Internal Quality Control and External Quality Assurance in The IVF Laboratory

Internal quality control and external quality
assurance in the IVF laboratory

Phillip L.Matson1
Concept Fertility Centre, King Edward Memorial Hospital, Bagot Road,
Subiaco, Western Australia 6008, and External Quality Assurance Schemes
for Reproductive Medicine, PO Box 1101, West Leederville,
Downloaded from https://academic.oup.com/humrep/article/13/suppl_4/156/611170 by guest on 01 June 2021

Western Australia 6901
'To whom correspondence should be addressed at: Concept Fertility Centre,
King Edward Memorial Hospital, Bagot Road, Subiaco, Western Australia 6008
The existence of internal quality control programmes and external quality

assurance schemes is important in enabling the maintenance of good service
to patients. All aspects of our work in laboratories involved in the diagnosis
and treatment of human infertility can benefit from such programmes and
schemes, moving the work from being a subjective art form to an objective
science. Equally, many clinical procedures are amenable to such scrutiny.
Acceptance and introduction of such schemes and programmes will rely
initially on the self-motivation of the laboratories themselves and then
pressure brought to bear by accrediting authorities.
Key words: control/IVF/quality assurance
Introduction
Despite recent clinical advances in ovarian stimulation regimens and surgical

procedures to collect gametes, successful assisted reproductive technology remains
the province of the clinical scientist in the organization and running of a high
grade laboratory. Poor laboratory technique will invariably reduce the number of
women that become pregnant, and sloppy techniques may have disastrous
consequences (Linden and Critser, 1995; van Kooij etai, 1997). The establishment
of formal training protocols for laboratory staff, particularly those in an in-vitro
fertilization (IVF) laboratory (Dawson et al., 1996), and the requirement by
regulatory authorities of standard operating procedures (Visscher, 1991; Pool,
1997) are ways in which the infrastructure of good practice can be set in place.
However, attention to quality is a dynamic exercise and the present review will
concentrate on tools currently available to help scientists within the IVF laboratory
maintain the required levels of performance.
Terminology and concepts used in the monitoring of performance
High quality work in the laboratory is not something that happens spontaneously,
but rather requires the development of skills and then continuous monitoring of
156 © European Society for Human Reproduction and Embryology Human Reproduction Volume 13 Supplement 4 1998
Quality control, quality assurance and IVF
performance. This attention to the quality of our work has many facets, and the
terms given below reflect the different perspectives that we may take although
their definitions often overlap.
Quality control
This includes the establishment of a quality standard or specifications for each

aspect of the testing procedure, determination of how close to the quality standard
the testing procedure is, and then taking any necessary corrective actions to bring
the procedures up to the required standard. In practice, internal quality control
is designed to check that a laboratory will produce the same result or outcome
if the test or procedure is done on different occasions (within-laboratory variation),
or by different technicians (between-operator variation).
Quality assurance
This is the overall surveillance of everything to do with quality throughout the
laboratory or clinic. This expansive term not only encompasses quality control
of the testing process, but involves monitoring and control of the ultimate
outcomes of the testing process, including aspects such as clinical relevance of
the tests, reporting accuracy and user satisfaction. In practice, external quality
assurance enables the laboratory to test the same samples or undertake the same
procedure as other laboratories and to evaluate their performance against that
of others.
Quality audit
This is the review of the prescribed procedures, often in the form of checking
the standard operating procedures or laboratory manual and re-arranging the
procedures if required.
Internal quality control
The monitoring of equipment to ensure performance at a set level is very

important, so that the temperature of incubators (Ambramcszuk and Lopata,
1986), accuracy of pipettes, environmental conditions (Cohen et al., 1997), etc,
are all measured and recorded at regular intervals. Moreover, schemes for the
appraisal of one's own performance on a day-to-day basis are commonplace in
routine pathology laboratories (Petersen et al., 1996), and usually take the simple
form of analysing an aliquot of a sample from a stored pool. The results obtained
should then be within accepted limits of variation. Many of the procedures
undertaken in the assisted reproduction laboratory were often regarded as more
art than science and, as such, were not amenable to scrutiny and evaluation.
However, the current view is that the same attention to detail and appraisal
157
P.L.Matson
should exist. Different approaches are often needed for the diagnostic and
therapeutic procedures undertaken in the laboratory, and examples are given
below of the assessment of variability with various commonplace techniques.
Diagnostic testing
Semen analysis is a test which is beset with problems, not least of which is the

