Epigenetic immune cell counting in human blood samples for immunodiagnostics

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Abstract

Immune cell profiles provide valuable diagnostic information for hematologic and immunologic diseases. Although it is the most widely applied analytical approach, flow cytometry is limited to liquid blood. Moreover, either analysis must be performed with fresh samples or cell integrity needs to be guaranteed during storage and transport. We developed epigenetic real-time quantitative polymerase chain reaction (qPCR) assays for analysis of human leukocyte subpopulations. After method establishment, whole blood from 25 healthy donors and 97 HIV+ patients as well as dried spots from 250 healthy newborns and 24 newborns with primary immunodeficiencies were analyzed. Concordance between flow cytometric and epigenetic data for neutrophils and B, natural killer, CD3+ T, CD8+ T, CD4+ T, and FOXP3+ regulatory T cells was evaluated, demonstrating substantial equivalence between epigenetic qPCR analysis and flow cytometry. Epigenetic qPCR achieves both relative and absolute quantifications. Applied to dried blood spots, epigenetic immune cell quantification was shown to identify newborns suffering from various primary immunodeficiencies. Using epigenetic qPCR not only provides a precise means for immune cell counting in fresh-frozen blood but also extends applicability to dried blood spots. This method could expand the ability for screening immune defects and facilitates diagnostics of unobservantly collected samples, for example, in underdeveloped areas, where logistics are major barriers to screening.

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Standardizing immunophenotyping for the Human Immunology Project.

Holden T. Maecker, J. Philip McCoy, Robert Burton Nussenblatt (2012)

The heterogeneity in the healthy human immune system, and the immunological changes that portend various diseases, have been only partially described. Their comprehensive elucidation has been termed the 'Human Immunology Project'. The accurate measurement of variations in the human immune system requires precise and standardized assays to distinguish true biological changes from technical artefacts. Thus, to be successful, the Human Immunology Project will require standardized assays for immunophenotyping humans in health and disease. A major tool in this effort is flow cytometry, which remains highly variable with regard to sample handling, reagents, instrument setup and data analysis. In this Review, we outline the current state of standardization of flow cytometry assays and summarize the steps that are required to enable the Human Immunology Project.

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Enzymes of the mevalonate pathway of isoprenoid biosynthesis.

Henri Miziorko (2011)

The mevalonate pathway accounts for conversion of acetyl-CoA to isopentenyl 5-diphosphate, the versatile precursor of polyisoprenoid metabolites and natural products. The pathway functions in most eukaryotes, archaea, and some eubacteria. Only recently has much of the functional and structural basis for this metabolism been reported. The biosynthetic acetoacetyl-CoA thiolase and HMG-CoA synthase reactions rely on key amino acids that are different but are situated in active sites that are similar throughout the family of initial condensation enzymes. Both bacterial and animal HMG-CoA reductases have been extensively studied and the contrasts between these proteins and their interactions with statin inhibitors defined. The conversion of mevalonic acid to isopentenyl 5-diphosphate involves three ATP-dependent phosphorylation reactions. While bacterial enzymes responsible for these three reactions share a common protein fold, animal enzymes differ in this respect as the recently reported structure of human phosphomevalonate kinase demonstrates. There are significant contrasts between observations on metabolite inhibition of mevalonate phosphorylation in bacteria and animals. The structural basis for these contrasts has also recently been reported. Alternatives to the phosphomevalonate kinase and mevalonate diphosphate decarboxylase reactions may exist in archaea. Thus, new details regarding isopentenyl diphosphate synthesis from acetyl-CoA continue to emerge. Copyright Â© 2010 Elsevier Inc. All rights reserved.