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Overview of Reticulocyte Count

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Overview of Reticulocyte Count Overview of Reticulocyte count Clinical utility of a parameter is the demonstration of its potential usefulness and added value to patient management decision-making. 1. Summary The enumeration of peripheral blood reticulocytes is essential in clinical hematology and thanks to the technology achievements may be considered now as a part of the CBC. The number of reticulocytes in peripheral blood reflects erythropoïetic activity of the bone marrow or some erythroïd diseases. Thus enumeration of peripheral blood reticulocytes is an essential part of the diagnosis and management of anemic and also oncologic patients since anemia is one of the most frequent side effects of anticancer treatments 2. Introduction Reticulocytes are generated in the bone marrow by enucleation of mature erythroblasts. The reticulocyte develops into a mature red blood cell within 4 days. As a rule, the reticulocyte remains in the bone marrow for 3 more days and for 1 day in the peripheral bloodstream. Reticulocytes undergo extensive membrane remodeling, volume changes, and eliminate endoplasmic reticulum and all internal membrane-bound organelles and ribosomes. These transformative processes ensure that critical cellular functions, such as hemoglobin production, oxygen transport, and deformability, are optimized in mature erythrocytes. MKT-PMA-SASU-2012-0010 July 2012– Philippe Milian / Stéphanie Ricaud– Marketing dept Page. 1 of 10 3. Definition and principles of measurement Definition In normal human blood, the reticulocytes represent 0.5 to 2.0% of the whole erythrocytes. Reticulocyte enumeration is currently performed by various methods. The CLSI Area Committee on Hematology and the International Council for Standardization in Hematology (ICSH) provided guidance (CLSI H44-A2) for the performance of reticulocyte counting by flow cytometry, automated hematology instruments, and supravital dyes. (1) Principles of measurement Conventional manual counting of reticulocytes by light microscopy with supravital dye remains a method of reticulocyte enumeration. The reticulocyte count is one of the last common hematology measurements still being performed using manual/visual methods. Thus for classifying and counting reticulocytes by manual method, a blood sample is mixed with a solution containing a basic dye such as new methylene blue and brilliant cresyl blue Photomicrograph of a new methylene blue-stained allowing staining of reticular mesh-like peripheral blood smear (1000x).Reticulocytes are structure. In 1931, Heilmeyer proposed differentiated from mature red blood cells by the classification of the stages of reticulocyte presence of brown-black intracytoplasmic precipitate. maturation on the basis of staining intensities. (reticulin) Heilmeyer’s classification The maturity level of reticulocytes can be assessed by their intensities to new methylene blue staining. The different stages will be discussed in a separate document and compared with hematology analyzers. Error rates for manual counting are quoted in the literature between 20 – 40% CV and higher, depending on the number of reticulocytes. Counting 2, 000 cells are recommended by the guideline CLSI H44-A2 as a routine method. Flow cytometer methods have been developed that significantly reduce the time to perform to perform this measurement and have been shown to be more precise than manual/visual methods Reticulocytes are immature red blood cells in the final stage that have been recently released from the bone marrow and still retain intracellular protein and RNA. Reticulocytes that are MKT-PMA-SASU-2012-0010 July 2012– Philippe Milian / Stéphanie Ricaud– Marketing dept Page. 2 of 10 stained with one of a number of different supravital stains, fluorochromes or monoclonal antibodies (CD71) are counted in a flow cytometer or automated blood cell counter that are specially designed, or otherwise modified or adjusted, for this procedure. Detection of reticulocyte by Flow cytometry using anti-CD71 monoclonal antibody can give both qualitative and quantitative information. CD71 also known as transferrin receptor is a type II transmembrane glycoprotein mediates iron uptake from transferrin. Automated reticulocyte counts may vary widely dependent on the methodology and instrumentation used and monitoring should be done using the same methodology over time. Technique Light Flurorescence Optical light microscopy detection scatter RNA-specific Monoclonal Marker Vital Dye Vital Dye fluorochrome antibody FluorescenceFluorescenc HematologyFlow Flow Hematology Instrument Microscope e microscope cytometerAnalyzer cytometer Analyzer microscope Horiba Siemens Manufacturer Sysmex BCI Abbott Analytic Methods of Reticulocyte Counters Horiba