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Reticulocytes are young, immature, anucleated erythroid cells which contain remnant cytoplasmic ribonucleic acid (RNA) and organelles such as mitochondria and ribosomes. They are slightly larger than mature red blood cells (9-10µm in diameter as compared to 6-8µm of mature red blood cells). Reticulocytes normally spend two to three days in the bone marrow and one day circulating in the peripheral blood before developing into mature erythrocytes. Reticulocytes can be visualized under light microscope using a supravital stain such as new methylene blue. When stained with new methylene blue, the ribosomal materials within the reticulocyte precipitate, forming purplish-blue colour granules in the cytoplasm. The intensity of reticulocyte is proportional to the RNA and cellular contents of the cell. From visual examination of a peripheral blood film so stained, the number of reticulocytes among 1000 RBCs is found and expressed as a percentage. This is termed the reticulocyte count. Under normal circumstances, approximately 1% of red blood cells (RBCs) are turned over each day; and erythrocyte contains RNA for approximately one day after leaving the marrow, therefore the normal reticulocyte count is approximately 1%.
Reticulocyte count is an important diagnostic tool in assessing the erythropoietic activity of the bone marrow and monitoring the effectiveness of therapy. For example, in patients with anemia, hemorrhage, and patients with disorder resulting in ineffective erythropoiesis. Traditional manual staining and visual reticulocyte counting method has been largely replaced by automated reticulocyte counting (e.g. flow cytometry) over the past few decades as manual determination of reticulocyte count is very imprecise, inaccurate and time-consuming; moreover, manual count is highly relied on the staining technique, counting skill and experience of the scientist. The imprecision and inaccuracy have been well documented in several studies, with coefficients of variation ranging form 25% to more than 50%. Many factors including stain variability, sample size, statistical sampling error and interobserver variation in reticulocyte definition can affect the results obtained from the manual counting method. Automated reticulocyte counting using flow cytometry on the other hand, provides a more accurate, precise and time-saving method. In addition, automated reticulocytes count 'removes much of the subjective interpretation involved in manual count, allows evaluation of large numbers of RBCs to avoid sampling error, and provides a standard and uniform analysis' . This article aims to review, compare and evaluate reticulocyte measurement by three high-end haematology analysers including the Beckman Coulter LH750 series, Sysmex XE-2100, and the Bayer ADVIA 120.
Beckman Coulter LH-750:
The Beckman Coulter LH-750 system is a fully automated on-line haematology analyzer for in vitro diagnostic use in most haematology laboratories. It provides both automated complete blood count (CBC), white blood cell differential and reticulocyte analysis. The maximum test throughput for the LH-750 is up to 105 samples per hour. A minimum of 200µL of specimen is required for an opened mode and a minimum of 300µL of specimen are required for a closed mode analysis; or minimum of 500µL of specimen for a closed mode analysis with slide-maker. The Coulter LH-750 performs reticulocyte analysis using combination of both the new methylene blue staining procedure and flow cytometry with Coulter's VCS (volume, conductivity, and scatter) technology. In Coulter's VCS technology, specimens suspended in an electrically conductive diluent are passed through an aperture in a glass tube using hydrodynamic focusing technique (sample stream is surrounded by a sheath fluids as it passes through the central axis of the aperture) . Cell volume and number are measured by impedance where a change on the electrical current is occurred when cells pass through the sensing aperture. Voltage pulses are generated and measured when the cell passes though the aperture and cause an interruption of the current, where the number of pulses is proportional to the number of cells counted and the size of the pulse is proportional to the size of the cells. The size of the nucleus, internal and density of each cell are measured with a high frequency current (conductivity). In addition, the conductivity signal is corrected for cellular volume, yielding a unique measurement called 'opacity'. Information for the internal structure, granularity, shape and the surface morphology of cells are generated using a helium-neon laser light with multiple-angle light scatter (range from 10 degrees to 70 degrees).
The following steps are obtained from a pamphlet published by Beckman Coulter TM which summarizes the reticulocyte analysis method on its GEN-S analyser. Note that the LH-750 series uses the same method as the GEN-S for reticulocyte analysis. The steps include:
A small segment of specimen is incubated in a heated chamber with a supravital stain, Coulter's special new methylene blue (NMB) solution. The NMB solution precipitates any residual RNA material within the erythrocyte;
A portion of the stained sample is then transferred to a second chamber together with a hypotonic clearing solution. The clearing solution will remove the haemoglobin within the erythrocyte but preserve the stained RNA within the cell;
The sample cells then processed through the VCS flow cells immediately for analysis by the same three independent probes used for differential analysis.
