Macrophages and Monocytes: "Trojan Horses" in COVID-19.

We aimed to explore whether variants of SARS-CoV-2 (Chinese-derived strain (D614, lineage A), Italian strain PV10734 (D614G, lineage B.1.1) and Alpha strain (lineage B.1.1.7)) were able to infect monocytes (MN) and monocyte-derived macrophages (MDM) and whether these infected cells may, in turn, be vectors of infection. For this purpose, we designed an in vitro study following the evolution of MN and MDM infection at different time points in order to confirm whether these cells were permissive for SARS-CoV-2 replication. Finally, we investigated whether, regardless of viral replication, the persistent virus can be transferred to non-infected cells permissive for viral replication. Thus, we co-cultured the infected MN/MDM with permissive VERO E6 cells verifying the viral transmission. This is a further in vitro demonstration of the important role of MN and MDM in the dissemination of SARS-CoV-2 and evolution of the COVID-19 disease.

Multiple Sclerosis: Microglia, Monocytes, and Macrophage-Mediated Demyelination.

This study examined the roles of microglia and monocytes in myelin destruction in patients with early multiple sclerosis (MS). Twenty-two cases were studied; the clinical duration was <9 weeks in 10 cases. Twenty myeloid cell subtypes or categories were identified including 2 cell types not known previously to occur in demyelinating diseases. Commencing myelin breakdown in plaques and in perivascular and subpial tissues occurred in the immediate presence of infiltrating monocytes and was effected by a homogeneous population of IgG-positive Fc receptor-bearing early phagocytes interacting with abnormal myelin. Oligodendrocyte apoptosis was observed in intact myelinated tissue bordering areas of active demyelination. Capillaries in the cerebral cortex plugged by large numbers of monocytes were common in acute cases of MS and in a patient with a neuromyelitis optica variant and extreme systemic recruitment of monocytes. In an MS patient with progressive disease, microglial nodules centered on MHC-II-positive capillaries plugged by monocytes were present in the cerebral cortex. This constitutes a new gray matter lesion in MS.

An advanced method for quantitative measurements of cholesterol crystallization.

BACKGROUND: Cholesterol crystallization within an atherosclerotic plaque significantly contributes to the acceleration of plaque rupture - a problematic event due to the current lack of specific treatments to prevent such formations. Modelling this pathogenic process is also difficult due to the lack of suitable experimental models that enable quantitative analysis of crystal formation and bioactivity screening of potential therapeutic compounds. AIM: To develop an in vitro human cell model of cholesterol crystallization combined with an imaging system that incorporates both quantitative analysis and real-time continuous imaging of cholesterol crystal formation. METHODS AND RESULTS: An enhanced in vitro model of cholesterol crystallization was developed through the use of acetylated low-density lipoprotein (AcLDL) and 7-ketocholesterol as agents of foam cell induction within a human THP-1 monocytic cell line. Advanced confocal and polarizing microscopies were incorporated into the model so as to allow for quantitation of cholesterol crystallization, with the lipid-loaded group producing significantly greater numbers of cholesterol crystals than the untreated group. The utility of this system was also demonstrated by investigating the effects of the cholesterol-lowering drug lovastatin and therapeutic bile compound ursodeoxycholic acid (UDCA), showing that these drugs influence different aspects of cholesterol crystal formation. CONCLUSIONS: The in vitro human THP-1 monocyte model of cholesterol crystallization provides an effective and efficient means of quantitating cholesterol crystallization in the pre-clinical stage of research. The model also allows for the screening of potentially therapeutic compounds that may be used in attenuating or preventing cholesterol crystallization.

Novel insight from the first lung transplant of a COVID-19 patient.

