News updates on www.cli-online.

com | April/May 2016 | Volume 40

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

Also in this issue :

Vacuum sample tube with
glycolysis inhibition Diagnosis and management Pharmacogenomics Porphyrias clinical and
of testicular cancer Pg. 6 in AML patient Pg. 14 diagnostic aspects Pg. 25
by Greiner Bio-One Pg.34
Delivering Smart Solutions
The GCMS-QP2020 and the GCMS Insight software package dramatically
improve the efficiency of daily analysis procedures
The Smart SIM creates optimal methods automatically
and enables a batch analysis of more than 400 compo-
nents, thereby reducing the number of measurements.

Simultaneous analysis of 434 pesticides using Smart SIM

With LabSolutions Insight software, quantitative results
for a complete series of data files can be displayed side-
by-side, enabling easy identification of any outliers, which
shortens analysis time.

www.shimadzu.eu
www.cli-online.com & search 27248
 EDITORS LETTER 3 April/May 2016

Call for action on diabetes

Frances Bushrod,
Ph.D.

This years annual World Health Day wait in situ for a result that reects the available for around 9 a unit, surely up of subjects with positive test
on 7th April highlighted the dra- average blood glucose level over the cost-eective if a result of prediabetes results but surely such screen-
matic rise in the prevalence of Type past three months is clearly preferable precipitates patient lifestyle changes, ing programmes are more likely
2 diabetes (T2DM) and urged global to measuring fasting or random glu- and a diagnosis of diabetes leads to to have an eect on the T2DM
action to contain the epidemic. The cose levels, tests which require patient follow-up care. epidemic than frequently over-
number of people suering from forethought, laboratory facilities, weight healthcare workers pon-
T2DM has approximately quad- larger samples and frequently repeat Of course one must develop ticating about healthy diets
rupled in three and a half decades; tests. POC A1c tests are currently clear guidelines for the follow and exercise?
currently 8.5% of the global adult
population is aected. Because
uncontrolled, elevated levels of blood
glucose can eventually result in car-
diovascular disease, kidney failure,
lower limb amputation and loss of
sight, as well as premature death, the
disease has major socioeconomic
STart Max Max
Accuracy
impacts in addition to health issues.
Yet it is unlikely, at least in Western
populations, that interventions to Max
promote more balanced diets and Practicality
less sedentary lifestyles will reduce
the widespread overweight and obe-
sity that fuels the T2DM epidemic.
The general public in the West is con- Max
tinuously informed about the ben- Innovation
ecial eects of healthy eating and
sucient physical exercise, but mod-

phot s - 03/2016
ern working environments, family Max
commitments and social activities Reliability

tual photos
often preclude compliance with

photo
good health advice. And many of us,

- 2015 Diagnostica Stago - All rights reserved - Non-contractual

on-contr actual
healthcare professionals included,
think its worth taking the risk of
eating and drinking (even smok-
ing) 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.

Many studies assessing the outcome

of T2DM screening have reported
minimal impact on prevalence. How- Simplicity born from Expertise
ever, some recent community-based
screening projects oering testing at Discover the STart Max, the new semi-automated
a variety of venues including sports instrument from the Max Generation.
grounds, shopping centres, pharma- Designed by Stago, the expert in Coagulation,
cies (and why not polling stations?) the STart Max remains simple to use but with true
innovation to achieve a maximum performance.
show promise. In such an approach
Start with Max, stay with Max!
it is clearly simpler to utilise point-of-
care capillary glycosylated haemoglo-
bin (A1c) tests. A nger stick to obtain
one drop of blood followed by a short
www.cli-online.com & search 27140
Contents SINCE 1977

Av. Princesse Elisabeth 176 B1030 Brussels, Belgium

Tel. +32-2-240 26 11 Fax: +32-2-240 26 18
FRONT COVER www.cli-online.com
Considerable rewards could be obtained from Managing Editor
early identication of Type 2 diabetes mellitus Alison Sleigh, Ph.D.
Contributing Editor
News updates on www.cli-online.com | April/May 2016 | Volume 40

Type 2 diabetes:
(T2DM). One of the most obvious, as suggested
biomarker models to predict risk
Frances Bushrod, Ph.D.
Pg.21
Compact hematology system
in a recent report on diabetes global burden, News Editor
by Beckman Coulter
Pg.31

would be better disease management. The re- Tony Spit, Ph.D.

port, by the University of East Anglia in the UK, Editorial Coordinator

Shirley Waring
Meningitis/encephalitis panel

by BioFire Diagnostics
Pg.32

concludes that early investments into preven-

Vacuum sample tube with
glycolysis inhibition
Editor in Chief/Publisher
Also in this issue :
tion and disease management may therefore be Bernard Lger, M.D.
Diagnosis and management Pharmacogenomics Porphyrias clinical and

Advertising Coordinator
of testicular cancer Pg. 6 in AML patient Pg. 14 diagnostic aspects Pg. 25

particularly worthwhile.
by Greiner Bio-One Pg.34

Jennifer Christophers
Circulation Manager
Arthur Lger
Publishing Executive / Advertising Manager
[6 - 13] TUMOUR MARKERS Astrid Wydouw
a.wydouw@panglobal.be
[6 - 10] The clinical chemistry laboratory in the diagnosis and management of
Webmaster
testicular cancer Jennifer Christophers
[11- 13] Use of serum free light chain analysis in screening for multiple myeloma
in primary care patients 2016 by PanGlobal Media bvba-sprl. Production &
Lay-out by Studiopress Communication, Brussels.

Circulation Controlled by Business of

Performing Audits, Shelton, CT, USA.
[14 - 16] PERSONALIZED MEDICINE
The publisher assumes no responsibility for opinions or state-
Pharmacogenomics in an acute myelogenous leukemia patient ments expressed in advertisements or product news items.
The opinions expressed in by-lined articles are those of the
author and do not necessarily reect those of the publisher. No
conclusion can be drawn from the use of trade marks in this
publication as to whether they are registered or not.
[17-24] DIABETES
ISSN 1373-1580
[17 - 20] Diagnosis of diabetes mellitus

[21 - 22] Type 2 diabetes - biomarker models promise new means to predict risk COMING UP IN CLI JUNE 2016
[23 -24] The use of point-of-care ketone meters to diagnose and monitor
Molecular diagnostics focus
diabetic ketoacidosis in pediatric patients
Kidney disease diagnosis

For submission of editorial material, contact

[25 - 28] HEMATOLOGY
the editors at CLIeditor@panglobal.be
Porphyrias: clinical and diagnostic aspects
For advertising information, go online to
www.cli-online.com, simply click on Magazine
and Media Information or contact
Astrid Wydouw at a.wydouw@panglobal.be
REGULARS
[3] Editors letter Free Subscription for
[29 - 30] Industry news clinical lab professionals
[31 - 34] Product news Clinical lab professionals are entitled to receive the
digital edition of CLI for the next 12 months completely
[34] Calendar of events
free of charge. To begin a new subscription or to
continue your existing free subscription go to

www.cli-online.com
Click on Free Subscription and follow instructions
 NOW
I CAN
OPTIMISE MY TEAMS
EFFICIENCY

With the DxN VERIS Molecular Diagnostics System.*

DxN VERIS consolidates extraction, amplication and detection onto
a single platform, saving hands-on time as technicians are not required
to pipette samples and reagents, and also saves vital laboratory space.
Simple to learn and easy to use, DxN VERIS enables your team to do
the work you want, when you want.

See how DxN VERIS can work for your team at:
www.beckmancoulter.com/moleculardiagnostics

*Not for sale or distribution in the U.S.; not available in all markets.
2016 Beckman Coulter, Inc. All rights reserved. Beckman Coulter, the stylized logo and the Beckman
Coulter product and service names mentioned herein are trademarks or registered trademarks
of Beckman Coulter, Inc. in the United States and other countries.

For Beckman Coulters worldwide office locations and phone numbers,

please visit Contact Us at www.beckmancoulter.com Move healthcare forward.

www.cli-online.com & search 27128

 April/May 2016 6 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 long-
term surveillance in patients with testicular cancer.
by Dr Angela Cooper and Dr Sen Costelloe
Incidence of testicular cancer United Kingdom and the United States
Testicular cancer (TC) is relatively rare, of America, TC is the most common
accounting for approximately 0.7% of type of cancer observed [2, 3, 6, 7].
all UK male cancers, with a worldwide
incidence estimated as ~7 per 100 000 Classication of TC
[1, 2]. Incidence of TC has noticeably Approximately 95% of malignant TCs
increased in industrialized countries originate from primordial germ cells,
over the last few decades, particularly also known as germ cell tumours
in white males of European descent, (GCTs) [3, 79]. However, rarely these
although the reasons for this remain malignancies may arise from extrago-
unclear [25]. Amongst younger men nadal primary sites such as the retro-
aged between 15 and 49 years in the peritoneum, mediastinum or pineal
Percentage of all
Cell type of origin Type of tumour
Germ cell tumours Intratubular germ cell neoplasias of the unclassied type
(IGCNU)
Other types
Tumours originating from Seminomas
one cell type Seminoma with syncytiotrophoblastic cells
Spermatocytic seminomas
Spermatocytic seminoma with sarcoma
Non-seminomatous germ cell tumours (NSGCT)
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
Tumours originating from Mixed embryonal carcinomas and teratomas
more than one cell type Mixed teratomas and seminomas
(mixed GCTs)
Choriocarcinomas and teratomas/embryonal carcinomas
Polyembryomas
Others
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, origi-
nate from multiple cell types such as
embryonal carcinomas, teratomas,
choriocarcinomas and yolk sac car-
cinomas. GCTs arising from mixed
germ cells comprise the remaining
20%. The World Health Organization
(WHO) classification system for tes-
ticular tumours (Table 1) define five
basic GCT types based on histological
examination:
Seminomatous GCTs
Non-seminomatous GCTs
(NSGCTs)
Embryonal cell carcinomas
Yolk sac tumours
Teratomas
Choriocarcinoma
GCTs
The vast majority of non-GCTs are sex
~20%
cord-gonadal stromal tumours involv-
ing the Sertoli or Leydig cells of the tes-
ticles, and are often benign [8, 9, 11].
~40%
Burned-out GCTs, or spontaneous
regression of a testicular GCT, is a very
rare phenomenon occasionally observed
in male patients presenting with meta-
static malignancy with an absence of
primary testicular tumour. Often, the
only remaining evidence of malignancy
are features such as homogeneous scar-
ring, hemorrhage, intratubular calcifi-
cation and testicular atrophy. This may
be associated with choriocarcinomas or
teratomas [5, 12].
Testicular GCTs exhibit very diverse his-
tology and immunostaining profiles, and
have varying clinical progression and
prognosis outcomes as demonstrated
by the numerous methods of GCT clas-
~40%
sification systems. It is outside the focus
of this paper to consider histology or
immunostaining used in the identifi-
cation and differentiation of GCTs, as
these topics has been extensively docu-
mented 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 April/May 2016

