The authors are grateful to the Andrology Laboratory of the Center for Reproductive Medicine, the Third Affiliated Hospital of Sun Yat-sen University, for providing the facilities for this study. No funding was involved in the preparation of this article.

This study has been reviewed and approved by the Medical Ethics Committee of The Third Affiliated Hospital of Sun Yat-sen University.
1. Preparation of the working solution
<ol>
	<li>Gather the following reagents essential for preparing the working solution- ortho-toluidine substrate solution, saturated ammonium chloride (NH4Cl) solution, 148 mmol/L ethylenediaminetetraacetic acid disodium salt (Na2EDTA) solution, and 6% (v/v) hydrogen peroxide solution. Place them on a room temperature (RT) lab bench along with the necessary tools, such as transfer pipettes (1000 &#181;L, 200 &#181;L, 10 &#181;L) and test tube racks. 
	CAUTION: Read the safety data sheet (SDS) before handling the above-mentioned reagents.</li>
	<li>Prepare an opaque brown reagent bottle to store and protect the prepared working solution from exposure to light.</li>
	<li>Transfer the reagents into the opaque brown reagent bottle as per the proportions specified below:
	<ol>
		<li>Utilize the 200 &#181;L transfer pipette and adjust the scale to 100 &#181;L by turning the knob. Then, transfer 100 &#181;L of the NH4Cl solution into the opaque brown reagent bottle.</li>
		<li>Replace the pipette tip and transfer 100 &#181;L of the 148 mmol/L Na2EDTA solution into the opaque brown reagent bottle using the 200 &#181;L transfer pipette mentioned above. Then, gently blow by pipetting and thoroughly homogenize the solution.</li>
		<li>Utilize the 1000 &#181;L transfer pipette and adjust the scale to 900 &#181;L by turning the knob. Then, transfer 900 &#181;L of the ortho-toluidine substrate solution into the opaque brown reagent bottle, gently blow by pipetting, and thoroughly homogenize the solution.</li>
		<li>Utilize the 10 &#181;L transfer pipette and adjust the scale to 5 &#181;L by turning the knob. Then, transfer 5 &#181;L of the 6% (v/v) hydrogen peroxide solution into the opaque brown reagent bottle.</li>
	</ol>
	</li>
	<li>Utilize the 1000 &#181;L transfer pipette and adjust the scale to 1000 &#181;L by turning the knob. Replace the pipette tip, gently blow by pipetting, and thoroughly homogenize the solution in the opaque brown reagent bottle.</li>
	<li>Store the ready-to-utilize working solution in the opaque brown reagent bottle at RT, away from light. The working solution is reactive for 24 h.</li>
	<li>Each semen sample requires 900 &#181;L of the working solution. Ensure that the quantity of the working solution prepared each day corresponds to the number of samples examined daily.</li>
</ol>
2. Preparation of semen sample
<ol>
	<li>Before collecting the semen sample, ensure the patient has abstained from ejaculation for 2 to 7 days as required by the World Health Organization (WHO) laboratory manual for examining and processing human semen (5th ed.)8. Masturbation is the preferred method for obtaining semen samples. To improve the accuracy of the examination, instruct the patient to collect all ejaculated semen.</li>
	<li>Turn on the constant temperature stand and heat up to 37 &#176;C in advance so that the subsequent liquefaction of the semen sample can proceed smoothly.</li>
	<li>Upon receipt of the patient&#39;s semen sample, meticulously record detailed information, including identification code, collection time, abstinence duration, and other pertinent information, on the outer wall of the polymer-based receptacle.</li>
	<li>Place the polymer-based receptacle housing the semen sample on the constant temperature stand preheated to 37 &#176;C, which is beneficial for the semen liquefaction.</li>
	<li>Draw the semen sample into a polymer-based pipette and observe the state of liquefaction every 5 min until the semen sample is fully liquefied.</li>
	<li>If the semen sample has not been fully liquefied within 30 min, do not perform the examination yet; continue to wait for another 30 min. If the semen is still not fully liquefied after 60 min, add an equal volume of phosphate buffer solution for a 1:1 dilution and gently agitate to promote liquefaction.</li>
