MMCSPERM Fully Automated Sperm Analysis

• MOTILITY
• CONCENTRATION
• MORPHOLOGY
• DNA FRAGMENTATION
• VITALITY

Here is a summary of the amazing functionality of this elegant and easy to use system:

• Acquisition of images and clips in AVI format from an imaging device provided with DirectShow drivers.
• Storage of textual data, images and clips in the built-in MultiMedia Catalog database. You can store patient data, analysis results, video clips, images and any other files in one record (accompanying documents of any office software like MS Office, Open Office etc., Adobe or similar applications for extended image processing). The database provides fast and flexible search and spectacular reporting with images, graphs and other features adjusted to your particular needs. Herewith our database can serve the needs of virtually any laboratory or research institution.
• Automated analysis of sperm concentration and motility on native samples according to WHO recommendations.
• Automated morphology analysis on stained samples according to strict Krueger’s criteria.
• Manual assessment of concentration of white blood cells, immature germ cells, round cells.
• Vitality. Estimation of the percentage of live spermatozoa is assessed by identifying those with an intact cell membrane, from dye exclusion or by hypotonic swelling.
• Manual measurements for special research or other individual tasks.

ADVANTAGES OVER MANUAL OBSERVATION METHODS

​Working with a camera system is much more comfortable than looking through eyepieces with far less strain.
Using MMC Sperm, you can record all the clips to your hard disk to repeat the motility analysis any time - without it you are restricted to the active lifetime of the spermatozoa under observation.
Morphology analysis becomes more accurate. The software will assess every cell that you have acquired fully automatically.
The use of the automated software for semen analysis helps to hugely decrease innacuracies and inconsistencies caused by manual interpretations . It is complicated to analyse, for instance, the form of sperm head by sight whereas with MMC Sperm you receive instant and consistent objective quantitative data: head length, width, area, form factor. The same applies to motility: estimation of sperm movement becomes more objective if you know its speed and exact track. But the final decision is always yours and you can correct the results of the automated analysis.By using a Computer Aided Sperm Analyser, it is much easier to train new staff in your laboratory and control the results of their work.The MultiMedia Catalog database provides the ability to create unlimited number of records with infinite number of fields to archive all the required patient information accurately and search for it fast.Having the ability to work over local network, you can select the suitable location of your database (e.g. on the firewall protected server with automatic backup procedures). Even if working on one PC, you are protected from data loss through two-stage deleting procedure in MultiMedia Catalog (if you delete a record, it goes to Recycle Bin first and can be restored).Selecting proper database field types helps you prevent misprints in your database (you may not enter text into numeric or time fields etc.) Drop-down lists will aid in fast and accurate filling the records with repeatable information. Special tool for insertion of predefined text blocks will aid you in filling the fields with related nomenclature, standard phrases etc.Your patients will enjoy informative reports with explanations, reference norm values, images (and whatever your creative imagination may produce).