as the use of cisplatin therapies [13], intersex patients have also been associ- immunostaining profiling as appro-
careful staging at diagnosis, early inter- ated with an increased TC risk [3, 5, 7]. priate, and in the majority of cases,
vention using multidisciplinary teams, treatment options should be based on
rigorous surveillance follow-up, and Presentation of TC is often a painless the histology results [10]. Biochemi-
salvage therapy, means that GCTs are lump in the testis body, but due to a fre- cal analysis should include initial con-
highly curable. Currently, expected cure quent lack of pain, medical opinion is centrations of serum tumour markers
rates of 95% are observed in patients frequently delayed. A testicular mass or (STMs). Metabolic biochemistry, liver
who receive a TC diagnosis, and cure swelling, or episodic diffuse pain may be function tests and a full blood count
rates of 80% in patients with a diagnosis observed. More rarely, metastatic symp- should be undertaken to determine
of metastatic TC [3, 13]. toms such back pain arising from ret- general organ function, and may dem-
roperitoneal lymph node involvement, onstrate evidence of metastasis [9].
or coughing, pain or hemoptysis due to
Causes and presentation of TC lung metastasis may be reported [3, 7, 8]. This collective information can be used
The causes of TC cancer are still to reference the Tumour-node-metasta-
unknown, although cryptochordism is Diagnosis and staging of TC sis (TNM) Classification of Malignant
the best-characterized risk factor asso- Clinical suspicion of TC, such as Tumours staging system (Table 2). This
ciated with TC. Research has shown altered testicular shape or non-painful cancer staging system is based on pri-
that timing of orchiopexy impacts on swelling, should prompt a full physical mary tumour site, nearby lymph node
future risk of TC development, suggest- examination and patient history, imag- involvement, and presence of distal
ing hormonal changes during puberty ing to include testicular and abdomi- metastatic spread from initial primary
are strongly associated with TC etiology nal ultrasound, as well as chest X-ray tumour site [4, 15]. The use of STMs as
in males. However, prenatal risk factors, [14]. If metastasis is suspected, chest, a fourth staging system has added diag-
environmental exposures in adulthood, abdominal and brain computerized nostic and prognostic value, independ-
male infertility, certain genetic or con- tomography (CT), and bone scintig- ent of the TNM system (Table 3) [9].
genital disorders such as Downs syn- raphy should be undertaken [9]. Final The decision for chemotherapy or radi-
drome, Klinefelters syndrome, human diagnosis and prognosis requires otherapy treatment for non-surgical
immunodeficiency virus infection and biopsy sampling for histology and metastatic disease is based on CT and/
or magnetic resonance imaging (MRI)
results, and concentrations of STMs [4].
pT Primary tumour
pTx No primary tumour able to be identied The majority of patients (~75%) pre-
pT0 No evidence of a tumour (e.g. scar) senting with a testicular mass are diag-
pTis IGCNU nosed at stage 1 [7, 8]. At this stage,
PT1 Limited to testis and epididymis. Absence of vascular or lymphatic invasion, may invade tunica treatment options are typically surgery
albuginea but not tunica vaginalis with an excellent cure rate. For meta-
pT2 pT1 with vascular, lymphatic or tunica vaginalis invasion static disease, combinations of surgery,
pT3 Invasion of spermatic cord with or without vascular or lymphatic invasion
chemotherapy or radiotherapy are
required depending on cancer mass,
pT4 Invasion of scrotum cord with or without vascular or lymphatic invasion
location and distal lymph node involve-
pN Regional lymph nodes
ment [13]. Greater than 80% of patients
pNx Regional lymph node involvement unable to be identied with metastatic GCTs are successfully
pN0 No lymph node metastasis identied treated and cured.
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 Treatment of TC
extranodal spread TC cells are extremely sensitive to
pN3 Lymph node mass >5 cm chemotherapy [9, 10]. Specifically, the
standard chemotherapy regime consists
M Distant metastasis
of 3 or 4 cycles of bleomycin, etoposide
Mx Distant metastasis unable to be identied
and cisplatin (BEP) chemotherapy, or
M0 No distant metastasis etoposide and cisplatin (EP) chemo-
M1 Distant metastasis identied therapy every 21 days [8, 9]. Surgery
M1a Non-regional nodal or lung metastasis identied may be considered to remove residual
M1b Distant metastasis identied (excluding non-regional nodal or lungs) masses post-chemotherapy. Data sug-
S Serum tumour markers (STMs)
gests a higher relapse rate in patients
with NSGCTs than seminomas follow-
Sx STMs not available or not undertaken
ing an initial chemotherapy regime.
S0 STMs concentrations within normal limits This relapse rate can be used to further
LDH (U/L) hCG (U/L) AFP classify patients into good, intermedi-
S1 <1.5 N and <5000 and <1000 ate and poor prognostic groups, using
S2 1.510 N or 500050 000 or 100010 000 a combination of STM concentrations
S3 >10 N or >50 000 or >10 000
and location of primary tumour or
Table 2. Tumour-node-metastasis (TNM) classication system for testicular tumours [Sourced from
metastases. Around 5099% of patients
1416].
can still expect to survive [8].
 April/May 2016 8 Tumour markers

Salvage therapy, often in combina-
tion 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
Advances in treatment
strategies, such as the
use of cisplatin therapies,
careful staging at diagnosis,
early intervention using
multidisciplinary teams, rigorous
surveillance follow-up, and
salvage therapy, means that
GCTs are highly curable
suffer a late relapse, i.e. >2 years post-
diagnosis but also potentially 10
years post-diagnosis. These patients
are less responsive to chemotherapy,
so are treated primarily with sur-
gery. Unfortunately, less than half
will remain disease-free following
surgical intervention [8, 9]. Chem-
otherapy-induced side effects are
governed by the dose and combina-
tion of drugs used. This has triggered
more recent trials designed at main-
taining a cure rate but with reduced
associated chemotoxicity [8].

Upper
Tissue Conditions causing
Marker reference Serum t Use in testicular cancer
origin elevated serum markers
limit
-fetoprotein (AFP) ~10 kiU/L 57 days Fetal yolk sac, Benign liver disease Not secreted by pure cell
liver, GI tract Certain malignancies seminomas irrespective of
Mixed cell NSGCTs histology, or pure cell teratomas
Hepatocellular carcinoma Secreted by NSGCTs except
Gastric, colon, gall bladder, for choriocarcinomas or pure
pancreatic, lung cancer embryonal cell carcinomas
Hepatotoxicity (drug or viral) Exceptionally high levels seen in
Ataxia telangiectasia (>95% yolk sac NSGCTs
patients)
Hereditary persistence of AFP
Gestational trophoblastic disease
Poorly differentiated
adenocarcinoma
Tyrosinemia

& human 5 U/L 1624 hours Placental GCTs (pure seminomas, NSGCTs) Secreted by all NSGCTs except
chorionic (males) tiotrophoblasts Hydatidiform moles teratomas. Always secreted by
gonadotrophin Primary hypogonadism choriocarcinoma NSGCTs
(hCG) Gonadotroph adenoma High concentrations (>5000
Poorly differentiated U/L) suggestive of mixed GCTs
hCG adenocarcinoma Pure seminomas secrete hCG in
-subunit (hCG) Choriocarcinomas 1015 % of cases
typically the subunit Pancreas, islet cell, small/
detected by most large bowel, liver, stomach,
commercial assays lung, ovarian, breast and renal
malignancy
Gestational trophoblastic disease
Marijuana use

Lactate Laboratory 48113 Every tissue Muscle disease, MI, pernicious Not useful if the only tumour
dehydrogenase specic hours cell of body. anaemia, leukaemia, thalassemia, marker measured as not specic
subtype 1 Highest PE for TC resulting in high false-
(LDH-1) concentrations In vitro haemolysis positive rates. Most helpful in
found in all conjunction with AFP and hCG,
GCTs
muscle types, or for surveillance in patients with
liver and brain advanced seminoma
Placental alkaline <100 iU/L ~1567 Placental Normal testis, cervix, thymus, lung Elevated in seminomas
phosphatase (PLAP) hours blasts activity (Should not be measured in
GCTs, ovarian & lung malignancy 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.
 A New Breakthrough in
Hemostasis Quality Management.

New ACL TOP Family 50 Series* The new ACL TOP Family 50 Series offers the most advanced
automation and quality management for routine and specialty
Extending standardization Hemostasis testing in mid- to high-volume labs. All models
are standardized and offer automated pre-analytical sample
beyond the analytical phase integrity checks to identify under-lled sample tubes, abnormal
sample aspiration potentially caused by clots, and assay-specic
interference from hemolysis, lipemia and bilirubin. Plus,
new lab accreditation tools make compliance easier. Better
efficiency and quality management for youbetter quality
care for your patients.

The ACL TOP Family 50 Series. Quality in. Quality out.

For more information, please contact your local Werfen

representative/distributor or visit www.werfen.com.
2016 Instrumentation Laboratory. All rights reserved.
* Not available in all countries.