	<li>After thorough agitation and consistent homogenizing, retain the fully liquefied semen sample for the subsequent examination procedures.</li>
</ol>
3. Staining of the peroxidase-positive leukocytes and analyzing the original semen sample
<ol>
	<li>Prior to sampling, utilize a polymer-based pipette to repeatedly blow by pipetting and thoroughly homogenize the semen sample under examination.</li>
	<li>Utilize the 200 &#181;L transfer pipette and adjust the scale to 100 &#181;L by turning the knob. Replace the pipette tip, and then transfer 100 &#181;L of the semen sample into a centrifuge tube.</li>
	<li>Utilize the 1000 &#181;L transfer pipette and adjust the scale to 900 &#181;L by turning the knob. Replace the pipette tip, then transfer 900 &#181;L of the above-mentioned working solution into the centrifuge tube.</li>
	<li>Repeatedly blow by pipetting and thoroughly homogenize the reactant, which consists of the semen sample and working solution, using the 1000 &#181;L transfer pipette.</li>
	<li>Position the centrifuge tube into a test-tube rack upon a two-dimensional shaker and subject it to continuous agitation at 200 rpm for 30 min at RT.</li>
	<li>While waiting for the staining reaction, analyze the semen sample utilizing the CASA system to determine the concentration of spermatozoa in the original semen sample in the polymer-based receptacle.
	<ol>
		<li>Turn on the power switch of the CASA system and double-click the SCA SCOPE icon to open the application software.</li>
		<li>Before sampling, repeatedly blow by pipetting and thoroughly homogenize the original semen sample again with a polymer-based pipette.</li>
		<li>Utilize the 10 &#181;L transfer pipette and adjust the scale to 10 &#181;L by turning the knob. Replace the pipette tip, pipette up 10 &#181;L of the original semen sample, and place it into the SCA counting chamber. Wait for 60 s for the semen sample to stop drifting.</li>
		<li>Position the SCA counting chamber onto the sample holder of the CASA system and set the patient&#39;s information in the dialog box of the application software to prepare for the formal examination.</li>
		<li>Examine the original semen sample in the SCA counting chamber with the CASA system by clicking the Start button and record the necessary data, such as the concentration of spermatozoa.</li>
	</ol>
	</li>
</ol>
4. Observation of the peroxidase-positive leukocytes and spermatozoa
<ol>
	<li>Utilize the 1000 &#181;L transfer pipette and adjust the scale to 1000 &#181;L by turning the knob. Replace the pipette tip, then repeatedly blow by pipetting and thoroughly homogenize the reactant, which consists of semen sample and working solution in the centrifuge tube again at the end of the 30 min reaction process.</li>
	<li>Utilize the 10 &#181;L transfer pipette and adjust the scale to 10 &#181;L by turning the knob. Replace the pipette tip, pipette up 10 &#181;L of the reactant mentioned above, and place it into the sperm counting chamber. Finally, cover the sperm counting chamber with a dedicated glass coverslip.</li>
	<li>Place the sperm counting chamber onto the microscopic stage and turn on the power switch of the optical microscope with two 10x eyepieces. Then, wait for 60 s for the reactant sample to stop drifting.</li>
	<li>Switch the microscopic converter to the 10x objective lens during the waiting time. Then adjust the coarse adjustment knob and the fine adjustment knob in sequence. Finally, observe the spermatozoa and round cells under a magnification of 10&#215;10.</li>
	<li>Switch the microscopic converter to the 40x objective lens after determining the field of view to be analyzed, and observe the spermatozoa and brown peroxidase-positive leukocytes under a magnification of 10 &#215; 40.</li>
	<li>Utilize the electronic counter system to count at least 200 spermatozoa and the number of brown peroxidase-positive leukocytes within the corresponding fields.</li>
</ol>
5. Calculation of the concentration of peroxidase-positive leukocytes
<ol>
	<li>Calculate the concentration of peroxidase-positive leukocytes through the using the following formula: 
	<img alt="Equation 1" src="/files/ftp_upload/66211/66211eq01v2.jpg" style="margin: auto;" /></li>
</ol>

