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Type 2 diabetes:
biomarker models to predict risk
Pg.21
Compact hematology system
by Beckman Coulter
Pg.31
Meningitis/encephalitis panel
by BioFire Diagnostics
Pg.32
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Vacuum sample tube with
glycolysis inhibition
by Greiner Bio-One
Pg.34
Diagnosis and management
Pharmacogenomics
of testicular cancer Pg.
in AML patient
Pg. 14
Porphyrias clinical and

diagnostic aspects
Pg. 25
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EDITORS LETTER
April/May 2016
Call for action on diabetes

Frances Bushrod,
Ph.D.
Many studies assessing the outcome

of T2DM screening have reported
minimal impact on prevalence. However, some recent community-based
screening projects oering testing at
a variety of venues including sports
grounds, shopping centres, pharmacies (and why not polling stations?)
show promise. In such an approach
it is clearly simpler to utilise point-ofcare capillary glycosylated haemoglobin (A1c) tests. A nger stick to obtain
one drop of blood followed by a short
wait in situ for a result that reects the

average blood glucose level over the
past three months is clearly preferable
to measuring fasting or random glucose levels, tests which require patient
forethought, laboratory facilities,
larger samples and frequently repeat
tests. POC A1c tests are currently
available for around 9 a unit, surely

cost-eective if a result of prediabetes
precipitates patient lifestyle changes,
and a diagnosis of diabetes leads to
follow-up care.
Of course one must develop
clear guidelines for the follow
STart Max
up of subjects with positive test

results but surely such screening programmes are more likely
to have an eect on the T2DM
epidemic than frequently overweight healthcare workers ponticating about healthy diets
and exercise?
Max
Accuracy
Max
Practicality
Max
Innovation
Max
Reliability
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on-contr actual
tual photos
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phot s - 03/2016
This years annual World Health Day

on 7th April highlighted the dramatic rise in the prevalence of Type
2 diabetes (T2DM) and urged global
action to contain the epidemic. The
number of people suering from
T2DM has approximately quadrupled in three and a half decades;
currently 8.5% of the global adult
population is aected. Because
uncontrolled, elevated levels of blood
glucose can eventually result in cardiovascular disease, kidney failure,
lower limb amputation and loss of
sight, as well as premature death, the
disease has major socioeconomic
impacts in addition to health issues.
Yet it is unlikely, at least in Western
populations, that interventions to
promote more balanced diets and
less sedentary lifestyles will reduce
the widespread overweight and obesity that fuels the T2DM epidemic.
The general public in the West is continuously informed about the benecial eects of healthy eating and
sucient physical exercise, but modern working environments, family
commitments and social activities
often preclude compliance with
good health advice. And many of us,
healthcare professionals included,
think its worth taking the risk of
eating and drinking (even smoking) what we really enjoy! However,
advice once a subject knows that s/
he has prediabetes or T2DM, or is at
higher risk because she has suered
from gestational diabetes, is much
more likely to be heeded. Thus mass
screening programmes are surely the
most eective way of curbing the
escalating T2DM epidemic.
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Considerable rewards could be obtained from
early identication of Type 2 diabetes mellitus
News updates on www.cli-online.com | April/May 2016 | Volume 40
Type 2 diabetes:
biomarker models to predict risk
Pg.21
(T2DM). One of the most obvious, as suggested

in a recent report on diabetes global burden,
by Beckman Coulter
Pg.31
would be better disease management. The report, by the University of East Anglia in the UK,
by BioFire Diagnostics
Pg.32
concludes that early investments into prevenAlso in this issue :
Vacuum sample tube with

glycolysis inhibition
by Greiner Bio-One
Pg.34
Diagnosis and management
Pharmacogenomics
of testicular cancer Pg.
in AML patient
Pg. 14
Managing Editor
Alison Sleigh, Ph.D.
Contributing Editor
Frances Bushrod, Ph.D.
News Editor
Tony Spit, Ph.D.
Editorial Coordinator
Shirley Waring
tion and disease management may therefore be
Editor in Chief/Publisher
Bernard Lger, M.D.
particularly worthwhile.
Advertising Coordinator
Porphyrias clinical and

diagnostic aspects
Pg. 25
Jennifer Christophers
Circulation Manager
Arthur Lger
[6 - 13]
TUMOUR MARKERS
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The clinical chemistry laboratory in the diagnosis and management of

testicular cancer
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Use of serum free light chain analysis in screening for multiple myeloma
in primary care patients
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Type 2 diabetes - biomarker models promise new means to predict risk
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[25 - 28] HEMATOLOGY

Porphyrias: clinical and diagnostic aspects
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Tumour markers
The clinical chemistry laboratory in the

diagnosis and management of testicular cancer
Cancer of the testicles, primarily the germ cells, is a highly
treatable disease common to young men. This article describes
how chemical biomarkers are central to the diagnosis,
characterization, therapeutic monitoring, prognosis and longterm surveillance in patients with testicular cancer.
by Dr Angela Cooper and Dr Sen Costelloe
Incidence of testicular cancer

Testicular cancer (TC) is relatively rare,
accounting for approximately 0.7% of
all UK male cancers, with a worldwide
incidence estimated as ~7 per 100 000
[1, 2]. Incidence of TC has noticeably
increased in industrialized countries
over the last few decades, particularly
in white males of European descent,
although the reasons for this remain
unclear [25]. Amongst younger men
aged between 15 and 49 years in the
Cell type of origin

Germ cell tumours
United Kingdom and the United States

of America, TC is the most common
type of cancer observed [2, 3, 6, 7].
Classication of TC
Approximately 95% of malignant TCs
originate from primordial germ cells,
also known as germ cell tumours
(GCTs) [3, 79]. However, rarely these
malignancies may arise from extragonadal primary sites such as the retroperitoneum, mediastinum or pineal
Type of tumour
Intratubular germ cell neoplasias of the unclassied type
Percentage of all
GCTs
~20%
(IGCNU)
Other types
Tumours originating from
one cell type
Seminomas
Seminoma with syncytiotrophoblastic cells
Embryonal carcinomas
Yolk sac tumours
Trophoblastic tumours
Choriocarcinomas
Trophoblastic neoplasms other than choriocarcinomas
Monophasic choriocarcinomas
Placental site trophoblastic tumours
Teratomas
Dermoid cysts (rare in testis)
Monodermal teratomas
Teratomas with somatic type malignancies
Mixed embryonal carcinomas and teratomas
Mixed teratomas and seminomas
(mixed GCTs)
Seminomatous GCTs
Non-seminomatous GCTs
(NSGCTs)
Embryonal cell carcinomas
Yolk sac tumours
Teratomas
Choriocarcinoma
The vast majority of non-GCTs are sex

cord-gonadal stromal tumours involving the Sertoli or Leydig cells of the testicles, and are often benign [8, 9, 11].
Burned-out GCTs, or spontaneous
regression of a testicular GCT, is a very
rare phenomenon occasionally observed
in male patients presenting with metastatic malignancy with an absence of
primary testicular tumour. Often, the
only remaining evidence of malignancy
are features such as homogeneous scarring, hemorrhage, intratubular calcification and testicular atrophy. This may
be associated with choriocarcinomas or
teratomas [5, 12].
Non-seminomatous germ cell tumours (NSGCT)
more than one cell type
~40%
Spermatocytic seminomas
Spermatocytic seminoma with sarcoma
Tumours originating from
gland [35, 8, 10]. Germ cell tumours

classified as seminomas (~40%) are
predominantly formed of uniform
cell types, whereas non-seminomatous
germ cell tumours (NSGCTs), also
accounting for ~40% of GCTs, originate from multiple cell types such as
embryonal carcinomas, teratomas,
choriocarcinomas and yolk sac carcinomas. GCTs arising from mixed
germ cells comprise the remaining
20%. The World Health Organization
(WHO) classification system for testicular tumours (Table 1) define five
basic GCT types based on histological
examination:
~40%
Choriocarcinomas and teratomas/embryonal carcinomas

Polyembryomas
Others
Testicular GCTs exhibit very diverse histology and immunostaining profiles, and
have varying clinical progression and
prognosis outcomes as demonstrated
by the numerous methods of GCT classification systems. It is outside the focus
of this paper to consider histology or
immunostaining used in the identification and differentiation of GCTs, as
these topics has been extensively documented in other review articles.
Table 1. World Health Organization (WHO) histological classication of testicular germ cell tumours
Treatment and cure rates in TC
(GCTs) [sourced from 3, 8, 9, 11].
Advances in treatment strategies, such
7
as the use of cisplatin therapies [13],
careful staging at diagnosis, early intervention using multidisciplinary teams,
rigorous surveillance follow-up, and
salvage therapy, means that GCTs are
highly curable. Currently, expected cure
rates of 95% are observed in patients
who receive a TC diagnosis, and cure
rates of 80% in patients with a diagnosis
of metastatic TC [3, 13].
Causes and presentation of TC

The causes of TC cancer are still
unknown, although cryptochordism is
the best-characterized risk factor associated with TC. Research has shown
that timing of orchiopexy impacts on
future risk of TC development, suggesting hormonal changes during puberty
are strongly associated with TC etiology
in males. However, prenatal risk factors,
environmental exposures in adulthood,
male infertility, certain genetic or congenital disorders such as Downs syndrome, Klinefelters syndrome, human
immunodeficiency virus infection and
intersex patients have also been associated with an increased TC risk [3, 5, 7].
Presentation of TC is often a painless
lump in the testis body, but due to a frequent lack of pain, medical opinion is
frequently delayed. A testicular mass or
swelling, or episodic diffuse pain may be
observed. More rarely, metastatic symptoms such back pain arising from retroperitoneal lymph node involvement,
or coughing, pain or hemoptysis due to
lung metastasis may be reported [3, 7, 8].
Diagnosis and staging of TC

Clinical suspicion of TC, such as
altered testicular shape or non-painful
swelling, should prompt a full physical
examination and patient history, imaging to include testicular and abdominal ultrasound, as well as chest X-ray
[14]. If metastasis is suspected, chest,
abdominal and brain computerized
tomography (CT), and bone scintigraphy should be undertaken [9]. Final
diagnosis and prognosis requires
biopsy sampling for histology and
pT Primary tumour
pTx
No primary tumour able to be identied
pT0
No evidence of a tumour (e.g. scar)
pTis
IGCNU
PT1
Limited to testis and epididymis. Absence of vascular or lymphatic invasion, may invade tunica
albuginea but not tunica vaginalis
pT2
pT1 with vascular, lymphatic or tunica vaginalis invasion
pT3
Invasion of spermatic cord with or without vascular or lymphatic invasion
pT4
Invasion of scrotum cord with or without vascular or lymphatic invasion
pNx
Regional lymph node involvement unable to be identied
pN0
No lymph node metastasis identied
pN1
Metastasis to 5 lymph nodes, no lymph nodes >2 cm OR no lymph node masses 2 cm
pN2
Metastasis to >5 lymph nodes, no lymph nodes >5 cm OR lymph node masses >2 cm but 5 cm OR
extranodal spread
pN3
Lymph node mass >5 cm
pN Regional lymph nodes
M Distant metastasis
Mx
Distant metastasis unable to be identied
M0
No distant metastasis
M1
Distant metastasis identied
M1a
Non-regional nodal or lung metastasis identied
M1b
Distant metastasis identied (excluding non-regional nodal or lungs)

S Serum tumour markers (STMs)
Sx
STMs not available or not undertaken
S0
STMs concentrations within normal limits

LDH (U/L)
hCG (U/L)
AFP
S1
<1.5 N and
<5000 and
<1000
S2
1.510 N or
500050 000 or
100010 000
S3
>10 N or
>50 000 or
>10 000
Table 2. Tumour-node-metastasis (TNM) classication system for testicular tumours [Sourced from
1416].
April/May 2016
immunostaining profiling as appropriate, and in the majority of cases,

treatment options should be based on
the histology results [10]. Biochemical analysis should include initial concentrations of serum tumour markers
(STMs). Metabolic biochemistry, liver
function tests and a full blood count
should be undertaken to determine
general organ function, and may demonstrate evidence of metastasis [9].
This collective information can be used
to reference the Tumour-node-metastasis (TNM) Classification of Malignant
Tumours staging system (Table 2). This
cancer staging system is based on primary tumour site, nearby lymph node
involvement, and presence of distal
metastatic spread from initial primary
tumour site [4, 15]. The use of STMs as
a fourth staging system has added diagnostic and prognostic value, independent of the TNM system (Table 3) [9].
The decision for chemotherapy or radiotherapy treatment for non-surgical
metastatic disease is based on CT and/
or magnetic resonance imaging (MRI)
results, and concentrations of STMs [4].
The majority of patients (~75%) presenting with a testicular mass are diagnosed at stage 1 [7, 8]. At this stage,
treatment options are typically surgery
with an excellent cure rate. For metastatic disease, combinations of surgery,
chemotherapy or radiotherapy are
required depending on cancer mass,
location and distal lymph node involvement [13]. Greater than 80% of patients
with metastatic GCTs are successfully
treated and cured.
Treatment of TC
TC cells are extremely sensitive to
chemotherapy [9, 10]. Specifically, the
standard chemotherapy regime consists
of 3 or 4 cycles of bleomycin, etoposide
and cisplatin (BEP) chemotherapy, or
etoposide and cisplatin (EP) chemotherapy every 21 days [8, 9]. Surgery
may be considered to remove residual
masses post-chemotherapy. Data suggests a higher relapse rate in patients
with NSGCTs than seminomas following an initial chemotherapy regime.
This relapse rate can be used to further
classify patients into good, intermediate and poor prognostic groups, using
a combination of STM concentrations
and location of primary tumour or
metastases. Around 5099% of patients
can still expect to survive [8].
Salvage therapy, often in combination with chemotherapy, is reserved

for patients who have relapsed, or for
patients where cancer progression
continues after following a standard
chemotherapy regime. High-dose
chemotherapy with autologous bone
marrow transplant is a controversial
approach for patients with a poor
prognosis, and where a standard
chemotherapy regime and salvage
therapy has been unsuccessful. Initial
studies are encouraging but further
trials are required. A small cohort
of patients have been identified who
Marker
-fetoprotein (AFP)
Tumour markers
April/May 2016
Upper
reference
limit
~10 kiU/L
Advances in treatment
strategies, such as the
use of cisplatin therapies,
careful staging at diagnosis,
early intervention using
multidisciplinary teams, rigorous
Serum t
57 days
surveillance follow-up, and

salvage therapy, means that
GCTs are highly curable
Tissue
origin
Fetal yolk sac,
liver, GI tract
Conditions causing
elevated serum markers
Not secreted by pure cell

seminomas irrespective of
histology, or pure cell teratomas
Secreted by NSGCTs except
for choriocarcinomas or pure
embryonal cell carcinomas
Exceptionally high levels seen in
yolk sac NSGCTs
GCTs (pure seminomas, NSGCTs)

Hydatidiform moles
Primary hypogonadism
Gonadotroph adenoma
Poorly differentiated
adenocarcinoma
Choriocarcinomas
Pancreas, islet cell, small/
large bowel, liver, stomach,
lung, ovarian, breast and renal
malignancy
Gestational trophoblastic disease
Marijuana use
Secreted by all NSGCTs except

teratomas. Always secreted by
choriocarcinoma NSGCTs
High concentrations (>5000
U/L) suggestive of mixed GCTs
Pure seminomas secrete hCG in
1015 % of cases
Every tissue
cell of body.
Highest
concentrations
found in all
muscle types,
liver and brain
Muscle disease, MI, pernicious

anaemia, leukaemia, thalassemia,
PE
In vitro haemolysis
Not useful if the only tumour

marker measured as not specic
for TC resulting in high falsepositive rates. Most helpful in
conjunction with AFP and hCG,
or for surveillance in patients with
advanced seminoma
Placental
blasts
Normal testis, cervix, thymus, lung

activity
GCTs, ovarian & lung malignancy
5 U/L
(males)
1624 hours
Placental
tiotrophoblasts
hCG
-subunit (hCG)
typically the subunit
detected by most
commercial assays
Lactate
dehydrogenase
subtype 1
(LDH-1)
Placental alkaline
phosphatase (PLAP)
Laboratory
specic
<100 iU/L
48113
hours
~1567
hours
Use in testicular cancer
Benign liver disease