MMC SPERM DATABASE FEATURES

• Our database is specially tailored for image analysis needs. You can catalogue virtually any digital assets corresponding with sperm analysis in one place and have fast and smart access to any files.
• The volume of semen analysis data, images, clips and documents in your database is restricted only by the size of your storage media.
• Your sperm analysis database can be stored on server in your local network which will enable all the security policies and backup procedures provided by Windows. Alternatively, you can also save your spermiogram database to any suitable storage media (internal/external HDD, flash, etc.) if you need to have access to it on different workstations.
• The internal archiving tool will help you to keep your important semen analysis and patient data save as well as to make archives with older information to free your storage space.
• There are many security tools and procedures to ensure the integrity of your sperm analyzer data. Database records are deleted to internal recycle bin and removed permanently only after you empty your bin. Images and videos are deleted to Windows recycle bin and can be restored. To prevent unwanted changes in the database records, all the records are locked for editing until you press a special button.
• To avoid unwanted changes by unauthorized personnel, you can enable PIN mode. The PIN mode will disable the following options until you enter correct PIN: edit database structure (add/delete fields), edit print templates, making changes in the autotext box, deleting database filters, changing default calibrations.
• Database structure is implemented as a tree of folders with separate technical files, images, videos, 3rd-party files for every record. This provides the advantage of loading only one record at a time, which makes your work with a large database fast and effective. Furthermore, storage of data in multiple files will protect you in case your PC is infected with a virus: if the virus destroys some files, you will lose only several records and in no case the whole database.
• The database is written in XML Unicode which provides wide options of integration of your data in 3rd-party databases as well as the ability to use your local language to fill in the database fields.
• Database structure can be easily adjusted by the user through a simple and visually clear interface. Add fields for storing text data (also multiline fields for large amounts of text, drop-down lists with predefined values or automatically generated lists for fast selection of repeating words and phrases), numerical data (e.g. parameter values), use special date tool with calendar. Move single fields or large groups of fields to organize your working space. Rename fields as you need, add any number of fields to store additional sperm analysis data along with automatically calculated parameters.
• Use fast search option to find required data in the fields or to retrieve images, video, documents.
• Apply extended filters to make selection of records according to any number of parameters (e.g. records created by certain doctor at certain time period having certain diagnosis etc.) Easy filters can be created fast, complicated multiparametric filters can be saved to filter list and reapplied any time.
• Numeric fields can be set up to count records in the database automatically according to required parameters (e.g. you can keep track of the number of records with certain diagnosis in your database etc.)
• Special autotext box allows you to make an extended tree of predefined text blocks (nomenclature, ready-made phrases describing the diagnosis, prescriptions etc.) Making an advanced tree will let you fill in the patient record with a few clicks without the need of typing long phrases again and again which saves your time and helps to avoid misprints in the record.
• Automatic diagnosis. Autotext box also contains special tool which combines database filters and autoinsertion of text blocks to required text fields. Create a filter describing a diagnosis (if a number of parameters meets normal limits) and select this filter as condition of insertion of the diagnosis into corresponding field. This will allow you to provide exact automatic diagnosis with one click without the need to look through a number of parameters and compare them to normal values.
• Multilanguage support. If your clinic provides consulting in several languages, using autotext insertion based on filters, you can add diagnosis and other text blocks in as many languages as you need. Add a field marking required language and translate your text with one button.
• Database can be displayed in the table mode which allows you to keep track of all the statistics and make your own local reference values of all the measured parameters.
• Reports All the data you have provided in the patient record along with automatically calculated parameters, corresponding bar or pie charts and images can be placed into your report. The reports are based on *.rtf format which is recognized by virtually any text editors. You can integrate your spermiogram into any local document form or use our default templates. Internal document editor will allow you to create your own *.rtf files, save the resulting reports to corresponding records. Create as many templates as you need for your clinic: brief ones containing only text and prescriptions, full reports with all the statistical data and images, comprehensive charts and visually supported explanations to patient’s diagnosis.
• WHO MMC Sperm software provides automated analysis of sperm concentration and motility on native samples according to WHO recommendations. The analysis is based on frame by frame detection of sperm heads on video clips and building precise tracks which reveal the nature of sperm movement and provide sperm concentration value. The total number of sperms is also calculated automatically. One frame with tracks is automatically saved to database record to be as visual support in the report or you can save a suitable frame yourself. Flexible tool for adjustment of sperm head detection will let you work with both bright field and phase contrast (negative phase contrast yields the best results) and not only human sperms but also many animal species.
•  Automated morphology analysis on stained samples according to strict Krueger’s criteria. Special detection tool will help you to adjust head and acrosome detection to your particular sample processing and staining procedure. Stains like Diff-Quik are required. Software is able to detect both samples prepared according to WHO recommendations for CASA including centrifugation (recommended) but also can be adjusted to easier procedures which provide higher background staining. For complicated cases, there is an option of manual drawing and deleting of objects.
• Sperm vitality is an important test, especially for samples with less than about 40% progressively motile sperms. The percentage of live spermatozoa is assessed by identifying those with an intact cell membrane, from dye exclusion or by hypotonic swelling. The dye exclusion method is based on the principle that damaged plasma membranes, such as those found in non-vital (dead) cells, allow entry of membrane-impermeant stains. The hypo-osmotic swelling test presumes that only cells with intact membranes (live cells) will swell in hypotonic solutions. Vitality results should be assessed in conjunction with motility results from the same semen sample. The presence of a large proportion of vital but immotile cells may be indicative of structural defects in the flagellum. A high percentage of immotile and non-viable cells (necrozoospermia) may indicate epididymal pathology. Using MMC Sperm, you can estimate vitality on saved or newly captured images fast and reliable while keeping track of the number of sperms analyzed to avoid statistical errors.
• Sperm DNA fragmentation test provides additional information on semen fertility. MMC Sperm software allows you to detect and calculate percentage of fragmented sperms automatically. Spermatozoa are automatically classified into sperms wit Big Halo, Medium Halo, Small Halo, Without Halo and Degraded. DFI (DNA fragmentation index) is calculated which represents the percentage of fragmented sperms (small halo, without halo, degraded).
• Interactive MMC Sperm is a fully interactive tool. For both motility and morphology you can see all the parameters calculated for a sperm by just pointing your mouse cursor on the detected object and make your decision to confirm or to deny the automatic result. In morphology, the software provides the reasons based on which the decision has been made (which parameters are out of classification limits). If you find out that some of standard limits are not compatible to your sample preparation procedure, you can easily adjust the classifier.
• Reference values can be changed in software options if you prefer to use values different to those of WHO 2010 or if you work with nonhuman sperms.
• Special automatic fields will indicate if the analysis has Passed/Failed the reference values. Based on this information you can adjust the automatic diagnosis tool.
• Manual assessment of concentration of white blood cells, immature germ cells, round cells is available.
• Statistics: mean values of all motility and morphology parameters by classes are calculated. After having made a number of analyses, you can calculate your own local limit values for all the parameters using database table mode which will make your system a unique and precise tool adjusted to your current hardware and sample preparation procedures.
• All the raw data calculated for every spermatozoon can be automatically uploaded to separate text files for your extended research purposes.
• Manual measurements for special research or other individual tasks are available. You can make measurements on a series of images and send statistical results to previously assigned database fields. Such results will allow you to adjust your classifier limits to any special cases.
• Most important image processing operations like cropping, image brightness, contrast and color adjustment, rotation and flip. Enhance images with filters.

