저자들은 이 연구를 위한 시설을 제공한 Sun Yat-sen University의 제3 부속 병원인 Center for Reproductive Medicine의 Andrology Laboratory에 감사드립니다. 이 기사를 준비하는 데 자금이 투입되지 않았습니다.

이 연구는 Sun Yat-sen University의 제 3 부속 병원 의료 윤리 위원회의 검토 및 승인을 받았습니다.
1. 작업 용액의 준비
<ol><li>작업 용액을 준비하는 데 필수적인 다음 시약인 오르토 톨루이딘 기질 용액, 포화 염화암모늄(NH4Cl) 용액, 148mmol/L 에틸렌디아민테트라아세트산 디소듐염(Na2EDTA) 용액 및 6%(v/v) 과산화수소 용액. 이송 피펫(1000 μL, 200 μL, 10 μL) 및 시험관 랙과 같은 필요한 도구와 함께 실온(RT) 실험실 벤치에 놓습니다. 주의 : 위에서 언급한 시약을 취급하기 전에 안전보건자료(SDS)를 읽으십시오.</li>
	<li>준비된 작업 용액을 저장하고 빛에 노출되지 않도록 보호하기 위해 불투명한 갈색 시약 병을 준비합니다.</li>
	<li>아래 지정된 비율에 따라 시약을 불투명한 갈색 시약 병에 옮깁니다.<ol><li>200 μL 이송 피펫을 사용하고 손잡이를 돌려 스케일을 100 μL로 조정합니다. 그런 다음 100μL의 NH4Cl용액을 불투명한 갈색 시약 병에 옮깁니다.</li>
		<li>피펫 팁을 교체하고 위에서 언급한 200μL 이송 피펫을 사용하여 148mmol/L Na2EDTA 용액 100μL를 불투명한 갈색 시약 병에 옮깁니다. 그런 다음 피펫팅으로 부드럽게 불어 용액을 완전히 균질화합니다.</li>
		<li>1000 μL 이송 피펫을 사용하고 손잡이를 돌려 스케일을 900 μL로 조정합니다. 그런 다음 900μL의 오르토-톨루이딘 기질 용액을 불투명한 갈색 시약 병에 옮기고 피펫팅으로 부드럽게 불어 용액을 완전히 균질화합니다.</li>
		<li>10 μL 이송 피펫을 사용하고 손잡이를 돌려 스케일을 5 μL로 조정합니다. 그런 다음 6%(v/v) 과산화수소 용액 5μL를 불투명한 갈색 시약 병에 옮깁니다.</li>
	</ol></li>
	<li>1000 μL 이송 피펫을 사용하고 손잡이를 돌려 스케일을 1000 μL로 조정합니다. 피펫 팁을 교체하고 피펫팅으로 부드럽게 불어낸 다음 불투명한 갈색 시약 병에 용액을 완전히 균질화합니다.</li>
	<li>바로 사용할 수 있는 작업 용액을 불투명한 갈색 시약 병에 담아 빛에서 멀리 떨어진 RT에 보관하십시오. 작업 솔루션은 24시간 동안 반응합니다.</li>
	<li>각 정액 샘플에는 900μL의 작업 용액이 필요합니다. 매일 준비되는 작업 용액의 양이 매일 검사되는 샘플의 수와 일치하는지 확인하십시오.</li>
</ol>2. 정액 샘플 준비
<ol><li>정액 샘플을 채취하기 전에 환자가 인간 정액 검사 및 처리를 위한 세계보건기구(WHO) 실험실 매뉴얼(5판)에서 요구하는 대로 2-7일 동안 사정을 자제했는지 확인하십시오. 8. 자위는 정액 샘플을 얻기 위해 선호되는 방법입니다. 검사의 정확도를 높이려면 환자에게 사정된 정액을 모두 채취하도록 지시합니다.</li>
	<li>항온 스탠드를 켜고 정액 샘플의 후속 액화가 원활하게 진행될 수 있도록 미리 37°C까지 가열합니다.</li>
	<li>환자의 정액 샘플을 받으면 식별 코드, 수집 시간, 금욕 기간 및 기타 관련 정보를 포함한 세부 정보를 폴리머 기반 용기의 외벽에 꼼꼼하게 기록합니다.</li>
	<li>정액 샘플을 수용하는 폴리머 기반 용기를 37°C로 예열된 항온 스탠드에 놓으면 정액 액화에 유리합니다.</li>
	<li>정액 샘플을 폴리머 기반 피펫에 넣고 정액 샘플이 완전히 액화될 때까지 5분마다 액화 상태를 관찰합니다.</li>
	<li>정액 샘플이 30분 이내에 완전히 액화되지 않으면 아직 검사를 수행하지 마십시오. 30분 더 기다립니다. 60분 후에도 정액이 완전히 액화되지 않으면 동일한 양의 인산염 완충 용액을 첨가하여 1:1로 희석하고 부드럽게 저어 액화를 촉진합니다.</li>