BACKGROUND: To reveal detailed histopathological changes, virus distributions, immunologic properties and multi-omic features caused by SARS-CoV-2 in the explanted lungs from the world's first successful lung transplantation of a COVID-19 patient. MATERIALS AND METHODS: A total of 36 samples were collected from the lungs. Histopathological features and virus distribution were observed by optical microscope and transmission electron microscope (TEM). Immune cells were detected by flow cytometry and immunohistochemistry. Transcriptome and proteome approaches were used to investigate main biological processes involved in COVID-19-associated pulmonary fibrosis. RESULTS: The histopathological changes of the lung tissues were characterized by extensive pulmonary interstitial fibrosis and haemorrhage. Viral particles were observed in the cytoplasm of macrophages. CD3+ CD4- T cells, neutrophils, NK cells, γ/δ T cells and monocytes, but not B cells, were abundant in the lungs. Higher levels of proinflammatory cytokines iNOS, IL-1ß and IL-6 were in the area of mild fibrosis. Multi-omics analyses revealed a total of 126 out of 20,356 significant different transcription and 114 out of 8,493 protein expression in lung samples with mild and severe fibrosis, most of which were related to fibrosis and inflammation. CONCLUSIONS: Our results provide novel insight that the significant neutrophil/ CD3+ CD4- T cell/ macrophage activation leads to cytokine storm and severe fibrosis in the lungs of COVID-19 patient and may contribute to a better understanding of COVID-19 pathogenesis.

Generating Bovine Monocyte-Derived Dendritic Cells for Experimental and Clinical Applications Using Commercially Available Serum-Free Medium.

Advances in fundamental and applied immunology research often originate from pilot studies utilizing animal models. While cattle represent an ideal model for disease pathogenesis and vaccinology research for a number of human disease, optimized bovine culture models have yet to be fully established. Monocyte-derived dendritic cells (MoDC) are critical in activating adaptive immunity and are an attractive subset for experimental and clinical applications. The use of serum-supplemented culture medium in this ex vivo approach is undesirable as serum contains unknown quantities of immune-modulating components and may induce unwanted immune responses if not autologous. Here, we describe a standardized protocol for generating bovine MoDC in serum-free medium (AIM-V) and detail the MoDC phenotype, cytokine profile, and metabolic signature achieved using this culture methodology. MoDC generated from adult, barren cattle were used for a series of experiments that evaluated the following culture conditions: medium type, method of monocyte enrichment, culture duration, and concentration of differentiation additives. Viability and yield were assessed using flow cytometric propidium iodide staining and manual hemocytometer counting, respectively. MoDC phenotype and T cell activation and proliferation were assessed by flow cytometric analysis of surface markers (MHC class II, CD86, CD14, and CD205), and CD25 and CFSE respectively. Cytokine secretion was quantified using a multiplex bovine cytokine panel (IL-1α, IL-1ß, IL-8, IL-10, IL-17A, IFN-γ, MIP-1α, TNF-α, and IL-4). Changes in cell metabolism following stimulation were analyzed using an Extracellular Flux (XFe96) Seahorse Analyzer. Data were analyzed using paired t-tests and repeated measures ANOVA. Immature MoDC generated in serum-free medium using magnetic-activated cell sorting with plate adhesion to enrich monocytes and cultured for 4 days have the following phenotypic profile: MHC class II+++, CD86+, CD205++, and CD14-. These MoDC can be matured with PMA and ionomycin as noted by increased CD86 and CD40 expression, increased cytokine secretion (IL-1α, IL-10, MIP-1α, and IL-17A), a metabolic switch to aerobic glycolysis, and induction of T cell activation and proliferation following maturation. Cultivation of bovine MoDC utilizing our well-defined culture protocol offers a serum-free approach to mechanistically investigate mechanisms of diseases and the safety and efficacy of novel therapeutics for both humans and cattle alike.

Advanced chronic kidney disease is associated with higher serum concentration of monocyte microparticles.

