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Development of a chemometric platform for forensic body fluid analysis using vibrational spectroscopy

髙村, 彩里 東京大学 DOI:10.15083/0002004749

2022.06.22

概要

1 序
 ケモメトリクスでは、数学的及び統計的手法を用いることで、複雑かつ大量の化学実験データから化学的解釈を見出し、そして分類や物性値推定などの分析を図る。現在、分析化学の諸分野で広く活用されており、中でも犯罪捜査のための科学分析法を追究する「法科学」分野では、定量的・客観的証拠提示が求められることから、その有効性が期待されている。
 法科学では、犯罪現場から回収される様々な試料を分析対象とするが、生体試料は紛れもなく最も重要な試料の一種である。特に体液試料(血液、唾液、精液など)は頻繁に遺留され、その分析は、犯行の立証や、個人DNAの由来を明らかにすることに繋がる。現行の体液検査法では、主に生化学的手法が用いられているが、貴重な試料の消費が避けられない点や、費用や時間を要する点などが、検査実務上の問題となっている。そこで近年新たに注目されているのが、振動分光手法(赤外分光法やラマン分光法)の利用である。振動分光手法は、非破壊かつ迅速に、そして様々な試料に対して汎用的に測定が可能という特長を持つ。しかし体液試料のスペクトルは、多様な成分由来の混合信号から成ると同時に、個人差等による変動も含む。そこで、ケモメトリクス手法を用いたスペクトル解析が有用となる。
 しかしながら、振動分光にケモメトリクスを組み合わせた既報の分析法は、特定の体液種や理想的な実験条件での適用に限られ、法科学実務で想定される要請に十分に応えるものではない。また、ケモメトリクスモデルの背景にある化学的解釈も、ほとんど示されていない。そこで本研究では、振動分光を用いた体液試料の法科学分析のため、実践性の向上と化学的知見の獲得を追究した、新規なケモメトリクス手法の開発を行った。さらには、通例の体液種分析に加え、より詳細な犯罪情報を提供するための高度な分析法の開拓を図った。

2 赤外分光を用いた多体液種識別(第3章)
 法科学的体液試料分析における第一の目標は、体液種の同定である。そこで、赤外分光法を用いた体液種同定法を開発した。既報の手法は、特定の体液種かつ新鮮な試料への適用に留まるものであった。本研究では、血液・唾液・精液・尿・汗の5種類の体液試料を用意し、数か月保存する間のスペクトル変化を観測した。各体液種試料のスペクトルは、それぞれ特徴的なパターンを持つ(図1a)と同時に、個体差や経時的劣化による変化を示した。体液種毎のスペクトル特徴の違いを効率的に捉えるため、クラスター解析による類似度評価の結果に基づき、階層的分類スキームを新規に開発した(図1b)。さらに、非体液試料を除去する機能として、Q-テストによる外れ値分析を組み込んだ。本手法は、各体液種試料に対して優れた分類精度を実現し、さらに、より多くの体液種への拡張可能性を提供するものである。

3 由来人物のフェノタイピング:赤外分光による尿試料からの性別識別(第4章)
 高度な分析の一つの方向性として、由来人物絞り込みのためのフェノタイピング、特に性別を尿試料から識別する手法を開発した。尿試料は細胞性成分が少なく、DNAによる個人識別が特に難しいとされる。男性尿・女性尿の赤外スペクトルは酷似しており(図2a)、同時に個人差によるばらつきも認められた。スペクトルに微分処理を施したのち、主成分分析を行ったところ、性別間でのわずかな特徴の違いが示唆された。そこで、微分スペクトルに対し部分最小二乗判別分析(PLS-DA)を適用したところ、約80%の性別識別正解率が得られた。さらに、遺伝的アルゴリズム(GA)による選択波数領域の最適化解析を行った結果、識別正解率は約97%まで向上した(図2b)。加えて、性別識別に有効な波数領域の化学的由来を検証したところ、筋肉の代謝物で男性尿により多いとされる「クレアチニン」の寄与が示唆された。

4 犯行経緯の推定分析(第5,6章)
 高度な分析のもう一つの方向性として、犯行経緯推定のための分析法開発を行った。犯行過程に関する情報が、体液試料中成分の変化として記録され、そしてスペクトルデータに反映される可能性がある。
 まず、赤外分光法を用いた血痕の由来(死体血(死亡後の出血)/生体血(生存時の出血))識別法を開発した。死体血試料に赤外分光を適用したのは本研究が初めてである。死体血と生体血の赤外スペクトルは1127cm−1に若干の強度の違いを示し(図3)、多変量識別分析法の適用により、これらの由来の識別に成功した。さらにGAによる波数選択解析の結果、1127cm−1に表される死後血中での乳酸の増加が、識別に寄与しているとの示唆を得た。
 二つ目に、近赤外(785nm励起)ラマン分光法を用い、血痕付着からの経過時間(陳旧度)推定法を開発した。これまでに統計モデルを用いた手法は提案されているが、化学的背景が不明であるため、多様な実験条件への適用性は十分に保障されていない。そこで本研究では、血中成分の存在量の経時変化を速度論的に記述した、多変量スペクトル分解モデルを新規に提案した。30℃、24℃及び16℃における血痕ラマンスペクトルの経時変化を、数カ月にわたり追跡した上(図4a)、多変量スペクトル分解解析により5つのスペクトル構成成分に分解できることを見出した(図4b)。このうち、ヘモグロビンの自動酸化(Comp1)と、タンパク質の変性を表す成分(Comp4)について、存在量の経時変化を化学反応速度式に当てはめた(図4c)。そして、この反応速度式への回帰計算を組み込んだ、多変量スペクトル分解最適化アルゴリズムを新規に開発した。本研究により、血痕中成分の経時による化学的変化の描像を包括的に捉え、また最終的に、任意の実験条件で利用可能な「血痕陳旧度指標」の提示に成功した。

5 担体由来干渉信号除去のためのスペクトル前処理(第7,8章)
 最後に、実践的かつ挑戦的な課題として、体液スペクトルに混入した担体由来信号を解析的に除去するための、スペクトル前処理法を検討した。これは、体液が衣服等に染み込んだ場合に有用となる。本研究では、考案手法Augmented principal components-least squares((APCLS)と、仮想添加多変量解析法(HAMAND(Ando, et al. 2015))について、近赤外ラマン分光法を用いた木綿生地上血痕の経過時間推定精度を指標に、比較を行った。作成から2か月にわたって測定した木綿生地上血痕のラマンスペクトルには、担体である木綿由来の強い信号の混入が観測された(図5a)。上記2種類の担体信号除去法を施したのち、事前に純粋血痕試料のラマンスペクトルで構築した経過時間推定モデルに当てはめた(図5b)。結果、APCLSの使用で、より高い推定精度が得られ、担体信号の微小な実験的変動を、前処理計算で考慮することの有効性が示唆された。さらには、識別分析への利用を指向した、APCLSの改良法を提案した。赤外分光を用いた、様々な生地上の死体血痕/生体血痕の識別に対し、改良法の利用での識別精度の向上を実証した。

6 今後の展望
 本研究の究極的目標は、開発したケモメトリクス手法を組み合わせ、体液試料の振動スペクトルから多様かつ包括的な犯罪情報の提供するための、分析プラットフォームを確立することである(図6)。本論文で報告した結果は、その骨子となる技術を実証し、また今後の拡張可能性を積極的に示唆するものである。

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