www.cli-online.com & search 27191

 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 clini-
cal 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 Oncol-
ogy recommend against using STMs as
a screening test for GCTs in asympto-
matic males. Given the low incidence
and mortality of TC combined with the
high cure rate, it is suggested a screen-
ing programme would be neither cost-
effective nor decrease mortality [10].
Furthermore, although STMs can be
helpful in combination with imaging
techniques in the diagnosis of TC, nor-
mal STMs alone do not exclude TC and
may also be raised in other conditions
[3, 810]. Routine testicular examina-
tion 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 embryo-
nal carcinoma, while conversely, AFP
is never elevated in pure seminomas.
Human chorionic gonadotrophin ele-
vations are associated with 1015 %
10 Tumour markers
of pure seminomas. Lactate dehydro-
genase 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 time-
table of STM measurements and radi-
ology imaging to detect disease recur-
rence depending on initial GCT type,
thereby avoiding relapse and presenta-
tion 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 diag-
nosed 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 prog-
nosis, patients who have relapsed fol-
lowing first-line chemotherapy and
patients who have a late relapse. Long-
term health consequences for patients
surviving TC, in particular side effects
associated with chemotherapy and radi-
otherapy such as cardiovascular disease,
impaired fertility and secondary can-
cers, continues to drive collaborative
studies nationally and internationally to
improve TC outcomes for the future.
References
1. Cancer registration statistics, first release, Eng-
land, 2014. Office for National Statistics 2014.
(http://web.ons.gov.uk/ons/rel/vsob1/cancer-
statistics-registrations--england--series-mb1-
/2014--first-release-/rpt-cancer-stats-registra-
tions.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 tes-
ticular 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, Mar-
tin 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 dis-
coveries 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 dif-
ferential diagnosis, newly appreciated, and con-
troversial 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-Recom-
mendations-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, Boke-
meyer C, Cohn-Cedermark G, Fizazi K, Horwich
A, Laguna MP, Nicolai N, Oldenburg J. Guide-
lines on testicular cancer. Eur Urol. 2015. (https://
uroweb.org/guideline/testicular-cancer/)
The authors
Angela Cooper* PhD, Sen Costelloe,
PhD
Derriford Combined Laboratory, Plym-
outh 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 tions took 6 months (33% >12 months)
diagnosis of multiple myeloma. Traditionally, this has been achieved from the onset of the rst related symp-
toms to referral [5]. Another study
using a combination of serum and urine electrophoresis, but this can
showed the time to diagnosis of MM can
result in incomplete investigation. The use of serum free light chains as be unacceptably prolonged [6] and the
an alternative screening test has been advocated to overcome this. pathway to diagnosis in MM was more
likely to include a string of repeated pri-
mary care consultations, infrequent use
by David Baulch and Beverley Harris
of urgent referral routes and increased
emergency presentation [7]. In particu-
lar, patients whose referral was delayed
Multiple myeloma cause uncontrolled proliferation of neo- by 6 months or more were more likely
Multiple myeloma (MM) accounts for plastic plasma cells, leading to plasma cell to suer a greater number of more sig-
1% of all cancers, with nearly 5000 peo- disorders (PCDs) such as MM [4]. Clonal nicant complications such as renal
ple in the UK being diagnosed each year. expansion of a plasma cell line under insuciency which, if swift diagnosis
The average age of presentation is 70 with such circumstances can cause overpro- had occurred, may have been reversible
only 15% of patients presenting at less duction of intact monoclonal Ig (IgG, [5]. This highlights the need not only to
than 60 years of age [1]. Its prevalence IgA, IgM, rarely IgD and IgE) or mono- raise awareness of disease symptoms,
has increased by 11% in the last decade, clonal free light chains (FLCs) kappa but to increase the sensitivity of labora-
due mainly to increased survival rates in and lambda. Although the classication tory detection.
those diagnosed [2]. Despite this, MM of PCDs is based on the immunoglobu-
still accounts for around 2700 deaths lin type secreted, 12% of MM cases are Laboratory investigation of
annually in the UK and over 70 000 classied as non-secretory. This may be multiple myeloma
worldwide with a median survival of due to an absence of secreted monoclo- In addition to clinical and hematological
only 34 years from diagnosis [3]. nal protein (M protein), or secretion at investigations, screening for MM within
a concentration below the limits of the the laboratory is based on the detection
MM is characterized by the accumulation laboratory methods used for detection. and classication of M proteins by serum
of clonal plasma cells, predominantly protein electrophoresis (the separation of
within the bone marrow, and subsequent Compared with other cancers, diagnosis serum proteins according to molecular
clonal expansion of the plasma cell line- of MM is challenging. Patients present size, hydrophobicity and electric charge
age [4]. It is almost always preceded by with a range of non-specic symptoms [8]), followed by immunoxation or
a premalignant, asymptomatic period of and as a result often have a string of immunotyping to identify and quantify
monoclonal gammopathy of undeter- primary care consultations resulting in the Ig isotypes. This method is less reliable
mined signicance (MGUS) [1]. The pro- diagnostic delay. Such delays signicantly for detecting disease when only FLCs are
cess of immunoglobulin (Ig) production impact the clinical course of MM [5], for secreted, as these are rapidly cleared by
by plasma cells is normally under a state which a complete cure remains elusive. the kidneys. Free light chains in the urine
of homeostasis, but random and non- [known as Bence Jones protein (BJP)]
random genetic aberrations, epigenetic Consequences of diagnostic delay can also be detected by electrophoresis
changes and atypical interactions within Studies have shown that over 50% of followed by immunoxation. However,
the bone marrow microenvironment can patients attending primary care institu- this methodology is time consuming and
may not detect low concentration BJP in
dilute urine samples [9]. Interpretation of
sEP and uEP sEP and sFLC the results can be dicult and should be
Sensitivity, % 81 (6989) 98 (91100) performed by appropriately qualied and
experienced laboratory sta. In addition,
Specicity, % 99 (99100) 89 (8592) obtaining both urine and serum samples
for screening can be problematic, with
PPV, % 96 (9899) 58 (4968)
some laboratories reporting that both
NPV, % 96 (9498) 100 (98100) samples are received for only ~17% of
MM screens.
Efciency, % 98 (9499) 90 (8693)
Table 1. Result Summary. The 95% condence interval limits are in parentheses. The highest performer There is growing evidence to support
for each statistical parameter is highlighted. the direct measurement and quantita-
PPV, positive predictive value; NPV, negative predictive value; sEP, serum protein electrophoresis and tion of serum kappa and lambda FLCs
reexed serum protein immunoxation; uEP, urine protein electrophoresis and reexed urine protein in diagnosis, monitoring and prognosis
immunoxation; sFLC, serum free light chain ratio. of MM and related PCDs [4]. The serum
 April/May 2016
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 anti-
bodies 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.
This sFLC assay can be used to replace
traditional urine methods for the labo-
ratory 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 nd-
ing by Dispenzieri et al. was that baseline
sFLC results can be used in prognostica-
tion 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.
Studies such as these have informed
changes to MM guidelines published
in 2016 [12] to acknowledge that sig-
nicantly abnormal FLC ratios, in the
absence of clinical features of end organ
damage, can be used in the diagnosis of
12 Tumour markers
MM [4]. This eliminates a traditional
major challenge with MM diagnosis in
that disease denition was clinicopatho-
logical. 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 laboratory-
dened rather than a symptom-dened
disease, allowing for earlier intervention.
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 consid-
erations to be made before such screen-
ing could be implemented.
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 per-
formed on 446 samples with no previous
history of, or investigations for, MM. The
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 alter-
native 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
The purpose of a medical screening pro-
gramme is to recognize a disease in its
preclinical phase to allow intervention at
an earlier stage. Such strategies have ben-
ets, risks and costs and the nal screen-
ing algorithm is often a compromise
between these three. However, a pro-
posed 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 addi-
tion 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 uncon-
rmed (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 suggest-
ing singular sEP testing is not sensitive
enough to detect minor abnormalities
in FLC production. This proposed com-
bination of screening tests increased
sensitivity by 17% when compared with
current protocols, indicating that the
sFLC assay is more sensitive than uri-
nalysis 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 pre-
sent with normal sEP and uEP, especially

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. Investi-
gation into this was beyond the scope of
our study, though the false positive rate
could potentially be reduced by employ-
ing 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 submit-
ting a single serum sample rather than
two separate specimens, increased use of
automation and reduction in subjectiv-
ity in reporting of results. However, it is
also important to consider the potential
increased cost of performing sFLC on all
samples submitted for myeloma screen-
ing, the importance of using appropriate
reference ranges and the need to develop
guidelines for interpretation of border-
line results. This latter point is particu-
larly important in order that unneces-
sary referrals are prevented, and should
involve close liaison with local hematol-
ogy 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 long-
term survival of patients diagnosed with multi-
ple myeloma in 20062010. Haematologica 2009;
94(2): 270275.
3. Rajkumar SV, Kyle RA, Therneau TM, Melton
LJ, III, Bradwell AR, Clark RJ, Larson DR, Ple-
vak 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.
13 April/May 2016
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, 11. Dispenzieri A, Kyle R, Merlini G, Miguel JS,
Blade J, Merlini G, Mateos MV, Kumar S, Hillen- Ludwig H, Hajek R, Palumbo A, Jagannath S,
gass J, Kastritis E, et al. International Myeloma Blade J, et al. International Myeloma Working
Working Group updated criteria for the diag- Group guidelines for serum-free light chain
nosis of multiple myeloma. Lancet Oncol. 2014; analysis in multiple myeloma and related disor-
15(12): e538548. ders. Leukemia 2009; 23(2): 215224.
5. Kariyawasan CC, Hughes DA, Jayatillake MM, 12. Myeloma: diagnosis and monitoring. National
Mehta AB. Multiple myeloma: causes and con- Institute for Health and Care Excellence (NICE)
sequences of delay in diagnosis. QJM 2007; 2016. (https://www.nice.org.uk/guidance/ng35)
100(10): 635640. 13. Wilson JM, Jungner YG. [Principles and prac-
6. Howell DA, Smith AG, Jack A, Patmore R, tice of mass screening for disease]. Bol Ocina
Macleod U, Mironska E, Roman E. Time-to- Sanit Panam. 1968; 65(4): 281393 (in Spanish).
diagnosis and symptoms of myeloma, lympho- 14. Jagannath S. Value of serum free light chain
mas and leukaemias: a report from the Haema- testing for the diagnosis and monitoring of
tological Malignancy Research Network. BMC monoclonal gammopathies in hematology. Clin
Hematol. 2013; 13(1): 9. Lymphoma Myeloma 2007; 7(8): 518523.
7. Elliss-Brookes L, McPhail S, Ives A, Greenslade 15. McTaggart MP, Lindsay J, Kearney EM. Replac-
M, Shelton J, Hiom S, Richards M. Routes to ing urine protein electrophoresis with serum
diagnosis for cancer determining the patient free light chain analysis as a rst-line test for
journey using multiple routine data sets. Br J detecting plasma cell disorders oers increased
Cancer 2012; 107(8): 12201226. diagnostic accuracy and potential health ben-
8. Bossuyt X. Separation of serum proteins by auto- et to patients. Am J Clin Pathol. 2013; 140(6):
mated capillary zone electrophoresis. Clin Chem 890897.
Lab Med. 2003; 41(6): 762772.
9. Kaplan IV, Levinson SS. Misleading urinary pro- The authors
tein pattern in a patient with hypogammaglobu- David Baulch* MSc, Beverley Harris MSc,
linemia: eects of mechanical concentration of FRCPath
urine. Clin Chem. 1999; 45(3): 417419. Department of Clinical Biochemistry,
10. Bradwell AR, Carr-Smith HD, Mead GP, Tang Royal United Hospitals Bath NHS Foun-
LX, Showell PJ, Drayson MT, Drew R. Highly dation Trust, Bath, UK
sensitive, automated immunoassay for immu-
noglobulin free light chains in serum and urine. *Corresponding author
Clin Chem. 2001; 47(4): 673680. 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 changed so that the ALL-type therapy was
levels of voriconazole while taking the antifungal prophylactically as discontinued and standard AML therapy
that included cytarabine, daunorubicin,
part of her treatment regimen in addition to standard chemotherapy
and etoposide was begun. To address other
for a leukocyte neoplasm. The usefulness of molecular diagnostic specic issues, this patient was treated
testing as an aid in voriconazole dosing is discussed. with multiple medications along with her
chemotherapy drugs, including Ambien,
Bactrim, Benadryl, cefepime, cyprohepta-
by S. Resaei, L. Collier and Dr S. Taylor
dine, hydroxyzine, meropenem, vancomy-
cin, and voriconazole.

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. Mar-
row aspiration was markedly hypercellular
with diuse clusters of blasts (Fig. 1). Flow
cytometry on the aspirate disclosed a sig-
nicant (50% of total sample) blast popu-
lation that exhibited CD33, CD13 (partial,
dim), CD34 (partial), CD15 (heterogene-
ous), 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 exhib-
ited by the circulating blasts in her periph-
eral blood. The co-expression of B-lym-
phoid and myeloid antigens prompted an
initial diagnosis of biphenotypic acute leu-
kemia. After multiple expert consultations,
it was decided to model the patients treat-
ment on therapy for acute lymphocytic
leukemia (ALL). Thus, the patient received
prednisone, vincristine, daunorubicin and
PEG asparaginase as induction chemo-
therapy, with vincristine and daunorubicin
administered again 7 days later.
Cytogenetic test results that were returned
on day 8, revealed a chromosomal trans-
location of (8;21)(q22;q22); RUNX1-
RUNX1T1, which changed the patients
diagnosis to an atypical form of acute mye-
logenous leukemia (AML). Accordingly,
the patients chemotherapy regimen was
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 atten-
tion turned to the medications that she
was receiving. All medications that seemed
likely to contribute to her neurologic prob-
lems were suspended and then reintro-
duced 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 neu-
rological 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 immuno-
suppressive chemotherapy. Patients with
hematologic malignancies are at high risk
of aspergillosis and candidiasis infec-
tions, because of the neutropenia that is
often caused by their chemotherapy regi-
mens [13].

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
Figure 1. Photomicrograph of patients bone marrow aspirate at 40 magnication (left) and 100 accepted as the key determinant in voricona-
magnication (right). Original diagnosis was biphenotypic acute leukemia, based upon blast zole clearance [46]. Variants of the CYP2C19
appearance and ow cytometry results. 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].

