SARS-CoV-2 Viral Load Predicts Mortality in Patients with and without Cancer Who Are Hospitalized with COVID-19.

Patients with cancer may be at increased risk of severe coronavirus disease 2019 (COVID-19), but the role of viral load on this risk is unknown. We measured SARS-CoV-2 viral load using cycle threshold (CT) values from reverse-transcription polymerase chain reaction assays applied to nasopharyngeal swab specimens in 100 patients with cancer and 2,914 without cancer who were admitted to three New York City hospitals. Overall, the in-hospital mortality rate was 38.8% among patients with a high viral load, 24.1% among patients with a medium viral load, and 15.3% among patients with a low viral load (p < 0.001). Similar findings were observed in patients with cancer (high, 45.2% mortality; medium, 28.0%; low, 12.1%; p = 0.008). Patients with hematologic malignancies had higher median viral loads (CT = 25.0) than patients without cancer (CT = 29.2; p = 0.0039). SARS-CoV-2 viral load results may offer vital prognostic information for patients with and without cancer who are hospitalized with COVID-19.

Detection and Genetic Characterization of Community-Based SARS-CoV-2 Infections - New York City, March 2020.

To limit introduction of SARS-CoV-2, the virus that causes coronavirus disease 2019 (COVID-19), the United States restricted travel from China on February 2, 2020, and from Europe on March 13. To determine whether local transmission of SARS-CoV-2 could be detected, the New York City (NYC) Department of Health and Mental Hygiene (DOHMH) conducted deidentified sentinel surveillance at six NYC hospital emergency departments (EDs) during March 1-20. On March 8, while testing availability for SARS-CoV-2 was still limited, DOHMH announced sustained community transmission of SARS-CoV-2 (1). At this time, twenty-six NYC residents had confirmed COVID-19, and ED visits for influenza-like illness* increased, despite decreased influenza virus circulation.† The following week, on March 15, when only seven of the 56 (13%) patients with known exposure histories had exposure outside of NYC, the level of community SARS-CoV-2 transmission status was elevated from sustained community transmission to widespread community transmission (2). Through sentinel surveillance during March 1-20, DOHMH collected 544 specimens from patients with influenza-like symptoms (ILS)§ who had negative test results for influenza and, in some instances, other respiratory pathogens.¶ All 544 specimens were tested for SARS-CoV-2 at CDC; 36 (6.6%) tested positive. Using genetic sequencing, CDC determined that the sequences of most SARS-CoV-2-positive specimens resembled those circulating in Europe, suggesting probable introductions of SARS-CoV-2 from Europe, from other U.S. locations, and local introductions from within New York. These findings demonstrate that partnering with health care facilities and developing the systems needed for rapid implementation of sentinel surveillance, coupled with capacity for genetic sequencing before an outbreak, can help inform timely containment and mitigation strategies.

Guidance from the Scientific and Standardization Committee for lupus anticoagulant/antiphospholipid antibodies of the International Society on Thrombosis and Haemostasis: Update of the guidelines for lupus anticoagulant detection and interpretation.

This guidance focuses on methodological aspects of lupus anticoagulant (LA) testing, as well as interpretation of results for clinicians. The main changes in how to test for LA compared with the International Society on Thrombosis and Haemostasis Scientific and Standardization Committee 2009 guidelines, in the preanalytical phase are more detailed recommendations on how to handle testing in anticoagulated patients, and the timing of testing. Also, routine coagulation tests are advised to obtain more information on the coagulation background of the patient, and when necessary, anti-Xa activity measurement for heparins or specific assays for direct oral anticoagulants should be performed. The three-step procedure with two test systems (diluted Russell's viper venom time and activated partial thromboplastin time [aPTT]) is essentially not changed. Silica remains the preferable activator in the aPTT assays, but ellagic acid is not excluded. We advise simultaneous performance of the mixing and confirmatory step, in each sample with a prolonged screening test. The confirmatory step can also be performed on a mixture of patient plasma and normal pooled plasma. Cutoff values should be established in-house on at least 120 normals, with transference of the manufacturer's cutoffs as an alternative. Reporting of results has not been changed, although more attention is focused on what clinicians should know. Patient selection for LA testing has been expanded.

Absence of Distinct Immunohistochemical Distribution of Annexin A5, C3b, C4d, and C5b-9 in Placentas From Patients With Antiphospholipid Antibodies, Preeclampsia, and Systemic Lupus Erythematosus.

