개의 림프종을 분자유전자학적으로 진단하고자 본 연구를 수행하였다. 이를 위하여 12개의 염색 및 고정된 도말 표본으로부터 DNA를 추출한 후, 임파육종에 특이적인 재배열된 항원 수용체 유전자를 중합효소연쇄반응으로 증폭시켰다. 그 결과 6개의 type-B 림프종에서 type-B 세포의 림프종에 특이적인 IgH (immunoglobulin H) 주유전자(major gene)와 type-B 림프종의 부유전자(minor gene)가 증폭되었다. 또한, 3개의 type-T 세포의 림프종의 경우에도 이에 특이적인 $TCR{\gamma}$ 유전자가 증폭되었다. 한편, 에를리키아증에 이환된 표본은 $TCR{\gamma}$ 유전자가 위양성으로 증폭되었지만, 활성화된 림프절 샘플의 경우에는 어떤 유전자의 재배열도 관찰되지 않았다. 따라서 림프종의 진단에 있어서 혈액 또는 조직 도말 표본의 DNA를 이용하여 재배열된 항원 수용체 유전자의 검출을 시도한다면, 특이적이고 객관적으로 림프종을 감별진단 할 수 있을 것이며, 나아가 회고적 분석에도 유용하게 활용될 수 있을 것으로 사료된다.
개의 림프종을 분자유전자학적으로 진단하고자 본 연구를 수행하였다. 이를 위하여 12개의 염색 및 고정된 도말 표본으로부터 DNA를 추출한 후, 임파육종에 특이적인 재배열된 항원 수용체 유전자를 중합효소연쇄반응으로 증폭시켰다. 그 결과 6개의 type-B 림프종에서 type-B 세포의 림프종에 특이적인 IgH (immunoglobulin H) 주유전자(major gene)와 type-B 림프종의 부유전자(minor gene)가 증폭되었다. 또한, 3개의 type-T 세포의 림프종의 경우에도 이에 특이적인 $TCR{\gamma}$ 유전자가 증폭되었다. 한편, 에를리키아증에 이환된 표본은 $TCR{\gamma}$ 유전자가 위양성으로 증폭되었지만, 활성화된 림프절 샘플의 경우에는 어떤 유전자의 재배열도 관찰되지 않았다. 따라서 림프종의 진단에 있어서 혈액 또는 조직 도말 표본의 DNA를 이용하여 재배열된 항원 수용체 유전자의 검출을 시도한다면, 특이적이고 객관적으로 림프종을 감별진단 할 수 있을 것이며, 나아가 회고적 분석에도 유용하게 활용될 수 있을 것으로 사료된다.
We performed the PARR (PCR to detect antigen receptor rearrangements) test on DNA isolated from twelve archival canine cytological slides including nine lymphoma, two reactive lymphocytes and one sample from Ehrlichia canis infected dog. As a result, our PCR control gene, $C{\mu}$, was su...
We performed the PARR (PCR to detect antigen receptor rearrangements) test on DNA isolated from twelve archival canine cytological slides including nine lymphoma, two reactive lymphocytes and one sample from Ehrlichia canis infected dog. As a result, our PCR control gene, $C{\mu}$, was successfully amplified from all of the DNA samples. Six out of nine lymphoma samples showed a clonal rearrangement of immunoglobulin gene whereas three samples did a clonal rearrangement of T cell receptor gamma ($TCR{\gamma}$) gene. However, we observed no visible or clear bands from PCR conducted using our antigen receptor rearrangement primers on DNA from a reactive lymphoid cell proliferation used as a negative control. False-positive amplification in $TCR{\gamma}$ gene was observed only in one sample from E. canis infection. The use of archival cytological specimens demonstrated in this study offers potential advantages for cost-effective specimen acquisition and efficient high-fidelity DNA analysis.
We performed the PARR (PCR to detect antigen receptor rearrangements) test on DNA isolated from twelve archival canine cytological slides including nine lymphoma, two reactive lymphocytes and one sample from Ehrlichia canis infected dog. As a result, our PCR control gene, $C{\mu}$, was successfully amplified from all of the DNA samples. Six out of nine lymphoma samples showed a clonal rearrangement of immunoglobulin gene whereas three samples did a clonal rearrangement of T cell receptor gamma ($TCR{\gamma}$) gene. However, we observed no visible or clear bands from PCR conducted using our antigen receptor rearrangement primers on DNA from a reactive lymphoid cell proliferation used as a negative control. False-positive amplification in $TCR{\gamma}$ gene was observed only in one sample from E. canis infection. The use of archival cytological specimens demonstrated in this study offers potential advantages for cost-effective specimen acquisition and efficient high-fidelity DNA analysis.
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문제 정의
In this study, we aimed at validating the diagnosis of B- and T- cell lymphomas in dogs using PARR analysis of DNA from archival cytological slides.
제안 방법
PCR was performed using primers for the positive DNA control, Cµ (lane C), Ig H major (lane H), Ig H minor (lane h) and TCRγ (lane T).
The PCR assay was optimized using a DNA thermal cycler (PTC-200, MJ research, USA) with the following reaction conditions: initial denaturation for 2 min at 95ºC, followed by 35 cycles of 95ºC for 8 seconds, 60ºC for 10 sec, and 72ºC for 15 sec.
대상 데이터
A total of twelve cytological slides referred to the Chonbuk National University for the diagnosis of PARR included in this study. Selected cases were as follow: nine samples from peripheral blood or specimens from lymph node, liver and kidney collected by fine needle aspiration (FNA) from suspected cases of lymphoma based on cytological evaluation; two samples showing reactive lymphocytes in cytological examination from lymph node; one sample showing positive for Ehrlichia canis ELISA test (SNAP 3Dx, IDEXX Laboratories, Westbrook, USA) without lymphoid malignancy from lymph node.
성능/효과
For the six cases showing B-cell malignancies, the ‘‘Ig H major’’ primers, which were specific for the majority of immunoglobulin gene rearrangements were amplified in 4 cases (case 4, 5, 7 and 8 in Fig. 1) whereas the ‘‘Ig H minor’’ primers, which are specific for a smaller proportion of immunoglobulin rearrangements, successfully amplified DNA in all cases.
In concluded, the use of archival cytological specimens demonstrated in this study offers potential advantages for cost-effective specimen acquisition and efficient high-fidelity DNA analysis.
The diagnostic method we used could improve retrospective cytological studies on archival clinical specimens and also be valuable for molecular diagnosis. The results showed that the sensitivity of the PARR for the diagnosis of the canine lymphoma was 100%, including B-cell malignancies and T-cell malignancies. In addition, clonal rearrangement of immunoglobulin and TCRγ were not observed in the cases of reactive lymphocytes.
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