Analysis of Semen for Peroxidase-Positive Leukocyte Cells

<ol>
	<li>Agarwal, A., Mulgund, A., Hamada, A., Chyatte, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+unique+view+on+male+infertility+around+the+globe.">A unique view on male infertility around the globe.</a> Reprod Biol Endocrinol. 13, 37 (2015).</li><li>Sharlip, I. D., 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=Best+practice+policies+for+male+infertility.">Best practice policies for male infertility.</a> Fertil Steril. 77 (5), 873-882 (2002).</li><li>Choy, J. T., Eisenberg, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Male+infertility+as+a+window+to+health.">Male infertility as a window to health.</a> Fertil Steril. 110 (5), 810-814 (2018).</li><li>Pellati, D., 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=Genital+tract+infections+and+infertility.">Genital tract infections and infertility.</a> Eur J Obstet Gynecol Reprod Biol. 140 (1), 3-11 (2008).</li><li>Rivero, M. J., Kulkarni, N., Thirumavalavan, N., Ramasamy, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+and+management+of+male+genital+tract+infections+in+the+setting+of+male+infertility:+an+updated+review.">Evaluation and management of male genital tract infections in the setting of male infertility: an updated review.</a> Curr Opin Urol. 33 (3), 180-186 (2023).</li><li>Long, S., Kenworthy, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Round+cells+in+diagnostic+semen+analysis:+A+guide+for+laboratories+and+clinicians.">Round cells in diagnostic semen analysis: A guide for laboratories and clinicians.</a> Br J Biomed Sci. 79, 10129 (2022).</li><li>Johanisson, E., Campana, A., Luthi, R., de Agostini, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+'round+cells'+in+semen+analysis:+a+comparative+study.">Evaluation of 'round cells' in semen analysis: a comparative study.</a> Hum Reprod Update. 6 (4), 404-412 (2000).</li><li>World Health Organization. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=WHO+Laboratory+Manual+for+the+Examination+and+Processing+of+Human+Semen.+5th+edn.">WHO Laboratory Manual for the Examination and Processing of Human Semen. 5th edn.</a> World Health Organization. , (2010).</li><li>Domes, T., 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=The+incidence+and+effect+of+bacteriospermia+and+elevated+seminal+leukocytes+on+semen+parameters.">The incidence and effect of bacteriospermia and elevated seminal leukocytes on semen parameters.</a> Fertil Steril. 97 (5), 1050-1055 (2012).</li><li>Aziz, N., Agarwal, A., Lewis-Jones, I., Sharma, R. K., Thomas, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+associations+between+specific+sperm+morphological+defects+and+leukocytospermia.">Novel associations between specific sperm morphological defects and leukocytospermia.</a> Fertil Steril. 82 (3), 621-627 (2004).</li><li>Alahmar, A. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+oxidative+stress+in+male+infertility:+An+updated+review.">Role of oxidative stress in male infertility: An updated review.</a> J Hum Reprod Sci. 12 (1), 4-18 (2019).</li><li>Schlegel, P. N., 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=Diagnosis+and+treatment+of+infertility+in+men:+AUA/ASRM+guideline+Part+I.">Diagnosis and treatment of infertility in men: AUA/ASRM guideline Part I.</a> J Urol. 205 (1), 36-43 (2021).</li><li>Ricci, G., Presani, G., Guaschino, S., Simeone, R., Perticarari, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Leukocyte+detection+in+human+semen+using+flow+cytometry.">Leukocyte detection in human semen using flow cytometry.</a> Hum Reprod. 15 (6), 1329-1337 (2000).</li><li>Brunner, R. J., Demeter, J. H., Sindhwani, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Review+of+guidelines+for+the+evaluation+and+treatment+of+leukocytospermia+in+male+infertility.">Review of guidelines for the evaluation and treatment of leukocytospermia in male infertility.</a> World J Mens Health. 37 (2), 128-137 (2019).</li><li>Amulic, B., Cazalet, C., Hayes, G. L., Metzler, K. D., Zychlinsky, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neutrophil+function:+from+mechanisms+to+disease.">Neutrophil function: from mechanisms to disease.</a> Annu Rev Immunol. 30, 459-489 (2012).</li><li>Khan, A. A., Alsahli, M. A., Rahmani, A. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myeloperoxidase+as+an+active+disease+biomarker:+recent+biochemical+and+pathological+perspectives.">Myeloperoxidase as an active disease biomarker: recent biochemical and pathological perspectives.</a> Med Sci (Basel). 6 (2), 33 (2018).</li><li>Khan, A. A., Rahmani, A. H., Aldebasi, Y. H., Aly, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biochemical+and+pathological+studies+on+peroxidases+-an+updated+review).">Biochemical and pathological studies on peroxidases -an updated review).</a> Glob J Health Sci. 6 (5), 87-98 (2014).</li><li>Liu, W. 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>