Volume which generates from the specimen is plotted against light scatter and against conductivity, correlating with opacity of the RBCs. Stained reticulocytes show greater optical scatter and greater opacity than mature RBCs . Parameters that are generated from the LH-750 analyzer for reticulocyte analysis include the relative and absolute reticulocyte count, mean reticulocyte volume (MRV) and the immature reticulocyte fraction (IRF), an index used to indicate the least mature fraction of reticulocytes. It is calculated by the sum of the populations of high and medium immaturity of the RBCs. The IRF has replaced the reticulocyte maturity index and became an internationally accepted term. The IRF can also be used as a superior parameter to monitor the erythropoietic activity in the bone marrow.
The Sysmex XE-2100 system is a fully-automated online haematology analyzer. It provides automated complete blood count, white blood cell count with immature granulocytes differential, fluorescent nucleated red blood cells analysis, fluorescent optical platelet analysis; as well as fluorescent reticulocyte count. The maximum test throughput for the XE-2100 analyzer is up to a maximum of 150 samples per hour. A minimum of 130µL of specimen is required for an open mode analysis; a minimum of 200µL of specimen is required for a closed mode analysis, and a minimum of 40µL of specimen is required for a capillary mode analysis.
In the XE-2100 analyzer, differentiation of different cell populations is based on the principle of flow cytometry [combination of both the low-voltage DC impedance and the high-voltage radio frequency (RF) resistance]. With the use of DC and RF techniques, the total volume and interior density such as the nuclear volume of cells can be measured. The total volume of the cell is proportional to the change in the low-voltage DC, whereas the interior density of the cell is proportion to the changes on the pulse size in the RF signal. In addition, information such as the nuclear to cytoplasmic ratio, cytoplasmic granulation and nuclear density are obtained using a high frequency electromagnetic probe (conductivity)  .
For reticulocyte analysis, the XE-2100 uses both fluorescence and light scattering technique. Unlike other Sysmex analyzers such as the SE9500 and the R-series which uses the Auramine O for cells staining process; the XE-2100 uses a unique supravital stain called the 'Polymethine' dye to stain the RNA of reticulated RBCs [  ,  ]. Cells from specimen are stained with ploymethine dye; each single is then passed through the beam of a semi-conductor diode laser, an argon laser. Signals are generated and measured when each single cell passes through the argon laser; these signals are expressed as the forward scatter and side fluorescence on a scattergram. Forward scatter intensity is correlates with the size of cells, which is plotted against the fluorescence intensity that is proportional to the RNA content of the stained cells . More than 30,000 cells are counted for each sample [  ].
Reticulocytes have a high contents of RNA and DNA material, hence, they have a lower fluorescence intensity compare to other nucleated cells such as leukocytes and nucleated red blood cells, or red blood cells containing Howell-Jolly bodies. Therefore, reticulocytes are easily being distinguished and separated from other nucleate cells [  ]. Both the percentage and absolute count for reticulocytes are produced by the XE-2100. Furthermore, this analyzer can also produce a reticulocyte differential based on the RNA content of the cell . The fluorescence-stained reticulocytes are further divided into 3 fractions based on the intensity of fluorescence. These 3 fractions are: (i) high fluorescence reticulocytes - HFR, (ii) medium fluorescence reticulocytes - MFR; and (iii) low fluorescence reticulocytes - LFR); the 3 fractions together with the mature RBC fraction are expressed on the RET scattergram obtained from the analyzer. Moreover, the IRF (immature red cell fraction) can be calculated by adding the HFR with the MFR. As stated earlier, the IRF indicates the ratio of immature reticulocytes to the total reticulocyte in the specimen sample.
Bayer ADVIA 120:
The ADVIA 120 system is a fully-automated haematology analyzer. It provides a complete hemogram and white blood cell differential as well as a fully automated reticulocyte analysis. Different reticulocyte parameters can be produced from the analyzer, including the MCVr (mean reticulocyte volume), RDWr (reticulocyte distribution width); and CHr (reticulocyte Hb content), etc. These parameters are useful for the prediction and assessment of the appropriateness of bone marrow response and iron status in patients and will be discussed further on the following paragraph. The maximum test throughout for the ADVIA 120 analyzer is up to a maximum of 120 samples per hour for both CBC and cell differential. A minimum of 157µL of specimen is required for both open and closed mode analysis. As highlighted by Chapman, [  ] the ADVIA 120 uses a combination of laser light-scatter and peroxidase-based cytochemistry and a second analytic basophil-nuclear lobulartiy channel, where cells undergo cytoplasmic stripping to bare nuclei. Series of cytograms are therefore can be produced using the information from these two channels.