Certain malignancies
Mixed cell NSGCTs
Hepatocellular carcinoma
Gastric, colon, gall bladder,
pancreatic, lung cancer
Hepatotoxicity (drug or viral)
Ataxia telangiectasia (>95%
patients)
Hereditary persistence of AFP
Gestational trophoblastic disease
Poorly differentiated
adenocarcinoma
Tyrosinemia
& human
chorionic
gonadotrophin
(hCG)
suffer a late relapse, i.e. >2 years postdiagnosis but also potentially 10
years post-diagnosis. These patients
are less responsive to chemotherapy,
so are treated primarily with surgery. Unfortunately, less than half
will remain disease-free following
surgical intervention [8, 9]. Chemotherapy-induced side effects are
governed by the dose and combination of drugs used. This has triggered
more recent trials designed at maintaining a cure rate but with reduced
associated chemotoxicity [8].
GCTs
Elevated in seminomas
(Should not be measured in
smokers)
Table 3. Commonly used serum tumour markers in the diagnosis and management of germ cell tumours in testicular cancer patients [Sourced from 3, 4, 9,
10, 16]. NSGCTs, non-seminomatous germ cell tumours.
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April/May 2016
The use of serum tumour

markers in TC
The discovery of serum and urine
tumour markers and the advent of
chemotherapy
have
significantly
improved cancer staging, management
and prognosis in patients with TC. The
benefit of initial STMs is predominantly
with regard to disease staging, whereas
serial STMs are particularly useful for
monitoring response to treatment after
surgery, chemotherapy or radiation
therapy. STMs are useful because they
are often detectable well before clinical radiological detection in patients.
Furthermore, concentrations can be
helpful to differentiate GCT type. The
detection of at least one elevated STM
occurs in ~85% of NSGCTs, and the
presence of elevated STMs occurs in
significant numbers of pure seminoma
cases [9, 10]. However, in rare cases
where patients present with evidence of
a testicular mass, radiographic evidence
of metastatic disease, with significantly
elevated alpha-fetoprotein (AFP) or
human chorionic gonadotrophin (hCG)
serum concentrations, it is advised that
treatment is not delayed while awaiting
histology results [10].
The American Society of Clinical Oncology recommend against using STMs as
a screening test for GCTs in asymptomatic males. Given the low incidence
and mortality of TC combined with the
high cure rate, it is suggested a screening programme would be neither costeffective nor decrease mortality [10].
Furthermore, although STMs can be
helpful in combination with imaging
techniques in the diagnosis of TC, normal STMs alone do not exclude TC and
may also be raised in other conditions
[3, 810]. Routine testicular examination via palpation is recommended in
all males from puberty up to ~45 years.
This is of particular importance for
males with a past medical history that
may suggest an increased GCT risk as
detailed previously.
Commonly employed serum markers
include: AFP and hCG as mentioned
previously, hCG beta-subunit (hCGb),
placental alkaline phosphatase (PLAP)
and lactate dehydrogenase (LDH).
Alpha-fetoprotein levels are elevated in
teratocarcinoma or testicular embryonal carcinoma, while conversely, AFP
is never elevated in pure seminomas.
Human chorionic gonadotrophin elevations are associated with 1015 %
Tumour markers
10
of pure seminomas. Lactate dehydrogenase is an enzyme found in all cell

types, meaning it is less specific for TC,
although it does have prognostic value
in advanced stage GCTs [3, 9]. A decline
in serial STM concentrations is useful to
detect the presence of residual disease
following surgery, or to assess response
to chemotherapy. In both scenarios, the
decline in STM concentrations should
follow the half-lives of each marker [9].
There are detailed STM surveillance
guidelines in place following surgery,
which recommend a meticulous timetable of STM measurements and radiology imaging to detect disease recurrence depending on initial GCT type,
thereby avoiding relapse and presentation at a later date with advanced stage
disease [8, 9].
The discovery of serum and

urine tumour markers and
the advent of chemotherapy
have signicantly improved
cancer staging, management
and prognosis in patients
with testicular cancer
Future focus
While the majority of patients diagnosed with TC will survive, challenges
still persist. Serum tumours markers
have been pivotal to improved outcomes
for patients with and without metastatic
disease. Future research is focused on
patients with an initial poorer prognosis, patients who have relapsed following first-line chemotherapy and
patients who have a late relapse. Longterm health consequences for patients
surviving TC, in particular side effects
associated with chemotherapy and radiotherapy such as cardiovascular disease,
impaired fertility and secondary cancers, continues to drive collaborative
studies nationally and internationally to
improve TC outcomes for the future.
References
1. Cancer registration statistics, first release, England, 2014. Office for National Statistics 2014.
(http://web.ons.gov.uk/ons/rel/vsob1/cancerstatistics-registrations--england--series-mb1/2014--first-release-/rpt-cancer-stats-registrations.html)
2. Hameed A, White B, Chinegwundoh F, Thwaini A,

Pahuja A. A review in management of testicular
cancer: single centre review. World J Oncol. 2011;
2: 94101.
3. Bosl GJ, Motzer RJ. Testicular germ-cell cancer. N
Engl J Med. 1997; 337: 242254.
4. Bahrami A, Ro JY, Ayala AG. An overview of testicular germ cell tumors. Arch Pathol Lab Med.
2007; 131: 12671280.
5. Sesterhenn IA,Davis, CJ. Pathology of germ cell
tumors of the testis. Cancer Control 2004; 11:
374387.
6. Wu X, Groves FD, McLaughlin CC, Jemal A, Martin J, Chen, VW. Cancer incidence patterns among
adolescents and young adults in the United States.
Cancer Causes Control. 2005; 3: 309320.
7. Hanna NH, Einhorn LH. Testicular cancer discoveries and updates. N Engl J Med. 2014; 371:
20052016.
8. Horwich A, Nicol D,Huddart R. Testicular germ
cell tumours. BMJ 2013; 347: f5526.
9. Barlow LJ, Badalato GM,McKiernan JM. Serum
tumor markers in the evaluation of male germ
cell tumours. Nat Rev Urol. 2010; 7: 610617.
10. Gilligan TD, Hayes DF, Seidenfeld J, Temin S.
ASCO clinical practice guideline on uses of
serum tumor markers in adult males with germ
cell tumors. J Clin Oncol. 2010; 6: 199202.
11. Eble JN, Sauter G, Epstein JI, Sesterhenn IA.
World Health Organization classification of
tumours. Pathology and genetics of tumours
of the urinary system and male genital organs.
IARC 2004.
12. Ulbright TM. Germ cell tumours of the gonads:
a selective review emphasizing problems in differential diagnosis, newly appreciated, and controversial issues. Mod Pathol. 2005; 18: S61S79.
13. Masters JR, Kberle B. Curing metastatic cancer:
lessons from testicular germ-cell tumours. Nat
Rev Cancer. 2003; 3:517525.
14. Suspected cancer: recognition and referral
guidelines [NG12]. National Institute for Health
and Care Excellence (NICE) 2015. (https://www.
nice.org.uk/guidance/NG12/chapter/1-Recommendations-organised-by-site-of-cancer)
15. Sobin LH, Gospodarowicz MK and Wittekind C.
TNM classification of malignant tumours (7th
ed). International Union against Cancer (UICC).
Wiley-Blackwell 2009.
16. Albers P. (Chair), Albrecht W, Algaba F, Bokemeyer C, Cohn-Cedermark G, Fizazi K, Horwich
A, Laguna MP, Nicolai N, Oldenburg J. Guidelines on testicular cancer. Eur Urol. 2015. (https://
uroweb.org/guideline/testicular-cancer/)
The authors
Angela Cooper* PhD, Sen Costelloe,
PhD
Derriford Combined Laboratory, Plymouth Hospital NHS Trust, Plymouth, UK
*Corresponding author
E-mail: angelacooper5@nhs.net
Tumour markers
11
April/May 2016
Use of serum free light chain analysis in screening

for multiple myeloma in primary care patients
Identication of a serum or urine paraprotein is a key element in the
diagnosis of multiple myeloma. Traditionally, this has been achieved
using a combination of serum and urine electrophoresis, but this can
result in incomplete investigation. The use of serum free light chains as
an alternative screening test has been advocated to overcome this.
by David Baulch and Beverley Harris
Multiple myeloma
Multiple myeloma (MM) accounts for
1% of all cancers, with nearly 5000 people in the UK being diagnosed each year.
The average age of presentation is 70 with
only 15% of patients presenting at less
than 60 years of age [1]. Its prevalence
has increased by 11% in the last decade,
due mainly to increased survival rates in
those diagnosed [2]. Despite this, MM
still accounts for around 2700 deaths
annually in the UK and over 70 000
worldwide with a median survival of
only 34 years from diagnosis [3].
MM is characterized by the accumulation
of clonal plasma cells, predominantly
within the bone marrow, and subsequent
clonal expansion of the plasma cell lineage [4]. It is almost always preceded by
a premalignant, asymptomatic period of
monoclonal gammopathy of undetermined signicance (MGUS) [1]. The process of immunoglobulin (Ig) production
by plasma cells is normally under a state
of homeostasis, but random and nonrandom genetic aberrations, epigenetic
changes and atypical interactions within
the bone marrow microenvironment can
cause uncontrolled proliferation of neoplastic plasma cells, leading to plasma cell

disorders (PCDs) such as MM [4]. Clonal
expansion of a plasma cell line under
such circumstances can cause overproduction of intact monoclonal Ig (IgG,
IgA, IgM, rarely IgD and IgE) or monoclonal free light chains (FLCs) kappa
and lambda. Although the classication
of PCDs is based on the immunoglobulin type secreted, 12% of MM cases are
classied as non-secretory. This may be
due to an absence of secreted monoclonal protein (M protein), or secretion at
a concentration below the limits of the
laboratory methods used for detection.
Compared with other cancers, diagnosis
of MM is challenging. Patients present
with a range of non-specic symptoms
and as a result often have a string of
primary care consultations resulting in
diagnostic delay. Such delays signicantly
impact the clinical course of MM [5], for
which a complete cure remains elusive.
Consequences of diagnostic delay

Studies have shown that over 50% of
patients attending primary care institu-
sEP and uEP
sEP and sFLC
Sensitivity, %
81 (6989)
98 (91100)
Specicity, %
99 (99100)
89 (8592)
PPV, %
96 (9899)
58 (4968)
NPV, %
96 (9498)
100 (98100)
Efciency, %
98 (9499)
90 (8693)
Table 1. Result Summary. The 95% condence interval limits are in parentheses. The highest performer
for each statistical parameter is highlighted.
PPV, positive predictive value; NPV, negative predictive value; sEP, serum protein electrophoresis and
reexed serum protein immunoxation; uEP, urine protein electrophoresis and reexed urine protein
immunoxation; sFLC, serum free light chain ratio.
tions took 6 months (33% >12 months)

from the onset of the rst related symptoms to referral [5]. Another study
showed the time to diagnosis of MM can
be unacceptably prolonged [6] and the
pathway to diagnosis in MM was more
likely to include a string of repeated primary care consultations, infrequent use
of urgent referral routes and increased
emergency presentation [7]. In particular, patients whose referral was delayed
by 6 months or more were more likely
to suer a greater number of more signicant complications such as renal
insuciency which, if swift diagnosis
had occurred, may have been reversible
[5]. This highlights the need not only to
raise awareness of disease symptoms,
but to increase the sensitivity of laboratory detection.
Laboratory investigation of
multiple myeloma
In addition to clinical and hematological
investigations, screening for MM within
the laboratory is based on the detection
and classication of M proteins by serum
protein electrophoresis (the separation of
serum proteins according to molecular
size, hydrophobicity and electric charge
[8]), followed by immunoxation or
immunotyping to identify and quantify
the Ig isotypes. This method is less reliable
for detecting disease when only FLCs are
secreted, as these are rapidly cleared by
the kidneys. Free light chains in the urine
[known as Bence Jones protein (BJP)]
can also be detected by electrophoresis
followed by immunoxation. However,
this methodology is time consuming and
may not detect low concentration BJP in
dilute urine samples [9]. Interpretation of
the results can be dicult and should be
performed by appropriately qualied and
experienced laboratory sta. In addition,
obtaining both urine and serum samples
for screening can be problematic, with
some laboratories reporting that both
samples are received for only ~17% of
MM screens.
There is growing evidence to support
the direct measurement and quantitation of serum kappa and lambda FLCs
in diagnosis, monitoring and prognosis
of MM and related PCDs [4]. The serum
April/May 2016
12
Tumour markers
sensitivity, specicity, positive predictive
value (PPV), negative predictive value
(NPV) and eciency were calculated for
our current screening tests (sEP and uEP)
and the use of sEP with sFLC as an alternative strategy. Figures 1 and 2 outline
the process for each of these screening
strategies and a summary of the results is
given in Table 1.
Conclusion
Figure 1. Screening strategy 1: serum and urine protein electrophoresis with reexed serum
immunotyping and urine immunoxation.
Number of patients in parentheses. EP, electrophoresis; IT, immunotyping; IF, immunoxation; M
protein, monoclonal protein; MGUS, monoclonal gammopathy of undetermined signicance; LCMM,
light chain multiple myeloma; NAD, no clinical abnormality detected; MM, multiple myeloma; ?MM;
likely but unconrmed multiple myeloma; WM, Waldenstrm macroglobulinaemia.
FLC (sFLC) assay (The Binding Site)

was rst developed in 2001 [10]. It is
an immunoturbidimetric method using
latex-enhanced polyclonal sheep antibodies targeted to epitopes on the light
chains of Ig that are exposed when the
light chain is free, i.e. not bound to heavy
chain Ig. Results are expressed as a ratio
of kappa : lambda light chains.
MM [4]. This eliminates a traditional

major challenge with MM diagnosis in
that disease denition was clinicopathological. The use of the sFLC ratio in this
way therefore marks a milestone in the
early detection of MM and highlights a
disease transition to being a laboratorydened rather than a symptom-dened
disease, allowing for earlier intervention.
This sFLC assay can be used to replace

traditional urine methods for the laboratory detection of FLCs. This practice
has the obvious benet of using a single
serum sample and eliminating the need
for a paired urine sample, which may not
always be supplied. In addition to the
reported increased diagnostic sensitivity
of the sFLC assay, an unexpected nding by Dispenzieri et al. was that baseline
sFLC results can be used in prognostication and risk stratication of MGUS [11].
Although the rationale for this is poorly
understood, it is thought that a greater
degree of abnormality in the sFLC ratio
reects an increasing tumour burden.
There is, however, controversy as to

whether the sFLC assay is indeed a robust
candidate for inclusion in PCD screening
strategies. There is currently only limited
guidance on how it should be used in
clinical practice [4] and there is ongoing
debate regarding result interpretation,
especially for those mildly abnormal
ratios. There are, therefore, many considerations to be made before such screening could be implemented.
Studies such as these have informed

changes to MM guidelines published
in 2016 [12] to acknowledge that signicantly abnormal FLC ratios, in the
absence of clinical features of end organ
damage, can be used in the diagnosis of
Study overview and results

Our real-time prospective study aimed
to assess the clinical utility of three index
laboratory investigations [serum and
urine protein electrophoresis (sEP and
uEP) and sFLC] to determine the most
eective rst-line testing strategy for
detecting PCDs in primary care patients.
These laboratory investigations were performed on 446 samples with no previous
history of, or investigations for, MM. The
The purpose of a medical screening programme is to recognize a disease in its

preclinical phase to allow intervention at
an earlier stage. Such strategies have benets, risks and costs and the nal screening algorithm is often a compromise
between these three. However, a proposed screening strategy should full the
criteria outlined by Wilson and Jungner
in 1968 [13]. Of note, criterion 4 suggests
there should be a detectable preclinical
stage, in this case MGUS, and criterion
5 suggests there should be a suitable test
for screening strategies. This real-time
prospective study presents evidence of
the clinical utility of the sFLC assay and
its use in developing a more sensitive
screening strategy for PCD detection.
Standard screening practice combining
sEP and uEP increased the sensitivity of
the constituent index tests (78% and 30%
respectively) to 81%, meaning the addition of urinalysis to sEP increased the
sensitivity by only 3%. This reinforces
the need for a more sensitive method
for detecting sFLC than sEP alone. This
combination also displayed a good PPV
without compromising eciency (98%).
Despite this, its use missed signicant
cases of PCDs including a light-chain
multiple myeloma, a possible but unconrmed (in the time frame of the study)
case of MM and 10 cases of MGUS,
highlighting its limitation as a rst line
screening investigation.
Combining sEP with sFLC analysis
increased the sensitivity from sEP alone
by 20% (data not shown), again suggesting singular sEP testing is not sensitive
enough to detect minor abnormalities
in FLC production. This proposed combination of screening tests increased
sensitivity by 17% when compared with
current protocols, indicating that the
sFLC assay is more sensitive than urinalysis for detecting PCDs. The sFLC
assay has been demonstrated to show a
high sensitivity for light chain MM and
non-secretory MM [14]. These often present with normal sEP and uEP, especially
13
April/May 2016
in low tumour burden stages when renal

function remains adequate, which may
explain the increased sensitivity of sFLC
over uEP.
The results of this study conrm also
those of others [15], which show that the
addition of sFLC analysis to sEP increases
the detection of MM and related PCDs.
In our case, there was a 17% increase in
patients with a PCD detected. However,
a concurrent rise in false positive results
(10%) was also seen when compared to
traditional screening protocols. Investigation into this was beyond the scope of
our study, though the false positive rate
could potentially be reduced by employing screening strategies that apply renal
reference intervals for the sFLC ratio for
those with renal insuciency.
Summary
On balance, there are several advantages
to replacing urinalysis with the sFLC
assay. These include increased clinical
sensitivity for detection of early-stage
disease, patient convenience in submitting a single serum sample rather than
two separate specimens, increased use of
automation and reduction in subjectivity in reporting of results. However, it is
also important to consider the potential
increased cost of performing sFLC on all
samples submitted for myeloma screening, the importance of using appropriate
reference ranges and the need to develop
guidelines for interpretation of borderline results. This latter point is particularly important in order that unnecessary referrals are prevented, and should
involve close liaison with local hematology teams to ensure that primary care
clinicians are given clear guidance for
further investigation and referral of their
patients.
References
1. Bird JM, Owen RG, DSa S, Snowden JA, Pratt
G, Ashcroft J, Yong K, Cook G, Feyler S, et al.
Guidelines for the diagnosis and management
of multiple myeloma 2011. Br J Haematol. 2011;
154(1): 3275.
2. Brenner H, Gondos A, Pulte D. Expected longterm survival of patients diagnosed with multiple myeloma in 20062010. Haematologica 2009;
94(2): 270275.
3. Rajkumar SV, Kyle RA, Therneau TM, Melton
LJ, III, Bradwell AR, Clark RJ, Larson DR, Plevak MF, Dispenzieri A, Katzmann JA. Serum free
light chain ratio is an independent risk factor
for progression in monoclonal gammopathy of
undetermined signicance. Blood 2005; 106(3):
812817.
Figure 2. Screening strategy 2: serum protein electrophoresis with reexed serum immunotyping and
serum free light chain analysis.
Number of patients in parentheses. EP, electrophoresis; IT, immunotyping; M protein, monoclonal
protein; MGUS, monoclonal gammopathy of undetermined signicance; LCMM, light chain
multiple myeloma; NAD, no clinical abnormality detected; MM, multiple myeloma; ?MM; likely
but unconrmed multiple myeloma; WM, Waldenstrm macroglobulinaemia; Normal FLC ratio,
(0.261.65).
4. Rajkumar SV, Dimopoulos MA, Palumbo A,