Advanced chronic kidney disease is associated with high rates of cardiovascular disease. Considering the crucial role of capillaries in renal function, our study aimed to evaluate microparticles related to vascular physiology examining the link between stages of chronic kidney disease with circulating endothelial (EMP), platelet (PMP) and monocytic (MMP) microparticles. Cross-sectional study with blinded endpoints included subjects of both sexes, aged 40-75 years (n = 247), with established cardiovascular disease (coronary heart disease, ischemic stroke, or peripheral artery disease). They were stratified 1:1 by the presence or absence of decreased glomerular filtration rate (GFR < 60 mL/min/1.73 m2) estimated by the CKD-EPI criteria, and according to the stages of CKD. Microparticles were quantified by flow-cytometry using specific antibodies to identify endothelial, platelet, and monocytic derived microparticles. Higher percentages of circulating MMP (p = 0.036), but not for EMP or PMP, were observed in subjects with reduced GFR. Circulating MMP were also related to the stages of chronic kidney disease (trend analysis across renal stages, p = 0.038). Higher percentages of circulating MMP were found in subjects with reduced GFR, and their percentages were progressively higher according to the stage of chronic renal function.

Immune modulation by complement receptor 3-dependent human monocyte TGF-ß1-transporting vesicles.

Extracellular vesicles have an important function in cellular communication. Here, we show that human and mouse monocytes release TGF-ß1-transporting vesicles in response to the pathogenic fungus Candida albicans. Soluble ß-glucan from C. albicans binds to complement receptor 3 (CR3, also known as CD11b/CD18) on monocytes and induces the release of TGF-ß1-transporting vesicles. CR3-dependence is demonstrated using CR3-deficient (CD11b knockout) monocytes generated by CRISPR-CAS9 genome editing and isolated from CR3-deficient (CD11b knockout) mice. These vesicles reduce the pro-inflammatory response in human M1-macrophages as well as in whole blood. Binding of the vesicle-transported TGF-ß1 to the TGF-ß receptor inhibits IL1B transcription via the SMAD7 pathway in whole blood and induces TGFB1 transcription in endothelial cells, which is resolved upon TGF-ß1 inhibition. Notably, human complement-opsonized apoptotic bodies induce production of similar TGF-ß1-transporting vesicles in monocytes, suggesting that the early immune response might be suppressed through this CR3-dependent anti-inflammatory vesicle pathway.

Systematic Investigation of Polyurethane Biomaterial Surface Roughness on Human Immune Responses in vitro.

It has been widely shown that biomaterial surface topography can modulate host immune response, but a fundamental understanding of how different topographies contribute to pro-inflammatory or anti-inflammatory responses is still lacking. To investigate the impact of surface topography on immune response, we undertook a systematic approach by analyzing immune response to eight grades of medical grade polyurethane of increasing surface roughness in three in vitro models of the human immune system. Polyurethane specimens were produced with defined roughness values by injection molding according to the VDI 3400 industrial standard. Specimens ranged from 0.1 µm to 18 µm in average roughness (Ra), which was confirmed by confocal scanning microscopy. Immunological responses were assessed with THP-1-derived macrophages, human peripheral blood mononuclear cells (PBMCs), and whole blood following culture on polyurethane specimens. As shown by the release of pro-inflammatory and anti-inflammatory cytokines in all three models, a mild immune response to polyurethane was observed, however, this was not associated with the degree of surface roughness. Likewise, the cell morphology (cell spreading, circularity, and elongation) in THP-1-derived macrophages and the expression of CD molecules in the PBMC model on T cells (HLA-DR and CD16), NK cells (HLA-DR), and monocytes (HLA-DR, CD16, CD86, and CD163) showed no influence of surface roughness. In summary, this study shows that modifying surface roughness in the micrometer range on polyurethane has no impact on the pro-inflammatory immune response. Therefore, we propose that such modifications do not affect the immunocompatibility of polyurethane, thereby supporting the notion of polyurethane as a biocompatible material.

Initial interaction of citrate-coated iron oxide nanoparticles with the glycocalyx of THP-1 monocytes assessed by real-time magnetic particle spectroscopy and electron microscopy.

