Name: Murine Nasal Lavage Fluid Collection without Blood Contamination (Video) | JoVE Journal
Uploaded: 2025-07-11T15:10:00.000+00:00
Duration: 5 min 12 s
Description: Learn a novel method for collecting murine nasal lavage fluid without blood contamination using pharyngeal lavage and cotton ball placement. This technique improves immunological accuracy and respiratory pathogen detection.

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5 minutes

We thank Seiichi Omura, Ph.D., Cong Thanh Nguyen, M.D., Ph.D., Kota Moriguchi, M.D., Ph.D., Reona Shiro, M.D., and Sandesh Rimal, M.S., Department of Microbiology, Kindai University Faculty of Medicine, and Hirotaka Ebina, Ph.D., Shinya Okamura, Ph.D., and Yasuo Yoshioka, Ph.D., Virus Vaccine Group, BIKEN Innovative Vaccine Research Alliance Laboratories, Research Institute for Microbial Diseases, Osaka University, for helpful discussions. We also express our gratitude to the members of the Life Science Research Institute, Kindai University, for their excellent technical assistance. This work was supported by the Ministry of Education, Culture, Sports, Science and Technology, Japan, through the Monbukagakusho (MEXT, 2021-2025) Scholarship (I.A.), Grant-in-Aid for Scientific Research KAKENHI from the Japan Society for the Promotion of Science (JSPS) JP23K06493 (F.S.), JP21K07287/JP24K10500 (A.-M. P.), and JP22K18378/JP24K10163 (I.T.), and Kindai University Research Enhancement Grant KD2406 (F.S.) and KD2506 (I.T.).

