Clinica Chimica Acta 423 (2013) 56–61

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Clinica Chimica Acta

journal homepage: www.elsevier.com/locate/clinchim

Invited critical review

Using an algorithmic approach to secondary amenorrhea: Avoiding

diagnostic error
Tiffany K. Roberts-Wilson a,⁎, Jessica B. Spencer b, Corinne R. Fantz a
a
Department of Pathology and Laboratory Medicine, Emory University, Atlanta, GA, United States
b
Department of Gynecology and Obstetrics, Emory University, Atlanta, GA, United States

a r t i c l e i n f o a b s t r a c t

Article history: Secondary amenorrhea in women of reproductive age may be an indication of an undiagnosed, chronic
Received 26 November 2012 condition and appropriate treatment is dependent upon accurate diagnosis of the underlying etiology. A
Received in revised form 2 April 2013 thorough clinical assessment and a few common laboratory tests can easily identify the most frequent causes
Accepted 3 April 2013
of secondary amenorrhea. However, once these have been ruled out, the more uncommon pathophysiologies
Available online 12 April 2013
can be difficult to diagnose due to similarities in presentation and appropriate laboratory testing and inter-
Keywords:
pretation become critical. In these cases, misdiagnosis is unfortunately common and often the result of
Secondary amenorrhea poor laboratory utilization in the form of a failure to employ indicated tests, the use of obsolete tests, or
Amenorrhea erroneous interpretation in the face of interfering factors or co-morbidities. Consequently, the algorithmic
Laboratory testing algorithm approach to laboratory evaluation in the context of secondary amenorrhea described in this review can
Hypothalamic amenorrhea minimize the risk of diagnostic error as well was decrease test volume, cost, and time to diagnosis.
Clinical laboratory utilization © 2013 Elsevier B.V. All rights reserved.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 56
2. Laboratory investigation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57
2.1. Pregnancy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58
2.2. Hypothyroidism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58
2.3. Hyperprolactinemia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58
2.4. Hypopituitarism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58
2.5. Polycystic ovary syndrome . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58
2.6. Late-onset congenital adrenal hyperplasia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59
2.7. Cushing's syndrome . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59
2.8. Primary ovarian insufficiency . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59
2.9. Hypothalamic amenorrhea . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59
3. Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60

1. Introduction experienced irregular menstrual cycles, secondary amenorrhea is the

Approximately 5% of women of reproductive age will experience
secondary amenorrhea [1]. In women who had previously experi-
enced regular menstrual cycles, secondary amenorrhea is the absence
of menstruation for three months [1]. In women who had previously
⁎ Corresponding author at: Department of Pathology and Laboratory Medicine, Emory
University School of Medicine, 1364 Clifton Rd., Rm. G163, Atlanta, GA 30322, United
States. Tel.: +1 404 712 1875.
E-mail address: tkrober@emory.edu (T.K. Roberts-Wilson).
absence of menstruation for a year (12 months) [1]. While short-term
absence of menses is rarely a cause for concern, amenorrhea of longer
duration may indicate the presence of disease or a chronic condition
that, left undiagnosed, could cause obesity, sexual dysfunction, infer-
tility, osteoporosis, endometrial hyperplasia, or endometrial cancer.
Secondary amenorrhea is a symptom that can be caused by many path-
ological states and some patients will not demonstrate an obvious
etiology, therefore, correct diagnosis requires a systematic evaluation.
Evaluating a patient for secondary amenorrhea and its underlying
cause begins with a careful history and physical examination. Family