It is intuitive that an individuals CYP2C19

genotype fundamentally contributes to
Figure 2. Timeline of the patients voriconazole treatment and response. Voniconizole was
voriconazole metabolism, elimination, and
administered beginning on day 8 at 200 mg/twice daily. Four days after she began to experience
therefore bioavailability of the drug [46].
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
Systemic exposure to voriconazole is gen-
voriconazole level plateaued at 2.0 g/mL.
erally higher in individuals with reduced
enzyme activity. Thus the CYP2C19*1 vari- functioning (*1) allele. Poor metaboliz- ability to metabolize and eliminate the
ant is the wild-type variant and exhibits nor- ers (PM) are individuals with an enzyme drug. Trough plasma concentrations of
mal enzyme activity. CYP2C19 *2, *3, *4, *5, activity phenotype that is less than optimal, voriconazole have been signicantly higher
*6, and *8 isotypes display loss of function- caused by a genotype consisting of loss-of- in people possessing PM phenotypes fol-
ality as they possess little or no activity, and function alleles (*2*8/*2*8 ). Ultrarapid lowed by individuals with an IM pheno-
the CYP2C19*17 variant is assigned gain-of- metabolizers (UM) are at the other end of type, with the lowest bioavailability of the
function status because of its robust enzyme the enzyme activity spectrum, they may drug detected in individuals with an EM or
activity (Table 1) [7, 8]. either be heterozygous ultrarapid metabo- UM phenotype [46, 8]. However, higher
lizers with a wild-type allele combined trough levels of voriconazole are not uni-
Individuals who possess a normal or with an gain-of-function allele (*1/*17 versally higher in individuals with reduced
wild-type drug metabolizing phenotype genotype), or they may be homozygous CYP2C19 activity [8, 10]. Voriconazole dis-
inherit two copies of the normal CYP2C19 ultrarapid metabolizers with only gain- plays expected pharmacokinetic behaviour
genotype (*1/*1), and are designated as of-function alleles (*17/*17) (Table 1) [7, according to genotype in healthy volun-
extensive metabolizers (EM). Intermedi- 8]. The drug metabolizing phenotype of teers, but there is often a marked depar-
ate metabolizers (IM) have any one of individuals with the gain-of-function allele ture from the customary dose/response
the *2*8 alleles coupled with a normally (*17) combined with a loss-of-function relationship in patients. Presumably this

M e d i z i n i s ch e
EUROIMMUN Labordiagnostika
AG

Full automation of IIFT with EUROPattern

BIOCHIP Mosaics for detailed patient antibody
proles Test kits
Highly exible and efcient incubation options
Incubation
Fast image acquisition and evaluation: up to
500 positions in 2.5 hours (18 sec/eld)
Pattern recognition and titer designation: ANCA,
ANA (incl. mixed patterns), CLIFT, EUROPLUS,
cell-based assays (e.g. anti-neuronal antibod-
ies), soon also tissues Data processing

Consolidation of analytical results for each

patient Image recording
EUROPattern
Complete integration into existing laboratory
software (LIS) and archiving of data
Pattern recognition
EUROPattern

For further information contact Dr. Konstantin Ens (k.ens@euroimmun.de, +49 451 5855 25721)

EUROIMMUN AG D-23560 Luebeck (Germany) Seekamp 31 Tel +49 451 58550 Fax 5855591 E-mail euroimmun@euroimmun.de www.euroimmun.com

www.cli-online.com & search 27189

 April/May 2016 16 Personalized medicine

deviation from expected pharmacokinetic its clearance is aected by circumstances individualised medicine arrived for antifungals? A
behaviour is due to drugdrug interac- such as patient sex, age, disease state, liver review of antifungal pharmacogenomics. Bone Mar-
tions and/or the pathological circum- function, obesity and the presence of row Transplant. 2012;47(7): 881894.
stances of the patient [5, 6]. Generally, it inammation [11, 13, 14]. 5. Dolton MJ, McLachlan AJ. Voriconazole pharma-
is expected that disease circumstances or cokinetics and exposure-response relationships:
drug side eects that reduce liver enzyme Conclusion assessing the links between exposure, ecacy
activity (especially of CYP2C19, CYP2C9 The pharmacodynamic behaviour of vori- and toxicity. Int J Antimicrob Agents. 2014;44(3):
and CYP3A4) will decrease metabolism conazole remains dicult to predict as 183193.
and clearance of voriconazole, and thus it displays considerable interpatient and 6. Dolton MJ, Mikus G, Weiss J, Ray JE, McLachlan AJ.
increase patient exposure to the drug. intrapatient variablility. Although TDM Understanding variability with voriconazole using
for patients receiving voriconazole is rec- a population pharmacokinetic approach: implica-
Therapeutic drug monitoring ommended, establishing a patients phar- tions for optimal dosing. J Antimicrob Chemother.
The United States Food and Drug Adminis- macogenomic prole can provide clini- 2014;69(6): 16331641.
tration and the Infectious Diseases Society cians with valuable information to aid in 7. Owusu OA1, Egelund EF, Alsultan A, Peloquin CA,
of America recommend therapeutic drug appropriate voriconazole dosing, especially Johnson JA. CYP2C19 polymorphisms and thera-
monitoring (TDM) for patients receiv- in the initial stages of therapy. Pharmacog- peutic drug monitoring of voriconazole: are we
ing voriconazole [7]. Numerous studies enomic information is likely to contribute ready for clinical implementation of pharmacog-
indicate that voriconazole trough values to the goal of rapidly attaining a therapeutic enomics? Pharmacotherapy. 2014;34(7): 703718.
should be maintained above 1.0 g/mL for concentration while avoiding toxicity. It is 8. Moriyama B, Kadri S, Henning SA, Danner RL,
fungal prophylaxis. Moreover, some stud- possible that our patient has a PM pheno- Walsh TJ, Penzak SR. Therapeutic drug monitoring
ies indicate that voriconazole is more e- type for voriconazole and that pharmacog- and genotypic screening in the clinical use of vori-
cacious when trough levels are maintained enomic testing might have minimized her conazole. Curr Fungal Infect Rep. 2015;9(2): 7487.
at 2.0 g/mL or higher [11, 12]. exposure to toxic levels of voriconazole that 9. Swen JJ, Nijenhuis M, de Boer A, Grandia L, Mait-
arose from standard voriconazole dosing. land-van der Zee AH, Mulder H, Rongen GA, van
It is important to dose voriconazole accu- Schaik RH, Schalekamp T, Touw DJ, van der Weide J,
rately, as voriconazole ecacy is dependent References Wilert B, Deneer VH, Guchelaar HJ. Pharmacoge-
on adequate exposure to the drug; how- 1. Barreto JN, Beach CL, Wolf RC, Merten JA, Tosh netics: from bench to byte-an update of guidelines.
ever, increased trough levels are associ- PK, Wilson JW, Hogan WJ, Litzow MR. The inci- Clin Pharmacol Ther. 2011; 89(5): 662673.
ated with numerous severe adverse eects dence of invasive fungal infections in neutropenic 10. Kim SH, Yim DS, Choi SM, Kwon JC, Han S, Lee
(SAE). Voriconazole has been linked to patients with acute leukemia and myelodysplastic DG, Park C, Kwon EY, Park SH, Choi JH, Yoo JH.
several adverse events including abnor- syndromes receiving primary antifungal prophy- Voriconazole-related severe adverse events: clini-
mal liver function tests, gastrointestinal laxis with voriconazole. Am J Hematol. 2013; 88(4): cal application of therapeutic drug monitoring
disturbances, rash and vomiting. Neuro- 283288. in Korean patients. Int J Infect Dis. 2011;15(11):
toxicity (visual disturbances, hallucina- 2. Mattiuzzi GN, Cortes J, Alvarado G, Verstovsek S, 753758.
tions) is somewhat infrequently observed Koller C, Pierce S, Blamble D, Faderl S, Xiao L, Her- 11. Davies-Vorbrodt S, Ito JI, Tegtmeier BR, Dadwal
[1, 2]. Since CYP2C19 is a key metabo- nandez M, Kantarjian H. Ecacy and safety of intra- SS, Kriengkauykiat J. Voriconazole serum concen-
lizer of voriconazole, it seems reasonable venous voriconazole and intravenous itraconazole trations in obese and overweight immunocom-
to predict a patients drug metabolizing for antifungal prophylaxis in patients with acute promised patients: a retrospective review. Phar-
phenotype based on their CYP2C19 geno- myelogenous leukemia or high-risk myelodysplastic macotherapy. 2013 Jan;33(1): 2230.
type, and to use this information to guide syndrome. Support Care Cancer. 2011; 19(1): 1926. 12. Smith J, Safdar N, Knasinski V, Simmons W, Bhav-
dosing. In practice, the drug metabolizing 3. Rping MJ, Mller C, Vehreschild JJ, Bhme A, nani SM, Ambrose PG, Andes D. Voriconazole
genotype alone is not sucient to predict Mousset S, Harnischmacher U, Frommolt P, Was- therapeutic drug monitoring. Antimicrob Agents
the metabolizing phenotype. Confounding smer G, Drzisga I, Hallek M, Cornely OA. Vori- Chemother. 2006;50(4): 15701572.
variables include the fact that voricona- conazole serum concentrations in prophylactically 13. van Wanrooy MJ, Span LF, Rodgers MG, van den
zole has a high propensity for drugdrug treated acute myelogenous leukaemia patients. Heuvel ER, Uges DR, van der Werf TS, Kosterink
interactions, a narrow therapeutic index, it Mycoses. 2011; 54(3): 230233. JG, Alenaar JW. Inammation is associated with
exhibits non-linear pharmacokinetics, and 4. Ashbee HR, Gilleece MH. Has the era of voriconazole trough concentrations. Antimicrob
Agents Chemother. 2014;58(12): 70987101.
14. Brggemann RJ, Antonius T, Heijst Av, Hooger-
CYP2C19 allele Allelic functional status
brugge PM, Burger DM, Warris A. Therapeutic
*1 Normal function; normal activity; wild-type drug monitoring of voriconazole in a child with
*2, *3, *4, *5, *6, *7, *8 Loss-of-function; no or decreased activity invasive aspergillosis requiring extracorpor-
*17 Gain-of-function; increased activity eal membrane oxygenation. Ther Drug Monit.
2008;30(6): 643646.
Genotype Metabolizing phenotype
*17/*17 Ultrarapid (homozygous) The authors
*1/*17 Ultrarapid (heterozygous) Sahar Resaei BS; Laura Collier MLS(ASCP);
*1/*1 Extensive Sara Taylor* PhD, MLS(ASCP)MB
*1/*2-*8 Intermediate
Tarleton State University, Fort Worth, TX,
USA
*17/*2-*8 Ultrarapid, extensive, intermediate, unknown
*2-*8/*2-*8 Poor *Corresponding author
Table 1. CYP2C19 gene variants and drug metabolizing activity. E-mail: sataylor@tarleton.edu