All experimental procedures were approved by the Institutional Animal Care and Use Committee (IACUC) of Kindai University Faculty of Medicine and performed according to the criteria outlined by the National Institutes of Health (NIH)20. 4-month-old male C57BL/6 mice were used in this study. Information on reagents and equipment used in this study are listed in the Table of Materials.
1. NLF collection
<ol>
	<li>Euthanize a mouse with 5% isoflurane21 following institutionally approved protocols.</li>
	<li>Place the mouse on its back on a surgical plate and fix it by pinning down all four limbs.</li>
	<li>Take blood from the heart using a 1 mL syringe.</li>
	<li>Open the abdominal cavity using scissors to expose the visceral organs and diaphragm.</li>
	<li>Incise the diaphragm and ribs using scissors to make the right atrium visible.</li>
	<li>Puncture the right atrium using scissors to remove blood further.</li>
	<li>Place three cotton balls into the mouth of the animal while pulling the tongue.</li>
	<li>Separate the lower jaw using scissors while raising the head (nose-up position) to inhibit blood contamination in NLF.</li>
	<li>Replace the cotton balls with new ones to avoid blood contamination.</li>
	<li>After bleeding has stopped, inject 200 &#181;L of sterile PBS into the choana and collect NLF ejected from the nostrils into a 1.5 mL tube.</li>
	<li>Repeat the step 1.10 once more.</li>
</ol>
2. Detection of blood contamination by luminol chemiluminescence
<ol>
	<li>Dissolve 1 mg of luminol and 5 mg of sodium peroxide (Na2O2) with 1 mL of sterile water using a 1.5 mL tube to make the stock solution. 
	NOTE: The final concentration is 1 mg/mL luminol and 5 mg/mL Na2O2.</li>
	<li>Cover the tube with aluminum foil and store it at 4 &#176;C until use.</li>
	<li>Dilute the stock solution 10-fold with deionized water to make a working solution. 
	NOTE: The final concentration is 0.1 mg/mL luminol and 0.5 mg/mL Na2O2.</li>
	<li>Pipette 2 &#181;L of the NLF samples into wells of a 96-well plate.</li>
	<li>Add 100 &#181;L of the diluted luminol solution to each well.</li>
	<li>Briefly shake the 96-well plate to mix the samples with the luminol solution.</li>
	<li>Directly observe luminol chemiluminescence in the dark.</li>
	<li>Semi-quantify the Hb concentration using a luminometer, if necessary.</li>
</ol>
3. Quantification of IgA and IgG by ELISAs
<ol>
	<li>Coat wells of 96-well plates with 100 &#181;L/well of a total anti-mouse IgA (20 ng/well; 200 ng/mL) or IgG (10 ng/well; 100 ng/mL) antibody&#160;(see Table of Materials).</li>
	<li>Seal the plates and incubate them at 4 &#176;C overnight.</li>
	<li>Wash the plates three times with 300 &#956;L/well of&#160;a washing buffer containing 0.05% Tween 20 in&#160;PBS each time, and then blot the plates on paper towels.</li>
	<li>Add 200 &#181;L of an assay diluent containing 10% fetal bovine serum (FBS) and 0.2% Tween 20 in PBS to each well.</li>
	<li>Incubate the plates at room temperature (RT) for 60 min.</li>
	<li>Aspirate the assay diluent and then blot the plates on paper towels.</li>
	<li>Prepare 100 ng/mL of a mouse IgA or IgG standard with the assay diluent and make two-fold serial dilutions: 50 ng/mL, 25 ng/mL, 12.5 ng/mL, 6.25 ng/mL, 3.13 ng/mL, 1.56 ng/mL, and 0.78 ng/mL. 
	NOTE: The required volume of each standard is 100 &#181;L per well.</li>
	<li>Dilute the samples with the assay diluent at the appropriate dilutions: serum (104 to 106), NLF (10, 50, 200, and 500), and BALF (101 to 104). 
	NOTE: The required volume of each sample is 100 &#181;L per well.</li>
	<li>Add 100 &#181;L of IgA or IgG standards and samples to the appropriate wells.</li>
	<li>Incubate the plates at RT for 75 min.</li>
	<li>Wash the plates three times with 300 &#181;L/well of the washing buffer each time, and then blot the plates on paper towels.</li>
	<li>Dilute a peroxidase-conjugated anti-mouse F(ab&#39;)2 antibody (see Table of Materials) with the assay diluent at the appropriate dilution: 104-fold dilution. 
	NOTE: The required volume is 100 &#181;L per well.</li>
	<li>Add 100 &#181;L of the diluted peroxidase-conjugated anti-mouse F(ab&#39;)2 antibody to each well.</li>
	<li>Incubate the plates at RT for 75 min.</li>
	<li>Wash the plates five times with 300 &#181;L/well of the washing buffer each time, and then blot the plates on paper towers.</li>
	<li>Add 100 &#181;L of a substrate solution containing tetramethylbenzidine (TMB) and hydrogen peroxide to each well.</li>
	<li>Incubate the plates at RT for 5 min.</li>
	<li>Add 50 &#181;L of a stop solution, 2 N sulfuric acid (2N H2SO4), to each well.</li>
	<li>Measure the absorbance at 450 nm using a microplate reader.</li>
	<li>Determine the concentrations of IgA and IgG in each sample by utilizing the standard curves.</li>
</ol>

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</ol>

The authors have nothing to disclose.

Nasal lavage is a valuable technique for acquiring biochemical, cytological, and biological information in respiratory diseases, such as influenza and COVID-1923. Besides identifying various infiltrating cells, the non-cellular composition of the respiratory tract can be examined from the NLF samples using various methods, such as ELISAs, immunoblot, and quantitative polymerase chain reaction1,19,24. Here...

