14th EFLM Continuous Postgraduate Course in Clinical

Chemistry and Laboratory Medicine:

“New trends in laboratory diagnosis and management of diabetes mellitus:
Diabetes mellitus revisited 14 years after the first Dubrovnik course”

Inter-University Centre Dubrovnik,

Dubrovnik,
Croatia

October 25-26, 2014

Organizers:
Croatian Society of Medical Biochemistry and Laboratory Medicine (CSMBLM)
European Federation of Clinical Chemistry and Laboratory Medicine (EFLM)
Slovenian Association for Clinical Chemistry (SZKK)
Inter-University Centre Dubrovnik (IUC)

Scientific and Organizing Committee:

Elizabeta Topic (chair), Croatia
Sverre Sandberg, Norway
Evgenija Homsak, Slovenia
Jasna Lenicek Krleza, Croatia
Pika Mesko Brguljan, Slovenia
Greta Strakl, Slovenia
Marina Njire Braticevic, Croatia

Course secretary:
Jasna Đogić
8th CSMBLM Congress
RIJEKA, CROATIA Hrvatsko društvo za medicinsku
biokemiju i laboratorijsku medicinu
HKD Conference Centre Croatian society of medical
biochemistry and laboratory medicine
22-26 September 2015

First announcement
Dear colleagues and friends,
We are happy to invite you to the 8th Congress of the Croatian Society of
Medical Biochemistry and Laboratory Medicine with International
Participation that will take place at the HKD Conference Centre (Hrvatski
kulturni dom na Sušaku) in Rijeka, J.J. Strossmayer Street 1, from 22 to 26
September 2015.
We are looking forward to seeing you next fall at the Congress!
Welcome to Rijeka!

HDMBLM
Congress organizers

Scientific Committee Organizing Committee

Lidija Biliæ-Zulle, chair Koraljka Mittel, chair

Karmela Barišiæ Dragana Antonèiæ
Lorena Honoviæ Merica Aralica
Nora Nikolac Vedrana Drvar
Daria Pašaliæ Nora Nikolac
Dunja Rogiæ Snježana Hrabriæ Vlah
Ana-Maria Šimundiæ Vesna Šupak Smolèiæ
Anðela Žic

info.Rijeka2015@hdmblm.hr
www.hdmblm.hr
 14th EFLM Continuous Postgraduate Course in Clinical Chemistry and Laboratory Medicine

Contents
Welcome note. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . S5

Lectures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . S7

Diabetes epidemics - classification and prevalence. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . S7

Grazyna Sypniewska
Type 2 diabetes and cardiovascular diseases: How biomarkers can support more tailored based approaches?. . . . . . . . . . . . . . . . . . . . . . . . . . . . . S11
Damien Gruson
The role of diabetes registries to monitor the treatment and complications of diabetes. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . S13
John A McKnight
Guidelines and recommendations for testing in diagnosis of DM: The role of HBA1c . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . S17
Elizabeta Topić
HbA1c analysing – challenges for the laboratory – internal and external QC. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . S21
Gunnar Nordin
Post-analytical factors – how should HbA1c results be communicated to clinicians?. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . S22
Andrea Mosca
Early recognition of gestational diabetes (Introduction of new guidelines and practice) – how should the routines be?. . . . . . . . . . . . . . . . . . . . S26
Andrej Zavratnik
How to diagnose the prediabetes?. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . S31
Grazyna Sypniewska
Diabetes as autoimmune disease - Diabetes type I . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . S35
Evgenija Homsak
POC testing instruments for diagnosing and monitoring diabetes in clinical settings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . S39
Sverre Sandberg
The impact of preanalytical factors on glucose concentration measurement. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . S41
Nora Nikolac
Self measurement of glucose – how useful is it and how can it be done. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . S45
Sverre Sandberg
Can glycated albumin assist in management of diabetes mellitus?. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . S47
Jasna Lenicek Krleza
Pro and Contra of Incretin therapy in Type 2 diabetes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . S53
Dario Rahelić
The practical issues in Type 2 diabetes management - pharmacogenomic consideration. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . S53
Sabina Semiz

Posters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . S57

P1- Quality improvement in point-of-care blood glucose testing with Cobas IT 1000. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . S57
Čeri A, Baršić I, Rogić D
P2 - Results of external quality control of HbA1c in Croatian medical laboratories indicated that POCT system differ from
immunoassay methods. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . S57
Hrabric Vlah S, Antoncic D, Grzunov A, Dobrijevic S,Vlasic-Tanaskovic J, Bilic-Zulle L, Lenicek Krleza J
P3 - Hemolysis effect on coagulation test results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . S58
Coen Herak D, Dolčić M, Vogrinc Ž
P4 - OGTT - National survey. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . S58
Gorenjak M, Jevšnikar B
P5 - Urinary protein analysis in diabetic patients . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . S59
Kostovska I, Cekovska S, Tosheska Trajkovska K, Labudovik D
P6 - Oxidized proteins and selenium in patients with type 2 diabetes. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . S60
Kozic I, Bakula M, Grubic P, Rumenjak V
P7 - Hyperglycaemia in children with cancer: Two case reports. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . S60
Lenicek Krleza J, Kozaj S, Grzunov A, Rajčić A, Stepan Giljević J, Jakovljević G

Biochemia Medica 2014;24(Suppl 1):S1-S78

S1
 European Commission
Joint Research Centre

Institute for Reference

Materials and Measurements

1st EFLM Strategic Conference

8th CIRME International Scientific Meeting

with the
auspices of

GENERAL INFORMATION
REGISTRATION FEE VENUE
EUR 305,00 (VAT 22% included) Atahotel Executive
Viale Luigi Sturzo, 45 - 20154 Milano, Italy
The registration fee includes:
Located in a strategic and privileged position, close to the Porta
Coffee break & lunch buffet as indicated in the programme
Garibaldi Railway Station and in the heart of Milan's nightlife
Certificate of participation
(Corso Como and Brera area). Well connected to public
Cancellations: transports, the underground stations (M2 Green line and M5 Lilac
- registrations cancelled within August 30, 2014 will result in a line) are only few steps from the hotel.
20% penalty For more information, please visit:
- cancellations between August 30 and September 30, 2014 will http://www.atahotels.it/en/executive
be subject to a 50% penalty
ACCOMMODATION
- afterwards, registrations will result in a 100% penalty The following hotels are all located walking distance from the
To make your registration, please access the following link: congress venue. To book your room please refer to the below
http://reg.mzcongressi.com/cmsweb/elenco.asp?oIDEvento=681&Lang=EN indicated hotel reservation system.
OFFICIAL LANGUAGE  c/o Atahotel Executive (conference venue)
The official language of the conference is English. http://www.atahotels.it/en/executive

ORGANIZING SECRETARIAT  c/o UNA Toqc Hotel (200 meters from the congress venue)
MZ Congressi srl http://www.unahotels.it/it/una_hotel_tocq/hotel_milano_corso_como.htm
Via Carlo Farini, 81 - 20159 Milano - ITALY  c/o Hotel AC Milano (500 meters from the congress venue)
Tel: +39 0266802323 ext 917 http://www.marriott.com/hotels/travel/milmi-ac-hotel-milano/
Ms Patrizia Sirtori  c/o Holiday Inn (700 meters from the congress venue)
e-mail: patrizia.sirtori@mzcongressi.com http://www.himilangaribaldi.com/en/

EFLM thanks the following companies for the kind and unconditional support
 14th EFLM Continuous Postgraduate Course in Clinical Chemistry and Laboratory Medicine

P8 - Urinary patterns analysis of type 2 diabetes mellitus patients by nuclear magnetic resonance spectroscopy method (1H-NMR) . . . . . . . S61
Stefan LI, Nicolescu A, Sandu M, Popa GS, Moţa M, Deleanu C
P9 - Urinary biochemistry of type 1 diabetes mellitus patients using Proton Nuclear Magnetic Resonance Spectroscopy Method (1H-NMR). . . . S62
Stefan LI, Nicolescu A, Sandu M, Popa GS, Moţa M, Deleanu C
P10 - Plasma cholinesterase method comparison. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . S63
Finderle P, Snoj N
P11 - Is there a role for HbA1c in diagnosing gestational diabetes in high-risk pregnancies? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . S63
Krhač M, Radišić Biljak V, Radeljak A, Božičević S, Prašek M, Vučić Lovrenčić M
P12 - Antioxidative Effect of Naringenin on the Activity of Superoxide Dismutase and Glutathione Peroxidase in HepG2 Cells under
Hyperglycaemic Conditions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . S64
Knežević T, Petlevski R
P13 - Performance of equations for estimating glomerular filtration rate in diabetic patients. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . S65
Šimetić L, Drmić S, Zibar L, Begić I, Šerić V
P14 - Serum Omentin-1 Level in Diabetic Patients on Haemodialysis: a pilot study . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . S65
Kocijancic M, Vujicic B, Racki S, Zaputovic L, Dvornik S
P15 - How well are the patients in Croatia informed about the OGTT procedure?. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . S66
Banković Radovanović P, Kocijančić M
P16 - Hb1Ac comparıson and evaluatıon ın patıents wıth cardıovascular dıseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . S67
Isiksacan N, Koser M
P17 - Evaluation of the analytical quality of B-ANALYST as a POCT analyzer for HbA1c determination. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . S67
Menéndez-Valladares P, Fernandez-Riejos P, Sanchez-Mora C, Perez-Perez A, Sanchez-Margalet V, Gonzalez-Rodriguez C
P18 - Application of the guidelines for the request of microalbuminuria in diabetic patients . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . S68
Lukic V
P19 - Plasma glucose concentration, Hba1c together in assessing stability of diabetes. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . S69
Nake A, Hatibi S
P20 - Dried Blood Spot: Could potassium solve hematocrit issue?. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . S69
Bilandžija I, Fumić K
P21 - Association of serum nesfatin-1/NUCB2 with metabolic risk factors in non-obese, normoglycemic subjects. . . . . . . . . . . . . . . . . . . . . . . . . . S70
Bergmann K, Olender K, Kretowicz M, Manitius J, Sypniewska G
P22 - Importance of the analysis of the fructosamine 3-kinase gene promoter region: experience in an Italian cohort of diabetic patients. . . . S70
Avemaria F, Carrera P, Lapolla A, Ferrari M, Mosca A
P23 - Evaluation of the performance of an immunoturbidimetric HbA1c reagent applied to the Siemens ADVIA 2400 automatic analyzer. . . . S71
Barbaro M, Carobene A, Ku-Chulim C, Cochrane R, Passerini G, Ceriotti F
P24 - Validation of five point-of-care glucometers. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . S72
Ćelap I, Milevoj Kopčinović L, Kosovec V, Grabusin P, Begčević I, Vrkić N
P25 - Estimation of the detection of gestational diabetes mellitus - do we need to resign from 1h50g load?. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . S73
Grudzień U, Panek A, Kozłowska D, Swadźba J
P26 - User verification for HbA1c on AU 480 Beckman Coulter . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . S73
Sablek J, Todorić Z, Sablek Lj
P27 - Low-grade inflammatory state in pregnancy complicated by diabetes. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . S74
Panek A, Grudzień U, Kozłowska D, Swadźba J
P28 - Correlation between two analytical method of HbA1c measuring in aligned and misaligned diabetic patients. . . . . . . . . . . . . . . . . . . . . . . S74
Stec K, Grudzień U, Panek A, Kozłowska D, Swadźba J
P29 - Testing paracetamol interference with glucose analysis in serum . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . S75
Trifunović J
P30 - Determination of potential urine biomarkers of kidney disease by LC-MS / MS. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . S76
Vojtova L, Přikryl P, Hruškova Z, Vokurka M,Tesař V, Zima T
P31 - Demand management of hemoglobin A1c testing according to the American Diabetes Association’s Standards of Care 2014 . . . . . . . . S76
Varo Sánchez GM, Munoz Calero M, Salas Herrero E, Montilla Lopez C
P32 - Activities of superoxide dismutase and glutathione peroxidase in plasma of patients with Balkan endemic nephropathy. . . . . . . . . . . . S77
Gnjidic T, Pavlovic D, Radic T, Coric V, Simic T, Savic-Radojevic A
P33 - Pharmacovigilance - case report. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . S77
Romić M