MOTILITY ROUTINE     
Sperm concentration assessment (M/ml) and motility analysis are performed on native samples by recording AVI clips (acquired live from your imaging device to PC memory or prerecorded to disk). The analysis strictly follows the requirements of the “WHO laboratory manual for the examination of human semen and sperm-cervical mucus interaction”. The analysis is based on frame by frame detection of sperm heads on video clips and building precise tracks which reveal the nature of sperm movement and provide sperm concentration value. The total number of sperms is also calculated automatically. One frame with tracks is automatically saved to database record to serve as visual support in the report or you can save a suitable frame yourself.

The following parameters are calculated:
VCL  =  curvilinear velocity (micron/s). Time-average velocity of a sperm head along its actual curvilinear path, as perceived in two dimensions in the microscope.
VSL =  straight line velocity  (micron/s). Time-average velocity of a sperm head along the straight line between its first detected position and its last.
VAP  =  average path velocity (micron/s). Time-average velocity of a sperm head along its average path. This path is computed by smoothing the actual path.
LIN = linearity. The linearity of a curvilinear path.
STR straightness. The linearity of the average path.
BCF = beat cross frequency (beats/s). The average rate at which the sperm's curvilinear path crosses its average path.
ALH = amplitude of lateral head displacement. Magnitude of lateral displacement of a sperm head about its average path.
WOB = wobble. A measure of oscillation of the actual path about the average path, VAP/VCL.
MAD mean angular displacement of the sperm head.
Elongation of sperm head.
Mean values of all the parameters are represented in statistics.

Based on the parameters mentioned above, the motility of each spermatozoon is graded A, B, C or D (WHO), according to whether it shows:
A = rapid progressive motility
B = slow or sluggish progressive motility
C = nonprogressive motility
D = immotility
A+B = progressive motility
A+B+C = total motiltiy

Additionally, the software enables the user to assess the concentration of:

• White blood cells
• Immature germ cells
• Round cells

MORPHOLOGY ROUTINE     
Morphology of the sperm head is an important criterion for the correct diagnosis. The so

ftware is set up to analyze still images of smears stained with the Diff-Quik stain according to strict Krueger’s criteria. We have selected Diff-Quik as worldwide recognized leader in rapid staining of sperm. With Diff-Quik, the head is stained pale blue in the acrosomal region and dark blue in the post-acrosomal region which is a good basis for precise image analysis. The following parameters are assessed for every spermatozoon:

