The QAPonline administrator largely defines the choice of method for assessments largely based upon experience and on feedback from participants. In some EQA Schemes there is no defined numeric based method such as in Embryology or Ultrasound. In others, the manufacturer has defined the methodology.
Notes
3.05.1
Internet Image Based Methodology versus Sample Based Methodology.
A primary departure in methodology for QAPonline is the use of image based processes as opposed to sample based processes. For some EQA disciplines such as Embryology or Ultrasound, a sample based EQA is not possible while for others such as Sperm DNA Fragmentation or AMH it is imperative. For others such as Andrology, there is an option to utilise either process. QAPonline has decided to use the image based process rather than the sample based processes for Andrology both to follow the line developed for Embryology and because of issues with samples currently in use for Adnrology EQA schemes elsewhere.
Updated: 1/11/2012
3.05.1.1
VISUAL basis for assessment. Unlike biochemical and similar analyses, Andrology and Embryology are largely visual based assessment. Even though there are methods for the computerisation of Semen Analysis, in may laboratories and indeed as the basis for all assessment is the visual interpretation of the sample rather than the mechanical preliminary steps prior to the inspection that are critical and where the majority of the variation lies in the generation of the final result.
3.05.1.1
An argument for sample based processes is that in the laboratory, a sample is mixed, pipetted, stained, etc before examination and the EQA scheme should reflect these technical steps in addition to the visual skills of the operator. In the assessment of sperm morphology, the WHO manual argues that an initial assessment is required for the determination of the amount of dilution that is required for the accurate measurement by haemocytometer. QAPonline acknowledges that only a sample based process can monitor this step and that some aspects of sample handling cannot be replicated by image based processes.
3.05.1.1
An argument against sample based processes primarily includes the variation due to the problem of repeated re-suspension a fixed sample where there has been significant agglutination of cells. Secondly, in the vast majority of samples, a standard dilution is used and the loading of the sample is relatively simple and uncomplicated. The largest variation is thought to be the counting and dilution calculations and this can be tested by image based processes.
3.05.1.1
A further issue involves the staining of sperm for morphology where there are varying methods for staining each of which may cause varying distortion of the sperm head. One argument is that is should be included in the EQA assessment since this is what happens on the bench. WHO recommends a standard staining method which is believed to generate cells of similar proportions and it is these methods that all standards are based. Quick staining methods are not encouraged for several reasons and any clinic using such methods must be able to justify the correlation between the standard methods and their own method. Regardless, the identification of a "normal" to an "abnormal" sample is based upon the WHO recommendation of shape.
3.05.1.1
Therefore, an image bases processes cannot replicate the few mechanical steps in assessment but does focus on the visual interpretive skills of the participant. Since many argue that this the largest source of variation between scientists and clinics, focusing on this aspect provides the greater opportunity to improve the clinics performance.
3.05.1.1
Finally, since QAPonline aims to deliver EQA services to an international clientele, delivery of sample based material internationally becomes prohibitively expensive.
3.05.2
Method Variables.
In 2011, QAPonline introduced a methods variable to each EQA Series. The list of current method variables available are found on the link below.
The concept for the introduction of the methods variable was developed in response to the AMH and the Sperm DNA Fragmentation Assay but was used also in the Sperm Morphology EQA Scheme.
3.05.2.2
The methods listed for the EQA Series is available at the time of enrolment and is allocated to a specific participant. The method can be changes at anytime if need be AND is allocated to the REPLY table at the time of data submission.
3.05.3
Andology.
The primary methodology for Semen Analysis is the World Health Organisation publication - WHO laboratory manual for the Examination and processing of human semen. There are various editions to this manual and disappointingly, there are some changes between the editions especially for the definition of sperm morphology. These variations exist largely in the definition of what is the normal range and not so much on methodology.
Concentration. 5th edition of WHO recommends only the use of haemocytometer suggesting all others tools are incorrect. QAPonline provides an image based haemocytometer EQA scheme and a Makler based EQA scheme since the majority of IVF clinics use a Makler for ease and speed. SIRT/NATA recognises this and indicates that if a tool other than a haemocytometer is used then the clinic needs to show a comparative dataset.
3.05.3.2
Motility. There were initially 4 grades of motility fast, average, slow, non-progressive and non-motile. Since the motility of a spermatozoon depends on temperature and space to swim, adn there was considerable variation in how one separated fast from average speed, WHO5 (5th edition) has removed the fast option and now has 4 speeds- progressive, slowly progressive, non-progressive and non motile. QAPonline has included all these estimates of motility as an educational tool and uses the % total motility as the KEY QAP question. This is the basic assessment of motility and common for WHO4 and WHO5.
3.05.3.3
Morphology is the most difficult parameter to assess in an ejaculate. Based upon experience and training, each operator needs to decide on shape alone if the sperm has normal morphology. There have been various definition in the past but that based upon sperm in cervical mucus (Kruger Method) has become the common standard. Implementation of this can be strict (in which cases few sperm are normal) or less strict (where more sperm are normal)is variable as is the rules for the normal range. QAPonline has 2 EQA schemes one for WHO4 (HAA - less Strict) and WHO5 (HAC - more strict) to allow participants using either of these two standards to participate independently. Minor society based variations are accommodated using the methods tool.
3.05.3.4
AntiSperm Antibodies in semen is assessed by the degree of binding of IGA,IGA beads to sperm if the sperm is coated with antibodies. Some EQA schemes use serum of either positive or negative antibody presence. QWO 4/5 indicate a normal sample is one where more than 60% of motile sperm have two or more beads attached. QAPonline has focused on the reading of the sample by supplying a video with immunobeads and motile sperm and where the KEY QAP Question is what is the % binding.