Blade J, Merlini G, Mateos MV, Kumar S, Hillengass J, Kastritis E, et al. International Myeloma
Working Group updated criteria for the diagnosis of multiple myeloma. Lancet Oncol. 2014;
15(12): e538548.
5. Kariyawasan CC, Hughes DA, Jayatillake MM,
Mehta AB. Multiple myeloma: causes and consequences of delay in diagnosis. QJM 2007;
100(10): 635640.
6. Howell DA, Smith AG, Jack A, Patmore R,
Macleod U, Mironska E, Roman E. Time-todiagnosis and symptoms of myeloma, lymphomas and leukaemias: a report from the Haematological Malignancy Research Network. BMC
Hematol. 2013; 13(1): 9.
7. Elliss-Brookes L, McPhail S, Ives A, Greenslade
M, Shelton J, Hiom S, Richards M. Routes to
diagnosis for cancer determining the patient
journey using multiple routine data sets. Br J
Cancer 2012; 107(8): 12201226.
8. Bossuyt X. Separation of serum proteins by automated capillary zone electrophoresis. Clin Chem
Lab Med. 2003; 41(6): 762772.
9. Kaplan IV, Levinson SS. Misleading urinary protein pattern in a patient with hypogammaglobulinemia: eects of mechanical concentration of
urine. Clin Chem. 1999; 45(3): 417419.
10. Bradwell AR, Carr-Smith HD, Mead GP, Tang
LX, Showell PJ, Drayson MT, Drew R. Highly
sensitive, automated immunoassay for immunoglobulin free light chains in serum and urine.
Clin Chem. 2001; 47(4): 673680.
11. Dispenzieri A, Kyle R, Merlini G, Miguel JS,

Ludwig H, Hajek R, Palumbo A, Jagannath S,
Blade J, et al. International Myeloma Working
Group guidelines for serum-free light chain
analysis in multiple myeloma and related disorders. Leukemia 2009; 23(2): 215224.
12. Myeloma: diagnosis and monitoring. National
Institute for Health and Care Excellence (NICE)
2016. (https://www.nice.org.uk/guidance/ng35)
13. Wilson JM, Jungner YG. [Principles and practice of mass screening for disease]. Bol Ocina
Sanit Panam. 1968; 65(4): 281393 (in Spanish).
14. Jagannath S. Value of serum free light chain
testing for the diagnosis and monitoring of
monoclonal gammopathies in hematology. Clin
Lymphoma Myeloma 2007; 7(8): 518523.
15. McTaggart MP, Lindsay J, Kearney EM. Replacing urine protein electrophoresis with serum
free light chain analysis as a rst-line test for
detecting plasma cell disorders oers increased
diagnostic accuracy and potential health benet to patients. Am J Clin Pathol. 2013; 140(6):
890897.
The authors
David Baulch* MSc, Beverley Harris MSc,
FRCPath
Department of Clinical Biochemistry,
Royal United Hospitals Bath NHS Foundation Trust, Bath, UK
E-mail: david.baulch@nhs.net
April/May 2016
14
Personalized medicine
Pharmacogenomics in an acute
myelogenous leukemia patient
This article examines the case of a patient who developed toxic
levels of voriconazole while taking the antifungal prophylactically as
part of her treatment regimen in addition to standard chemotherapy
for a leukocyte neoplasm. The usefulness of molecular diagnostic
testing as an aid in voriconazole dosing is discussed.
by S. Resaei, L. Collier and Dr S. Taylor
Case report
The patient was a 14-year-old female who
was referred to the emergency department
with a 10-day history of generalized bone
pain and progressively worsening fatigue.
An initial complete blood count (CBC)
revealed a white blood cell (WBC) count
that was well within the normal range, and
only slight anemia and thrombocytopenia.
However, because marked neutropenia and
elevated numbers of leukemic blasts were
noted in the dierential, a bone marrow
(BM) examination was performed. Marrow aspiration was markedly hypercellular
with diuse clusters of blasts (Fig. 1). Flow
cytometry on the aspirate disclosed a signicant (50% of total sample) blast population that exhibited CD33, CD13 (partial,
dim), CD34 (partial), CD15 (heterogeneous), CD19 (dim), CD10 (dim), HLA-DR,
CD64 (partial, dim), CD71 (dim), CD117,
CD123, CD58, CD38, cytoplasmic CD79a,
CD45 (dim), Tdt, and myeloperoxidase

markers. These same markers were exhibited by the circulating blasts in her peripheral blood. The co-expression of B-lymphoid and myeloid antigens prompted an
initial diagnosis of biphenotypic acute leukemia. After multiple expert consultations,
it was decided to model the patients treatment on therapy for acute lymphocytic
leukemia (ALL). Thus, the patient received
prednisone, vincristine, daunorubicin and
PEG asparaginase as induction chemotherapy, with vincristine and daunorubicin
administered again 7 days later.
Cytogenetic test results that were returned
on day 8, revealed a chromosomal translocation of (8;21)(q22;q22); RUNX1RUNX1T1, which changed the patients
diagnosis to an atypical form of acute myelogenous leukemia (AML). Accordingly,
the patients chemotherapy regimen was
changed so that the ALL-type therapy was

discontinued and standard AML therapy
that included cytarabine, daunorubicin,
and etoposide was begun. To address other
specic issues, this patient was treated
with multiple medications along with her
chemotherapy drugs, including Ambien,
Bactrim, Benadryl, cefepime, cyproheptadine, hydroxyzine, meropenem, vancomycin, and voriconazole.
On day 16, 8 days after the start of her new
pharmacology regimen, the patient began
to experience uctuating confusion and
auditory/visual hallucinations. Screening
tests revealed no abnormalities that could
explain her altered mental status, so attention turned to the medications that she
was receiving. All medications that seemed
likely to contribute to her neurologic problems were suspended and then reintroduced gradually with no adverse eect.
Voriconazole was not suspected of being
contributory to her altered mental status,
and was not interrupted. This antifungal
was rst administered to the patient on day
8 of her ordeal, at 200 mg/twice daily. She
continued to receive this dose from day 8
onwards, until 4 days after her initial neurological trouble (day 20). At this time, her
plasma voriconazole level was determined
to be >10.0 g/mL [normal range (NR):
1.06.0 g/mL]. The patients 200 mg twice
a day dosing regimen was reduced to 100
mg twice a day. Her plasma concentration
of voriconazole was monitored regularly
until its level plateaued at 2 g/mL (Fig. 2).
Pharmacogenomics
Voriconazole is an ecient triazole agent
used as an antifungal prophylactic in this
patient as she was receiving immunosuppressive chemotherapy. Patients with
hematologic malignancies are at high risk
of aspergillosis and candidiasis infections, because of the neutropenia that is
often caused by their chemotherapy regimens [13].
Figure 1. Photomicrograph of patients bone marrow aspirate at 40 magnication (left) and 100
magnication (right). Original diagnosis was biphenotypic acute leukemia, based upon blast
appearance and ow cytometry results.
Voriconazole is extensively metabolized in

the liver, primarily by CYP2C19 and, to a
lesser extent, by CYP2C9 and CYP3A4 liver
enzymes. The CYP2C19 genotype is generally
accepted as the key determinant in voriconazole clearance [46]. Variants of the CYP2C19
genotype have been identied and assigned
15
April/May 2016
allele (*2*8) is less clear. There is a certain

amount of dissention in the literature as to
how these individuals should be classied,
that is, various researchers classify them
as ultrarapid, extensive, intermediate, or
unknown metabolizers [7, 9].
Figure 2. Timeline of the patients voriconazole treatment and response. Voniconizole was
administered beginning on day 8 at 200 mg/twice daily. Four days after she began to experience
hallucinations her plasma voriconazole level was determined to be >10.0 g/mL and her
voriconazole dosage was reduced to 100 mg twice a day. By day 27, her plasma concentration of
voriconazole level plateaued at 2.0 g/mL.
enzyme activity. Thus the CYP2C19*1 variant is the wild-type variant and exhibits normal enzyme activity. CYP2C19 *2, *3, *4, *5,
*6, and *8 isotypes display loss of functionality as they possess little or no activity, and
the CYP2C19*17 variant is assigned gain-offunction status because of its robust enzyme
activity (Table 1) [7, 8].
Individuals who possess a normal or
wild-type drug metabolizing phenotype
inherit two copies of the normal CYP2C19
genotype (*1/*1), and are designated as
extensive metabolizers (EM). Intermediate metabolizers (IM) have any one of
the *2*8 alleles coupled with a normally
EUROIMMUN
functioning (*1) allele. Poor metabolizers (PM) are individuals with an enzyme
activity phenotype that is less than optimal,
caused by a genotype consisting of loss-offunction alleles (*2*8/*2*8 ). Ultrarapid
metabolizers (UM) are at the other end of
the enzyme activity spectrum, they may
either be heterozygous ultrarapid metabolizers with a wild-type allele combined
with an gain-of-function allele (*1/*17
genotype), or they may be homozygous
ultrarapid metabolizers with only gainof-function alleles (*17/*17) (Table 1) [7,
8]. The drug metabolizing phenotype of
individuals with the gain-of-function allele
(*17) combined with a loss-of-function
It is intuitive that an individuals CYP2C19

genotype fundamentally contributes to
voriconazole metabolism, elimination, and
therefore bioavailability of the drug [46].
Systemic exposure to voriconazole is generally higher in individuals with reduced
ability to metabolize and eliminate the
drug. Trough plasma concentrations of
voriconazole have been signicantly higher
in people possessing PM phenotypes followed by individuals with an IM phenotype, with the lowest bioavailability of the
drug detected in individuals with an EM or
UM phenotype [46, 8]. However, higher
trough levels of voriconazole are not universally higher in individuals with reduced
CYP2C19 activity [8, 10]. Voriconazole displays expected pharmacokinetic behaviour
according to genotype in healthy volunteers, but there is often a marked departure from the customary dose/response
relationship in patients. Presumably this
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16
April/May 2016
deviation from expected pharmacokinetic

behaviour is due to drugdrug interactions and/or the pathological circumstances of the patient [5, 6]. Generally, it
is expected that disease circumstances or
drug side eects that reduce liver enzyme
activity (especially of CYP2C19, CYP2C9
and CYP3A4) will decrease metabolism
and clearance of voriconazole, and thus
increase patient exposure to the drug.
Therapeutic drug monitoring

The United States Food and Drug Administration and the Infectious Diseases Society
of America recommend therapeutic drug
monitoring (TDM) for patients receiving voriconazole [7]. Numerous studies
indicate that voriconazole trough values
should be maintained above 1.0 g/mL for
fungal prophylaxis. Moreover, some studies indicate that voriconazole is more ecacious when trough levels are maintained
at 2.0 g/mL or higher [11, 12].
It is important to dose voriconazole accurately, as voriconazole ecacy is dependent
on adequate exposure to the drug; however, increased trough levels are associated with numerous severe adverse eects
(SAE). Voriconazole has been linked to
several adverse events including abnormal liver function tests, gastrointestinal
disturbances, rash and vomiting. Neurotoxicity (visual disturbances, hallucinations) is somewhat infrequently observed
[1, 2]. Since CYP2C19 is a key metabolizer of voriconazole, it seems reasonable
to predict a patients drug metabolizing
phenotype based on their CYP2C19 genotype, and to use this information to guide
dosing. In practice, the drug metabolizing
genotype alone is not sucient to predict
the metabolizing phenotype. Confounding
variables include the fact that voriconazole has a high propensity for drugdrug
interactions, a narrow therapeutic index, it
exhibits non-linear pharmacokinetics, and
Personalized medicine
its clearance is aected by circumstances

such as patient sex, age, disease state, liver
function, obesity and the presence of
inammation [11, 13, 14].
Conclusion
The pharmacodynamic behaviour of voriconazole remains dicult to predict as
it displays considerable interpatient and
intrapatient variablility. Although TDM
for patients receiving voriconazole is recommended, establishing a patients pharmacogenomic prole can provide clinicians with valuable information to aid in
appropriate voriconazole dosing, especially
in the initial stages of therapy. Pharmacogenomic information is likely to contribute
to the goal of rapidly attaining a therapeutic
concentration while avoiding toxicity. It is
possible that our patient has a PM phenotype for voriconazole and that pharmacogenomic testing might have minimized her
exposure to toxic levels of voriconazole that
arose from standard voriconazole dosing.
References
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Allelic functional status
*1
Normal function; normal activity; wild-type
*2, *3, *4, *5, *6, *7, *8
Loss-of-function; no or decreased activity
*17
Gain-of-function; increased activity

Genotype
Metabolizing phenotype
individualised medicine arrived for antifungals? A

review of antifungal pharmacogenomics. Bone Marrow Transplant. 2012;47(7): 881894.
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and toxicity. Int J Antimicrob Agents. 2014;44(3):
183193.
6. Dolton MJ, Mikus G, Weiss J, Ray JE, McLachlan AJ.
Understanding variability with voriconazole using
a population pharmacokinetic approach: implications for optimal dosing. J Antimicrob Chemother.
2014;69(6): 16331641.
7. Owusu OA1, Egelund EF, Alsultan A, Peloquin CA,
Johnson JA. CYP2C19 polymorphisms and therapeutic drug monitoring of voriconazole: are we
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8. Moriyama B, Kadri S, Henning SA, Danner RL,
Walsh TJ, Penzak SR. Therapeutic drug monitoring
and genotypic screening in the clinical use of voriconazole. Curr Fungal Infect Rep. 2015;9(2): 7487.
9. Swen JJ, Nijenhuis M, de Boer A, Grandia L, Maitland-van der Zee AH, Mulder H, Rongen GA, van
Schaik RH, Schalekamp T, Touw DJ, van der Weide J,
Wilert B, Deneer VH, Guchelaar HJ. Pharmacogenetics: from bench to byte-an update of guidelines.
Clin Pharmacol Ther. 2011; 89(5): 662673.
10. Kim SH, Yim DS, Choi SM, Kwon JC, Han S, Lee
DG, Park C, Kwon EY, Park SH, Choi JH, Yoo JH.
Voriconazole-related severe adverse events: clinical application of therapeutic drug monitoring
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753758.
11. Davies-Vorbrodt S, Ito JI, Tegtmeier BR, Dadwal
SS, Kriengkauykiat J. Voriconazole serum concentrations in obese and overweight immunocompromised patients: a retrospective review. Pharmacotherapy. 2013 Jan;33(1): 2230.
12. Smith J, Safdar N, Knasinski V, Simmons W, Bhavnani SM, Ambrose PG, Andes D. Voriconazole
therapeutic drug monitoring. Antimicrob Agents
Chemother. 2006;50(4): 15701572.
13. van Wanrooy MJ, Span LF, Rodgers MG, van den
Heuvel ER, Uges DR, van der Werf TS, Kosterink
JG, Alenaar JW. Inammation is associated with
voriconazole trough concentrations. Antimicrob
Agents Chemother. 2014;58(12): 70987101.
14. Brggemann RJ, Antonius T, Heijst Av, Hoogerbrugge PM, Burger DM, Warris A. Therapeutic
drug monitoring of voriconazole in a child with
invasive aspergillosis requiring extracorporeal membrane oxygenation. Ther Drug Monit.
2008;30(6): 643646.
*17/*17
Ultrarapid (homozygous)
The authors
*1/*17
Ultrarapid (heterozygous)
*1/*1
Extensive
*1/*2-*8
Intermediate
*17/*2-*8
Ultrarapid, extensive, intermediate, unknown
Sahar Resaei BS; Laura Collier MLS(ASCP);