In both clinical and basic research, appropriate sample collection methods are most crucial to evaluating the results. Nasal lavage is an irrigation technique used to analyze the cellular and humoral components in the respiratory tract1,2,3. Clinically, nasal lavage fluid (NLF) has been used as an effective noninvasive sample for biomarker discovery, with several studies supporting its use across various respiratory diseases, including sinusitis, cystic fibrosis, and general respiratory disorders4,5,6,7. In experimental animals, NLF has also been used to study respiratory disease models, including influenza, coronavirus disease-2019 (COVID-19), and allergic disease3,8,9. This method is widely used to determine key inflammatory biomarkers, such as polymorphonuclear cells and cytokines, which are often examined as indicators of responses to allergens, asthma, and respiratory infections10,11,12. More recently, to investigate the efficacy and safety of potential intranasal vaccine candidates, NLF samples have often been collected from experimental animals, and the levels of pathogen and immunoglobulin (Ig) in NLF samples have been quantified as indicators13.
In experimental mice, NLF can be collected using the trans-pharyngeal or trans-tracheal method. In the trans-pharyngeal method, the murine head, including the palatopharyngeal region, was separated from the larynx, and then a catheter was inserted through the pharyngeal opening into the choana to inject phosphate-buffered saline (PBS) and collect NLF from the nostril14. The trans-tracheal method gained access to the respiratory tract through the trachea without severing the head, and NLF was collected from the nostril, minimizing the risk of blood contamination. Although these two primary methods have been employed for NLF collection in experimental mice, both conventional methods have their own limitations. The trans-tracheal method has been demonstrated to yield lower amounts of NLF than the trans-pharyngeal method due to the leakage of trans-tracheal lavage fluid into the oral cavity. The previous study showed that, following injection of PBS, the recovered fluid volumes in NLF were 70%-80% and 90%-100% in the trans-tracheal and trans-pharyngeal methods, respectively14. Although the trans-pharyngeal method can be considered better with larger amounts of NLF than the trans-tracheal method, blood contamination during the procedure was inevitable to some extent14,15. Similarly, several studies have reported that NLF samples were often contaminated with high levels of blood serum proteins16,17,18. This can interfere with the detection and quantification of NLF-specific biomarkers for respiratory diseases19. This may also affect the concentrations of Ig, particularly IgA and IgG, in NLF samples since blood contains larger amounts of IgA and IgG than NLF.
To address these challenges, this study aimed to establish a unique, simple, and reliable trans-pharyngeal method for collecting NLF samples from experimental mice, maximizing sample yield and minimizing blood contamination. During the experimental processes, cotton balls were placed into the mouth of the mouse to inhibit blood contamination, and then PBS was injected to collect NLF samples from the nostril. The levels of blood contamination in NLF were determined using a forensic technique, luminol-based chemiluminescence, that enables semi-quantification of hemoglobin (Hb) levels in NLF samples; the luminol reaction in NLF collected by the novel method was negative. Thus, the current novel method can improve the NLF sample quality for various biological experiments, such as antibody enzyme-linked immunosorbent assays (ELISAs), cytokine profiles, immunological and medical biomarker determinations, and murine respiratory disease models.