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 T.K. Roberts-Wilson et al. / Clinica Chimica Acta 423 (2013) 56–61 57

history of genetic anomalies, diabetes, thyroid disease, etc. can be
especially helpful in reaching a correct diagnosis and often will great-
ly narrow the differential in these patients. Physical examination is
primarily performed to rule out any sort of anatomical cause, but
can be critical in identifying other causes as well. For example, overall
physical condition and body mass index (BMI) can be helpful in iden-
tifying functional hypothalamic amenorrhea, also known as “the
female athlete triad”. Furthermore, signs of hirsutism or virilization
can be indicative of polycystic ovary syndrome (PCOS). Nonetheless,
laboratory testing is necessary to definitively diagnose the underlying
cause of secondary amenorrhea.
2. Laboratory investigation
The most common causes of secondary amenorrhea can be easily
identified and/or ruled out with laboratory testing of human chorionic
gonadotropin (hCG), thyroid stimulating hormone (TSH), luteinizing
hormone (LH), follicle stimulating hormone (FSH), and prolactin levels
as discussed below (Fig. 1). Once these causes have been ruled out,
more complex laboratory investigation becomes necessary. Approxi-
mately 44% of diagnostic errors are made at the level of laboratory test-
ing [2]. In some cases, the required follow-up testing is not ordered at all
or, when it is, tests that are obsolete or even inappropriate may be in-
cluded [3,4]. With the number of orderable laboratory tests increasing
at an alarming rate, it can be a challenge for front-line clinicians to
maintain familiarity with tests that are not utilized frequently. Unfortu-
nately, in the context of secondary amenorrhea misdiagnosis of the un-
derlying pathology is not infrequent [5–9]. An algorithmic approach to
the laboratory evaluation of secondary amenorrhea, in which negative
results for first-line tests will reflex to more complex testing, would
simplify ordering for the physician and would be more convenient for
the patient [10,11]. This approach would also decrease costs as only
one office visit would be required even if more complex testing was
needed. Furthermore, the length of time to results and diagnosis