Biochemia Medica 2014;24(Suppl 1):S1-S78

S3
 14th EFLM Continuous Postgraduate Course in Clinical Chemistry and Laboratory Medicine
Welcome note
14th EFLM Continuous Postgraduate
Course in Clinical Chemistry and
Laboratory Medicine:
“New trends in laboratory diagnosis
and management of diabetes mellitus:
Diabetes mellitus revisited 14 years after
the first Dubrovnik course”
In 2001, the EFLM launched the postgraduate sci-
entific courses of continuing education in clinical
chemistry and laboratory medicine, organized
yearly at Inter University Centre Dubrovnik for
EFLM member societies. These Courses, hosted by
Croatian Society of Medical Biochemistry and Lab-
oratory Medicine (CSMBLM) and Slovenian Associ-
ation of Clinical Chemistry and Laboratory Medi-
cine (SACCLM) presents continuation of postgrad-
uate courses aimed for Continuing Professional
Development in clinical chemistry and laboratory
medicine.
The common title of all the courses is EFLM Con-
tinuous Postgraduate Courses in Clinical Chemis-
try and Laboratory Medicine: New trends in Classi-
fication, Diagnosis and Management, each of
them dedicated to a particular medical entity such
as Diabetes Mellitus (2001), Cardiovascular Diseas-
es (2002), Neurological Diseases (2003), Tumor Dis-
eases (2004), Autoimmune Diseases (2005), Meta-
bolic Syndrome (2006), Molecular Diagnosis (2007),
and Kidney Diseases (2008) Thyroid diseases
(2009), Thrombophilia (2010), Infection (2011), Gas-
trointestinal diseases (2012) and POC testing and
monitoring (2013).
The subject of 14th EFLM Continuing Postgraduate
Course in Clinical Chemistry and Laboratory Medi-
cine “New trends in laboratory diagnosis and man-
agement of diabetes mellitus: Diabetes mellitus
revisited 14 years after the first Dubrovnik course”
is dedicated to diabetes mellitus similar to the first
organized course, and will bring new information
documenting the progress in the field of diabetes
mellitus after almost one and a half decade.
S5
The Scientific and Organizing Committee have
done a great job in gathering recognized interna-
tional speakers in the field, who  will present the
latest news in their field.
The Scientific Program will discuss many new as-
pects in diagnosis of type I and type II diabetes as
well as early markers of pre-diabetes and diabetic
complications.
We draw your particular attention to the session
dealing with new guidelines and recommendation
for HbA1c testing in diagnosis and control of dia-
betes as well as how HbA1c results should be com-
municated to  clinicians and how EQAS results
should be communicated to laboratories.
Analytical consideration will be discussed through
the lectures such as POC testing, Instruments for
diagnosing and monitoring diabetes in clinical set-
tings , The impact of preanalytic factors of glucose
measurement, Self measurement of glucose – how
useful is it and how can it be done . Disease man-
agement topics are Can glycated albumin assist in
management of diabetes mellitus? and Pro and
Contra of Incretin therapy in Type 2 diabetes, Prac-
tical issues in Type 2 diabetes management - phar-
macogenomic consideration will be elaborated.
In conclusion: One of the most important aspects
of this event is that it brings together some of Eu-
rope’s specialist in laboratory medicine as well as
physicians focused on Diabetes Mellitus and its
complications. Thus, this is an opportunity to lis-
ten and discuss with experts in the field. In addi-
tion the poster presentations of young scientist
will be given ample spare and will reflect their
contribution to diabetic care across Europe.  We
are proud that bursaries were available for
young (<40) course participants.
The Course is credited for Continuing Professional
Development  by Croatian, Slovenian and Serbian
professional Chamber and certified with hours of
lectures by Inter University Centre Dubrovnik.
Elizabeta Topic,
Scientific and Organising Committee Chair
Biochemia Medica 2014;24(Suppl 1):S1-S78
In 2001, the EFLM launched the postgraduate scientific courses of continuing education in clinical chemistry
and laboratory medicine and subject of them was diabetes mellitus.
Lectures
Diabetes epidemics - classification and
prevalence
Grazyna Sypniewska
Department of Laboratory Medicine, Collegium Medicum,
Nicolaus Copernicus University, Bydgoszcz, Poland
Corresponding author: grazynaodes@interia.pl
Classification of diabetes
Diabetes mellitus is a chronic metabolic disease
characterized by the presence of hyperglycaemia
that occurs due to defective insulin production
and secretion by the pancreas, defective insulin
action resulting from no response of the cells to in-
sulin or both. High blood glucose leads to typical
clinical symptoms: weight loss, polyuria, increased
thirst, weakness. According to the American Dia-
betes Association (ADA) there are four main types
of diabetes mellitus: type 1, type 2, gestational dia-
betes mellitus and other specific types.
Type 1 diabetes (T1D), named immune-mediated
diabetes, account for 5-10% of all diabetic cases, oc-
curs more commonly in the younger persons and
results from an immune-mediated depletion of
pancreatic beta-cells with very sudden onset, espe-
cially in infants and children, that causes lifelong de-
pendence on exogenous insulin (1). This form of dia-
betes was previously referred to as “insulin-depend-
ent juvenile diabetes mellitus” due to more frequent
diagnosis in children. However, according to recent
ADA position statement the majority of individuals
with T1D are adults (1). T1D is traditionally diag-
nosed based on clinical symptoms suggestive of in-
sulin deficiency: polyuria, increased thirst, tiredness,
constant hunger, sudden decrease of weight, slow-
healing wounds, recurrent infections, blurred vision
and hyperglycemia resistant to oral hypoglycemic
agents. Severe insulinopenia predisposes patients
with T1D to diabetic ketoacidosis which may be the
first manifestation of the disease. The characteristic
of T1D are five different pancreatic autoantibodies :
anti-glutamic acid decarboxylase antibodies (GADA
or GAD65), islet cell antibodies (ICA), insulin autoan-
tibodies (IAA), protein tyrosine phoshphatases anti-
S7
bodies (IA-2A and IA-2β) and zinc transporter pro-
tein (ZnT8) (1,2). In 85-90% of individuals one or
more type of these antibodies are present at diag-
nosis of fasting hyperglycemia. This type of diabe-
tes has multiple genetic predispositions and is relat-
ed to not yet defined environmental factors. Indi-
viduals with T1D are also prone to other disorders of
autoimmune background. Some forms of T1D may
be idiopathic without autoimmune background.
Individuals with type 2 diabetes are characterized
by a spectrum of disturbances from „predominantly
insulin resistance with relative insulin deficiency to
predominantly an insulin secretory defect with in-
sulin resistance” (2). There are several important risk
factors associated with the development of type 2
diabetes (T2D) including obesity, poor diet, low
physical activity, advancing age, family history of di-
abetes, ethnicity, prior high blood glucose in preg-
nancy, dyslipidemia and hypertension. Most cases
with T2D have insulin resistance (with concomittant
hypertriglyceridemia and low HDL-cholesterol) and
concurrent insulin deficiency and glucagon excess.
T2D can go unnoticed and undiagnosed for years.
Those affected by T2D are unaware of the long-
term damage being caused by the disease. As a re-
sult up to 25% of patients with T2D have already de-
veloped one or more microvascular complications
by the time of diagnosis. T2D accounts for approx.
90-95% of all diabetic cases. There are more than
sixty distinct genetic disorders associated with glu-
cose intolerance or diabetes but substantial com-
plex genetic factors which contribute to risk of type
2 diabetes are not defined yet. In spite of that rou-
tine genetic testing in T2D is not recommended.
In the recent observational study the long-term
outcomes were compared in type 1 and 2 diabetic
patients in Australia who had disease onset be-
tween 15 and 30 years of age (2). The two groups
differed significantly on important characteristics
such as age at onset, diabetes duration, and ethnic-
ity. In the type 2 diabetes cohort significantly high-
er concentrations of serum triglycerides, lower HDL-
cholesterol and higher blood pressure were found
which may support the notion that T2D is by nature
a cardiovascular condition. Macrovascular compli-
cations were much more common in the T2D co-
hort (ischemic heart disease and stroke), but there
Biochemia Medica 2014;24(Suppl 1):S1-S78
14th EFLM Continuous Postgraduate Course in Clinical Chemistry and Laboratory Medicine
was no difference in retinopathy or nephropathy.
Death was also more common in patients with T2D
and occurred after shorter disease duration (3).
Third main type is gestational diabetes (GDM)
which occurs around the 24th week of pregnancy
when women without a previous diagnosis of dia-
betes develop insulin-resistance and subsequent
hyperglycaemia or glucose intolerance. This can
lead to serious risks to the mothers and their infants
(macrosomia, neonatal hypoglycemia, increased
risk of perinatal mortality) and increase the risk for
developing T2D 5-10 years later. In 50-60% of cases,
however, glucose metabolism returns to normal af-
ter delivery. The risk to babies is more severe for
those whose mother had type 1 or type 2 diabetes
before pregnancy, a condition named diabetes in
pregnancy, which is substantially distinct from
GDM. GDM accounts for up to 4% of diabetic cases.
The other specific types of diabetes include genet-
ic defects of pancreatic β-cell function, endo-
crinopathies, drug induced and infection related.
Maturity onset diabetes of the young (MODY) has
a genetic background with mutations in HNF-1A-
hepatic nuclear transcription factor (MODY 3),
HNF-4B (MODY 1) or glucokinase genes (MODY 2)
as well as transient or permanent neonatal diabe-
tes diagnosed in infants before 6 months of life. In
the latter the diagnosis of genetic mutations may
be valuable. Other genetic defects may lead to dis-
turbed insulin action. Diabetes may also occur due
to point mutations in mitochondrial DNA (2).
Pancreatitis, trauma, infection or cancer that can
damage the pancreas can also cause diabetes.
Other factors which may cause the development
of diabetes are endocrinopathies (Cushing syn-
drome, acromegaly, phaeochromocytoma, gluca-
gonoma and hyperthyroidism), pharmacotherapy
(nicotinic acid, glucocorticoids, thyroid hormones,
tiazides) or toxic chemicals and infections (Rubella,
cytomegalovirus).
Among uncommon forms of immune-mediated
diabetes the presence of anti-insulin receptor anti-
bodies which block insulin binding to the cell re-
ceptors or act as insulin agonists should be noted.
These antibodies occur in individuals with other
autoimmune diseases (2).
Biochemia Medica 2014;24(Suppl 1):S1-S78
S8
All types of diabetes should be treated in order to
prevent long-term micro- and macrovascular com-
plications such as: retinopathy, nephropathy, neu-
ropathy and cardiovascular diseases (ischemic
heart disease, stroke) to prevent or limit mortality.
Prediabetes is the term used to describe people
with impaired fasting glucose (IFG) or impaired
glucose tolerance (IGT). Subjects with IGT are at in-
creased risk of developing T2D but evidence sup-
ports the effectiveness of lifestyle changes to pre-
vent diabetes (4).
Prevalence of diabetes
Diabetes is one of the fastest-growing health prob-
lem in the world, reaching epidemic proportion in
some regions, as a consequence of life-style, lack
of exercise, unhealthy diet, obesity and over-
weight. The estimated health costs are enormous
reaching in 2013 almost 11% of the total worldwide
health budget. The global prevalence of diabetes
in adult population (20-79 years old) in 2013 is esti-
mated as 8.3% (382 million people) [5]. According
to these estimates the region with the higher prev-
alence of 11% is North America and the Caribbean
followed by the Middle East and North Africa with
the prevalence of 9.2% and Western Pacific regions
with 8.6% that is close to the world prevalence.
There are three countries with the estimated prev-
alence of diabetes over 30% : Tokelau (37.5%), Fed-
erated States of Micronesia (35%) and Marshall Is-
lands (34.9%) followed by at least seven countries
with the estimated prevalence of diabetes over
20% including Saudi Arabia (23.9%), Kuwait (23.1%)
and Qatar (22.9%). Of the total 219 countries 16%,
located mainly in Western Pacific, Middle East and
North Africa regions have very high prevalence of
diabetes, over 12% (4).
In spite of the fact, that North Africa and three other
African countries have high diabetes prevalence
over 10%, this is the region with the lower estimated
prevalence (4.9%). Mali, a small Middle-West African
country with a population of 15 millions, has the low-
est prevalence of diabetes of 1.6%. These numbers
indicate that geographic distribution of the preva-
lence of diabetes mellitus is very heterogeneous.
 14th EFLM Continuous Postgraduate Course in Clinical Chemistry and Laboratory Medicine
Europe has the 8.5% prevalence, similar to the
global, having Turkey in upper extreme with
14.9%, Montenegro with 10.1%, Serbia 9.9% and
Bosnia Hercegovina with 9.7% of prevalence. The
lowest prevalence of diabetes in Europe is in Nor-
way, Sweden, United Kingdom (4-5%) followed by
Croatia, Finland, France, Greece and Poland (5-6%).
According to newly released data from the UK Na-
tional Diabetes Audit (July 2014), each day 738
people are being diagnosed with type 2 diabetes,
among them 30 people with T1DM.
The North America and Caribbean region not only
show the highest average prevalence of diabetes
but also the highest prevalence of impaired glu-
cose tolerance (IGT) with a median of 12%. Global-
ly, countries with high prevalence of diabetes tend
to have higher IGT prevalence.
The new estimates show an increasing and worri-
some trend towards younger generations develop-
ing diabetes and possible ~50% increase in its preva-
lence within next generation (5). The burden of dia-
betes is reflected in the growing number of prema-
ture deaths due to diabetes. In 2013 almost 50% of all
deaths due to diabetes were noted in people under
the age of 60. The greatest unfavourable changes
are expected in the developing countries due to in-
creasing life expectancy and rapid life style changes.
Trends in the prevalence of type 2 and type
1 diabetes in the United States.
The prevalence of diabetes in the United States has
increased substantially over the last two decades,
paralleling an increase in the prevalence of obesity
(6). The prevalence of confirmed diabetes in US in-
creased from 6.2% in 1988 to 9.9% in 2010 but si-
multaneously from 1994 to 2005 the prevalence of
undiagnosed diabetes, defined by either HbA1c or
fasting glucose, decreased from 16% to 11%.
The prevalence of type 2 diabetes in all major ra-
cial/ethnic groups of American teenagers, has enor-
mously increased by 35% over an 8-year period (7).
The greatest change in T2D were found in Hispanic
children, on the contrary no significant changes
were observed among Asian Pacific Islanders or
American Indians. This trend in T2D reflects the cur-
rent obesity epidemic and has also the long-term
S9
impact of higher gestational diabetes rates. It was
emphasized that some young people with type 2
diabetes have a more aggressive form of the dis-
ease than is seen in adults, with a high risk for com-
plications such as early nephropathy leading to
chronic kidney disease and cardiovascular disease.
The global prevalence of T1D is not known but in
the US reaches approx. 9% (up to 3,3 mlns in youths
aged 0-19 years). The prevalence of paediatric type 1
diabetes in US increased by 30% from 2001 to 2009
and this was mostly seen in white youth however,
the increasing burden of type 1 diabetes is experi-
enced by youth of minority racial/ethnic groups as
well (7). The potential causes may be „lack of certain
viral or bacterial triggers at an early age, changes in
early diet that might negatively affect the develop-
ing gut microenvironment, and increased rates of
obesity in the general population” (7). The incidence
of T1DM varies considerably among countries: East
Asia and American Indians have the lowest inci-
dence rates compared to Finland with the highest
incidence rate (>64.2/100.000 per year, 8-fold high-
er) (1). Worldwide prevalence of T1D increases due to
the rising number of new-onset cases of T1D diag-
nosed in adults, including those diagnosed with
LADA (latent autoimmune diabetes of adults), as
well as to longer lifetime of subjects with childhood-
onset diabetes as a consequence of better care (1).
Recent study from Sweden showed that the inci-
dence of type 1 diabetes among 0-34 years old
was two to three times higher than previously re-
ported (8). Contrary to this, new findings from Fin-
land, which has the highest incidence of T1D in the
world, suggest that it appears to be levelling off
[9]. Since 2006 the overall incidence rate of T1D
which was before 3.6% per year has not increased.
In 2011 the incidence increased only in boys but
continued to decline among girls. Interestingly,
among the factors which might be behind these
changes increased vitamin-D consumption is
mentioned and the possibility that the T1D onset
may have shifted to an older age group.
Trends in incidence of diabetes in pregnancy
The incidence of diabetes in pregnancy (named
pre-GDM) and gestational diabetes is the rapidly-
Biochemia Medica 2014;24(Suppl 1):S1-S78
14th EFLM Continuous Postgraduate Course in Clinical Chemistry and Laboratory Medicine
growing concern. Recent large population-based
study performed in Ontario, Canada compared
several aspects of this issue including the trends in
rates of diabetes in pregnancy over the past 14
years [10]. It was found that the age-adjusted rate
doubled from 1996 to 2010 for GDM (2.7–5.6%,
p<0.001) and for pre-GDM (0.7–1.5%, p<0.001).
Among factors responsible for the increasing rates
of GDM, obesity, decreased physical activity, diet
and increasing prevalence of type 2 diabetes were
listed. The rise of diabetes in pregnancy has also
been found in German and UK studies. Presented
data indicated that in pregnant women >30 years
of age almost 10% had diabetes with the preva-
lence of GDM of 7.4% and pre-GDM of 1.9%.
In the United States the prevalence of GDM seems
to be even higher, up to 9.2%, based on the study
performed between 2007 and 2010 (11).
Hidden diabetes
Recent data published by the Danish authors have
shown that increasing severity of heart failure in
References
1. Chiang JL, Kirkman MS, Laffel RMB, Peters AL. Type 1 Dia-
betes through the lifespan: a position statement of the
American Diabetes Association. Diabetes Care 2014,2034-
2054; DOI: 10.2337/dc14-1140.
2. ADA position statement: diagnosis and classification of
diabetes mellitus. Diabetes Care 2013;36,S1:S67-S74.
3. Constantino MI, Molyneaux L, Limacher-Gisler F, Al-Saeed
A, Luo C, Wu T et al. Long-term Complications and Morta-
lity in Young-Onset Diabetes: Type 2 Diabetes Is More Ha-
zardous and Lethal Than Type 1 Diabetes. Diabetes Care
2013;36:3863-9.
4. Tominaga M, Eguchi H, Manaka H, Igarashi K, Kato T, Seki-
kawa A. Impaired glucose toerance is a risk factor for cardi-
ovascular disease but not impaired fasting glucose.Diabet
Care 1999;22, 920-4.
5. International Diabetes Federation. IDF Diabetes Atlas, 6th
ed. Brussels, Belgium: 2013. Available at: http://www.idf.
org/diabetesatlas Accessed on 28th August 2014.
6. Selvin E, Parrinello CM, Sacks DB, Coresh J. Trends in pre-
valence and control of diabetes in the United States, 1988-
1994 and 1999-2010. Ann Intern Med 2014; 160:517-25.
7. Dabelea D, Mayer-Davis EJ, Saydah S, Imperatore G, Lin-
der B, Divers J et al. Prevalence of type 1 and type 2 diabetes
Biochemia Medica 2014;24(Suppl 1):S1-S78
S10
Danish patients with a mean age of about 75 yrs
was associated with an increasing risk of type 2 di-
abetes [12]. During a 10-year follow-up, patients
with the most severe heart failure were three
times more likely to develop diabetes than pa-
tients with the least severe heart failure. Type 2
diabetes is also a common co morbidity in pa-
tients hospitalized with an acute myocardial in-
farction and sometimes this hospitalization repre-
sents the first opportunity to recognize the dis-
ease.
Conclusion
The enormous increase in the prevalence of diabe-
tes worldwide should shift the focus from improv-
ing treatment to much better diabetes prevention
strategies in the next decades and reducing the
human and health care costs associated with this
condition. More efforts should be put on screen-
ing and earlier diagnosis of diabetes to limit high
risk for heart attack, stroke, blindness, kidney dis-
ease and amputation.
among children and adolescents from 2001 to 2009. JAMA
2014;311:1778-86.
8. Rawshani A, Landin-Olsson M, Svensson AM, Nystrom
L, Arnqvist HJ, Bolinder J et al. The incidence of diabetes
among 0-34 year olds in sweden: new data and better met-
hods. Diabetologia 2014; 57:1375-81
9. Harjutsalo V, Sund R, Knip M, Groop PH. Incidence of type 1
diabetes in Finland. JAMA 2013;31:427-8.
10. Feig DS, Hwee J, Shah BR, Booth GL, Bierman AS, Lipscombe
LL. Trends in Incidence of Diabetes in Pregnancy and Serio-
us Perinatal OutcomesA Large, Population-Based Study in
Ontario, Canada, 1996-2010. Diabetes Care 2014;37:1590-