	<li>철저한 교반과 일관된 균질화 후 후속 검사 절차를 위해 완전히 액화된 정액 샘플을 보관합니다.</li>
</ol>3. 과산화효소 양성 백혈구의 염색 및 원본 정액 샘플 분석
<ol><li>샘플링 전에 폴리머 기반 피펫을 사용하여 피펫팅으로 반복적으로 불고 검사 중인 정액 샘플을 완전히 균질화합니다.</li>
	<li>200 μL 이송 피펫을 사용하고 손잡이를 돌려 스케일을 100 μL로 조정합니다. 피펫 팁을 교체한 다음 정액 샘플 100μL를 원심분리 튜브로 옮깁니다.</li>
	<li>1000 μL 이송 피펫을 사용하고 손잡이를 돌려 스케일을 900 μL로 조정합니다. 피펫 팁을 교체한 다음 위에서 언급한 작업 용액 900μL를 원심분리기 튜브에 옮깁니다.</li>
	<li>피펫팅으로 반복적으로 불고 1000μL 이송 피펫을 사용하여 정액 샘플과 작업 용액으로 구성된 반응물을 완전히 균질화합니다.</li>
	<li>원심분리기 튜브를 2차원 셰이커의 시험관 랙에 놓고 RT에서 200분 동안 30rpm으로 연속 교반합니다.</li>
	<li>염색 반응을 기다리는 동안 CASA 시스템을 사용하여 정액 샘플을 분석하여 폴리머 기반 용기의 원래 정액 샘플에서 정자 농도를 결정합니다.<ol><li>CASA 시스템의 전원 스위치를 켜고 SCA SCOPE 아이콘을 두 번 클릭하여 응용 프로그램 소프트웨어를 엽니다.</li>
		<li>샘플링 전에 피펫팅으로 반복적으로 불고 폴리머 기반 피펫으로 원래 정액 샘플을 다시 완전히 균질화합니다.</li>
		<li>10 μL 이송 피펫을 사용하고 손잡이를 돌려 스케일을 10 μL로 조정합니다. 피펫 팁을 교체하고 원래 정액 샘플의 10μL를 피펫팅한 다음 SCA 계수 챔버에 넣습니다. 정액 샘플이 표류를 멈출 때까지 60초 동안 기다립니다.</li>
		<li>SCA 계수 챔버를 CASA 시스템의 샘플 홀더에 배치하고 애플리케이션 소프트웨어의 대화 상자에서 환자 정보를 설정하여 공식 검사를 준비합니다.</li>
		<li>시작 버튼을 클릭하여 CASA 시스템이 있는 SCA 계수 챔버에서 원본 정액 샘플을 검사하고 정자 농도와 같은 필요한 데이터를 기록합니다.</li>
	</ol></li>
</ol>4. 과산화효소 양성 백혈구와 정자의 관찰
<ol><li>1000 μL 이송 피펫을 사용하고 손잡이를 돌려 스케일을 1000 μL로 조정합니다. 피펫 팁을 교체한 다음 피펫팅으로 반복적으로 불고 30분 반응 과정이 끝나면 원심분리기 튜브에서 정액 샘플과 작업 용액으로 구성된 반응물을 완전히 균질화합니다.</li>
	<li>10 μL 이송 피펫을 사용하고 손잡이를 돌려 스케일을 10 μL로 조정합니다. 피펫 팁을 교체하고 위에서 언급한 반응물 10μL를 피펫팅한 다음 정자 계수 챔버에 넣습니다. 마지막으로 전용 유리 커버슬립으로 정자 계수 챔버를 덮습니다.</li>
	<li>정자 계수 챔버를 현미경 스테이지에 놓고 두 개의 10x 접안렌즈로 광학 현미경의 전원 스위치를 켭니다. 그런 다음 반응물 샘플이 표류를 멈출 때까지 60초 동안 기다립니다.</li>
	<li>대기 시간 동안 현미경 변환기를 10x 대물 렌즈로 전환하십시오. 그런 다음 거친 조정 노브와 미세 조정 노브를 순서대로 조정합니다. 마지막으로 10×10의 배율로 정자와 둥근 세포를 관찰합니다.</li>
	<li>분석할 시야를 결정한 후 현미경 변환기를 40x 대물 렌즈로 전환하고 10× 40의 배율에서 정자 및 갈색 과산화효소 양성 백혈구를 관찰합니다.</li>
	<li>전자 계수기 시스템을 활용하여 해당 필드 내에서 최소 200개의 정자와 갈색 과산화효소 양성 백혈구의 수를 계산합니다.</li>
</ol>5. 과산화효소 양성 백혈구의 농도 계산
<ol><li>다음 공식을 사용하여 과산화효소 양성 백혈구의 농도를 계산합니다. <img alt="Equation 1" src="/files/ftp_upload/66211/66211eq01v2.jpg" style="margin: auto;"></li>
</ol>