INTRODUCTION: In pregnancy, the presence of preeclampsia (PEC), systemic lupus erythematosus (SLE), and/or antiphospholipid antibody syndrome (APLS) is characterized by poor obstetric outcomes, with potential adverse effects for both mother and fetus. Although the histopathologic changes observed in these entities have been well established, the pathogenic mediators associated with tissue injury are poorly understood. METHODS: Forty placentas were evaluated, including 10 patients with preeclampsia, 9 with SLE, 11 with APLS, and 10 disease-free controls. Each case was subjected to a panel of immunohistochemical markers including C3b, C4d, Annexin A5, and C5b-9. Staining was graded on intensity and distribution. RESULTS: C4d staining was distinctly different among disease groups and controls. Moreover, 6/10 PEC cases, 3/9 SLE cases, and 4/11 APLS cases showed at least focal staining for C4d. All controls were negative. Annexin A5 (AnxA5) staining showed intrinsic variability in all disease groups, while 10/10 controls showed diffuse, strong staining (2+ or 3+). C3b staining was heterogeneous among groups. DISCUSSION: Previously, antiphospholipid antibody (aPLA)-associated pregnancy complications have been thought to be a consequence of a unique aPLA-mediated pathogenic mechanism. However, the immunohistochemical similarity (increased complement and decreased AnxA5 staining) observed in placentas from patients with APLS, PEC, and SLE suggests that aPLA-associated pregnancy complications may reflect a more general autoimmune mechanism.

Reimagining the antiphospholipid syndrome, an enigmatic thrombophilic disorder, through the looking glass of microscopic imaging.

The antiphospholipid syndrome (APS) is an autoimmune thrombophilic disorder that was described as a diagnostic entity over 30 years ago. And yet the pathogenic mechanisms that are responsible for its clinical manifestations remain to be definitively established. The syndrome is defined by (1) the concurrence of vascular thrombosis and/or pregnancy complications together with (2) positivity for immunoassays and coagulation tests that were derived from clinical observations of two anomalous laboratory test results-specifically, false positivity for syphilis infection in uninfected individuals and the finding of inhibitors of blood coagulation in patients who lacked any bleeding tendencies. Over the years, these were standardized into immunoassays and coagulation assays for APS. Here, we describe how prior knowledge of the immunologic and coagulation aspects of the disorder led to research involving a range of imaging modalities including light microscopy, immunohistochemistry, confocal scanning laser microscopy, transmission and scanning electron microscopy, and atomic force microscopy. In turn, the results from those studies led to a "reimagining" of APS that has advanced the understanding of pathogenic mechanisms of the disorder and has led to the development of novel mechanistically based diagnostics along with potential new treatment approaches that target disease mechanisms.

The effect of hydroxychloroquine on haemostasis, complement, inflammation and angiogenesis in patients with antiphospholipid antibodies.

Objectives: HCQ has been described as having a beneficial effect in patients with APS but its mechanism of action is unclear. We hypothesized that HCQ may have effects on subnormal angiogenesis, inflammation and haemostatic biomarkers seen in APS. The aim of our study was to assess laboratory markers [annexin A5 (AnxA5) anticoagulant activity, tissue factor (TF) levels, thromboelastography (TEG), CRP, Bb, C3a and VEGF] in HCQ-naïve patients with aPL at baseline and after commencing HCQ. Methods: Twenty-two patients with aPL [20 female, 2 male, median age 55 (range 18-70) years] had blood taken pre- and 3 months after starting HCQ 200 mg daily. Results: Soluble TF levels were significantly reduced comparing baseline and 3 months after HCQ commencement [401.8 (152.8) vs 300.9 (108) pg/ml (P = 0.010)]. No significant changes were found in the following [reported as pre- and post-HCQ commencement, mean (s.d.)]: AnxA5 anticoagulant ratio [187.1 (29.5) vs 193 (31) (P = 0.157)], anti-domain1 ß2 glycoprotein1 IgG activity [1.8 (2) vs 1.2 (1.4) µg/ml (P = 0.105)], complement C3a-des-Arg [147.8 (84.5) vs 154.4 (88.1) ng/ml (P = 0.905)], complement Bb [1.3 (0.7) vs 1.1 (0.7) µg/ml (P = 0.422)], VEGF [68.8 (40) vs 59.4 (19.6) pg/ml (P = 0.454)] and CRP [7 (3.5) vs 7 (3.9) µg/ml (P = 0.917)]. TEG results including TEG reaction time, achievement of clot firmness, TEG maximum amplitude and TEG percentage lysis 30 and 60 min after maximum amplitude showed no significant difference. Conclusion: HCQ significantly reduced soluble TF levels in patients with aPL. No significant change was observed in AnxA5 activity, anti-domain 1 IgG activity, TEG, CRP, complement Bb and C3a-des-Arg, and VEGF. Further studies of a larger patient cohort are needed.