96.
11. DeSisto CL, Kim SY, Sharma AJ. Prevalence estimates of
gestational diabetes mellitus in the United States, Pre-
gnancy Risk Assessment Monitoring System (PRAMS),
2007-2010. Prev Chronic Dis. 2014;11:E104. doi: 10.5888/
pcd11.130415.
12. Demant MN, Gislason GH, Køber L, Vaag A, Torp-Pedersen
C, Andersson C. Association of heart failure severity with
risk of diabetes: a Danish nationwide cohort study. Diabe-
tologia 2014;57:1595-600.
 14th EFLM Continuous Postgraduate Course in Clinical Chemistry and Laboratory Medicine
Type 2 diabetes and cardiovascular
diseases: How biomarkers can
support more tailored based
approaches?
Damien Gruson
Department of Laboratory Medicine, Unit of Diabetes and
Nutrition, St-Luc University Hospital and Catholic University,
Louvain, Brussels, Belgium
Corresponding author: gruson_damien@yahoo.fr
Abstract
The rapid increase in type 2 diabetes mellitus
(T2DM) and metabolic syndrome (MetS) preva-
lence is alarming, affecting all age groups across
most ethno-geographical boundaries. This in-
creased prevalence of T2DM and MetS, which con-
stitutes a health threat to the individual and a ma-
jor burden for health economy, is due to popula-
tion growth, ageing, urbanization, unhealthy life-
styles, and reduced muscle mass. Among the vast
array of associated co-morbidities, T2DM and
MetS are associated with higher risk for develop-
ing cardiovascular complications and microangi-
opathy, and patients with these condition often
require throughout life multi-target pharmacolog-
ical interventions as a result of overlapping occur-
rence of truncal fat distribution, overweight, hy-
pertension, atherogenic dyslipidemia, systemic in-
flammation, insulin resistance (IR), a pro-coagu-
lant/hypofibrinolytic state, and hyperglycaemia in
the subset of subjects with impaired fasting glu-
cose, impaired glucose tolerance and T2DM.
T2DM and MetS are significant risk factors for micro
and macrovascular complications. Yet despite the
well-established epidemiological association be-
tween MetS and heightened cardiovascular disease
risk, little is known about the underlying relation-
ship between MetS and neurohormonal profile in
T2DM patients. This is an especially relevant issue in
regards to the known effects of insulin on the pro-
duction and release of key vascular tone regulators
and the potential role of vasoconstrictor peptides
in the pathogenesis of vascular complications.
S11
Circulating neurohormonal biomarkers are in-
creasingly used to stratify patients at risk or with
established cardiovascular disease, as well as for
predicting morbidity and mortality outcomes. The
use of these non-invasive markers for assessing
cardiovascular risk is also advocated to stratify
high-risk patients into primary or secondary pre-
vention. This is of particular importance in T2DM
subjects with or without MetS who are recognized
to be a high-risk population.
The role of peptides involved in vascular remodel-
ling such as galectin-3, aldosterone and endothe-
lin-1, in the pathogenesis and progression of various
cardio-metabolic and renal conditions is well estab-
lished. Recent studies have underlined their puta-
tive role in human atherosclerosis progression.
Thus, measurement of their plasma levels might
contribute to the risk stratification of T2DM patients.
Osteocalcin (OC) has also demonstrated recently a
new face that might allow its integration in the
management of T2DM patients. OC, one of the os-
teoblast-specific proteins, has several hormonal
features and is secreted in the general circulation
from osteoblastic cells. Recent data have suggest-
ed that OC might act as hormone that regulates
glucose metabolism and fat mass and Lee et al.
have showed that mice lacking OC display de-
creased beta-cell proliferation, glucose intoler-
ance, and insulin resistance. Iglesias and coworkers
have reported that patients with T2DM exhibited
OC serum levels significantly lower than those
found in subjects with normal glucose tolerance.
They also showed that OC concentration was inde-
pendently related to 2-h plasma glucose. Hwang
et al. have demonstrated that OC levels were in-
versely associated with the development of T2DM
independent of age, gender, body mass index,
and fasting plasma glucose and plasma adiponec-
tin levels. Furthermore, serum OC levels were in-
dependently associated with glucose intolerance
and abdominal obesity as the components of
metabolic syndrome and T2DM in postmenopau-
sal women. The link between OC and atheroscle-
rosis is also emerging in T2DM patients as serum
OC levels were associated with parameters of ath-
erosclerosis, such as intima-media thicknesses, ca-
rotid plaques and aortic calcifications.
Biochemia Medica 2014;24(Suppl 1):S1-S78
14th EFLM Continuous Postgraduate Course in Clinical Chemistry and Laboratory Medicine
Therefore, several biomarkers appear as potential
cardio-metabolic risk marker/factor in subjects with
T2DM and/or MetS. Such biomarkers might also
References
1. Wild S, Roglic G, Green A, Sicree R, King H. Global prevalen-
ce of diabetes: estimates for the year 2000 and projections
for 2030. Diabetes Care 2004;27:1047-53.
2. Wild SH, Forouhi NG. What is the scale of the future diabe-
tes epidemic, and how certain are we about it? Diabetolo-
gia 2007;50:903-5.
3. Grundy SM, Cleeman JI, Daniels SR, Donato KA, Eckel RH,
Franklin BA et al. Diagnosis and management of the meta-
bolic syndrome: an American Heart Association/National
Heart, Lung, and Blood Institute Scientific Statement. Cir-
culation 2005;112:2735-52.
4. Meetoo D, McGovern P, Safadi R. An epidemiological over-
view of diabetes across the world. Br J Nurs 2007;16:1002-7.
5. Cooper ME, Bonnet F, Oldfield M, Jandeleit-Dahm K.
Mechanisms of diabetic vasculopathy: an overview. Am J
Hypertens 2001;14:475-86.
6. Cooper ME, Johnston CI. Optimizing treatment of hyper-
tension in patients with diabetes. JAMA 2000;283:3177-9.
7. Third Report of the National Cholesterol Education Pro-
gram (NCEP) Expert Panel on Detection, Evaluation, and
Treatment of High Blood Cholesterol in Adults (Adult Tre-
atment Panel III) final report. Circulation 2002;106:3143-
421.
Biochemia Medica 2014;24(Suppl 1):S1-S78
S12
help strategizing the treatment selection of T2DM
patients. The understanding of their regulation al-
lows the identification of new therapeutic targets.
8. Collinson PO, Gaze DC. Biomarkers of cardiovascular da-
mage and dysfunction--an overview. Heart Lung Circ
2007;16 Suppl 3:S71-82.
9. Revkin JH, Shear CL, Pouleur HG, Ryder SW, Orloff DG. Bio-
markers in the prevention and treatment of atherosclerosis:
need, validation, and future. Pharmacol Rev 2007;59:40-53.
10. Zaninotto M, Mion MM, Novello E, Altinier S, Plebani M.
New biochemical markers: from bench to bedside. Clin
Chim Acta 2007;381:14-20.
11. Lee NK, Sowa H, Hinoi E, Ferron M, Ahn JD, Confavreux C, at
al. Endocrine regulation of energy metabolism by the skele-
ton. Cell 2007;130: 456-69.
12. Schmidt-Gayk H, Spanuth E, Kötting J, Bartl R, Felsenberg
D, Pfeilschifter J, at al. Performance evaluation of automa-
ted assays for beta-CrossLaps, N-MID-Osteocalcin and in-
tact parathyroid hormone (BIOROSE Multicenter Study).
Clin Chem Lab Med 2004;42: 90-5.
13. Vietri MT, Sessa M, Pilla P, Misso M, Di Troia D, Sorriento A at
al. Serum osteocalcin and parathyroid hormone in healthy
children assessed with two new automated assays. J Pedia-
tr Endocrinol Metab 2006;19: 1413-9.
 14th EFLM Continuous Postgraduate Course in Clinical Chemistry and Laboratory Medicine
The role of diabetes registries to monitor
the treatment and complications of
diabetes
John A McKnight
Metabolic Unit, Western General Hospital, Edinburgh, Scotland,
United Kingdom
Corresponding author: john.mcknight@nhs.net
Introduction
The management of diabetes requires input from
many different healthcare professionals and car-
ers, as well as a major input form the patients
themselves. There is a strong evidence base for
many interventions, a direct link between the risk
of complications and many measured parameters,
(e.g. HbA1c, BP, cholesterol) and a need for a struc-
ture to ensure clinical measures are performed in a
timely fashion, without duplication, for all patients.
In this modern age of information technology
there is an opportunity to develop systems to re-
cord and review the core data relating to the care
of people with diabetes in a comprehensive diabe-
tes register. This should include the whole popula-
tion with diabetes in any defined area, be efficient
(e.g. automatic collection of data from laborato-
ries) and easy to use as possible and provide some
reward (e.g. generation of formatted letters, easy
local audit summaries) to those who have to make
any extra effort to record data.
Scotland
The health service in Scotland is a National Health
Service, free to the whole population. The respon-
sibility for delivering this is through 14 NHS Boards
who organise the care for their population. The
population of Scotland is 5.3 million. The size of
NHS Board areas varies from 21,000 to 122,000.
The diabetes care within NHS Boards is supported
by Managed Clinical Networks that are multidisci-
plinary and work across usual NHS boundaries
S13
(e.g. primary care and secondary care) to coordi-
nate services.
We have developed a clinical management system
and register for Scotland called SCI-Diabetes. This
database automatically collects information de-
fined in our core dataset for diabetes from many
different systems in the NHS in Scotland, including
all primary care IT systems, the NHS Hospital dia-
betes clinic systems, all the laboratories, from po-
diatry foot screening and the eye screening digital
photography system. This system started around
2001 and took some time to mature into the cur-
rent form. The data has been robust for the whole
population for the last 8 years or so.
The data is analysed to assess the overall perfor-
mance of the whole service and for each element
of the service, with data available;
• for all of Scotland,
• for each of 14 NHS Board areas,
• for individual Primary Care Practices,
• for Hospital Specialist Services
• in a form for the individual patient with for ex-
ample graphic presentation of an individuals
HbA1c for the last 15 or more years.
The national data has been published annually as
the Scottish Diabetes Survey (1), the latest report is
for 2013 and is available on line.
Scottish Diabetes Survey 2013
In the Scottish Diabetes Survey 2013, we report
that:
• There were 268,154 people diagnosed with dia-
betes in Scotland recorded on local diabetes
registers at the end of 2013. This represents
5.0% of the population;
• Crude prevalence of diabetes ranged from
4.34% to 5.80% across NHS Boards;
• 88.2% (236,605) of all people registered with
diabetes had type 2 diabetes;
• 10.9% of all registered people had type 1 dia-
betes. The number of people registered with
type 1 diabetes increased from 26,294 in 2006
to 29,261 in 2014;
Biochemia Medica 2014;24(Suppl 1):S1-S78
14th EFLM Continuous Postgraduate Course in Clinical Chemistry and Laboratory Medicine
• 0.85% (2,288) were recorded as having “other”
types of diabetes, including maturity onset dia-
betes of the young (MODY) and those with un-
known diabetes type;
• 37.5% of patients with a recorded BMI and type
1 diabetes and 31.8% of those with a recorded
BMI and type 2 diabetes were overweight (BMI
25-30 kg/m2), while 24.8% of those with type 1
and 55.0% of those with type 2 were obese
(BMI 30 kg/m2 or above);
• 88.7% (type 1) and 93.9% (type 2) had an HbA1c
recorded in the previous 15 months. Of these,
21.5% and 61.1% had a result < 58mmol/mol
(7.5%), the target reported in previous surveys;
• 86.8% of those with type 1 and 94.9% of those
with type 2 diabetes had their blood pressure
recorded in the previous 15 months. Of these,
47.6% and 33.8% respectively had a systolic BP
measurement of ≤ 130/80 mmHg;
• Cholesterol was recorded in 91.0% of patients
within the previous 15 months, and the target
of ≤5 mmol/l was achieved in 71.3% of those
with type 1 and 80.8% of those with type 2 dia-
betes;
• 23.5% (type 1) and 18.1% (type 2) were current
smokers;
• 1,060 (3.6%) of those with type 1 and 23,546
(10.0%) of those with type 2 diabetes have had
a myocardial infarction and survived, and 2.6%
and 7.3% respectively have undergone cardiac
revascularisation;
• 357 (1.2%) of those with type 1 and 1210 (0.5%)
of those with type 2 diabetes have a record of
having end stage renal failure;
• 86.7% of people with diabetes had had eye
screening in the previous 15 months.
Quality Improvement
We now have a good understanding of the chal-
lenges facing us in organising care for the increas-
ing number of people with diabetes. The total reg-
istered prevalence has increased from 210,000 in
2007 to 268,000 in 2013. Despite this our screening
performance improves each year for most param-
eters and as described above is above 85 % for
Biochemia Medica 2014;24(Suppl 1):S1-S78
S14
most measures. Our data can help with future
planning. For example we know how many new
cases of type 1 and type 2 diabetes occur in each
NHS Board area, enabling some planning for new
patient education. We can use our system to de-
crease duplication of work as the data from one el-
ement of the service is available to others.
The provision of the reports for each NHS Board is
a real driver for improvement and change as the
comparison with others in the system stimulates a
degree of competition to do better. This applies at
all levels of the service. There are many examples
where reviewing data has resulted in a change of
clinical practice. In 1996 a comparison of the dia-
betes data between two clinics in NHS Lothian re-
vealed similar data other than the mean cholester-
ol at one was 5.6 and another 5.1 mmol/l. The 4S
trial had been recently published and there was
some discussion of the lipid results. Within 6
months the mean result at both clinics had de-
creased to 5.0 mmol/l. We also have the ability to
compare local Primary Care practices or groups.
Again comparison with peer groups is of major in-
fluence in stimulating change.
In recent years we have been concerned about the
control of our patients with type 1 diabetes in
Scotland. Through contacts in various countries
we have organised an international comparison of
HbA1c in 324,000 people with type 1 diabetes
from registers in 19 countries. This has stimulated
more work locally. We now regularly review the
data from each of more than 20 diabetes centres
in Scotland and are developing projects using im-
provement methodology to improve diabetes
control in this important group of patients. Our
register has also enabled us to identify that a
group that is not accessing care (HbA1c and retin-
opathy screening) in our current service is that
aged 19 to 27 years and we are actively investigat-
ing ways to address this issue.
Epidemiological Research
The population-based diabetes register, which
contains a unique patient identifier used through-
out NHS Scotland, has enabled us, through the
 14th EFLM Continuous Postgraduate Course in Clinical Chemistry and Laboratory Medicine
Scottish Diabetes Epidemiology Group, to study
many other aspects of diabetes in Scotland.
We have studied the effect of Type 2 diabetes on
mortality in different socioeconomic groups (SES)
(2). Absolute mortality from all causes among peo-
ple with type 2 diabetes increased with increasing
age and socioeconomic deprivation and was high-
er for men than women. Relative risk for mortality
associated with type 2 diabetes was highest for
women aged 35–64 years in the least deprived
quintile with diabetes duration < 2 years at 4.83
(95% CI 3.15–7.40) and lowest for men aged 65–84
years in the most deprived quintile with diabetes
duration ≥ 2 years at 1.13 (1.03–1.24) (2).
In type 1 diabetes the age-adjusted incident rate
ratio (IRR) for first CVD event associated compared
to the non-diabetic population was higher in
women (3.0: 95% CI 2.4–3.8, p,0.001) than men
(2.3: 2.0–2.7, p,0.001) while the IRR for all-cause
mortality associated with T1DM was comparable
at 2.6 (2.2–3.0, p,0.001) in men and 2.7 (2.2–3.4,
p,0.001) in women (3). We have therefore shown
that the risks of cardiovascular disease and mortal-
ity in type 1 diabetes seem to have declined com-
pared to previous studies. We have been able to
show that a high percentage of deaths in the un-
der 40 year age group are due directly to diabetes,
whereas after the age of 40 cardiovascular disease
becomes a major factor.
Further work has focussed on hospital admissions
due to diabetes. Diabetes inpatient expenditure
accounted for 12% of the total Scottish inpatient
expenditure, whilst people with diabetes account
for 4.3% of the population (4). Of the modifiable
risk factors, HbA1c was the most important driver
of cost in type 1 diabetes
People with type 1 diabetes with highest and low-
est mean HbA1c values were associated with in-
creased odds of admission (5). The lowest odds of
S15
admission were associated withHbA1c 7.7–8.7%
(61–72mmol/mol). People with high HbA1c
(>10.8%/95mmol/mol) were at particularly high
risk (relative risk of 2.8 compared to the lowest risk
decile. There is the need to develop effective inter-
ventions to reduce this risk.
Women, smokers, those with high HbA1c and
those living in more deprived areas have an in-
creased risk of admission to hospital for diabetic
ketoacidosis (6). This work highlights that those liv-
ing in poorer areas of the community with high
HbA1c represent a group who might be usefully
supported to try to reduce hospital admission.
Linkage has also enabled us to investigate the
changing incidence of amputation in our popula-
tion (8) and to consider the best interval for screen-
ing for diabetic retinopathy (9)
Our data has also enabled us to develop a tool to
estimate the risk of developing diabetes following
hospital admission based on age and a random
glucose taken during the admission (7).
Conclusion
An important element of developing a register for
people with diabetes is the automatic collection
and assimilation of core diabetes data as part of
routine clinical care without, or with minimal extra
work for front line clinicians.
The rewards for achieving this include the use of
up to date clinical data for audit and quality im-
provement purposes and opportunities for epide-
miological research that will in turn influence clini-
cal care.
I would encourage all those involved in diabetes
care to develop, support and use a register for their
patient group. It has been extremely interesting
and rewarding to be involved in this in Scotland.
Biochemia Medica 2014;24(Suppl 1):S1-S78
14th EFLM Continuous Postgraduate Course in Clinical Chemistry and Laboratory Medicine
References
1. Scottish Diabetes Survey 2013. Available at: www.diabe-
tesinscotland.org.uk/Publications/SDS%202012.pdf , Acce-
ssed on 20th August 2014.
2. Walker JJ, Livingstone SJ, Colhoun HM, Lindsay RS,
McKnight JA, Morris AD, at al. The Scottish Diabetes Rese-
arch Network Epidemiology Group. Effect of socio-econo-
mic status on mortality among people with type 2 diabe-
tes: a study from the Scottish Diabetes Research Network
epidemiology group. Diabetes Care 2011; 34:1127-32.
3. Livingstone SJ, Looker HC, Hothersall EJ, Wild SH, Lindsay
RS, Chalmers J at al. Risk of cardiovascular disease and to-
tal mortality in adults with type 1 diabetes: Scottish regi-
stry linkage study. Epub PLoS Med 2012;9:e1001321. doi:
10.1371/journal.pmed.1001321.
4. Govan L, Wu O, Briggs A, Colhoun HM, McKnight JA, Morris
AD et al. Inpatient costs for people with type 1 and type 2
diabetes in Scotland: a study from the SDRN epidemiology
group. Diabetologia 2011;54:2000-8.
5. Govan L, Wu O, Briggs A, Colhoun HM, Fischbacher CM,
Leese GP et al. Achieved Levels of HbA1c and Likelihood of
Hospital Admission in People With Type 1 Diabetes in the
Scottish Population. A study from the Scottish Diabetes Re-
search Network Epidemiology Group. Diabetes Care 2011;
34:1992-7.
Biochemia Medica 2014;24(Suppl 1):S1-S78
S16
6. Govan L, Maietti E, Torsney B, Wu O, Briggs A, Colhoun
HM et al. The effect of deprivation and HbA1c on admissi-
on to hospital for diabetic ketoacidosis in type 1 diabetes.
Scottish Diabetes Research Network Epidemiology Group.
Diabetologia 2012;55:2356-60.
7. McAllister D, Hughes KA, Lone N, Mills NL, Sattar N,
McKnight JA, Wild SH. Stress hyperglycaemia in hospita-
lised patients and their 3-year risk of diabetes: A Scottish
Retrospective cohort study. Available at: www.plosme-
dicine.org/article/info%3Adoi%2F10.1371%2Fjournal.
pmed.1001708, Accessed on: 20th August 2014.
8. Kennon B, Leese G, Cochrane L, Colhoun H, Wild S, Stang D
et al. Reduced incidence of lower extremity amputations in
people with diabetes in Scotland: a nationwide study. Dia-
betes Care 2012;35:2588-90. ISSN 0149-5992.
9. Looker HC, Nyangoma SO, Cromie DT, Olson JA, Leese GP,
Philip S et al. Scottish Diabetes Research Network Epidemi-
ology Group and the Scottish Diabetic Retinopathy Scree-
ning Collaboration: Predicted impact of extending the scre-
ening interval for diabetic retinopathy: the Scottish Dia-
betic Retinopathy Screening programme. Diabetologia
2013;56:1716–25.
 14th EFLM Continuous Postgraduate Course in Clinical Chemistry and Laboratory Medicine
Guidelines and recommendations for
testing in diagnosis of diabetes mellitus:
The role of HbA1c
Elizabeta Topić
Croatian Society of Medical biochemistry and Laboratory