Peroxidase-Positive Leukocyte Cells에 대한 정액 분석

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Q., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myeloperoxidase-derived+hypochlorous+acid+promotes+ox-LDL-induced+senescence+of+endothelial+cells+through+a+mechanism+involving+beta-catenin+signaling+in+hyperlipidemia.">Myeloperoxidase-derived hypochlorous acid promotes ox-LDL-induced senescence of endothelial cells through a mechanism involving beta-catenin signaling in hyperlipidemia.</a> Biochem Biophys Res Commun. 467 (4), 859-865 (2015).</li><li>Nicholls, S. J., Hazen, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myeloperoxidase+and+cardiovascular+disease.">Myeloperoxidase and cardiovascular disease.</a> Arterioscler Thromb Vasc Biol. 25 (6), 1102-1111 (2005).</li><li>Wolff, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+biologic+significance+of+white+blood+cells+in+semen.">The biologic significance of white blood cells in semen.</a> Fertil Steril. 63 (6), 1143-1157 (1995).</li><li>Chen, Y., Hashiguchi, N., Yip, L., Junger, W. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hypertonic+saline+enhances+neutrophil+elastase+release+through+activation+of+P2+and+A3+receptors.">Hypertonic saline enhances neutrophil elastase release through activation of P2 and A3 receptors.</a> Am J Physiol Cell Physiol. 290 (4), C1051-C1059 (2006).</li><li>Weiss, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue+destruction+by+neutrophils.">Tissue destruction by neutrophils.</a> N Engl J Med. 320 (6), 365-376 (1989).</li></ol>

저자는 공개할 것이 없습니다.

