Improvement Of Reticulated Platelets Detection And Its Clinical Application In Blood Disorders

BackgroundIn 1969 when Ingrain and Cooper smith dyed the circulated blood of losingblood Beagle dog with brilliant cresyl blue, they observed that there were partiallythick platelets like network structure, so-called reticulated platelet (RP). Reticulatedplatelets originate from megakaryocytes as anucleate cells which were the youngestplatelets were a fraction of newly released circulating elements characterized by aresidual amount of RNA and rough endoplasmic reticulum. They retain the ability tosynthesize small amounts of protein. Recently with the developing of measuringmethod of the reticulated platelets and the clinical application of flow cytometry, ithas been suggested that the reticulated platelet count, providing an estimate ofthrombopoiesis in the same way as erythrocyte reticulocyte count is a measure oferythropoiesis, may be useful in the study of thrombocytopenic diseases. Manydifferent modifications of the analytical protocol have been published f but consensuson the method has not yet been established, and they were significantly discrepancysamong the reference values. It was limited in the assessment of patients under clinicaltrial. ObjectiveWe design experiments to approach several contents below with theinvestigation background we mentioned before.1. Evaluated a method for detecting thiazole orange-positive (TO+) reticulatedplatelets in blood and platelet-rich plasma using flow cytometry, and determinedthe optimal measuring condition and simplified measuring methods of reticulatedplatelets.2. Established normal reticulated platelets percentages in men and women, anddistinguished their difference.3. Investigated the association of reticulated platelets and the related parameters ofplatelets (MPV, PDW or P-LCR).4. Determination of the percentage of thiazole orange (TO)- reticulated platelets byflow cytometry has been advocated as an aid in the diagnosis ofthrombocytopenic disorders, as well as distinguished the cause of differentthrombocytopenic disease.Methods1. Evaluate a method for detecting reticulated platelets using flow cytometry.Evaluate a method for detecting reticulated platelets using flow cytometry.Improve measuring methods of reticulated platelets to obtain the appropriate samplerequirement, different TO concentration and optimal incubation time for the assay.Labeled anti-CD42b monoclonal antibody with platelets in blood and platelet-richplasma, and applied thiazole orange-staining of reticulated platelets with residualmRNA, so we can detect of the percentage of reticulated platelets by flow cytometry.Also we compared the discrepancies of reticulated platelets in blood with inplatelet-rich plasma at the same assay condition. To check the reproducibility of this assay, we measured the same sample for 10 times continuously in order to obtainCV%.2. Ensure the reference of reticulated platelets in normal.100 normal subjects including 50 men and 50 women were selected to measurethe percent of reticulated platelets, and compared the difference between normalmen and women for this measurement with statistics analysis.3. Analyse the correlation of reticulated platelets and the related parameters ofplatelets (MPV, PDW or P-LCR).Analysis the correlation of reticulated platelet and mean platelet volume(MPV),platelet distribution width(PDW),P-LCR in 147 specimens were measured.Searched for difference of RP% in group (MPV＞11.0%) and in group (MPV＜11.0%).4. The clinical significance of reticulated platelets was researched in the diagnosticclassification of patients with thrombocytopenia.In order to find out the value of the diagnostic classification of patients withthrombocytopenia, we determine the percent of RP and its absolute counts inpatients with thrombocytopenia of different reasons, and analyze the reasons ofthrombocytopenia. Specimens come from the patients with with immunethrombocytopenic purpura (ITP, n=12), aplastic anemia(AA, n=12), acutelymphocytic leukemia(ALL, n=36), acute non-lymphocytic leukemia(ANLL,n=30), myelodysplastic syndrome(MDS, n=5), chronic myelocyticleukemia(CML, n=8) and normal subjects (control, n=100). We determine thepercent of RP and its absolute counts in those patients and compare the results withthe normal subjects. Compared RP% in patients of different hematopathy directlyto evaluate RP% how to reflect the thrombocytopoiesis in bone marrow.Results 1. The sample of blood or platelet-rich plasma was incubated with thiazole orange,and flow cytometric analysis was performed. The results of reticulated platelets reinfluence by the concentration of TO and the incubation time. Along with theconcentration of TO raising and time lengthening, the results of reticulated plateletwere obviously elevated. So we concluded that the important points for effectivestaining were: 1:2 dilution of TO and incubation for 1 hour. At this condition, we candistinguish the reticulated platelets from the TO negative platelets better and theresult of reticulated platelets is stable. We stained reticulated platelet samples inblood and platelet-rich plasma by the above method, and we selected randomly 18results to compare. There was a significant correlation between RP% in blood and inplatelet-rich plasma (r=0.997). Then we achieved the commendable reproducibleresults (CV 1.32%). and it suggested that our method was stable and can beeffectively utilized for the assay the percent of reticulated platelets in clinical.2. 