Sara Taylor* PhD, MLS(ASCP)MB
Tarleton State University, Fort Worth, TX,
USA
*2-*8/*2-*8
Poor
Table 1. CYP2C19 gene variants and drug metabolizing activity.
E-mail: sataylor@tarleton.edu
Diabetes
17
April/May 2016
Diagnosis of diabetes mellitus

Diabetes is characterized by hyperglycemia, but diagnosis no
longer depends exclusively on plasma glucose measurements. The
endorsement of glycated hemoglobin as a diagnostic test for diabetes
has seen its widespread adoption for this purpose: it is vital that its
application in this role is appropriate and its limitations understood.
by Dr Shirley Bowles
Introduction
The term diabetes mellitus encompasses
several diseases of abnormal carbohydrate metabolism that are characterized
by hyperglycemia associated with relative or absolute defects in insulin secretion and varying degrees of peripheral
resistance to its action [1]. Diabetes is
the most common metabolic disorder:
in 2014, 422 million people in the world
had diabetes, a prevalence of 8.5% in the
adult population [2].
The fact that various pathogenetic processes may be involved in the development of diabetes is illustrated by the etiological classification outlined in Table
1 but, in fact, the vast majority of cases
are categorized as either Type 1 (510%)
or Type 2 (9095%). Type 1 diabetes is
usually due to cellular-mediated autoimmune destruction of the pancreatic
-cells, with absolute loss of insulin
secretion, and, although the rate of cell
destruction is variable, most individuals will ultimately become dependent

on exogenously administered insulin for
survival, and are at risk of ketoacidosis.
In contrast, patients with Type 2 diabetes often have insulin levels that appear
normal, or even elevated, but secretion
is considered defective because it is
insufficient to compensate for varying
degrees of insulin resistance, which may
be attributable to the obesity found in
most of these patients. In Type 2 diabetes, treatment with insulin is not essential for survival, although it may eventually prove necessary to achieve glycemic
control [3].
The majority of individuals with Type
2 diabetes are largely asymptomatic,
diagnosed only after laboratory evaluation, whereas those with Type 1 are
more likely to present with the classical
symptoms of hyperglycemia: polyuria,
polydipsia, blurred vision and weight
Type of diabetes
Comments
I. Type 1 diabetes (510% of cases)
-cell destruction: absolute insulin deficiency
II. Type 2 diabetes (9095% of cases)
-cell destruction: absolute insulin deficiency
III. Other specific types:

A. Genetic defects of -cell function
B. Genetic defects in insulin action
C. Diseases of the exocrine pancreas*
e.g. Pancreatitis, cystic fibrosis,

hemochromatosis
D. Endocrinopathies*
e.g. Acromegaly, Cushings syndrome,

glucagonoma
E. Drug- or chemical-induced*
e.g. Glucocorticoids, thiazides, thyroid

hormones
F. Infections*
e.g. Congenital rubella, cytomegalovirus
G. Uncommon forms of immune-related

diabetes*
e.g. Stiff-man syndrome
H. Other genetic syndromes sometimes

associated with diabetes
e.g. Downs syndrome, Turners syndrome
IV.
Gestational diabetes mellitus
*Considered secondary diabetes
Table 1. Etiological classication of diabetes mellitus [4].
loss. There may also be acute life-threatening consequences of uncontrolled

diabetes: diabetic ketoacidosis in Type
1 diabetes and non-ketotic hyperosmolar syndrome in Type 2 [1]. Both forms
are associated with a number of characteristic long-term complications, usually considered to be a consequence of
microvascular disease, including retinopathy, with potential loss of vision;
nephropathy, leading to kidney failure;
peripheral and autonomic neuropathy.
However, in reality, the major determinant of the reduced life expectancy seen
in diabetes is the significantly increased
incidence of macrovascular atherosclerotic disease, which causes myocardial
infarction or angina, stroke or peripheral vascular disease [4].
Diagnostic criteria
1. Blood glucose measurements

For decades, the diagnosis of diabetes was based exclusively on glucose
measurements but, as blood glucose is
a continuous variable, cut-off points
for diagnosis are necessarily somewhat
arbitrary, and information derived
from research and clinical practice has
prompted periodic re-evaluation of the
diagnostic criteria. By 1997, the diagnosis of diabetes, as defined by the World
Health Organization (WHO), required
a fasting plasma glucose (FPG) of
7.8 mmol/L or a plasma glucose (PG)
of at least 11.1 mmol/L, in either a random blood specimen or in one collected
2 hours after a standard 75-g glucose
load, as part of an oral glucose tolerance
test (OGTT). An at-risk category was
also recognized: impaired glucose tolerance (IGT), which was identified on
the basis of an OGTT 2-hour PG of 7.8
11.0 mmol/L. These values were chosen,
based on the risk of future symptoms of
uncontrolled hyperglycemia [5].
As the major objective of diagnosing
diabetes is to intervene so as to prevent
premature mortality and morbidity, it
seemed logical to consider diagnosis in
terms of risk of complications: following
the recommendations of the National
Diabetes Data Group in 1997 [6], the
WHO revised the diagnostic threshold,
with respect to the fasting glucose, based
on the observed association between
glucose levels and the risk of developing the microvascular complication of
Diabetes
18
April/May 2016
Test
Diagnosis
Normal
Impaired glucose
regulation or
Pre-diabetes
Diabetes
HbA1c** (mmol/mol)
<42
4247
48*
Fasting glucose (mmol/L)
<6.1
6.16.9
7.0*
2hr glucose in OGTT

(mmol/L)
<7.8
7.811.0
11.1*
Random glucose (mmol/L)
11.1*
*If the patient is asymptomatic, a repeat test is required to conrm the diagnosis.
** HbA1c is not appropriate for diagnosis of diabetes:
Symptoms suggestive of Type 1 diabetes (e.g. weight loss, ketonuria)
Symptoms of diabetes for less than 2 months
Pregnancy
All those under 18 years of age
Treatment with corticosteroids, antipsychotics or immunosuppressants
Acute pancreatic damage (e.g. pancreatitis or pancreatic surgery)
Conditions that may prevent accurate measurement of HbA1c: hemoglobinopathies;
hemolytic anemia; iron-deciency anemia; splenomegaly; antiretroviral drugs; splenectomy;
chronic kidney disease (when associated with renal anemia).
This exclusion only apples to patients on short-term therapy with these drugs: if a patient
is on such treatment for more than 3 months, HbA1c would be an appropriate test to
screen for diabetes.
Table 2. Criteria for diagnosing diabetes [4, 12, 15].
retinopathy. The OGTT 2-hour PG of

11.1 mmol/L closely approximates
to a point at which the prevalence of
microvascular complications increases
dramatically. However, only approximately 25% of those who exceed this
2-hour threshold will also have a FPG
7.8 mmol/L, whereas almost all individuals with FPG 7.8 mmol/L have a
2-hour OGTT level 11.1 mmol/L. Thus,
this earlier FPG cut-off defined a greater
degree of hyperglycemia, a discrepancy
that was considered undesirable: both
fasting and 2-hour cut-off points should
reflect a similar degree of hyperglycemia
and risk of adverse outcomes. In addition, due to the inconvenience of undertaking OGTTs, the FPG alone was often
performed, meaning that a substantial
number of individuals, who were at
increased risk of microvascular complications, would not have been detected.
The revised FPG cut-off of 7.0 mmol/L
was shown to have a similar predictive
value for adverse outcomes as the 11.1
mmol/L 2-hour OGTT threshold, which
validated the use of this simpler test for
diagnostic purposes.
It was at this stage that a secondary

criterion for the at-risk category was
recognized: impaired fasting glycemia
(IFG), a FPG of 6.16.9 mmol/L. Both
IFG, and the previously described IGT,
have been referred to as pre-diabetes,
indicating a relatively high risk of future
diabetes. Studies have demonstrated an
approximately 510% annualized risk
of progression to diabetes in individuals
with either IFG or IGT and 1015% in
those with both abnormalities [7].
2. Glycated hemoglobin
Glycated hemoglobin (HbA1c), formed
as a consequence of a non-enzymatic,
irreversible reaction between glucose
and the N-terminal valine residue of the
globin chains of hemoglobin, reflects
average blood glucose levels over the
preceding 812-week period (the lifespan of a red blood cell) and its potential
as an indicator of glycemic control was
recognized in 1977 [8]. Over the intervening years, supported by evidence
from the Diabetes Control and Complications Trial (Type 1 diabetes) [9] and
the United Kingdom Prospective Diabetes Study (Type 2 diabetes) [10], which
validated the direct relationship between
glycated hemoglobin levels and clinical outcomes, it has had a vital role in
monitoring diabetes. With respect to the
diagnosis of diabetes, however, although
epidemiological studies also showed a
clear relationship between HbA1c and
retinopathy, variation in methodology
and standardization, and concern about
the confounding effect of factors affecting erythrocyte turnover, seemed to preclude its use for this purpose [8]. This
situation has changed in recent years, as
a result of a number of HbA1c standardization programmes, culminating in
the work of the IFCC Working Group
on Standardization of HbA1c, which
established true International Reference Methods for HbA1c and provided
a preparation of pure HbA1c, against
which manufacturers could standardize
their calibrators [11].
In 2011, in response to this global
standardization of HbA1c methods, the
WHO stated that HbA1c could be used
as a diagnostic test for diabetes mellitus,
provided that stringent quality assurance methods are in place, assays are
standardized to criteria aligned to the
international reference values and there
are no conditions present that preclude
its accurate measurement [12]. Based
on the DETECT-2 pooled data analysis, which examined the association
between diabetes-specific retinopathy
and glycemic measures, an HbA1c of
48 mmol/mol was recommended as the
cut-off point for diagnosing diabetes
[13]. As with glucose measurements,
there is a range of HbA1c levels below
this diagnostic value, which indicates an
increased risk of future diabetes and/
or cardiovascular disease: a systematic
review indicated that HbA1c values
between 37 and 48 mmol/mol are associated with a substantially increased risk
of diabetes [14]. The WHO did not provide specific guidance on HbA1c criteria for pre-diabetes but the 2009 International Expert Committee concluded
that individuals with an HbA1c of
4247 mmol/mol should be considered
at high risk of progression to diabetes
[15] (estimated 5-year risk of 2550%
[14]), a range that was endorsed by a UK
Expert Position Statement [16].
Current recommendations
The current criteria for the diagnosis of
diabetes and pre-diabetes, in accordance
19
with WHO recommendations, are summarized in Table 2. OGTTs, which are
time-consuming, inconvenient and
show poor reproducibility, are increasingly confined to the diagnosis of gestational diabetes. HbA1c confers definite
advantages over FPG (and OGTT): no
patient preparation; lower biological
variation; less fluctuation in acute stress
and illness, and standardization of measurement is now better than for glucose,
which has no internationally recognized
reference method. However, there are a
number of situations, in which the use
of HbA1c for diagnosis is not appropriate (Table 2): as a measure of chronic
hyperglycemia, HbA1c should not be
used where rapidly developing hyperglycemia is suspected and results will be
unreliable in the presence of any factors
affecting erythrocyte lifespan [12].
Regardless of the test used, in an
asymptomatic patient, a diagnostic
result should be confirmed by repeat
testing on a separate day, preferably
using the same test, in order to increase
the likelihood of concordance. In the
same way that there is less than 100%
concordance between the results of
FPG and 2-hour OGTT PG, there is

not full concordance between HbA1c
and glucose measurements: these three
different measures of glycemia represent different physiological processes
and, therefore, inevitably, they identify somewhat different populations of
patients [17]. In fact, although HbA1c
performs equally well as a predictor of
retinopathy risk, in most populations,
its use results in a lower diabetes prevalence (the OGTT 2-hour PG is the most
sensitive test). A study including 6890
adults from the US National Health and
Nutrition Examination Survey (1999
2006) indicated that the prevalence of
undiagnosed diabetes was 2.3% using
HbA1c, compared to 3.6% using FPG
[18]. Other studies have confirmed this
discrepancy although, in fact, the magnitude of the difference appears to vary
between populations, perhaps reflecting geographical or ethnic differences
in hemoglobin glycation rates or the
distribution of certain forms of anemia
or hemoglobinopathy. It is anticipated
that, in practice, the lower sensitivity of
HbA1c will be mitigated by its ease of
use, which will facilitate its wider application [3].
April/May 2016
For those individuals with pre-diabetes,

structured lifestyle intervention, aimed
at increasing physical activity and
achieving a loss of body weight, may
prevent, or at least delay, the development of diabetes. Within this category,
for all three tests, the risk of future diabetes is curvilinear, extending below the
lower limit of the range and becoming
disproportionately greater at the higher
end: accordingly, intervention and follow-up should be most aggressive for
those considered at particularly high
risk [3]. The associated increased risk
of cardiovascular disease should also be
targeted, with appropriate management
of other relevant risk factors (smoking,
lipids, blood pressure).
From glucose measurements

to HbA1c in the diagnosis of
diabetes mellitus: one UK
laboratorys experience of
the change in clinical practice
Guidance, outlining the WHOs position on the use of HbA1c in the diagnosis of diabetes, was issued to local
clinicians in 2012. Subsequently, in
September 2014, updated guidance was
disseminated, advocating the use of
April/May 2016
HbA1c as a diagnostic test for diabetes mellitus, except where inappropriate, and providing advice on follow-up.
This was supported by modification of
the requesting process, which allowed a
distinction to be made between HbA1c
requests made for monitoring established diabetes (designated HbA1cM)
and those being used for diagnosis (designated HbA1cD). This facilitated the
provision of additional targeted guidance in the form of interpretative comments and, importantly, for HbA1cD
requests, allowed flagging, as abnormal, results that indicated pre-diabetes
(4247 mmol/mol).
The pattern of fasting glucose, OGTT
(excluding those from maternity services) and HbA1c requesting between
April 2012 and March 2016 is summarized in the Figure 1. Between late 2012
and September 2014, there was a steady
increase in HbA1c requests, which was
mirrored by a decrease in the number of
fasting glucoses requested and OGTTs
performed. Since the introduction of
the two separate requests, HbA1cD and
HbA1cM, in September 2014, it can be
seen that, with regard to monitoring,
the number of requests has remained
at around 2200 per month, about 10%
higher than the number being done
early in 2012 (when all such requests
were for this purpose). In contrast,
those requested for diagnostic purposes increased rapidly and, since late
2015, the number of HbA1cD requests
has been similar to the total number of
HbA1c requests/month in 2014.
20
Diabetes
Summary
Local experience indicates an enthusiastic uptake in the use of HbA1c for
diagnosing diabetes and a concurrent
fall in glucose measurements (FPG and
2-hour OGTT PG) for this purpose. As
anticipated, the convenience of this test
has led to increased screening for diabetes but there is concern that this ease
of use may mean that the limitations
of HbA1c as a diagnostic test are overlooked, resulting in its application in
circumstances when glucose measurements would, in fact, be indicated. There
is a clear role for laboratory staff in the
provision of ongoing education of clinicians, in order to ensure the appropriate
use and interpretation of these tests.
References
1. McCulloch DK. Clinical presentation and diagnosis of diabetes mellitus in adults. UpToDate. (http://
uptodate.com/contents/clinical-presentation-anddiagnosis-of-diabetes-mellitus)
2. Global Report on Diabetes. World Health Organization
2016.
(http://apps.who.int/iris/bitstr
eam/10665/204871/1/9789241565257_eng.pdf)
3. American Diabetes Association Position Statement.
Diagnosis and classication of diabetes mellitus.
Diabetes Care 2011; 34(Suppl 1): S62S69.
4. Report of a WHO Consultation. Denition, diagnosis and classication of diabetes mellitus and its
complications. World Health Organization 1999.
(https://www.sta.ncl.ac.uk/philip.home/who_dmg.
pdf)
5. Denition and diagnosis of diabetes mellitus and
intermediate hyperglycemia. World Health Organization 2006. (http://www.who.int/diabetes/publications/Definition%20and%20diagnosis%20of%20
diabetes_new.pdf)
6. Expert Committee on the Diagnosis and Classication of Diabetes Mellitus. Report of the Expert Committee on the diagnosis and classication of diabetes
mellitus. Diabetes Care 1997; 20: 11831197.
7. Inzucchi SE. Diagnosis of diabetes. N Engl J Med.
2012; 367(6): 542550.
8. Day A. HbA1c and diagnosis of diabetes. The test has
nally come of age. Ann Clin Biochem. 2012; 49: 78.
9. The Diabetes Control and Complications Trial
Research Group. The eect of intensive treatment
of diabetes on the development and progression of
long-term complications in insulin-dependent diabetes. N Engl J Med. 1993: 329: 977986.
10. United Kingdom Prospective Diabetes Study
(UKPDS) Group. Intensive blood glucose control
with sulphonylureas or insulin compared with conventional treatment and risk of complications in
patients with type 2 diabetes (UKPDS 33). Lancet
1998; 352: 837853.
11. The American Diabetes Association, European
Association for the Study of Diabetes, International
Federation of Clinical Chemistry and Laboratory
Medicine and the International Diabetes Federation Consensus Committee. Consensus statement on the worldwide standardisation of the
HbA1c measurement. Diabetologia 2007; 50(10):
20422043.
12. Use of glycated haemoglobin (HbA1c) in the diagnosis of diabetes mellitus. Abbreviated report of a
WHO consultation. World Health Organization
2011. (http://www.who.int/diabetes/publications/
report-hba1c_2011.pdf)
13. Colagiuri S, Lee CMY, Wong TW, Balkau B, Shaw
JE, Borch-Johnsen K. Glycemic thresholds for diabetes-specic retinopathy: implications for diagnostic criteria for diabetes. Diabetes Care 2011; 34:
145150.
14. Zhang X, Gregg EW, Wiliamson DF, Barker LE,
Thomas W, Imperatore G, Williams DE, Albright
AL. A1c level and future risk of diabetes: a systematic review. Diabetes Care 2010; 33(7): 16651673.
15. International Expert Position Report on the role of
the A1C assay in the diagnosis of diabetes. Diabetes
Care 2009; 32: 13271334.
16. Expert Position Statement: Use of HbA1c in the
diagnosis of diabetes mellitus in the UK. The implementation of World Health Organization guidance
2011. Diabetic Medicine 2012; 29: 13501357.
17. American Diabetes Association. Classication and
diagnosis of diabetes. Diabetes Care 2015; 38(Suppl
1): S8S16.
18. Carson AP, Reynolds K, Fonseca VA, Muntner P.
Comparison of A1C and fasting glucose criteria to
diagnose diabetes among U.S. adults. Diabetes Care
2010; 33: 9597.
The author
Figure 1. Pattern of diabetes test requesting: Fasting glucoses, oral glucose tolerance tests (OGTT) and
HbA1c, 20122016. (For a laboratory in a UK District General Hospital, serving a population of
~ 260 000).
Shirley A. Bowles MB ChB, MSc, FRCPath