오르토톨루이딘과 과산화수소는 감광성을 가지며 빛에 노출되면 분해될 수 있습니다. 테스트 시약의 효과를 보장하려면 준비된 작동 유체를 어두운 곳에 보관하는 것이 좋습니다. 
정액 샘플은 자연적으로 이질적인 특성을 나타냅니다. 정액 샘플의 준비 과정에서 정액의 액화 상태를 정확하게 판단하고 각 샘플링 전에 정액 샘플을 완전히 균질화하는 것은 테스트 결과의 신?...

불임은 가임기 부부의 약 15%에 영향을 미치는 글로벌 공중 보건 문제로 부상했습니다. 전체 난임 사례의 약 50%가 남성 요인에 기인하며, 거의 20%에서 30%는 남성 요인에 기인할 수 있다 1,2,3. 남성 생식기 감염(MGTI)은 남성 불임의 중요한 원인 중 하나로, 전체 불임 사례의 약 15%를 차지한다 4,5.
대부분의 개인은 정액에 백혈구를 가지고 있으며, 이는 비정자 세포의 13%를 구성하며, 호중구 12%, 대식세포 0.9%, 림프구 0.1%6,7의 차등 비율이 있습니다. 세계보건기구(WHO)의 인간 정액 검사 및 처리 실험실 매뉴얼(5판)에 따르면, 백혈구 정자증은 일반적으로 정액에 >1 × 106 cells/mL 백혈구가 존재하는 것으로 정의된다8. 이 질환은 환경 독소, 약물 남용, 정맥류, MGTI 등과 같은 다양한 요인에 의해 발생할 수 있으며, 이 모든 요인은 정액 내 백혈구 농도의 비정상적인 증가로 이어질 수 있다9. 백혈구는 정액의 활성산소종(ROS) 수치를 높여 단백질과 DNA에 지질 과산화 및 산화적 손상을 일으켜 정자 운동성 감소, 정자 형태학적 이상 증가, 정자 DNA 단편화 지수 상승, 정자 아크로솜 기능 손상, 심지어 배아 발달에 부정적인 영향을 미칠 수 있다10,11.
현재 남성의학 전문의들은 생식기 염증을 확인할 때 정액의 둥근 세포나 정액 혈장의 엘라스타제를 검사하는 것이 일반적입니다. 그러나 무분별하고 불필요한 항생제 사용을 줄이는 데 기여하지 않는 정자 생성 세포와 생식관 상피 세포를 구별하는 것은 종종 어렵다6. 후자의 검사 방법은 상대적으로 복잡하고 시간이 많이 걸리며 결과 보고가 느리기 때문에 MGTI와 같은 질병의 조기 진단 및 치료에 도움이 되지 않습니다. 미국 비뇨기과학회(American Urological Association, AUA)는 정액 분석에서 원형 세포 농도가 1 × 106 cells/mL12 이상일 때 생식기에서 백혈구와 축출된 세포를 추가로 구별해야 한다고 제안한다. 일부 남성학 실험실에서는 백혈구를 검사하기 위해 유세포 분석(flow cytometry)13 또는 백혈구 항원(le-g., CD45) 면역세포화학(immunocytochemistry)14 을 이용한다. 이러한 방법은 정확하지만 비용과 시간이 많이 소요되어 특히 개발도상국에서 대규모 임상 구현이 어렵습니다.
과산화효소는 다양한 유형의 세포에 널리 분포되어 있으며 산화 스트레스 손상에 대한 저항력에 중요한 역할을 합니다. 골수화효소(MPO)는 일반적으로 면역 세포에서 발현되는 과산화효소 아과(subfamily)의 구성원입니다. 호중구의 azurophilic granules 15,16에서 가장 많이 발현되며 림프구 17,18, monocyte 및 macrophage 19에서도 발현됩니다. 정액에서 백혈구, 특히 호중구의 농도는 과산화효소 양성 7,20인 둥근 세포를 검사하여 얻을 수 있습니다. 정액 내 백혈구 검사 과정을 경제적이고 편리하며 효율적으로 만들기 위해 검사 방법을 재설계했습니다. CASA(Computer-Assisted Semen Analysis) 시스템의 도움을 받아 60분 이내에 백혈구 농도를 얻을 수 있습니다. 이 새로운 방법은 환자의 검사 비용과 결과를 얻기 위한 대기 시간을 줄이고 실험실 기술자의 작업량을 줄이며 의사의 진단 및 치료 대기 시간을 단축합니다.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>148 mmol/L ethylenediamine tetraacetic acid disodium (Na2EDTA) solution</td>
			<td>ShenZhen HuaKang Biomedical Engineering Co.,LTD</td>
			<td>20230301</td>
			<td>Agentia B</td>
		</tr>
		<tr>
			<td>6% (v/v) hydrogen peroxide solution</td>
			<td>ShenZhen HuaKang Biomedical Engineering Co.,LTD</td>
			<td>20230301</td>
			<td>Agentia D</td>
		</tr>
		<tr>
			<td>Automatic Sperm Class Analyzer &#8211; SCA SCOPE&#160;</td>
			<td>Microptic S.L.</td>
			<td>1222996; Model type: SCA-SCOPE-H</td>
			<td>Computer-Assisted Semen Analysis (CASA) equipment</td>
		</tr>
		<tr>
			<td>Disposable centrifugal tube (1.5 mL)</td>
			<td>Zhejiang Gongdong Medical Equipment Co., LTD</td>
			<td>2210009</td>
			<td>Centrifugal tube for Staining process</td>
		</tr>
		<tr>
			<td>Disposable transfer pipette (3 mL)</td>
			<td>Jiangsu Kangjian Medical Supplies Co., LTD</td>
			<td>20221101</td>
			<td>Polymer-based pipette</td>
		</tr>
		<tr>
			<td>Electronic counter</td>
			<td>None</td>
			<td>None</td>
			<td>A multichannel counter</td>
		</tr>
		<tr>
			<td>Electronic scales</td>
			<td>Shanghai Liangping Instrument Co., LTD</td>
			<td>D9008084</td>
			<td>MAX = 100 g, e = 10 d, d = 0.01 g</td>
		</tr>
		<tr>
			<td>Makler counting chamber</td>
			<td>Makler</td>
			<td>MQ30004</td>
			<td>0.01 sq.mm, 10 &#956;m deep</td>
		</tr>
		<tr>
			<td>Optical microscope</td>
			<td>Olympus</td>
			<td>3G41067201307</td>
			<td>CX31, 10&#215;40</td>
		</tr>
		<tr>
			<td>Ortho-toluidine substrate solution</td>
			<td>ShenZhen HuaKang Biomedical Engineering Co.,LTD</td>
			<td>20230301</td>
			<td>Agentia C</td>
		</tr>
		<tr>
			<td>Pipet tips(1 mL)</td>
			<td>Coming Life Sciences (Wujiang) Co.,Ltd</td>
			<td>02923205</td>
			<td>1000 tips/unit, 5 units/case</td>
		</tr>
		<tr>
			<td>Pipet tips(10 &#181;L)</td>
			<td>Coming Life Sciences (Wujiang) Co.,Ltd</td>
			<td>00323961</td>
			<td>1000 tips/unit, 20 units/case</td>
		</tr>
		<tr>
			<td>Pipet tips(200 &#181;L)</td>
			<td>Coming Life Sciences (Wujiang) Co.,Ltd</td>
			<td>32822810</td>
			<td>1000 tips/unit, 20 units/case</td>
		</tr>
		<tr>
			<td>Saturated ammonium chloride (NH4Cl) solution</td>
			<td>ShenZhen HuaKang Biomedical Engineering Co.,LTD</td>
			<td>20230301</td>
			<td>Agentia A</td>
		</tr>
		<tr>
			<td>SCA counting chamber</td>
			<td>Microptic S.L.</td>
			<td>102230608</td>
			<td>10 &#181;m, 2 Chambers</td>
		</tr>
		<tr>
			<td>The container for semen sample</td>
			<td>Jiangsu Kangjian Medical Supplies Co., LTD</td>
			<td>20230701</td>
			<td>Polymer-based receptacle for semen sample</td>
		</tr>
		<tr>
			<td>Thermostatic table</td>
			<td>Shenzhen MIAOQUAN Instrument Co., LTD</td>
			<td>MQ30004</td>
			<td>MQ-300</td>
		</tr>
		<tr>
			<td>Transfer pipette (10 &#181;L)</td>
			<td>Eppendoff</td>
			<td>3121000.015</td>
			<td></td>
		</tr>
		<tr>
			<td>Transfer pipette (1000 &#181;L)</td>
			<td>Eppendoff</td>
			<td>3121000.12</td>
			<td></td>
		</tr>
		<tr>
			<td>Transfer pipette (200 &#181;L)</td>
			<td>Eppendoff</td>
			<td>3121000.082</td>
			<td></td>
		</tr>
		<tr>
			<td>Two-dimensional shaker</td>
			<td>DragonLab</td>
			<td>822000010000</td>
			<td>VC5A002205</td>
		</tr>
	</tbody>
</table>

