Table 4.

1 (Continued)
Adult reference hormone

Range

Units

Range

Unit

Early follicular phase

70-600

pmol/L

19-160

pg/mL

Mid-cycle

700-1900

pmol/L

188-371

pg/mL

Luteal phase

300-1250

pmol/L

81-337

pg/mL

Pancreatic polypetide (fasting)

0-100

pmol/L

0-418.5

pg/mL

Parathyroid hormone (PTH)

0-4.4

pmol/L

0-41.5

pg/mL

Prolactin

80-500

mU/L

3.8-23.6

ng/mL

Progesterone (day 21, luteal phase)

>30

nmol/L

>9.4

ng/mL

Renin (am; out of bed for 2 h; seated

5-15 min)ag

2-30

mU/L

0.9-13.6

pg/mL

Females

40-120

nmol/L

Males

20-60

nmol/L

0-150

pmol/L

0-245

pg/mL

Males

8-35

nmol/L

230-1000

ng/mL

Females

0.7-3.0

nmol/L

20-85

ng/mL

Thyroglobulin

1.5-30

pmol/L

1-20

g/L

Thyroid-stimulating hormone (TSH)

0.3-5.0

mU/L

Thyroxine, free (fT4)

9-23

pmol/L

0.7-1.8

ng/dL

Tri-iodothyronine, free (fT3)

3.1-7.7

pmol/L

0.2-0.5

ng/dL

Vasoactive intestinal polypeptide

(fasting)

0-30

pmol/L

102

pg/mL

Vitamin D (25-OH-cholecalciferol)

4-40

nmol/L

1.6-16

ng/mL

Vitamin D (1,25-OH-cholecalciferol)

48-110

pmol/L

20-45.8

pg/mL

Sex hormone-binding globulin

Somatostatin (fasting)
Testosterone

Ranges shown are for serum unless otherwise stated. Ranges vary slightly between laboratories due to differences in the
methods employed. These examples are only intended to be illustrative and readers should check with their local
laboratories.
a
Most informative as part of the aldosterone:renin ratio (see Chapter 6). bAge-dependent. Low values
indicate poor ovarian reserve.
c
Salivary assays are variable and require establishment of local normal ranges.
d
Greater suppression from glucose load can be demonstrated using newer more sensitive immunoradiometric or chemiluminescent assays.
e
The World Health Organization and the American Diabetes Association have endorsed HbA 1c for the diagnosis of diabetes above or equal to these
values.
f
IGF-I values are approximate as age- and sex-adjusted ranges are required.
g
Renin is also measured as plasma renin activity when 1 mU/L equates to 1.56 pmol/L/min (0.12 ng/mL/h).

56 / Chapter 4: Investigations in endocrinology and diabetes

Box 4.2 Dynamic investigation in

endocrinology
If underactivity suspected: try to stimulate it
If overactivity suspected: try to suppress it

Cell and molecular biology as

diagnostic tools
Karyotype
Karyotype refers to the number and
microscopic
appearance of chromosomes arrested at
metaphase
(see Chapter 2). The word also describes
the
com
plement of chromosomes within an
individuals
cells, i.e. the normal karyotype for
females
is
46,XX
and for males is 46,XY. A karyogram is
the
reorgan
ized
depiction
of
metaphase
chromosomes
as
pairs
in ascending number order. An
abnormal
total
number of chromosomes is called
aneuploidy
(common
in
malignant
tumours).
More
detail
comes from Giemsa (G) staining of
metaphase
chromosomes,
where
each
chromosome
can
be
identified by its particular staining
pattern,
called
G-banding.
Ascertaining the karyotype can be
useful
in
con
genital endocrinopathy, such as genital
ambiguity
(i.e. is it 46,XX or 46,XY?), or if there is
concern
over Turner syndrome (45,XO) or
Klinefelter
syn
drome (47,XXY) (see Chapter 7). G-

banding
allows
experienced cytogeneticists to resolve
chromosomal
deletions, duplications or translocations
(when
frag
ments are swapped between two
chromosomes)
to
within a few megabases. Sometimes,
there
is
evi
dence of mosaicism when cells from
the
same
person show more than one karyotype.
This
implies
that
something
went
wrong
downstream
of
the
first
cell division such that some cell
lineages
have
a
normal karyotype while others are
abnormal.
Fluorescence in situ hybridization
When a syndrome is suspected, for
which
the
causa
tive gene or locus (genomic position)
is known,

probe detects
fluorescence in situ hybridization (FISH)
allows
assessment of duplications, deletions or
transloca
tions on a smaller scale. For instance, a
locus
for
congenital hypoparathyroidism, as part of
DiGeorge
syndrome, exists on the long arm of
chromosome
22 (22q). FISH utilizes the principle that
comple
mentary DNA sequences will hybridize
together
by
hydrogen bonding. Stretches of DNA
from
the
region of interest are fluorescently
labelled
and
hybridized to the patients DNA. The
fluorescence
is visible as a dot on each sister
chromatid
of
each relevant chromosome (Figure 4.4).
Therefore,
normal autosomal copy number is viewed
as
two
pairs of two dots; one pair indicates a
deletion;
and
three pairs indicate either duplication or
potentially
a translocation breakpoint (where the