Area of the head.
FFC = form factor circle. The degree of similarity of the sperm head to a circle.
Perimeter of the head.
Brightness.
ELL_B = Big axis of ellipse outlining the sperm head, the length of the sperm head.
ELL_S = Small axis of ellipse outlining the sperm head, the width of the sperm head.
Elng = elongation of the sperm head.
FFE = form factor ellipse. The degree of similarity of the sperm head to an ellipse.
Acrosome = Percentage of the acrosomal region.
Mean values of all the parameters are represented in statistics.

The software classifies spermatozoa into Norm and Head Pathology classes automatically based on head parameters. You can easily correct the results manually and also specify other anomalies (Tail Pathology, Neck Pathology). An extended morphology classifier is available which allows you to specify the following spermatozoa structure abnormalities indicating potential infertility: tapered, pyriform, round, amorphous, vacuolated, small acrosome, double head, pinhead, bent neck, asymmetrical neck, thick insertion, thin neck, short tail, bent tail, coiled tail, excess residual cytoplasm (ERC).

In case you can not receive a good image of stained smear suitable for automated detection (e.g. because of incorrect sample preparation), there is a special tool which allows you to outline the cells manually.

STATISTICS     

Statistics on all the parameters which have been measured in Motility and Morphology is displayed in the database (mean values of parameters). Statistics provides additional information on sperm quality. Corresponding database fields are filled in automatically during analysis like those for motility and morphology results. Statistics can be printed out to represent it to patient if required. Extended report template should be selected for this purpose (see below). Should the statistical data not be required for the patient, a brief report without statistics can be created.
All the raw data calculated for every spermatozoon can be automatically uploaded to separate text files for your extended research purposes.

VITALITY     
Sperm vitality is an important test, especially for samples with less than about 40% progressively motile sperms. The percentage of live spermatozoa is assessed by identifying those with an intact cell membrane, from dye exclusion or by hypotonic swelling. The dye exclusion method is based on the principle that damaged plasma membranes, such as those found in non-vital (dead) cells, allow entry of membrane-impermeant stains. The hypo-osmotic swelling test presumes that only cells with intact membranes (live cells) will swell in hypotonic solutions. Sperm vitality should be assessed as soon as possible after liquefaction of the semen sample, preferably at 30 minutes, but in any case, within 1 hour of ejaculation, to prevent observation of deleterious effects of dehydration or of changes in temperature on vitality. It is clinically important to know whether immotile spermatozoa are alive or dead. Vitality results should be assessed in conjunction with motility results from the same semen sample. The presence of a large proportion of vital but immotile cells may be indicative of structural defects in the flagellum. A high percentage of immotile and non-viable cells (necrozoospermia) may indicate epididymal pathology.

DNA FRAGMENTATION

Sperm DNA fragmentation test provides additional information on semen fertility potential that can not be achieved by means of standard motility, morphology and vitality tests. We have selected the Sperm Chromatin Dispersion (SCD) method as simple, fast, accurate, and highly reproducible method for the analysis of sperm DNA fragmentation in semen and processed sperm (Fernández et. al., Journal of Andrology 2003 Jan-Feb;24(1):59-66). The SCD test utilizes the idea that after acid denaturation and removal of nuclear proteins sperm with non-fragmented DNA produce the well seen halo of dispersed DNA loops while spermatozoa with fragmented DNA fail to produce such halo. When somatic cells or spermatozoa with nonfragmented DNA are immersed in an agarose matrix and directly exposed to lysing solutions, the resulting deproteinized nuclei show extended halos of DNA dispersion. The halos correspond to relaxed DNA loops attached to the residual nuclear structure. These deproteinized nuclei are called "nucleoids". The presence of DNA breaks promotes the expansion of the halo of the nucleoid and is the basis for the halo test to detect DNA damage when sperm are treated with an acid solution prior to lysis buffer, the DNA dispersion halos that are observed in sperm nuclei with nonfragmented DNA after the removal of nuclear proteins are either minimally present or not produced at all in sperm nuclei with fragmented DNA. You can use Halosperm staining or contact us for cheaper alternatives. 

