Recommendations for using artificial intelligence in clinical flow cytometry.

Flow cytometry is a key clinical tool in the diagnosis of many hematologic malignancies and traditionally requires close inspection of digital data by hematopathologists with expert domain knowledge. Advances in artificial intelligence (AI) are transferable to flow cytometry and have the potential to improve efficiency and prioritization of cases, reduce errors, and highlight fundamental, previously unrecognized associations with underlying biological processes. As a multidisciplinary group of stakeholders, we review a range of critical considerations for appropriately applying AI to clinical flow cytometry, including use case identification, low and high risk use cases, validation, revalidation, computational considerations, and the present regulatory frameworks surrounding AI in clinical medicine. In particular, we provide practical guidance for the development, implementation, and suggestions for potential regulation of AI-based methods in the clinical flow cytometry laboratory. We expect these recommendations to be a helpful initial framework of reference, which will also require additional updates as the field matures.

Deep learning-based light scattering microfluidic cytometry for label-free acute lymphocytic leukemia classification.

The subtyping of Acute lymphocytic leukemia (ALL) is important for proper treatment strategies and prognosis. Conventional methods for manual blood and bone marrow testing are time-consuming and labor-intensive, while recent flow cytometric immunophenotyping has the limitations such as high cost. Here we develop the deep learning-based light scattering imaging flow cytometry for label-free classification of ALL. The single ALL cells confined in three dimensional (3D) hydrodynamically focused stream are excited by light sheet. Our label-free microfluidic cytometry obtains big-data two dimensional (2D) light scattering patterns from single ALL cells of B/T subtypes. A deep learning framework named Inception V3-SIFT (Scale invariant feature transform)-Scattering Net (ISSC-Net) is developed, which can perform high-precision classification of T-ALL and B-ALL cell line cells with an accuracy of 0.993 ± 0.003. Our deep learning-based 2D light scattering flow cytometry is promising for automatic and accurate subtyping of un-stained ALL.

Discrimination of Head and Neck Squamous Cell Carcinoma Patients and Healthy Adults by 10-Color Flow Cytometry: Development of a Score Based on Leukocyte Subsets.

BACKGROUND: Leukocytes in peripheral blood (PB) are prognostic biomarkers in head and neck squamous cell carcinoma cancer patients (HNSCC-CPs), but differences between HNSCC-CPs and healthy adults (HAs) are insufficiently described. METHODS: 10-color flow cytometry (FCM) was used for in-depth immunophenotyping of PB samples of 963 HAs and 101 therapy-naïve HNSCC-CPs. Absolute (AbsCC) and relative cell counts (RelCC) of leukocyte subsets were determined. A training cohort (TC) of 43 HNSCC-CPs and 43 HAs, propensity score (PS)-matched according to age, sex, alcohol, and smoking, was used to develop a score consecutively approved in a validation cohort (VC). RESULTS: Differences in AbsCC were detected in leukocyte subsets (p < 0.001), but had low power in discriminating HNSCC-CPs and HAs. Consequently, RelCC of nine leukocyte subsets in the TC were used to calculate 36 ratios; receiver operating characteristic (ROC) curves defined optimum cut-off values. Binary classified data were combined in a score based on four ratios: monocytes-to-granulocytes (MGR), classical monocytes-to-monocytes (clMMR), monocytes-to-lymphocytes (MLR), and monocytes-to-T-lymphocytes (MTLR); ≥3 points accurately discriminate HNSCC-CPs and HAs in the PS-matched TC (p = 2.97 × 10-17), the VC (p = 4.404 × 10-178), and both combined (p = 7.74 × 10-199). CONCLUSIONS: RelCC of leukocyte subsets in PB of HNSCC-CPs differ significantly from those of HAs. A score based on MGR, clMMR, MLR, and MTLR allows for accurate discrimination.

A novel direct co-culture assay analyzed by multicolor flow cytometry reveals context- and cell type-specific immunomodulatory effects of equine mesenchymal stromal cells.