Department of Blood Sciences, Countess
of Chester Hospital NHS Foundation
Trust, Chester, UK
E-mail: shirleybowles@nhs.net
Diabetes
21
April/May 2016
Type 2 diabetes - biomarker models

promise new means to predict risk
Considerable rewards could be obtained from early identication
of Type 2 diabetes mellitus (T2DM). One of the most obvious, as
suggested in a recent report on diabetes global burden, would be
better disease management. The report, by the University of East
Anglia in the UK, concludes that early investments into prevention
and disease management may therefore be particularly worthwhile.
Risk factors
Such perspectives are strengthened by
evidence that the onset of T2DM can be
delayed by behaviour modication. A
study in the British Medical Journal in
2007 noted that lifestyle changes could be
at least as eective as drug treatment in
slowing the onset of diabetes. It concluded
that the only barrier to the eectiveness
of such a strategy was to identify diabetes
quickly enough.
Much is now known about the risk factors
associated with T2DM such as parental
history, age, body mass index and elevated
blood glucose levels. Combining these
with measurable indicators of metabolic
syndrome - high blood pressure, LDL and
HDL cholesterol and excess triglyceride can result in a credible degree of prediction. However, there are several barriers to
the process.
(FPG). However, the tests specicity is

poor. Two decades ago, the so-called Hoorn
study at Amsterdam warned about signicant levels of variation in blood glucose
levels. Although many individuals are
identied as having impaired fasting glucose (IFG), their absolute risk of conversion to diabetes is a mere 5 to 10% per year.
Over this period, dierences have also
emerged about how best to measure glucose. In the year 2000, while some experts
(including the American Diabetes Association) recommended the use of fasting
plasma glucose (FPG) alone, others noted
that many diabetic subjects would have
been classied as non-diabetic on the
FPG test. As a result, they recommended
use of the two-hour oral glucose tolerance test (OGTT). Nevertheless, in spite of
its greater accuracy, OGTT is rarely used
since it requires two hours to perform and
is an unpleasant experience for the patient.
Fasting glucose and oral glucose Glucose tolerance only one risk
tolerance
indicator
The typical method for assessing T2DM
risk is to measure fasting plasma glucose
The above factors have provoked a search

for new approaches to predict T2DM.
Some beliefs about OGTT have been

brought into question, too. In 2002, clinical
epidemiologists at the University of Texas
Health Center in San Antonio published
the results of a prospective cohort study to
identify people at high risk of T2DM.
The results were unequivocal. Impaired
glucose tolerance was only one indicator
of risk. Persons at high risk for T2DM, the
study concluded, were better identied
by using a simple prediction model than
by relying exclusively on the results of a
2-hour oral glucose tolerance test.
Predictive models
Subsequent years have been witness to
signicant eorts to develop and rene
predictive models for T2DM. However,
ve years after the San Antonio study, the
choices are still less than wholly clear.
In 2007, the Framingham Ospring study
in the US estimated seven-year T2DM risk
based on a pyramid of metrics consisting at the base - of age, sex, parental history and
body mass index. This was followed by the
inclusion of simple clinical measurements
on metabolic syndrome traits, and thereafter, the 2-hour post-oral glucose tolerance
test, fasting insulin and C-reactive protein levels. At its most complex, the model
used the Gutt insulin sensitivity index or a
homoeostasis model of insulin resistance.
For proponents of new alternatives to
impaired glucose tolerance, the conclusions of the Framingham study were stark.
Complex clinical models, it stated, were
not superior to the simple one, and in spite
of the denite existence of T2DM prediction rules, we lack consensus for the most
eective approach.
The limitations of biotech

More recently, investigations at the frontiers of biotech have also faced challenges
to clear-cut answers. Although it is clear
that multiple genetic loci are associated
with the risk of T2DM, researchers have
not managed to connect the genetics
underlying a family history of diabetes
with predictability.
In 2008, researchers at Harvard/Massachusetts General and Emory University
published results of a study on 18 singlenucleotide polymorphisms (SNPs) known
to have associations with the risk of T2DM,
April/May 2016
to predict new cases in a large, prospectively examined, community-based cohort.

However, the outcome, in terms of risk
prediction, was less than encouraging. In
reality, it proved to be only slightly better
at making a prediction than did traditional
risk factors on their own. The authors concluded: Our ndings underscore the view
that identication of adverse phenotypic
characteristics remains the cornerstone of
approaches to predicting the risk of type 2
diabetes.
Adiponectin and ferritin

Meanwhile, the eort to identify and validate alternate biomarkers for prediction
and screening continue. Two especially
promising ones appear to be adiponectin,
an adipocyte-derived, insulin-sensitizing
peptide, and ferritin, a protein that binds
to iron and accounts for most of the iron
stored in the body.
Studies in the early 2000s in the US and
Germany conrmed that adiponectin was
independently associated with a reduced
risk of type 2 diabetes.
Interest in this area goes back a long time,
to a cross-sectional and longitudinal study
of Arizonas Pima Indians, who have the
worlds highest reported prevalence and
incidence of non-insulin-dependent diabetes mellitus (NIDDM). The study dates
to the early 1980s when it sought to document the sequence of metabolic events
occurring with the transition from normal
to impaired glucose tolerance and then to
diabetes.
In 2004, a prospective study within the US
Nurses Health Study investigated iron storage, given a belief that T2DM was a manifestation of hemochromatosis, due to iron
overload. Researchers have established that
higher iron store (reected by an elevated
ferritin concentration and a lower ratio of
transferrin receptors to ferritin) is associated with increased T2DM risk in healthy
women, independent of known diabetes
risk factors.
However, there still are reasons for caution. In July 2014, or more than a decade
after the US Nurses Health Study, a metaanalysis of T2DM risk and ferritin in the
journal Diabetes/Metabolism Research
and Reviews warned that though evidence
suggested a causal link, publication bias
and unmeasured confounding cannot be
excluded.
Nevertheless, ferritin and adiponectin
do appear to play a key role in predicting
T2DM when combined with other selected
biomarkers.
22
Diabetes
The Danish model

One predictive model that has emerged in
Denmark selected a panel of six biomarkers out of a total of 64, to assess T2DM risk.
The selected biomarkers include adiponectin and ferritin, as well as four of their
more common counterparts: glucose and
insulin, as well as the inammation markers C-reactive protein (CRP) and interleukin-2 receptor A (IL2RA).
The model was developed by a research
team from Copenhagens Glostrup Hospital and Steno Diabetes Centre, along with
the Copenhagen and Aarhus universities,
and Tethys Bioscience of the US.
The researchers used the so-called Inter99
cohort, a study of about 6,600 Danes with
the primary outcome of 5-year conversion
to T2DM, to select 160 individuals who
developed T2DM and 472 who did not.
They carefully measured several clinical
variables and candidate biomarkers from a
multitude of diabetes-associated pathways,
using an ultrasensitive immunoassay microsample molecular counting technology.
Their eort ultimately led to six biomarkers
that gave a Diabetes Risk Score. This, they
concluded in a July 2009 issue of Diabetes
Care, provided an objective and quantitative estimate of the 5-year risk of developing type 2 diabetes, performs better than
single risk indicators and a noninvasive
clinical model, and provides better stratication than fasting plasma glucose alone.
Expert acclaim
The researchers who developed the Danish Diabetes Risk Score are modest in their
claims. In an appendix to their report in
Diabetes Care, they point out that their
selection process for biomarkers may not
have identied the best possible model, but
do state that they identied a good model.
Some outside observers are however less
circumspect, given what many acknowledge to be one of the most exhaustive and
profound selection eorts to date. James
Meigs of Harvard Medical School calls
the Danish Diabetes Risk Score the most
robust multimarker prediction model
possible.
Beyond Europeans to Chinese

One of the only major caveats in the Danish eort consisted of demographics. The
report on the Danish model in Diabetes
Care noted that it may only apply to white
Northern Europeans enrolled in a lifestyle
intervention trial and that it was an open
question whether the model would produce the same biomarkers or discriminate
well in race/ethnicity populations that are
dierentially aected by diabetes.
Answers to these are still emerging. In

2013, a study on 2,198 community-living
Chinese by the Shanghai Institutes for
Biological Sciences endorsed the use of
ferritin as a biomarker. Though the focus
of the research was on iron storage, two of
three other biomarkers used in the eort
were the same as those in the Danish study,
namely adiponectin and CRP (the fourth
was -glutamyltransferase).
Biomarker search continues

Meanwhile, the search for TD2M biomarkers continues.
Two endothelial dysfunction biomarkers
being investigated for T2DM risks consist
of E-selectin and ICAM-1. The US Nurses
Health Study mentioned above also found
that signicantly elevated levels of the latter predicted incident diabetes in women
independent of traditional risk factors
such as BMI, family history, diet and activity. In addition, adjustment for baseline
levels of C-reactive protein, fasting insulin,
and hemoglobin A (1c) did not alter these
associations.
Incretins and melatonin

Incretins, metabolic hormones which
lower blood glucose by causing an increase
in insulin after eating, are another potentially signicant biomarker. An incretin
eect is associated with the fact that oral
glucose elicits a higher insulin response
than does intravenous glucose. There are
two hormones responsible for the incretin
eect: glucose-dependent insulinotropic
hormone (GIP) and glucagon-like peptide-1 (GLP-1).
In patients with type 2 diabetes, the incretin eect is reduced. In addition, about
half rst-degree relatives of patients with
T2DM show reduced responses toward
GIP, without any signicant change in GIP
or GLP-1 secretion after oral glucose. To
some researchers, this opens the possibility that a reduced responsiveness to GIP is
an early step in the pathogenesis of type 2
diabetes.
Variation in the Circadian system has also
drawn a great deal of attention.
Reverse transcription polymerase chain
reaction (RT-PCR) analyses, led by a team
at the University of Lille in France, investigated melatonin receptor 2 (MT2 transcripts) in neural tissues and MT2 expression in human pancreatic islets and beta
cells. Their ndings suggest a link between
circadian rhythm regulation and glucose
homoeostasis through the melatonin signalling pathway.
Diabetes
23
April/May 2016
The use of point-of-care ketone meters

to diagnose and monitor diabetic
ketoacidosis in pediatric patients
Children presenting with diabetic ketoacidosis (DKA) require prompt
assessment and treatment initiation to prevent serious complications.
The use of point-of-care (POC) analysers to assess blood ketones
is beginning to replace the traditional analysis of urine ketones,
but some questions remain as to their optimal utilization.
by Dr A.M. Ferguson, Dr J. Michael, Prof. S. DeLurgio and Dr M. Clements
Introduction
Diabetic ketoacidosis (DKA) is an acute
complication of uncontrolled diabetes mellitus resulting from insulin deciency. It is
biochemically dened as hyperglycemia
(blood glucose >200 mg/dL) with metabolic acidosis (venous pH <7.3 or bicarbonate <15 mmol/L), ketonemia, and ketonuria [1]. The clinical picture of the patient
can include fatigue, polydipsia, polyuria,
dehydration, abdominal pain, vomiting
and altered mental status (Box 1). DKA
can occur in known diabetics and can be
the presenting symptom prior to diagnosis.
Children who are on insulin pump therapy,
who have unstable family situations, or
have limited access to healthcare are at an
increased risk of DKA [1], and DKA is the
most common cause of diabetes-related
mortality in children.
Assessing urine ketones has been part of
the standard practice when assessing if
a patient has DKA, but this has multiple
Box 1. Clinical symptoms of
diabetic ketoacidosis
Fatigue
Polydipsia
Polyuria
Dehydration
Abdominal pain
Nausea and vomiting
Rapid breathing
Altered mental status
Fruity odor of breath
issues. There are three types of ketones: acetoacetate, acetone, and -hydroxybutyrate
(BHB). BHB is the predominant ketone
produced during DKA and can be present
at up to 10 times the amount of acetoacetate. The urine dipsticks that are commonly
used to assess ketonuria utilize a nitroprusside reagent that reacts with acetoacetate
and acetone but not at all with BHB. This is
problematic because the major ketone produced in DKA is not detected, which can
lead to false negative urine ketone testing.
Additionally, as ketosis resolves, BHB is
converted to acetoacetate, increasing urine
ketones during the recovery phase, potentially leading the clinician to believe that
the ketosis is worsening instead of resolving. An added obstacle is the diculty of
getting a urine specimen from a young
child, especially one in nappies. Measuring
serum ketones, specically BHB, is a solution to both of these issues.
Clinical measurement of
serum ketones
As the methodology for measuring serum
BHB became more automated, the test
moved from being used only on a research
basis to being available for clinical use. Initial studies were done to see how serum
BHB functioned for the diagnosis of DKA.
A large retrospective study looking at
simultaneous measurements of BHB and
bicarbonate found that BHB levels of 3
and 3.8 mmol/L in children and adults,
respectively, could be used to diagnose
DKA and provides a more specic assessment of DKA than bicarbonate alone [2].
When assessing patients for DKA, it is
critical to make the diagnosis as quickly as
possible to initiate treatment and prevent
the patient from decompensating further.
The commercial availability of point-ofcare (POC) meters to assess serum ketones

allows the patient to be tested immediately
on presentation at the bedside. There have
been multiple studies performed in adults
showing that use of POC BHB meters in
the emergency room can aid in diagnosis
and treatment of DKA. Arora et al. compared POC BHB and urine ketone dipstick
results in 54 patients with DKA presenting
to the emergency department [3]. They
found that both methods were equally sensitive for detecting DKA at 98.1%, but that
BHB with a cut-o of 1.5 mmol/L is more
specic for DKA compared to urine dipsticks (78.6 vs 35.1%) and could cut down
on unnecessary DKA work ups in hyperglycemic patients. Another study found
that a BHB value of 3.5 mmol/L yielded
100% sensitivity and specicity for the
diagnosis of DKA [4].
Use of POC testing in pediatrics