MMC Sperm software allows you to detect and calculate percentage of fragmented sperms automatically. The method is adjusted for the use of bright field 40x lens, no expensive fluorescence staining and equipment required. Spermatozoa are automatically classified into sperms wit Big Halo, Medium Halo, Small Halo, Without Halo and Degraded.

DFI (DNA fragmentation index) is calculated which represents the percentage of fragmented sperms (small halo, without halo, degraded). The DFI below 15% represents fine fertility potential, DFI between 15% and 30% is associated with medium fertility while DFI more than 30% is correlated with bad fertility potential of semen. The distribution of fragmented sperms is represented with bar or pie chart. Special field is used to compare the result to user-defined reference value. For details, see description of measured parameters below.

MANUAL MEASUREMENTS     
Manual measurements for special research or other individual tasks are available. You can make measurements on a series of images and send statistical results to previously assigned database fields. Such results will allow you to adjust your classifier limits to any special cases.
Most important image processing operations are available like cropping, image brightness, contrast and color adjustment, rotation and flip, resize, background subtraction etc. Enhance images with filters.

Use any 3rd-party image editors for extended processing and adding comments, figures etc. while still keeping your images in our database. An option allows you to assign external software for editing images and video.

VETERINARY AND ANIMAL BREEDING     
MMC Sperm sperm quality analyzer can be successfully used in veterinary, animal breeding, bee breeding, fish breeding as well as in zoos for semen quality control and calcualtion of the number of doses for artificial insemination that can be produced from current sperm sample. Artificial insemination provides several advatages over natural mating: long range of transportation for frozen sperm, more effective use of semen of high quality thoroughbred animals, the use with the purpose of overcoming mating refusal (e.g., in national parks and zoos to breed endangered animal species). Success of artificial insemination directly depends on semen quality, therefore, application of computer assisted sperm analysis in breeding farms is consistently growing all over the world.     
MMC Sperm semen analyzer provides special features for calculation of doses. You can set the following parameters in software options:

• Dose volume - volume of the semen container to be used (straws, tubes etc.)
• Number of sperm per dose - required number of sperms in a dose.
• By default, the calculation is based on Motility routine results: percentage of progressively moving sperms (A+B%).
• Consider vitality - calculation with respect to percentage of live spermatozoa in a the sample if sperm vitality has been tested.
• Consider morphology - calculation with respect to percentage of sperms with normal morphology if sperm morphology has been tested.

By the end of the semen quality analysis, the software calculates corresponding number of doses for artificial insemination and volume of extender that should be added to current sperm sample to get this number of doses.     
           
REPORTING     
All the data you have provided in the patient record along with automatically calculated parameters, corresponding bar or pie charts and images can be placed into your report (spermiogram). Spermiograms are based on *.rtf format which is recognized by virtually any text editors. You can integrate your spermiogram into any local document form or use our default templates. Internal document editor will allow you to create your own *.rtf files, save the resulting reports to corresponding records. Create as many templates as you need for your clinic: brief ones containing only text and prescriptions, full reports with all the statistical data and images, comprehensive charts and visually supported explanations to patient’s diagnosis. If you have some special requirements, please contact us. We will help to adjust the report.

HARDWARE REQUIREMENTS     
     Since MMC Sperm acts as a part of Computer Aided Sperm Analysis system, there are some special hardware requirements that you should consider. Generally, it is not so complicated to gather all the components of the system by yourself but we highly recommend contacting us to ask for a local distributor in your area to get all the equipment from one hand. If there is no distributor in your area, we will be happy to share direct contacts to reliable hardware manufacturers that we have collected during the years of our work. Our target is to earn on our software product, therefore we are ready to provide you any help in buying other hardware parts at the best price.     
           
A Computer Aided Sperm Analyzer (CASA) consists of a PC with sperm analysis software installed, a microscope with imaging device attached to it and a special sperm counting chamber.    

