MATERIAL FOR SPERM ANALYSIS - Automatic Diagnostic Systems
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1. multiply by 100 gt Total number of sperms X 100 of rapid progressive sperms N rapid progressive gt Total number of sperms x 100 of motile no progressive sperms N motile no progressive gt Total number of sperms x 100 7 of motile in situ sperms N motile in situ gt Total number of sperms x 100 of static sperms N static sperms lf the number of sperms is very low count the sperms in all the grid and then divide the number obtained for 10 gt Total sperm number counted in all makler x 10 6 total sperm concentration 10 Attention count only the head of the sperms inside the squares and the heads on two of the square s sides chosen err by the user Se ee eee 4 il co www automaticdiagnosticsystems com 6 Automatic Diagnostic Systems Pin heads should be counted separately and commented in the report form Immature germ cells should not be counted these will be assessed in differential morphology count but round cells and inflammatory cells should be counted in the same way as the sperms and their number added in the final report Borderline cases only spermatozoa whose head is located on the upper or left limiting lines marked OK in Fig 1 should be counted as belonging to that square Thus do not count spermatozoa located on the lower or right limiting lines marked out in Fig 1 gt Clear the Makler chamber with tap water and dry very well if the Makler chamber is2. one drop of semen with two drops of 1 Eosin Y After 30 seconds add three drops of 10 nigrosin solution and mix Place a drop of the semen eosin nigrosin mixture on a slide and make a smear within 30 seconds of adding the nigrosin The smears should not be too thick Allow to air dry and examine under oil immersion 1000 with a light microscope The live spermatozoa are white and the dead are stained red The nigrosin provides a dark background which makes the slides easier to assess www automaticdiagnosticsystems com 12 Automatic Diagnostic Systems MORPHOLOGY ANALYSIS Microscope 100x oil objective Materials Diff Quik staining solution 3 bottles protocol or Cell Vu prestained slide for morphology protocol or Sperm Blue staining solution Positive displacement Micropipette 1 DIFF QUIK Diff Quik fixative Solution Diff Quik Solution 1 red Diff Quik Solution 2 blue Distilled water STEP BY STEP DESCRIPTION If the raw sample is less than 1 M ml you can concentrate according to the following procedure 1 Determine the concentration of the sample C 2 Transfer 500 ul of sample to a centrifuge tube and add 1000 2000 ul of washing medium e g Hams F10 1 BSA 3 Mix gently by swirling or agitate by running tube once along the top of an empty test tube rack no vortex 4 Centrifuge sample for 5 minutes at 200 g 5 Carefully aspirate the supernatant being extremely careful The volume
3. very damaged and scratched must be changed with a new one 2 PROCEDURE WITH LEJA SLIDE Automatic Analysis E Before performing the analysis leave the Leja slide 10 minutes in the incubator at 37 C or on the heating stage Using the positive displacement micropipette pick up 1 ul of semen and release slowly the sample in the right space of the Leja slide waiting until is filled by capillarity Put the specimen slide in the microscope focus and analyze with the SCA motility amp Concentration module Leja 10 is for 4 analysis one analysis for each chamber in the slide IMPORTANT In case of high concentration in the sample gt 90 millions ml the W H O recommends to dilute the sample in order to better analyze the concentration and motility After the counting remember to arrange the values to the dilution if you are working with SCA you just have to insert the dilution rate in the window that will appear on the screen before the analysis SPERM SAMPLE DILUITIONS Table 1 Dilution of the ejaculate Spermatozoa per field of vision 40X Semen uL Diluent uL objective Quality Control www automaticdiagnosticsystems com T Automatic Diagnostic Systems Duplicate counting should be done to detect random errors in sampling and counting chamber Calibration of the positive displacement pipettes some times called PCR pipette and ordinary pipettes for measurements of diluents should be done regularly Pipe