100 normal subjects including 50 men and 50 women were measured the percent ofreticulated platelets. The value was 9.72% +/- 2.59% (S.D., n=100). The value was9.15% +/- 2.01% (S. D.) in men and 10.29% +/- 2.97% (S. D.) in women. There was asignificant (P＜0.05) difference between normal men and women for thismeasurement. The absolute value of reticulated platelet was 21.29+/-6.75(×10~9/L) in100 normal subjects; The value was 19.46+/-5.52(×10~9/L) in men and23.13+/-7.39(×10~9/L) in women. The results in women was higher than the results inmen. There was a significant (P＜0.05) difference between normal men and womenfor this measurement.3. In the 147 samples there was significant correlation between RP% and MPV(thegroup of men:r=0.407, P＜0.001; the group of women:γ=0.452, P＜0.001), aswell as there was significantly difference(p＜0.01) of RP% between ingroup(MPV＞11.0%)and in group (MPV＜11.0%). There was s significant correlation between RP% and PDW, P-LCR.4. The results of RP% in ITP patients (n=12) is 28.87% +/-8.91%(range from15.16%-44.96%). RP% was significantly elevated in ITP patients (n=12) than in thecontrols (P＜0.05). The result of cured patients in ITP was 9.06% +/-3.87%, and therewere no significant difference with normal subjects. The results of RP% in AApatients (n=12) is 5.96% +/-3.01% (range from2.50%-14.49%). RP% was lower thanin the controls. The result of RP% in ALL patients (n=36) was 12.20% +/-5.33%. Thepercentage values did not differ significantly from those of the control group (P＞0.05).Similarly, the result of RP% in ANLL patients (n=30) was 10.83% +/-5.16%, andthere were no significant difference with normal subjects (P＞0.05). The absolutenumber of reticulated platelets in CML patients (n=8) was 139.67+/-80.06(×10~9/L).The absolute number of reticulated platelets was higher than normal..There wassignificantly difference of RP percentage between the patients with MDS(n=5) andnormal controls, the result of RP% in MDS patients (n=5) was 21.75% +/-7.37%.5. Evaluated the correlation of RP% and megakaryocyte in bone marrow: comparedRP% with the cell population of megakaryocyte in bone marrow(n=6), we findthat the result of RP% has high consistency with the amount of megakaryocyte inbone marrow.Conclusions1. We concluded that the optimal measuring condition was: 1:2 dilution of To andincubation for 1 hour. At this condition, we can distinguish the reticulated plateletsfrom the TO negative platelets better and the measuring condition was controlledeasily. It suggested that our method was stable and can be effectively utilized for theassay the percent of reticulated platelets in clinical.2. There was a significant correlation between RP% in blood and in platelet-rich plasma (r=0.997). Both of them can be utilized for the assay RP% in clinical.Measuring RP with whole blood may simplify laboratory procedure and reduceanother interference when we measure.3. RP% was significantly higher in normal women than in normal men.4. There was significant correlation between MPV, PDW, P-LCR and RP%. And RP%was higher in group(MPV＞11.0%) than in group (MPV＜11.0%).5. Overall, our data demonstrate that flow cytometric analysis of the plateletpopulation in whole blood after staining with TO is a valuable diagnostic aid in theevaluation of thrombocytopenic disorders. RP% was significantly elevated in ITPpatients than in the controls, and decreased in AA patients. The absolute number ofRP was significantly elevated in CML patients than in the controls. These results hadcorrelation with the amount of megakaryocyte in bone marrow. Though themeasurement of reticulated platelets, we can indirectly evaluate the status ofmegakaryocyte in bone marrow and analyze the reasons of thrombocytopenia.Main newly idea1. Improved measuring methods of reticulated platelets using flow cytometry toobtain the optimal measuring condition, and double labeled thiazole orange andanti-CD42b monoclonal antibody with reticulated platelets in whole blood firstly inChina.2. Established reference value of this method in normal.3. Compared RP% with P-LCR to search for their dependability firstly in China.4. Compared RP% with the cell population of megakaryocyte in bone marrowdirectly to confirm the application of reticulated platelets in clinical diagnosis firstlyin China.5. Observed the result of RP and RP% in ITP group,AA group,and CML group, provided some reference value in the cause of different thrombocytopenic disease.