Fig. 1. Algorithm for laboratory evaluation of secondary amenorrhea. Laboratory investigation of secondary amenorrhea should be done in a step-wise fashion. The suggested order
of evaluation is shown here and it is recommended that a panel of basic tests be performed at the initial visit to allow more specific testing, or perhaps a diagnosis, upon the
following visit in the secondary care setting. This figure represents a simplified schematic of recommended laboratory investigations and clinical correlation is necessary for
accurate diagnosis.
58 T.K. Roberts-Wilson et al. / Clinica Chimica Acta 423 (2013) 56–61
would be decreased [11,12]. Individual laboratories should consider
partnering with physician colleagues to develop a testing algorithm
similar to the one outlined in this review (Fig. 1).
2.1. Pregnancy
The first laboratory test in the evaluation of secondary amenor-
rhea should always be human chorionic gonadotropin (hCG) to rule
out pregnancy, which is the most common cause of cessation of men-
ses in women of reproductive age. Clinically, this is usually performed
during the first office visit with a serum or urine hCG test prior to
ordering additional studies to evaluate amenorrhea.
hCG is a glycoprotein hormone produced by the placenta and, there-
fore, is normally undetectable in non-pregnant, pre-menopausal women.
hCG testing may be qualitative, which is usually sufficient to screen for
pregnancy, or quantitative, which is the gold standard [13]. Furthermore,
quantitative assessment of the rate of hCG doubling can be used to assess
viability of a pregnancy and, therefore, play an important role in diagnos-
ing a suspected ectopic pregnancy or a failing early pregnancy [14].
While hCG is a very common test, it is plagued by confounding fac-
tors that can complicate interpretation, disposing it to diagnostic error.
Perhaps the most pervasive confounding factor is due to the use of im-
munoassay methods to measure hCG concentrations in serum and/or
urine. Various laboratories often use assorted assays with different anti-
bodies and this can lead to discordant results [15,16]. Patients may have
circulating heterophilic antibodies, resulting in false positive results in
both serum quantitative and qualitative assays [15,16]. In these cases,
analysis by a urine method or by observing non-parallel dilutions of
the serum specimens can confirm the presence of an interfering
substance. Urinary measurements can be impacted by use of dilute
urine, caused by large fluid intake or use of diuretics, which may
cause false-negative results. Lastly, hCG isoforms, particularly β-core
fragment, can cause false negatives in urine assays [17,18]. High con-
centrations of β-core fragment in the urine of pregnant women beyond
5–8 weeks gestation have been shown to cause a negative interference
when using urine POC devices to test for hCG [19].
Elevated levels of hCG indicate that pregnancy is a likely cause for
secondary amenorrhea. However, elevated hCG can also be the result
of trophoblastic and ovarian germ cell tumors, such as choriocarcino-
ma and hydatidiform mole, both of which can be very invasive and
are derived from the normal invasion process by which embryo
implantation takes place. Therefore, hCG serves as a tumor marker
for these types of cases with the amount of tumor present being
directly proportional to the amount of circulating hCG [20]. In these
cases, hCG concentrations are typically very high, much higher than
expected in a normal pregnancy. Clinicians are undoubtedly familiar
with hCG testing, nonetheless interpretation of results can be a chal-
lenge for even the most experienced. In 1999 the USA hCG Reference
Service was started in response to a large number of false positive
hCG results and the need to accurately differentiate these [21]. During
the first four years, the service identified 83 false positive cases, 62 of
which were misdiagnosed and subjected to needless chemotherapy
or hysterectomy [21]. The service continues to consult on over 100
cases annually to help avoid these kinds of diagnostic error in
complex cases.
2.2. Hypothyroidism
If the hCG is negative, the next line of laboratory tests in the algo-
rithm should include thyroid stimulating hormone (TSH), prolactin,
luteinizing hormone (LH), and follicle stimulating hormone (FSH)
[1,22] (Fig. 1). Elevated TSH is suggestive of hypothyroidism; howev-
er, it does not indicate the underlying cause of thyroid dysfunction
[23]. In this case, reflective testing of free thyroxine (fT4) and thyroid
autoantibody testing is necessary to determine the ultimate cause
and to guide treatment [24,25] (Fig. 1).
Direct measurement of fT4 is now readily available and has
replaced the free thyroxine index (FTI), which has traditionally been
calculated from total T4 and T3 uptake, making these tests obsolete
[23,26]. High fT4 levels can indicate thyroid hormone resistance or a