The immunomodulatory potential of multipotent mesenchymal stromal cells (MSC) provides a basis for current and future regenerative therapies. In this study, we established an approach that allows to address the effects of pro-inflammatory stimulation and co-culture with MSC on different specific leukocyte subpopulations. Equine peripheral blood leukocyte recovery was optimized to preserve all leukocyte subpopulations and leukocyte activation regimes were evaluated. Allogeneic labeled equine adipose-derived MSC were then subjected to direct co-culture with either non-stimulated, concanavalin A (ConA)-activated or phosphate 12-myristate 13-acetate and ionomycin (PMA/I)-activated leukocytes. Subsequently, production of the cytokines interferon-γ (IFN- γ), interleukin-1 (IL-1) and tumor necrosis factor-α (TNF-α) and presence of FoxP3 were determined in specific cell populations using multicolor flow cytometry. Prostaglandin E2 (PGE2) was measured in the supernatants. ConA-stimulation induced mild activation of leukocytes, whereas PMA/I-stimulation led to strong activation. In T cells, PMA/I promoted production of all cytokines, with no distinct suppressive effects of MSC. However, increased numbers of CD25/FoxP3-positive cells indicated that MSC supported regulatory T cell differentiation in PMA/I-activated leukocyte cultures. MSC also reduced numbers of cytokine-producing B cells and granulocytes, mostly irrespective of preceding leukocyte activation, and reversed the stimulatory effect of ConA on IFN-γ production in monocytes. Illustrating the possible suppressive mechanisms, higher numbers of MSC produced IL-10 when co-cultured with non-stimulated or ConA-activated leukocytes. This was not observed in co-culture with PMA/I-activated leukocytes. However, PGE2 concentration in the supernatant was highest in the co-culture with PMA/I-activated leukocytes, suggesting that PGE2 could still mediate modulatory effects in strongly inflammatory environment. These context- and cell type-specific modulatory effects observed give insight into the interactions between MSC and different types of immune cells and highlight the roles of IL-10 and PGE2 in MSC-mediated immunomodulation. The approach presented could provide a basis for further functional MSC characterization and the development of potency assays.

Dendritic Cells in the Context of Human Tumors: Biology and Experimental Tools.

Dendritic cells (DC) are the most potent and versatile antigen-presenting cells (APC) in the immune system. DC have an exceptional ability to comprehend the immune context of a captured antigen based on molecular signals identified from its vicinity. The analyzed information is then conveyed to other immune effector cells. Such capability enables DC to play a pivotal role in mediating either an immunogenic response or immune tolerance towards an acquired antigen. This review summarizes current knowledge on DC in the context of human tumors. It covers the basics of human DC biology, elaborating on the different markers, morphology and function of the different subsets of human DC. Human blood-borne DC are comprised of at least three subsets consisting of one plasmacytoid DC (pDC) and two to three myeloid DC (mDC) subsets. Some tissues have unique DC. Each subset has a different phenotype and function and may induce pro-tumoral or anti-tumoral effects. The review also discusses two methods fundamental to the research of DC on the single-cell level: multicolor flow cytometry (FCM) and image-based cytometry (IC). These methods, along with new genomics and proteomics tools, can provide high-resolution information on specific DC subsets and on immune and tumor cells with which they interact. The different layers of collected biological data may then be integrated using Immune-Cytomics modeling approaches. Such novel integrated approaches may help unravel the complex network of cellular interactions that DC carry out within tumors, and may help harness this complex immunological information into the development of more effective treatments for cancer.

Inflammation in tissue engineering: The Janus between engraftment and rejection.

Tissue engineering (TE) for tissue and organ regeneration or replacement is generally performed with scaffold implants, which provide structural and molecular support to in vitro seeded or in vivo recruited cells. TE implants elicit the host immune response, often resulting in engraftment impediment or rejection. Besides this negative effect, however, the immune system components also yield a positive influence on stem cell recruitment and differentiation, allowing tissue regeneration and healing. Thus, a balanced cooperation between proinflammatory and proresolution players of the immune response is an essential element of implant success. In this context, macrophage plasticity plays a fundamental role. Therefore modulating the immune response, instead of immune suppressing the host, might be the best way to successfully implant TE tissues or organs. In particular, it is becoming evident that the scaffold, immune, and stem cells are linked by a three-way interaction, and many efforts are being made for scaffold-appropriate design and functionalization in order to drive the inflammation process toward regeneration, vascularization, and implant success. This review discusses current and potential strategies for inflammation modulation to aid engraftment and regeneration, supporting the concept that quality, and not quantity, of inflammation might influence implant success.

Assessment of Immunological Biomarkers in the First Year after Heart Transplantation.