the examination of peroxidase-positive leukocytes in semen

백혈구 정자증은 정자 운동성 감소, 정자 형태학적 이상 증가, 정자 DNA 단편화 지수 증가, 정자 아크로솜 기능 손상, 심지어 배아 발달에 영향을 미칠 수 있습니다. 임상에서 흔히 볼 수 있는 남성학적 질환이며 남성 불임의 중요한 원인 중 하나입니다. 남성 생식기 염증의 존재 여부를 결정할 때, 남성의학 전문의는 종종 임상 진단 근거로 정액의 원형 세포 또는 정액 혈장 엘라스타제를 검사합니다. 그러나 원형 세포의 검사는 무분별하고 불필요한 항생제 사용을 줄이는 데 기여하지 않는 정자 생성 세포와 생식관 상피 세포에 의해 쉽게 영향을 받습니다. 동시에 엘라스타제의 검출 과정은 상대적으로 복잡하고 시간이 많이 걸리며 결과 보고가 느리기 때문에 남성 생식기 감염(MGTI)과 같은 질병의 조기 진단 및 치료에 도움이 되지 않습니다. 백혈구 정자증의 진단 기준으로 CASA(Computer-Assisted Semen Analysis) 시스템을 통해 정액에서 과산화효소 양성 백혈구 검사를 혁신적으로 적용하여 이러한 문제를 성공적으로 해결하였습니다. 이 검사는 4개의 시약으로 구성된 사용 유체를 시료에 추가하기만 하면 되며 실온에서의 총 반응 시간은 20-30분 이내에 제어할 수 있습니다. 후속 도말 및 현미경 검사를 통해 총 60분 이내에 정액 내 과산화효소 양성 백혈구 농도를 얻을 수 있으며, 이는 남성 생식관의 염증 존재 여부를 진단하는 데 사용할 수 있습니다.

앞서 언급한 절차를 구현하면 원심분리 튜브의 반응물이 반투명하고 유백색 모양으로 나타나는 경우가 많습니다(그림 1). 명백한 갈색을 검출하면 백혈구의 잠재적인 탈과립이 드러나며, 이로 인해 액체 염색이 발생할 수 있습니다. 결과적으로, 염색 과정이 실패할 수 있으며, 재검사를 위해 다시 염색하거나 새로운 정액 샘플을 채취해야 할 수 있다21.
<...

Watch this Scientific Journal Video about A Simple and Cost-Effective Method for Leukocyte Analysis in Semen at JoVE.com

저자 스포트라이트: 정액의 백혈구 분석을 위한 간단하고 비용 효율적인 방법 

이 논문은 정액에서 과산화효소 양성 백혈구를 검사하기 위한 경제적이고 효율적인 프로토콜을 제시합니다. CASA(Computer-Assisted Semen Analysis) 시스템의 도움으로 정액의 과산화효소 양성 백혈구 농도를 총 60분 이내에 얻을 수 있어 남성학 실험실 및 남성학자의 효율성을 효과적으로 향상시킬 수 있습니다.

정액에서 과산화효소 양성 백혈구 검사

Leukocytospermia can lead to decreased spermatozoa motility, increased spermatozoa morphological abnormalities, elevated spermatozoa DNA fragmentation ...

남성 생식 의학은 저의 주요 연구 초점이며, 우리의 프로토콜은 주로 정액에서 과산화효소 양성 백혈구를 분석하는 속도와 비용 문제를 다룹니다. 정액은 본질적으로 이질적이어서 정액 분석 결과의 안정성에 큰 영향을 미칩니다. 따라서 검사 전에 정액을 액화하고 완전히 혼합하는 것이 중요합니다.당사의 프로토콜은 정자 농도를 활용하여 백혈구 농도를 계산하기 위해 수동 및 자동 방법을 결합하여 시간을 절약하고 즉시 계수하는 백혈구에 비해 비용 효율적입니다.

the-examination-of-peroxidase-positive-leukocytes-in-semen

Research

JoVE Journal

Medicine

The Examination of Peroxidase-Positive Leukocytes in Semen

3.6K 조회수.  The Third Affiliated Hospital of Sun Yat-sen University. 남성 생식 의학은 저의 주요 연구 초점이며, 우리의 프로토콜은 주로 정액에서 과산화효소 양성 백혈구를 분석하는 속도와 비용 문제를 다룹니다. 정액은 본질적으로 이질적이어서 정액 분석 결과의 안정성에 큰 영향을 미칩니다. 따라서 검사 전에 정액을 액화하고 완전히 혼합하는 것이 중요합니다.당사의 프로토콜은 정자 농도를 활용하여 백혈구 농도를 계산하기 위...

비디오: 저자 스포트라이트: 정액의 백혈구 분석을 위한 간단하고 비용 효율적인 방법 

Making Sense of Listening: The IMAP Test Battery

The ability to hear is only the first step towards making sense of the range of information contained in an auditory signal. Of equal importance are the abilities to extract and use the information encoded in the auditory signal. We refer to these as listening skills (or auditory processing AP). Deficits in these skills are associated with delayed language and literacy development, though the nature of the relevant deficits and their causal connection with these delays is hotly debated.
When a child is referred to a health professional with normal hearing and unexplained difficulties in listening, or associated delays in language or literacy development, they should ideally be assessed with a combination of psychoacoustic (AP) tests, suitable for children and for use in a clinic, together with cognitive tests to measure attention, working memory, IQ, and language skills. Such a detailed examination needs to be relatively short and within the technical capability of any suitably qualified professional. Current tests for the presence of AP deficits tend to be poorly constructed and inadequately validated within the normal population. They have little or no reference to the presenting symptoms of the child, and typically include a linguistic component. Poor performance may thus reflect problems with language rather than with AP. To assist in the assessment of children with listening difficulties, pediatric audiologists need a single, standardized child-appropriate test battery based on the use of language-free stimuli.
We present the IMAP test battery which was developed at the MRC Institute of Hearing Research to supplement tests currently used to investigate cases of suspected AP deficits. IMAP assesses a range of relevant auditory and cognitive skills and takes about one hour to complete. It has been standardized in 1500 normally-hearing children from across the UK, aged 6-11 years. Since its development, it has been successfully used in a number of large scale studies both in the UK and the USA. IMAP provides measures for separating out sensory from cognitive contributions to hearing. It further limits confounds due to procedural effects by presenting tests in a child-friendly game-format. Stimulus-generation, management of test protocols and control of test presentation is mediated by the IHR-STAR software platform. This provides a standardized methodology for a range of applications and ensures replicable procedures across testers. IHR-STAR provides a flexible, user-programmable environment that currently has additional applications for hearing screening, mapping cochlear implant electrodes, and academic research or teaching.