TSH-secreting adenoma of the pituitary [24,25]. Low levels of fT4
can indicate primary hypothyroidism [24,25]. Low or normal levels
of fT4 should be followed-up with thyroid autoantibody testing,
which would aid in a diagnosis of Hashimoto's Disease [24,25].
2.3. Hyperprolactinemia
The second most common cause of secondary amenorrhea occurring
in ~30% of patients is hyperprolactinemia, which is indicated by elevat-
ed prolactin levels [27]. Hyperprolactinemia is frequently due to a
prolactinoma, a benign tumor of the pituitary gland that results in ex-
cess production of prolactin [28]. However, the most common cause
of hyperprolactinemia is the use of prescription drugs, such as estrogen
therapies (oral contraceptive pills [OCPs]), tricyclic antidepressants,
opiates, amphetamines, anti-hypertension drugs (reserpine, verapamil,
methyldopa), and some drugs used to treat gastroesophageal reflux
(cimetidine) [1,28]. Prolactin levels of 30–200 ng/mL may be due to pre-
scription drugs [28]. However, a serum prolactin level of ≥200 ng/mL is
essentially diagnostic for prolactinoma, but an MRI is always
recommended for confirmation [28]. Many prolactinomas co-secrete
growth hormone; therefore Insulin-like Growth Factor (IGF)-1 levels
should also be obtained to exclude acromegaly.
When the clinical suspicion is high, it is important to consider the
“hook effect” where exceedingly high concentrations of prolactin are
above the measurement range of the assay and yield a false-negative
result [29,30]. Homogenous immunoassays are particularly sensitive
to the “hook effect”. When large amounts of the antigen are present,
it can overwhelm the capture antibody and no signal is generated
even though there is an excess of the antigen present. In these
cases, the specimens frequently require dilution for accurate results.
Therefore, if clinical symptoms are present (e.g. galactorrhea) and
levels of prolactin are unexpectedly low, the clinician should suspect
the “hook effect” and request that the patient sample be diluted and
retested [29,30].
2.4. Hypopituitarism
In addition to hyperprolactinemia, other conditions associated
with hypopituitarism can result in menstrual disturbances and
amenorrhea, often referred to hypogonadotropic hypogonadism [31].
Depressed levels of LH and FSH along with decreased estradiol, in
women of child-bearing age, are sufficient to confirm a diagnosis of hy-
popituitarism [32]. However, LH and FSH levels can be suppressed by an
elevated prolactin and, therefore, should not be interpreted unless
prolactin is low or normal [32] (Fig. 1). Furthermore, administration of
OCPs will also result in a low serum FSH, LH and estradiol, so it is impor-
tant to make sure that the patient is not taking hormonal contraception
or to have a sufficient wash out period before performing the tests.
2.5. Polycystic ovary syndrome
Polycystic ovary syndrome (PCOS) is a common endocrine disor-
der, affecting a minimum of 6% of women of reproductive age [33].
Clinical presentation of PCOS is characterized by irregular menses or
amenorrhea, infertility, hirsutism, and metabolic disturbance often
manifesting as type 2 diabetes mellitus, insulin resistance, or meta-
bolic syndrome accompanied by obesity [33,34]. Traditionally, labora-
tory confirmation of a diagnosis of PCOS relied on the results of a
progestin challenge but this test has become obsolete as reliable
methods to measure estrogen levels are now readily available. In
fact, evidence suggests that withdrawal bleeding associated with
the progestin challenge correlates poorly with estrogen status and
 T.K. Roberts-Wilson et al. / Clinica Chimica Acta 423 (2013) 56–61
the progestin test actually imposes a delay on the diagnostic process
[22,35]. Furthermore, estrogen concentrations do not effectively
distinguish between PCOS and hypothalamic amenorrhea because
the levels tend to fluctuate and each condition is associated with
both normal and low estrogen production [22]. Elevated LH in the
presence of a normal FSH level (LH/FSH ratio > 2.5) is suggestive of
PCOS, although further testing is required to confirm this diagnosis
and not all women with PCOS have this characteristic finding [36].
Current confirmatory laboratory diagnosis of PCOS involves test-
ing levels of total and/or free testosterone [37] (Fig. 1). Previously,
levels of dehydroepiandrosterone sulfate (DHEA-S) and sex hormone
binding globulin (SHBG) were also recommended; however, these
tests have also become somewhat obsolete as a testosterone level
alone is now considered sufficient [38]. Moderately elevated testos-
terone is suggestive of PCOS; however, highly elevated levels of
testosterone may indicate an ovarian or adrenal tumor [37,39]. If
levels of testosterone are consistent with PCOS, then exclusion of
other etiologies is necessary to confirm the diagnosis [37,39] (Fig. 1).
