IPHT-Institutskolloquium

Recent advances in in vivo flow cytometry

Prof. Vladimir P. Zharov

University of Arkansas for Medical Sciences

The sensitivity of conventional flow cyt ometry is limited by the small volume of blood collected, in which no less than one disease-specific markers can be detected. It leads to missing many thousands of abnormal cells in the whole blood volume (~5 L in adults), which can be sufficient for disease progression to barely treatable or incurable stage. For example, despite enormous efforts to detect circulating tumor cells (CTCs) responsible for 90% of all cancer deaths as a result of the development of deadly metastases, the mortality rates for metastatic cancer have still been almost flat. To solve this problem we introduced in vivo flow cytometry for detection of rare circulating biomarkers directly in the bloodstream using blood vessels as natural tubes with native cell flow. In this technology, laser irradiates vessels of interests followed by the detection of photoacoustic (PA) waves from circulating objects using ultrasound transducer attached to the skin. The PA waves are generated either using intrinsic contrast agents (e.g., melanin) or absorbing low toxic plasmonic nanoprobes functionalized to specific markers. In this report we summarize recent advances of new generation of in vivo multicolor flow cytometry platform using multispectral lasers, fiber-based transducers, label-free and/or multiplex molecular targeting, plasmonic probes with ultrasharp PA spectral resonances, in vivo magnetic capturing of CTCs, and combination of PA diagnosis with photothermal (PT) elimination of abnormal cells. The capacity of this new technology was first demonstrated in preclinical models for real-time detection of bulk and stem CTCs, S. aureus, and sickle cells. Then, the clinical prototype of in vivo PA flow cytometry (PAFC) provided the assessment of ~1 L of patient blood during 30-60 minutes. The first clinical studies focused on detection of CTCs and cancer-associated clots in melanoma patients. The PAFC data in vivo were validated in vitro with conventional flow cytometry, immunohistochemical staining, and magnetic kits. The obtained results demonstrate that PAFC is more sensitivity (≥100-fold) , accurate and rapid (≤ 1 hour) that conventional technique. Unlike typical blood sampling involving extraction of a volume of blood ranging from 10 μL (drop) to a few mL (CTC assays), in vivo examination involves nearly the entire volume of blood passing through 1–2-mm-diameter peripheral vessels over 0.5–1 h and thus will enable a dramatic increase in diagnostic sensitivity, ultimately up to 103–105 times, reflecting the ratio of the volume of blood sampled in vivo to that in vitro. According to pilot clinical results, the portable personal flow cytometer can provide a breakthrough in early diagnosis of cancer, infection and cardiovascular disorders with a potential to inhibit, if not prevent, metastasis, sepsis, and strokes or heart attack through the use of well-timed personalized therapy.