A test battery (IMAP) for performing an in-depth assessment of auditory and cognitive abilities contributing to listening skills is described. It is quick to administer, child-friendly and free from linguistic confounds. Stimulus generation and protocol management are controlled via a software platform (IHR-STAR) to ensure replicable procedures.

IMAP 테스트 배터리 : 듣기의 감각을 만들기

The ability to hear is only the first step towards making sense of the range of information contained in an auditory signal. Of equal importance are the ...

연구

의학

Ex Vivo Culture of Patient Tissue &amp; Examination of Gene Delivery

This video describes the use of patient tissue as an ex vivo model for the study of gene delivery. Fresh patient tissue obtained at the time of surgery is sliced and maintained in culture. The ex vivo model system allows for the physical delivery of genes into intact patient tissue and gene expression is analysed by bioluminescence imaging using the IVIS detection system. The bioluminescent detection system demonstrates rapid and accurate quantification of gene expression within individual slices without the need for tissue sacrifice. This slice tissue culture system may be used in a variety of tissue types including normal and malignant tissue and allows us to study the effects of the heterogeneous nature of intact tissue and the high degree of variability between individual patients. This model system could be used in certain situations as an alternative to animal models and as a complementary preclinical mode prior to entering clinical trial.

Ex Vivo Culture of Patient Tissue & Examination of Gene Delivery

This article describes the culture of patient tissue slices for gene delivery studies and subsequent analysis of gene expression using IVIS bioluminescence imaging.

환자 조직 및 유전자 전달의 시험 전의 VIVO 문화

This video describes the use of patient tissue as an ex vivo model for the study of gene delivery.  Fresh patient tissue obtained at the time of surgery ...

A Strategy to Identify de Novo Mutations in Common Disorders such as Autism and Schizophrenia

There are several lines of evidence supporting the role of de novo mutations as a mechanism for common disorders, such as autism and schizophrenia. First, the de novo mutation rate in humans is relatively high, so new mutations are generated at a high frequency in the population. However, de novo mutations have not been reported in most common diseases. Mutations in genes leading to severe diseases where there is a strong negative selection against the phenotype, such as lethality in embryonic stages or reduced reproductive fitness, will not be transmitted to multiple family members, and therefore will not be detected by linkage gene mapping or association studies. The observation of very high concordance in monozygotic twins and very low concordance in dizygotic twins also strongly supports the hypothesis that a significant fraction of cases may result from new mutations. Such is the case for diseases such as autism and schizophrenia. Second, despite reduced reproductive fitness1 and extremely variable environmental factors, the incidence of some diseases is maintained worldwide at a relatively high and constant rate. This is the case for autism and schizophrenia, with an incidence of approximately 1% worldwide. Mutational load can be thought of as a balance between selection for or against a deleterious mutation and its production by de novo mutation. Lower rates of reproduction constitute a negative selection factor that should reduce the number of mutant alleles in the population, ultimately leading to decreased disease prevalence. These selective pressures tend to be of different intensity in different environments. Nonetheless, these severe mental disorders have been maintained at a constant relatively high prevalence in the worldwide population across a wide range of cultures and countries despite a strong negative selection against them2. This is not what one would predict in diseases with reduced reproductive fitness, unless there was a high new mutation rate. Finally, the effects of paternal age: there is a significantly increased risk of the disease with increasing paternal age, which could result from the age related increase in paternal de novo mutations. This is the case for autism and schizophrenia3. The male-to-female ratio of mutation rate is estimated at about 4&#x2013;6:1, presumably due to a higher number of germ-cell divisions with age in males. Therefore, one would predict that de novo mutations would more frequently come from males, particularly older males4. A high rate of new mutations may in part explain why genetic studies have so far failed to identify many genes predisposing to complexes diseases genes, such as autism and schizophrenia, and why diseases have been identified for a mere 3% of genes in the human genome. Identification for de novo mutations as a cause of a disease requires a targeted molecular approach, which includes studying parents and affected subjects. The process for determining if the genetic basis of a disease may result in part from de novo mutations and the molecular approach to establish this link will be illustrated, using autism and schizophrenia as examples.

Molecular genetic strategy for finding de novo mutations causing common disorders such as autism and schizophrenia.

같은 자폐증과 정신 분열증과 같은 일반적인 장애에 드 노보 변이를 식별하는 전략

There are several lines of evidence supporting the role of de novo mutations as a mechanism for common disorders, such as autism and schizophrenia. First, ...