<table><tbody><tr><td>Animal fixation needle</td><td>Natsume Seisakusho Co., Ltd., Tokyo, Japan</td><td>KN-358-A</td><td>We fixed a mouse with animal fixation needles. Stainless steel; total length, 23 mm; and needle tip, 15 mm.</td></tr><tr><td>BD OptEIA&lt;sup&gt;TM&lt;/sup&gt;&amp;nbsp;TMB Substrate Reagent Set</td><td>BD Biosciences, San Diego, CA, USA</td><td>555214</td><td>We added 100 &amp;mu;L of TMB substrate solution per well.</td></tr><tr><td>Bell Jar</td><td>NA</td><td>NA</td><td>We used a bell jar for anesthesia.</td></tr><tr><td>Clear Flat-Bottom Immuno Nonsterile 96-Well Plates&amp;nbsp;</td><td>ThermoFisher Scientific, Rochester, NY, USA</td><td>442404</td><td>We used Clear Flat-Bottom Immuno Nonsterile 96-Well Plates with MaxiSorp surface treatment for enzyme-linked immunosorbent assays.</td></tr><tr><td>Cotton ball</td><td>Sansho Co., Ltd., Tokyo, Japan</td><td>94-1951</td><td>We used cottons balls to avoid blood contamination. The diameter of cotton balls was 7 mm.</td></tr><tr><td>Fetal bovine serum</td><td>Sigma-Aldrich Co., St. Louis, MO, USA</td><td>172012-500 ML</td><td>We added 10% of fetal bovine serum to the assay diluent.</td></tr><tr><td>Forceps (Micro tweezers for research experiments, curved tip)</td><td>Sansho Co., Ltd., Tokyo, Japan</td><td>91-1506</td><td>We used forceps for disection. Model, HSC 551-10; and overll all length, 105 mm.</td></tr><tr><td>Forceps (Precision tweezers)</td><td>Sansho Co., Ltd., Tokyo, Japan</td><td>91-2772</td><td>We used forceps for disection. Model, HSC 607-12; overll all length, 120 mm; and tip diameter, 0.3 mm.</td></tr><tr><td>Goat Anti-Mouse IgA-UNLB</td><td>SouthernBiotech, Birmingham, AL, USA</td><td>1040-01</td><td>We coated Clear Flat-Bottom Immuno Nonsterile 96-Well Plates with 100 &amp;mu;L/well (20 ng/well) of total immunoglobulin A.</td></tr><tr><td>Isoflurane&amp;nbsp;</td><td>Viatris Inc., Canonsburg, PA, USA</td><td>NA</td><td>We used isoflurane for anesthesia.</td></tr><tr><td>Luminol Rection Reagent Set</td><td>FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan</td><td>124-06511</td><td>We used a luminol reaction reagent set to detect hemoglobin in NLF samples.</td></tr><tr><td>Microtube</td><td>Watson Co., Ltd., Tokyo, Japan</td><td>131-7155C</td><td>We used&amp;nbsp; microtubes for collecting NLF and BALF (1.5-mL microtube with lockining-cap, round bottom, soft touch-cap).</td></tr><tr><td>Milli-Q water</td><td>Merck KGaA, Darmstadt, Germany</td><td>Milli-Q Direct 8</td><td>We used MilliQ water to make a luminol rection solution.</td></tr><tr><td>Mini-Shaker PSU-2T</td><td>Biosan Ltd., Riga, Latvia</td><td>PSU-2T</td><td>We shaked 96-well plates using the Mini-Shaler PSU-2T.</td></tr><tr><td>Mouse strain</td><td>CLEA Japan, Inc., Tokyo, Japan</td><td>NA</td><td>we used 4-month-old C57BL/6 male mice.</td></tr><tr><td>Nunc MicroWell 96-Well, Nunclon Delta-Treated, Flat-Bottom Microplate</td><td>Thermo Fisher Scientific Nunc A/S . Kamstrupvej,Roskilde, Denmark</td><td>136101</td><td>We used the Nunc&lt;sup&gt;TM&lt;/sup&gt; MicroWell&lt;sup&gt;TM&lt;/sup&gt; 96-Well, Nunclon Delta-Treated, Flat-Bottom Microplate for the luminol test.</td></tr><tr><td>Peroxidase-conjugated AffiniPure F(ab&#039;)&lt;sub&gt;2&lt;/sub&gt; Fragment Goat Anti-Mouse IgG, F(ab&#039;)&lt;sub&gt;2&lt;/sub&gt; Fragment Specific (minimal cross-reaction to Human, Bovine, and Horse Serum Proteins)</td><td>Jackson ImmunoResearch Laboratories, Inc., West Grove, PA, USA</td><td>115-036-072</td><td>We added 100 &amp;mu;L of peroxidase-conjugated AffiniPure F(ab&#039;)2 fragment goat anti-mouse IgG, F(ab&#039;)&lt;sub&gt;2&lt;/sub&gt; fragment specific, as the detection antibody (10,000-fold dilution) to each well.</td></tr><tr><td>Phosphate-buffered saline</td><td>Sigma-Aldrich Co., St. Louis, MO, USA</td><td>D8537-500ML</td><td>We used Dulbecco&#039;s Phosphate-Buffered Saline (modified without calcium chloride and magnesium chloride, liquid, sterile-filtered, suitable for cell cullture) for NLF and BALF collection.</td></tr><tr><td>Pipette&amp;nbsp;</td><td>Watson Co., Ltd., Tokyo, Japan</td><td>NA</td><td>We used a 200-&amp;mu;L pipette for nasal lavage.</td></tr><tr><td>Pipette tip</td><td>Watson Co., Ltd., Tokyo, Japan</td><td>NA</td><td>We used a 200-&amp;mu;L multifit pipette tip for nasal lavage.</td></tr><tr><td>Polyoxyethylene(20) Sorbitan Monolaurate (Tween 20)</td><td>FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan</td><td>167-11515</td><td>We added 0.05% of Tween 20 to the washing buffer and 0.2% of Tween 20 to the assay diluent.</td></tr><tr><td>Purified Goat anti-mouse IgG (minimal x-reactivity) Antibody</td><td>BioLegend, San Diego, CA, USA</td><td>405301</td><td>We coated Clear Flat-Bottom Immuno Nonsterile 96-Well Plates with 100 &amp;mu;L/well (10 ng/well) of total immunoglobulin G.</td></tr><tr><td>Small scissors, double-pointed</td><td>Sansho Co., Ltd., Tokyo, Japan</td><td>91-7005</td><td>We used scissors for disection. Model, S-4A; and overll all length, 115 mm.</td></tr><tr><td>Sulfuric acid</td><td>Wako Pure Chemical Industries, Ltd, Tokyo, Japan</td><td>192-04755</td><td>We added 50 &amp;mu;L of 2 N sulfuric acid to each well to stop the reaction.</td></tr><tr><td>Surflo intravenous catheter&amp;nbsp;</td><td>Terumo Corporation, Tokyo, Japan&amp;nbsp;</td><td>SR-FS2032</td><td>We used a surflo intravenous catheter and injected PBS into the trachea for bronchoalveolar lavage. Catheter, 20G; catheter length, 32 mm; catheter inner diameter, 0.80 mm; catheter outer diameter, 1.1 mm; and inner needel, 22G.</td></tr><tr><td>Surgical straight scissors</td><td>Sansho Co., Ltd., Tokyo, Japan</td><td>91-7004</td><td>We used scissors for disection. Model, S-3B; and overll all length, 120 mm.</td></tr><tr><td>Surgilcal plate</td><td>Natsume Seisakusho Co., Ltd., Tokyo, Japan</td><td>KN-321-B</td><td>We used a cork board for dissection. Depth, 20 cm; width, 15 cm; and height 3 cm.</td></tr><tr><td>Synergy H1 Hybrid Multi-Mode Microplate Reader&amp;nbsp;</td><td>Agilent Technologies, Inc., Santa Clara, CA, USA</td><td>NA</td><td>We measured the absorbance of samples at 450 nm, using the Synergy H1 Hybrid Multi-Mode Microplate Reader.</td></tr><tr><td>Syringe</td><td>Terumo Corporation, Tokyo, Japan&amp;nbsp;</td><td>SS-01T</td><td>We used a 1-mL syringe for blood collection from the heart.</td></tr><tr><td>Thread</td><td>&amp;nbsp;Niccho Kogyo Co., Ltd. Tokyo Japan</td><td>NA</td><td>We tied a catheter with thread to ensure proper positioning during the procedure.</td></tr></tbody></table>