The authors have nothing to disclose.

Ortho-toluidine and hydrogen peroxide have photosensitive properties and can decompose when exposed to light. To ensure the effectiveness of the test reagents, it is recommended that the prepared working fluid be stored in a dark place. 
Semen samples naturally exhibit heterogeneous characteristics. In the preparation process of semen samples, correctly judging the liquefaction state of semen and thoroughly homogenizing the semen samples before each sampling are crucial for improving the relia...

Infertility has emerged as a global public health issue affecting approximately 15% of couples in their reproductive age. Male factors are contributed by around 50% of total subfertility cases, and nearly 20% to 30% can be attributed solely to male factors1,2,3. Male genital tract infections (MGTIs) constitute one of the significant causes of male infertility, accounting for about 15% of cases4,5.
Most individuals have leukocytes in their semen, constituting 13% of non-spermatozoan cells, with differential proportions being neutrophils 12%, macrophages 0.9%, and lymphocytes 0.1%6,7. According to the World Health Organization (WHO) laboratory manual for the examination and processing of human semen (5th ed.), leukocytospermia is usually defined as the presence of &#62;1 &#215; 106 cells/mL leucocytes in semen8. This condition can be caused by numerous factors such as environmental toxins, substance abuse, varicoceles, MGTIs, etc., all potentially leading to an abnormal increase in leukocyte concentration in semen9. Leukocytes can elevate reactive oxygen species (ROS) levels in semen, causing lipid peroxidation and oxidative damage to protein and DNA, which results in decreased spermatozoa motility, increased spermatozoa morphological abnormalities, elevated spermatozoa DNA fragmentation index, impairment of the spermatozoa acrosome function, and even negative impacts on embryo development10,11.
Currently, andrologists commonly choose to examine round cells in semen or elastase in seminal plasma when identifying genital tract inflammation. However, it&#39;s often challenging to distinguish sloughed spermatogenic cells and reproductive tract epithelial cells, which do not contribute to reducing the indiscriminate and unnecessary use of antibiotics6. The latter examination method is relatively complicated and time-consuming, with slow result reporting, which is not beneficial for early diagnosis and treatment of diseases such as MGTIs. The American Urological Association (AUA) suggests further differentiation between leukocytes and sloughed cells from the genital tract should be made when the round cell concentration in semen analysis is greater than 1 &#215; 106 cells/mL12. Some andrology labs utilize flow cytometry13 or leukocyte antigen (e.g., CD45) immunocytochemistry14 to examine leukocytes. While these methods are precise, they are expensive and time-consuming, making large-scale clinical implementation difficult, especially in developing countries.
Peroxidase is widely distributed in various types of cells and plays a crucial role in the resistance to oxidative stress damage. Myeloperoxidase (MPO) is a member of the peroxidase subfamily that is generally expressed in immune cells. It is most highly expressed in neutrophils&#39; azurophilic granules15,16 and is also expressed in lymphocytes17,18, monocytes, and macrophages19. The concentration of leukocytes, especially neutrophils, in semen can be obtained by examining round cells that are peroxidase-positive7,20. To make the examination process of leukocytes in semen economical, convenient, and efficient, we reengineered the examination method. Assisted by the computer-assisted semen analysis (CASA) system, the concentration of leukocytes can be obtained within 60 min. This new method reduces the patients&#39; examination costs and waiting time for obtaining results, alleviates the workload of lab technicians, and shortens the doctor&#39;s diagnosis and treatment waiting period.