Technical Aspects of the Mouse Aortocaval Fistula

Technical aspects of creating an arteriovenous fistula in the mouse are discussed. Under general anesthesia, an abdominal incision is made, and the aorta and inferior vena cava (IVC) are exposed. The proximal infrarenal aorta and the distal aorta are dissected for clamp placement and needle puncture, respectively. Special attention is paid to avoid dissection between the aorta and the IVC. After clamping the aorta, a 25 G needle is used to puncture both walls of the aorta into the IVC. The surrounding connective tissue is used for hemostatic compression. Successful creation of the AVF will show pulsatile arterial blood flow in the IVC. Further confirmation of successful AVF can be achieved by post-operative Doppler ultrasound.

The goal is to produce an arteriovenous fistula that is simple and reproducible. This method does not use sutures or glue adhesive. Therefore the samples can be used with the least amount of foreign materials for analysis.

마우스 Aortocaval 누관의 기술적 측면

Technical aspects of creating an arteriovenous fistula in the mouse are discussed. Under general anesthesia, an abdominal incision is made, and the aorta ...

Clinical Examination Protocol to Detect Atypical and Classical Scrapie in Sheep

The diagnosis of scrapie, a transmissible spongiform encephalopathy (TSEs) of sheep and goats, is currently based on the detection of disease-associated prion protein by post mortem tests. Unless a random sample of the sheep or goat population is actively monitored for scrapie, identification of scrapie cases relies on the reporting of clinical suspects, which is dependent on the individual&#39;s familiarization with the disease and ability to recognize clinical signs associated with scrapie. Scrapie may not be considered in the differential diagnosis of neurological diseases in small ruminants, particularly in countries with low scrapie prevalence, or not recognized&#160;if it presents as nonpruritic form like atypical scrapie. To aid in the identification of clinical suspects, a short examination protocol is presented to assess the display of specific clinical signs associated with pruritic and nonpruritic forms of TSEs in sheep, which could also be applied to goats. This includes assessment of behavior, vision (by testing of the menace response), pruritus (by testing the response to scratching), and movement (with and without blindfolding). This may lead to a more detailed neurologic examination&#160;of reporting animals as scrapie suspects. It&#160;could also be used in experimental TSE studies of sheep or goats to evaluate disease progression or to identify clinical end-point.

A short clinical examination protocol is presented to aid in the detection of classical and atypical scrapie in sheep and goats.

양에서 비정형 및 고전적인 스크레이키를 감지하는 임상 시험 프로토콜

The diagnosis of scrapie, a transmissible spongiform encephalopathy (TSEs) of sheep and goats, is currently based on the detection of disease-associated ...

Collection and Extraction of Saliva DNA for Next Generation Sequencing

The preferred source of DNA in human genetics research is blood, or cell lines derived from blood, as these sources yield large quantities of high quality DNA. However, DNA extraction from saliva can yield high quality DNA with little to no degradation/fragmentation that is suitable for a variety of DNA assays without the expense of a phlebotomist and can even be acquired through the mail. However, at present, no saliva DNA collection/extraction protocols for next generation sequencing have been presented in the literature. This protocol optimizes parameters of saliva collection/storage and DNA extraction to be of sufficient quality and quantity for DNA assays with the highest standards, including microarray genotyping and next generation sequencing.

DNA extraction from saliva can provide a readily available source of high molecular weight DNA, with little to no degradation/fragmentation. This protocol provides optimized parameters for saliva collection/storage and DNA extraction to be of sufficient quality and quantity for downstream DNA assays with high quality requirements.

차세대 시퀀싱에 대한 수집 및 타액 DNA의 추출

The preferred source of DNA in human genetics research is blood, or cell lines derived from blood, as these sources yield large quantities of high quality ...

A Neuroscientific Approach to the Examination of Concussions in Student-Athletes

Concussions are occurring at alarming rates in the United States and have become a serious public health concern. The CDC estimates that 1.6 to 3.8 million concussions occur in sports and recreational activities annually. Concussion as defined by the 2013 Concussion Consensus Statement &#8220;may be caused either by a direct blow to the head, face, neck or elsewhere on the body with an &#8216;impulsive&#8217; force transmitted to the head.&#8221; Concussions leave the individual with both short- and long-term effects. The short-term effects of sport related concussions may include changes in playing ability, confusion, memory disturbance, the loss of consciousness, slowing of reaction time, loss of coordination, headaches, dizziness, vomiting, changes in sleep patterns and mood changes. These symptoms typically resolve in a matter of days. However, while some individuals recover from a single concussion rather quickly, many experience lingering effects that can last for weeks or months. The factors related to concussion susceptibility and the subsequent recovery times are not well known or understood at this time. Several factors have been suggested and they include the individual&#8217;s concussion history, the severity of the initial injury, history of migraines, history of learning disabilities, history of psychiatric comorbidities, and possibly, genetic factors. Many studies have individually investigated certain factors both the short-term and long-term effects of concussions, recovery time course, susceptibility and recovery. What has not been clearly established is an effective multifaceted approach to concussion evaluation that would yield valuable information related to the etiology, functional changes, and recovery. The purpose of this manuscript is to show one such multifaceted approached which examines concussions using computerized neurocognitive testing, event related potentials, somatosensory perceptual responses, balance assessment, gait assessment and genetic testing.

There is great variability in an individual&#8217;s risk for concussion and their corresponding recovery. A multifaceted approach to concussion evaluation is warranted; including baseline testing of athletes before participation in sport and timely evaluation post injury. The goal of this protocol is to provide an appropriate multifaceted approach to examine concussions.