Medicine, Zagreb, Croatia
Corresponding author: elizabeta.topic@gmail.com
Background
The first arbitrary criteria for diagnosis of diabetes
mellitus (standardized by the World Health Organ-
isation, WHO) appeared in 1980 (1). These criteria
were based on blood glucose measurement fast-
ing and 2 h after oral ingestion of a certain amount
of glucose in non-pregnant adults (OGTT). In 1997,
these criteria were supplemented with the value
of fasting plasma glucose (FPG) more central to
the diagnosis (2). Twelve years later, in 2009, the In-
ternational Expert Committee for Diagnosis and
Management of Diabetes recommended that
HbA1c be used as the preferred test for diagnosing
type 2 diabetes (T2D). The diagnosis of diabetes
should be made solely on the basis of an HbA1c
value 48 mmol/mol (≥6.5%) (3).
HbA1c term
Although the IUPAC-IUB Joint Commission on Bio-
chemical Nomenclature (JCBN) and Nomenclature
Commission of IUB (NC-IUB) drew attention to the
issue of nomenclature related to glycated haemo-
globin as early as 1984, explaining the difference
between the two terms ‘glycated’ and glycosylat-
ed (or glucosylated) haemoglobin’, unfortunately,
the improper use of the term can still be found in
the literature. The term glycated haemoglobin re-
fers to haemoglobin that has been modified by
the non-enzymatic addition of glucose, i.e. non-
enzymatic reactions between glucose or other
sugars and free amino groups of proteins. The
compounds so formed result from the Schiff base
formation followed by Amadori rearrangement.
S17
The term glycosylated (glucosylated) is an enzy-
matic process resulting in glycoside (glucoside)
compounds (4).
The currently acceptable term for glycation of hae-
moglobin in general is ‘glycated haemoglobin’
(GHb). HbA1c is a specific glycated species that is
modified by glucose on the N terminus of the hae-
moglobin β chain. ‘HbA1c’ is also the internation-
ally accepted term for reporting all GHb results.
Methodology
Roughly 100 different GHb methods from low-
throughput manual minicolumn methods to high-
throughput automated systems for HbA1c meas-
urement have been used for 35 years. Related to
the principle, the methods can be classified mostly
into two groups: methods that quantify GHb
based on charge differences between glycated
and nonglycated components (cation-exchange
chromatography, agar-gel electrophoresis), and
methods that separate glycated and nonglycated
components based on structural differences (bo-
ronate affinity chromatography, immunoassay).
Most charge-based and immunoassay methods
measure HbA1c, whereas other methods quantify
‘total glycated haemoglobin’ (5-7).
It is not strange that GHb results reported for the
same blood sample can differ considerably among
methods.
In 1995, the IFCC established a working group
(IFCC WG-HbA1c) to achieve international stand-
ardisation of HbA1c measurement by establish-
ment of reference measurement procedure with
purified primary calibrators, by development of a
network of reference laboratories and implemen-
tation of traceability to the IFCC reference system.
The analyte measured by IFCC reference method
has been defined as βN1-deoxifructosyl-
haemoglobin and recommended units are mmol/
mol.
The IFCC WG-HbA1c recommended the term
HbA1c to be used in clinical practice (8).
In 1996, the National Glycohemoglobin Standardi-
sation Program (NGSP) initiated to standardise
Biochemia Medica 2014;24(Suppl 1):S1-S78
14th EFLM Continuous Postgraduate Course in Clinical Chemistry and Laboratory Medicine
GHb test results among laboratories to The Diabe-
tes Control and Complication Trial (DCCT)-equiva-
lent values (9). It was developed under the auspic-
es of the AACC and endorsed by ADA recommen-
dation that laboratories use only the methods that
are certified by the NGSP as traceable to the DCCT
reference. The manufacturers of HbA1c assays
should also follow traceability to the IFCC refer-
ence method. The NGSP web site contains de-
tailed information on the process of certification
and maintains a list of certified methods (monthly
updated) and factors known by now to interfere
with specific methods (10).
In 1997, the IFCC formed a committee to develop a
higher-order reference method and reference ma-
terials for HbA1c analysis; the method was ap-
proved in 2001. Since the preparing and measur-
ing samples with this method is laborious, very ex-
pensive, and time-consuming, the method serves
to manufacturers for standardisation of the meth-
od.
The advantage of a new reference method to
standardize the HbA1c results, along with the an-
ticipated documentation that the assay does in-
deed indicate average blood glucose resulted in a
variety of proposed changes in the reporting of
HbA1c test results worldwide.
Related to this, an international consensus agree-
ment among the American Diabetes Association
(ADA), the European Association for the Study of
Diabetes (EASD), the International Diabetes Feder-
ation (IDF), and the International Federation of
Clinical Chemistry and Laboratory Medicine (IFCC)
was signed in Milan, Italy, in 2007 (11).
The implications of the activities according to the
agreement were as follows:
1. HbA1c test results should be standardized
worldwide, including the reference system and
reporting of results.
2. The new IFCC reference system for HbA1c rep-
resents the only valid anchor to implement
standardization of the measurement.
3. HbA1c results are to be reported worldwide in
IFCC units (mmol/mol) and derived NGSP units
(%), using the IFCC-NGSP master equation.
Biochemia Medica 2014;24(Suppl 1):S1-S78
S18
4. If the ongoing ‘average plasma glucose study’
fulfils its a priori specified criteria, a HbA1c-de-
rived average glucose (ADAG) value calculated
from the HbA1c result will also be reported as
an interpretation of the HbA1c results.
5. In clinical setting, glycemic goals are expressed
in IFCC units and derived NGSP units.
Since June 2011, the way of HbA1c reporting has
switched from percentage to mmol/mol as the
equivalent to NGSP DCCT-aligned results. Both
NGSP and IFCC units were recommended to be
used, but the decision to report was left to the dis-
cretion of individual countries.
To make sense of the new HbA1c units and com-
pare these with old units and vice versa, a convert-
er has been developed based on so-called master
equation (12).
International consideration and
recommendation
An International Expert Committee comprising
members appointed by the ADA, EASD and IDF
published their report on the role of HbA1c in di-
agnosis of diabetes in 2009 (13), in which they rec-
ommend that diagnosis in type 2 diabetes should
be made solely on the basis of an HbA1c confirmed
to be 48 mmol/mol (≥6.5%), without the need to
measure plasma concentration in the subject. A
sub-diabetic high risk state would exist for sub-
jects with an HbA1c of 42-46 mmol/mol (6.0%-
6.4%).
Kilpatrick and Vinocour in the article on the Asso-
ciation of British Clinical Diabetologist (ABCD) po-
sition statement on haemoglobin A1c for the diag-
nosis of diabetes, on behalf of the ABCD, endorsed
by the Association for Clinical Biochemistry (ACB),
emphasis the advantages and disadvantages of
using HbA1c to diagnose diabetes (14). Advantag-
es include non-fasting samples, low biological var-
iability, the measure of previous (prior) glycaemia,
as well as analytical postulates enabling to bring
results from different laboratories closer together.
Disadvantages are abnormal haemoglobin, anae-
mias, age and ethnicity, as well as technological
 14th EFLM Continuous Postgraduate Course in Clinical Chemistry and Laboratory Medicine
limitations that may, despite strong advantages of
using HbA1c in the diagnosis of diabetes, give a
misleading indication of glycaemia in an individu-
al and so lead to an inappropriate or missed diag-
nosis.
While the current glucose criteria for diagnosis are
arbitrary and the testing process itself has well
documented limitations, at the moment it seems
that there also are unresolved concerns related to
the HbA1c, i.e. it may appear that HbA1c is much
more likely than glucose to completely misdiag-
nose an individual as having or not having diabe-
tes. The possible requirement for using HbA1c
alone in the diagnosis of diabetes mellitus is previ-
ous exclusion of the conditions such as anaemia or
haemoglobinopathies, while taking in considera-
tion the factors of patient age and ethnicity. In this
way, diagnostic workup would be simplified con-
siderably.
The National Academy of Clinical Biochemistry:
Laboratory Medicine Practice Guidelines pub-
lished in 2011 Guidelines and Recommendation
for laboratory analysis in the diagnosis and man-
agement of diabetes mellitus by David Sacks, en-
dorsed by AACC and ADA.
The Guidelines revealed important recommenda-
tions concerning HbA1c; HbA1c should be meas-
ured routinely in all patients with diabetes to doc-
ument their degree of glycemic control. Laborato-
ries should use only HbA1c methods that are certi-
fied by the NGSP as traceable to the DCCT refer-
ence. The manufacturers of HbA1c assays should
also show traceability to the IFCC reference meth-
od. Related to the reference intervals, it is recom-
mended that a laboratory should determine its
own reference interval even if the manufacturer
has provided one.
In clinical settings, patient samples with HbA1c re-
sults below the lower limit of reference interval or
140 mmol/mol (>15%) HbA1c should be verified by
repeat testing, i.e. HbA1c values that are inconsist-
ent with clinical presentation should be investigat-
ed further.
The Guidelines also emphasise the importance of
HbA1c interpretation, which requires close labora-
tory-physician interaction.
S19
Use of HbA1c
Use of HbA1c to diagnose diabetes
Although the proposed diagnostic cut-off value
for HbA1c of 48 mmol/mol is recommended, many
studies have implicated that fewer patients will be
newly diagnosed if HbA1c at this level is used
alone, as compared with plasma glucose value.
The prevalence of undiagnosed diabetes using
any glucose criterion (fasting or 2 h) was 5.1%, and
upon including HbA1c it rose to 5.4%. So, the prev-
alence differs markedly between the Expert Com-
mittee (1.6%) and both WHO (5.1%), and ADA rec-
ommendation (5.4%).
Summarised, 25% of individuals with a ‘positive’
OGTT had 48mmol/mol, while 45% of individuals
that exceeded both the fasting and 2 h glucose
criteria were not diagnosed with diabetes using
HbA1c. The additional effect of ethnicity and aging
has an important role in these proportions.
Using HbA1c to identify the risk of
microvascular complications
It seems that the International Expert Committee
agreed that HbA1c 48mmol/mol could be at least
predictive in identifying patients at risk of devel-
oping microvascular complications, particularly
retinopathy.
Using HbA1c to identify the risk of
macrovascular complications
One of the most common macrovascular compli-
cations is cardiovascular system (CV) involvement.
Several risk factors along with hyperglycaemia
should be managed in diabetes patients; so many
patients are prescribed antihypertensives and lipid
lowering drugs. At the HbA1c 48 mmol/mol cut-off,
this treatment would automatically be considered
in fewer individuals. The HbA1c is also known to be
poor in the group of patients with impaired glu-
cose tolerance and in identifying patients with im-
paired fasting glucose. With regard to CV risk pre-
diction, there is evidence that HbA1c may be supe-
rior to fasting glucose alone in predicting future
CV events. There is evidence for a relationship be-
tween the increasing HbA1c and increasing CV risk.
Biochemia Medica 2014;24(Suppl 1):S1-S78
14th EFLM Continuous Postgraduate Course in Clinical Chemistry and Laboratory Medicine
Using HbA1c to identify type 1 diabetes
The ADA makes HbA1c applicable to patients with
type 1 diabetes, but there is a concern that rapidly
evolving hyperglycaemia in these patients may
not be immediately reflected in a raised HbA1c. So,
it could result in delaying the diagnosis of type 1
diabetes.
Using HbA1c in monitoring of diabetes
The HbA1c testing should be performed at least
biannually in all diabetic patients and quarterly in
patients whose therapy has changed or who failed
to meet treatment goals. These tastings are rec-
ommended for non pregnant patients with either
type 1 or type 2 diabetes.
When using HbA1c in monitoring of diabetic pa-
tients, it is of utmost importance the consider the
race- and age-specific HbA1c target, as well as dif-
References
1. World Health Organisation. Diabetes Mellitus report of
WHO Study Group (Tech Rep Ser. No 727). Geneva: WHO,
1985.
2. Report of the Expert Committee on the diagnosis and classi-
fication of diabetes mellitus. Diabetes Care 1997;20:1183-
97.
3. International Expert Committee Report on the role of the
HbA1c assay in the diagnosis of diabetes. Diabetes Care
2009;32:1327-34.
4. IUPAC-IUB Joint Commission on Biochemical Nomenclatu-
re (JCBN), and Nomenclature Commission of IUB (NC-IUB).
Arch Biochem Biophys 1984;229:237-45.
5. Goldstein DE, Little RR, Wiedmeyer HM, England JD, Mc-
Kenzie EM. Glycated hemoglobin: methodologies and clini-
cal applications. Clin Chem 1986;32:B64-70.
6. Sacks DB. Diabetes mellitus. In: Burtis CA, Ashwood ER,
Bruns DE, eds. Tietz textbook of clinical chemistry and mo-
lecular diagnostics. 5th ed. St. Louis: Elsevier Saunders, 2012.
7. Goldstein DE, Little RR, Lorenz RA, Malone JI, Nathan D, Pe-
terson CM, Sacks DB. Tests of glycemia in diabetes. Diabe-
tes Care 2004;27:1761-73.
8. Mosca A, Goodall I, Hoshino T, Jeppsson JO, Garry John W,
Little RR, et al. Clin Chem Lab Med 2007;45:1077-80.
Biochemia Medica 2014;24(Suppl 1):S1-S78
S20
ferent causes leading to misinterpretation of
HbA1c, such as haemoglobinopathies, anaemia,
renal failure, HIV infection, etc.
Using HbA1c in emergency
Point-of-care (POC) HbA1c is not sufficiently accurate
to use it for the diagnosis of diabetes. Although sev-
eral POC HbA1c assays are NGSP certified, due to the
lack objective and ongoing documentation of per-
formance proficiency testing POC HbA1c instru-
ments should not be used for diagnosis or screening.
In conclusion, the existence of formal recommen-
dations is crucial for standardization of the criteria,
methods and procedures in various clinical condi-
tions, however, a number of questions remain that
require additional research for the recommenda-
tions to resolve all the shortcomings observed to
date.
9. The Diabetes Control and Complication Trial research gro-
up. The effect of intensive treatment of diabetes on the
development and progression of long-term complicati-
ons in insulin-dependent diabetes mellitus. N Engl J Med
1993;329:977-86.
10. Harmonising Haemoglobin A1c testing. Available at:
http://www.ngsp.org. Accessed on 28th August 2014.
11. Hicks JMB, Müller MM, Panteghini M, John WG, Deeb L,
Buse J at al. Consensus Statement on the Worldwide Stan-
dardization of the Hemoglobin A1c Measurement. Diabe-
tes Care 2007;30:2399-400.
12. Weykamp C, John WG, Mosca A, Hoshino T, Little R, Jepps-
son JO, et al. The IFCC Reference Measurement System for
HbA1c: a 6-year progress report. Clin Chem 2008;54:240–8.
13. Kilpatrick E, Bloomgarden Z, Zimmet P. Is haemoglo-
bin A1c a step forward for diagnosing diabetes?  BMJ
2009;339:1288-90.
14. Kilpatrick ES, Vinocour PH. ABCD position statement of
haemoglobin A1c for the diagnosis of diabetes. Pract Diab
Int 2010;27(6):1-5.
15. Sacks DB, Arnold M, Bakris GL, Bruns DE, Horvath AR, Kir-
kman MS, at al. Guidelines and Recommendations for La-
boratory Analysis in the Diagnosis and Management of
Diabetes Mellitus. Diabetes Care 2011;34:e61–e99.
 14th EFLM Continuous Postgraduate Course in Clinical Chemistry and Laboratory Medicine
HbA1c analysing – challenges for the
laboratory – internal and external QC
Gunnar Nordin
External Quality Assessment for Clinical Laboratory
Investigations (Equalis), Uppsala, Sweden
Corresponding author: gunnar.nordin@equalis.se
The uncertainty of a laboratory result is usually
higher than expected by the general consumer of
laboratory results, including the patients. It is a
challenge for the laboratory to explain why a re-
sult never is exactly correct, but still useful for an
intended clinical purpose.
The uncertainty of a result is estimated through a
complete error analysis, including pre-analytical
error components. The laboratory continuously
monitor the performance of the assay as such in
order to guarantee that the uncertainty of the re-
ported results are within claimed limits. Internal
quality control (QC) samples (expected values are
determined and known prior to test) and external
QC samples (for the laboratory blinded values) are
used to check and describe the accuracy of the
major analytic steps of a method over time. The in-
ternal and external QC are the pillars on which the
monitoring of laboratory performance relies.
Assays for HbA1c have developed over years from
assays with large uncertainties, to the current tests
with a high degree of precision and trueness. The
HbA1c results are now also used for the diagnosis
of diabetes.
The external quality control material must be as
commutable as possible and should have a target
value achieved with a reference method proce-
dure. For HbA1c are pools of fresh frozen native
whole blood the best available sample material
and is, with few exceptions, fully commutable. The
pools should contain samples from at least 5 dif-
ferent patients. HbA1c is stable for up to a week in
a cooled blood sample. If the logistics for sampling
and distribution of samples to participating labo-
ratories can be arranged within this period, fresh
material can be used for external quality assess-
S21
ment. If logistics for collection and distribution of
samples does not allow the use of fresh material,
lyophilized material might have to be used, in
spite of lack of commutability.
For tests used for the monitoring of diseases, the
precision of the tests are often more important than
the trueness. Long time stability of the internal QC
material is therefore of prime importance. Lyophi-
lized materials are often used because they meet
the requirements of stability. Due to lack of com-
mutability of lyophilized materials, method depend-
ent target values and acceptance limits have to be
assigned to the materials. Sometimes locally defined
target values and acceptance limits have to be ap-
plied. Stabilized sample materials used for internal
QC often show higher values for reproducibility than
native samples do. Despite such shortcomings, sta-
bilized materials are proper to use in order to identi-
fy changes over time in the performance of the as-
say. Internal QC material is usually provided by the
manufacturer of the device. An identified problem is
the lack of available internal QC material with target
values close to the diagnostic limit 48 mmol/mol.
For a quantitative assays used to diagnose a condi-
tion defined by an agreed cut off level, the true-
ness of the assay might be more important than
the precision. Diabetes is now diagnosed accord-
ing to WHO by the finding of a repeated HbA1c
value > 48 mmol/mol. A small bias of the HbA1c
assay, e.g. 2 mmol/mol, will affect the prevalence
of diabetes with 5 %.
The acceptance limits for external QC results might
be decided with respect to uncertainty of the target
value, and the allowable bias and imprecision of the
assays. For Equalis EQA scheme the acceptance lim-
its is defined as deviations less than +/- 3,5 mmol/
mol from the target value with 95 % probability at
the diagnostic limit 48 mmol/mol. This level of ac-
curacy can be achieved with both hospital methods
and point of care methods under good control.
A requirement for an accredited EQA scheme is
that target values are set by accredited methods.
Target values for HbA1c are offered by the Europe-
an Reference Laboratory for Glycohemoglobin
(ERL) in Holland, who offer measurements with the
IFCC reference methods.
Biochemia Medica 2014;24(Suppl 1):S1-S78
14th EFLM Continuous Postgraduate Course in Clinical Chemistry and Laboratory Medicine
Post-analytical factors – how should
HbA1c results be communicated to
clinicians?
Andrea Mosca
Department of Physiopathology and Transplantation, Center
for Metrological Traceability in Laboratory medicine (CIRME),
University of Milano, Milano, Italy
Corresponding author: andrea.mosca@unimi.it
Post-analytical errors in laboratory medicine usu-
ally are poorly discussed, but this is certainly one
of the issues needing also particular attention, be-
cause clinical decisions are expected to be taken
on the basis of a laboratory result. In the case of
glycated hemoglobin, the main post-analytical
factors to be considered are those related to the
measurement units and to the interpretation of
the result of HbA1c, particularly in relation to the
glycometabolic control of the subject. Other
source of post-analytical errors, including a longer
turnaround time, errors in keyboard entering of
the data, failures in the follow-up and documenta-
tion of laboratory data, will not be discussed here.
Measurement units
With regard to the units of measurement, in 1998 the
European Union introduced a directive on in vitro di-
agnostics (Directive 98/79/EC) requiring that labora-
tory tests be traceable to a “higher-order method”. It
has also to be reminded that in 2007 an international
consensus statement was signed, saying that “A1c
test results should be standardized worldwide, in-
cluding the reference system and results reporting”