BACKGROUND: Pharmacodynamic biomarkers that detect changes of immunological functions have been recognized as a helpful tool to increase the efficacy of immunosuppressive drug therapies. However, physiological changes of immunological biomarkers following transplantation are not investigated. Therefore, we assessed frequently used immunological biomarkers of the circulating blood in the first year following heart transplantation (HTx). METHODS: Activation markers CD25 and CD95, intracellular cytokines IL-2 and IFNγ, chemokines IP10 and MIG, and subsets of dendritic cells as well as antibodies against human leukocyte antigens (HLA) and major histocompatibility complex class I-related chain A (MICA) antigens were analyzed at different time points using flow cytometry and Luminex xMAP technology. RESULTS: Expression of IL-2, IFNγ, and plasmacytoid dendritic cells (pDCs) significantly increased (p < 0.01) during the first year. Anti-HLA antibodies decreased continuously, while anti-MICA antibodies showed minor increase within the first year. An association between percentage of pDCs and anti-MICA antibody positivity was proven. pDCs, IFNγ-producing T cells, and IP10 concentration were associated in a stronger way with age and gender of HTx recipients than with antibodies against HLA or MICA. CONCLUSIONS: We conclude that certain immunological biomarkers of the circulating blood change during the first year after HTx. These changes should be considered for interpretation of biomarkers after transplantation.

Detection of gold nanorods uptake by macrophages using scattering analyses combined with diffusion reflection measurements as a potential tool for in vivo atherosclerosis tracking.

In this study, we report a potential noninvasive technique for the detection of vulnerable plaques using scatter analyses with flow cytometry (FCM) method combined with the diffusion reflection (DR) method. The atherosclerotic plaques are commonly divided into two major categories: stable and vulnerable. The vulnerable plaques are rich with inflammatory cells, mostly macrophages (MΦ), which release enzymes that break down collagen in the cap. The detection method is based on uptake of gold nanorods (GNR) by MΦ. The GNR have unique optical properties that enable their detection using the FCM method, based on their scattering properties, and using the DR method, based on their unique absorption properties. This work demonstrates that after GNR labeling of MΦ, 1) the FCM scatter values increased up to 3.7-fold with arbitrary intensity values increasing from 1,110 to 4,100 and 2) the DR slope changed from an average slope of 0.196 (MΦ only) to an average slope of 0.827 (MΦ labeled with GNR) (P<0.001 for both cases). The combination of FCM and DR measurements provides a potential novel, highly sensitive, and noninvasive method for the identification of atherosclerotic vulnerable plaques, aimed to develop a potential tool for in vivo tracking.

Nanoparticle uptake by macrophages in vulnerable plaques for atherosclerosis diagnosis.

The composition of atherosclerotic (AS) plaques is crucial concerning rupture, thrombosis and clinical events. Two plaque types are distinguished: stable and vulnerable plaques. Vulnerable plaques are rich in inflammatory cells, mostly only M1 macrophages, and are highly susceptible to rupture. These plaques represent a high risk particularly with the standard invasive diagnosis by coronary angiography. So far there are no non-invasive low-risk clinical approaches available to detect and distinguish AS plaque types in vivo. The perspective review introduces a whole work-flow for a novel approach for non-invasive detection and classification of AS plaques using the diffusion reflection method with gold nanoparticle loaded macrophages in combination with flow and image cytometric analysis for quality assurance. Classical biophotonic methods for AS diagnosis are summarized. Phenotyping of monocytes and macrophages are discussed for specific subset labelling by nanomaterials, as well as existing studies and first experimental proofs of concept for the novel approach are shown. In vitro and in vivo detection of NP loaded macrophages (MΦ). Different ways of MΦ labelling include (1) in vitro labelling in suspension (whole blood or buffy coat) or (2) labelling of short-term MΦ cultures with re-injection of MΦ-NP into the animal to detect migration of the cells in the plaques and (3) in vivo injection of NP into the organism.

Agonist-induced ß2-adrenoceptor desensitization and downregulation enhance pro-inflammatory cytokine release in human bronchial epithelial cells.

It is not clear whether increased asthma severity associated with long-term use of ß2-adrenoceptor (ß2-AR) agonists can be attributed to receptor degradation and increased inflammation. We investigated the cross-talk between ß-AR agonist-mediated effects on ß2-AR function and expression and cytokine release in human bronchial epithelial cells. In 16HBE14o(-) cells grown in the presence and absence of ß-AR agonists and/or antagonists, the ß2-AR density was assessed by radioligand binding; the receptor protein and mRNA was determined using laser scanning cytometer and RT-PCR; cAMP generation, the cytokines IL-6 and IL-8 release were determined using AlphaScreen Assay and ELISA, respectively. Isoprenaline (ISO) and salbutamol (Salbu) induced a concentration- and time-dependent significant decrease in ß2-AR density. Both Salbu and ISO reduced cAMP generation in a concentration-dependent manner while in same cell culture the IL-6 and IL-8 release was significantly enhanced. These effects were antagonized to a greater extent by ICI 118.551 than by propranolol, but CGP 20712A had no effect. Reduction of the ß2-AR protein and mRNA could be seen when cells were treated with ISO for 24 h. Our findings indicate a direct link between cytokine release and altered ß2-AR expression and function in airway epithelial cells. ß2-AR desensitization and downregulation induced by long-term treatment with ß2-AR agonists during asthma may account for adverse reactions also due to enhanced release of pro-inflammatory mediators and should, thus, be considered in asthma therapy.