4. with alcohol Rinse with distilled water and wipe dry Warm slides on the slide warmer set to 37 C 2 Place a 5 ul droplet of sperm near the frosted end of the microscope slide 3 Using another slide held obliquely to the first approximately 30 angle touch it against the drop of sample away from the frosted end 4 After the droplet has spread along the junction of the slide gently pull the second slide away from the drop along the length of the first slide Note If specimen has a very low initial concentration 10 M ml or less the droplet should be spread in a streak down the center of the slide to conserve sufficient sperm density Placed prepared smear on the slide warmer and allow to air dry approximately 15 minutes Staining with Diff Quik recommendable www automaticdiagnosticsystems com 14 Automatic Diagnostic Systems Reagents amp Supplies Needed Diff Quik Kit consisting of Fixative Stain Solution 1 and Stain Solution 2 Distilled Water Coplin Staining Jars Paper towels Diff Quik Staining Protocol ae oN Fix smear by immersing slide in Diff Quik Fixative 1 second 4 times Stain sperm by immersing fixed slide in Diff Quik Solution 1 1 second 4 times Stain sperm by immersing slide in Diff Quik Solution 2 1 second 4 times Rinse slide gently in distilled water Allow to dry If re examination of slides is required coverslipping will help prevent deterioration of the smear due to repeated ex
5. 00 g sec for 10 minutes 1400 g sec if the test tube is the conical one 5 When the centrifugation time is finished take off all the upper liquid part leaving the bottom with the semen 6 Swim up Add gently on the surface maintaining the pipette attached to the internal side of the test tube no drops directly 0 5 ml of G sperm 7 Leave the test tube close but not completely the stopper only above not pressed on in the incubator at 37 C for 1 hour 8 After 1 hour pick up from the surface without touching the pellet below 0 25 ml and put in a smaller test tube with the name of the patient and the concentration of the sample this last one after the swim up Take a small aliquot from the main test tube and control the concentration N B A normal Swim up made with a clean sample is prepared in a test tube putting on the bottom 1 ml of semen and then adding gently 1 ml of G sperm on the surface maintaining the pipette attached to the internal side of the test tube no drops directly www automaticdiagnosticsystems com 24 Automatic Diagnostic Systems Put the sample in the incubator at 37 C with the stopper not completely closed it has to move After 1 hour pick up 0 5 ml of liquid from the surface the liquid has to be transparent and clean don t touch the semen below and put it in a smaller test tube Pick up a small aliquot and control the concentration of the swimming sperm The meaning of the swim up is to provide a se
6. Automatic Diagnostic Systems MATERIAL FOR SPERM ANALYSIS Laboratory equipment Plastic Gloves Recommended without Talcum Slides Recommended Leja 10 human leja 20 animal Or Makler chamber ATTE CURLER th Temperature control keep the chamber to the 37 C using a heating stage or inside the incubator Use a micropipette with a particular volume of sample different for each analysis chamber see instructions of use With the Makler chamber you have to capture the fields images and videos outside the grid and avoid line of scratching Neubauer counting chamber only for manual counting Sample sterile collection container e g Sarstedt 100 mL www automaticdiagnosticsystems com 1 Automatic Diagnostic Systems Phase contrast microscope 10x Ph for human Ph for animals 40X 100x phase contrast positive objectives Coverslips 18 x 18 mm 15 thickness 22 x 22 mm 1 Automatic Pipettes for making wet preparation Air displacement positive displacement recommended 6 uL morphology smears air displacement positive displacement recommended 8 15 yL vitality smears air displacement positive displacement recommended 15 50 uL www automaticdiagnosticsystems com 2 Piston Unprotected air space t Aerosol contamination Filter 39 Aerosol Vr V T Regular air displacement pipette Gilson Pipetman P200 Automatic Diagnostic System
7. developed to stain all the components of the sperm acrosome head midpiece and main piece of tail differentially in different intensities of blue The staining procedure is extremely simple and only involves two main steps fixing in one medium and staining in a second medium It works equally well for smears of raw semen as well as swim up Percoll Pure Sperm gradient preparations using most culture media Contents of Sperm Blue All Sperm Blue packages contain vials bottles with equal volumes of fixative Clear or transparent solution in buffered salt and a dark blue stain dark blue staining solution in buffered salt Sperm Blue is packaged in three variations www automaticdiagnosticsystems com 20 Automatic