Interaction with biological material can alter physicochemical parameters of magnetic nanoparticles and might thereby change their magnetic behavior with potentially important implications for various nanoparticle applications. Little is known about changes of the magnetic behavior that occur during the initial phase of cell binding and uptake. We investigate the magnetic behavior of very small superparamagnetic iron-oxide nanoparticles (VSOP) during initial contact with THP-1 monocytes. We combine real-time magnetic particle spectroscopy (MPS), a fast and sensitive method for specific detection of magnetic nanoparticles in biological specimen with high-pressure-freezing/freeze-substitution transmission electron microscopy (HPF/FS-TEM), enabling us to generate snapshots of the interaction of VSOP with the cellular glycocalyx. MPS reveals significant changes of the dynamic magnetic behavior within seconds after VSOP injection into monocyte suspensions that correlate with the formation of nanoparticle clusters in the glycocalyx. The combination of real-time MPS and HPF/FS-TEM provides an ideal platform to analyze magnetic behaviors of nanoparticles upon interaction with cells and tissues.

Gold Nanoparticles Modulate BCG-Induced Innate Immune Memory in Human Monocytes by Shifting the Memory Response towards Tolerance.

Innate immune memory is characterized by a modulation in the magnitude with which innate immune cells such as monocytes and macrophages respond to potential dangers, subsequent to previous exposure to the same or unrelated agents. In this study, we have examined the capacity of gold nanoparticles (AuNP), which are already in use for therapeutic and diagnostic purposes, to modulate the innate memory induced by bacterial agents. The induction of innate memory was achieved in vitro by exposing human primary monocytes to bacterial agents (lipopolysaccharide -LPS-, or live Bacille Calmette-Guérin -BCG) in the absence or presence of AuNP. After the primary activation, cells were allowed to return to a resting condition, and eventually re-challenged with LPS. The induction of memory was assessed by comparing the response to the LPS challenge of unprimed cells with that of cells primed with bacterial agents and AuNP. The response to LPS was measured as the production of inflammatory (TNFα, IL-6) and anti-inflammatory cytokines (IL-10, IL-1Ra). While ineffective in directly inducing innate memory per se, and unable to influence LPS-induced tolerance memory, AuNP significantly affected the memory response of BCG-primed cells, by inhibiting the secondary response in terms of both inflammatory and anti-inflammatory factor production. The reprogramming of BCG-induced memory towards a tolerance type of reactivity may open promising perspectives for the use of AuNP in immunomodulatory approaches to autoimmune and chronic inflammatory diseases.

Phenotypic landscape of granulocytes and monocytes by multiparametric flow cytometry: A prospective study of a 1-tube panel strategy for diagnosis and prognosis of patients with MDS.

BACKGROUND: Multiparametric flow cytometry (MFC) was recently reported to be a helpful additional tool in the diagnosis of myelodysplastic syndromes (MDS). However, numerous aberrancies have been reported that makes their evaluation difficult as part of a routine diagnosis. METHODS: Here, we validated a 1-tube panel for the evaluation of granulocytic and monocytic maturation by MFC and correlated our findings with diagnosis and prognosis of MDS. A total of 251 samples with MDS suspicion were prospectively analyzed and compared to an internal reference database leading to the calculation of the Diff score. RESULTS: The associated specificity and sensitivity values of this scoring system were 92.1% and 60.4% in a first learning cohort and 96.7% and 65.2% in a second independent validation cohort. The combination of the Diff score with the concomitantly calculated Ogata score increased the sensitivity to 74.2% and 78.3% in the learning and validation cohorts, respectively. Finally, a normal Diff score in MDS patients was associated with a significant prolonged progression-free survival. CONCLUSIONS: Taken together, the present data indicate that our strategy is a sensitive and specific MFC tool for the diagnosis of MDS-related cytopenia(s) which could be also useful for predicting evolution of these diseases.

Phagolysosome resolution requires contacts with the endoplasmic reticulum and phosphatidylinositol-4-phosphate signalling.