At the low levels of testosterone seen in women, children, and
hypogonadal men, measurement challenges exist, including lack of ac-
curacy and precision, particularly when using an immunoassay [40,41].
Furthermore, methods of measuring testosterone levels are not harmo-
nized and results from different laboratories may appear discordant, fur-
ther complicating the interpretation of these results [42,43]. However,
liquid chromatography–tandem mass spectrometry (LC–MS/MS) has
greatly improved the measurement of testosterone over traditional im-
munoassays. Using this more sensitive technique, we can quantify even
low levels of testosterone in women, children and hypogonadal men
[41,42].
2.6. Late-onset congenital adrenal hyperplasia
Late-onset or non-classical congenital adrenal hyperplasia (NCAH)
can present very similarly to PCOS and must be excluded before a
diagnosis of PCOS is made [37,44]. NCAH results from a deficiency of
21-hydroxylase. The absence of this enzyme prevents proper synthe-
sis of aldosterone and cortisol. 17-Hydroxyprogesterone (17-OHP) is
then shunted to androgen synthesis, leading to increased masculini-
zation in female patients. A diagnosis of NCAH is usually confirmed
by discovering extreme elevations of 17-OHP along with moderately
high testosterone levels [45] (Fig. 1). An ACTH stimulation test may
be needed in mild cases, but usually a random level of 17-OHP is
enough to confirm the diagnosis [45,46].
2.7. Cushing's syndrome
Cushing's syndrome may also be considered in the differential as the
underlying cause of amenorrhea in the context of hyperandrogenism,
especially in the setting of hypertension and obesity [47,48]. Tradition-
ally, the two most common tests for diagnosis of Cushing's syndrome
are the dexamethasone suppression test and a 24 h urinary free cortisol
[49]. Both of these tests are thought to be equally sensitive, though the
sensitivity and specificity of these tests remain unclear in light of the
broad clinical spectrum covered by Cushing's syndrome [49,50].
In the dexamethasone suppression test the patient is given a low
dose of dexamethasone, a glucocorticoid that simulates cortisol
action and provides negative feedback to the pituitary gland. If
there is no suppression of cortisol levels after dexamethasone admin-
istration, then Cushing's syndrome is likely [49,51]. Cortisol levels
above the normal limits in the 24 h urine sample may indicate
Cushing's syndrome [49,51] (Fig. 1).
More recently, late night salivary measurements of cortisol have
been used [52–54]. Cortisol production is usually suppressed at
night, but not in Cushing's patients. An elevated cortisol level in a
late-night sample of either saliva or plasma/serum is suggestive of
Cushing's syndrome [52,54]. Although salvia is easy to collect, it is
59
not a routine matrix in most clinical laboratories [55]. It requires spe-
cialized handling and assays are typically not as sensitive as plasma/
serum based methods [51,53,55–59].
Cushing's, NCAH, and PCOS can all have very similar clinical pre-
sentations and even experts may have difficulty making an accurate
diagnosis. For this reason, laboratory evaluation is critical. However,
a 2008 meta-analysis found that the tests commonly used to diagnose
Cushing's, while appearing highly accurate in referral practices with
samples enriched with patients with Cushing's, are of questionable
value in general clinical practice [50,60]. In response to this evidence,
the Task Force of the Endocrine Society proposed clinical practice
guidelines for the diagnosis of Cushing's [50,61]. However, the best
practices in terms of testing remain vague as demonstrated by a
poll of 32 top experts in the field, which found considerable variabil-
ity in the choice of first line testing [50,62]. It is in these types of com-
plex cases that an algorithmic approach to laboratory utilization,
including reflective testing, can contribute to improved diagnostic
efficiency.
2.8. Primary ovarian insufficiency
Primary or premature ovarian insufficiency (POI) is the cessation
of menses due to ovarian follicle dysfunction or depletion before
age 40 and is accompanied by hypoestrogenism and elevated gonad-
otropins [63,64]. The condition affects approximately 0.1% of women
by age 30 and 1% by age 40 [63,64]. Elevated levels of LH and FSH in
the presence of secondary amenorrhea are indicative of POI (Fig. 1).
Ovarian biopsy and anti-ovarian antibody testing have not been
shown to be of clinical benefit in the diagnosis of POI [64,65].
Patients younger than 30 or those women under 40 with short
stature, who are diagnosed with POI, should have a karyotype to
rule out Turner syndrome (45,X), with fluorescent in situ hybridiza-
tion (FISH) for the presence of Y chromosome material if indicated
because of their risk of gonadoblastoma. Women with POI should