Diagnostic Systems omall 1 8ml vials consisting of 50 vials containing fixative and 50 vials containing the stain Allow fixing and staining of 200 smears on slides Approximately 0 45 ml sufficient to stain one slide Two bottles containing 100 ml each of fixative and 100 ml stain Allow staining of 200 smears on glass slides Two bottles containing 250 ml each of fixative and 250 ml of stain Sufficient to stain 500 sperm smears on slides If fixative and stains are stored at 4C it will last for one year Room temperature storage not guaranteed Staining Procedure 1 2 Make sperm smear using 5ul of semen or 5 to 10 ul of swim up sperm adapt volume to concentration of sperm and allow to air dry Use a p
8. ion i Thick midpiece I Thin midpiece v C Tail defects D Cytoplasmic droplet K Short Cut 1 Bent m Coiled www automaticdiagnosticsystems com 23 Automatic Diagnostic Systems DNA FRAGMENTATION Kit Halomax halosperm halotech Om Kit to Assess Sperm DNA Fragmentation in Human T Warning prototype For research use only Do not Z use for diagnoses of disease in humans or animals cR 5 0 1 Protocol included in the Halosperm kit for 10 analysis see the attached file Is always recommendable do swim up of the semen sample before any analysis motility morphology or DNA fragmentation analysis to avoid the interference with the debris of the sample Protocol for the ICSI semen preparation cleaning 1 Put all the volume of the sample in a test tube conical bottom if the sample has an high concentration otherwise normal test tubes and add gently on the surface maintaining the pipette attached to the internal side of the test tube no drops directly 6 ml of G Sperm with an air pipette Close the test tube 2 Put the test tube in the centrifuge 1500 g sec for 10 minutes 1400 g sec if is the conical one 3 When the centrifugation time is finished take off all the upper liquid part leaving the bottom with the semen 4 Add gently on the surface maintaining the pipette attached to the internal side of the test tube no drops directly 3 ml of G Sperm and repeat the centrifugation 15
9. lastic disposable pipette to put 0 45 to 0 5ml of clear fixative solution over smear Roll slide from side to side regularly to ensure proper fixation over smear surface Fix for 10 minutes Wash by dipping slide gently 2 to 3 times in beaker containing distilled water Use a plastic pipette to put 0 45 to 0 5ml of blue stain on fixed sperm smear Roll slide from side to side regularly and stain for 10 minutes Dip slides very gently 3 to 5 times in beaker containing distilled water until most of blue colour disappears on slide Air dry Mount slide with DPX or equivalent synthetic medium for permanent slides When mounting medium is dry view under oil immersion x1000 OR Do not mount cover slip after air drying but put immersion oil directly on slide and study immediately using oil immersion optics at x1000 Discard slide after use not permanent Precautions All cytological stains are toxic and handle with care Always work with gloves and preferably in a fume cupboard preferable but not essential for Sperm Blue Only stain when sperm are fixed dead Do not use for live unfixed cells www automaticdiagnosticsystems com 21 Automatic Diagnostic Systems W H O SPERM MORPHOLOGY A Head defects a Brush b Pyriform c Round Without Acrosome Small d Vacuolated e Reduced Acrosome www automaticdiagnosticsystems com 22 Automatic Diagnostic Systems B Neck and Midpiece defects g Bent neck h Asymmetrical insert
10. lection of the best motile spermatozoa If is a preparation for an intrauterine insemination the protocol is the same just change the G sperm with the sperm washing Gradients of density Cleaning put in a test tube gt 1 ml of LOWER solution 1 ml of UPPER solution gt 2 ml the semen sample if the volume is higher use two Different test tubes Put the test tube in the centrifuge at 1500 g sec for 10 minutes Recuperate the pellet in the bottom of the test tube through away the rest and put it in a small test tube with 0 5 ml of sperm washing solution Mix gently with the pipette Take a small aliquot of the final sample and count concentration the count represent the final concentration 2 because the volume is 0 5 ml www automaticdiagnosticsystems com 25