PC Hardware Requirements     
PC requirements are mostly determined by the need of recording high resolution video clips at 60 fps speed in motility analysis. Other analyses are not that demanding. If you already have a modern PC that you would like to use, the first step would be ordering the required digital camera. MMC Sperm software provides a tool to test real PC performance with camera even in demo software mode. If you want to buy a new PC, follow the instructions below:

• 64 bit Windows 10 (Windows 7, 8 supported but no recommended).OR LATER OS
• If possible, avoid laptops since their components provide less performance, use desktop PCs.
• Intel chipsets (no AMD chipsets), chipsets with Z mark recommended, Z170 and higher recommended. Check currently available processors and chipsets on Intel web page
• Intel i5, i7 processors, 6th generation and higher, Intel HD Graphics 530 and higher.
• Minimum 6 Gb operating memory, DDR4 recommended.
• If you have to buy a laptop, look among "gamers" laptops, they provide better components. Look for Intel processors of 6th generation and higher with HQ mark (e.g. i7-6770HQ) and Nvidia discrete graphic cards. No AMD processors and video cards! Always check real specifications of selected laptop model on manufacturer's web site.

Counting Chambers     
Special counting chambers with fixed depth should be used for motility analysis: Leja, Makler, MicroCell, Cell-Vu etc. Such chambers provide standard volume and monolayered distribution of sperms in one focal plane. We offer a handy and cost-effective alternative to well-known Makler counting chamber: reusable MMC-SK sperm counting chamber.

Counting slides are useful for a Computer Aided Sperm Analysis System if the clinic or laboratory has got a large volume of analyses.

GT Vision offer a wide range in disposable counting chambers for standard semen analysis. There are slides with 2 or 4 chambers and with chamber depths of 10 or 20 micron. The Leja slides have a standard low level of quantification which implies that even low sperm counts can be determined. The clinical implication of this is that you are well equipped to determine a threshold to choose between IUI, IVF, or ICSI for example. Leja slides are very easy to work with and allow a laboratory technician to quickly learn to perform an accurate count thus improving efficiency and quality.

SAMPLE PREPARATION     
     For details on sample preparation protocol, please refer to the WHO laboratory manual for the examination and processing of human semen (5th edition 2010). This manual provides updated, standardized, evidence-based procedures and recommendations for laboratory managers, scientists and technicians to follow in examining human semen in a clinical or research setting. Detailed protocols for routine, optional and research tests are elaborated. Visit WHO web page for printed version.     
           
Motility     
Sperm concentration is estimated using special counting chambers with fixed depth to provide standard volume and distribute spermatozoa in one focal plane to let them move free and stay in focus all the time they are being tracked. Sperm counting chambers provide the ability to work with undiluted specimen. The preparation consists usually in mixing the sperm sample carefully while avoiding bubbles. If the specimen is highly mucous, it can be aspirated with syringe several times to make it more uniform.     
           
Morphology     
To achieve good quality images of stained spermatozoa for morphology assessment, seminal plasma should be diluted and removed after centrifugation. The sperm pellet is resuspended in an appropriate volume to obtain the highest sperm concentration possible, but not exceeding 80 x 10^6/ml. An aliquot of 0.2 to 0.5 ml of semen, depending on sperm concentration, is diluted to 10 ml with normal saline at room temperature. The tube is centrifuged at 800g for 10 minutes and most of the supernatant tipped off. The pellet is resuspended in the remaining saline, typically 20-40 µl, by gently tapping the tube. Then 5-10 µl of this suspension is placed on a glass microscope slide and the drop is spread across the slide with a pipette to make a smear as described below.

Preparation of smears. At least two smears should be made from the fresh semen sample for duplicate assessment and in case of problems with staining. The slides should first be thoroughly cleaned, washed in 70% ethanol and dried, before a small drop of semen (5 to 20 µl) is applied to the slide. If the sperm concentration is over 20 x 10^6/ml, then 5 µl of semen can be used: if the sperm concentration is less than 20 X 10^6/ml, then 10 to 20 µl of semen should be used. The 'feathering' technique whereby the edge of a second slide is used to drag a drop of semen along the surface of the cleaned slide may be used to make smears of spermatozoa, but care must be taken not to make the smears too thick. Feathering works well when viscosity is low but is often unsuitable for viscous semen. Alternatively, a drop of semen can be placed in the middle of a slide and then a second slide, face down, placed on top so that the semen spreads between them: the two slides are then gently pulled apart to make two smears simultaneously.

