the lungs. Three of the four cases had histologic evidence of hemophagocytosis within pulmonary hilar/mediastinal lymph nodes. One case showed hemophagocytosis in the spleen but none showed hemophagocytosis in liver or bone marrow. Lymphophagocytosis was the predominant form of hemophagocytosis observed. One patient showed diagnostic features of HLH with an H-score of 217 while a second patient was likely HLH with a partial H-score of 145 due to missing triglyceride level. Both patients exhibited high fever and early onset rise in serum ferritin; however, neither bicytopenia, pancytopenia, nor hypofibrinogenemia were observed in either. The remaining two patients had H-scores of 131 and 96. Interpretation: This is the first report of SARS-CoV-2 associated HLH. Identification of HLH in a subset of patients with severe COVID-19 will inform clinical trials of therapeutic strategies.

Whitfield and colleagues in a recent letter in this journal used survey and focus group data from internal medicine trainees to get their input into how supervision could be most helpful; they highlight the value of supervisors being approachable, maintaining regular contact with trainees to develop a more rounded view of the trainee, knowing more than just their names and avoiding a ‘tick box’ approach to supervision.1 In another study among internal medicine and emergency medicine resident physicians, supportive factors within the domain of interaction with attending physicians accounted for half of the modifiable items in the work environment felt to be most important to reduce burn-out.2 A s a haematologist assuming a transfusion medicine position within the pathology department 17 years ago, I had barely interacted with pathology trainees in my training and working experience to date, let alone supervised any. Teaching the residents was mentioned as an afterthought by the pathology chairman during the interview, …

Late occurrence of antibody-mediated rejection (AMR), defined as occurring 6 months after transplantation, is associated with poor renal allograft survival, compared to early acute AMR and acute cellular rejection. The proteasome inhibitor bortezomib has multiple immunomodulatory effects on plasma cells, the source of donor-specific HLA antibodies which mediate AMR. Consecutive patients who presented with biopsy-proven AMR and donor-specific anti-HLA antibodies (DSA) at a single institution between July 2011 and February 2015 were included. They received rituximab 375 mg/m2 on day 1, bortezomib 1.3 mg/m2 and methylprednisolone on days 1, 4, 8, 11, and plasmapheresis on days -1, 4, 8, 11, 14, 15, 17, with herpes zoster prophylaxis. The primary outcome was graft survival independent of dialysis. Patients were prospectively assessed with serial monitoring of renal function and proteinuria, and neuropathy symptoms. Toxicity determination was made by medical record review for hospitalizations within 3 months of therapy, or documentation of opportunistic infection. Eleven patients were treated for late AMR (diagnosed at a median of 38 months post renal transplant) with this bortezomib-based protocol; 2 patients underwent the regimen twice. Of the 11 patients, 9 have functioning allografts (82% graft survival) with a median creatinine of 2.1 mg/dL (range 1.1 - 3.4 mg/dL), at a median follow-up of 50 months after AMR therapy (range 24 - 63 months). One patient was re-transplanted at 4 years post AMR treatment with no AMR recurrence to date at 2-years' follow-up, and a second patient re-initiated dialysis at 2 years post AMR treatment. Patient survival is 91% (10/11): 1 patient relocated out of state and was reported to have died from complications of hypertensive encephalopathy. The majority of patients (7/11) had several class I and class II DSA specificities; of these, 4 patients had negative class I DSA but persistent class II DSAs at 2 - 3 months post therapy. Only 1 patient who was positive for class II DSAs alone (DR53 and DQ2) converted to negative DSA, although DSA testing was delayed to 2 years' follow-up. Two patients were hospitalized within 1 month of the protocol, 1 for ileus and 1 for urinary tract infection and ruptured ovarian cyst. One other patient had herpes zoster. Renal allograft survival was 82% at 4 years after bortezomib-based therapy for late onset AMR; toxicity profile of this regimen was acceptable. Eradication of DSAs may not be necessary for meaningful and durable renal response. .

Focal segmental glomerulosclerosis (FSGS) is a leading cause of end-stage renal disease and has a high recurrence rate after kidney transplantation, attributed to a circulating permeability factor. Plasmapheresis is the treatment of choice after recurrence to remove the circulating factor. We present a case of recurrent FSGS 6 years after transplantation. It is instructive because proteinuria did not respond to intensive plasmapheresis—combined with rituximab—until the possibility of ineffective apheresis secondary to multiple aneurysms in the arteriovenous fistula (AVF) was considered. Proteinuria improved soon after alternative access for plasmapheresis was secured and AVF aneurysms were surgically repaired.