11. odule under fluorescence DUO VITAL KIT FOR SPERM VITALITY ASSESMENT IN ANIMAL SPECIES The assessment of sperm vitality is one of the basic elements of semen analysis Vital stains can help to differentiate live from dead sperm Results of vitality test are linked to the integrity of the plasma membrane of viable sperm an intact plasma membrane is able to exclude certain stains either for bright field microscopy or fluorescence microscopy Sperm vitality evaluated with the combined use of fluorescent dyes correlates with that determined by eosin nigrosin stains bright field microscopy but opens a new range of possibilities for automatic counting using flow citometry or fluorescence microscopy and image analysis data collection The DUO VITAL kit is for the evaluation of sperm vitality from fresh or unthaws semen samples The kit provides the user with specific adjusted solutions of two reagents to evaluate sperm vitality by means of a dual emission fluorescent signal Green Live Red dead Protocol Step 1 Dilute the sperm sample to a final concentration of approximately 10 15M ml Step 2 Add 1 microliter of the diluted sperm sample onto a clean slide Add 1 microliter of the fluorochrome solution red and 1 microliter of the fluorochrome solution green to the diluted sperm sample and mix Cover with a clean coverslip and observe under the fluorescence microscope Score green as live cells and red as dead cells The analysis is a
12. of the remaining pellet should be approximately 10 ul 6 Resuspend the pellet using the volume determined by the following equation V 5C yl Thus if a sample had an initial concentration C of 20 M ml then V 5 20 100 ul So 100 ul of washing medium would be used to resuspend the pellet Samples that are concentrated using the above procedure may not have to be washed However if the smear shows excessive debris additional washing will be necessary Washing the sample If the sample shows a lot of detritus is convenient to wash it 1 Place 500 ul of sample into a microcentrifuge or conical tube www automaticdiagnosticsystems com 13 Automatic Diagnostic Systems 2 Add 500 ul of washing medium Hams F10 1 BSA If Percoll is used the specimen must be washed completely Percoll free before staining otherwise staining will be incomplete 3 Swirl or Repeat pipette gently to mix 4 Centrifuge for 5 minutes at 200 g 5 Remove supernatant by aspiration 6 Resuspend pellet in 500 ul of washing medium 7 Repeat pipette gently to mix 8 Centrifuge a second time for 3 minutes at 500 g 9 Remove supernatant by aspiration 10 Resuspend pellet with 125 ul of washing medium 11 Repeat pipette gently to mix The sample is now ready to use to make a smear If sample is extremely debris laden repeat steps 8 11 again Prepare at least two smears from each sample as follows 1 Wash pre cleaned microscope slides
13. posure to immersion oil It will also help to maintain a clean objective Though coverslipping is optional it is recommended Mounting a coverslip onto the slide requires the use of a mounting medium such as the Eukitt mounting medium Coverslipping Procedure ROMA Place 3 4 drops of mounting medium along the center of the dried slide Carefully place a clean coverslip 50mm x 22mm on the slide Press the coverslip gently to distribute the mounting medium Allow to dry sufficiently www automaticdiagnosticsystems com 15 Automatic Diagnostic Systems Advises for morphology analysis with Sperm Class Analyzer The following images show different kind of captures 1 Correct image Clean background Light stain Optimum illumination control Separated spermatozoa 2 Incorrect image Two spermatozoa heads very close together 3 Incorrect image www automaticdiagnosticsystems com 16 Automatic Diagnostic Systems Background with a lof of detritus Dirty background Incorrect image Too light image some of the midpices cannot be analysed www automaticdiagnosticsystems com 17 Automatic Diagnostic Systems Incorrect image Too dark field not enough contrast Incorrect image Too stained acrosome cannot be detected www automaticdiagnosticsystems com 18 Automatic Diagnostic Systems Incorrect image One spermatozoon s head is very close together with the
14. rm while the dead sperm and debris are caught up in the gradient media The pellet is then resuspended in washing media and centrifuged twice The final pellet is resuspended in a final volume of approximately 0 5 mls of media There are several commercially available kits Simple Wash Method The simple centrifuge sperm wash should be performed on a sample that has a decreased concentration and or motility A sample containing round cells and debris should not be prepared by this method Sperm washing media is added to the specimen and centrifuged The pellet is recovered resuspended and centrifuged again The final pellet is resuspended in approximately 0 5 mls of media In each of the above cases the laboratory