Fewer studies have been done in pediatric patients. One such study by Ham et al.
determined that using a POC meter in the
hospital setting could aid in monitoring
the resolution of DKA in pediatric patients
[5]. The BHB values from the POC meter
correlated with BHB values from the laboratory for most of the meters measurement range. Use of the meter had both a
strong positive predictive value (PPV, 0.85)
as well as negative predictive value (NPV,
1.0) for indicating the presence or absence
of DKA at a meter value of 1.5 mmol/L [5].
Noyes et al. used POC ketone testing to
identify the endpoint of an integrated care
pathway when treating DKA in children
[6]. They compared their current treatment endpoint of pH >7.3 and no presence
of urine ketones with an endpoint dened
by pH >7.3 and two successive POC ketone
measurements of <1 mmol/L. The study
measured time of treatment in 35 patient
episodes in children ranging in age from
114 years. The time to completion of
treatment using POC ketone measurement was 17 hours, compared to 28 hours
using measurement of urine ketones to
end treatment [6] . They found that occasionally a value below 1 mmol/L would be
followed by a value above 1 mmol/L, but
this never occurred after two subsequent
24
April/May 2016
Diabetes
values under 1 mmol/L, leading them to recommend waiting for

the two successive low values before ending treatment. In addition to allowing an earlier treatment endpoint, this approach enables less time to be spent in the ICU, with decreased cost associated with treatment. Using a POC ketone meter can also result
in fewer tests being ordered overall. Rewers and colleagues asked
whether monitoring serum BHB values at the bedside could result
in a decrease in laboratory testing in pediatric patients [7]. Their
results indicated that the real-time changes observed in POC
serum BHB values correlated strongly with changes in pH, bicarbonate, and pCO2 and also had good correlation with the laboratory BHB method. While initial measurement of pH, bicarbonate
and pCO2 is encouraged, following up the patient with POC BHB
can replace serial laboratory measurements of those analytes and
decrease the amount of laboratory testing [7]. Similarly, a separate
study showed that use of a POC BHB meter at home decreased
diabetes-related hospital visits and hospitalizations of pediatric
diabetics when compared to urine ketone testing by allowing earlier identication of ketosis and initiation of treatment [8].
Most of the studies mentioned are close to 10 years old, but measuring
serum BHB to diagnose DKA or monitor its resolution has not become
standard practice. A recent review of the standard treatment guidelines
for DKA in children and adolescents raises the question of whether
blood ketones should be evaluated during management of DKA [9].
The authors recommend using serum BHB measurement, either from
the laboratory or at the point of care, to both diagnose DKA and monitor treatment. Despite the inaccuracies of POC meters seen at high
BHB values [57], use of a diagnostic cut-o of >3 mmol/L is well
within the accurate range of the meters and can be used to condently
diagnose DKA and monitor the patients response to treatment.
Conclusions
Despite the increasing body of knowledge indicating that measurement of serum BHB can aid in both diagnosis and management of DKA, a study conducted in 2014 indicated that although
89% of pediatric emergency medicine and critical care providers
responding to a survey stated that they had a DKA protocol at
their institution, 67% perceived no clinical advantage in the use
of serum ketone measurements [10]. This suggests that evaluation of serum ketone monitoring during DKA management from
a quality improvement and research perspective may be necessary
before clinical adoption is widespread. The next iteration of DKA
management guidelines should address the potential utility of
serum ketone monitoring.
References
rly registratio
Deadline for ea
gust 2016
fees: 2 Au
XXXI International Congress

of the International Academy
of Pathology
and
th
28 Congress of the European

Society of Pathology
Predictive Pathology, Guiding
and Monitoring Therapy
25 29 September 2016
Congress-Centrum Ost Koelnmesse, Cologne, Germany
www.iap2016.com
www.esp-congress.org
jointly organised by
e German Division of the IAP
e European Society of Pathology
1. Wolfsdorf J, Craig ME, et al. Diabetic ketoacidosis in children and adolescents with
diabetes. Pediatr Diabetes 2009; 10(Suppl 12): 118133.
2. Sheikh-Ali M, Karon BS, et al. Can serum beta-hydroxybutyrate be used to diagnose
diabetic ketoacidosis? Diabetes Care 2008; 31(4): 643647.
3. Arora S, Henderson SO, et al. Diagnostic accuracy of point-of-care testing for diabetic ketoacidosis at emergency-department triage: {beta}-hydroxybutyrate versus
the urine dipstick. Diabetes Care 2011; 34(4): 852854.
4. Charles RA, Bee YM, et al. Point-of-care blood ketone testing: screening for diabetic
ketoacidosis at the emergency department. Singapore Med J. 2007; 48(11): 986989.
5. Ham MR, Okada P, White PC. Bedside ketone determination in diabetic children
with hyperglycemia and ketosis in the acute care setting. Pediatr Diabetes 2004; 5(1):
3943.
6. Noyes KJ, Crofton P, et al. Hydroxybutyrate near-patient testing to evaluate a new
end-point for intravenous insulin therapy in the treatment of diabetic ketoacidosis
in children. Pediatr Diabetes 2007; 8(3): 150156.
7. Rewers A, McFann K, Chase HP. Bedside monitoring of blood beta-hydroxybutyrate
levels in the management of diabetic ketoacidosis in children. Diabetes Technology
& Therapeutics 2006; 8(6): 671676.
8. Lael LM, Wentzell K, et al. Sick day management using blood 3-hydroxybutyrate
(3-OHB) compared with urine ketone monitoring reduces hospital visits in young
people with T1DM: a randomized clinical trial. Diabet Med. 2006; 23(3): 278284.
9. Wolfsdorf JI. The International Society of Pediatric and Adolescent Diabetes guidelines for management of diabetic ketoacidosis: Do the guidelines need to be modied? Pediatr Diabetes 2014; 15(4): 277286.
10. Clark MG, Dalabih A. Variability of DKA management among pediatric emergency room and critical care providers: a call for more evidence-based and costeective care? J Clin Res Pediatr Endocrinol. 2014; 6(3): 190191.
The authors
Angela M. Ferguson*1 PhD, DABCC, FACB; Jeery Michael1 D.O.,
FAAP; Stephen DeLurgio2 PhD; Mark Clements1 MD, PhD, CPI
1
Childrens Mercy Hospital, Kansas City, MO, USA
2
Bloch School, University of Missouri, Kansas City, MO, USA
E-mail: amferguson@cmh.edu
Hematology
25
April/May 2016
Porphyrias: clinical and diagnostic aspects

Porphyrias are a group of disorders of the heme biosynthetic
pathway which clinically manifest with acute neurovisceral
attacks and cutaneous lesions. Diagnosis of porphyrias is based
on the accurate and precise measurement of various porphyrins
and precursor molecules in a range of samples. In addition,
molecular diagnostic assays can provide denitive diagnosis.
by Dr Vivion E. F. Crowley, Nadia Brazil, and Sarah Savage
What are porphyrias?

Porphyrias are a group of rare disorders each of which results from a deciency of an individual enzyme within
the heme biosynthetic pathway (Fig. 1)
[13]. With the exception of an acquired
form of porphyria cutanea tarda (PCT),
all porphyrias are inherited as monogenic
autosomal dominant, autosomal recessive
or X-linked genetic disorders, with varying degrees of penetrance and expressivity and this impacts on the prevalence
and incidence of clinically manifest porphyrias [4]. The biochemical consequence
of each porphyria is the overproduction
within the heme biosynthetic pathway of
specic porphyrin intermediates and/or
the porphyrin precursor molecules deltaaminolevulinic acid (ALA) and porphobilinogen (PBG) [23]. This in turn has
implications for the clinical manifestation
of these disorders, their overall classication and their diagnosis (see Table 2).
Clinical presentation
Porphyrias may present clinically with
either or both of two symptom patterns.
The rst is the acute neurovisceral attack,

which is a potentially life threatening
episode related to excessive hepatic generation of ALA and PBG, and which is
a feature only in acute intermittent porphyria (AIP), variegate porphyria (VP),
hereditary coproporphyria (HCP) and
the very rare ALA dehydratase deciency
porphyria (ADP) [57]. These attacks are
characterized principally by autonomic
dysfunction, including non-specic but
severe abdominal pain, constipation,
diarrhoea, nausea, vomiting, tachycardia,
hypertension or occasionally postural
hypotension. In addition, other features
may include a predominantly motor
peripheral neuropathy which, if left undiagnosed, may extend to respiratory failure
reminiscent of GuillainBarr syndrome,
as well as cerebral dysfunction, which
can vary from subtle alterations in mental state, to posterior reversible encephalopathy syndrome (PRES). Hyponatremia,
most likely due to SIADH [syndrome
of inappropriate antidiuretic hormone
(ADH) secretion] may also contribute
to CNS-related morbidity. The complex
neuropathic manifestations appear to be

primarily related to axonal degeneration
due to direct neurotoxicity by ALA, which
structurally resembles the neurotransmitter gamma-aminobutyric acid (GABA) [3,
57].
The second clinical presentation paradigm is cutaneous photosensitivity
caused by the interaction of ultraviolet
light with photoactive porphyrins in the
skin resulting in the production of reactive oxygen species (ROS) and an associated inammatory response [3]. In PCT,
VP and HCP the skin lesions typically
occur post-pubertally and consist of skin
fragility, vesicles, bullae, hyperpigmentation and hypertrichosis aecting sun
exposed areas, most usually the face and
dorsum of hands [13]. In erythropoietic
protoporphyria (EPP) and X-linked protoporphyria (XLP), which may present in
childhood, there is usually no blistering
but instead erythema, edema and purpura feature in the more acute setting,
with subsequent chronic skin thickening
noted, whereas congenital erythropoietic porphyria (CEP) is characterized by
severe cutaneous photosensitivity often
occurring in early infancy with bullae and
vesicles rupturing and being prone to secondary infection, with resultant scaring,
bone resorption, deformation and mutilation of sun-exposed skin [1, 2, 8].
Classication
The classication of porphyrias (Table 1)
has traditionally been determined either
on the basis of clinical manifestations, i.e.
acute or non-acute (cutaneous), or on the
primary organ of porphyrin overproduction, i.e. hepatic or erythropoietic [1, 3,
8]. A combined classication has recently
been proposed which takes account of
both of these elements [2]. However,
whichever classication is adopted there
should be a realization that VP, and to a
lesser extent HCP, can manifest with both
acute and cutaneous features either simultaneously or separately.
Clinical and biochemical

diagnosis
Figure 1. Heme biosynthetic pathway and associated porphyrias.
The clinical manifestations of porphyrias,

particularly the acute hepatic porphyrias,
are protean and consequently, patients
with a clinically active porphyria could
initially present to a relatively wide
spectrum of clinical specialties includ-
April/May 2016
ing, gastroenterology, acute medicine,

dermatology, neurology, endocrinology
and hematology amongst others [2]. In
general, cutaneous porphyrias should
not pose a diagnostic diculty for an
experienced dermatologist used to investigating photosensitive skin disorders,
but biochemical testing is still required
to dene the type of porphyria present.
However, denitive diagnosis of an initial
acute hepatic porphyria attack is critically dependent on biochemical testing, as
symptoms are often non-specic in nature
(Tables 1 & 2).
The diagnosis of an acute hepatic porphyria attack is founded on demonstrating an increase in urine PBG levels in
direct temporal association with the characteristic acute symptom complex, the
minimum level of increase being between
2- and 5-fold [9, 10]. The urine PBG may
be measured either as a random sample,
where it should be reported as urine PBG
to creatinine ratio or as a 24-hour urine
collection, where total PBG is reported.
The former has proven to be clinically
ecacious and has the advantage of timeliness, reduced within-subject variation
and convenience over the requirement for
a 24 hour urine collection [9]. If the urine
PBG is not elevated this eectively rules
out an acute porphyria attack at the time
of sampling, however, there are certain
caveats to this. Thus it is important to note
26
Hematology
that if specic treatment with either heme

preparations or carbohydrate loading
has been instigated prior to the test these
interventions could reduce the urine PBG
level signicantly, including normalization [3]. Furthermore, if the measurement
of urine PBG is delayed or undertaken at a
time removed from the actual acute clinical presentation e.g. by weeks or months,
then the nding of a normal urine PBG at
that later stage cannot eectively rule out
acute porphyria [3]. In this authors experience another important caveat concerns
patients with a previous conrmed diagnosis acute porphyria who present with
symptoms suggestive of recurrent acute
attack. In many instances these patients
have a perpetually elevated urine PBG,
even in between attacks, and therefore
an elevated urine PBG cannot eectively
guide diagnosis. In these situations a decision to treat as an acute attack has to be
made on the basis of clinical ndings.
Therefore, a clinically eective service
for acute porphyria diagnosis requires
that a timely, quality assured laboratory
method for urine PBG should be available
for analysis [11]. Although a qualitative
method for urine PBG may suce for the
purposes of establishing a diagnosis this
should be supported by the availability
of a conrmatory quantitative method
for urine PBG. The lack of availability of
urine PBG assay is very often the basis for
Table 1. Overview of porphyrias including genes involved, inheritance pattern and basic clinical features.
misdiagnosis or indeed delayed diagnosis

of acute porphyria attacks [10].
In conjunction with PBG, urine ALA
is often measured simultaneously and
although also elevated it does not tend
to reach the levels of PBG in acute
porphyrias. The one exception is the
extremely rare instance of autosomal
recessive ADP due to defective ALA synthase 2 (ALAS2) activity, where markedly
elevated urine ALA levels are reported
while PBG may be normal or only slightly
elevated [2, 3]. In addition, a similar pattern of urine ALA predominance relative
to PBG (although not as elevated) may be
observed in the context of lead poisoning,
wherein patients may also present with
abdominal pain and neuropathy [1, 3].
Once the diagnosis of acute porphyria
has been made based on the urine PBG
the next phase involves determining the
type of porphyria present. This is very
much dependent on the specic pattern
of porphyrin overproduction observed in
samples of urine, feces, plasma and erythrocytes. It is critically important that the
laboratory analytical methods available
extend beyond the sole measurement of
total porphyrin levels [1012]. In particular, it is essential that individual porphyrin analysis and isomer fractionation in
both urine and feces is available to facilitate the identication of the porphyria-
27
specic patterns of porphyrin overproduction [1012]. In many
instances non-porphyria disorders aecting the gastrointestinal
and hepatobiliary systems or certain dietary factors may cause
non-specic secondary elevations in porphyrins, e.g. coproporphyrinuria, which can be diagnostically misleading [3]. In such
cases urine PBG levels will not be elevated and the pattern of
porphyrins observed will not be indicative of any one of the specic porphyrias per se. Therefore, it is important to realize that
a nding of elevated porphyrin levels does not automatically
equate to a diagnosis of underlying porphyria. This further highlights the importance of developing specialist porphyria centres
to ensure that the appropriate repertoire of quality assured testing and expert interpretation and support are available for diagnosis and management of porphyria patients [11, 13].
The diagnosis of cutaneous (non-acute) porphyrias is also very
much based on the specic patterns of porphyrins observed in
urine and feces. In addition, the pattern of free and zinc protoporphyrin in erythrocytes can be useful in the diagnosis of CEP,
EPP and the related disorder, XLP. Moreover, the identication
of the porphyria subtype, either acute or cutaneous, may also be
enhanced by identifying characteristic plasma porphyrin uorescence emission peaks, e.g. VP emission peak between 625 and
628 nm [13]. Finally, it is essential that all samples for porphyrin and precursor measurement are protected from light prior
to analysis.
Role of genetic diagnosis