biological variability seen between samples from the same patient (Schwartz
et al, 1979) and the poor correlation between the results and the prediction of
fertility (Polansky and Lamb, 1988). Nevertheless, there is an absolute number
of spermatozoa in the fluid of each ejaculate and so an accurate and precise
analysis is possible (Comhaire et al, 1992), as is the use of internal quality
control programmes (Clements et al, 1995). Technical variables clearly influence
the results obtained and so the initial choice of methodology is important; such
concerns have resulted in the introduction of a series of international manuals to
help standardize methodology (e.g. World Health Organization, 1992). Specific-
ally, the choice of counting chamber is important in the determination of sperm
concentration (Ginsburg and Armant, 1990; Imade et al., 1993; Shiran et ah,
1995; Seaman et al., 1996; Mahmoud et al., 1997). Latex beads supplied at a
defined concentration can then be used to validate one's own counting chamber
(Peters et al., 1993). The criteria for sperm normality affect the proportion of
spermatozoa with normal morphology (Comhaire etal., 1994), and the temperature
at analysis affects sperm motility characteristics (Birks et al., 1995). Once a
particular method has been introduced in the laboratory, care must be taken to
ensure that errors are not introduced over time. There are many instances in the
literature in which errors have been detected in the performance of semen
analysis by internal quality control procedures and then corrected (Mortimer
et al., 1986; Dunphy et al, 1989; Knuth et al, 1989; Cooper et al, 1992;
Clements et al, 1995). The detection of antisperm antibodies is equally amenable
to standard internal quality control checks by the use of positive and negative
control samples at each assay (Junk et al, 1986), as well as other tests such as
the acrosome reaction to ionophore challenge (ARIC) test (Troup et al, 1994).
Therapeutic procedures
The control of methods to count spermatozoa accurately is also important in
treatments whereby a given number of spermatozoa are to be used for insemina-
tion, e.g. IVF and gamete intra-Fallopian transfer (GIFT), or the precise number
used is to be documented, e.g. during intrauterine insemination or donor
insemination. However, the main thrust of quality control in therapeutic procedures
is to ensure that pregnancy rates are kept at a maximum level. Pregnancy rates
are often reviewed at regular time intervals, but this form of audit has major
limitations, namely: (i) the chance of pregnancy can be influenced by the
underlying pathology of the patients; (ii) a drop in pregnancy rates means that a
158
problem was encountered at the time of treatment, usually a few weeks before,
and (iii) the actual problem is not revealed.
There has thus been the introduction of a range of bioassays to detect toxicity
and sub-optimal culture condition for gametes and embryos, such as human
sperm survival studies which identify the demise of spermatozoa over several
days in culture (Critchlow et al, 1989), hamster sperm survival which can
identify problems within a matter of h due to the increased sensitivity of these
spermatozoa (Bavister and Andrews, 1988), and the culture of mouse embryos

from either the 1-cell or 2-cell stage (Ackerman et al, 1984; Davidson et al,
1988; McDowell et al, 1988; Fitzgerald Scott et al, 1993) or somatic cell lines
(Bertheussen, 1989). These bioassays are just as informative and far more ethical
than animal testing (Wilsnak et al, 1973). Whilst these systems are not assured
of detecting subtle levels of toxicity, and the mouse embryo culture system in
particular has been shown to have limitations (Fleming et al, 1987; George
et al, 1989), there are several instances reported where the bioassays did help
to identify specific problems (Howell and McDermott, 1983; Kristensen et al,
1985; Kruger et al, 1985; Ray et al, 1987; Schiewe et al, 1988; Stewart-Savage
and Bavister, 1988; Harrison et al, 1990; Montoro et al, 1990). The general
principle seems to be that all new items introduced into the IVF laboratory
should be screened for toxicity using a bioassay or similar (Parinaud et al,
1987); laboratories should not relax, simply because many products designed for
use in assisted reproduction are now purchased from reputable suppliers.
External quality assurance
Participation within a recognized external quality assurance (EQA) scheme has