performs an analysis on both the fresh specimen and washed specimen This will include an assessment of the count motility volume and viscosity www automaticdiagnosticsystems com 9 Automatic Diagnostic Systems WHEN THE SAMPLE IS VERY DIRTY YOU HAVE TO CLEAN IT IN ORDER TO PERFORME A GOOD ANALYSIS The recommended medium is Pure Sperm 1 One uses a 40 solution on top and a 80 solution at the bottom in a centrifuge tube 2 Then put 2ml of sperm on top and centrifuge at x600 for 20 minutes 3 At bottom a pellet with beautifully clean sperm and 100 motility www automaticdiagnosticsystems com 10 Automatic Diagnostic Systems VITALITY ANALYSIS STAINING FOR VITALITY TEST Duo vital to work with the SCA Vitality m
15. s amp Protected air space No aerosol Capillary d e Microman positive displacement pipette Gilson Microman M50 Figure 1 Two pipetting concepts air displacement using standard filter tips and positive displacement as applied in the Microman pipette Pipette tips Plastic for air displacement pipettes volume 5 50 uL Air pipettes www automaticdiagnosticsystems com Automatic Diagnostic Systems Centrifuge 1000 3000g Incubator with humidity and CO2 control or heated chamber 37 C Medium for sperm washing sample cleaning www automaticdiagnosticsystems com 4 Automatic Diagnostic Systems HOW TO PERFORME THE ANALYSIS Basic Guide lines for human semen analysis Abstinence days W H O recommended a period is between 2 7 days Collection In sterile container maintained at 20 37 C important to avoid rapid temperature changes and stored in an incubator at 37 C protected from light The sample must be analyzed between 30 and 60 minutes from the collection time 60 minutes maximum TIMING OF PERFORMING ANALYSIS 0 5 minutes collect all the sample details and put the container in the incubator at 37 C 0 30 minutes Check the liquefaction of the sample the presence of prostatic crystals and all the macroscopic parameters In case of CS put 1 ml of sample in a test tube and overlayed it with 1 ml of swim up solution and wait 30 minutes Perform
16. t can easily be handled by the uterus There are three main methods of sperm washing the swim up density gradient wash and simple centrifugation wash The type of wash used depends on the individual characteristics of each semen specimen Swim Up Method The swim up is most successful when performed on patients with a normal semen analysis and is not recommended for samples of high viscosity with high numbers of round cells or with a high content of debris In this procedure the washing media is gently placed over the semen in a conical tube The specimen is then placed in an incubator for approximately one hour During this time the sperm are allowed to swim up into the media with the purpose of collecting the most motile normal sperm free of debris The supernatant is collected and centrifuged twice with sperm washing media The final pellet is then resuspended in approximately 0 5 mls of media Modified sperm washing media must be used to process the sample Density Gradient Wash Method The discontinuous density gradient method should be used on samples containing round cells or debris or those with increased viscosity but a relatively normal concentration and motility The gradient is achieved by layering media of two different densities in a conical tube The semen is then placed on top of the gradient and the tube is then spun to allow the specimen to proceed through the gradient The resulting pellet should contain the motile normal spe
17. tail of another www automaticdiagnosticsystems com 19 Automatic Diagnostic Systems 2 Cell VU Prestained Morphology Slides Direction for use l Apply a small drop approximately 2 4 microliters of specimen on the center of the slide 2 Place a longer coverglass 24 x 60 mm on top of the slide exerting slight pressure on the coverglass Note Do not slide the coverglass as this will remove the stain Note You can place covered slide coverglass side down on paper towel and press gently 3 Allow slide to stand at room temperature for 15 minutes before microscopic observation is made Place loaded slide on heating plate for 1 minute if you do not want to wait Expected Findings These slides are for evaluating sperm head acrosome size differentiation of white blood cells in peripheral blood and cytology of body fluids Notes The stain absorbs into living or recently dead sperm If the sperm were dead at the time of collection they would be in some state of degradation and no longer able to absorb the stain but if you not prepared your slide immediately and the sperm has just died it will still stain You can archive for 30 days Let air dry remove coverglass and store in dark place You will loose the tails but should still be able to see the heads fine If you need to reread a drop of stain on the slide will refresh the slide 3 Sperm Blue Staining Procedure Background The stain has been