also be offered testing for the fragile X premutation (FMR1), especial-
ly if they have a family history of POI or mental retardation [64,66,67].
POI has a strong association with autoimmune disorders, particu-
larly Addison disease, type 1 diabetes mellitus, and hypothyroidism
and patients should be evaluated for each of these conditions [63–66].
20–40% of patients with POI will develop an autoimmune disorder
and should undergo periodic screening [63–66].
2.9. Hypothalamic amenorrhea
When all other causes of secondary amenorrhea have been diag-
nostically excluded, hypothalamic amenorrhea may be considered
[68,69]. Hypothalamic amenorrhea is characterized by abnormalities
in gonadotropin releasing hormone (GnRH) pulsatile secretion with
resultant disruption of the hypothalamic–pituitary–ovarian (HPO)
axis and failure to stimulate sufficient synthesis and secretion of
FSH and LH [31,70]. While complete failure of gonadotropin secretion
should be detectable in peripheral blood samples, many clinical labo-
ratories use assays for FSH and LH that are imprecise when measuring
low concentrations [31,69]. Patients with hypothalamic amenorrhea
may therefore appear to have low normal serum concentrations of
FSH, LH and estradiol in the setting of amenorrhea [31,69] (Fig. 1).
Functional hypothalamic amenorrhea (FHA) is often due to weight
loss with disordered eating, excessive exercise, and psychological
stress [71,72]. The most common causes are anorexia nervosa and
bulimia nervosa; however, a similar etiology is observed in female
athletes.
Brain imaging should be considered in a woman with hypotha-
lamic amenorrhea that is not explained by excessive exercise, diet
or stress. Brain tumors causing pituitary stalk compression may
prevent delivery of GnRH to the pituitary causing amenorrhea [31].
The stalk compression may also cause hyperprolactinemia. Isolated
60 T.K. Roberts-Wilson et al. / Clinica Chimica Acta 423 (2013) 56–61
congenital gonadotropin deficiency is most often due to Kallmann
syndrome, which is associated with defects in olfactory bulb develop-
ment, and causes anosmia as well as amenorrhea and GnRH deficien-
cy [73]. Mutations in GnRH receptor genes may present similarly [74].
Other rare causes of hypothalamic amenorrhea include chronic debil-
itating diseases, such as uncontrolled juvenile diabetes, end-stage
renal disease (ESRD), malignancy, AIDS, and malabsorption, but are
uncommon in women of reproductive age [22].
Though not necessarily useful for the initial diagnosis, endocrine
stimulation tests might be helpful to better understand the physio-
pathological condition for a specific patient. Among them the most
useful are: the pulsatility study of LH and FSH (sampling every 10–
15 min for 4–6 h) to assess the gonadotropin profile and to classify
the type of LH pulses; the GnRH stimulation test to evaluate the LH
and FSH pituitary responses; and the naloxone test to assess whether
the opioidergic tone is responsible for the gonadotropin dysfunction
[69]. These kinds of studies are best performed in specific centers
where dynamic testing can be organized. In women with FHA, LH
response to naloxone infusion is considered positive when LH is
increased more than two times the baseline levels [69]. A negative
response does not exclude the presence of the opioidergic hypertone
since it might be higher than the amount of naloxone infused and is
therefore not effective in counteracting it [69].
3. Summary
Secondary amenorrhea is relatively uncommon in reproductive
medicine, but can be an indicator of a serious underlying chronic
condition. Treatment of secondary amenorrhea depends on the un-
derlying cause. Therefore, accurate diagnosis is necessary for success-
ful treatment and can only be attained by careful clinical assessment
and laboratory investigation. However, both selection of diagnostic
tests as well as result interpretation can be a complex and bewilder-
ing task. This is particularly true as a greater reliance is placed on
primary care physicians to initiate laboratory investigation [75,76].
Poor laboratory utilization in the context of complex pathologies con-
tributes to a high rate of diagnostic error, including delayed diagnosis
and misdiagnosis, as well as increased costs in both testing and defen-
sive medicine in the face of diagnostic uncertainty [75,76]. In this re-
view, we present an algorithmic approach to secondary amenorrhea
in which a panel of laboratory tests is ordered to rule out the most
common causes. The algorithm then allows for reflective testing,
based on the results of the initial panel, to efficiently ascertain more
complex etiologies [11,77]. An algorithmic approach to testing, as
reviewed here, could drastically reduce the risk of diagnostic error,
thereby decreasing costs and streamlining the diagnostic process for
clinicians.
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