many benefits, including information on the relative performance of different
methods, and knowledge about one's own ability to perform tests and report
results accurately, as well as gaining the confidence of clinicians and patients.
Diagnostic procedures
Manuals are available to help standardize the use of diagnostic tests in the
investigation of the infertile couple (Rowe et al, 1993). However, despite this,
practices are still variable (Helmerhorst et al, 1995) and the interpretation of
the results can vary dramatically for the same technique, as illustrated by the
example of the post-coital test (Oei, 1996). An attempt to help laboratories
compare their performance of semen analysis against others was first made by
Neuwinger et al. (1990). Swim-up preparations of spermatozoa were resuspended
in phosphate-buffered saline and distributed for the estimation of sperm concentra-
tion; fixed slides of spermatozoa were also prepared and distributed for assessment
of sperm morphology. The shipment of frozen spermatozoa between laboratories
has enabled laboratories to analyse aliquots of the same sample (Walker, 1992).
The first major attempt to establish an EQA scheme for semen analysis was then
159
P.L.Matson
12
10
8
Makler Haemocytomete sr
6
4
2 _ _ ^
v
0 mmd I ! mMmm
10 (15,20] (25,30] (35,40] (45,50] <= 10 (15,20] (25,30] (35,40] (45,50]
(10,15] (20,25] (30,35] (40,45] > 50 (10,15] (20,25] (30,35] (40,45] > 50

10 (15,20] (25,30] (35,40] (45,50]
(10,15] (20,25] (30,35] (40,45] > 50
sperm concentration (millions per ml)
Figure 1. The frequency distribution of results for sperm concentration when 39 laboratories analysed the
same semen sample. Results are expressed according to the counting chamber used.
made by Matson (1994), who distributed samples of semen containing preservative

for the assessment of sperm concentration and morphology. Serum samples were
also sent for the estimation of antisperm antibodies. A similar and more extensive
scheme has been established in Australia following a successful pilot study, and
details can be found at the website of Scientists in Reproductive Technology
(http://argo.net.au/tigers), the group of scientists within the Fertility Society of
Australia. When different laboratories are asked to analyse the same semen
sample, there is variability in the concentration of spermatozoa found as illustrated
in one of the results from the Australian pilot study shown in Figure 1. A more
striking variation was seen in the proportion of normal forms in the same sample
as an estimation of sperm morphology reported by the different laboratories
(Figure 2). However, the good correlation when two replicate samples were
analysed by the laboratories (Figure 3) would suggest real differences in the
notion of a normal spermatozoon, despite the supposed use of the same criteria,
rather than the use of sloppy technique. A perennial problem for these schemes
is determining what is the correct answer and then judging who is right and who
is wrong in their reported results. One must be careful in not simply taking the
results of a few selected and trusted laboratories and holding them up as the
'gold standard'. Differences in methodology can account for real differences in
results over and above that due to poor quality of work. This was typified by
the EQA schemes offered for the measurement of luteinizing hormone, in which
radioimmunoassay was regarded as the method of choice with immunometric
assays being new. Immunometric assays initially gave results that were generally
higher and regarded with suspicion, although the merits of immunometric assays
and the problems of radioimmunoassays have subsequently been recognized
160
r WHO RCPA In-house
-1 n
<= 10 (20,30] (40,50] (60,70] (80,90] <= 10 (20,30] (40,50] (60,70] (80,90] <= 10 (20,30] (40,50] (60,70) (80,90]
(10,20] (30,40] (50,60] (70,80] > 90 (10,20] (30,40] (50,60] (70,80] > 90 (10,20] (30,40] (50,60] (70,80] > 90

<= 10 (20,30] (40,50] (60,70] (80,90] <= 10 (20,30] (40,50] (60,70] (80,90]
(10,20] (30,40] (50,60] (70,80] > 90 (10,20] (30,40] (50,60] (70,80] > 90
Proportion of sperm with normal morphology (%)
Figure 2. The frequency distribution of results for sperm morphology when 39 laboratories analysed the
same semen sample. Laboratories were asked to report the proportion of spermatozoa with normal
morphology, and the results are expressed according to the criteria of normality used.
y=0.944x+0.076
110
10 20 30 40 50 60 70 80 90
sample CM0005 (% normal)
Figure 3. A scattergram showing the proportion of normal forms when sperm morphology was assessed on
the same sample twice. Laboratories were told they were different samples.
(Seth et al., 1989). Results for the antisperm antibody samples in the Australian
scheme would suggest that there are differences in the detection of antibody
activity by different methods. For instance, Table I shows the results when a
serum sample and a seminal plasma sample from a vasectomized man were
analysed. Some methods, like the mixed agglutination reaction (MAR) test which
detects only immunoglobulin G (IgG), identified antibodies in the serum but not
the semen whereas the immunobead tests found antibodies in both. Obviously,
161
P.L.Matson
Table I. The proportion of laboratories in the Australian external quality assurance (EQA)
schemes that analysed samples and found significant levels of antisperm antibodies present.
Results are expressed according to the method used when testing serum and seminal plasma
from a man vasectomized 3 years previously
Method used Serum Seminal plasma
Immunobead test 7/13 10/14

