Hayney M S, Dimanlig P, Lipsky J J, Poland G A
Clinical Pharmacology Unit, Mayo Clinic Rochester, MN 55905, USA.
Mayo Clin Proc. 1995 Oct;70(10):951-4. doi: 10.4065/70.10.951.
To demonstrate the utility of a "swish and spit" technique as a nucleated cell source for transporter associated with antigen processing (TAP) haplotype determination by molecular methods in large-scale clinical trials.
Twenty normal volunteers were recruited for this prospective feasibility study. From each subject, buccal or blood cells (or both) were collected for use in TAP haplotype assignment by molecular methods and subjected to various storage conditions.
As an alternative to use of lymphocytes obtained by venipuncture, we developed a swish and spit technique for collecting buccal cells for assigning TAP haplotype. For this technique, the subject vigorously swishes isotonic saline in the mouth and expectorates it into a collection container. DNA is extracted from the buccal cells by proteinase K digestion, phenol-chloroform extraction, and ethanol precipitation. In addition, we compared DNA extracted from mouthwash specimens stored under various conditions to which a specimen might be exposed if mailed.
DNA extracted from buccal cells obtained by the swish and spit technique provided excellent templates for the polymerase chain reaction (PCR), and subject acceptance of this method was universal. In all cases, assigning TAP haplotype by PCR amplification of specific alleles with use of buccal or blood-derived specimens was successful. The integrity of the specimens was unaffected by storage at -20 degrees C, 4 degrees C, 25 degrees C, or 37 degrees C, and we were able to use the DNA from cells stored under any of these conditions for TAP haplotying.
We conclude that DNA from buccal cells collected by the swish and spit technique for TAP haplotype assignment is an excellent substitute for DNA obtained from nucleated blood cells, and the technique is useful for large-scale clinical studies that require DNA from subjects geographically distant from the research site.
在大规模临床试验中，证明“漱吐法”作为一种有核细胞来源，用于通过分子方法确定抗原加工相关转运体（TAP）单倍型的实用性。
招募20名正常志愿者进行这项前瞻性可行性研究。从每个受试者收集颊黏膜或血液细胞（或两者），通过分子方法用于TAP单倍型分型，并使其经受各种储存条件。
作为使用静脉穿刺获得淋巴细胞的替代方法，我们开发了一种用于收集颊黏膜细胞以进行TAP单倍型分型的漱吐技术。对于该技术，受试者在口腔中用力漱动等渗盐水，然后将其吐入收集容器中。通过蛋白酶K消化、酚-氯仿提取和乙醇沉淀从颊黏膜细胞中提取DNA。此外，我们比较了从在各种条件下储存的漱口水标本中提取的DNA，这些条件是标本邮寄时可能会暴露于其中的。
通过漱吐技术获得的颊黏膜细胞提取的DNA为聚合酶链反应（PCR）提供了出色的模板，并且该方法被受试者普遍接受。在所有情况下，使用颊黏膜或血液来源的标本通过PCR扩增特定等位基因来确定TAP单倍型均获成功。标本的完整性不受在-20℃、4℃、25℃或37℃储存的影响，并且我们能够使用在这些条件下任何一种储存的细胞中的DNA进行TAP单倍型分型。
我们得出结论，通过漱吐技术收集的颊黏膜细胞的DNA是从有核血细胞获得的DNA的极佳替代品，并且该技术对于需要来自地理上远离研究地点的受试者的DNA的大规模临床研究很有用。