A novel 2-stage approach that detects complement activation in patients with antiphospholipid antibody syndrome.

INTRODUCTION: The antiphospholipid syndrome (APS) is marked by autoantibodies that recognize anionic phospholipids in a cofactor-dependent manner. A role for complement has been implicated in the pathophysiology, however, elevations of complement activation markers have not been consistently demonstrated in clinical studies. We therefore designed a proof-of-principle study to determine whether complement activation might be detectable in APS by first exposing plasmas to phospholipid vesicles. METHODS: We examined complement activation markers in patients with APS, non-APS thrombosis, systemic lupus erythematosus, cancer, patients with antiphospholipid antibodies without thrombosis (APL) and healthy controls. Direct measurements of plasma C5a and sC5b-9 levels were compared to levels that were generated in normal serum by phospholipid vesicles that had been pre-incubated with the same plasmas. We then determined the effects of the C5 inhibitor, eculizumab, examined the complement pathways involved, and determined whether the effects could be reproduced with purified IgGs and ß2-glycoprotein I (ß2GPI). RESULTS: Plasma levels of C5a and sC5b-9 were higher, but not significantly increased in APS patients compared to healthy controls. In contrast, phospholipid vesicles pre-incubated with APS plasmas generated significantly higher levels than healthy controls and the other groups, except for APL patients. Complement activation was abrogated by addition of eculizumab. The results with substrate sera indicated that the alternative and classical/lectin pathways were involved. The results were reproducible with purified IgGs and ß2GPI. CONCLUSION: This proof-of-principle study confirms a role for complement in APS and opens the possibility of monitoring complement activation by including phospholipid vesicles in assay systems.

Visualization of macro-immune complexes in the antiphospholipid syndrome by multi-modal microscopy imaging.

The antiphospholipid syndrome (APS) is an autoimmune thrombotic condition that is marked by autoantibodies against phospholipid-binding proteins. The mechanism(s) of thrombogenesis has (have) resisted elucidation since its description over thirty years ago. Nevertheless, a defining aspect of the disorder is positivity for clinical laboratory tests that confirm antibody binding to anionic phospholipids. It is remarkable that, to our knowledge, the binding of proteins from plasmas of APS patients to phospholipid has not been previously imaged. We therefore investigated this with high resolution microscopy-based imaging techniques that have not been previously used to address this question, namely atomic force microscopy and scanning electron microscopy. Atomic force microscopy imaging of APS plasmas incubated on an anionic planar phospholipid layer revealed the formation of distinct complex three-dimensional structures, which were morphologically dissimilar to structures formed from control plasmas from healthy patients. Likewise, scanning electron microscopy analysis of phospholipid vesicles incubated with APS plasmas in suspension showed formation of layered macro-immune complexes demonstrated by the significant agglomeration of a complex proteinaceous matrix from soluble plasma and aggregation of particles. In contrast, plasmas from healthy control samples bound to phospholipid vesicles in suspension generally displayed a more flattened, mat-like appearance by scanning electron microscopy. Scanning electron microscopy of plasma samples incubated on planar phospholipid layers and previously imaged by atomic force microscopy, corroborated the results obtained by mixing the plasmas with phospholipids in solution. Analysis of the incorporated proteins by silver stained SDS-polyacrylamide gel electrophoresis indicated considerable heterogeneity in the composition of the phospholipid vesicle-adsorbed proteins among APS patients. To our knowledge, these results provide the first images of plasma-derived APS immune complexes at high resolution, and show their consistent presence and heterogeneous compositions in APS patients. These findings demonstrate how high resolution microscopic techniques can contribute to advancing the understanding of an enigmatic disorder and may lay additional groundwork for furthering mechanistic understanding of APS.

Cardiovascular risk factors are major determinants of thrombotic risk in patients with the lupus anticoagulant.