18. the motility and vitality analysis and prepare the stained slide for the morphology analysis After 30 minutes Analyze the morphology slide has to be dry and the sperms has to be dead and fixed MATERIAL FOR MOTILITY ANALYSIS Microscope 10x positive phase contrast objective Ph negative for animals Disposables Materials Leja slides 4 chambers 10 microns or Makler chamber or New bauer Automatic pipettes and pipettes tips 1 Procedure with Makler Subjective analysis REMEMBER TO HEAT THE MAKLER AT 37 C BEFORE USING IT 10 MINUTES IN THE INCUBATOR OR ON HEATING STAGE 1 Homogenize the sample moving gently the container 2 Take 5 ul of sample and put it in the center of the makler chamber in the center of the circle then cover with the cover round slide of the chamber pressing gently to homogenize the volume of sample on the surface must have no air bubbles in the chamber 3 With a 20x 40x objective count the spermatozoa in ten squares random or on the diagonal www automaticdiagnosticsystems com 5 Automatic Diagnostic Systems Organize the counting in motile progressive sperms motile no progressive sperms motile in situ sperms static sperms The total number of counted sperms represents the total concentration to obtain the total concentration multiply by 1x10 6 To obtain the percentage of the four categories of sperms divide the total number for the number of each category and
19. ttes showing erroneous results should be amended according to instructions of the manufacturer or sent for repair and adjustments Results of calibration controls as well as repairs and adjustments should be recorded in a separate list for each pipette Great care is needed when mixing and diluting semen mixing the diluted sample and preparing counting chambers attachment of coverslip Counting chambers should be checked for accuracy when new and regularly after use risk for wear and changed chamber depth Values for semen Variables Normospermia Normal ejaculate values Oligospermia Sperm concentration 20 Mil ml Asthenospermia Forward progression type A B sperms concentration lt 50 or lt 25 rapid progressive type A spermsconcentration Teratozoospermia Percentage of normal morphology 309 Oligoasthenoteratozoospermia Signifies disturbance of all three variables concentration progressive sperms normal morphology Azoospermia No spermatozoa in the ejaculate Aspermia No ejaculate www automaticdiagnosticsystems com 8 Automatic Diagnostic Systems SPERM WASHING operm washing is the process that prepares a semen sample for an intrauterine insemination IUI For an IUI to be performed the semen sample must be washed free of debris white blood cells and prostaglandins which can cause the uterus to contract The processing also removes dead sperm and concentrates the sperm into a small volume tha
20. utomatic with the CASA Sperm Class Analyzer SCA Vitality In some cases green red fluorescent sperm nuclei are observed This is mainly due to an incipient loss of membrane integrity Score this sperm nuclei as dead Used under these conditions DUO VITAL provides a High Effective Fluorescent Signal for confident visual analysis of sperm vitality The combination of DUO VITAL with Computer Assisted Sperm Analysis CASA Leja or alternatives multiple chamber slides allows the simultaneous assessment of sperm concentration vitality and motility A CASA system for such purposes is available SCA Vital Microptic SL Barcelona Spain www automaticdiagnosticsystems com 11 Automatic Diagnostic Systems 2 Eosine To analyze with SCA Video counter Reagents Eosine Y gt Prepare the staining solution mixing a 0 5 5g l solution of Eosin Y C I 45380 in a 0 9 9 g l aqueous sodium chloride solution Procedure 1 Mix one drop 10 15 ul of fresh semen with the same volume of Eosin solution on a microscope slide and cover with a coverslip 2 After 1 minute observe the preparation with 20x or 40x objective under bright light or phase contrast 3 Count unstained sperm white as alive and the stained sperm rose as dead 3 Eosin nigrosin To analyze with SCA Video Counter Reagents Eosin Y C I 45380 10 g l in distilled water i e 1 Nigrosin C I 50420 100 g l in distilled water i e 10 Procedure Mix
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