Phosphoinositides have a pivotal role in the maturation of nascent phagosomes into microbicidal phagolysosomes. Following degradation of their contents, mature phagolysosomes undergo resolution, a process that remains largely uninvestigated. Here we studied the role of phosphoinositides in phagolysosome resolution. Phosphatidylinositol-4-phosphate (PtdIns(4)P), which is abundant in maturing phagolysosomes, was depleted as they tubulated and resorbed. Depletion was caused, in part, by transfer of phagolysosomal PtdIns(4)P to the endoplasmic reticulum, a process mediated by oxysterol-binding protein-related protein 1L (ORP1L), a RAB7 effector. ORP1L formed discrete tethers between the phagolysosome and the endoplasmic reticulum, resulting in distinct regions with alternating PtdIns(4)P depletion and enrichment. Tubules emerged from PtdIns(4)P-rich regions, where ADP-ribosylation factor-like protein 8B (ARL8B) and SifA- and kinesin-interacting protein/pleckstrin homology domain-containing family M member 2 (SKIP/PLEKHM2) accumulated. SKIP binds preferentially to monophosphorylated phosphoinositides, of which PtdIns(4)P is most abundant in phagolysosomes, contributing to their tubulation. Accordingly, premature hydrolysis of PtdIns(4)P impaired SKIP recruitment and phagosome resolution. Thus, resolution involves phosphoinositides and tethering of phagolysosomes to the endoplasmic reticulum.

H7N9 influenza A virus activation of necroptosis in human monocytes links innate and adaptive immune responses.

We previously demonstrated that avian influenza A H7N9 virus preferentially infected CD14+ monocyte in human peripheral blood mononuclear cells (PBMCs), which led to apoptosis. To better understand H7N9 pathogenesis in relation to monocyte cell death, we showed here that extensive phosphorylation of mixed lineage kinase domain-like (MLKL) protein occurred concurrently with the activation of caspases-8, -9 and -3 in H7N9-infected monocytes at 6 h post infection (hpi), indicating that apoptosis and necroptosis pathways were simultaneously activated. The apoptotic morphology was readily observed in H7N9-infected monocytes with transmission electron microscopy (TEM), while the pan-caspase inhibitor, IDN6556 (IDN), accelerated cell death through necroptosis as evidenced by the increased level of pMLKL accompanied with cell swelling and plasma membrane rupture. Most importantly, H7N9-induced cell death could only be stopped by the combined treatment of IDN and necrosulfonamide (NSA), a pMLKL membrane translocation inhibitor, but not by individual inhibition of caspase or RIPK3. Our data further showed that activation of apoptosis and necroptosis pathways in monocytes differentially contributed to the immune response of monocytes upon H7N9 infection. Specifically, caspase inhibition significantly enhanced, while RIPK3 inhibition reduced the early expression of type I interferons and cytokine/chemokines in H7N9-infected monocytes. Moreover, culture supernatants from IDN-treated H7N9-infected monocyte promoted the expression of co-stimulatory molecule CD80, CD83 and CD86 on freshly isolated monocytes and monocyte-derived dendritic cells (MDCs) and enhanced the capacity of MDCs to induce CD3+ T-cell proliferation in vitro. In contrast, these immune stimulatory effects were abrogated by using culture supernatants from H7N9-infected monocyte with RIPK3 inhibition. In conclusion, our findings indicated that H7N9 infection activated both apoptosis and necroptosis in monocytes. An intact RIPK3 activity is required for upregulation of innate immune responses, while caspase activation suppresses the immune response.

Intravital 2-photon imaging reveals distinct morphology and infiltrative properties of glioblastoma-associated macrophages.

