Acute Promyelocytic Leukemia: Molecular Diagnostic Procedures

ACUTE PROMYELOCYTIC LEUKEMIA: MOLECULAR DIAGNOSTIC PROCEDURES
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(02, September, 2010)
Acute Promyelocytic Leukemia: Molecular Diagnostic Procedures
Raymond, et al, (2008) and Massey, et al. (2006) says that on Cell Surface-Antigen Immunophenotyping, the separation of borne marrow mononuclear cells was by Ficoll-Hypaque density sedimentation and analysis of peripheral-blood was by a whole blood with the Q-prep system. By the use of dual-color fluorescence of the cells gating depending on size and the forward-scatter analyzed on the profile flow cytometer of EPICS while the expression of a panel of the antigens, including CD33 and CD16. All sample cells were analyzed up to three gate clusters.
In the method of Molecular Cytogenetics (Fish), part of material fixed in carnoy to make fish slides carried out in accordance with the probe supplier`s instructions. Slides analysis was by fluorescent microscope with rhodamine, DAPI, FITC and the triple band pass filters. The best images were taken by the CCD camera as shown if figure 3. Slide with having above 50% cell with fluorescent dots were selected and analyzed by two observers. There were two types of count; normal count, without rearrangement and present rearrangement. Cases with above 10% were looked at as positive. (Ghaffari, 2006)
With the method of PCR, quantitative Real-time PCR, the extracted RNA were subjected to 2 different quantitative real-time reverses transcription-polymarase chain reaction methods to detect the two types of PML/RAR-α fusion transcripts abl amplification is done as the control and reference for relative quantification. If PML/RARA (15:17) translocation is shown then the result is positive if the ratio is 0 then the result is negative. (Jürgen et al, 2010).
Results
The cell surface immunophenotyping demonstrated the progression of immature makers on the neoplastic cells at the presentation toward the form of mature granulocytic makers during the remission. In many cases there were observations of morphological maturing cells in the peripheral blood that at the same time showed mature and immature makers. The serial studies of surface makers` results of a patient with newly diagnosed disease are shown in figure 1. (Raymond, et al, 2008)
The fish results are reported as % and 12 diagnosis were positive except case 9 has no enough analysis materials. Positivity had a mean of 56%. There was a decrease in the % of rearrangement cells in the samples from evaluated patients after therapy except 4th and 6th cases. In the group of control, there was a presentation of 5.8% cells with rearrangement.
For the real-time PCR, the fluorescence intensity was plotted against the cycle numbers, showing the number of cycle at which the exponential increase in the fluorescence as they occurred in every sample. The results showed that the sample with large number of copies of the original target DNA sequence (the line at the top), reached the exponential increase at an earlier cycle (16th cycle). (Chauffaille, et al, 2007). This is shown in figure 2 below.
Figure 1. Surface-maker studies of peripheral-blood results. From:
/doi/full/10.1056/NEJM199105163242002
Fig2. Amplification of curve generated by the real-time PCP instrument. From: /pmc/articles/PMC1214554
Fig3. Images captured by CCD camera coupled with computer equipped with Keryotyping and fish software. (a) In situ fluorescent hybridi...