For reticulocyte analysis, specimens are stained with a nuclei acid dye called the 'oxazine 750'. Flow cytometric light scattering method is used to measure the size, volume; cellular contents and internal complexity of cells when they pass through the sheath-stream flow cell. Three different detectors include the (i) low-angle scatter (2-3degrees); (ii) high-angle scatter (5-15 degrees); and (iii) absorbance are used to generate information on each single cell as it passes through the 670nm laser diode. Three cytograms are generated:
cytogram of low-angle scatter versus high-angle scatter (also known as the RBC map);
cytogram of high-angle scatter versus absorption; and
cytogram of volume versus haemoglobin concentration
Cells that have taken up the dye (e.g. cells that contain RNA/ DNA materials) absorb more light than mature red cells, which contain no RNA/ DNA materials; and therefore, cell populations can be distinguished. Furthermore, the reticulocytes population are divided into three different subgroups: low-, medium- and high-absorbing cells, based on the amount of stains the cells absorbed. Similar to other high-end analyzers, the IRF is generated from the sum of medium-absorbing and high-absorbing cells.
Parameters used on different analyzers:
Parameters use on reticulocyte analysis varies between different analyzers. Of which the immature reticulocyte fraction (IRF) are used by all three analyzers. The IRF indicates the ratio of immature reticulocytes to the total reticulocytes in the specimen. It is calculated by the sum of the middle-fluorescence and high-fluorescence ratios. Clinical study has proven that the IRF is a reliable early indicator of changes in erythropoietic activity. Other parameter such as the RET-He is used by the Sysmex XE-2100 analyzer. This RET-He is a parameter measured in the reticulocyte channel and is used to measure the incorporation of iron into erythrocyte haemoglobin. The RET-He parameter has been adopted by the National Kidney foundation in the United States as for the assessment of anemia and is an established parameter which used in the Kidney Disease Outcomes Quality Initiative guidelines for assessing the initial iron status for patients.
The ADVIA 120 analyzer however, has its unique reticulocyte parameters in addition to the IRF. These parameters include: the MCVr (mean reticulocyte volume); CHr (mean haemoglobin content of reticulocytes); CHDWr (corresponding distribution width), etc. In the CHr parameter, haemoglobin content of each single reticulocyte is calculated as the product of the cell volume and the cell Hb concentration. A single-parameter histogram of CHr is then constructed, with a corresponding distribution width (CHDWr) calculated. These unique parameters provided by the analyzer is can be used as: (i) guidance of iron and erythropoietin (EPO) therapy in hemodialysis patient; (ii) diagnosis of iron deficiency in patients with inflammation and chronic disease; and (iii) diagnosis of iron deficiency in early childhood, etc.
Stability of samples:
Guder [  ] defines sample stability as "the capability of a sample to retain the initial value of a measured quantity for a defined period within specific limits when stored under defined conditions". Very few studies have been done in regards to the sample stability while in 2008, Imeri et al [  ] have performed a study on sample stability by compares 3 different analyzers (Coulter's LH-750, Sysmex XE-2100 and ADVIA 120).
Imeri et al states that "reticulocyte count showed a better stability at 4-8 degrees, and was found to be 72 hours with exception of Sysmex XE-2100 (48 hours). At room temperature the stability for ADVIA 120 was only 10 hours, and 48 hours for the other analyzers tested". The study has illustrated that not only storage time and storage temperature, but the measurement principle (i.e. use of analyzer) has an impact on sample stability (i.e. reticulocytes exhibit a better stability if stored at 4-8 degrees).
Three different high-end haematology analyzers (the Beckman Coulter LH-750, Sysmex XE-2100 and the ADVIA 120) have been chosen for the study on reticulocyte analysis. These three analyzers all have different methods for reticulocyte counting and they all have their unique parameters. These parameters are useful in the diagnosis and management of diseases such as iron deficiency, inflammation and chronic disease, etc. Of the 3 analyzers, I have found the ADVIA is more useful comparing to the other two analyzers, as it can measure the cellular contents of each single cell. Moreover, it provides the most numbers of parameters which as mentioned earlier, is helpful in the diagnosis and management of diseases in patients.
As stated by Chapman, "with the wide range of analyzers available, high quality of patient results, good sensitivity and specificity, reliability, service, safety, ease of operation and training, and cost effectiveness should continue to be important considerations in selecting a haematology system". More importantly, limitations and interferences can occur anytime within the laboratory, while blood film examination still being the most appropriate alternative way in diagnosis of disease in the world of haematology.