Tray agglutination test 0/1 0/1
MAR test (IgG) 2/3 0/3

Sperm immobilization test 0/4 0/3
Gelatin agglutination test 1/2 2/2
MAR = mixed agglutination reaction; Ig = immunoglobulin.
Table II. The results of one distribution from the Australian external quality assurance (EQA)
scheme for cytotoxicity testinga
Method used 1 ml syringe barrel 1 ml syringe plunger
Human sperm survival 1/4 3/4

Mouse 2-cell embryos 0/2 2/2
Total no. of laboratories 1/6 5/6
detecting toxicity
a
Six laboratories were sent parts from a 1 ml syringe and asked to test the items using their
bioassay, and the results show the number of laboratories which detected toxicity. Four
laboratories used human sperm survival studies, and two laboratories cultured 2-cell mouse
embryos to blastocyst.
the presence of IgG in serum but not seminal plasma, as with the MAR test,
may be physiologically correct.
There is obviously much work to be done in the standardization of methods
and results, with EQA schemes being a tool to help the laboratories resolve these
issues. The diagnostic implications for patients are obvious, but the validity of
many of the scientific papers that claim to compare results of treatment for
different diagnostic categories must be viewed with a certain suspicion. Is this
one reason why agreement in so many areas is not seen?
Therapeutic procedures
Internal quality control procedures have been outlined above which can be used
to identify problems of toxicity in the laboratory, but are they reliable and do
they each have the same ability to identify low levels of toxicity? This is now
able to be addressed by the use of an EQA scheme run in Australia whereby
items are distributed to laboratories for testing (enquiries to EQASRM, PO Box
1101, West Leederville, Western Australia 6901). An example of the results
obtained are given in Table II, where laboratories were asked to test the
components of a 1 ml syringe, finding the plunger toxic but not the barrel.
Greater disagreement between methods may well be seen with different test
samples, as the sensitivity of the different methods probably differ. Another EQA
162
Table III. Results from the Australian external quality assurance (EQA) scheme for assessment
of embryo gradinga
Description Human embryo Human embryo Human embryo Human embryo

frozen at 4-cell frozen at 4-cell frozen at 2-cell frozen at 2-cell
No. cells seen:

0 0 2 0 0
1 12 10 0 12
2 0 0 12 0

You would transfer this embryo:
alone 5/12 5/12 12/12 11/12
with a good one 6/12 5/12 12/12 10/12
a
Videos of four frozen/thawed human embryos were sent to 12 laboratories, and the laboratories
asked how many intact cells there were and whether the embryo would be transferred if it were
alone or if it was accompanying a good embryo. Results show the number of laboratories giving
a particular response.
scheme operating from Australia helpful to the therapeutic laboratory is that of

embryo grading, and an example of the results is given in Table III. In essence,
videos of human embryos were distributed and laboratories asked specific
questions. It can be seen that in this instance there are clear differences in
strategy, such that approximately half the laboratories would transfer a poor
quality embryo (i.e. one out of four cells surviving) and half not, but all would
transfer a good embryo (one out of two or two out of two cells surviving).
Conclusions
Laboratories staffed with properly trained personnel, operating with a close

attention to issues of quality, are crucial in maintaining high pregnancy rates
following assisted reproduction. The use of internal quality control procedures
and external quality assurance programmes are useful tools in monitoring the
performance of the laboratory.
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Downloaded from https://academic.oup.com/humrep/article/13/suppl_4/156/611170 by guest on 01 June 2021

The existence of internal quality control programmes and external quality

Despite recent clinical advances in ovarian stimulation regimens and surgical

Terminology and concepts used in the monitoring of performance

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