Implementation of flow cytometry testing on rare matrix samples: Special considerations and best practices when the sample is unique or difficult to obtain.

The publication of Clinical and Laboratory Standards Institute's guideline H62 has provided the flow cytometry community with much-needed guidance on development and validation of flow cytometric assays (CLSI, 2021). It has also paved the way for additional exploration of certain topics requiring additional guidance. Flow cytometric analysis of rare matrices, or unique and/or less frequently encountered specimen types, is one such topic and is the focus of this manuscript. This document is the result of a collaboration subject matter experts from a diverse range of backgrounds and seeks to provide best practice consensus guidance regarding these types of specimens. Herein, we define rare matrix samples in the setting of flow cytometric analysis, address validation implications and challenges with these samples, and describe important considerations of using these samples in both clinical and research settings.

DNA damage response as a biomarker in treatment of leukemias.

Early assessment of cancer response to the treatment is of great importance in clinical oncology. Most antitumor drugs, among them DNA topoisomerase (topo) inhibitors, target nuclear DNA. The aim of the present study was to explore feasibility of the assessment of DNA damage response (DDR) as potential biomarker, eventually related to the clinical response, during treatment of human leukemias. We have measured DDR as reported by activation of ATM through its phosphorylation on Ser 1981 (ATM-S1981(P)) concurrent with histone H2AX phosphorylation on Ser139 (gammaH2AX) in leukemic blast cells from the blood of twenty patients, 16 children/adolescents and 4 adults, diagnosed with acute leukemias and treated with topo2 inhibitors doxorubicin, daunomycin, mitoxantrone or idarubicin. Phosphorylation of H2AX and ATM was detected using phospho-specific Abs and measured in individual cells by flow cytometry. The increase in the level of ATM-S1981(P) and gammaH2AX, varying in extent between the patients, was observed in blasts from the blood collected one hour after completion of the drug infusion with respect to the pre-treatment level. A modest degree of correlation was observed between the induction of ATM activation and H2AX phosphorylation in blasts of individual patients. The number of the studied patients (20) and the number of the clinically non-responding ones (2) was too low to draw a conclusion whether the assessment of DDR can be clinically prognostic. The present findings, however, demonstrate the feasibility of assessment of DDR during the treatment of leukemias with drugs targeting DNA.

Dynamics of circulating microparticles in liver transplant patients.

BACKGROUND & AIMS: Microparticles are small membrane vesicles released from the cell plasma membrane, particularly in cell stress, apoptosis and altered cellular viability. Hepatocellular carcinoma (HCC) is a hypervascular neoplasm with high levels of apoptosis and necrosis. We investigated the levels of circulating microparticles of both tumor and endothelial origins in liver transplant patients with hepatitis C (HepC) cirrhosis with and without HCC and compared them with healthy people and patients with partial hepatectomy. METHODS: Using immunolabeling of microparticles of different origin and flow cytometry-based enumeration of microparticles, the levels of circulating microparticles were studied in 8 patients with HepC and 8 patients with both HepC and HCC before and within two weeks after the transplant. RESULTS: The initial levels of circulating microparticles were increased in patients with HepC and HCC as compared to patients with HepC alone. They were also increased in liver transplant patients as compared to patients with partial hepatectomy or healthy people. Levels of circulating microparticles were dynamically changing after the transplant, showing an initial increase with a subsequent decrease by the end of the second week after surgery. In some patients with a complicated clinical outcome, the levels of microparticles were continuously increasing after the surgery. CONCLUSION: The levels of circulating microparticles of endothelial and hepatic origin in liver transplant patients dynamically change after surgery and correlate with the clinical outcome. Perspectively, the levels of circulating microparticles may be used in clinical practice as a marker of the functional status of the transplanted liver.

Discontinuous fragmentation of nuclear DNA during apoptosis revealed by discrete "sub-G1" peaks on DNA content histograms.

BACKGROUND: Internucleosomal DNA fragmentation is one of the hallmarks of apoptosis. Because the low molecular weight DNA fragments are extracted during cell staining in aqueous solutions, apoptotic cells can be identified on DNA content frequency histograms as cells with fractional ("sub-G(1)") DNA content. The aim of the present study was to explore whether in situ DNA fragmentation during apoptosis is discontinuous or progresses incessantly and if it is discontinuous, to define the resistant to cleavage fraction of DNA that remains stainable with the fluorochrome. MATERIALS AND METHODS: The model of activation-induced apoptosis of human lymphocytes was chosen as it provides uniform cell population with identical DNA content (DI = 1.00) that undergo apoptosis. Their apoptosis was induced by multivalent mitogen phytohemagglutinin (PHA) in the absence and presence of geldanamycin (GA), the benzoquinone ansamycin antibiotic which binds to Hsp90 (Heat Shock Protein 90) and alters its function. The cells were stained with acridine orange, the metachromatic fluorochrome that differentially stains cellular DNA and RNA. RESULTS: A sharp, discrete peak representing the subpopulation of "sub-G(1)" cells with highly reproducible DI = 0.42 +/- 0.02 (CV = 5.5 +/- 1.2) was observed on DNA content histograms of lymphocytes whose apoptosis was induced by PHA alone. Two distinct peaks, one representing cell subpopulations with DI = 0.42 (as above) and another, with DI = 0.79 +/- 0.04 (CV = 5.8 +/- 0.4), respectively, were seen in apoptotic cells from cultures stimulated with PHA in the presence of GA. The frequency of cells represented by the sub-G(1) peaks varied depending on time of induction of apoptosis and GA concentration. CONCLUSIONS: Apoptosis-induced DNA fragmentation is discontinuous; approximately 42% of DNA is relatively stable and remains within the cell. The data suggest that the stable DNA is associated with nuclear matrix while the degradable fraction represents DNA in loop domains. A transient DNA stabilization is apparent in the presence of GA as evidenced by the presence of cell subpopulations with 79% of DNA retained in the cell. The observed discontinuity of DNA fragmentation appears to reflect sequential involvement of different nucleases and may also be modulated by chromatin structure.