Characterized by a dismal survival rate and limited response to therapy, glioblastoma (GBM) remains one of the most aggressive human malignancies. Recent studies of the role of tumor-associated macrophages (TAMs) in the progression of GBMs have demonstrated that TAMs are significant contributors to tumor growth, invasion, and therapeutic resistance. TAMs, which include brain-resident microglia and circulating bone marrow derived-monocytes (BMDMs), are typically grouped together in histopathological and molecular analyses due to the lack of reliable markers of distinction. To develop more effective therapies aimed at specific TAM populations, we must first understand how these cells differ both morphologically and behaviorally. Furthermore, we must develop a deeper understanding of the mechanisms encouraging their infiltration and how these mechanisms can be therapeutically exploited. In this study, we combined immunocompetent lineage tracing mouse models of GBM with high-resolution open-skull 2-photon microscopy to investigate the phenotypical and functional characteristics of TAMs. We demonstrate that TAMs are composed of 2 morphologically distinct cell types that have differential migratory propensities. We show that BMDMs are smaller, minimally branched cells that are highly migratory compared with microglia, which are larger, highly branched stationary cells. In addition, 2 populations of monocytic macrophages were observed that differed in terms of CX3CR1 expression and migratory capacity. Finally, we demonstrate the efficacy of anti-vascular endothelial growth factor A blockade for prohibiting TAM infiltration, especially against BMDMs. Taken together, our data clearly define characteristics of individual TAM populations and suggest that combination therapy with antivascular and antichemotaxis therapy may be an attractive option for treating these tumors.

Infection and nuclear interaction in mammalian cells by 'Candidatus Berkiella cookevillensis', a novel bacterium isolated from amoebae.

BACKGROUND: 'Candidatus Berkiella cookevillensis' and 'Ca. Berkiella aquae' have previously been described as intranuclear bacteria of amoebae. Both bacteria were isolated from amoebae and were described as appearing within the nuclei of Acanthamoeba polyphaga and ultimately lysing their host cells within 4 days. Both bacteria are Gammaproteobacteria in the order Legionellales with the greatest similarity to Coxiella burnetii. Neither bacterium grows axenically in artificial culture media. In this study, we further characterized 'Ca. B. cookevillensis' by demonstrating association with nuclei of human phagocytic and nonphagocytic cell lines. RESULTS: Transmission electron microscopy (TEM) and confocal microscopy were used to confirm nuclear co-localization of 'Ca. B. cookevillensis' in the amoeba host A. polyphaga with 100% of cells having bacteria co-localized with host nuclei by 48 h. TEM and confocal microscopy demonstrated that the bacterium was also observed to be closely associated with nuclei of human U937 and THP-1 differentiated macrophage cell lines and nonphagocytic HeLa human epithelial-like cells. Immunofluorescent staining revealed that the bacteria-containing vacuole invaginates the nuclear membranes and appears to cross from the cytoplasm into the nucleus as an intact vacuole. CONCLUSION: Results of this study indicate that a novel coccoid bacterium isolated from amoebae can infect human cell lines by associating with the host cell nuclei, either by crossing the nuclear membranes or by deeply invaginating the nuclear membranes. When associated with the nuclei, the bacteria appear to be bound within a vacuole and replicate to high numbers by 48 h. We believe this is the first report of such a process involving bacteria and human cell lines.

Nonglucuronidated Ezetimibe Disrupts CD13- and CD64-Coassembly in Membrane Microdomains and Decreases Cellular Cholesterol Content in Human Monocytes/Macrophages.