(1). So the post-analytical issue was fully addressed at
that time. It was also said that “A1c results are to be
reported worldwide in IFCC units (mmol/mol; SI
units) and derived NGSP units (%)”. These statements
were reinforced 3 years later but unfortunately these
expectations have not been met yet.
Indeed, several countries have shifted to the SI units
in various times between 2010 to present (Australia,
Check Republic, Finland, Germany, Hungary, Italy,
Biochemia Medica 2014;24(Suppl 1):S1-S78
S22
New Zeland, The Netherlands, Serbia, Sweden, UK),
some have kept the NGSP % units (Canada and US),
and the majority of the rest of the world apparently
did not take any official position about. Most of the
countries who have chosen to move to the SI units
have had a period of dual reporting, where both
original and SI units have been used together before
switching to the single SI units. Two countries, Japan
and Sweden, who did develop in the past their own
reference system, decided to change too. Sweden
moved to the SI units and Japan had double report-
ing (NGSP and Japan Diabetes Society units) up to
2013, and then kept the NGSP units only (2). In the
US it is very unlikely that the change to SI units will
be done, since for many test traditional units are still
used. In some cases however (i.e. for glucose) both SI
units (mmol/L) and traditional units (mg/dL) are used
alternatively or both at the same time, so apparently
the situation is at the same confused and conflicting.
Studies are still on the way in order to prove that
the new units could improve the outcome of the
patients, a topic very difficult to be proven. Indeed,
a recent study by Kilpatrick has shown that over 2
years after switching to the SI units did not lead to
any marked short-term deterioration in glycemia
or a different HbA1c outcome in patients with ini-
tial poor glucose control (3). So, it seems that the
mayor problem for those countries who did not
take a decision yet, especially in the case of less de-
veloped countries, efforts should be directed to-
ward adopting higher-quality and standardized
methods, possibly meeting the required desirable
standards of analytical imprecision and trueness
already clearly defined (4). In any case, the decision
to move to the SI units should be taken by involv-
ing all the stakeholders (diabetologists, family doc-
tors, endocrinologists, pediatricians, nurses, pa-
tients associations, head officials of the National
and/or Regional health systems, manufacturers of
diagnostics) and providing sufficient information.
In the countries where this change was done no
particular problems came to the light. With regard
to the publications, the already mentioned con-
sensus statement (1), editors of journals and other
printed material were strongly recommended to
require that submitted manuscripts report HbA1c
in both SI (IFCC) and NGSP/DCCT units.
 14th EFLM Continuous Postgraduate Course in Clinical Chemistry and Laboratory Medicine
Finally, the conversion of the HbA1c data between
different units has to be accomplished by using the
so-called master equation (NGSP = 0.09148(IFCC) +
2.152 or IFCC = 10.93(NGSP) – 23.50), which is de-
rived by the studies of the IFCC Network of Refer-
ence laboratories, which are performed regularly
twice a year, and who did prove that this equation
is stable over a long period of time (5) [exactly for
more than 17 years, according to the last report of
the Network Coordinator (Cas Weykamp, personal
communication)]. Moreover, comparisons between
the NGSP and IFCC networks continue to be con-
ducted twice a year, thus validating the stability
and reliability of the networks, and ensuring that
results can be converted from DCCT/NGSP units to
SI units and vice versa. In order to assist patients
and doctors, various facilities have been devel-
oped, either by using the Internet community, such
the one developed by the NGSP (http://www.ngsp.
org/convert1.asp), where a table and a calculator
are available, or also the one developed by the UK
Diabetes organization (http://www.diabetes.co.uk/
HbA1c-units-converter.html). Various applications
(“App”) are also available by the smart phones.
Two caveats should be always taken into account:
a) Since any value below 2.15 % is zero in SI units
(due to the lower specificity of the NGSP meth-
od respect to the IFCC reference measure-
ments procedure), various of these facilities
either report negative values (with no physi-
ological meaning) or simply do not allow the
calculation below a certain threshold (in the
case of the UK calculator the values that can
be converted should stay in the range 4 to 24
%, in terms of NGSP units).
b) Due to the normal use of just one decimal
place to calculate the HbA1c concentration in
the NGSP units, and to the rounding automati-
cally performed using the above mentioned
calculators, some values in SI units correspond
to the same HbA1c values in NGSP units (i.e. 35
and 36 mmol/mol are both equivalent to 5.4 %;
47 and 48 mmol/mol: 6.5 %; 58 and 59 mmol/
mol: 7.5 %; 70 and 71 mmol/mol: 8.6 %; just to
quote the most frequent values in the physio-
pathological range of HbA1c).
S23
Interpretation of an HbA1c result
The principal use of HbA1c is certainly the one re-
lated to the assessment of glycemic control in dia-
betic patients. Indeed, from the Diabetes Compli-
cation and Control Trial (DCCT) (6) and the United
Kingdom Prevention Diabetes Study (UKPDS) (7)
we have learn that a reduction in HbA1c level
caused a decrease in the incidence of the complica-
tions of diabetes mellitus, mostly retinopathy, with
optimal reduction achieved at an HbA1c value of
7% (53 mmol/mol). Based on these studies, HbA1c
testing may be used to monitor the effectiveness
of therapy or patient compliance (8). From these
studies a first target of 7.0 % (53 mmol/mol) for in-
dividuals with Type 1 or Type 2 diabetes to reduce
microvascular and macrovascular complications
was established by the American Diabetes Associa-
tion (ADA), followed later on by the Canadian Dia-
betes Association (CDA). Other thresholds were rec-
ommended by the CDA guidelines, as follows:
a) A target HbA1c of 6.5 % (48 mmol/mol) in indi-
viduals with Type 2 diabetes to reduce the risk
of nephropathy, taking into account the pos-
sible risk of hypoglycemia.
b) A target HbA1c value of 8.5 % (69 mmol/mol)
in children under 5, or of 8.0% (64 mmol/mol)
in children 6 to 12 years old and of ≤7.0 % (≤53
mmol/mol) in children 13 to 18 years of age.
c) A target HbA1c value of ≤7.0 % (≤53 mmol/
mol) in pre-pregnancy.
d) A target HbA1c value of ≤6.0 % (≤42 mmol/mol;
or within the reference range), in pregnancy.
In 2008 the results of an international trial aimed
to calculate HbA1c-derived average glucose values
calculated from the HbA1c results (ADAG) was
published (9). This trial confirmed that for an aver-
age increase in HbA1c of 1 % (10 mmol/mol) a
mean worsening of plasma glucose was about 29
mg/dL (1.6 mmol/L), but the reporting of an esti-
mated average glucose (eAG) calculated from the
HbA1c value was not included among the consen-
sus statements above mentioned, due to a set of
limitations in the ADAG study.
Another potential use of HbA1c is for the diagnosis
of diabetes. The threshold to this end (6.5 %, or 48
Biochemia Medica 2014;24(Suppl 1):S1-S78
14th EFLM Continuous Postgraduate Course in Clinical Chemistry and Laboratory Medicine
mmol/mol) was derived from an accurate analysis
of the third National Health and Nutrition Examina-
tion Study (NHANES III). This recommendation has
been then adopted by ADA and other Associations,
and finally in 2010 by the World Health Organiza-
tion. A number of papers have been published since
then, with controversial opinions about the sensitiv-
ity of HbA1c for detecting diabetes. At present, not
enough data are available to support the use of
HbA1c for the screening of diabetes, and some Na-
tional Associations (i.e. in UK and in Germany) have
proposed different flowcharts where HbA1c is
measured in association with fasting plasma glu-
cose or with oral glucose tolerance test (OGTT) to
diagnose diabetes in high-risk individuals.
Finally, HbA1c has been found to be the best pre-
dictor of 10 year fatal and non-fatal cardiovascular
events and all-cause mortality, compared to fast-
ing and 2 h post prandial glucose, in individuals
between 50 and 75 years of age without diabetes
(10). Another study, the North American Athero-
sclerosis Risk in Communities (ARIC) study, proved
that in non-diabetics HbA1c was a better predictor
of diabetes and cardiovascular disease compared
to fasting glucose.
About reporting
As far as I know, there is no consensus on how
HbA1c should be reported after a blood exam,
since almost every laboratory has its own format
and tradition. According to what is recommended
in most of the classical textbooks of laboratory
References
1. Consensus Committee. Consensus statement on the
worldwide standardization of the hemoglobin A1c measure-
ment: the American Diabetes Association, European Associa-
tion for the Study of Diabetes, International Federation of Cli-
nical Chemistry and Laboratory Medicine, and the Internati-
onal Diabetes Federation. Diabetes Care 2007;30:2399–2400.
2. Sacks DB. Measurement of hemoglobin A1c. A new twist on
the path to harmony. Diabetes Care 2012;35:2674-80.
3. Kilpatrick ES, Rigby AS, Atkin SL, Barth JH. Glycemic control
in the 12 months following a change to SI hemoglobin A1c
reporting units. Clin Chem 2013;59:1457-60.
Biochemia Medica 2014;24(Suppl 1):S1-S78
S24
medicine, it is a normal praxis to present, together
with the numerical result and measurement units
of the observed HbA1c value, the reference inter-
vals. In my opinion such a practice should be aban-
doned, since, as for other analytes such as total
cholesterol, it is now more important to report a
desirable level, or some target values.
A possible example for reporting the result of a
determination of glycated hemoglobin in human
blood, could then be the following: b-glycated
hemoglobin (HbA1c): 38 mmol/mol (desirable val-
ue: <39 mmol/mol; cut-off for the diagnosis of dia-
betes: >47 mmol/mol; therapeutic target: <53
mmol/mol).
Since most of the methods are now standardized, I
do not believe that the type of the method used
(i.e. HPLC or immunochemistry) should be report-
ed any more.
Conclusions
Long time has passed since the discovery of HbA1c
and its introduction in the laboratory practice relat-
ed to the management of diabetes mellitus. Great
improvements have been achieved on the analytical
side, but still a lot of work has to be done to achieve
a world-wide standardization of this important labo-
ratory test with regard to the post-analytical phase. I
believe that much effort should be now pushed at
the level of international associations (such as IFCC,
EFLM or WHO) in order to promote the creation, the
diffusion and finally the application of other ad-hoc
consensus documents or guidelines.
4. Braga F, Dolci A, Montagnana M, Pagani F, Paleari R, Gu-
idi GC, at al. Revaluation of biological variation of glyca-
ted hemoglobin (HbA1c) using an accurately designed pro-
tocol and an assay traceable to the IFCC reference system.
Clin Chim Acta 2011;412:1412-6.
5. Weykamp C, John WG, Mosca A, Hoshino T, Little R, Jepps-
son JO, et al. The IFCC Reference Measurement System for
HbA1c: a 6-year progress report. Clin Chem 2008;54:240–8.
6. The Diabetes Control and Complications Trial resear-
ch group. The effect of intensive treatment of diabetes on
the development and progression of long-term complica-
 14th EFLM Continuous Postgraduate Course in Clinical Chemistry and Laboratory Medicine
tions in insulin-dependent diabetes mellitus. N Engl J Med
1993;329:977–86.
7. U.K. Prospective Diabetes effect of intensive blood glucose
control with sulphonylureas or insulin compared with con-
ventional treatment and risk of complications in patients
with Type 2 diabetes (ULPDS33) U.K. Prospective Diabetes
Study (UKPDS) Group. Lancet 1998;352:837–53.
8. Higgins T. HbA1c - An analyte of increasing importance.
Clin Biochem 2012;45:1038-45.
S25
9. Nathan DM, Kuenen J, Borg R, Zheng H, Schoenfeld D, He-
ine RJ. Translating the A1c assay into estimated average
glucose values. Diab Care 2008;31: 1473– 8.
10. Van’t Riet E, Rijkelijkhuizen JM, Alssema M, Nijpels G, Ste-
nouwer CDA, Heine RJ, et al. HbA1c is an independent pre-
dictor of nonfatal cardiovascular disease in a Caucasian
population without diabetes: a 10 year follow up of the Ho-
orn Study. Eur J Cardiovasc Prev Rehabil 2012;19:23–31.
Biochemia Medica 2014;24(Suppl 1):S1-S78
14th EFLM Continuous Postgraduate Course in Clinical Chemistry and Laboratory Medicine
Early recognition of gestational diabetes
(Introduction of new guidelines and
practice) – how should the routines be?
Andrej Zavratnik
Department of Endocrinology, University Clinic Centre Maribor,
Maribor, Slovenia
Corresponding author: andrej.zavratnik@ukc-mb.si
Introduction
Diabetes in pregnant women may be pregesta-
tional, where the diabetes, type 1 or type 2 was di-
agnosed before pregnancy, or hyperglycemia can
be first recognised during the pregnancy that
comprise two distinct categories - gestational dia-
betes mellitus (GDM) and previously unrecognised
prepregnancy diabetes, so called »overt« diabetes
(1). Women who are in poor glycemic control dur-
ing the period of fetal organogenesis, which is
nearly complete by twelfth week postconception,
have a high incidence of spontaneous abortion
and fetuses with congenital anomalies. The risk in-
crease exponentially with increasing glycosylated
haemoglobin (HbA1c). On the other hand, the mal-
formation rates are similar to the background pop-
ulation of around 2% when the early pregnancy
HbA1c is within normal range (2). Therefore, im-
portance of preconceptional evaluation and coun-
selling of women with pregestational diabetes
mellitus cannot be overstated. Later in pregnancy,
poor glycemic control increases the risk of mac-
rosomia and its sequelae by two to four times
(3,4,5). Additionally, perinatal mortality rates (still-
births and first-week neonatal deaths) among
women who are diabetic remain approximately
two to four time higher as those observed in the
nondiabetic population, and perinatal morbidity
(neonatal hypoglycemia, respiratory distress, hy-
perbilirubinemia and jaundice, hypocalcaemia, hy-
pomagnesaemia, polycythemia, transient hyper-
trophic cardiomyopathy with congestive cardiac
failure) is higher as well (2,5). In addition to fetal
complications, pregnancy in women with preges-
tational diabetes can adversely influence maternal
Biochemia Medica 2014;24(Suppl 1):S1-S78
S26
health. In particular, women can suffer from treat-
ment-induced hypoglycemia and worsening of
pre-existing micro- and macro-vascular complica-
tions, such are retinopathy, nephropathy, neurop-
athy and cardiovascular disease (2). Gestational hy-
pertension and pre-eclampsia are two to twelve
times more common (3,4,5). Finally, the long-term
impact on offspring of exposure to hyperglycemia
in utero result in a greater risk of obesity, metabol-
ic syndrome and type 2 diabetes later in life, due
to epigenetic modifications of gene expression (2).
Early recognition of overt diabetes in pre-preg-
nancy unrecognized type 2 diabetic women, and
recognition of gestational diabetes with unde-
layed achievement of normoglycemia is therefore
crucial for optimizing maternal and fetal outcomes
in all women with hyperglycaemia during preg-
nancy, regardless of the type of diabetes.
Diagnosis of hyperglycemia during
pregnancy
Before the International Association of Diabetes
and Pregnancy Study Group (IADPSG) recommen-
dations have been launched, the absence of a uni-
versally accepted “gold standard” for the diagnosis
of GDM has resulted in a variety of recommended
diagnostic thresholds that have been endorsed by
different organisations in their guidelines. None of
those diagnostic criteria were based on fetal or
maternal outcomes of the pregnancy. To over-
come this shortage the Hyperglycemia and Ad-
verse Pregnancy Outcome (HAPO) study was de-
signed. The study involved 25505 pregnant wom-
en from 15 centres in nine countries. It was the first
study, designed to clarify risks of fetal and mater-
nal adverse outcomes associated with degrees of
maternal glucose intolerance less severe than
those with overt diabetes during pregnancy.
Women were tested using 75-g oral glucose toler-
ance test (OGTT) at 24-32 weeks. The study found
a continuous positive association with increasing
glucose levels and birth weight >90th percentile,
cord C-peptide >90th percentile, primary Cesarean
section and neonatal hypoglycemia. The study
also found positive associations between increas-
 14th EFLM Continuous Postgraduate Course in Clinical Chemistry and Laboratory Medicine
ing plasma glucose levels and each of the five sec-
ondary outcomes examined: premature delivery,
shoulder dystocia or birth injury, intensive neona-
tal care, hyperbilirubinemia and preeclampsia. The
associations were strongest for birth weight and
blood serum C-peptide levels (6). Results from the
HAPO study was the basis for IADPSG recommen-
dations launched in 2010.
Historically, the term “gestational diabetes” was
used to define women with onset or first recogni-
tion of abnormal glucose tolerance during preg-
nancy. After the IADPSG consensus it is prudent to
distinguish women with probable pre-existing di-
abetes that is first recognised during pregnancy –
»overt diabetes« from those with transient hyper-
glycemia due to pregnancy related insulin resist-
ance – »GDM«. The rationale for this recommenda-
tion is based on the fact that an increasing propor-
tion of young women have as yet overt but unrec-
ognized type 2 diabetes, due to the increasing
prevalence of obesity, and lack of routine glucose
screening/testing in this age group. Beside this,
nowadays women decide for pregnancy later in
life, what is inevitably related to a higher incidence
of diabetes type 2. Identifying overt diabetes early
in pregnancy as a distinct group is important be-
cause these women are at increased risk of con-
genital anomalies in offspring, are at risk of diabe-
tes complications (nephropathy and retinopathy),
and require rapid treatment of hyperglycaemia
during pregnancy to ensure prompt restoration of
normal glycemia and close follow-up during preg-
nancy. Early identification of overt diabetes and
treatment of hyperglycemia may reduce these
risks and provide an opportunity to optimize preg-
nancy outcome (1).
The overall strategy recommended by the IADPSG
Consensus Panel for detection and diagnosis of
hyperglycemic disorders in pregnancy include
two discrete phases (Figure 1). The purpose of the
first phase is detection of women with overt dia-
betes not previously diagnosed or treated outside
of pregnancy. Detection and diagnosis of overt di-
abetes during pregnancy should be made during
the initial visit for prenatal care. Universal early
testing in populations with a high prevalence of
type 2 diabetes is recommended by some organi-
S27
sations (1,7). In health care systems not deciding
for universal testing, screening for undiagnosed
type 2 have to be performed in those with risk fac-
tors, such as listed in Table 1 (8,9). The diagnosis of
overt diabetes is confirmed using standard diag-
nostic criteria: fasting plasma glucose ≥ 7,0 mmol/L
or HbA1c ≥ 6,5% or random plasma glucose ≥ 11,1
mmol/L plus confirmation (Table 2). If results indi-
cate overt diabetes treatment and follow-up as for
pre-existing diabetes is mandatory. If results are
not diagnostic of overt diabetes and fasting plas-
ma glucose is ≥5,1 mmol/l (92 mg/dl) but <7,0
mmol/l (126 mg/dl), diagnose immediately as
GDM. In all other women, not found to have overt
diabetes or GDM at early testing, the second phase
is one step 75-g OGTT performed at 24–28 weeks’
gestation. According to IADPSG recommendations
GDM should be diagnosed if one or more values
from a 75-g OGTT equal or exceed those listed in
Table 3 (1). As a result of IADPSG consensus, many
organizations have published new recommenda-
tions for screening and diagnosis of diabetes in
pregnancy. World Health Organisation (WHO) (10)
and Endocrine society (7), adopt IADPSG criteria
(75-g OGTT as one step approach), while American
Diabetes Association (ADA) (8) and Canadian Dia-
betes Association (9) allow using two approaches
(75-g OGTT as one step approach or 50-g OGTT
followed with confirmation testing using either
100-g OGTT in USA or 75-g OGTT in Canada as two
step approach), NICE guidelines are in develop-
ment.
However since there is a continuous risk of adverse
outcomes with increasing glycemia in HAPO study,
diagnostic thresholds are somewhat arbitrary. The
IADPSG Consensus Panel decided to define diag-
nostic values listed in Table 3 on the basis of an
odds ratio of 1,75 for adverse neonatal outcomes.
Based on HAPO study population the total inci-
dence of GDM would be around 17,8% of all preg-
nant women (1). The one step approach proposed
by the IADPSG, is anticipated to significantly in-
crease the prevalence of GDM (from 5-6% to ∼15-
20%), primarily because the recommended glucose
cut-off values for GDM correspond to those pro-
posed by IADPSG are lower than those recom-
mended by earlier guidelines, and because the
Biochemia Medica 2014;24(Suppl 1):S1-S78
14th EFLM Continuous Postgraduate Course in Clinical Chemistry and Laboratory Medicine