Given the heritable nature of porphyrias it is not surprising
that molecular genetic analysis has also become an important
diagnostic adjunct. There is an extensive allelic heterogeneity of
pathogenic mutations among the implicated genes for each porphyria disorder, which means that most mutations are uniquely
conned to one or at most a few kindreds. There are, however, a
few exceptions to this trend, most notably in relation to founder
mutations among the Swedish population and the Afrikaner
population in South Africa. The general approach in the application of genetic diagnostic strategies is rstly to characterize
the causative mutation in a known aected individual (proband)
using a mutation scanning approach [14]. Once a putative mutation has been identied its pathogenicity for a particular porphyria should be armed and then more extensive family cascade genetic screening can be organized based on the analysis of
this kindred-specic mutation [14].
This approach has important implications in the diagnosis of
porphyria susceptibility, particularly for the autosomal dominant
acute hepatic porphyrias, where both penetrance and expressivity of the disorders is low [3, 4]. Thus the penetrance among AIP,
VP and HCP is between 10 and 40%, implying that the majority of patients with an autosomal dominant acute hepatic porphyria will not manifest with an acute attack (or indeed cutaneous lesions in the case of VP and HCP) in their lifetime [3, 4].
Moreover, this lack of penetrance may also extend to the absence
of subclinical biochemical abnormalities indicative of an underlying autosomal dominant acute porphyria, demonstrating the
limited sensitivity of biochemical testing in identifying asymptomatic family members.
Currently there is no clear-cut mechanism for discriminating
between those who will manifest a clinical and/or biochemical
phenotype and those who will not. While the role of environmental precipitating factors, e.g. porphyrinogenic medications,
April/May 2016
stress, prolonged fasting, menstruation [13], have long been

recognized in triggering acute porphyria attacks, it is the presence of a pathogenic mutation which is still the single most
important factor determining the overall susceptibility for an
acute porphyria episode. Therefore, all patients carrying a pathogenic mutation should be regarded as pre-symptomatic carriers, i.e. capable of developing an acute attack, and one of the key
applications of genetic analysis in the area is in identifying presymptomatic carriers to allow for appropriate counselling and
management advice to prevent attacks [3, 14].
In this authors experience another useful role for molecular
diagnostics in porphyrias is in relation to those patients with
an historic diagnosis of acute hepatic porphyria in whom the
biochemical abnormalities have subsequently normalized over
years. In such instances genetic analysis can provide a denitive diagnosis for the type of porphyria and will accommodate
a more extensive family screening programme for potential presymptomatic carriers.
The current methods of genetic analysis vary but usually involve
a conrmatory step using direct nucleotide sequencing of the
putative pathogenic variants as the gold standard. However, the
emergence of next generation sequencing platforms has further
galvanized the diagnostic possibilities in this area. Overall, in
autosomal dominant acute hepatic porphyrias, approximately
95% of mutations are identiable [3, 14]. This sensitivity includes
the application of additional methods such as multiplex ligationdependent probe amplication (MLPA) and gene dosage analysis for identifying complex mutations, such large gene deletions,
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28
Hematology
Table 2. Biochemical diagnosis of porphyrias using porphyrin and porphyrin precursor analysis in urine, feces, plasma and red blood cells.
which may not be detected using standard

sequencing-based approaches [14].
In autosomal recessive porphyrias including ADP, CEP and EPP, the clinical penetrance approaches 100%. These disorders
also display a level of genetic heterogeneity. In the case of EPP the presence of a
relatively common low expression single
nucleotide polymorphism (SNP) located
in the ferrochetalase gene, FECH (IVS348C), appears to be essential for the clinical expression of the cutaneous phenotype
in the vast majority of cases [15].
The application of molecular genetics has
provided a means of establishing denitive
porphyria susceptibility, however, similar
to the situation for biochemical testing
services any genetic diagnostic services in
this area must be quality assured to a high
standard and need to adopt appropriate
mutation scanning assay validation protocols in accordance with international
standards and best practice recommendations [1114].
References
1. Puy H, Gouya L, Deybach JC. Porphyrias. Lancet
2010; 375(9718): 924937.
2. Balwani M, Desnick RJ. The Porphyrias: advances
in diagnosis and treatment. Blood 2012; 120:
44964504.
3. Badminton MN, Elder GH. The porphyrias: inherited disorders of haem synthesis. In: Marshall
W, Lapsley M, Day A, Ayling R, editors. Clinical
Biochemistry Metabolic and Clinical Aspects.

Churchill Livingstone Elsevier 2014; pp. 533549.
4. Elder G, Harper P, Badminton M, Sandberg S, Deybach JC. The incidence of inherited porphyrias in
Europe. J Inherit Metab Dis. 2013; 36: 849857.
5. Simon NG, Herkes GK. The neurologic manifestations of the acute porphyrias. J Clin NeuroSci.
2011; 18: 11471153.
6. Sonderup MW, Hift RJ. The neurological manifestations of the acute porphyrias. S Afr Med J. 2014;
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7. Crimlisk HL. The little imitator-porphyria: a neuropsychiatric disorder. J Neurol Neurosurg Psychiatry. 1997; 62: 319328.
8. Siegesmund M, van Tuyll van Serooskerker AM,
Poblete-Gutierrez P, Frank J. The acute hepatic
porphyrias: Current status and future challenges.
Best Pract Res Gastroenterol. 2010; 24: 593605.
9. Aarsand AK, Petersen PH, Sandberg S. Estimation
and application of biological variation of urinary
delta-aminolevulinic acid and porphobilinogen in
healthy individuals and in patients with acute intermittent porphyria. Clin Chem. 2006; 52: 650656.
10. Kauppinen R, von und zu Fraunberg M. Molecular and biochemical studies of acute intermittent
porphyria in 196 patients and their families. Clin
Chem. 2002; 48: 18911900.
11. Aarsand AK, Villanger JH, Stle E, Deybach JC,
Marsden J, To-Figueras J, Badminton M, Elder
GH, Sandberg S. European specialist porphyria
laboratories: diagnostic strategies, analytical
quality, clinical interpretation and reporting as
assessed by an external quality assurance programme. Clin Chem. 2011; 57: 15141523.
12. Whatley S, Mason N, Woolf J, Newcombe R,
Elder G, Badminton M. Diagnostic strategies
for autosomal dominant acute porphyrias: Retrospective analysis of 467 unrelated patients
referred for mutational analysis of HMBS, CPOX
or PPOX gene. Clin Chem. 2009; 55: 14061414.
13. Tollnes MC, Aarsand AK, Villanger JH, Stle E,
Deybach JC, Marsden J, To-Figueras J, Sandberg
S; European Porphyria Network (EPNET). Establishing a network of specialist porphyria centres
eects on diagnostic activities and services.
Orphanet J Rare Dis. 2012; 7: 93.
14. Whatley SD, Badminton MN. The role of genetic
testing in the management of patients with
inherited porphyria and their families. Ann Clin
Biochem. 2013; 50: 204216.
15. Gouya L, Puy H, Robreau AM, Bourgeois M,
Lamoril J, Da Silva V, Grandchamp B, Deybach
JC. The penetrance of dominant erythropoietic
protoporphyria is modulated by expression of
wildtype FECH. Nat Genet. 2002; 30: 2728.
The authors
Vivion E. F. Crowley*1 MB MSc FRCPath
FFPath(RCPI) FRCPI, Nadia Brazil2 BA
(Mod) FAMLS, Sarah Savage3 BSc MSc
1
Consultant Chemical Pathologist, Head of
Department, Biochemistry Department, St
Jamess Hospital, Dublin 8, Ireland
2
Porphyrin Laboratory, Biochemistry
Department, St Jamess Hospital, Dublin 8,
Ireland
3
Molecular Diagnostic Laboratory, Biochemistry Department, St Jamess Hospital,
Dublin 8, Ireland
E-mail: vcrowley@stjames.ie
April/May 2016
29
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as human papillomavirus
(HPV), chlamydia and gonorrhea, among others tests.
Abbott demonstrated a prototype of the companys nextgeneration molecular diagnostics platform at a recent
scientific event hosted for its
customers from across the
globe. At the event, molecular laboratory directors and
researchers had hands-on
interaction with the prototype and were able to provide
additional feedback on the
system prior to further stages
of development.
Abbotts new system is currently being designed from
the ground up based on
extensive input from laboratory customers. For example,
health systems around the
world are often challenged
with higher testing volumes
with staffing and budget
constraints, including in the
molecular laboratories.
Our molecular lab customers tell us they are facing
pressures to do more with
less, said John Carrino,
divisional vice president,
research and development,
Molecular
Diagnostics,
Abbott. Abbotts next-generation molecular system
is being designed to have
a faster turnaround time,
greater flexibility to run any
test at any time, an ability
to run higher volumes and
automation to increase lab
efficiency all without compromising the testing performance and quality for which
our organization is highly
regarded.
Additionally,
customer
insights suggest a need for
a broad testing menu in the
molecular lab. Abbott currently offers one of the broadest molecular testing menus
Abbotts molecular diagnostics can provide the information needed to help guide
some of lifes most important
health decisions, said Andrea

Wainer, president, Molecular Diagnostics, Abbott. Our
accurate, reliable and quality tests could allow clinicians to make more informed
treatment decisions to help
improve patient care.
In addition to the new molecular system, Abbott will be
launching next-generation
systems in blood screening,
immunoassay, clinical chemistry, hematology and point

of care testing in the near
future. All of the systems will
be built on the same software
and hardware platforms to
enable more automation and
to simplify the user experience for Abbotts customers.
www.abbottmolecular.com
Glucose Stabilisation Right from the Beginning

The birth of the new 9$&8(77( FC Mix tube
For the diagnosis of diabetes mellitus
and gestational diabetes
Unique additive mixture in the tube
Immediate stabilisation based on
the in vivo value for 48 hours
Prevents false negative diagnoses
Longer sample stabilisation enables
longer transport
Stabilisation in whole blood, no
immediate centrifugation required
Greiner Bio-One GmbH | Bad Haller Strae 32 | A-4550 Kremsmnster
Phone: (+43) 75 83 67 91-0 | Fax: (+43) 75 83 63 18 | E-mail: office@at.gbo.com
www.gbo.com/preanalytics
April/May 2016
30
INDUSTRY NEWS
Clinical application handbook

such as the iMScope TRIO. It combines
an optical microscope with a mass spectrometer for insights on the molecular
level.
For next-generation brain science, Shimadzu provides LABNIRS, an imaging
technology for visualization of brain functions by functional near-infrared spectroscopy (fNIRS).
Shimadzu has released the rst Application Handbook Clinical. It contains most
advanced technologies and solutions such
as chromatography, mass spectrometry,
spectroscopy and life sciences instruments.
With nearly 140 pages, the Application
Handbook Clinical covers 47 real life
applications related to hot subjects such as
Vitamin D, steroids, immunosuppressants,
catecholamines and amino acids analysis. The book is free of charge and can be
downloaded (17 MB) at www.shimadzu.
eu/clinical.
In clinical applications, analytical
instruments unfold a multitude of benets. They support the quality of human
life. The concentration of medications in
Therapeutic Drug Monitoring (TDM) is
assured, even though this may change
according to age and health conditions
and is dependent on gender, genetic
constitution or interferences with other
drugs. They help to save lives, particularly when it comes to time-critical
situations, e.g. through acute intoxication, medical or drug abuse. They analyse over- and undersupply of vitamins,
minerals and trace elements. They are
applied in genomics, proteomics and
metabolomics and also uncover fraud in
sports, particularly in animal or human
doping. At the same time, analytical systems support health protection of animals and humans, even in the long-term.
Clinical applications benet from Shimadzus complete portfolio covering chromatography and mass spectrometry (GC,
GC-MS, GC-MS/MS, HPLC, UHPLC,
LC-MS, LC-MS/MS); spectroscopy (UVVis, FTIR, AAS, EDX, ICP-OES); life
sciences (MALDI-(TOF)-MS); microchip-electrophoresis; biopharmaceutical
(aggregate sizer); observation of medical microbubbles in targeted drug delivery using the HPV-X2 ultra high-speed
camera.
Shimadzu breaks new grounds by
rethinking the use of mature technologies to develop new unique systems
Some analytical technologies used

in the clinical world
Chromatographic separation in gas
phase for analysis of volatile and semi
volatile components is in use in the clinical eld since many years. Gas chromatography is a key technique for quantitative analysis of alcohol in blood.
HPLC and UHPLC systems are able
to quantitatively analyse substances in
blood, serum, plasma and urine containing multiple compounds by separating and detecting target substances.
Shimadzu oers a wide variety of application-specic systems such as automated sample pretreatment systems for
amino acid analysis or on-line sample
trapping for quantication of drugs or
metabolites.
Gas chromatography-mass spectrometry (GC-MS) is a hyphenated technique
combining the separating power of GC
with the detection power of MS to identify dierent substances within a sample.
Mass spectrometry is a wide-ranging
analytical technique which involves the
production, subsequent separation and
identication of charged species according to their mass to charge (m/z) ratio. It
is well known for analysis of drug abuse.
Liquid chromatography-mass spectrometry (LC-MS) is an analytical chemistry
technique that combines the physical
separation capabilities of LC with the
mass analysis capabilities of MS, bringing together very high sensitivity and
high selectivity. Its application is oriented towards the separation, general
detection and potential identication of
compounds of particular masses in the
presence of other chemicals (e.g. complex mixtures like blood, serum, plasma
or urine). Its use is spreading in the
clinical eld (research and routine) as a
replacement of immunoassays thanks
to the capability of multiplexing analysis and reduced risk of cross-reaction in
immuno-assays.
www.shimadzu.eu/clinical
Sysmex and Siemens extend

long-standing global partnership
in hemostasis testing
Sysmex Corporation and Siemens Healthcare Laboratory Diagnostics announced
on April 13, 2016 an extension to their
long-standing partnership through at
least 2020. The contract extension adds a
minimum of two additional years to the
global supply, distributorship, and sales
and service agreement for hemostasis
products. The partnership enables laboratory customers around the world to
continue to benefit from the largest portfolio of hemostasis systems and reagents.
The companies, which began collaborating more than 20 years ago, also agreed to
continue joint hemostasis product development activities that will streamline and
optimize testing in laboratories throughout the world.
Siemens Healthcare and Sysmex provide hemostasis products used to test
for blood clotting disorders, preoperative bleeding risk management, and the
monitoring of patients on anticoagulant
therapy medications. In the past few
years alone, the companies have introduced several cutting-edge INNOVANCE reagents and multiple new platforms for various laboratory settings,
including the recent worldwide launch
of the Sysmex CS-2500 System, and
the U.S. launch of the Sysmex CS-5100
System with optional track-based
automation.
We are pleased to extend our longstanding partnership with Siemens Healthcare, said Hisashi Ietsugu, Chairman
and CEO, Sysmex Corporation. With the
aging population, hemostasis testing has
become even more important. Our partnership provides our customers with the
innovative technologies needed to manage the increase in testing volumes, while
providing accurate results for improved
patient care.
The continued collaboration and
twenty-year partnership between Siemens and Sysmex is rare in the rapidly changing world of diagnostics,
said Franz Walt, President, Siemens
Healthcare Laboratory Diagnostics.
As a leader in hemostasis testing, our
combined mission to offer best-in-class
solutions has enabled us to meet the
needs of diverse laboratories throughout the world.
www.siemens.com
PRODUCT NEWS
31
Prep automation in culture-independent pathogen PCR testing
Zika Virus real time PCR

detection kit
Micro-Dx enables the cultureindependent

diagnosis
of
pathogens
in
various clinical
samples. MicroDx is the first
product combining walk-away automated human DNA removal and pathogen DNA extraction with broad-range
rDNA Real-Time PCR and sequencing
into a rapid diagnostic system for bacteria and fungi. Prominent advantages
of molecular testing are the time gain
compared to culturing and detection of
pathogens that do not grow for reasons
of fastidious nutrition requirements
or growth inhibition due to antibiotic
treatment of patients. Extraction of 1
to 12 samples is operated in the SelectNAplus instrument, which saves tedious
manual handling and time. At the end of
the procedure an exact differentiation of
the species is obtained. Micro-Dx operates a wide range of specimens, including
EDTA blood, CSF, BAL, aspirates from
joints, swabs from wounds and abscesses
and tissue biopsies from heart valves,
liver and brain.
The growing
concern about
the proliferation of Zika
infections in
the Caribbean,
Central and South America, has turned the
ght against the virus in a global health emergency. The infection is mainly transmitted
through the bite of the Aedes spp mosquitoes and presents symptoms like mild fever,
arthralgia, myalgia, asthenia, headache and
maculopapular rash clinical symptoms, plus
additional symptoms like conjunctivitis, retroorbital pain, lymphadenopathy and diarrhea.
There is widespread concern about the association of the Zika Virus with increasing cases
of congenital microcephaly. Other research
indicate that the virus can cause brain damage. CerTest Biotec has developed a new kit
for the identication of Zika virus in patients
presenting symptoms of the disease. This new
ready-for-use product contains all the necessary components and reagents to perform a
test that detects viral Zika RNA using the real
time PCR technique. It is very important to
complete the identication of the virus in the
early stages of infection; therefore, it is recommended that samples of blood, serum and/or
saliva are collected during the rst 5 days after
the onset of symptoms. The VIASURE Zika
Virus real time PCR detection kit is designed
for specic identication and quantication
MOLZYM

The DxH 500 hematology system with
CE Mark is an openvial instrument oering a throughput of
up to 60 samples per
hour. It is the rst
analyser in a new
range of workowecient hematology analysers able to deliver
accurate, robust results from a nger prick of
blood. The DxH 500 analyser has been specically designed for low-volume hematology workloads and to promote rapid specimen turnaround and reduce patient wait
times in small- and medium-sized clinics.
Smaller than a standard microwave, the new
instrument is able to provide a complete
blood count (CBC) plus 5-part dierential
from as little as 12L of whole blood or from
20L of whole blood for pre-dilute analysis.
This makes the DxH 500 ideal for pediatric

and geriatric patients, for whom sample taking can be dicult. It is part of Beckman
Coulters line of DxH hematology instruments (the DxH Workcell, DxH 800 Cellular Analysis System and DxH Slidemaker
Stainer) incorporating the companys multidimensional, high-denition ow cytometric technology. As part of the multi-site clinical reliability study to test its performance,
uptime and workow eciencies, 36,000
samples were run across 26 sites in ve continents. The DxH 500 exhibited less than or
equal to one service call per year, providing
uptime of more than 98%. In addition to
high reliability, the system has several additional features to provide maximum uptime,
with automatic start-up, fast reagent changes,
no soft tubing, and minimum moving parts.
It operates like a mobile phone, using touch
screen technology so there is no need to add
a PC and monitor. Low power consumption
also reduces operational costs, with LED
April/May 2016
of Zika virus in clinical samples from patients

with signs and symptoms of Zika virus infection. This test is intended for use as an aid in
the diagnosis of the Zika virus in humans in
combination with clinical and epidemiological risk factors. RNA is extracted from specimens, amplied using RT-amplication and
detected using uorescent reporter dye probes
specic for Zika virus.
CERTEST BIOTEC
New PTH Control for QC Portfolio

