3차원 회절광 현미경을 이용한 골수이형성증후군 환자의 적혈구 분석
- Myelodysplastic syndrome (MDS) is a clonal hematopoietic neoplasm which its etiology and pathogenesis are unclear. Moreover, vague pathogenesis, various phenotypes, and genetic mutations make it hard to diagnose. Currently, there is no definite clue to rule out MDS from other hematologic diseases from the patient’s peripheral blood. Bone marrow examination must be done to diagnose MDS. Still, there are limitations to distinguish from other disease and there are no definitive diagnostic criteria that can clearly predict the prognosis of MDS.
Optical diffraction tomography (ODT) uses interferometric microscopy technique which can measure 3-D refractive index (RI) distribution of any samples directly without labeling procedure. This can also measure important parameters of individual RBC with the programmed algorithm such as corpuscular hemoglobin content (CH), corpuscular hemoglobin concentration (CHC) and corpuscular volume (CV). Also, unique individual RBC parameters can be measured which is not possible to obtain from commercially automated hematology analyzer, such as diameter, membrane fluctuation, surface area (SA) and surface index (SI) of an RBC. SI represents the sphericity of a cell defined as a normalized volume-to-surface area ratio. It ranges from 0 to 1, flat disks to perfect spheres, respectively. The fluctuation is a very exclusive parameter to get it under the microscope. It is obtained by continuously and fast recording 2-D RI phase images using a high-speed camera. The fluctuation of RBC membranes is acutely affected by diverse pathophysiological conditions. Therefore, we predicted the fluctuations from the MDS RBCs will be decreased because there should be unnoticeable damages done to RBC membranes caused by dyserythropoiesis.
With help of this microscope, we tried to find characteristics or different aspects of MDS RBCs in peripheral blood samples which can be helpful to diagnose MDS or to elucidate pathogenesis little more than the present.
We collected two groups of blood samples from EDTA anticoagulant tube which were left-overs from complete blood count tests done by Asan laboratory and diagnostic medicine department (Seoul, Korea). One group was from patients diagnosed with MDS and the other group was from healthy individuals who visited our hospital for a regular health check-up. We also collected samples with patients having cytopenia on their complete blood count (CBC) results including MDS patients to find out where there are any different morphologic features that we cannot distinguish on the routine blood smear examination using Wright staining method and light microscope. RI tomograms were measured using ODT setup microscope, HT-1S, and HT-1H (Tomocube Inc., Daejeon, South Korea). For the visualization of the measured 3-D RI tomograms and obtaining RBC parameters, commercial software (Tomostudio, Tomocube, Inc., Daejeon, South Korea) was used.
Correlation maps were used between RBC parameters. And two sample Welch's t-test was used for comparison of RBC parameters between MDS patients and healthy individuals.
Total 220 MDS RBCs, 240 normal RBC cells were analyzed to obtain 6 RBC parameters. Each parameter was separately visualized (Fig. 1) and analyzed (Table 1). There were statistical differences in CH, CHC, CV and SI (P<0.05). The means in CH, CV, CHC, and SI were higher in MDS RBCs. The SA and membrane fluctuation were not statistically different.
The relationship between two parameters of the six in individual RBC was visualized. CH-CV, CH-CHC, CH-SA, CV-SA were directly proportional and SA-SI was inversely proportional to each other. The result was same when analyzed normal RBCs and MDS RBCs separately. More RBCs must be analyzed to get trustworthy results.
There was a distinct morphological difference in RBCs obtained from one of our MDS patients who had dyserythropoietic features in the bone marrow. Unlike other anemic features of RBC that we were aware of such as target cells or increased central pallor area, it was a new morphologic peculiarity which shaped like a cup. This could be the result of decreased membrane elasticity of RBC membrane.
Individual RBC parameters could be measured easily and fast using ODT setup microscope. The correlations between these parameters were rational. However further study with more test samples is needed to obtain reliable measurements.
Being able to observe 3-D images of RBCs was a great strength of the ODT microscope. It wouldn’t be possible to discover our new morphologic characteristics during our routine peripheral blood smear examinations which can be viewed only the upper part of the RBCs.
Therefore, ODT setup microscope is a powerful and fast tool to investigate the morphology of cells including erythrocytes. This microscope could aid commercially automated hematology analyzer when the CBC results and peripheral blood smear slides are doubtful and need to be reviewed by hematology experts. Furthermore, it can elucidate the pathogenesis of MDS by obtaining 3-D images from live blood cells.
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