Immunomodulation in stem cell differentiation into neurons and brain repair.

Immunomodulators regulate stem cell activity at all stages of development as well as during adulthood. Embryonic stem cell (ESC) proliferation as well as neurogenic processes during embryonic development are controlled by factors of the immune system. We review here immunophenotypic expression patterns of  different stem cell types, including ESC, neural (NSC) and tissue-specific mesenchymal stem cells (MSC), and focus on immunodulatory properties of these cells. Immune and inflammatory responses, involving actions of cytokines as well as toll-like receptor (TLR) signaling are known to affect the differentiation capacity of NSC and MSC. Secretion of pro- and anti-inflammatory messengers by MSC have been observed as the consequence of TLR and cytokine activation and promotion of differentiation into specified phenotypes. As result of augmented differentiation capacity, stem cells secrete angiogenic factors including vascular endothelial growth factor, resulting in multifactorial actions in tissue repair. Immunoregulatory properties of tissue specific adult stem cells are put into the context of possible clinical applications.

Flow cytometric evaluation of T cell activation markers after cardiopulmonary bypass.

Background. Cardiopulmonary bypass surgery (CPBS) is associated with an increased risk for infections or with subsequent organ dysfunction. As T cell activation is a central mechanism during inflammatory processes, we developed an assay to evaluate T cell activation pathways in patients undergoing CPBS. Methods. Blood was obtained from eleven patients undergoing CPBS preoperatively, on postoperative day (POD)-3, and on POD-7 and was stimulated with different concentrations of Concanavalin A (ConA). Cyclosporine and sirolimus, inhibiting different pathways of the T cell cycle, were added to blood ex vivo. Expression of T cell activation markers CD25 and CD95 was analyzed by flow cytometry. Results. In untreated blood, expression of CD25 and CD95 significantly increased with higher ConA concentrations (P < 0.05) and decreased for all ConA concentrations for both antigens over the study time (P < 0.05). Independently from the ConA concentration, inhibition of CD25 and CD95 expression was highest preoperatively for sirolimus and on POD-3 for cyclosporine. At all time points, inhibition of CD25 and CD95 expression was significantly higher after cyclosporine compared to sirolimus treatment (P < 0.001). Conclusion. Our results showed that different pathways of T cell activation are impaired after CPBS. Such knowledge may offer the opportunity to identify patients at risk for postoperative complications.

Comparative immunophenotyping of equine multipotent mesenchymal stromal cells: an approach toward a standardized definition.

Horses are an approved large animal model for therapies of the musculoskeletal system. Especially for tendon disease where cell-based therapy is commonly used in equine patients, the translation of achieved results to human medicine would be a great accomplishment. Immunophenotyping of equine mesenchymal stromal cells (MSCs) remains the last obstacle to meet the criteria of the International Society for Cellular Therapy (ISCT) definition of human MSCs. Therefore, the surface antigen expression of CD 29, CD 44, CD 73, CD 90, CD 105, CD 14, CD 34, CD 45, CD 79α, and MHC II in equine MSCs from adipose tissue, bone marrow, umbilical cord blood, umbilical cord tissue, and tendon tissue was analyzed using flow cytometry. Isolated cells from the different sources and donors varied in their expression pattern of MSC-defining antigens. In particular, CD 90 and 105 showed most heterogeneity. However, cells from all samples were robustly positive for CD 29 and CD 44, while being mostly negative for CD 73 and the exclusion markers CD 14, CD 34, CD 45, CD 79α and MHC II. Furthermore, it was evident that enzymes used for cell detachment after in vitro-culture affected the detection of antigen expression. These results emphasize the need of standardization of MSC isolation, culturing, and harvesting techniques. As the equine MSCs did not meet all criteria the ISCT defined for human MSCs, further investigations for a better characterization of the cell type should be conducted.