Diff-Quik     
The software is adjusted to be used with Diff-Quik staining. Diff-Quik is a rapid staining kit recommended by WHO and popular worldwide. Rapid staining methods are particularly useful for clinical laboratories that have high throughput. A Diff-Quik kit consists of:     
     
Fixative reagent (triarylmethane dye dissolved in methanol).
Staining solution 1, red (eosinophilic xanthene).
Staining solution 2, blue (basophilic thiazine).
 
How to use Diff-Quik:     
     

• Immerse slides prepared as described above in triarylmethane fixative for 15 seconds.
• Drain the excess solution by placing slides vertically on absorbent paper.
• Immerse in rapid stain solution 1 for 10 seconds.
• Drain the excess solution by placing slides vertically on absorbent paper.
• Immerse in rapid stain solution 2 for 5 seconds.
• Immerse the slide in running tap water 10 to 15 times to remove excess stain.

 
     To achieve a good staining, please follow the instructions supplied with your Diff-Quik kit!     
           
Vitality     
MMC Sperm semen quality analyzer provides two modes for sperm vitality analysis: automated and manual. In manual mode any available staining can be used: eosin, eosin-nigrosin, hypo-osmotic swelling (HOS) or fluorescence staining. Automated sperm vitality analysis method is based on eosin alone (Eosin Y, colour index 45380). This is the fastest and cheapest method of spermatozoa staining. Preparation of reagents:

• NaCl, 0,9% (w/v): dissolve 0,9 g NaCl in 100 ml purified water.
• Eosin Y, 0.5% (w/v): dissolve 0.5 g of eosin Y (colour index 45380) in 100 ml of 0.9% NaCl.
• Some commercially available eosin solutions are hypotonic aqueous solutions that will stress the spermatozoa and can yield false-positive results. To use such a solution, add 0.9 g of NaCl to 100 ml of solution to raise the osmolality.

How to prepare vitality staining:

• Mix the sample well but avoid bubbles.
• Mix 5 microliter of semen with 5 microliter of eosin solution.
• Prepare a smear as described above for sperm morphology smears.

Nomenclature related to semen quality
     Aspermia - no semen (no or retrograde ejaculation).     
     Asthenozoospermia - percentage of progressively motile spermatozoa (A+B classes) below the lower reference limit.     
     Asthenoteratozoospermia - percentages of both progressively motile (A+B classes) and morphologically normal spermatozoa below the lower reference limits.     
     Azoospermia - no spermatozoa in the ejaculate.     
     Cryptozoospermia - spermatozoa absent from fresh preparations but observed in a centrifuged pellet.     
     Haemospermia (haematospermia) - presence of erythrocytes in the ejaculate.     
     Leukospermia (leukocytospermia, pyospermia) - presence of leukocytes in the ejaculate above the threshold value.     
     Necrozoospermia - low percentage of live, and high percentage of immotile, spermatozoa in the ejaculate.     
     Normozoospermia - total number or concentration of spermatozoa, and percentages of progressively motile (A+B classes) and morphologically normal spermatozoa, equal to or above the lower reference limits.     
     Oligoasthenozoospermia - total number or concentration of spermatozoa, and percentages of progressively motile (A+B classes), below the lower reference limits.     
     Oligoasthenoteratozoospermia - total number or concentration of spermatozoa, and percentages of both progressively motile (A+B classes)and morphologically normal spermatozoa, below the lower reference limits.     
     Oligoteratozoospermia - total number or concentration of spermatozoa, and percentage of morphologically normal spermatozoa, below the lower reference limits.     
     Oligozoospermia - total number or concentration of spermatozoa below the lower reference limit.     
     Teratozoospermia - percentage of morphologically normal spermatozoa below the lower reference limit.

Automated measurements of sperm quality parameters and database fields     
This software measures a large set of semen quality parameters which we update on the regular basis. To send a parameter value to your database you have to create a numeric field with certain reserved field ID (internal field name that is being given while you create a new field, not to be confused with field label, which you can edit in field properties anytime). So, to add a new automeasurement result, you not only need to update the software (during updates, your database is not being changed automatically for security purposes) but also to add a new numeric field with given field name. This field starts to receive new measurement results. The table below represents the list of actual parameters being measured by the software and corresponding database fields.