<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

Leukocytospermia can lead to decreased spermatozoa motility, increased spermatozoa morphological abnormalities, elevated spermatozoa DNA fragmentation index, impairment of the spermatozoa acrosome function, and even affected embryonic development. It is a common andrological disease in clinical practice and one of the important causes of male infertility. When determining whether male reproductive tract inflammation exists, andrologists often choose to examine round cells or seminal plasma elastase in the semen as a clinical diagnostic basis. However, the examination of round cells is easily influenced by sloughed spermatogenic cells and reproductive tract epithelial cells, which do not contribute to reducing the indiscriminate and unnecessary use of antibiotics. At the same time, the detection process of elastase is relatively complicated, time-consuming, and slow in reporting results, which is not beneficial for early diagnosis and treatment of diseases such as male genital tract infections (MGTIs). We have innovatively applied the examination of peroxidase-positive leukocytes in semen assisted by a computer-assisted semen analysis (CASA) system as a diagnostic criterion for leukocytospermia, successfully solving these problems. This examination only requires the addition of the operating fluid consisting of four reagents into the specimen, and the total reaction time at room temperature can be controlled within 20-30 min. With the subsequent smear and microscopic examination, the concentration of peroxidase-positive leukocytes in semen can be obtained within a total of 60 min, which can be used to diagnose whether the inflammation of the male reproductive tract existed.

Upon implementing the aforementioned procedure, the reactant in the centrifuge tube frequently manifests a subtranslucent, opalescent appearance (Figure 1). Detecting an obviously brown color discloses potential degranulation of leukocytes, which may result in liquid staining. Consequently, the staining process could fail, rendering it necessary to either re-stain or collect a new semen sample for retesting21.
Peroxidase-positive leukocytes...

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

Author Spotlight: A Simple and Cost-Effective Method for Leukocyte Analysis in Semen 

This paper presents an economical and efficient protocol for examining peroxidase-positive leukocytes in semen. With the assistance of a computer-assisted semen analysis (CASA) system, the concentration of peroxidase-positive leukocytes in semen can be obtained within a total of 60 min, effectively improving the efficiency of andrology laboratory and andrologists.

The Examination of Peroxidase-Positive Leukocytes in Semen

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

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Research

JoVE Journal

Medicine

3.4K Views.  The Third Affiliated Hospital of Sun Yat-sen University. This paper presents an economical and efficient protocol for examining peroxidase-positive leukocytes in semen. With the assistance of a computer-assisted semen analysis (CASA) system, the concentration of peroxidase-positive leukocytes in semen can be obtained within a total of 60 min, effectively improving the efficiency of andrology laboratory and andrologists.

Video: Author Spotlight: A Simple and Cost-Effective Method for Leukocyte Analysis in Semen 

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.

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.

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.

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.

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.

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.

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.

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

Observational Study Protocol for Repeated Clinical Examination and Critical Care Ultrasonography Within the Simple Intensive Care Studies

Longitudinal evaluations of critically ill patients by combinations of clinical examination, biochemical analysis and critical care ultrasonography (CCUS) may detect adverse events of interventions such as fluid overload at an early stage. The Simple Intensive Care Studies (SICS) is a research line that focuses on the prognostic and diagnostic value of combinations of clinical variables.
The SICS-I specifically focused on the use of clinical variables obtained within 24 h of acute admission for prediction of cardiac output (CO) and mortality. Its sequel, SICS-II, focuses on repeated evaluations during ICU admission. The first clinical examination by trained researchers is performed within 3 h after admission consisting of physical examination and educated guessing. The second clinical examination is performed within 24 h after admission and includes physical examination and educated guessing, biochemical analysis and CCUS assessments of heart, lungs, inferior vena cava (IVC) and kidney. This evaluation is repeated at days 3 and 5 after admission. CCUS images are validated by an independent expert, and all data is registered in an online secured database. Follow-up at 90 days includes registration of complications and survival status according to patient&#39;s medical charts and the municipal person registry. The primary focus of SICS-II is the association between venous congestion and organ dysfunction.
The purpose of publishing this protocol is to provide details on the structure and methods of this on-going prospective observational cohort study allowing answering multiple research questions. The design of the data collection of combined clinical examination and CCUS assessments in critically ill patients are explicated. The SICS-II is open for other centers to participate and is open for other research questions that can be answered with our data.