FIRST PHASE – First prenatal visit:

Measure FPG, HbA1c or random glucose

(on all or only high-risk women)

YES Overt diabetes? as per Table 2

Dg: Overt diabetes NO

Treatment and follow-up as for

preexisting diabetes

FPG ≥5.1 mmol/l (92 mg/dl) but FPG ≥5.1 mmol/l (92 mg/dl)
<7.0 mmol/l (126 mg/dl

Dg: GDM SECOND PHASE:

Test all for GDM from 24 to 28
Treatment and follow-up as for GDM weeks’ gestation with 75-g OGTT

Dg: Treatment and follow-up as

for GDM if fulfilled the criteria
from Table 3

Figure 1. Strategy for the detection and diagnosis of hyperglycemic disorders in pregnancy.

Table 1. Risk factors for hyperglycemia during pregnancy (from a reference 8).
Testing should be considered in all women who are overweight (BMI ≥25 kg/m2) and have additional risk factors:
1. physical inactivity
2. first-degree relative with diabetes
3. high-risk race/ethnicity (e.g., African American, Latino, Native American, Asian,American, Pacific Islander)
4. women who delivered a baby weighing 4,1 kg or were diagnosed with GDM
5. hypertension (140/90 mmHg or on therapy for hypertension)
6. HDL cholesterol level <0.90 mmol/L and/or a triglyceride level >2.82 mmol/L
7. women with polycystic ovarian syndrome
8. A1c ≥5.7%, IGT, or IFG on previous testing
9. other clinical conditions associated with insulin resistance (e.g., severe obesity, acanthosis nigricans)
10. history of CVD
BMI – Body mass index, IGT – impaired glucose tolerance, IFG – impaired fasting glucose, CVD – cardiovascular disease