Ezetimibe (EZE) and glucuronidated EZE (EZE-Glu) differentially target Niemann-Pick C1-like 1 (NPC1L1) and CD13 (aminopeptidase-N) to inhibit intestinal cholesterol absorption and cholesterol processing in other cells, although the precise molecular mechanisms are not fully elucidated. Cellular effects of EZE, EZE-Glu, and the low-absorbable EZE-analogue S6130 were investigated on human monocyte-derived macrophages upon loading with atherogenic lipoproteins. EZE and S6130, but not EZE-Glu disturbed the colocalization of CD13 and its coreceptor CD64 (Fcγ receptor I) in membrane microdomains, and decreased the presence of both receptors in detergent-resistant membrane fractions. Biotinylated cholesterol absorption inhibitor C-5 (i.e., derivative of EZE) was rapidly internalized to perinuclear tubular structures of cells, resembling endoplasmic reticulum (ER), but CD13 was detected on extracellular sites of the plasma membrane and endolysosomal vesicles. Administration of EZE, but not of EZE-Glu or S6130, was associated with decreased cellular cholesteryl ester content, indicating the sterol-O acyltransferase 1 (SOAT1)-inhibition by EZE. Furthermore, EZE decreased the expression of molecules involved in cholesterol uptake and synthesis, in parallel with increased apolipoprotein A-I-mediated cholesterol efflux and upregulation of efflux-effectors. However, NPC1L1 the other claimed molecular target of EZE, was not detected in macrophages, thereby excluding this protein as target for EZE in macrophages. Thus, EZE is very likely a CD13-linked microdomain-disruptor and SOAT1-inhibitor in macrophages leading to in vitro anti-atherosclerotic effects through a decrease of net cellular cholesterol content. © 2019 International Society for Advancement of Cytometry.

Immobilization of Denosumab on Titanium Affects Osteoclastogenesis of Human Peripheral Blood Monocytes.

Immobilization of proteins has been examined to improve implant surfaces. In this study, titanium surfaces were modified with nanofunctionalized denosumab (cDMAB), a human monoclonal anti-RANKL IgG. Noncoding DNA oligonucleotides (ODN) served as linker molecules between titanium and DMAB. Binding and release experiments demonstrated a high binding capacity of cDMAB and continuous release. Human peripheral mononuclear blood cells (PBMCs) were cultured in the presence of RANKL/MCSF for 28 days and differentiated into osteoclasts. Adding soluble DMAB to the medium inhibited osteoclast differentiation. On nanofunctionalized titanium specimens, the osteoclast-specific TRAP5b protein was monitored and showed a significantly decreased amount on cDMAB-titanium in PBMCs + RANKL/MCSF. PBMCs on cDMAB-titanium also changed SEM cell morphology. In conclusion, the results indicate that cDMAB reduces osteoclast formation and has the potential to reduce osteoclastogenesis on titanium surfaces.

Unraveling avian and reptilian hematology: An optical and electron microscopic study of the buffy coat.

BACKGROUND: Blood centrifugation and buffy coats are at the cornerstone of hematology. In mammals, the buffy coat has a layered disposition (from bottom to top) with neutrophils on top of erythrocytes, followed by monocytes/lymphocytes, and platelets. In nonmammals, this distribution is unknown. Recently, the cell tube block (CTB) technique was developed to study the buffy coat, but it was never applied to nonmammal buffy coats. OBJECTIVES: This study aimed to evaluate using the CTB technique to study reptilian and avian buffy coats and to propose its use for clinical applications. METHODS: Blood from five birds and eight reptiles of different species was obtained to make CTBs that were processed for optical/electron microscopy. H&E, Sirius red, and immunohistochemistry staining against CD3 (to label T lymphocytes) were applied to the CTBs. RESULTS: In birds, the buffy coat had a layered appearance with the granulocyte layer containing granulocytes (heterophils and eosinophils) and nucleated erythrocytes followed by a mononuclear cell layer containing lymphocytes, monocytes, and thrombocytes. In some animals, a nucleated erythrocyte layer was observed admixed with the granulocyte/mononuclear cell layer. A small clot within the buffy coat was seen in seven reptiles, and less definition of layers occurred in reptiles, with only one or two layers. Lymphocytes appeared toward the top of the buffy coat. CONCLUSIONS: From a comparative hematology perspective, the buffy coat of mammals differs from that of birds and more from that of reptiles. The CTB technique can be used to study these differences in avian and reptilian hematology, especially to study atypical circulating cells, hemoparasites, or blood cell proportions in health and disease.