This document discusses an automated method for counting reticulated platelets using a Sysmex XE-2100 hematology analyzer. The method uses fluorescent dyes and flow cytometry to stain and detect residual RNA in immature platelets, known as reticulated or high fluorescent platelets (HFP). The HFP percentage provides information about bone marrow platelet production and can help distinguish between consumptive and non-consumptive causes of thrombocytopenia. The automated method is rapid, reproducible and provides advantages over manual methods of detecting reticulated platelets.
This document discusses an automated method for counting reticulated platelets using a Sysmex XE-2100 hematology analyzer. The method uses fluorescent dyes and flow cytometry to stain and detect residual RNA in immature platelets, known as reticulated or high fluorescent platelets (HFP). The HFP percentage provides information about bone marrow platelet production and can help distinguish between consumptive and non-consumptive causes of thrombocytopenia. The automated method is rapid, reproducible and provides advantages over manual methods of detecting reticulated platelets.
This document discusses an automated method for counting reticulated platelets using a Sysmex XE-2100 hematology analyzer. The method uses fluorescent dyes and flow cytometry to stain and detect residual RNA in immature platelets, known as reticulated or high fluorescent platelets (HFP). The HFP percentage provides information about bone marrow platelet production and can help distinguish between consumptive and non-consumptive causes of thrombocytopenia. The automated method is rapid, reproducible and provides advantages over manual methods of detecting reticulated platelets.
This document discusses an automated method for counting reticulated platelets using a Sysmex XE-2100 hematology analyzer. The method uses fluorescent dyes and flow cytometry to stain and detect residual RNA in immature platelets, known as reticulated or high fluorescent platelets (HFP). The HFP percentage provides information about bone marrow platelet production and can help distinguish between consumptive and non-consumptive causes of thrombocytopenia. The automated method is rapid, reproducible and provides advantages over manual methods of detecting reticulated platelets.
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• Moderator : Dr.
Jyoti Shukla • Speaker : Dr. Shashikant C.U. Patne HFPF% or IPF%
• Automated method for counting reticulated
platelets. • Automated method uses XE-2100 automated blood cell counter equipped with a laser and upgraded software. • Extension of a standard method used to measure retic and retic fraction on the XE- 2100 automated haematology analyzer. THROMBOCYTOPENIA IN PBS - WHY??
• Increased platelet consumption
OR
• Decreased platelet production
↑Platelet consumption ↓ ↑Thromobopoietin production ↓ ↑Platelet production by megakaryocytes ↓ Residual RNA present in early platelets (Reticulated platelets analogous to Retic) ↓ RNA can be detected by RNA binding dyes ↓ Reticulated platelets-measure of adequate functionality of bone marrow METHODS OF DETECTING RETICULATED PLATELETS • Easily detected with supravital dye staining (e.g. new methylene blue) on blood films, • More commonly with fluorescent dyes (e.g thiazole orange) and flow cytometry. • Automated haematology analyzer with fluorescent dye and flow cytometry. CLINICAL APPLICATIONS • A measure of bone marrow platelet production and is sensitive and specific enough to discriminate between consumptive and aplastic causes of thrombocytopenia. • Recovery of platelet production/kinetics prior to normalization of the platelet count during various therapeutic regimes, e.g. the recovery of platelet count during treatment of thrombocytopenia or bone marrow transplantation. • In patients receiving growth factor therapy CLINICAL APPLICATIONS…
• Early indicator of increased platelet
destruction as seen in patients with pre- eclampsia or thrombotic thrombocytopenic purpura (TTP). • Analysis of the reticulocyte fraction in platelet concentrates for transfusion may improve the viability, function and storage conditions of these products, and could lead to the preparation of a reticulocyte enriched product for refractory patients. SYSMEX XE-2100 PRINCIPLE • The analyzer uses optical properties and proprietary fluorescent nucleic acid dyes which stain residual RNA of the platelets. • Fluorescent flow cytometer separates reticulated platelets and reticulocytes from other cells and from each other. • The analyzer arbitrarily but reproducibly defines HFP as those with highest 3% intensity of fluorescence. METHOD
• The blood is collected in K2-EDTA vial.
• In the fully automated haematology analyzer (Sysmex XE-2100), HFPF% is reliably quantified in the optical (fluorescence) reticulocyte/ platelet channel using upgraded software. • The blood cells are stained with proprietary fluorescent dye containing ploymethine and oxazine. METHOD… • The dyes penetrate the cell membrane and stain the RNA of RBC and platelets. • The stained cells are passed through a semiconductor diode laser beam. • The resulting forward scatter light (cell volume) and fluorescence intensity (RNA content) is measured. • A computer algorithm discriminates the mature platelet and immature platelet . METHOD… • Mature platelets appear as blue dots and the immature platelets are displayed as green dots. • Immature platelet fraction (IPF) or High fluorescent platelet fraction is usually expressed as a propotional value of the total optical platelet count. RESULTS & INTERPRETATIONS Subjects HFPF% value Interpretation
and HFPF% is demonstrated in patients with autoimmune thrombocytopenia.
• Low platelet count → High HFPF% value and
vice versa. ADVANTAGES OF HFPF% • Non invasive. • Fully automated, rapid method. • Staining of platelet RNA and its analysis in whole blood without preparatory steps. • Reproducibility/precision of results – no intraobserver or interobserver variation. • Stability of the sample up to 48 hrs. • Reference range is available for healthy subjects.