Biochemia Medica 2014;24(Suppl 1):S1-S78

S28
 14th EFLM Continuous Postgraduate Course in Clinical Chemistry and Laboratory Medicine
Table 2. Threshold values for diagnosis of overt diabetes in
pregnancy (from a reference 1). Decision to perform blood test-
ing for evaluation of glycemia on all pregnant women or only
on women with characteristics indicating a high risk for diabe-
tes is to be made on the basis of the background frequency of
abnormal glucose metabolism in the population and on local
circumstances.
Measure of glycemia Consensus threshold
FPG ≥7.0 mmol/l (126 mg/dl)
≥6.5% (DCCT/UKPDS
A1c
standardized)
≥11.1 mmol/l (200 mg/dl) +
Random plasma glucose
confirmation
only one abnormal value, not two, is sufficient to
make the diagnosis (8,10). Increased prevalence of
GDM would have significant impact on the costs,
medical infrastructure capacity, and potential for
increased “medicalisation” of pregnancies previ-
ously categorised as normal (8). Nevertheless,
IADPSG diagnostic criteria for the treatment of
GDM seem to be reasonable. Two randomized con-
trolled trials, Australian Carbohydrate Intolerance
Study in Pregnant Women (ACHOIS) and a multi-
center, randomized trial of treatment for mild ges-
tational diabetes (MFMU trial) comparing active
treatment versus standard obstetric care for mild
GDM have been conducted during the years in
which the HAPO study was carried out. In both tri-
als, treatment, achieved primarily by diet/lifestyle
modification, resulted in reduced risks of fetal over-
growth, shoulder dystocia, cesarean delivery, and
hypertensive disorders (3,4). Recruitment processes
and glycemic values of participants were not iden-
tical in the mentioned two randomized controlled
trials and the HAPO observational study. However,
there was substantial overlap between glucose val-
ues used for inclusion in the randomized controlled
trials and those recommended in IADPSG report as
new threshold values. Furthermore, frequencies of
outcomes such as LGA or birth weight >90th per-
S29
Table 3. Threshold values for diagnosis of GDM (from a refer-
ence 1).
Glucose measure Glucose concentration threshold*
mmol/l mg/dl
FPG 5.1 92
1-h plasma glucose 10.0 180
2-h plasma glucose 8.5 153
* One or more of these values from a 75-g OGTT must be equaled or
exceeded for the diagnosis of GDM.
centile and preeclampsia in usual care versus treat-
ment arms of the randomized controlled trials are
similar to those observed in the HAPO study among
women with one or more glucose values that meet
or exceed the threshold, compared with those with
all values below threshold. Although not directly
comparable, it was concluded that results of the
two randomized controlled trials and HAPO are
highly complementary (1).
Conclusion
Current evidence supports direct causal role be-
tween maternal glycaemia and fetal/offspring and
maternal adverse outcomes. Glucose testing early
in pregnancy to detect overt diabetes and again
with a 75-g OGTT at 24–28 weeks’ of gestation in
all pregnancies not already diagnosed with overt
diabetes or GDM by early testing represents fun-
damental changes in strategies for detection and
diagnosis of hyperglycemia in pregnancy. Detec-
tion and diagnosis of hyperglycemic disorders in
pregnancy based on IADPSG criteria will substan-
tially increase the frequency of hyperglycemic dis-
orders in pregnancy. If results indicate overt diabe-
tes treatment and follow-up as for pre-existing di-
abetes should be started. If results are diagnostic
for GDM the pregnant women should be follow-
up closely during pregnancy.
Biochemia Medica 2014;24(Suppl 1):S1-S78
14th EFLM Continuous Postgraduate Course in Clinical Chemistry and Laboratory Medicine
References
1. Metzger BE, Gabbe SG, Persson B, Buchanan TA, Catalano
PA, Damm P, et al. International Association of Diabetes
and Pregnancy Study Groups Consensus Panel, Diabetes
Care. International association of diabetes and pregnancy
study groups recommendations on the diagnosis and cla-
ssification of hyperglycemia in pregnancy. Diabetes Care
2010; 33: 676–82.
2. Ali S, Dornhorst A. Diabetes in pregnancy: health risks and
management. Postgrad Med J 2011; 87:417–27.
3. Landon MB, Spong CY, Thom E, Carpenter MW, Ramin SM,
Casey B, et al. A multicenter, randomized trial of treatment
for mild gestational diabetes. N Engl J Med. 2009 ; 361:
1339–48.
4. Crowther CA, Hiller JE, Moss JR, McPhee AJ, Jeffries WS, Ro-
binson JS. Australian Carbohydrate Intolerance Study in
Pregnant Women (ACHOIS) Trial Group. Effect of treatment
of gestational diabetes mellitus on pregnancy outcomes. N
Engl J Med 2005; 352: 2477–86.
Biochemia Medica 2014;24(Suppl 1):S1-S78
S30
5. Evers IM, de Valk HW, Visser GH. Risk of complications of pre-
gnancy in women with type 1 diabetes: nationwide pros-
pective study in the Netherlands. BMJ. 2004;328(7445):915.
6. HAPO Study Cooperative Research Group. Hyperglyce-
mia and adverse pregnancy outcomes. N Engl J Med.
2008;358:1991-2002.
7. Blumer I, Hadar E, Hadden DR, Jovanovič L, Mestman JH,
Murad MH, et al. Diabetes and pregnancy: an endocrine
society clinical practice guideline. J Clin Endocrinol Metab
2013;98:4227-49.
8. American Diabetes Association. Diagnosis and classifi-
cation of diabetes mellitus. Diabetes Care 2014; 37 Suppl
1:S81-90.
9. Canadian Diabetes Association Clinical Practice Guidelines
Expert Committee. Diabetes and pregnancy. Can J Diabe-
tes 2013;37 Suppl 1:S168-83.
10. WHO: Diagnostic Criteria and Classification of Hyper-
glycaemia First Detected in Pregnancy. Available at: http://
www.who.int/diabetes/publications/Hyperglycaemia_In_
Pregnancy/en/, Accessed on 28th August 2014.
 14th EFLM Continuous Postgraduate Course in Clinical Chemistry and Laboratory Medicine
How to diagnose the prediabetes?
Grazyna Sypniewska
Department of Laboratory Medicine, Collegium Medicum,
Nicolaus Copernicus University, Bydgoszcz, Poland
Corresponding author: grazynaodes@interia.pl
Diabetes is a chronic metabolic disease character-
ized by the presence of hyperglycemia that occurs
due to defective insulin production and secretion
by the pancreas, defective insulin action resulting
from no response of the cells to insulin or both. High
blood glucose leads to typical clinical symptoms :
weight loss, polyuria, increased thirst, weakness (1).
Prediabetes is the term used to describe the con-
dition with impaired fasting glucose (IFG), glucose
concentrations higher than normal but below the
established threshold for diabetes, or impaired
glucose tolerance (IGT) recognized based on the
results of a 2-hour oral glucose tolerance test
(OGTT). People with prediabetes are at high-risk
for developing diabetes and associated complica-
tions. It is estimated that each year 5-10% of indi-
viduals with prediabetes will develop T2 diabetes
[8]. Occurrence of IFG and/or IGT is asociated with
abdominal or visceral obesity, high serum triglyc-
erides and/or low HDL-cholesterol andhyperten-
sion (1). In subjects with IGT lifestyle changes such
as increase of physical activity, loosing weight (5-
10%) and pharmacological therapy may prevent or
delay the development of diabetes (2).
Diagnosis of prediabetes
World Health Organization (WHO) criteria for im-
paired fasting glucose differ from the American
Diabetes Association (ADA) criteria, because the
normal range of glucose is defined differently.
ADA lowered the upper limit of normal to a fasting
glucose under 100 mg/dL (5.6 mmol/L) as higher
fasting plasma glucose (FPG) has been shown to
increase complication rates significantly. WHO de-
cided to keep FPG upper limit of normal at under
110 mg/dL (2).
S31
Prediabetes is diagnosed when FPG is of 110 - 125
mg/dL (6.1 mmol/L - 6.9 mmol/L) according to
WHO criteria or 100 - 125 mg/dL (5.6 mmol/L - 6.9
mmol/L) according to ADA criteria and/or when
2-hour glucose tolerance test after ingesting the
standardized 75 g of glucose solution indicates
glucose concentration of 140 -198 mg/dL (7.8 - 11.0
mmol/L) and/or when hemoglobin A1c (HbA1c) is
between 5.7 - 6.4% ( 39-47 mmol/mol) (3).
According to ADA criteria published in 2010, the
recommended order for prediabetes testing is as
follows: HbA1c, FPG and/or OGTT (3). There is some
controversy on whether HbA1c can be used as the
only test and 2-hour post load glucose brings and
added value. Moreover, HbA1c-defined prediabe-
tes ranges are also a subject of debate, with some
favouring 6.0- 6.4% (42- 47 mmol/mol) instead of
5.7-6.4% (4). FPG is recommended to be measured
in venous plasma.
It should be mentioned that the analytical meth-
ods for glucose and HbA1c may affect the interpre-
tation of results. It is important to note that both
FPG and glucose tolerance test reflect distinct pro-
cesses. Normal FPG reflects maintaining the ade-
quate insulin secretion and control of hepatic glu-
cose output whereas normal glucose tolerance in-
dicates adequate secretion of insulin and insulin
sensitivity in the target tissues. This explains why
an individual with impaired fasting glucose may
have IGT or even diabetes and individual with nor-
mal FPG may have IGT.
According to ADA and WHO, screening for predia-
betes in the general population should add FPG
for those with HbA1c of 6.0-6.5%. However there
are some controversies concerning specific popu-
lations (Asian population) whether using only
HbA1c and FPG without OGTT for detecting predi-
abetes, leads to false negative results (2). It seems
clear that individuals with HbA1c of 6.0-6.5% are in
the high-risk group and require lifestyle changes
and management of risk factors that will be not
changed based on the further information from
OGTT. On the other hand, the estimated risk of
mortality and CVD showed the strongest associa-
tion with the results of OGTT than FPG or HbA1c
(2).
Biochemia Medica 2014;24(Suppl 1):S1-S78
14th EFLM Continuous Postgraduate Course in Clinical Chemistry and Laboratory Medicine
OGTT, comparing to HbA1c, is more difficult to
perform and time-consuming. HbA1c concentra-
tion may be affected by red blood cell survival
time and renal function but has several advantag-
es. Comparing to fasting glucose, HbA1c has a
small intraindividual variability, good stability after
blood collection and no need for fasting.
Recent data from the Centre for Disease Control
showed that ~30% of adults in United States have
prediabetes defined based on fasting plasma glu-
cose (FPG 100-125 mg/dL) or glycated hemoglobin
values (HbA1c 5.7- 6.4%). Without weight loss and
increased physical activity 15-30% of Americans
with prediabetes will progress to T2D within five
years.
Prevalence of prediabetes in adults
In the study of Korean population the prevalence
of diabetes and prediabetes was evaluated ac-
cording to FPG alone or the two tests - FPG and
HbA1c in combination (5). HbA1c in this study was
measured with high performance liquid chroma-
tography method. Using both tests a greater num-
ber of individuals with diabetes and prediabetes
was detected. The prevalence of prediabetes was
19.3% (23.8% in men and 14.9% in women) when
FPG only was used but increased up to 38.3% (41%
in men and 35.7% in women) when HbA1c was
added as the second test. It was concluded that
adding HbA1c as complementary test to FPG allow
to avoid underestimation of the diabetes and pre-
diabetes prevalence.
From the practical point of view, screening with
HbA1c is easier to perform however, it was sug-
gested that fewer cases of prediabetes are detect-
ed than with OGTT. It may be understandable as
both measurements reflect different physiological
processes. In their study Vlaar et al (6) screened
South Asian individuals aged 18-60 years perform-
ing OGTT and HbA1c measurements. Out of 353
cases meeting HbA1c prediabetes criteria only 62
met OGTT criteria (18%). In cases with prediabetes,
defined on the basis of OGTT, the AUC (the area
under the curve in ROC chracteristics) for HbA1c
was 0.73 whereas in cases with diabetes it was
Biochemia Medica 2014;24(Suppl 1):S1-S78
S32
0.86. The optimal threshold for HbA1c for predict-
ing prediabetes in this study was 5.8-6.3% (40-45
mmol/mol Hb). Individuals with prediabetes iden-
tified with HbA1c criteria had a high body mass in-
dex, hypertension and low insulin sensitivity. The
authors conclude that each test identifies partially
different group of subjects and that HbA1c only
should not be used for detection of prediabetes.
The other study aimed to identify the optimal
threshold of HbA1c and to evaluate the predictive
performance of HbA1c in diagnosing prediabetes,
detected previously with OGTT, in a middle-aged
and elderly Han Chinese population from north-
west China (7). HbA1c with the threshold of 6.1%
showed to be an effective and convenient test for
identifying prediabetes in this population.
Recently, the report from England was presented
on the prevalence of prediabetes in individuals 16
-75 years in the period from 2003 to 2011 (8). The
data are worrisome as the prevalence of prediabe-
tes, defined as HbA1c value between 5.7- 6.4%, in-
creased markedly in this period, from 11.6% to
35.3%. Moreover, within these eight years 50.6% of
overweight individuals over 40 years developed
prediabetes. These findings were not affected by
the methodological issues as the HbA1c measur-
ing devices were calibrated (8).
Morris et al (4) published a meta-analysis of 70 pro-
spective observational studies in which partici-
pants had prediabetes at baseline defined by dif-
ferent criteria : ADA-defined IFG (100 -125 mg/dL
or 5.6-6.9 mmol/l), WHO-defined IFG (110 – 125
mg/dL or 6.1-6.9 mmol/l), IGT (140-198 mg/dL or
7.8-11.0 mmol/l) or elevated HbA1c (6.0 -6.4% or
42-47 mmol/mol). It was found that HbA1c 6.0-
6.4% identify prediabetes most similarly to ADA-
defined IFG but with nonsignificantly lower rate
than IFG combined with IGT. Clearly, the definition
of prediabetes effects the incidence rates and it
seems that HbA1c 6.0-6.4% identify individuals at
a lower diabetes risk.
Another study from England on community based
population compared diagnostic accuracy of HbA1c
in screening for impaired fasting glucose with
standard criteria 5.6-6.9 mmol/l (100-125 mg/dL) (9).
Defining prediabetes at a lower HbA1c threshold of
 14th EFLM Continuous Postgraduate Course in Clinical Chemistry and Laboratory Medicine
39 mmol/mol (5.7%) instead of 47 mmol/mol (6.4%)
increases its sensitivity in diagnosing IFG that al-
lows to detect 40% more prediabetics.
Very recent report performed on adults without
known diabetes from the National Health and Nu-
trition Examination Survey (NHANES) assessed
ROC curves of HbA1c pertaining to the diagnosis
of prediabetes by FPG and/or 2-hr OGTT (10).
When patients were diagnosed using both FPG
and OGTT the false-negative rate for HbA1c in
identifying prediabetes was 64.9% but decreased
markedly (9.2%) when HbA1c was combined with
FPG for diagnosis.
In the above presented studies the performance of
hemoglobin A1c, advocated for the diagnosis of di-
abetes and prediabetes, has been assessed in cor-
roboration with FPG or with the combination of
FPG and 2-hr OGTT glucose values. It appears that
regarding recent American Diabetes Association
and joint European Society of Cardiology and Euro-
pean Association for the Study of Diabetes guide-
lines, it is important to point out that HbA1c below
5.7% do not reliably exclude the presence of predi-
abetes. The above presented data support the idea
for greater use of oral glucose tolerance tests in
combination with FPG for diagnosis of dysglycemia.
Prediabetes in children and adolescents
The prevalence of prediabetes have increased also
among overweight and obese youth that has im-
plications for long-term health. Haemer et al re-
viewed the available literature on current screen-
ing programs, diagnosis, and treatment of predia-
betes at 25 childhood obesity hospital treatment
centers (11). They found that current prediabetes
diagnostic criteria are derived from adult-based
studies and are not modified in respect to the low-
er age group. There is however, some evidence on
beneficial effects of preventing programs in chil-
dren which result in a high proportion of obese
with prediabetes returning to normoglycemia
without pharmacotherapy.
Li et al investigated Chinese adolescents (11-16
years of age) without known diabetes and evalu-
S33
ated the performance of HbA1c in detecting pre-
diabetes as well its association with cardiometa-
bolic risk (12). Prediabetes was diagnosed accord-
ing to ADA criteria with HbA1c, FPG and OGTT in
all individuals with HbA1c within 5.7-6.4%. ROC
curve for HbA1c to identify prediabetes diagnosed
by OGTT had an AUC of 0.53 only. These findings
indicate a weak agreement between HbA1c and
FPG or OGTT in detecting prediabetes.
Future perspectives
Secreted frizzled-related protein 4 (SFRP4), a re-
cently discovered adipocytokine, has been de-
scribed as a potential biomarker of early pancreat-
ic β-cells dysfunction (13). Expression of SFRP4
mRNA and secretion of this protein from visceral
adipose tissue is increased in obesity and corre-
lates with insulin resistance. A significant inverse
correlation of SFRP4 expression in human pancre-
atic islets with insulin secretion and positive rela-
tionship with glycated hemoglobin level was ob-
served. A novel purpose for SFRP4 investigation as
a biomarker of the pancreatic islet dysfunction
was indicated. SFRP4 is elevated in the serum sev-
eral years before clinical diagnosis of diabetes has
been made and its presence increases the risk of
diabetes up to five times. Therefore, SFRP4 might
be used as an early risk predictor of prediabetes/
diabetes, especially in apparently healthy individu-
als. The above mentioned cytokine should meet
general requirements for biomarkers prior to its
application in routine clinical practice. The assay
should be standardized (preferably adapted for
automatic analyzers) with proven analytical per-
formance. Clinical performance and evaluation of
clinical efectiveness is necessary in order to con-
firm the diagnostic goal and prognostic value in
assessing risk of prediabetes/T2D. Another impor-
tant aspect is a cost-effectiveness analysis which
compares the change in costs and in health effects
of introducing new test. Despite the promissing
results on the contribution of SFRP 4 in the patho-
genesis of T2D, more detailed large population-
based studies are needed to evaluate its diagnos-
tic and clinical utility.
Biochemia Medica 2014;24(Suppl 1):S1-S78
14th EFLM Continuous Postgraduate Course in Clinical Chemistry and Laboratory Medicine
References
1. ADA Position statement: Diagnosis and classification of
diabetes mellitus.Diabetes Care 2013;36, S1, S67-74.
2. ESC Guidelines on diabetes, prediabetes and cardiovascu-
lar diseases developed in collaboration with the EASD. Eur
Heart J 2013; 34:3035-87.
3. Zhang X, Gregg EW, Williamson DF, Barker LE, Thomas W,
Bullard KW et al: A1c level and future risk of diabetes: a
systematic review. Diabetes Care 2010;33:1665-73.
4. Morris DH, Khunti K, Achana F, Srinivasan B, Gray LJ, Davies
MJ et al : Progression rates from HbA1c 6.0-6.4% and other
prediabetes definitions to type 2 diabetes: a meta-analysis.
Diabetologia 2013; 56:1498-93.
5. Yeon JY, Ko SH, Kwon HS, Kim NH, Kim JH, Kim CS: Prevalen-
ce of diabetes and prediabetes according to fasting plasma
glucose and HbA1c. Diabetes Metab J 2013; 37:349-57.
6. Vlaar EMA, Admiraal W, Busschers W, Holleman F, Nierkens
V, Middelkoop BJC et al: Screening South Asians for type 2
diabetes and prediabetes:(1) comparing oral glucose tole-
rance and hemoglobin A1c test results and (2) comparing
the two sets of metabolic profiles of individuals diagnosed
with these two tests. BMC Endocrine Disord 2013;13:1-8.
7. Wu S, Zhou C, Yi F, Zhu Y, Tuniyazi Y, Huang L et al: HbA1c
and the diagnosis of diabetes and prediabetes in a middle-
aged and elderly Han population from northwest China. J
Diabetes 2013;5:282-90.
Biochemia Medica 2014;24(Suppl 1):S1-S78
S34
8. Mainous AG, Tanner RJ, Baker R, Zayas CE, Harle CA: Pre-
valence of prediabetes in England from 2003 to 2011: po-
pulation-based cross-sectional study. BMJ Open 2014:4;
doi:10.1136/bmjopen-2014-005002.
9. Kumaravel B, Bachmann MO, Murray N, Dhatariya K, Fe-
nech M, John WG et al: Use of haemoglobin A1c to detect
impaired fasting glucose or Type 2 diabetes in a United
Kingdom community based population. Disabetes Res Clin
Pract 2012;96:211-6.
10. Guo F, Moellering DR, Garvey WT: Use of HbA1c for diagno-
ses of diabetes and prediabetes: comparison with diagno-
ses based on fasting and 2-hr glucose values and effects
of gender, race, and age. Metab Syndr Relat Disord 2014;
12:258-68.
11. Haemer MA, Grow HM, Fernandez C, Lukasiewicz GJ, Rho-
des ET, Shaffer LA. Addressing Prediabetes in Childhood
Obesity Treatment Programs: Support from Research and
Current Practice. Child Obesity 2014; 10:292-303.
12. Li P, Jiang R, Li L, Li L, Wang Z, Li X et al: Diagnostic perfor-
mance of hemoglobin A1c for prediabetes and associati-
on with cardiometabolic risk factors in Chinese adolescents
without diabetes. J Investig Med 2012;60:888-94.
13. Bergmann K, Sypniewska G.: Secreted frizzled-related pro-
tein (SFRP4) and fractalkine (CX3CL1)- potential new bio-
markers for beta-cell dysfunction in diabetes. Clin Biochem
2014;47:529-32.
 14th EFLM Continuous Postgraduate Course in Clinical Chemistry and Laboratory Medicine
Diabetes as autoimmune disease -
Diabetes type I
Evgenija Homsak
Department for Laboratory Diagnostics, University Clinical
Centre Maribor, Maribor, Slovenia
Corresponding author: evgenija.homsak@ukc-mb.si
Introduction
Type 1 diabetes mellitus (T1D), also known as insu-
lin - dependent diabetes mellitus, is a chronic im-
mune mediated disease that is characterized by
selective loss of insulin producing β cells of the
pancreatic islets in genetically susceptible sub-
jects. The majority (95%) of cases are attributable
to an autoimmune-mediated destruction of β cells
(type 1a) while a small minority (5%) of cases re-
sults from an idiopathic destruction or failure of β
cells (type 1b) (1). T1D is observed in approximate-
ly 5-10% of diabetes mellitus patient. It may be
present at any age and with equal affection of
both sexes. It appears most typically in early life
with a peak around the puberty, but one-fourth of
cases are diagnosed in adults. T1D remains the
most common form of diabetes in childhood, ac-
counting for approximately two-thirds of new di-
agnoses of diabetes in patients ≤19 years of age in
the United States, despite the increasing rate of
type 2 diabetes.  The incidence of T1D varies 50-
100 fold around the world, with the highest rates
in northern Europe, with 57.4 cases/100.000 per
year in Finland and with relatively low incidence of
0.6/100.000 in China (2). The incidence of child-
hood T1D is rising rapidly in all population, espe-
cially in the age under 5 years, that suggests a
strong environmental contribution. Lately,
through several studies, there are strong efforts to
understand and explain pathogenesis and find
the new therapeutic options of the disease ac-
cording to potential auto-antigens (insulin) and
different environmental factors, as an important
key in the development of T1D. But the role of spe-
cific factors such as viruses or ingested food (milk)
remains controversial (3,4). Diagnosis of clinical
S35
manifested T1D is based traditionally on diabetes
mellitus typical clinical symptoms: polyuria, in-
creased thirst, weight loss, weakness, hunger, re-
current infection and in severe insulinopenia the
patient are prone to diabetic ketoacidosis. Impor-
tant diagnostic parameters are also for T1D char-
acteristic HLA gene and especially different auto-
antibodies against antigens of β cells, which could
be present several years before clinical manifesta-
tion of disease.
Pathogenesis
T1D is an autoimmune disorder against the β cells
of the pancreatic islets, that remain only 10-20%
functioning at the time of diagnosis. For T1D is
characteristic subclinical prodrome of variable du-
ration, as the pathogenetic process begins years
before the clinical onset, when the tolerance to
self-autoantigens is lost (5). The whole process and
factors that contribute to and influence the de-
struction of the β cells is still not known. Still limit-
ed current knowledge of pathogenesis of T1D as
autoimmune disease is based on several impor-
tant known facts, which have been gained
through several studies, mostly using animal mod-
els and confirmed by human clinical trials.   This
process occurs in genetically susceptible subjects,
is probably triggered by one or more environmen-
tal agents, and usually progresses over many
months or years during which the subject is
asymptomatic and euglycemic. Important genetic
predisposition in the presence of not yet defined
triggers and modulators from the environment
could influence on modulation of immune system
to lose the tolerance to auto-antigens and results
with several autoimmune reactions, local inflam-
mation (insulitis), specific T cell (LyT) and B cell
(LyB) responses, autoantibody production, and cell
destructions. The proposed mechanism of autoim-
mune inflammation process start with presenta-
tion of self antigens on antigen presenting cells
(dendritic, LyB, macrophage), that after releasing
interleukin (IL)-12 activate LyT (CD4 T) that produce
important key cytokine INF-γ. They inhibit Th2 cy-
tokine production, enhance production of toxic
cytokine (IL-β, TNF-α) and free radical by mac-
Biochemia Medica 2014;24(Suppl 1):S1-S78
14th EFLM Continuous Postgraduate Course in Clinical Chemistry and Laboratory Medicine
rophage, and activate cytotoxic CD8T cells that af-
fects the β cells. Important connection between
LyT and LyB causes activation of LyB for produc-
tion of auto-antibodies. Auto-antibodies, several
cytokines, cytotoxic CD8T cells with releasing per-
forine, granzymes or by Fas-mediated apoptosis
affect and progressively destroy the β cells. Addi-
tionally local chemokine production attracts auto-
reactive lymphocytes that potentiate the destruc-
tive autoimmune process. (4,5,6) In pathogenesis
of T1D we could find some similarities with other
autoimmune diseases, like celiac disease; the com-
mon genetic susceptibility, unknown hypothetical