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 carbohy-
drate metabolism that are characterized
by hyperglycemia associated with rela-
tive or absolute defects in insulin secre-
tion 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 pro-
cesses may be involved in the develop-
ment of diabetes is illustrated by the eti-
ological 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 auto-
immune destruction of the pancreatic
-cells, with absolute loss of insulin
secretion, and, although the rate of cell
Type of diabetes
I. Type 1 diabetes (510% of cases)
II. Type 2 diabetes (9095% of cases)
III. Other specific types:
A. Genetic defects of -cell function
B. Genetic defects in insulin action
C. Diseases of the exocrine pancreas*
D. Endocrinopathies*
E. Drug- or chemical-induced*
F. Infections*
G. Uncommon forms of immune-related
diabetes*
H. Other genetic syndromes sometimes
associated with diabetes
IV. Gestational diabetes mellitus
*Considered secondary diabetes
Table 1. Etiological classication of diabetes mellitus [4].
Diabetes 17
destruction is variable, most individu-
als will ultimately become dependent
on exogenously administered insulin for
survival, and are at risk of ketoacidosis.
In contrast, patients with Type 2 diabe-
tes 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 diabe-
tes, treatment with insulin is not essen-
tial for survival, although it may eventu-
ally prove necessary to achieve glycemic
control [3].
The majority of individuals with Type
2 diabetes are largely asymptomatic,
diagnosed only after laboratory evalu-
ation, whereas those with Type 1 are
more likely to present with the classical
symptoms of hyperglycemia: polyuria,
polydipsia, blurred vision and weight
Comments
-cell destruction: absolute insulin deficiency
-cell destruction: absolute insulin deficiency
e.g. Pancreatitis, cystic fibrosis,
hemochromatosis
e.g. Acromegaly, Cushings syndrome,
glucagonoma
e.g. Glucocorticoids, thiazides, thyroid
hormones
e.g. Congenital rubella, cytomegalovirus
e.g. Stiff-man syndrome
e.g. Downs syndrome, Turners syndrome
April/May 2016
loss. There may also be acute life-threat-
ening consequences of uncontrolled
diabetes: diabetic ketoacidosis in Type
1 diabetes and non-ketotic hyperosmo-
lar syndrome in Type 2 [1]. Both forms
are associated with a number of charac-
teristic long-term complications, usu-
ally considered to be a consequence of
microvascular disease, including retin-
opathy, with potential loss of vision;
nephropathy, leading to kidney failure;
peripheral and autonomic neuropathy.
However, in reality, the major determi-
nant of the reduced life expectancy seen
in diabetes is the significantly increased
incidence of macrovascular atheroscle-
rotic disease, which causes myocardial
infarction or angina, stroke or periph-
eral vascular disease [4].
Diagnostic criteria
1. Blood glucose measurements
For decades, the diagnosis of diabe-
tes 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 diagno-
sis 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 ran-
dom 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 tol-
erance (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 develop-
ing the microvascular complication of
 April/May 2016 18 Diabetes

the United Kingdom Prospective Diabe-

Test Diagnosis tes Study (Type 2 diabetes) [10], which
validated the direct relationship between
Impaired glucose glycated hemoglobin levels and clini-
Normal regulation or Diabetes cal outcomes, it has had a vital role in
Pre-diabetes monitoring diabetes. With respect to the
diagnosis of diabetes, however, although
HbA1c** (mmol/mol) <42 4247 48* epidemiological studies also showed a
Fasting glucose (mmol/L) <6.1 6.16.9 7.0* clear relationship between HbA1c and
retinopathy, variation in methodology
2hr glucose in OGTT
<7.8 7.811.0 11.1* and standardization, and concern about
(mmol/L)
the confounding effect of factors affect-
Random glucose (mmol/L) 11.1* ing erythrocyte turnover, seemed to pre-
*If the patient is asymptomatic, a repeat test is required to conrm the diagnosis. clude its use for this purpose [8]. This
situation has changed in recent years, as
a result of a number of HbA1c stand-
** HbA1c is not appropriate for diagnosis of diabetes: ardization programmes, culminating in
Symptoms suggestive of Type 1 diabetes (e.g. weight loss, ketonuria) the work of the IFCC Working Group
Symptoms of diabetes for less than 2 months
on Standardization of HbA1c, which
established true International Refer-
Pregnancy ence Methods for HbA1c and provided
All those under 18 years of age a preparation of pure HbA1c, against
Treatment with corticosteroids, antipsychotics or immunosuppressants which manufacturers could standardize
their calibrators [11].
Acute pancreatic damage (e.g. pancreatitis or pancreatic surgery)
Conditions that may prevent accurate measurement of HbA1c: hemoglobinopathies; In 2011, in response to this global
hemolytic anemia; iron-deciency anemia; splenomegaly; antiretroviral drugs; splenectomy; standardization of HbA1c methods, the
chronic kidney disease (when associated with renal anemia).
WHO stated that HbA1c could be used
as a diagnostic test for diabetes mellitus,
This exclusion only apples to patients on short-term therapy with these drugs: if a patient provided that stringent quality assur-
is on such treatment for more than 3 months, HbA1c would be an appropriate test to ance methods are in place, assays are
screen for diabetes. standardized to criteria aligned to the
international reference values and there
Table 2. Criteria for diagnosing diabetes [4, 12, 15].
are no conditions present that preclude
its accurate measurement [12]. Based
retinopathy. The OGTT 2-hour PG of on the DETECT-2 pooled data analy-
11.1 mmol/L closely approximates It was at this stage that a secondary sis, which examined the association
to a point at which the prevalence of criterion for the at-risk category was between diabetes-specific retinopathy
microvascular complications increases recognized: impaired fasting glycemia and glycemic measures, an HbA1c of
dramatically. However, only approxi- (IFG), a FPG of 6.16.9 mmol/L. Both 48 mmol/mol was recommended as the
mately 25% of those who exceed this IFG, and the previously described IGT, cut-off point for diagnosing diabetes
2-hour threshold will also have a FPG have been referred to as pre-diabetes, [13]. As with glucose measurements,
7.8 mmol/L, whereas almost all indi- indicating a relatively high risk of future there is a range of HbA1c levels below
viduals with FPG 7.8 mmol/L have a diabetes. Studies have demonstrated an this diagnostic value, which indicates an
2-hour OGTT level 11.1 mmol/L. Thus, approximately 510% annualized risk increased risk of future diabetes and/
this earlier FPG cut-off defined a greater of progression to diabetes in individuals or cardiovascular disease: a systematic
degree of hyperglycemia, a discrepancy with either IFG or IGT and 1015% in review indicated that HbA1c values
that was considered undesirable: both those with both abnormalities [7]. between 37 and 48 mmol/mol are asso-
fasting and 2-hour cut-off points should ciated with a substantially increased risk
reflect a similar degree of hyperglycemia 2. Glycated hemoglobin of diabetes [14]. The WHO did not pro-
and risk of adverse outcomes. In addi- Glycated hemoglobin (HbA1c), formed vide specific guidance on HbA1c crite-
tion, due to the inconvenience of under- as a consequence of a non-enzymatic, ria for pre-diabetes but the 2009 Inter-
taking OGTTs, the FPG alone was often irreversible reaction between glucose national Expert Committee concluded
performed, meaning that a substantial and the N-terminal valine residue of the that individuals with an HbA1c of
number of individuals, who were at globin chains of hemoglobin, reflects 4247 mmol/mol should be considered
increased risk of microvascular compli- average blood glucose levels over the at high risk of progression to diabetes
cations, would not have been detected. preceding 812-week period (the lifes- [15] (estimated 5-year risk of 2550%
The revised FPG cut-off of 7.0 mmol/L pan of a red blood cell) and its potential [14]), a range that was endorsed by a UK
was shown to have a similar predictive as an indicator of glycemic control was Expert Position Statement [16].
value for adverse outcomes as the 11.1 recognized in 1977 [8]. Over the inter-
mmol/L 2-hour OGTT threshold, which vening years, supported by evidence Current recommendations
validated the use of this simpler test for from the Diabetes Control and Compli- The current criteria for the diagnosis of
diagnostic purposes. cations Trial (Type 1 diabetes) [9] and diabetes and pre-diabetes, in accordance

with WHO recommendations, are sum-
marized in Table 2. OGTTs, which are
time-consuming, inconvenient and
show poor reproducibility, are increas-
ingly confined to the diagnosis of gesta-
tional 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 meas-
urement 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 appropri-
ate (Table 2): as a measure of chronic
hyperglycemia, HbA1c should not be
used where rapidly developing hyper-
glycemia 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
19
FPG and 2-hour OGTT PG, there is
not full concordance between HbA1c
and glucose measurements: these three
different measures of glycemia repre-
sent different physiological processes
and, therefore, inevitably, they iden-
tify 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 preva-
lence (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 mag-
nitude of the difference appears to vary
between populations, perhaps reflect-
ing 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 appli-
cation [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 develop-
ment of diabetes. Within this category,
for all three tests, the risk of future dia-
betes is curvilinear, extending below the
lower limit of the range and becoming
disproportionately greater at the higher
end: accordingly, intervention and fol-
low-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 posi-
tion on the use of HbA1c in the diag-
nosis of diabetes, was issued to local
clinicians in 2012. Subsequently, in
September 2014, updated guidance was
disseminated, advocating the use of
www.cli-online.com & search 27063
 April/May 2016
HbA1c as a diagnostic test for diabe-
tes mellitus, except where inappropri-
ate, 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 estab-
lished diabetes (designated HbA1cM)
and those being used for diagnosis (des-
ignated HbA1cD). This facilitated the
provision of additional targeted guid-
ance in the form of interpretative com-
ments and, importantly, for HbA1cD
requests, allowed flagging, as abnor-
mal, results that indicated pre-diabetes
(4247 mmol/mol).
The pattern of fasting glucose, OGTT
(excluding those from maternity ser-
vices) and HbA1c requesting between
April 2012 and March 2016 is summa-
rized 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 pur-
poses increased rapidly and, since late
2015, the number of HbA1cD requests
has been similar to the total number of
HbA1c requests/month in 2014.
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).
20 Diabetes
Summary
Local experience indicates an enthu-
siastic 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 dia-
betes but there is concern that this ease
of use may mean that the limitations
of HbA1c as a diagnostic test are over-
looked, resulting in its application in
circumstances when glucose measure-
ments would, in fact, be indicated. There
is a clear role for laboratory staff in the
provision of ongoing education of clini-
cians, in order to ensure the appropriate
use and interpretation of these tests.
References
1. McCulloch DK. Clinical presentation and diagno-
sis of diabetes mellitus in adults. UpToDate. (http://
uptodate.com/contents/clinical-presentation-and-
diagnosis-of-diabetes-mellitus)
2. Global Report on Diabetes. World Health Organi-
zation 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, diag-
nosis 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 Organi-
zation 2006. (http://www.who.int/diabetes/publica-
tions/Definition%20and%20diagnosis%20of%20
diabetes_new.pdf)
6. Expert Committee on the Diagnosis and Classica-
tion of Diabetes Mellitus. Report of the Expert Com-
mittee 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 dia-
betes. 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 con-
ventional 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 Fed-
eration Consensus Committee. Consensus state-
ment on the worldwide standardisation of the
HbA1c measurement. Diabetologia 2007; 50(10):
20422043.
12. Use of glycated haemoglobin (HbA1c) in the diag-
nosis 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 dia-
betes-specic retinopathy: implications for diag-
nostic 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 system-
atic 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 imple-
mentation 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
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 Some beliefs about OGTT have been
of Type 2 diabetes mellitus (T2DM). One of the most obvious, as brought into question, too. In 2002, clinical
epidemiologists at the University of Texas
suggested in a recent report on diabetes global burden, would be
Health Center in San Antonio published
better disease management. The report, by the University of East the results of a prospective cohort study to
Anglia in the UK, concludes that early investments into prevention identify people at high risk of T2DM.
and disease management may therefore be particularly worthwhile. The results were unequivocal. Impaired
glucose tolerance was only one indicator
of risk. Persons at high risk for T2DM, the
Risk factors (FPG). However, the tests specicity is study concluded, were better identied
Such perspectives are strengthened by poor. Two decades ago, the so-called Hoorn by using a simple prediction model than
evidence that the onset of T2DM can be study at Amsterdam warned about signi- by relying exclusively on the results of a
delayed by behaviour modication. A cant levels of variation in blood glucose 2-hour oral glucose tolerance test.
study in the British Medical Journal in levels. Although many individuals are
2007 noted that lifestyle changes could be identied as having impaired fasting glu- Predictive models
at least as eective as drug treatment in cose (IFG), their absolute risk of conver- Subsequent years have been witness to
slowing the onset of diabetes. It concluded sion to diabetes is a mere 5 to 10% per year. signicant eorts to develop and rene
that the only barrier to the eectiveness Over this period, dierences have also predictive models for T2DM. However,
of such a strategy was to identify diabetes emerged about how best to measure glu- ve years after the San Antonio study, the
quickly enough. cose. In the year 2000, while some experts choices are still less than wholly clear.
(including the American Diabetes Asso-
Much is now known about the risk factors ciation) recommended the use of fasting In 2007, the Framingham Ospring study
associated with T2DM such as parental plasma glucose (FPG) alone, others noted in the US estimated seven-year T2DM risk
history, age, body mass index and elevated that many diabetic subjects would have based on a pyramid of metrics consisting -
blood glucose levels. Combining these been classied as non-diabetic on the at the base - of age, sex, parental history and
with measurable indicators of metabolic FPG test. As a result, they recommended body mass index. This was followed by the
syndrome - high blood pressure, LDL and use of the two-hour oral glucose toler- inclusion of simple clinical measurements
HDL cholesterol and excess triglyceride - ance test (OGTT). Nevertheless, in spite of on metabolic syndrome traits, and thereaf-
can result in a credible degree of predic- its greater accuracy, OGTT is rarely used ter, the 2-hour post-oral glucose tolerance
tion. However, there are several barriers to since it requires two hours to perform and test, fasting insulin and C-reactive pro-
the process. is an unpleasant experience for the patient. tein levels. At its most complex, the model
used the Gutt insulin sensitivity index or a
Fasting glucose and oral glucose Glucose tolerance only one risk homoeostasis model of insulin resistance.
tolerance indicator For proponents of new alternatives to
The typical method for assessing T2DM The above factors have provoked a search impaired glucose tolerance, the conclu-
risk is to measure fasting plasma glucose for new approaches to predict T2DM. sions 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 predic-
tion rules, we lack consensus for the most
eective approach.