BACKGROUND: Patients with the lupus anticoagulant (LA) are at an increased risk of thrombotic events, which in turn increase the risk of death. Understanding the determinants of thrombotic risk in patients with LA may pave the way towards targeted thromboprophylaxis. In the Vienna Lupus Anticoagulant and Thrombosis Study (LATS), we systematically evaluate risk factors for thrombotic events in patients with LA. METHODS: We followed 150 patients (mean age: 41.3 years, female gender: n = 122 (81.3%), history of thrombosis or pregnancy complications: n = 111 (74.0%)), who tested repeatedly positive for LA until development of thrombosis, death, or censoring. The primary endpoint was a composite of arterial or venous thrombotic events (TEs). RESULTS: During a median follow-up of 9.5 years (range: 12 days-13.6 years) and 1076 person-years, 32 TEs occurred (arterial: n = 16, venous: n = 16; cumulative 10-year TE incidence: 24.3%). A prolonged lupus-sensitive activated partial thromboplastin time (aPTT-LA) (adjusted subdistribution hazard ratio (SHR) = 2.31, 95% CI: 1.07--5.02), diabetes (adjusted SHR = 4.39, 95% CI: 1.42-13.57), and active smoking (adjusted SHR = 2.31, 95% CI: 1.14-5.02) emerged as independent risk factors of both arterial and venous thrombotic risk. A risk model that includes a prolonged lupus-sensitive aPTT, smoking, and diabetes enabled stratification of LA patients into subgroups with a low, intermediate, and high risk of thrombosis (5-year TE risk of 9.7% (n = 77), 30.9% (n = 51), and 56.8% (n = 22). CONCLUSIONS: Long-term thrombotic risk in patients with LA is clustered within subjects harboring typical cardiovascular risk factors in addition to a prolonged lupus-sensitive aPTT, whereas patients with none of these risk factors represent a large subgroup with a low risk of thrombosis.

Inverted erythrocyte membranes demonstrate ß2GPI-antiphospholipid antibody interactions and membrane crosslinking.

INTRODUCTION: The antiphospholipid syndrome (APS) is an acquired autoimmune disorder predisposing patients to thrombosis or pregnancy complications. Since inverted erythrocyte membranes (iEMs) might provide a physiologically relevant source of anionic phospholipids, we studied the interactions of phospholipid-binding proteins and APS antibodies using iEMs. MATERIALS & METHODS: iEMs were prepared from packed erythrocytes by hypotonic lysis. Phosphatidylserine (PS) exposure was confirmed by annexin A5 (A5) binding using fluorescence microscopy and flow cytometry. Binding of ß2-glycoprotein I (ß2GPI)-IgG immune complexes to iEMs was investigated with gel electrophoresis, western blot and flow cytometry. Functional involvement in coagulation was documented in the thrombin generation assay. RESULTS: iEMs readily precipitated purified ß2GPI as well as ß2GPI from normal plasma and APS plasma. The plasma of APS patients provided higher levels of IgG binding to iEMs relative to healthy controls. Thrombin generation increased with increasing concentrations of iEMs, documenting that coagulation proteins bound to the exposed phospholipids. The LA effect was also distinguished in thrombin generation when comparing APS patients, as indicated by an increased lag time. Agglutination was observed after incubation with APS patient plasma and this was augmented by anti-human globulin. CONCLUSIONS: In conclusion, iEMs can provide a more physiological approach than phospholipid vesicle-based tests for investigating APS and are more amenable to standardization than platelet membranes.

A novel, rapid method to compare the therapeutic windows of oral anticoagulants using the Hill coefficient.

A central challenge in designing and administering effective anticoagulants is achieving the proper therapeutic window and dosage for each patient. The Hill coefficient, nH, which measures the steepness of a dose-response relationship, may be a useful gauge of this therapeutic window. We sought to measure the Hill coefficient of available anticoagulants to gain insight into their therapeutic windows. We used a simple fluorometric in vitro assay to determine clotting activity in platelet poor plasma after exposure to various concentrations of anticoagulants. The Hill coefficient for argatroban was the lowest, at 1.7 ± 0.2 (95% confidence interval, CI), and the Hill coefficient for fondaparinux was the highest, at 4.5 ± 1.3 (95% CI). Thus, doubling the dose of fondaparinux from its IC50 would decrease coagulation activity by nearly a half, whereas doubling the dose of argatroban from its IC50 would decrease coagulation activity by merely one quarter. These results show a significant variation among the Hill coefficients, suggesting a similar variation in therapeutic windows among anticoagulants in our assay.

Efficacy of solvent/detergent plasma after storage at 2-8 °C for 5 days in comparison to other plasma products to improve factor V levels in factor V deficient plasma.