3.05.3.5
Sperm DNA Fragmentation estimates the degree that ejaculated sperm DNA is fragmented. This is a recent test but is included on NATA list of test that may be accredited. There are several methods including TUNEL (several methods), Sperm Chromatin decondensation Assay (SCSA), Halosperm Assay and COMET assay. The halosperm assay is an image based EQA scheme (see below) while the others are a sample based EQA system.
3.05.3.6
Halosperm Assay for Sperm DNA Fragmentation. This is a commercial assay from Spain and involves the dispersion and staining of Sperm DNA in a gel where the measure is the diameter of the spreading of the DNA in the gel. the greater the spread, the less DNA fragmentation is present. QAPonline presents a slideshow of the stained slides and ask for the % sperm with DNA Fragmentation as per the manufacturers recommendations. The EQA does not cover the initial preparation.
3.05.4
Embryology
There are no clear standards for describing embryos even though there have been several books illustrating what are considered to be healthy and abnormal embryos at each stage of their development. The real problem is that morphology is loosely linked to pregnancy and that the methodology is not described in publications to indicate the rules for assessment. Neither has there been a tool to convert embryo assessment into a numeric description that will allow analysis. QAPonlines appoach is to use the commonest descriptors for an embryo at each stage of it's development and using weighted options construct a value to a value between 0 and 100 and use this construct as the KEY QAP Question for each stage. QAPonline has an EQA Scheme for each stage of embryo preimplantation development.
Updated: 7/11/2012
3.05.4.1
Oocytes. QAPonline uses the ESHRE/ALPHA guidelines for the description of oocytes, using these parameters to build a calculated numeric KEY QAP value.
3.05.4.2
PRONCULEAR stage (day 1 post fertilisation). Oocytes. QAPonline uses the ESHRE/ALPHA guidelines and those described by Lyn Scott for the description of pronuclear, using these parameters to build a calculated numeric KEY QAP value.
3.05.4.3
EARLY CLEAVAGE (between days 2 and 3). QAPonline uses the ESHRE/ALPHA guidelines for the description of day 2 and day 3 cleavage stage embryos, using these parameters to build a calculated numeric KEY QAP value.
3.05.4.4
Fragmentation. There are no rules for assessing fragmentation even though it is the cornerstone for all embryo grading schemes. QAPonline asks a simple question - what is the % fragmentation as the KEY QAP Question.
3.05.4.5
Advanced Cleavage (Morula and Blastocyst days 4 - 6). QAPonline uses the ESHRE/ALPHA guidelines for the description of day 4 and day 5/6 cleavage stage embryos, using these parameters to build a calculated numeric KEY QAP value.
3.05.4.6
Ranking of Oocytes and Embryos. Introduced as a trial scheme in 2012, the Ranking EQA scheme seeks to emulate and quantitate the ranking efficiency of embryologists. The scheme covers oocytes, pronuclear, cleavage and blastocysts stages.
3.05.5
Ultrasound
There are no other EQA schemes examining follicle diameter variability. QAPonline has developed a multiple image based system where individuals needs to rotate through he images to select one that best illustrates the largest diameter (similar to what an ultrasonographer does with the US probe). When selected QAPonline provides tools to measure the distance between 2 points (using a pixel calculator) and a screen standardisation tool.
Updated: 5/11/2012
3.05.5.1
There is no standard method for measuring follicle diameter nor endometrial thickness. Rather there are publications where methods are described and used in most clinics. In real time ultrasound, the technician may re-orientate the probe between several orientations to perform measurements. However, in most clinics with severe time pressures, most will select one perspective that gives what they consider to represent the largest diameter and make all their measures form the static image.
3.05.5.1
Follicle diameter EQA. QAPonline provides for up to 4 diameters to be made on a 2D image before being averaged. From experience, many scanners perform only 1 diameter and there is no data to suggest more than 2 diameters improves the estimate.
3.05.5.1
Endometrial thickness is normally measured from the two outer echogenic bands that describe the outer vascular layer on the endometrium. QAPonline provides up to 4 estimations to measure thickness even though most operators usually only perform 1 estimate. The pattern of endometrial develop follows commonly used terminology of 2-3 patterns loosely describing the number of echogenic layers and the intensity of the central layer.
3.05.5.4
The users of the Ultrasound EQA scheme are largely nurses charged with the responsibility of performing follicle and endometrial scans. Very few ultrasonographers (if any) participate , it being the administrators opinion they cannot see the value in it since they think they are always correct! Attempts to procure the participation of skilled ultrasonographers to act as peer participants have been successfully unsuccessful!!
3.05.6
Endocrinology (AMH online)
QAPonline was asked to set up a AMH (Anti Mullarian Hormone) assay in 2011 on a trial basis following the rapid use in IVF programmes to predict the likelihood of hyperstimulation.
Updated: 5/11/2012
3.05.6.1
The Scheme is made available only to interested parties to check how their assays compared to other clinics ON A TRIAL BASIS ONLY. It used pooled IVF human follicle phase serum samples to generate a number of samples that covered the range of normal samples expected from an IVF population. The samples came from women who have been tested for HIV and Hepatitis B & C during their routine monitoring and stabilised using the bacterostatic ProClin 150 (Sigma). The AMH of each serum sample was known and those samples of similar value were pooled to produce 10 pooled populations.
3.05.6.1
Each pooled primary sample was then aliquoted into smaller 0.8ml samples in 2l Cryovials. Sets of 10 test samples were then stored at -20C until dispatch which was usually in the first 2 weeks of January by overnight courier in special post approved sample tubes with frozen ice pack. The overnight delivery was confirmed the following days.
3.05.6.2
There were 10 monthly samples for the months February to November and were repeated in a different order between 2011 and 2012.