Structured protocols are necessary to provide answers on research questions in critically ill patients. The Simple Intensive Care Studies (SICS) provides an infrastructure for repeated measurements in critically ill patients including clinical examination, biochemical analysis and ultrasonography. SICS projects have specific focus but the structure is flexible to other investigations.

Longitudinal evaluations of critically ill patients by combinations of clinical examination, biochemical analysis and critical care ultrasonography (CCUS) ...

Transthoracic Echocardiographic Examination in the Rabbit Model

Large animal models such as the rabbit are valuable for translational preclinical research. Rabbits have a similar cardiac electrophysiology compared to that of humans and that of other large animal models such as dogs and pigs. However, the rabbit model has the additional advantage of lower maintenance costs compared to other large animal models. The longitudinal evaluation of cardiac function using echocardiography, when appropriately implemented, is a useful methodology for preclinical assessment of novel therapies for heart failure with reduced ejection fraction (e.g. cardiac regeneration). The correct use of this non-invasive tool requires the implementation of a standardized examination protocol following international guidelines. Here we describe, step by step, a detailed protocol supervised by veterinary cardiologists for performing echocardiography in the rabbit model, and demonstrate how to correctly obtain the different echocardiographic views and imaging planes, as well as the different imaging modes available in a clinical echocardiography system routinely used in human and veterinary patients.

Here we describe, step by step, a detailed protocol for performing echocardiography in the rabbit model. We show how to correctly obtain the different echocardiographic views and imaging planes, as well as the different imaging modes available in a clinical echocardiography system routinely used in human and veterinary patients.

Large animal models such as the rabbit are valuable for translational preclinical research. Rabbits have a similar cardiac electrophysiology compared to ...

Evaluation of a Point-of-Care Testing Analyzer for Measuring Peripheral Blood Leukocytes

White blood cell (WBC) is an important indicator of inflammation in the body, and it can help distinguish between bacterial and viral infections. At present, most primary medical institutions in China have a poor percentage of adoption of blood-testing technology, and a hematology detection system with a high price to performance ratio and easy operation is urgently needed in primary healthcare centers. This paper introduces the principle and operation procedures of a point-of-care testing (POCT) card-based leukocyte analyzer (evaluated system), which was used to detect WBC indexes such as neutrophils, lymphocytes, and intermediate group cells (including eosinophils, basophils, and monocytes) in whole blood. The results from the evaluated system were compared to those from two commercial automatic hematology analyzers (reference system). The correlation and consistency between the evaluated system and the commercial reference systems were analyzed. The results showed that WBC count and number of granulocytes detected by the evaluated and reference systems showed a strong positive correlation (rs = 0.972 and 0.973, respectively), while the number of lymphocytes showed a relatively low correlation (rs = 0.851). A Bland-Altman plot showed that the major difference between the values detected by the evaluated system and the reference systems is within 95% limits of agreement (LoA), indicating that the two systems are in good agreement. In conclusion, the evaluated system has an excellent correlation, robust consistency, and a reliable comparison with the results of the widely used automatic hematology analyzers. It is ideal for WBC detection in primary medical institutions where a full-automatic five-category hematology analyzer is unavailable, especially during the COVID-19 normalized prevention and control period.

A protocol to measure peripheral blood leukocytes using a POCT card-based leukocyte analyzer is presented here. Same blood samples were tested by two automated hematology analyzers to evaluate the consistency and accuracy of the results. The results showed that the evaluated analyzer had a good correlation with the reference system.

White blood cell (WBC) is an important indicator of inflammation in the body, and it can help distinguish between bacterial and viral infections. At ...