OBJECTIVES: Factor V (FV) plays an important role in coagulation. As no purified concentrate is available to restore critical FV levels, the main blood product used to replace FV is plasma. The aim of the present in vitro study was to compare the efficacy of the different available plasma products on the reversal of moderate and severe FV deficiency as assessed by ROTEM® and FV levels. METHODS: Five different plasma products (6 batches of each) were compared to determine their effectiveness in replacing FV in plasma moderately or severely deficient in FV. Effectiveness was measured using the ROTEM® EXTEM clotting time (CT) and a factor V assay. RESULTS: FFP, plasma frozen within 24 hours (FP24), Octaplas (solvent/detergent treated pooled plasma), as well as Octaplas and FP24 thawed and stored for 5 days (Octaplas TP and TP), were all used for in vitro replacement of FV. TP was significantly less effective at reversing a prolonged EXTEM CT and FV levels in FV deficient plasma than other tested products. There were no significant differences in EXTEM CT between Octaplas and Octaplas TP, while factor V activity was significantly lower in the Octaplas TP. There was no significant difference between Octaplas and FFP for EXTEM CT or FV activity. CONCLUSIONS: Octaplas and Octaplas TP appear to have an equivalent ability to improve the EXTEM CT and could be considered as a treatment alternative to FFP in patients with FV deficiency.

Relation of antiphospholipid antibodies to postmortem brain infarcts in older people.

BACKGROUND: There are few data on the relationship of antiphospholipid antibodies (aPLs) to pathologically proven brain infarcts. We tested the hypothesis that aPLs are associated with a higher odds of brain infarcts among older, community-dwelling individuals who came to autopsy. METHODS AND RESULTS: Specimens and clinical and pathological data were derived from 607 deceased subjects (mean age at death, 89 years; 66% women) who were participating in 1 of 2 cohort studies of aging (Rush Memory and Aging Project and Religious Orders Study) and had agreed to brain autopsy. Brain infarcts were identified on gross and microscopic examinations, and severity of cerebral vessel disease (atherosclerosis, arteriolosclerosis) was graded. Four clinically used aPLs were measured longitudinally: 3 in serum (anticardiolipin antibodies, ß2-glycoprotein I, and anti-phosphatidyl-serine) and 1 in plasma (lupus anticoagulant). A quarter of subjects (142 of 607, 23%) had at least 1 aPL present at baseline (median time interval from baseline to death, 4.6 years), and three quarters of these subjects had persistently positive measures over time. In a logistic regression analysis, baseline aPL positivity did not increase the odds of brain infarcts (odds ratio=1.08; 95% confidence interval, 0.74-1.58; P=0.19) or of gross or microscopic infarcts separately. Findings were essentially unchanged when considering number of baseline aPLs, aPLs proximate to death, and persistence of aPLs. Associations did not differ among subjects with increased severity of vessel disease. CONCLUSION: Overall, we did not find evidence that aPLs increase the odds of pathological brain infarcts in older people.

Antiphospholipid antibodies promote tissue factor-dependent angiogenic switch and tumor progression.

Progression to an angiogenic state is a critical event in tumor development, yet few patient characteristics have been identified that can be mechanistically linked to this transition. Antiphospholipid autoantibodies (aPLs) are prevalent in many human cancers and can elicit proangiogenic expression in several cell types, but their role in tumor biology is unknown. Herein, we observed that the elevation of circulating aPLs among breast cancer patients is specifically associated with invasive-stage tumors. By using multiple in vivo models of breast cancer, we demonstrated that aPL-positive IgG from patients with autoimmune disease rapidly accelerates tumor angiogenesis and consequent tumor progression, particularly in slow-growing avascular tumors. The action of aPLs was local to the tumor site and elicited leukocytic infiltration and tumor invasion. Tumor cells treated with aPL-positive IgG expressed multiple proangiogenic genes, including vascular endothelial growth factor, tissue factor (TF), and colony-stimulating factor 1. Knockdown and neutralization studies demonstrated that the effects of aPLs on tumor angiogenesis and growth were dependent on tumor cell-derived TF. Tumor-derived TF was essential for the development of pericyte coverage of tumor microvessels and aPL-induced tumor cell expression of chemokine ligand 2, a mediator of pericyte recruitment. These findings identify antiphospholipid autoantibodies as a potential patient-specific host factor promoting the transition of indolent tumors to an angiogenic malignant state through a TF-mediated pathogenic mechanism.

Atomic force microscopy: High resolution dynamic imaging of cellular and molecular structure in health and disease.