The limitations of biotech

More recently, investigations at the fron-
tiers 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/Massa-
chusetts General and Emory University
published results of a study on 18 single-
nucleotide polymorphisms (SNPs) known
to have associations with the risk of T2DM,
 April/May 2016
to predict new cases in a large, prospec-
tively 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 con-
cluded: 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 vali-
date 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 dia-
betes mellitus (NIDDM). The study dates
to the early 1980s when it sought to docu-
ment 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 stor-
age, given a belief that T2DM was a mani-
festation 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 associ-
ated with increased T2DM risk in healthy
women, independent of known diabetes
risk factors.
However, there still are reasons for cau-
tion. In July 2014, or more than a decade
after the US Nurses Health Study, a meta-
analysis 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 biomark-
ers out of a total of 64, to assess T2DM risk.
The selected biomarkers include adiponec-
tin and ferritin, as well as four of their
more common counterparts: glucose and
insulin, as well as the inammation mark-
ers C-reactive protein (CRP) and interleu-
kin-2 receptor A (IL2RA).
The model was developed by a research
team from Copenhagens Glostrup Hospi-
tal 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 micro-
sample 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 quantita-
tive estimate of the 5-year risk of develop-
ing type 2 diabetes, performs better than
single risk indicators and a noninvasive
clinical model, and provides better strati-
cation than fasting plasma glucose alone.
Expert acclaim
The researchers who developed the Dan-
ish 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 acknowl-
edge 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 Dan-
ish 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 pro-
duce 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 biomark-
ers 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 lat-
ter predicted incident diabetes in women
independent of traditional risk factors
such as BMI, family history, diet and activ-
ity. 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 poten-
tially 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 pep-
tide-1 (GLP-1).
In patients with type 2 diabetes, the incre-
tin 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 possibil-
ity 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, inves-
tigated melatonin receptor 2 (MT2 tran-
scripts) in neural tissues and MT2 expres-
sion in human pancreatic islets and beta
cells. Their ndings suggest a link between
circadian rhythm regulation and glucose
homoeostasis through the melatonin sig-
nalling pathway.

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 mel-
litus resulting from insulin deciency. It is
biochemically dened as hyperglycemia
(blood glucose >200 mg/dL) with meta-
bolic acidosis (venous pH <7.3 or bicarbo-
nate <15 mmol/L), ketonemia, and ketonu-
ria [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
Diabetes 23
issues. There are three types of ketones: ace-
toacetate, acetone, and -hydroxybutyrate
(BHB). BHB is the predominant ketone
produced during DKA and can be present
at up to 10 times the amount of acetoace-
tate. The urine dipsticks that are commonly
used to assess ketonuria utilize a nitroprus-
side reagent that reacts with acetoacetate
and acetone but not at all with BHB. This is
problematic because the major ketone pro-
duced 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, poten-
tially leading the clinician to believe that
the ketosis is worsening instead of resolv-
ing. 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 solu-
tion 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. Ini-
tial 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 assess-
ment 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.
April/May 2016
The commercial availability of point-of-
care (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. com-
pared 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 sen-
sitive 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 dip-
sticks (78.6 vs 35.1%) and could cut down
on unnecessary DKA work ups in hyper-
glycemic 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 pediat-
ric 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 labo-
ratory for most of the meters measure-
ment 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 treat-
ment 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 measure-
ment was 17 hours, compared to 28 hours
using measurement of urine ketones to
end treatment [6] . They found that occa-
sionally a value below 1 mmol/L would be
followed by a value above 1 mmol/L, but
this never occurred after two subsequent
 April/May 2016 24
values under 1 mmol/L, leading them to recommend waiting for
the two successive low values before ending treatment. In addi-
tion to allowing an earlier treatment endpoint, this approach ena-
bles less time to be spent in the ICU, with decreased cost associ-
ated 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, bicar-
bonate, and pCO2 and also had good correlation with the labora-
tory 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 ear-
lier identication of ketosis and initiation of treatment [8].
n
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
Diabetes
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 moni-
tor 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 meas-
urement of serum BHB can aid in both diagnosis and manage-
ment 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 evalua-
tion 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
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 dia-
betic 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 guide-
lines for management of diabetic ketoacidosis: Do the guidelines need to be modi-
ed? Pediatr Diabetes 2014; 15(4): 277286.
10. Clark MG, Dalabih A. Variability of DKA management among pediatric emer-
gency room and critical care providers: a call for more evidence-based and cost-
eective 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
*Corresponding author
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 neuropathic manifestations appear to be
pathway which clinically manifest with acute neurovisceral primarily related to axonal degeneration
due to direct neurotoxicity by ALA, which
attacks and cutaneous lesions. Diagnosis of porphyrias is based
structurally resembles the neurotransmit-
on the accurate and precise measurement of various porphyrins ter gamma-aminobutyric acid (GABA) [3,
and precursor molecules in a range of samples. In addition, 57].
molecular diagnostic assays can provide denitive diagnosis.
The second clinical presentation para-
digm is cutaneous photosensitivity
by Dr Vivion E. F. Crowley, Nadia Brazil, and Sarah Savage caused by the interaction of ultraviolet
light with photoactive porphyrins in the
skin resulting in the production of reac-
What are porphyrias? The rst is the acute neurovisceral attack, tive oxygen species (ROS) and an associ-
Porphyrias are a group of rare disor- which is a potentially life threatening ated inammatory response [3]. In PCT,
ders each of which results from a de- episode related to excessive hepatic gen- VP and HCP the skin lesions typically
ciency of an individual enzyme within eration of ALA and PBG, and which is occur post-pubertally and consist of skin
the heme biosynthetic pathway (Fig. 1) a feature only in acute intermittent por- fragility, vesicles, bullae, hyperpigmen-
[13]. With the exception of an acquired phyria (AIP), variegate porphyria (VP), tation and hypertrichosis aecting sun
form of porphyria cutanea tarda (PCT), hereditary coproporphyria (HCP) and exposed areas, most usually the face and
all porphyrias are inherited as monogenic the very rare ALA dehydratase deciency dorsum of hands [13]. In erythropoietic
autosomal dominant, autosomal recessive porphyria (ADP) [57]. These attacks are protoporphyria (EPP) and X-linked pro-
or X-linked genetic disorders, with vary- characterized principally by autonomic toporphyria (XLP), which may present in
ing degrees of penetrance and expressiv- dysfunction, including non-specic but childhood, there is usually no blistering
ity and this impacts on the prevalence severe abdominal pain, constipation, but instead erythema, edema and pur-
and incidence of clinically manifest por- diarrhoea, nausea, vomiting, tachycardia, pura feature in the more acute setting,
phyrias [4]. The biochemical consequence hypertension or occasionally postural with subsequent chronic skin thickening
of each porphyria is the overproduction hypotension. In addition, other features noted, whereas congenital erythropoi-
within the heme biosynthetic pathway of may include a predominantly motor etic porphyria (CEP) is characterized by
specic porphyrin intermediates and/or peripheral neuropathy which, if left undi- severe cutaneous photosensitivity often
the porphyrin precursor molecules delta- agnosed, may extend to respiratory failure occurring in early infancy with bullae and
aminolevulinic acid (ALA) and porpho- reminiscent of GuillainBarr syndrome, vesicles rupturing and being prone to sec-
bilinogen (PBG) [23]. This in turn has as well as cerebral dysfunction, which ondary infection, with resultant scaring,
implications for the clinical manifestation can vary from subtle alterations in men- bone resorption, deformation and mutila-
of these disorders, their overall classica- tal state, to posterior reversible encepha- tion of sun-exposed skin [1, 2, 8].
tion and their diagnosis (see Table 2). lopathy syndrome (PRES). Hyponatremia,
most likely due to SIADH [syndrome Classication
Clinical presentation of inappropriate antidiuretic hormone The classication of porphyrias (Table 1)
Porphyrias may present clinically with (ADH) secretion] may also contribute has traditionally been determined either
either or both of two symptom patterns. to CNS-related morbidity. The complex on the basis of clinical manifestations, i.e.
acute or non-acute (cutaneous), or on the
primary organ of porphyrin overproduc-
tion, 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 simul-
taneously or separately.

