Enzyme-enhanced immunocytochemical detection of cytoplasmic proteins in human leukocytes sorted by surface antigens using anti-CD antibody microarray

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Abstract

Anti-cluster-of-differentiation (anti-CD) antibody microarray is an attractive multiplex analogue to leukocyte immunocytochemistry for surface markers. The transparent plastic microarray support permits to sort leukocytes by their surface lineage-specific markers making them available for subsequent high-resolution morphology examination. The combined data on the pathologic cells’ immunophenotype, morphology and cytochemistry from the microarray is sufficient to suggest preliminary diagnosis in many leukemia types. However, in some cases additional staining for internal markers is required, i.e. in T cell acute leukemia the T cell origin of the blasts can only be proved by the presence of cytoplasmic CD3 (cytCD3). Here we describe a new protocol of immunocytochemical detection of internal proteins in mononuclear cells captured by mouse anti-CD antibodies on the microarray surface. The protocol uses primary mouse FITC-conjugated (FITC – fluorescein isothiocyanate) antibodies against the target, secondary anti-FITC antibodies conjugated with alkaline phosphatase and colorimetric alkaline phosphatase substrate, BCIP/NBT. We show on normal leukocytes and leukemic cell lines that the protocol is sensitive and specific. The percentages of cytCD3-positive cells determined by this method in bone marrow aspirates of two patients with T cell acute lymphoblastic leukemia are in excellent agreement with flow cytometry results. This method expands the diagnostic capabilities of anti-CD antibody microarray.

About the authors

Olga S. Fedyanina

Dmitry Rogachev National Medical Research Centre for Pediatric Hematology, Oncology and Immunology, Ministry of Health of Russia; Centre for Theoretical Problems of Physicochemical Pharmacology, Russian Academy of Sciences; N.D. Zelinsky Institute of Organic Chemistry, Russian Academy of Sciences

Email: foshope@yandex.ru
ORCID iD: 0000-0001-7131-8006
Russian Federation, 1 Samory Mashela St., 117198 Moscow; 30 Srednyaya Kalitnikovskaya St., Moscow 109029; 47 Leninsky Prospekt, Moscow 119991

Anna O. Zakirova

Dmitry Rogachev National Medical Research Centre for Pediatric Hematology, Oncology and Immunology, Ministry of Health of Russia; Centre for Theoretical Problems of Physicochemical Pharmacology, Russian Academy of Sciences

Email: a-doronina@mail.ru
ORCID iD: 0000-0001-9475-3318
Russian Federation, 1 Samory Mashela St., 117198 Moscow; 30 Srednyaya Kalitnikovskaya St., Moscow 109029

Alvina I. Khamidullina

Institute of Gene Biology, Russian Academy of Sciences; Ufa Federal Research Center, Russian Academy of Sciences

Email: alvina@genebiology.ru
ORCID iD: 0000-0001-8137-0625

Laboratory of Molecular Oncobiology; Ufa Institute of Chemistry

Russian Federation, 34/5 Vavilova St., Moscow 119334; 71 Oktyabrya Prospekt, Ufa 450054

Alina N. Khvastunova

Dmitry Rogachev National Medical Research Centre for Pediatric Hematology, Oncology and Immunology, Ministry of Health of Russia; Centre for Theoretical Problems of Physicochemical Pharmacology, Russian Academy of Sciences

Email: alina_shunina@mail.ru
ORCID iD: 0000-0002-7117-0168
Russian Federation, 1 Samory Mashela St., 117198 Moscow; 30 Srednyaya Kalitnikovskaya St., Moscow 109029

Ivan V. Grebennik

Dmitry Rogachev National Medical Research Centre for Pediatric Hematology, Oncology and Immunology, Ministry of Health of Russia; Centre for Theoretical Problems of Physicochemical Pharmacology, Russian Academy of Sciences

Email: iv.grebennik@gmail.com
ORCID iD: 0009-0008-9607-101X
Russian Federation, 1 Samory Mashela St., 117198 Moscow; 30 Srednyaya Kalitnikovskaya St., Moscow 109029

Alexander V. Filatov

Institute of Immunology, Federal Medical and Biological Agency of Russia

Email: avfilat@yandex.ru
ORCID iD: 0000-0002-6460-9427
Russian Federation, Bld. 2, 24 Kashirskoe Shosse, Moscow 115478

Sofya A. Kuznetsova

Dmitry Rogachev National Medical Research Centre for Pediatric Hematology, Oncology and Immunology, Ministry of Health of Russia; Centre for Theoretical Problems of Physicochemical Pharmacology, Russian Academy of Sciences

Author for correspondence.
Email: kuznetsova.sonya@gmail.com
ORCID iD: 0000-0001-5946-0026
Russian Federation, 1 Samory Mashela St., 117198 Moscow; 30 Srednyaya Kalitnikovskaya St., Moscow 109029

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Copyright (c) 2026 Fedyanina O.S., Zakirova A.O., Khamidullina A.I., Khvastunova A.N., Grebennik I.V., Filatov A.V., Kuznetsova S.A.

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