Figure 4.4 Fluorescent in situ hybridization in a

patient with congenital hypoparathyroidism due to
DiGeorge syndrome causing hypocalcaemia and
congenital heart disease. Metaphase chromosomes
were hybridized with a fluorescent probe from
chromosome 22q11. The two bright dots indicate
hybridization on the sister chromatids of the normal
chromosome 22. The arrow points to the other
chromosome 22 that lacks signal, indicating a deletion.
Images kindly provided by Professor David Wilson,
University of Southampton.

Chapter 4: Investigations in endocrinology and diabetes / 57

sequence either side of the breakpoint

on different chromosomes).

Genome-wide microarray-based
technology
Applying the principles of FISH on a
genome-wide
scale in a microarray format is called
array
com
parative genomic hybridization (array
CGH).
Short
stretches of the genome are printed as
thousands
of
microscopic spots on a glass slide (the
microarray).
The
patients
genomic
DNA
is
fluorescently
labelled
and hybridized to the spots on the slide.
According
to the strength of the fluorescent signal,
microdele
tions or duplications anywhere in the
whole
genome
can be detected in one experiment with a
resolution
of several kilobases.
Single
nucleotide
polymorphism
(SNP)
arrays
are being used similarly. Spread across
the
entire
genome, there are millions of very subtle
variations
(polymorphisms) at specific nucleotides
between
different individuals. On SNP arrays,
the
spots
on
the glass slide represent the different
sequences
at
each SNP. As an individuals paired
chromosomes
come one from each parent, this means
that
at
any
one SNP, there are often two different
sequences
(one from the mother, one from the
father;
this
is
called heterozygosity). Across stretches
of
DNA,
SNP arrays can identify regions

showing
loss
of
heterozygosity (i.e. there is no
variation
in
the
signal), which is indicative of deletion
of
either
the
maternal or paternal copy, or altered
ratio
of
signals
indicative of duplication.
Diagnosing mutations in single genes
by polymerase chain reaction and
sequencing
With the discovery of diseasecausing
genes
in
monogenic disorders (i.e. a single gene
is
at
fault),
genetic testing is expanding rapidly
into
clinical
endocrinology
and
diabetes.
Increasingly
precise
prediction is becoming possible from
correlating
genotype (i.e. the gene and the
position
in
a
gene
that a specific mutation lies) and
phenotype
(i.e.
the
clinical course of a patient). For
instance,
certain
mutations in the RET proto-oncogene
in type 2

multiple endocrine neoplasia (MEN2; see

Chapter 10) have never been associated
with phaeochromo cytoma, normally one
of the commonest features of MEN2.
However, other RET mutations predict
medullary carcinoma of the thyroid at a
very young age, thus instructing when
total
thyroidectomy
is
needed.
Genetically defining certain forms of
monogenic diabetes is now dictating
choice of therapy (see Case history
11.3).
Polymerase chain reaction (PCR) and
sequenc
ing is used to identify a mutation in a
specific
gene
(Figure 4.5). Using DNA isolated from the
patients
white blood cells, PCR amplifies the
exons
of
the
gene of interest in a reaction catalyzed
by
bacterial
DNA polymerases that withstand high
temperature
(>90C). These enzymes originate from
microor
ganisms that replicate in hot springs. A
second
modified PCR reaction provides the
base
pair
sequence of the DNA, demonstrating
whether
or
not the gene is mutated.
Since sequencing the human genome in
the
last
decade,
technology
has
advanced
enormously,
greatly bringing down cost. What was once
achieved
by cutting-edge multi-million pound

international
consortia is now possible within an
individual
labo
ratory in a matter days or weeks for a
few
thousand
pounds.
In
addition
to
ethical
implications
of
holding these whole genome datasets,
the
bioinfor
matics required for their analysis is
massive.
Nevertheless, by next generation
sequencing
on
exome arrays (i.e. all exons of nearly
all
genes),
defining a patients genome is fast
becoming
a
diag
nostic reality.

Imaging in endocrinology
Ultrasound
Ultrasound travels as sound waves
beyond
the
range
of human hearing and, according to the
surface
encountered, is reflected back towards
the
emitting
source (the ultrasound probe). Different
tissues
have different reflective properties. By
knowing
the
speed of the waves and the time
between
emission
and detection, the distance between the
reflective
surfaces and the source can be
calculated.
These
data allow a two-dimensional image to be
generated