Randox Quality
Control
announces
a
further
expansion to
their comprehensive QC
portfolio, the
Acusera PTH Control. This new control
has been designed with convenience in
mind, providing the laboratory with a true
third party solution for the measurement
of PTH. The assayed liquid control has
been developed with an extended open
vial stability of 30 days and 2-year shelf
life, reducing waste and ensuring consistency for this notoriously unstable assay.
RANDOX
lighting replacing traditional lasers. The

DxH 500 uses 50% less reagent volume per
sample compared to other low-volume analysers so that a single set of reagent bottles
can support hundreds of tests. Further, the
DxH 500 needs only three reagents, which
take less than two minutes each to replace,
making better use of sta time and supporting a consistent workow throughout the
day. By providing non-toxic, cyanide-free
and formaldehyde-free reagents, labs can
reduce the cost of disposal and more easily
meet environmental and regulatory compliance standards. Additionally, the DxH 500
supports laboratories paperless eorts with
a bidirectional laboratory information system (LIS) interface for better data keeping.
This integrated LIS interface can potentially
help reduce data errors that occur during
manual processes.
BECKMAN COULTER
April/May 2016
BioFire
Diagnostics FilmArray
Meningitis/
Encephalitis (ME)
Panel is now available in the countries which recognize CE marking. The ME
Panel provides highly benecial medical
value, as it addresses the critical unmet
need for quick and accurate identication
of central nervous system (CNS) infectious
agents. The comprehensive ME Panel tests
cerebrospinal uid (CSF) for the 14 most
common pathogens (6 bacteria, 7 viruses
and 1 yeast) responsible for community
acquired meningitis or encephalitis in
about an hour. Currently, testing CSF for
multiple organisms can take days and is
not always possible because it can be difcult to obtain enough uid from each
patient to run multiple tests.
The ME Panel received a de novo clearance
by the U.S. Food and Drug Administration (FDA) in October 2015. The ME Panel
New assay range including

automated TSI
A range of
new assays has
been released
by
Siemens
for use on the
ADVIA Centaur and IMMULITE XPi 2000 systems.
The range includes anti-CCP used as an aid
in the evaluation of Rheumatoid Arthritis
(RA) and the rst automated quantitative
thyroid stimulating immunoglobulin (TSI)
assay used in the diagnosis of Graves disease. The ADVIA Centaur anti-CCP assay is
for use in the semi-quantitative determination of the IgG class of autoantibodies specic to cyclic citrullinated peptide (CCP) in
human serum and plasma, aiding with the
diagnosis of RA. The assay provides 96%
specicity for an early accurate diagnosis
of RA, ensuring improved patient care by
allowing timely intervention and treatment.
The Siemens IMMULITE 2000 XPi thyroid
stimulating immunoglobulin (TSI) assay
specically detects thyroid stimulating antibodies, which are the hallmark of Graves
disease, unlike the commonly used TRAb
assay which detects both stimulating and
blocking antibodies. This makes the assay
highly specic, to aid in the diseases diagnosis. With a clinical sensitivity and specicity of 98.3% and 99.7% respectively, it
32
PRODUCT NEWS
brings a unique opportunity to test simultaneously and rapidly for most bacteria,
viruses and fungi found in those pathologies that can be extremely severe and
sometimes lethal. Such an approach will
positively impact the management of those
patients by helping clinicians and biologists speed the diagnosis of these potentially severe conditions and make much
faster decisions on appropriate therapy
to prevent complications. More than 1.2
million people every year are aected by
meningitis worldwide, resulting in 120,000
deaths globally from bacterial meningitis.
Bacterial meningitis can occur suddenly
in healthy people and even with prompt
diagnosis and treatment, approximately
10% of patients may die and up to 20% or
more may sustain permanent damage and
disability. The ME Panel is cleared for the
FilmArray and FilmArray 2.0 systems and
is commercially available around the globe.
BIOFIRE DIAGNOSTICS
ensures laboratories can provide a fast, easy

and specic diagnosis. The addition of the
new assays to the existing extensive range
will help laboratory sta integrate testing
into routine workow, reducing the need for
send-away testing. This enables laboratories
to reduce operational costs and time, as well
as becoming more productive and ecient.
SIEMENS HEALTHCARE
Automated evaluation of ANA,

ANCA, CLIFT and cell-based assays
The
FDA-approved
EUROPattern
system
provides fully automated
evaluation of indirect
immunof luores cence
tests for anti-nuclear
antibodies (ANA), antineutrophil
antibodies
(ANCA) and now also Crithidia luciliae
(CLIFT), EUROPLUS antigens and cellbased assays (e.g. anti-neuronal antibodies). The EUROPattern system consists of
a fully automated microscope with slide
magazine and advanced diagnostic software for rapid recording, interpretation
and archiving of IFT images. Up to 500
analyses can be processed in 2.5 hours, corresponding to 18 seconds per eld. Slides
are correctly identied by means of matrix

codes. The controlled LED in the microscope provides over 50,000 hours of constant light intensity, ensuring highly reproducible results. Positive and negative results
for the substrates are clearly dierentiated
by the powerful software. Crithidiae evaluation is based on specic kinetoplast uorescence rather than just dark-light classication, increasing reliability. Dierent
ANA, anti-cytoplasmic and ANCA patterns are reliably identied, even if more
than one antibody is present. Furthermore,
the software provides titre designations
with condence values. Images from further substrates such as liver, kidney and
stomach or the new anti-Zika assay can
be automatically recorded and archived.
Images and results are viewed at the PC
screen, and can be checked retrospectively
at the microscope if necessary. The software consolidates all results into one report
per patient and also compares new ndings
with previous records. Dierent user levels
provide a hierarchical review of results,
thus increasing security. The software can
be fully integrated into existing laboratory
software (LIS) for a streamlined laboratory routine.
EUROIMMUN
Hemoglobin meter
QDx Hemostat is a
compact handheld
POC hemoglobin
meter for measuring hemoglobin
from a nger prick
of whole blood. This hemoglobin measuring system is intended to help people manage their hemoglobin levels. It also provides
healthcare professionals with helpful information by measuring hemoglobin in fresh
capillary whole blood as well as venous
blood. Calibration is done by simply inserting the code key into the test meter. The virtually painless test requires only 1 L sample
volume and provides quick results in ve
seconds. The device features a large display, a
100-test memory and a measuring range of
5 - 26 g/dL. Battery life allows 3,000 tests to
be performed. By using Hemostat, a check of
both quantitative hemoglobin and hematocrit will give quick results within 5 seconds.
Only 1 L sample volume is needed which
makes it nearly painless for the patient. The
ergonomic design allows comfortable usage.
DIASYS
PRODUCT NEWS
33
Automated urinalysis tailored to the laboratorys workload

The
Iris
iRICELL
workcell
integrates urine
chemistry
and
microscopy into
a fully automated
walk-away
urinalysis
system
that is easy to
use and maintain.
The integrated workcell combines the
iQ200 Series automated microscopy
system with the iChemVELOCITY
automated urine chemistry system.
The iQ200 Series delivers clear, clinically relevant urine particle images
that are auto-classified for more objective and consistent results. This automated microscopy system is designed
for all volume workloads, delivering
a shorter turnaround time and standardizing results. The iQ200 Series are
available either as a stand-alone system or connected to an iChemVELOCITY urine chemistry analyser to
form an automated iRICELL workcell.
The iQ200SPRINT is one of the fastest automated systems on the market,
meeting high-volume productivity and
workload requirements. It can handle 101 microscopic samples an hour.

The IQ200ELITE manages medium- to
high-volume workloads running 70
microscopic samples an hour; with the
iQ200SELECT more suitable for lowvolume workloads running 40 microscopic samples an hour. iChemVELOCITY provides a fully automated,
high-capacity urine chemistry analysis with excellent low-end sensitivity.
The system delivers a high throughput
of 210 samples an hour, with the continuous strip loading and a capacity
of 300 strip loads. It has a pad on the
strip designed to detect and measure
the presence of ascorbic acid. This provides clinically significant information
about potential interference with key
chemistry assays. The system evaluates
all standard urine chemistry parameters, including glucose, protein, bilirubin, urobilinogen, pH, specific gravity,
blood, ketones, nitrite and leukocyte
esterase.
BECKMAN COULTER
April/May 2016
Utility of reex urine culture based

on results of urinalysis and automated microscopy
Specimens submitted for urine culture
in hospital settings are frequently negative for bacteria. Various approaches have
been developed to select urine samples in
the laboratory to improve the eciency of
handling these samples. At a time of concern for cost containment, utilization of a
reex testing policy using specic screening criteria would be benecial to eliminate
unnecessary urine cultures. With this in
mind, an evaluation of Beckman Coulters
IRIS iQ200 system (urinalysis and automated urine microscopy) was carried out
in the US at the Johns Hopkins Bayview
Medical Center (JHBMC) clinical microbiology laboratories. The study prospectively collected and reviewed 1248 clinical
urine specimens submitted for urinalysis
and/or urine culture. It investigated the
IRIS iQ200s utility in aiding a predictive
algorithm for the implementation of reex
urine cultures. Findings showed that these
test parameters, separate or combined, may
be a useful screening method to determine
the need for a reex urine culture.
April/May 2016
34
PRODUCT NEWS
CALENDAR OF EVENTS
Vacuum sample tube with glycolysis inhibition
The rapid breakdown of glucose
(glycolysis)
in
venous
blood
samples is very
signicant for the
diagnosis of both diabetes mellitus and gestational diabetes which should be detected at
an early stage to avoid complications such as
infections, premature births and long-term
eects for the mother and child. In order
to have a reliable diagnosis, it is necessary
to inhibit glucose breakdown immediately
after collecting blood. Various institutions
have drafted guidelines, which recommend
the addition of a citrate-uoride additive to
maintain the in vivo glucose level. The special feature of the new VACUETTE FC Mix
tube from Greiner Bio-One is the powder
additive. It stabilizes the glucose level immediately after collection for 48 hours. This
allows for reliable diagnosis of diabetes conditions and avoids false negatives. The stabilization is carried out in the whole blood
and therefore does not require immediate
Walk-away 25-OH vitamin

D2/D3 UHPLC analyser
Zivak Technologies
supplies ready to
use LC-MS/MS and
HPLC analysis kits
with its own fully
automated sample
preparation and injection system which
enables laboratories to make efficient
use of their LC-MS/MS as well as HPLC
instruments. Many scientific studies and
papers show the inaccuracy of measuring
total vitamin D with commercial immunoassays without separating and measuring vitamin D2 and vitamin D3 metabolites individually. HPLC and LC-MS/
MS methods are accepted as the Gold
standard for separate analysis of 25-OH
metabolites of vitamin D. Usage of these
chromatographic methods is increasing
significantly in clinical laboratories in
recent years. Many of these laboratories
cannot use their systems effectively due to
the fact that these systems require qualified staff, complex sample preparation
steps, high kit prices/ operation costs and
do not allow walk-away operation. The
VD-200 was especially designed for routine vitamin D2/D3 testing clinical labs.
VD-200 enables to analyse vitamin D2/
centrifugation. Unlike in tubes where liquid is added, the nely granulated additive
does not cause a dilution eect. There is no
need to convert the measurement result.
The citrate/citric acid buer reduces the pH
value in the sample. As a result, the enzymes
needed for the glycolysis process are inhibited and the actual in vivo level is stabilized
from the start. The additive is completely dissolved, and therefore optimally mixed with
the sample, after swivelling ten times. In the
case of storage between 4C and room temperature, a further sodium uoride additive
ensures long-term stabilization for 48 hours.
The VACUETTE FC Mix tube is available
with both a grey and pink security cap and
therefore allows for dierentiation from
standard glucose tubes. The cap is particularly easy to open and allows for hygienic
working in the laboratory. The VACUETTE
FC Mix tube is made of highly-transparent
PET plastic and is shatter-proof.
May 21-24, 2016

European Human
Genetics Conference
2016 (ESHG 2016)
Barcelona, Spain
www.eshg.org/
home2016.0.html
June 14-17, 2016

ESGAR 2016
Prague
Czech Republic
www.esgar.org
June 21-23, 2016

JIB Journes
Franaises de
Biologie
Paris, France
www.jib-sdbio.fr/
GREINER BIOONE
D3 samples fully automatically without

any human intervention - from primer
sample tube. The company offers higher
sensitivity than entry level LC- MS/MS
thanks to the newly designed Special D
detector. This system is fully validated
with the Zivak vitamin D2/D3 UHPLC
analysis kit which includes all necessary
reagents, calibrator and controls. It offers
a complete solution to clinical laboratories performing routine vitamin D2/
D3 assays on HPLC-UV and LC-MS/MS
systems. As they have a large number of
vitamin D2/D3 samples, they cannot run
all the other LC-MS/MS specific analyses
on their LC-MS/MS systems effectively.
The VD-200 has been designed especially
for this kind of laboratory, providing a
cost-effective and fully automated system
that frees expensive LC-MS/MS systems
for specific tests which have to be run
on LC-MS/MS systems. The innovative
design of the VD-200 enables barcode
reading, reagent adding, vortex mixing,
centrifuge and injection processes to be
done by robotic arms. The system provides 240 accurate results in 12 hours
fully automatically.
ZIVAK
July 31-August 4,
2016
AACC
Atlanta, GA, USA
www.aacc.org
September
12-15, 2016
MSACL EU
Salzburg, Austria
www.msacl.org
September
14-17, 2016
19th Annual
ESCV Meeting
Lisbon, Portugal
www.escv2016.com
September 2124, 2016

EFLM-UEMS
Congress
Warsaw, Poland
www.em-uems.
warsaw2016.eu
September 2530, 2016

European Congress of Pathology
Cologne, Germany
www.esp-congress.org/2016
September 29-1
October, 2016
British Society for
Allergy & Clinical
Immunology
Telford, Shropshire, UK
www.bsaci.org
Location and date
TBC
CMEF Autumn
2016
www.cmef.com.
cn/g1250.aspx
November 14-17,
2016
MEDICA
Dsseldorf,
Germany
www.medica.de
January 23-26,
2017
MEDLAB at Arab
Health
Dubai, UAE
www.arabhealthonline.com
October 22-25,
2017
IFCC WorldLab
Durban 2017
Durban,
South Africa
www.durban2017.org
For more events see:

www.cli-online.com/events/
Dates and descriptions of future events have been obtained
from ofcial industrial sources. CLi cannot be held
responsible for errors, changes or cancellations.
AV NO
AI W
LA
BL
E
CELL-DYN Emerald 22
The Information You Need
The Size You Want
CELL-DYN Emerald 22, a compact, easy-to-use 5-PART DIFFERENTIAL

hematology analyzer, offers big lab results with small lab requirements. A small
footprint, low reagent consumption and easy-to-use features like touch-screen
technology, automatic startup, shutdown and cleaning make it the ideal choice
where space and specialized staff are limited. Ask your Abbott representative about
our growing portfolio or visit abbottdiagnostics.com for more information.
ADD-00057330E

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The clinical chemistry laboratory in the

Incidence of testicular cancer

Cell type of origin

United Kingdom and the United States

The vast majority of non-GCTs are sex

Non-seminomatous germ cell tumours (NSGCT)

more than one cell type

Tumours originating from

gland [35, 8, 10]. Germ cell tumours

Choriocarcinomas and teratomas/embryonal carcinomas

Treatment and cure rates in TC

(GCTs) [sourced from 3, 8, 9, 11].

Advances in treatment strategies, such

Causes and presentation of TC

Diagnosis and staging of TC

No primary tumour able to be identied

No evidence of a tumour (e.g. scar)

pT1 with vascular, lymphatic or tunica vaginalis invasion

Invasion of spermatic cord with or without vascular or lymphatic invasion

Invasion of scrotum cord with or without vascular or lymphatic invasion

Regional lymph node involvement unable to be identied

No lymph node metastasis identied

Metastasis to 5 lymph nodes, no lymph nodes >2 cm OR no lymph node masses 2 cm

Lymph node mass >5 cm

allele (28) is less clear. There is a certain

2, 3, 4, 5, 6, 7, *8