murine nasal lavage fluid collection without blood contamination

Murine Nasal Lavage Fluid Collection without Blood Contamination

Upper airway samples, including nasal lavage fluid (NLF) and nasal swabs, are useful in detecting pathogens, including viruses and bacteria, which can cause respiratory diseases. NLF has been used to examine cellular and humoral components in the respiratory system, for example, in evaluating the induction of immunoglobulin (Ig) A following mucosal vaccinations. In experimental rodents, the NLF samples can be collected by either the trans-pharyngeal or trans-tracheal route. Although the trans-pharyngeal route has been reported to be more efficient in collecting the NLF samples than the trans-tracheal route, the NLF samples collected by the trans-pharyngeal route were often contaminated with blood, affecting the levels of cellular and humoral contents in the original NLF samples. Thus, this study aimed to establish a novel NLF collection method in experimental mice to minimize blood contamination. Briefly, before the lower jaw of the mouse was separated, cotton balls were placed in the mouth to absorb blood. When the bleeding was stopped, the NLF samples were collected by inserting a micropipette into the choana and flushing with 200 &#181;L of phosphate-buffered saline twice (final volume: 400 &#181;L/mouse). To detect blood contamination, a simple and sensitive forensic luminol test that detects hemoglobin was used. The luminol test demonstrated that blood contamination was detectable in the NLF samples harvested by the conventional method (without cotton balls), but not those harvested by the novel method (with cotton balls). Due to the contamination of blood, the total IgA and IgG concentrations in the NLF samples were higher in the conventional method than in the novel method; blood has been known to contain much higher levels of IgA and IgG than NLF. Therefore, this unique method can be used as a simple and sensitive method to collect NLF samples from experimental mice to prevent blood contamination.

Using the conventional or novel method, NLF samples were collected from C57BL/6 mice through the trans-pharyngeal route. As shown in Figure 1, the NLF samples collected by the conventional method were slightly pink-tinged (Figure 1A); the NLF samples collected by the novel method were clear (Figure 1B). Since the difference seemed to result from blood contamination in the NLF samples by the conventional method, blood contamination i...