Clinical and biochemical

diagnosis
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
Figure 1. Heme biosynthetic pathway and associated porphyrias. 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 inves-
tigating 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 criti-
cally dependent on biochemical testing, as
symptoms are often non-specic in nature
(Tables 1 & 2).
The diagnosis of an acute hepatic por-
phyria attack is founded on demonstrat-
ing an increase in urine PBG levels in
direct temporal association with the char-
acteristic 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 time-
liness, 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
Table 1. Overview of porphyrias including genes involved, inheritance pattern and basic clinical features.
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 normaliza-
tion [3]. Furthermore, if the measurement
of urine PBG is delayed or undertaken at a
time removed from the actual acute clini-
cal 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 expe-
rience another important caveat concerns
patients with a previous conrmed diag-
nosis 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 deci-
sion 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
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 syn-
thase 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 pat-
tern 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 eryth-
rocytes. It is critically important that the
laboratory analytical methods available
extend beyond the sole measurement of
total porphyrin levels [1012]. In particu-
lar, it is essential that individual porphy-
rin analysis and isomer fractionation in
both urine and feces is available to facili-
tate the identication of the porphyria-

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. copropor-
phyrinuria, 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 spe-
cic 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 high-
lights the importance of developing specialist porphyria centres
to ensure that the appropriate repertoire of quality assured test-
ing and expert interpretation and support are available for diag-
nosis 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 proto-
porphyrin 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 uo-
rescence emission peaks, e.g. VP emission peak between 625 and
628 nm [13]. Finally, it is essential that all samples for porphy-
rin 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 por-
phyria 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 appli-
cation 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 muta-
tion has been identied its pathogenicity for a particular por-
phyria should be armed and then more extensive family cas-
cade 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 expressiv-
ity of the disorders is low [3, 4]. Thus the penetrance among AIP,
VP and HCP is between 10 and 40%, implying that the major-
ity of patients with an autosomal dominant acute hepatic por-
phyria will not manifest with an acute attack (or indeed cutane-
ous 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 under-
lying autosomal dominant acute porphyria, demonstrating the
limited sensitivity of biochemical testing in identifying asymp-
tomatic 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 environ-
mental precipitating factors, e.g. porphyrinogenic medications,
27 April/May 2016
stress, prolonged fasting, menstruation [13], have long been
recognized in triggering acute porphyria attacks, it is the pres-
ence 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 path-
ogenic mutation should be regarded as pre-symptomatic carri-
ers, i.e. capable of developing an acute attack, and one of the key
applications of genetic analysis in the area is in identifying pre-
symptomatic 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 deni-
tive diagnosis for the type of porphyria and will accommodate
a more extensive family screening programme for potential pre-
symptomatic 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 ligation-
dependent probe amplication (MLPA) and gene dosage analy-
sis for identifying complex mutations, such large gene deletions,
Hemostat
Worlds Fastest Hemoglobin Meter
Hemoglobin and hematocrit measurement
5 seconds to result
1 L sample volume only needed
Electrochemical biosensor technology
Unique test strip
Compact hand-held meter
Point of care. Anytime.
Anywhere.
www.diasys-diagnostics.com
www.cli-online.com & search 27208
 April/May 2016
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 includ-
ing ADP, CEP and EPP, the clinical pen-
etrance approaches 100%. These disorders
also display a level of genetic heterogene-
ity. In the case of EPP the presence of a
relatively common low expression single
nucleotide polymorphism (SNP) located
in the ferrochetalase gene, FECH (IVS3-
48C), appears to be essential for the clini-
cal 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 pro-
tocols in accordance with international
standards and best practice recommenda-
tions [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: inher-
ited disorders of haem synthesis. In: Marshall
W, Lapsley M, Day A, Ayling R, editors. Clinical
28 Hematology
Biochemistry Metabolic and Clinical Aspects.
Churchill Livingstone Elsevier 2014; pp. 533549.
4. Elder G, Harper P, Badminton M, Sandberg S, Dey-
bach JC. The incidence of inherited porphyrias in
Europe. J Inherit Metab Dis. 2013; 36: 849857.
5. Simon NG, Herkes GK. The neurologic manifes-
tations of the acute porphyrias. J Clin NeuroSci.
2011; 18: 11471153.
6. Sonderup MW, Hift RJ. The neurological manifes-
tations of the acute porphyrias. S Afr Med J. 2014;
104: 285286.
7. Crimlisk HL. The little imitator-porphyria: a neu-
ropsychiatric disorder. J Neurol Neurosurg Psy-
chiatry. 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 inter-
mittent porphyria. Clin Chem. 2006; 52: 650656.
10. Kauppinen R, von und zu Fraunberg M. Molecu-
lar 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 pro-
gramme. 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: Ret-
rospective 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). Estab-
lishing 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, Bio-
chemistry Department, St Jamess Hospital,
Dublin 8, Ireland
*Corresponding author
E-mail: vcrowley@stjames.ie
 April/May 2016 29 INDUSTRY NEWS

Abbott demonstrates next-generation molecular diagnostics prototype

for infectious diseases such health decisions, said Andrea immunoassay, clinical chem-
as HIV, hepatitis and tuber- Wainer, president, Molecu- istry, hematology and point
culosis, as well as sexually lar Diagnostics, Abbott. Our of care testing in the near
transmitted infections such accurate, reliable and qual- future. All of the systems will
as human papillomavirus ity tests could allow clini- be built on the same software
(HPV), chlamydia and gon- cians to make more informed and hardware platforms to
orrhea, among others tests. treatment decisions to help enable more automation and
improve patient care. to simplify the user experi-
Abbott demonstrated a pro- Abbotts molecular diagnos- In addition to the new molec- ence for Abbotts customers.
totype of the companys next- tics can provide the infor- ular system, Abbott will be
generation molecular diag- mation needed to help guide launching next-generation www.abbottmolecular.com
nostics platform at a recent some of lifes most important systems in blood screening,
scientific event hosted for its
customers from across the
globe. At the event, molecu-
lar laboratory directors and
researchers had hands-on
interaction with the proto-
type and were able to provide
additional feedback on the
system prior to further stages
of development.

Abbotts new system is cur-

rently being designed from
the ground up based on
extensive input from labora-
tory 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 custom-
ers 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-gen-
eration molecular system
is being designed to have
a faster turnaround time,
Glucose Stabilisation Right from the Beginning
greater flexibility to run any The birth of the new 9$&8(77( FC Mix tube
test at any time, an ability
to run higher volumes and For the diagnosis of diabetes mellitus
automation to increase lab and gestational diabetes
efficiency all without com- Unique additive mixture in the tube
promising the testing perfor-
Immediate stabilisation based on
mance and quality for which the in vivo value for 48 hours
our organization is highly
regarded. Prevents false negative diagnoses
Longer sample stabilisation enables
Additionally, customer longer transport
insights suggest a need for Stabilisation in whole blood, no
a broad testing menu in the immediate centrifugation required
molecular lab. Abbott cur- Greiner Bio-One GmbH | Bad Haller Strae 32 | A-4550 Kremsmnster
rently offers one of the broad- Phone: (+43) 75 83 67 91-0 | Fax: (+43) 75 83 63 18 | E-mail: office@at.gbo.com www.gbo.com/preanalytics

est molecular testing menus

www.cli-online.com & search 27177
 April/May 2016
Clinical application handbook
Shimadzu has released the rst Applica-
tion 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 analy-
sis. 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 ben-
ets. 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, particu-
larly when it comes to time-critical
situations, e.g. through acute intoxica-
tion, medical or drug abuse. They ana-
lyse 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 sys-
tems support health protection of ani-
mals and humans, even in the long-term.
Clinical applications benet from Shi-
madzus complete portfolio covering chro-
matography and mass spectrometry (GC,
GC-MS, GC-MS/MS, HPLC, UHPLC,
LC-MS, LC-MS/MS); spectroscopy (UV-
Vis, FTIR, AAS, EDX, ICP-OES); life
sciences (MALDI-(TOF)-MS); micro-
chip-electrophoresis; biopharmaceutical
(aggregate sizer); observation of medi-
cal microbubbles in targeted drug deliv-
ery using the HPV-X2 ultra high-speed
camera.
Shimadzu breaks new grounds by
rethinking the use of mature technolo-
gies to develop new unique systems
30 INDUSTRY NEWS
such as the iMScope TRIO. It combines
an optical microscope with a mass spec-
trometer for insights on the molecular
level.
For next-generation brain science, Shi-
madzu provides LABNIRS, an imaging
technology for visualization of brain func-
tions by functional near-infrared spec-
troscopy (fNIRS).
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 clini-
cal eld since many years. Gas chroma-
tography is a key technique for quantita-
tive analysis of alcohol in blood.
HPLC and UHPLC systems are able
to quantitatively analyse substances in
blood, serum, plasma and urine con-
taining multiple compounds by sepa-
rating and detecting target substances.
Shimadzu oers a wide variety of appli-
cation-specic systems such as auto-
mated sample pretreatment systems for
amino acid analysis or on-line sample
trapping for quantication of drugs or
metabolites.
Gas chromatography-mass spectrom-
etry (GC-MS) is a hyphenated technique
combining the separating power of GC
with the detection power of MS to iden-
tify dierent substances within a sample.
Mass spectrometry is a wide-ranging
analytical technique which involves the
production, subsequent separation and
identication of charged species accord-
ing to their mass to charge (m/z) ratio. It
is well known for analysis of drug abuse.
Liquid chromatography-mass spectrom-
etry (LC-MS) is an analytical chemistry
technique that combines the physical
separation capabilities of LC with the
mass analysis capabilities of MS, bring-
ing together very high sensitivity and
high selectivity. Its application is ori-
ented towards the separation, general
detection and potential identication of
compounds of particular masses in the
presence of other chemicals (e.g. com-
plex 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 analy-
sis 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 Health-
care 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 labo-
ratory customers around the world to
continue to benefit from the largest port-
folio of hemostasis systems and reagents.
The companies, which began collaborat-
ing more than 20 years ago, also agreed to
continue joint hemostasis product devel-
opment activities that will streamline and
optimize testing in laboratories through-
out the world.
Siemens Healthcare and Sysmex pro-
vide hemostasis products used to test
for blood clotting disorders, preopera-
tive bleeding risk management, and the
monitoring of patients on anticoagulant
therapy medications. In the past few
years alone, the companies have intro-
duced several cutting-edge INNO-
VANCE reagents and multiple new plat-
forms 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 longstand-
ing partnership with Siemens Health-
care, said Hisashi Ietsugu, Chairman
and CEO, Sysmex Corporation. With the
aging population, hemostasis testing has
become even more important. Our part-
nership provides our customers with the
innovative technologies needed to man-
age the increase in testing volumes, while
providing accurate results for improved
patient care.
The continued collaboration and
twenty-year partnership between Sie-
mens and Sysmex is rare in the rap-
idly 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 through-
out the world.
www.siemens.com
 PRODUCT NEWS
Prep automation in culture-inde-
pendent pathogen PCR testing
Micro-Dx ena-
bles the culture-
independent
diagnosis of
pathogens in
various clinical
samples. Micro-
Dx is the first
product combining walk-away auto-
mated human DNA removal and patho-
gen DNA extraction with broad-range
rDNA Real-Time PCR and sequencing
into a rapid diagnostic system for bac-
teria 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 Select-
NAplus instrument, which saves tedious
manual handling and time. At the end of
the procedure an exact differentiation of
the species is obtained. Micro-Dx oper-
ates 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.
MOLZYM
www.cli-online.com & search 27261
Compact hematology system
The DxH 500 hema-
tology system with
CE Mark is an open-
vial instrument oer-
ing a throughput of
up to 60 samples per
hour. It is the rst
analyser in a new
range of workow-
ecient hematology analysers able to deliver
accurate, robust results from a nger prick of
blood. The DxH 500 analyser has been spe-
cically designed for low-volume hematol-
ogy workloads and to promote rapid speci-
men 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.
31
Zika Virus real time PCR
detection kit
The growing
concern about
the prolifera-
tion of Zika
infections in
the Caribbean,
Central and South America, has turned the
ght against the virus in a global health emer-
gency. The infection is mainly transmitted
through the bite of the Aedes spp mosqui-
toes and presents symptoms like mild fever,
arthralgia, myalgia, asthenia, headache and
maculopapular rash clinical symptoms, plus
additional symptoms like conjunctivitis, retro-
orbital pain, lymphadenopathy and diarrhea.
There is widespread concern about the asso-
ciation of the Zika Virus with increasing cases
of congenital microcephaly. Other research
indicate that the virus can cause brain dam-
age. 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 nec-
essary 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 recom-
mended 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
This makes the DxH 500 ideal for pediatric
and geriatric patients, for whom sample tak-
ing can be dicult. It is part of Beckman
Coulters line of DxH hematology instru-
ments (the DxH Workcell, DxH 800 Cel-
lular Analysis System and DxH Slidemaker
Stainer) incorporating the companys multi-
dimensional, high-denition ow cytomet-
ric technology. As part of the multi-site clin-
ical reliability study to test its performance,
uptime and workow eciencies, 36,000
samples were run across 26 sites in ve con-
tinents. 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 addi-
tional 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 infec-
tion. This test is intended for use as an aid in
the diagnosis of the Zika virus in humans in
combination with clinical and epidemiologi-
cal risk factors. RNA is extracted from speci-
mens, amplied using RT-amplication and
detected using uorescent reporter dye probes
specic for Zika virus.
CERTEST BIOTEC
www.cli-online.com & search 27238
New PTH Control for QC Portfolio
Randox Qual-
ity Control
announces
a further
expansion to
their compre-
hensive 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 consist-
ency for this notoriously unstable assay.
RANDOX
www.cli-online.com & search 27266
lighting replacing traditional lasers. The
DxH 500 uses 50% less reagent volume per
sample compared to other low-volume ana-
lysers 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 support-
ing 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 compli-
ance standards. Additionally, the DxH 500
supports laboratories paperless eorts with
a bidirectional laboratory information sys-
tem (LIS) interface for better data keeping.
This integrated LIS interface can potentially
help reduce data errors that occur during
manual processes.
BECKMAN COULTER
www.cli-online.com & search 27263
 April/May 2016
Meningitis/encephalitis panel
BioFire Diag-
nostics FilmAr-
ray Meningitis/
Encephalitis (ME)
Panel is now avail-
able in the coun-
tries 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 dif-
cult 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 Administra-
tion (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 Cen-
taur 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 dis-
ease. The ADVIA Centaur anti-CCP assay is
for use in the semi-quantitative determina-
tion of the IgG class of autoantibodies spe-
cic 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 anti-
bodies, 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 diag-
nosis. With a clinical sensitivity and speci-
city of 98.3% and 99.7% respectively, it
32 PRODUCT NEWS
brings a unique opportunity to test simul-
taneously and rapidly for most bacteria,
viruses and fungi found in those patholo-
gies that can be extremely severe and
sometimes lethal. Such an approach will
positively impact the management of those
patients by helping clinicians and biolo-
gists speed the diagnosis of these poten-
tially 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
www.cli-online.com & search 27264
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
www.cli-online.com & search 27255
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), anti-
neutrophil antibodies
(ANCA) and now also Crithidia luciliae
(CLIFT), EUROPLUS antigens and cell-
based assays (e.g. anti-neuronal antibod-
ies). The EUROPattern system consists of
a fully automated microscope with slide
magazine and advanced diagnostic soft-
ware for rapid recording, interpretation
and archiving of IFT images. Up to 500
analyses can be processed in 2.5 hours, cor-
responding to 18 seconds per eld. Slides
are correctly identied by means of matrix
codes. The controlled LED in the micro-
scope provides over 50,000 hours of con-
stant light intensity, ensuring highly repro-
ducible results. Positive and negative results
for the substrates are clearly dierentiated
by the powerful software. Crithidiae evalu-
ation is based on specic kinetoplast uo-
rescence rather than just dark-light clas-
sication, increasing reliability. Dierent
ANA, anti-cytoplasmic and ANCA pat-
terns are reliably identied, even if more
than one antibody is present. Furthermore,
the software provides titre designations
with condence values. Images from fur-
ther 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 soft-
ware 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 labora-
tory routine.
EUROIMMUN
www.cli-online.com & search 27259
Hemoglobin meter
QDx Hemostat is a
compact handheld
POC hemoglobin
meter for measur-
ing hemoglobin
from a nger prick
of whole blood. This hemoglobin measur-
ing system is intended to help people man-
age their hemoglobin levels. It also provides
healthcare professionals with helpful infor-
mation by measuring hemoglobin in fresh
capillary whole blood as well as venous
blood. Calibration is done by simply insert-
ing the code key into the test meter. The vir-
tually 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 hemato-
crit 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
www.cli-online.com & search 27258
 PRODUCT NEWS 33 April/May 2016