BCI Abbott Siemens Sysmex Thiazole New Methylene CD4K530 Oxazine Polymethine Dye Orange Blue Thiazole like-dye 750 Fluorescence Optical light Fluorescence Optical Fluorescence Technique detection scatter detection light detection scatter RET# Ret# RETC # RETIC #RET RET% Ret% %R % RETIC RET% parameter RI CRC RPI CRC: Corrected Reticulocyte Count RETC:Reticulocyte absolute concentration MKT-PMA-SASU-2012-0010 July 2012– Philippe Milian / Stéphanie Ricaud– Marketing dept Page. 3 of 10 Pentra DX: RET measurement Method: Scattered light Fluorescence Focused flow impedance. Wavelength: 530nm Reticulocytes measuring principles The blood sample is mixed with ABX Fluocyte. This reagent contains a fluorescent stain which is specific to nucleic acids: thiazole orange. The solution is then transferred to the laser optical bench to be measured. The laser optical bench simultaneously measures the fluorescence of the cells passing through the measuring point into the flowcell, and volume by impedance. A cell passing through the flowcell gives 2 types of information: The size of the cell measured by resistivity CIS (Cell Impedance Signal) The Orthogonal Fluorescence Light (OFL). OFL: The fluorescence is collected using a lens focused on the optical flowcell and located at 90° from the light beam, an interferential filter specific to the thiazole orange stain selecting only the fluorescent wavelength and a photomultiplier tube. RET M atrix The Reticulocytes matrix is generated from 2 measurements: resistivity volume (CIS) and orthogonal fluorescence (OFL) of cells according on the X and Y axes respectively. Mature red blood cells without RNA show little or no fluorescent signal. They are located at the bottom of the matrix and horizontally distributed according to their MCV and RDW. Reticulocytes are separated from the red blood cells by their fluorescence which is proportional to the RNA content and their maturity. The most fluorescent elements Fluorescence Orthogonal OFL Fluorescence which are saturated at the top of the matrix are the most immature. Nucleated Red Blood Cells may also be found in this area. CIS Cell Impedance Signal Parameters RET%: Percentage of counted reticulocytes RET#: Absolute value of reticulocytes according to the number of RBC counted in the RBC channel (RBC/PLT chamber). RET# = (RET% x RBC#)/100 CRC (Corrected Reticulocytes count): CRC% = Ret% x (patient HCT/normal HCT) MKT-PMA-SASU-2012-0010 July 2012– Philippe Milian / Stéphanie Ricaud– Marketing dept Page. 4 of 10 Advia 2120: forward light scatter using the Mie theory The laser optical assembly consists of the illuminator, flowcell, and detector assemblies. This optical assembly is shared by the RBC/Plt, retic, and baso/lobularity channels. The use of a sheath stream in the flowcell allows cell-by-cell measurement of low- and high-angle light scattering and absorption. A laser diode, housed in the illuminator assembly, is used as the light source. Briefly, the image of a slit illuminated by light from the laser diode is focused into the flowcell. RBCs and platelets are isovolumetrically sphered using ADVIA 120 auto RETIC reagent. ADVIA 120 auto RETIC also contains a cationic dye, Oxazine 750 that stains cells according to their RNA content. A constant volume of the RBCs and reticulocytes suspension from the retic reaction chamber passes through the flowcell: The RBCs and reticulocytes that pass through the slit image in the flowcell scatter light at low and high angles; the stained reticulocytes also absorb a percentage of the light. The scattered light is detected by the two scatter photodiodes and generates the following signals: The low-angle (2° to 3°), and high-angle light (5° to 15°) scatter signatures are proportional to cell size and hemoglobin concentration. Light absorption is proportional to RNA content; the stained reticulocytes will absorb more light than the mature RBCs. RETIC Scatter Abs cytogram It is formed from the paired high-angle, low- gain light scatter and the high-gain absorption data. Sample-specific thresholds separate the cell populations 1 RTC Platelet threshold 2 RTC Coincidence threshold 3 RTC threshold 4 Low/Medium RTC threshold 5 Medium/High RTC threshold A Mature RBCs B Low absorption retics C Medium absorption retics D High absorption retics concentration Hemoglobin E Platelets F Coincidence events Light absorption DxH 800 Reticulocytes are stained with supravital dye new methylene blue. The blood sample preparation for retic analysis occurs in the module of VC. A device used to guide particles as they pass one at the time through a laser beam in a stream of fluid called sheath. In the flow cell, low frequency, direct current measures volume, while high frequency (RF) current senses cellular internal content through measuring changes in conductivity. As the particles
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