The present protocol describes a novel method for avoiding blood contamination in murine nasal lavage fluid (NLF). Since this method ensures the NLF collection without blood contamination, it allows for more accurate detections of immunological components and various respiratory pathogens.

Upper airway samples, including nasal lavage fluid (NLF) and nasal swabs, are useful in detecting pathogens, including viruses and bacteria, which can ...

murine-nasal-lavage-fluid-collection-without-blood-contamination

Research

JoVE Journal

Immunology and Infection

Source: Venuprasad, K., et al. Intra-tracheal Administration of Haemophilus influenzae in Mouse Models to Study Airway Inflammation. J. Vis. Exp. (2016)This video demonstrates the collection and processing of bronchoalveolar lavage fluid from a euthanized mouse infected with non-typeable Haemophilus influenzae for downstream analysis.

Bronchoalveolar Lavage Fluid Collection from a Mouse Model Infected with Non-typeable Haemophilus influenzae

This video demonstrates the use of flow cytometry to identify and count various immune cells in bronchoalveolar lavage fluid. Initially, single immune cells are identified using a viability dye, followed by differential staining of surface marker proteins, enabling the precise enumeration of each cell type.

Identification and Enumeration of Immune Cells in Bronchoalveolar Lavage Fluid by Flow Cytometry

Colonization of the murine nasopharynx with Streptococcus pneumoniae and the subsequent extraction of adherent or recruited cells is described. This technique involves flushing the nasopharynx and collection of the fluid through the nares and is adaptable for various readouts, including differential cell quantification and analysis of mRNA expression in situ.

Characterization of Inflammatory Responses During Intranasal Colonization with Streptococcus pneumoniae

The health status of the lung is reflected by the type and number of immune cells that are present in the bronchioles of the lung. We describe a bronchoalveolar lavage technique that allows the isolation and study of nonadherent cells and soluble factors from the lower respiratory tract of mice.

Bronchoalveolar Lavage of Murine Lungs to Analyze Inflammatory Cell Infiltration

This protocol describes a noninvasive technique for the sampling of undisturbed mucosal lining fluid from the upper airways. It can be used to perform the quantification of in vivo levels of protein mediators, such as cytokines and chemokines, in subjects of all ages.

Noninvasive Sampling of Mucosal Lining Fluid for the Quantification of In Vivo Upper Airway Immune-mediator Levels

This manuscript describes the use of nasal and bronchial absorption techniques, specifically using synthetic absorptive matrices (SAM) to sample the mucosal lining fluid (MLF) of the upper and lower airway. These methods provide better standardization and tolerability than existing respiratory sampling techniques.

Absorption of Nasal and Bronchial Fluids: Precision Sampling of the Human Respiratory Mucosa and Laboratory Processing of Samples

This protocol describes an improved technique for the abundant collection of cerebrospinal fluid (CSF) with no contamination from blood. With greater sample collection and purity, more analyses can be performed using CSF to further our understanding of diseases that affect the brain and spinal cord.

An Improved Method for Collection of Cerebrospinal Fluid from Anesthetized Mice

This communication describes methodologies for isolation and culture of alveolar macrophages from humans and murine models for experimental purposes.

Isolation and In Vitro Culture of Murine and Human Alveolar Macrophages

Saline nasal lavage can be used to sample the canine nasal immune microenvironment. Because the approach is relatively non-invasive and does not disrupt the nasal tissues, it can be performed serially. Cells and proteins collected from the nasal lavage technique can be processed for various laboratory analyses.

Saline Lavage for Sampling of the Canine Nasal Immune Microenvironment

Here, we present a protocol for an optimized blood collection technique in murine subjects through facial vein venipuncture using a lancet. We use this technique alongside flow cytometric assessment to characterize cellular phenotypes of transgenic, congenic, and reporter strains. It is also suitable for serum cytokine analysis and diabetes studies.

Facial Vein Venipuncture for Murine Blood Collection

Learn a novel method for collecting murine nasal lavage fluid without blood contamination using pharyngeal lavage and cotton ball placement. This technique improves immunological accuracy and respiratory pathogen detection.

Murine Nasal Lavage Fluid Collection without Blood Contamination

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