Automated urinalysis tailored to the laboratorys workload

The Iris iRI-
CELL workcell
integrates urine
chemistry and
microscopy into
a fully automated
walk-away uri-
nalysis 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, clini-
cally relevant urine particle images
that are auto-classified for more objec-
tive and consistent results. This auto-
mated microscopy system is designed
for all volume workloads, delivering
a shorter turnaround time and stand-
ardizing results. The iQ200 Series are
available either as a stand-alone sys-
tem or connected to an iChemVE-
LOCITY urine chemistry analyser to
form an automated iRICELL workcell.
The iQ200SPRINT is one of the fast-
est automated systems on the market,
meeting high-volume productivity and
workload requirements. It can han-
dle 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 low-
volume workloads running 40 micro-
scopic samples an hour. iChemVE-
LOCITY provides a fully automated,
high-capacity urine chemistry analy-
sis with excellent low-end sensitivity.
The system delivers a high throughput
of 210 samples an hour, with the con-
tinuous 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 pro-
vides clinically significant information
about potential interference with key
chemistry assays. The system evaluates
all standard urine chemistry parame-
ters, including glucose, protein, biliru-
bin, urobilinogen, pH, specific gravity,
blood, ketones, nitrite and leukocyte
esterase.
BECKMAN COULTER
www.cli-online.com & search 27267
Utility of reex urine culture based
on results of urinalysis and auto-
mated microscopy
Specimens submitted for urine culture
in hospital settings are frequently nega-
tive 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 con-
cern for cost containment, utilization of a
reex testing policy using specic screen-
ing criteria would be benecial to eliminate
unnecessary urine cultures. With this in
mind, an evaluation of Beckman Coulters
IRIS iQ200 system (urinalysis and auto-
mated urine microscopy) was carried out
in the US at the Johns Hopkins Bayview
Medical Center (JHBMC) clinical micro-
biology laboratories. The study prospec-
tively 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 break- centrifugation. Unlike in tubes where liq-
down of glucose uid is added, the nely granulated additive May 21-24, 2016 September 25-
(glycolysis) in does not cause a dilution eect. There is no European Human 30, 2016
venous blood need to convert the measurement result. Genetics Conference European Con-
samples is very The citrate/citric acid buer reduces the pH 2016 (ESHG 2016) gress of Pathology
signicant for the value in the sample. As a result, the enzymes Barcelona, Spain Cologne, Ger-
diagnosis of both diabetes mellitus and ges- needed for the glycolysis process are inhib- www.eshg.org/ many
tational diabetes which should be detected at ited and the actual in vivo level is stabilized home2016.0.html www.esp-con-
an early stage to avoid complications such as from the start. The additive is completely dis- gress.org/2016
infections, premature births and long-term solved, and therefore optimally mixed with
eects for the mother and child. In order the sample, after swivelling ten times. In the June 14-17, 2016
to have a reliable diagnosis, it is necessary case of storage between 4C and room tem- ESGAR 2016 September 29-1
to inhibit glucose breakdown immediately perature, a further sodium uoride additive Prague October, 2016
after collecting blood. Various institutions ensures long-term stabilization for 48 hours. Czech Republic British Society for
have drafted guidelines, which recommend The VACUETTE FC Mix tube is available www.esgar.org Allergy & Clinical
the addition of a citrate-uoride additive to with both a grey and pink security cap and Immunology
maintain the in vivo glucose level. The spe- therefore allows for dierentiation from Telford, Shrop-
cial feature of the new VACUETTE FC Mix standard glucose tubes. The cap is particu- June 21-23, 2016 shire, UK
tube from Greiner Bio-One is the powder larly easy to open and allows for hygienic JIB Journes www.bsaci.org
additive. It stabilizes the glucose level imme- working in the laboratory. The VACUETTE Franaises de
diately after collection for 48 hours. This FC Mix tube is made of highly-transparent Biologie Location and date
allows for reliable diagnosis of diabetes con- PET plastic and is shatter-proof. Paris, France TBC
ditions and avoids false negatives. The sta- www.jib-sdbio.fr/ CMEF Autumn
bilization is carried out in the whole blood GREINER BIOONE 2016
and therefore does not require immediate www.cli-online.com & search 27257 www.cmef.com.
July 31-August 4, cn/g1250.aspx
2016
Walk-away 25-OH vitamin D3 samples fully automatically without AACC
D2/D3 UHPLC analyser any human intervention - from primer Atlanta, GA, USA November 14-17,
Zivak Technologies sample tube. The company offers higher www.aacc.org 2016
supplies ready to sensitivity than entry level LC- MS/MS MEDICA
use LC-MS/MS and thanks to the newly designed Special D Dsseldorf,
HPLC analysis kits detector. This system is fully validated September Germany
with its own fully with the Zivak vitamin D2/D3 UHPLC 12-15, 2016 www.medica.de
automated sample analysis kit which includes all necessary MSACL EU
preparation and injection system which reagents, calibrator and controls. It offers Salzburg, Austria
enables laboratories to make efficient a complete solution to clinical labora- www.msacl.org January 23-26,
use of their LC-MS/MS as well as HPLC tories performing routine vitamin D2/ 2017
instruments. Many scientific studies and D3 assays on HPLC-UV and LC-MS/MS September MEDLAB at Arab
papers show the inaccuracy of measuring systems. As they have a large number of 14-17, 2016 Health
total vitamin D with commercial immu- vitamin D2/D3 samples, they cannot run 19th Annual Dubai, UAE
noassays without separating and measur- all the other LC-MS/MS specific analyses ESCV Meeting www.arab-
ing vitamin D2 and vitamin D3 metabo- on their LC-MS/MS systems effectively. Lisbon, Portugal healthonline.com
lites individually. HPLC and LC-MS/ The VD-200 has been designed especially www.escv2016.com
MS methods are accepted as the Gold for this kind of laboratory, providing a
standard for separate analysis of 25-OH cost-effective and fully automated system October 22-25,
metabolites of vitamin D. Usage of these that frees expensive LC-MS/MS systems September 21- 2017
chromatographic methods is increasing for specific tests which have to be run 24, 2016 IFCC WorldLab
significantly in clinical laboratories in on LC-MS/MS systems. The innovative EFLM-UEMS Durban 2017
recent years. Many of these laboratories design of the VD-200 enables barcode Congress Durban,
cannot use their systems effectively due to reading, reagent adding, vortex mixing, Warsaw, Poland South Africa
the fact that these systems require quali- centrifuge and injection processes to be www.em-uems. www.dur-
fied staff, complex sample preparation done by robotic arms. The system pro- warsaw2016.eu ban2017.org
steps, high kit prices/ operation costs and vides 240 accurate results in 12 hours
do not allow walk-away operation. The fully automatically.
For more events see:
VD-200 was especially designed for rou- www.cli-online.com/events/
tine vitamin D2/D3 testing clinical labs. ZIVAK Dates and descriptions of future events have been obtained
VD-200 enables to analyse vitamin D2/ from ofcial industrial sources. CLi cannot be held
www.cli-online.com & search 27256 responsible for errors, changes or cancellations.
www.cli-online.com & search 27241
 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 www.cli-online.com & search 27253