SAMi is a CASA (computer assisted semen analysis) system developed by reproductive scientists for routine use in an assisted conception or pathology lab setting or for research.
Sperm quality data is validated and supported by peer reviewed publication showing parity with the haemocytometer (World Health Organization recommended). Manpower savings of on average 14-15 minutes per sample are achievable using SAMi when compared to WHO recommended (WHO, 2010) methods for sperm concentration and motility. With the bonus of considerably improved reproducibility and objective measures of motility.
- Microscope with phase contrast (usually x10 objective)
- Dedicated video camera (60 frames per second)
- PC (choice of mini hub, touchscreen all in one to suit laboratory)
- Edit screen before analysis (eliminates non sperm cells, optimizes detection) gives unrivalled sperm counting for CASA
- Improved detection improves reliability of motility
- Simple reporting system and storage of tracked or untracked video
- Availability of EQA (external quality assessment) scheme for users
Why automate semen analysis?
Almost all manual semen analysis methods lack accuracy and reproducibility and with poor standardization across the globe do not give confidence to users of any laboratory service whether they are patients or clinicians. Even those recommended by the World Health Organisation (WHO, 2010) are prone to error with especially motility being nothing more than an ‘educated guess’. The WHO (2010) continue to recommend the haemocytometer as method of choice and whilst the haemocytometer is not only relatively inexpensive and reliable, like any method it is not without limitations and associated error including: Differences in manufacturing standards, age and quality of chambers, application of the cover glass, to dilution and mathematics. All of these contribute to the ‘often’ poor results shown in external quality assurance (EQA) scheme where often wide-ranging sperm counts can be given for the same sample (see below)
EQA scheme in the UK showing a wide range of sperm counts for the same sample
In the absence of any other recommendation a good quality CASA system should be demonstrated as giving results which equate to the haemocytometer which is exactly what SAMi can do – see validation below.
Sperm motility is the only measure of sperm function incorporated into routine semen analysis yet recommendations for its estimation has arguably become less accurate over time. Uncertainty arises from the subjective grading of sperm swimming speed ‘by eye’ and the lack of an industry ‘gold standard’ methodology or suitable calibrant which would form the basis of any validation exercise. Even the experienced operator, studying motile sperm using microscopy cannot avoid focusing on a moving object or moreover studying the field for several minutes during which time many motile sperm will have entered and left. This leaves only the non-progressive fraction enumerated with any accuracy and an over-counting of motile sperm, which gets compounded in samples with higher density and sperm with high velocity.
Despite the demonstration over the years of the importance of sperm motility, the current WHO recommendation of reporting of only 3 grades of motility is misleading since it offers only the % of sperm swimming forward but not how well they swim and as a consequence reduced the measurement’s its clinical value. Reliable CASA such as SAMi is the only sensible means of obtaining objective measurements of sperm motility.
Any CASA has to demonstrate a high degree of ‘parity’ with measurements made by the haemocytometer as ‘gold standard’. This is not the same as correlation and any manufacturer of a sperm counting device which only shows correlation with the gold standard should be disregarded and must demonstrate sample by sample comparison.
Fundamentally if CASA cannot count sperm properly there are implications for the evaluation of other parameters as it reflects the inability to accurately identify objects under the microscope. Of course, during validation not every measurement will turn out the same for both methods just like they do not when you provide repeated haemocytometer readings as there are inherent errors associated with most methods which cannot be avoided. This is why in general we suggest that any unit trying to validate its CASA against the haemocytometer follows strict guidelines for reducing error and make use of the protocols listed in the WHO manual. Any taking of shortcuts or use of an alternative chamber to the haemocytomter will not be appropriate for validation purposes. Done carefully results similar to those shown in the figure below.
How can we validate motility? We are under no illusions that this is difficult since there is no traceable standard which we may use as a basis for calibration. If as we are to believe from the literature, motility grades should be based on swimming speeds then at least using the video each sperm. Essentially the still image from the below which represents a 1 second video loop can be played and the track length verified against the 25 micron scale (bottom right).