The atomic force microscope (AFM), invented in 1986, and a member of the scanning probe family of microscopes, offers the unprecedented ability to image biological samples unfixed and in a hydrated environment at high resolution. This opens the possibility to investigate biological mechanisms temporally in a heretofore unattainable resolution. We have used AFM to investigate: (1) fundamental issues in cell biology (secretion) and, (2) the pathological basis of a human thrombotic disease, the antiphospholipid syndrome (APS). These studies have incorporated the imaging of live cells at nanometer resolution, leading to discovery of the "porosome," the universal secretory portal in cells, and a molecular understanding of membrane fusion from imaging the interaction and assembly of proteins between opposing lipid membranes. Similarly, the development of an in vitro simulacrum for investigating the molecular interactions between proteins and lipids has helped define an etiological explanation for APS. The prime importance of AFM in the success of these investigations will be presented in this manuscript, as well as a discussion of the limitations of this technique for the study of biomedical samples.

Current diagnostic and therapeutic approaches to patients with acquired von Willebrand syndrome: a 2013 update.

Acquired von Willebrand syndrome (AVWS) is an acquired bleeding disorder, first reported in 1968, with clinical and laboratory features similar to inherited von Willebrand disease. This rare bleeding disorder occurs mainly in patients with underlying lymphoproliferative, cardiovascular, myeloproliferative, and immunologic disorders. In contrast to acquired hemophilia A, AVWS is rarely associated with measurable anti-von Willebrand factor inhibitors. In most instances, AVWS is identified because of bleeding complications: in fact, more than 80% of the patients with this syndrome are active bleeders. Recurrent bleeding episodes occur in approximately 20 to 33% of patients with AVWS, especially following major trauma and surgery. Because of the heterogeneous mechanisms of AVWS, more than one therapeutic approach is often required to prevent or treat acute bleedings. Remission from some forms of AVWS can be obtained when the underlying disorders are treated.

Antiphospholipid antibodies, brain infarcts, and cognitive and motor decline in aging (ABICMA): design of a community-based, longitudinal, clinical-pathological study.

The overall goal of the Antiphospholipid Antibodies, Brain Infarcts, and Cognitive and Motor Decline in Aging study is to test the hypothesis that antiphospholipid antibodies (aPL) are associated with an increased risk of pathologically proven brain infarcts and are related to cognitive and motor decline in aging. Putative biologic mechanisms underlying the association of aPL with infarcts and the relation of aPL with clinical outcomes of cognitive and motor impairment, including vascular and other processes, will be examined. The design of this longitudinal, clinical-pathologic study involves quantifying four aPL assays, and relating these to brain infarcts, and to cognitive and motor decline. Vascular mechanisms assessed using antemortem magnetic resonance neuroimaging and postmortem neuropathology, as well as nonvascular mechanisms of inflammation and blood-brain barrier permeability alterations will be examined as plausible mediators of the relation of aPL to cognitive and motor impairment. We will take advantage of antemortem biological specimens (longitudinally collected sera and plasma from which aPL, annexins, C-reactive protein, and matrix metalloproteinases will be quantified), and clinical, neuroimaging, and postmortem neuropathologic data from about 800 elderly, community-dwelling women and men who have agreed to brain autopsy at the time of death, participating in one of two ongoing studies of aging: the Religious Orders Study and the Memory and Aging Project.

Viewing dynamic interactions of proteins and a model lipid membrane with atomic force microscopy.

The information covered in this chapter will present a model homogenous membrane preparation technique and dynamic imaging procedure that can be successfully applied to more than one type of lipid study and atomic force microscope (AFM) instrument setup. The basic procedural steps have been used with an Asylum Research MFP-3D BIO and the Bruker (formerly, Veeco) BioScope. The AFM imaging protocol has been supplemented by procedures (not to be presented in this chapter) of ellipsometry, standardized western blotting, and dot-blots to verify appropriate purity and activity of all experimental molecular components; excellent purity and activity level of the lipids, proteins, and drug(s) greatly influence the success of imaging experiments in the scanning probe microscopy field. The major goal of the chapter is to provide detailed procedures for sample preparation and operation of the Asylum Research MFP-3D BIO AFM. In addition, one should be cognizant that our comprehensive description in the use of the MFP-3D BIO's functions for successful image acquisitions and analyses is greatly enhanced by Asylum Research's (AR's) accompanying extensive manual(s), technical notes, and AR's users forum. Ultimately, the stepwise protocol and information will allow novice personnel to begin acquiring quality images for processing and analysis with minimal supervision.