학생 운동 선수에 진탕의 시험에 대한 신경 과학적 접근

Concussions are occurring at alarming rates in the United States and have become a serious public health concern. The CDC estimates that 1.6 to 3.8 ...

Diagnosis of Musculus Gastrocnemius Tightness - Key Factors for the Clinical Examination

Common foot and ankle pathologies have been linked to isolated Musculus gastrocnemius tightness (MGT). Various examination techniques have been described to assess MGT. Still, a standardized examination procedure is missing. Literature argues for weightbearing examination but the degree of knee flexion needed to eliminate the restraining effect of the M. gastrocnemius on ankle dorsiflexion (ADF) is unknown. This manuscript investigates the effect of knee flexion on ankle dorsiflexion and provides a detailed description of a standardized examination protocol. Examination on 20 healthy individuals revealed, that 20&#176; of knee flexion is sufficient to fully eliminate the influence of the M. gastrocnemius on ADF. This builds the prerequisite for a standardized examination for MGT. Non-weightbearing and weightbearing examination of ADF has to be conducted with the knee fully extended and at least 20&#176; flexed. Two investigators should conduct non-weightbearing testing with the subject in supine position. In order to obtain reliable results, the axis of the fibula should be marked. One examiner can conduct weightbearing examination with the subject in lunge stance. Isolated MGT is present if ADF is impaired with the knee fully extended and knee flexion results in a significant ADF increase. The herein presented standardized examination is the prerequisite for future studies aiming at establishing norm values.

Diagnosis of Musculus Gastrocnemius Tightness - Key Factors for the Clinical Examination

Isolated Musculus gastrocnemius tightness is a common cause for foot and ankle pathologies. Currently no standardized examination procedure exists. This manuscript demonstrates that 20 degree of knee flexion eliminates the restraining effect of the M. gastrocnemius on ankle dorsiflexion and presents a video description of a standardized examination protocol.

진단<em&gt;의 musculus 비복근</em&gt; 기밀 - 임상 시험을위한 중요한 요인

Common foot and ankle pathologies have been linked to isolated Musculus gastrocnemius tightness (MGT). Various examination techniques have been described ...

Fluorescent Dye Labeling of Erythrocytes and Leukocytes for Studying the Flow Dynamics in Mouse Retinal Circulation

The retinal and choroidal blood flow dynamics may provide insight into the pathophysiology and sequelae of various ocular diseases, such as glaucoma, diabetic retinopathy, age-related macular degeneration (AMD) and other ocular inflammatory conditions. It may also help to monitor the therapeutic responses in the eye. The proper labeling of the blood cells, coupled with live-cell imaging of the labeled cells, allows for the investigation of the flow dynamics in the retinal and choroidal circulation. Here, we describe the standardized protocols of 1.5% indocyanine green (ICG) and 1% sodium fluorescein labeling of mice erythrocytes and leukocytes, respectively. Scanning laser ophthalmoscopy (SLO) was applied to visualize the labeled cells in the retinal circulation of C57BL/6J mice (wild type). Both methods demonstrated distinct fluorescently labeled cells in the mouse retinal circulation. These labeling methods can have wider applications in various ocular disease models.

Live-cell imaging of the labeled blood cells in ocular circulation can provide information about inflammation and ischemia in diabetic retinopathy and age-related macular degeneration. A protocol to label blood cells and image the labeled cells in the retinal circulation is described.

마우스 망막 순환에서 유동 역학을 연구하기위한 적혈구 및 백혈구의 형광 염료 표지

The retinal and choroidal blood flow dynamics may provide insight into the pathophysiology and sequelae of various ocular diseases, such as glaucoma, ...

Echocardiographic and Histological Examination of Cardiac Morphology in the Mouse

An increasing number of genetically modified mouse models has become available in recent years. Moreover, the number of pharmacological studies performed in mice is high. Phenotypic characterization of these mouse models also requires the examination of cardiac function and morphology. Echocardiography and magnetic resonance imaging (MRI) are commonly used approaches to characterize cardiac function and morphology in mice. Echocardiographic and MRI equipment specialized for use in small rodents is&#160;expensive and requires a dedicated space. This protocol describes cardiac measurements in mice using a clinical echocardiographic system with a 15 MHz human vascular probe. Measurements are performed on anesthetized adult mice. At least three image sequences are recorded and analyzed for each animal in M-mode in the parasternal short-axis view. Afterwards, cardiac histological examination is performed, and cardiomyocyte diameters are determined on hematoxylin-eosin- or wheat germ agglutinin (WGA)-stained paraffin sections. Vessel density is determined morphometrically after Pecam-1 immunostaining. The protocol has been applied successfully to pharmacological studies and different genetic animal models under baseline conditions, as well as after experimental myocardial infarction by the permanent ligation of the left anterior descending coronary artery (LAD). In our experience, echocardiographic investigation is limited to anesthetized animals and is feasible in adult mice weighing at least 25 g.

Echocardiographic examination is frequently used in mice. Expensive high-resolution ultrasound devices have been developed for this purpose. This protocol describes an affordable echocardiographic procedure combined with histological morphometric analyses to determine cardiac morphology.

마우스의 심장 형태학의 echocardiographic 및 조직학 검사

An increasing number of genetically modified mouse models has become available in recent years. Moreover, the number of pharmacological studies performed ...