Limitations, help and optimizing performance
As with any methods of laboratory analysis there are always limitations of a CASA and understanding these will help users get the best out of the system. First and foremost, problems with image quality and especially microscopy will affect object recognition and therefore evaluation of every parameter. It is surprising just how many centres do not offer basic microscopy training and complain of poor image quality on their CASA but simply have poorly adjust phase contrast. Images such as the one shown below showing good contrast between background and white sperm heads are ideal
A clear limitation to its use in routine practice is related to the inherent biological variation in semen composition and the effect that this has on the image recorded by the video system. Indeed, the image obtained by the microscope and camera is key to the satisfactory detection of sperm, even to the level of how grey is the background field. Therefore analysis of, for example washed sperm suspensions will often be very clear and consistent with excellent sperm detection but, adjustments must be made for any system to account for the effect that seminal plasma and other contaminants may have on image quality.
Specimens will demonstrate variation in
- Viscosity, presence of gelatinous bodies or mucin
- Sperm head size, shape, optical appearance and consequently - detectability
- Dynamics of chamber filling
- Presence of non sperm cells including leucocytes and germinal cells
- Presence of agglutination, aggregation and debris
- Higher sperm concentration/motility
The operator must have some idea of how all of these factors may affect the eventual outcome. Sperm must be allowed to settle adequately before evaluation takes place in order that immotile sperm are not inappropriately classed as motile. The presence of non-sperm cells or debris will in some cases be erroneously identified as sperm and must have trackers removed within review screen, if they are not excluded adequately by ‘gaiting’. Agglutination and aggregation will affect the degree of homogenisation and therefore accuracy of any analysis, be it manual or automated and in the worst-case scenario no reliable analysis can be made no matter what method is used.
Accuracy will also be affected by having too few sperm evaluated, too many to evaluate in a single field or indeed the motility is so vigorous as to cause excessive collisions either sperm with other sperm or sperm to non sperm cells.
All testing has been completed using a sample minimum of 200 sperm in line with WHO recommendations and have clearly provided adequate results. However it is also patently clear that increasing the cell number per analysis is permitted with automation and the following statements should be made clear to the user
Reliability of measurements will increase if
- The system is operated by trained scientific staff
- The settings are checked and the system regularly calibrated using QC (quality control) beads
- The microscope image is optimised to obtain suitable contrast
- The number of individual slides/samples taken from the specimen is increased
- The number of microscopic fields analysed is increased
- The number of sperm analysed per test is increased
- Specimens/samples are well mixed
- Samples are diluted in a suitable medium (homologous seminal plasma recommended) if the initial sperm concentration measurement exceeds 75-80 millions per ml
- User intervention during the review screen is increased, particularly where there are high levels of cell debris, agglutination/aggregation and non sperm cells.
Special features of new SAMi
SAMi is available on a very flexible platform and we can provide a PC/microscope combination which best suits your lab. Micro PCs which can be easily located under benches with screen attached to a wall. Alternatively, you can use a conveniently sized all-in-one touchscreen PC.
Recent additions to functionality include
- Edit feature to maximise sperm detection and sperm count accuracy
- Variable sensitivity - improves pick-up rate and reduces operator intervention*
- Embedded image into report page showing tracked sperm (below)
*With any CASA there is sometimes a ‘trade-off’ between detecting ALL sperm in a field and ensuring that debris is not detected in error. This feature allows moves the ‘gates’ to either ignore debris completely or occasionally if sperm heads are so small as to resemble debris the gates may be changed as shown in the figure below.
A simple database is provided with SAMi but data can be exported into its simplest format (.csv) for incorporation into other data management software
Summary reports can also be accessed in a separately stored folder alongside video stored for every analysis
Saved data for any analysis in the form of a. video files b. summary notepad and c. track data - showing X,Y plots for 60 frames of video for every sperm per field analysed (very useful for researchers wanting to examine basic kinematic parameters in its most raw form and without pre-definition
EQA scheme - see Sperminator/SAMi EQA
The Sperminator/SAMi EQA (external quality assessment) scheme is for users of this CASA system only. EQA schemes which send out physical semen specimens have demonstrated poor levels of agreement between centres and as a result fail to give the intended level of confidence in the service. This is partly due to the variation associated with different methods or application of methods but also because of the significant increase in the level of aggregation observed in pooled fixed specimens as shown below.
Highly aggregated EQA specimen which cannot be assessed accurately by an method which relies on homogeneous cell distribution. CASA users should examine aggregation-free fields but this results in method bias.
With this in mind we have developed a simple scheme based on the analysis of short video clips which have been generated by previous tests.
To find out more please contact the team at IVFsynergy on email@example.com