trigger from environment, infection, driving auto-
antigen, autoantibodies and outcome (1/5 of with
HLA conferred susceptibility progresses to clinical
disease).
Genetics: Disease susceptibility is highly associat-
ed with the inheritance or presence of certain hu-
man leukocyte antigen (HLA), which is characteris-
tic for autoimmune diseases. HLA molecules are
responsible for presentation of peptides (also au-
to-antigens) to Ly T cells and are involved in thym-
ic selection of new generated T cell repertoire
(central tolerance), to avoid potential autoimmune
clones that could be released into the periphery.
Therefore, a defect within the thymus or presence
of specific HLA molecules allows autoimmune T
cells to escape central tolerance. HLA genes on
short arm of chromosome 6p21.3 with alleles DR3
and DR4 as well as the associated alleles DQ2 and
DQ8, that are expressed either as DR3DQ2 or
DR4DQ8, are present in more than 90% of individ-
uals with T1D (7). Remain 10% of T1D might have
influence of 20 non-HLA genes. Among them are
important polymorphisms on insulin locus on
chromosome 11p5,5 (PTPN22 and INS VNTR) that
contributes approximately 10% to the familial ag-
gregation of disease (8). As with HLA, peripheral T
cell repertoires may be significantly influenced by
polymorphisms in the insulin gene affecting thy-
mocyte selection. Recent studies identifies auto-
immune disease associated polymorphisms of T
cell regulatory gene CTLA-4 (chromosome 2q22),
that reduce the efficiency of regulatory function of
LyT4CD4 cells (9). Understanding the genetics of
T1D as well the determination of susceptible pop-
Biochemia Medica 2014;24(Suppl 1):S1-S78
S36
ulation is important for predicting disease, accura-
cy of diagnosis, prognosis and treatment.
LyT and LyB involvement: Autoimmunity and in-
volvement of LyT is further supported with the
presence of specific LyT infiltrates within inflamed
islets of pancreas of patient with T1D, according to
Imagawa and co-workers, who found close corre-
lation between serological and histological mark-
ers and histological evidence of cellular autoim-
munity (10). Insulitis as autoimmune inflammation
could be present years before hyperglycemia is
evident. Ly B cells also serve as antigen presenting
cells and as autoantibodies producing cells (6).
Both Ly as important actors of disease could be
target of the future therapeutic options.
Autoantibodies: There are five disease related
autoantibodies: islet cell antibodies (ICA), insulin
autoantibodies (IAA) with epitope on B-chain of
insulin molecule, autoantibodies against 65-kDa
isoform of glutamic acid decarboxilase (GAD65),
tyrosine phosphatase related IA-2 molecule or in-
sulinoma associate antigen-2 antibodies (IA-2) and
zinc transporter protein (ZnT8). Presence of auto-
antibodies are evident years before clinical onset
and are mostly the first sign of autoimmune pro-
cess, that will or not progress to T1D. Several stud-
ies confirm their important role in prediction of
the disease development and appearance accord-
ing to the detection of different specific autoanti-
bodies (4,5,11). Mrena and co-workers in the Finn-
ish DIPP study observed that presence of positivity
for only a single autoantibody specificity for sev-
eral years represents in most cases harmless non-
progresive β cell autoimmunity, whereas the pres-
ence of two or more autoantibodies reflects a pro-
gressive process (10). But their direct pathogenetic
role is controversial, since transfer of autoantibod-
ies, using serum of diabetic humans, alone did not
reconstitute disease and that plasmapheresis pro-
vides little therapeutic benefit (6,12).
Environment factors as a triggers and drivers
of disease: There is still unexplained cause of ini-
tiation of the autoimmune process and why the
auto-antigens become auto-antigenic. The factors
that control progression from insulitis (inflamma-
tion of the β cells) to diabetes remain largely un-
 14th EFLM Continuous Postgraduate Course in Clinical Chemistry and Laboratory Medicine
known. Understanding this may provide new op-
portunities for preventing disease among popula-
tion with high risk for developing T1D or halt pro-
gression of the β cells lost.
There are recently several approaches for pre-
venting studies that might contribute to knowl-
edge of disease process. The lack of complete
concordance among monozygotic twins (only 20-
40%) indicates that both genetic and environ-
mental factors contribute to the pathogenesis of
T1D. There are several environmental candidates
that might trigger and modulate this autoim-
mune process: early introduction of milk food
(bovine insulin, milk casein), entero-viruses (rubel-
la, coxsackie), infection, vitamin D. But unfortu-
nately resent studies gave us controversial results
and have not been able yet to confirm their une-
quivocal role. And there are also several autoanti-
gens as trigger candidates: insulin, GAD65, which
could represent the potential future therapeutic
target. (4,5,6)
Diagnosis
The presence of autoantibodies confirms the T1D.
But they are also capable to identify insulin-requir-
ing older patients who are initially diagnosed with
type 2 diabetes. They typically have GAD65 or IA-
2. These adult patients have a form of latent auto-
immune diabetes in adult (LADA) or slow progres-
sive insulin dependent diabetes mellitus (SPIDDM)
(6). They can have pronounced hyperglycemia and
after therapy with oral hypoglycemic agents for
several months or years, they may become insulin
dependent.
Diagnosis of T1D is still based on typical clinical
symptoms, as the consequences of the end stage
of progressed disease. Determination and moni-
toring of glucose levels by measuring concentra-
tion of glucose, glycosylated haemoglobin
(HbA1c), fructosamine, according to ADA and WHO
criteria, is important. But promising screening
marker for general population would be genetics
and autoantibodies that could define the risk pop-
ulation for possible preventing treatment in the
future.
S37
Thus, genetic markers for T1D are present from
birth, immune markers are detectable after the
onset of the autoimmune process, and metabolic
markers can be detected with sensitive tests once
enough β cell damage has occurred, but before
the onset of symptomatic hyperglycemia. This
long latent period is a reflection of the large num-
ber of functioning β cells that must be lost before
hyperglycemia occurs.
New therapeutic options
The complete puzzle of pathogenetic process of
T1D, with known all (f)actors, will give us the op-
portunity for new therapeutic options. Recent
studies are concern on immune therapy at three
different stages. Primary prevention is treatment
of individuals at increased genetic risk, without
known presence of autoantibodies. There were
and still are several ongoing studies that try to find
out probable environment factors (nonautoanti-
gens) and autoantigens as well, that could as ther-
apeutic agent reduce T1D incidence in genetically
predisposed infants (hydrolyzed casein milk for-
mula, Vitamin D, insulin). Secondary prevention is
targeted at individuals with persistent islet au-
toantibodies. Ongoing trials involve non-autoanti-
gen and autoantigen specific therapies (Bacillus
Calmette-Guerin vaccine, anti-CD3 monoclonal
antibodies, oral and nasal insulin, recombinant
GAD65). Trial interventions at onset of T1D also in-
cluded non- and autoantigen approaches (proin-
suline peptide). According to current results, pri-
mary prevention studies are the major goal in the
future as would aim to induce immunological tol-
erance to islet autoantigens. Unfortunately com-
pleted secondary prevention and intervention tri-
als show little promise of achieving the preserva-
tion of β cell function. (5)
There are, as already used and as a future direction
for managing the onset T1D disease, new ap-
proaches of different alternative source of islet
cells: as pancreas transplant, islet cell transplant,
xenogenic islet cells (humanized pig islet cells), ex-
pansion and trans-differentiation of pancreatic
duct cells, fetal pancreatic stem cells and β cell
Biochemia Medica 2014;24(Suppl 1):S1-S78
14th EFLM Continuous Postgraduate Course in Clinical Chemistry and Laboratory Medicine
precursors, embryonic stem cells, and engineering
other cells to produce insulin (duodenal K cells,
hepatocytes, pituitary cells have been successfully
transfected). (6)
Conclusion
Diagnosis of T1D is no longer the matter of only
young population, despite the incidence in early
childhood rising in world population. The recogni-
tion of LADA type is becoming important also for
the adult patients, who have been previously not
References
1. American Diabetes Association. Diagnosis and classifica-
tion of diabetes mellitus. Diabetes Care. 2013; 36(Suppl 1):
S67-S74.
2. Patterson CC, Dahlquist GG, Gyürüs E, Green A, Soltész G,
EURODIAB Study Group. Incidence trends for childhood
type 1 diabetes in Europe during 1989–2003 and predicted
new cases 2005–20. Lancet 2009;373(9680):2027-33.
3. Gale EAM. Spring harvest? Reflections on the rise in type 1
diabetes. Diabetologia 2005;48:2445-50.
4. Knip M, Veijola R, Virtanen SM, Hyöty H, Vaarala O,
Åkerblom HK et al. Environmental triggers and determi-
nants of type 1 diabetes. Diabetes 2005; 54 (Suppl.2): S125-
S136.
5. Lenmark Ä, Larsson HE. Immune therapy in type 1 diabetes
mellitus. Nat Rev Endocrinol 2013; 9: 92-103.
6. Khardori R, Pauza ME. Type 1 diabetes mellitus: Pathoge-
nesis and advances in therapy. Int.J.Diab. Dev. Countries.
2003; 23: 106-19.
7. Pociot F, McDermott MF. Genetics of type 1 diabetes melli-
tus. Genes Immun 2002(5): 235-49.
Biochemia Medica 2014;24(Suppl 1):S1-S78
S38
diagnosed. Both populations need reasonable
treatment and possible prevention steps as well to
prevent the disease progress to the end stage. Im-
portance of understanding the complete patho-
genic process of T1D is very important not only for
diagnoses approaches, but also for prediction and
probable prevention of disease in genetic suscepti-
ble or general population. According to these
knowledge there would be the possibility for crea-
tion and developing the new therapeutic approach-
es that will help manage the disease on several
points of its development, especially to prevent islet
autoimmunity or halt progressive β cell destruction.
8. Bennet ST, Todd JA. Human type 1 diabetes and the insu-
lin gene: principles of mapping polygenes. Ann Rev Genet
1996; 30: 343-70.
9. Ueda H1, Howson JM, Esposito L, Heward J, Snook H,
Chamberlain G at al. Association of the T-cell regulatory
gene CTL4 with susceptibility to autoimmune disease. Na-
ture 2003; 423 (6939): 506-11.
10. Imagawa A, Hanafusa T, Tamura S, Moriwaki M, Itoh N, Ya-
mamoto K at al. Pancreatic biopsy as a procedure for de-
tecting in situ autoimmune phenomena in type 1 diabetes:
close correlation between serological markers and histo-
logical markers and histological evidence of cellular auto-
immunity. Diabetes 2001; 50(6): 1269-73.
11. Mrena S, Savola K, Kulmala P, Akerblom HK, Knip M. The
Childhood Diabetes in Finland Study Group: Natural cour-
se of preclinical type 1 diabetes in siblings of affected chil-
dren. Acta Pediatr 2003; 92: 1403-10.
12. Lenmark A. Contolling the controls: Gad65 autoreactive T
cells in type 1 diabetes. J clin Invest 2002; 109: 869-70.
 14th EFLM Continuous Postgraduate Course in Clinical Chemistry and Laboratory Medicine
POC testing instruments for diagnosing
and monitoring diabetes in clinical
settings
Sverre Sandberg
The Norwegian Quality Improvement of Primary Care
Laboratories (NOKLUS), University of Bergen, Bergen, Norway
Corresponding author: sverre.sandberg@isf.uib.no
Abstract
POC HbA1c is commonly used for monitoring of di-
abetes mellitus. In a monitoring situation more em-
phasis is usually placed on precision and less on
trueness. Concerning HbA1c, however, one can ar-
gue that the same quality specifications should be
used both for monitoring and diagnosing since
many of the recommendations for good practice
deal with absolute HbA1c numbers and not only
“improvement” or “deterioration” of the diabetic
condition. An expert committee officially recom-
mended to use HbA1c for the diagnosis of diabetes
in 2009 (1). There are several advantages using
HbA1c compared with glucose such as pre-analyti-
cal stability of the sample and low within-subject
biological variation of HbA1c (1). Furthermore,
HbA1c is stable throughout the day and fasting and
dietary restrictions are therefore avoided. World
Health Organization (WHO) recommends an HbA1c
level of 6.5% (48 mmol/mol) as the cut off point for
diabetes, and the assays must be “standardized to
criteria aligned to the international reference val-
ues” (2). The College of American Pathologist (CAP)
recommends that the EQA acceptable limits for ac-
curacy should be 7% in 2012 and 6% in 2013 com-
pared to a target value (6). Furthermore, NACB rec-
ommends a within-laboratory CV <2% and a single
method should have a between-laboratory CV
<3% (3). All CVs are based on the DCCT (Diabetes
Control and Complications Trial)/NGSP units (3). It is
well known that these CVs will be higher if IFCC
(mmol/mol) units are used since the NGSP units are
not on the ratio scale. NACB recommends using
hospital laboratory HbA1c instruments for diagno-
S39
sis of diabetes since POC HbA1c assays are “current-
ly not sufficiently accurate for this purpose” (3). It is
interesting that when using glucose for diagnosing
and monitoring diabetes no quality specifications
have be set except for glucometers for self-moni-
toring and for glucometers in critical care units.
Whether the POC instruments should be used for
monitoring or diagnosing, within-subject biologi-
cal variation is of great importance, both from
“healthy” people and from stable diabetic patients.
We have found that the within-subject variation for
HbA1c is 1.2% and 1.7% for healthy and diabetes
patients respectively. The corresponding numbers
for glucose is 5.4 and 30.5% (4). To be able to judge
if POC instruments can be used for diagnosing and
monitoring, it is important to look at results from
studies where a) the instruments are evaluated un-
der optimal conditions, b) the instruments are eval-
uated in the hands of the users and c) the instru-
ments are evaluated after they have been on the
market for some time, for example the perfor-
mance in an EQA scheme.
A recently published study evaluated 7 HbA1c POC
instruments and concluded that Afinion, DCA Van-
tage, Cobas B101, and B-analyst instruments met
the generally accepted performance criteria for
HbA1c. Quo-Test, Quo-Laboratory, and InnovaStar
met the criteria for precision but not for bias. The
paper also concluded that roficiency testing
should be mandated for users of HbA1c POC as-
says to ensure quality (5). This study was based on
laboratory experiments following the CLSI EP-5
guidelines and not longitudinal results from clini-
cal practice. In a recent study (6) results from 13
HbA1c external quality assurance surveys (EQAS)
during six years in from both GPs offices using
POC instruments and from hospital laboratory in-
struments were compared with the recommend-
ed analytical quality specifications for using HbA1c
diagnostically for diabetes mellitus. All General
Practice and hospital laboratories measuring
HbA1c in Norway participated in the EQAS. Be-
tween 60 - 90% of Afinion and DCA users and hos-
pital laboratories performed HbA1c measurements
within the quality specifications for both trueness
(6.0%) and imprecision (CV ≤ 2.0%) in two levels in
each EQA survey.
Biochemia Medica 2014;24(Suppl 1):S1-S78
14th EFLM Continuous Postgraduate Course in Clinical Chemistry and Laboratory Medicine
In conclusion, results indicate that POC instru-
ments for HbA1c can be very useful for monitoring
and diagnosing diabetes mellitus. The same is true
for POC glucometers although usually not quality
specifications are set for the use of these instru-
References
1. The International Expert Committee. International expert
committee report on the role of the A1c assay in the dia-
gnosis of diabetes. Diabetes Care 2009;32:1327–34.
2. World Health Organization. Use of glycated haemoglobin
(HbA1c) in the diagnosis of diabetes mellitus. Abbreviated
report of a WHO consultation. WHO/NMH/CHP/CPM/11.1
ed. Geneva, World Health Organization. WHO; 2011
3. Sacks DB, Arnold M, Bakris GL, Bruns DE, Horvath AR, Kir-
kman MS, et al. Guidelines and recommendations for labo-
ratory analysis in the diagnosis and management of dia-
betes mellitus. Clin Chem 2011;57:e1–e47.
Biochemia Medica 2014;24(Suppl 1):S1-S78
S40
ments in all different clinical situations. However, a
presupposition for using these POC instruments
for diagnosing and monitoring diabetes mellitus is
that a stringent quality assurance program is es-
tablished to monitor the quality.
4. Carlsen S, Petersen PH, Skeie S, Skadberg Ø, Sandberg S.
Within-subject biological variation of glucose and HbA(1c)
in healthy persons and in type 1 diabetes patients. Clin
Chem Lab Med 2011;49:1501–7.
5. Lenters-Westra E, Slingerland RJ. Three of 7 Hemoglo-
bin A1c Point-of-Care Instruments do not meet generally
accepted analytical performance criteria. Clin Chem. 2014,
in press
6. Sølvik UØ, Røraas T, Christensen NG, Sandberg S. Diagno-
sing diabetes mellitus: performance of hemoglobin A1c
point-of-care instruments in general practice offices. Clin
Chem. 2013;59:1790–801.
 14th EFLM Continuous Postgraduate Course in Clinical Chemistry and Laboratory Medicine
The impact of preanalytical factors on
glucose concentration measurement
Nora Nikolac
University Department of Chemistry, Medical School University
Hospital Sestre Milosrdnice, Zagreb, Croatia
Corresponding author: nora.nikolac@gmail.com
Glucose is the most commonly ordered test in a
clinical chemistry laboratory accounting for about
30-40% of the total laboratory workload. Measure-
ment of glucose concentration is done in all types
of samples: capillary and venous whole blood and
plasma, serum, pleural fluid, ascites, cerebrovascu-
lar fluid and urine. Most of these measurements
are included into healthcare of patients with dia-
betes mellitus. Diabetes mellitus is diagnosed
based on the well-established cut-off values origi-
nating from the worldwide-accepted guidelines.
When performing glucose tolerance tests, chang-
es in glucose concentration indicate the degree of
glucose metabolism impairment. Therefore, all
factors influencing glucose concentration variabil-
ity have to be minimized in order to obtain accu-
rate results. Fortunately, nowadays, there are prac-
tically no analytical challenges for glucose concen-
tration measurement. Automated methods used
in the laboratories fulfil strict criteria with very low
analytical variability. However, the biggest pitfall,
as for all other laboratory measurements, lies in
the preanalytical phase, which is, almost exclusive-
ly, responsible for the errors in glucose concentra-
tion measurement.
A large number of preanalytical factors like sample
type, transport conditions, time from blood sam-
pling, temperature and type of test tube influence
glucose concentration. Each of these factors intro-
duces a certain degree of variability. Their cumula-
tive effect can result with the error of the measure-
ment that can wrongly be attributed to the clinical
condition.
Glucose can be determined in the venous and in
the capillary sample. It is widely accepted that fast-
ing glucose concentration is similar in the venous
S41
and capillary blood. However, postprandial glu-
cose and glucose measured after glucose load in
tolerance tests differ significantly between venous
and capillary sample. Capillary plasma glucose is
higher because of the rate of the glucose con-
sumption in the tissues (1,2). This difference can be
as high as 19%, as observed in our recently per-
formed study of the quality of the capillary sample
in non-fasting healthy volunteers (3). Therefore,
currently used recommendations issued by the
American Diabetes Association do not support us-
ing capillary sample for diagnosing diabetes in
non-pregnant adults (4). However, many laborato-
ries still use capillary and venous sample inter-
changeably for the random glucose concentration
measurement, especially in children. To compli-
cate things even further, the practice of alternat-
ing between serum and plasma samples, especial-
ly in the emergency services, is also widespread.
Studies have confirmed that there are significant
differences in glucose concentration between se-
rum and plasma (5). Glucose is higher in plasma
than in serum because of the lower water content
of the plasma. Additionally, some authors specu-
late that glucose is consumed when the clotting
occurs. Interchangeable usage of different sam-
ples that significantly differ in glucose concentra-
tion, can lead to numerous errors. This practise
can cause repeated testing, unnecessary perform-
ing of glucose tolerance tests and result in pro-
longed turn-around-time and delay in diagnosis.
Therefore, single sample type, ideally venous plas-
ma, has to be used for glucose concentration
measurement. If other types of samples are used,
this has to be recorded on the test report, with the
explanation of the potential influence on the re-
sult.
The critical point in glucose preanalytical variabili-
ty is the continuation of the ex vivo glycolysis in
the test tube. The rate of glycolysis is enhanced
with the high number of leukocytes, causing large
interindividual differences in the degree of loss of
glucose. During the years, many efforts have been
done in order to minimize this process. Several
types of additives have been investigated. Up to
recently, tubes containing Iodoacetate have been
used. Iodoacetate acts as a glycolysis inhibitor by
Biochemia Medica 2014;24(Suppl 1):S1-S78
14th EFLM Continuous Postgraduate Course in Clinical Chemistry and Laboratory Medicine
inactivating glyceraldehydes-3-phosphate dehy-
drogenase. D-mannose is a competitive hexoki-
nase inhibitor in the first glycolysis reaction, but
this effect of inhibition lasts for a very short period
of time. Additionally, mannose interferes with the
glucose measurement if glucose-oxidase or
hexokinase methods are used. The procedure that
effectively inhibits ex vivo glycolysis is placing the
tube on ice immediately after collection and sepa-
rating the sample from the cells. However, be-
cause of the technical inability, especially of the
distant phlebotomy sites to perform this process,
test tubes containing sodium fluoride are nowa-
days routinely used to stop the glycolysis. In the
presence of inorganic phosphates, fluoride forms
a fluorophosphates ion that is bound to the mag-
nesium in the enolase enzyme. This reaction inhib-
its the enzyme and stops glycolysis.
Although, this was a gold standard practise used
for many years and implemented in all guidelines,
numerous studies have confirmed that fluoride
does not stop glycolysis in the first two hours after
the sampling. Glucose concentration gradually de-
creases during that period and stabilisation is ob-
tained after 90-120 minutes. When comparing glu-
cose concentration between heparin plasma sam-
ples centrifuged at phlebotomy site and sodium
fluoride plasma samples centrifuged after trans-
port to the central laboratory, Shi et al. discovered
that glucose was approximately 0.39 mmol/L
(~5%) higher in lithium heparin plasma sample,
proving that glycolysis still occurs in fluoride sam-
ple (6). Waring et al. did a similar experiment using
serum separator tubes (SST tubes) (7). They have
also confirmed that the glucose concentration was
5% higher in the serum than in the sodium fluo-
ride plasma. Both of these authors, and many oth-
ers, have concluded that the separation from the
cells and refrigeration of the sample are superior
to sodium fluoride for stopping glycolysis (8,9).
Fernandez et al. have observed another interesting
finding concerning sodium fluoride tubes. When
comparing NaF tubes with SST tubes, they have dis-
covered that 86.2% of the sodium fluoride samples
had free haemoglobin concentration > 0.15 g/L. In-
terestingly, in the SST tubes that rate was only 2.2%.
This very high hemolysis rate can potentially influ-
Biochemia Medica 2014;24(Suppl 1):S1-S78
S42
ence glucose concentration, since red blood cells
contain lower glucose concentration than serum.
Catalase that is released from red blood cells during
hemolysis can further lower glucose concentration
in hemolysed samples (10).
It has been postulated that the 30-90 minutes de-
lay of inhibition of glycolysis in the sodium fluo-
ride tube can be explained by the period that is
necessary for the fluoride ion to enter the red
blood cells. However, Mikesh LM and Burns DE
have disproven this hypothesis (11). They have in-
vestigated influence of the sodium fluoride on
the change of lactate and glucose concentration
and discovered that fluoride almost instantly in-
hibits the production of lactate, while the glucose
concentration slowly decreases. If there was a de-
lay in fluoride ion influx to the cell, the concentra-
tion of the lactate would not be stabilised imme-
diately. Therefore, the authors conclude that fluo-
ride inhibits enolase within of 5 minutes of addi-
tion to the blood, while enzymes in the upstream
path of glycolysis remain active. Production of
lactate is inhibited because piruvate cannot be
formed, but glucose is still metabolized into glu-
cose 6-phosphate and other phosphorylated me-
tabolites that are accumulating within the cell.
Other pathways may metabolise phosphorylated
sugars, until steady state is obtained. Phosphoryl-
ation of glucose will continue until there is availa-
ble supply of ATP in the cell. Supply of ATP is ex-
hausted about 60 minutes after addition of the
fluoride and then glucose concentration stabilises
in the tube (12).
Based on the study of Mikesh and other authors, it
is evident that fluoride alone cannot effectively
stop the glycolysis and new solutions have to be
implemented. In the year 1986, Uchida K, Okuda S,
and Tanaka K by Terumo Corporation, Japan has
reported a patent where they have proven that
acidification of blood immediately stops ex-vivo
glycolysis. Low pH value effectively inhibits
hexokinase and phosphofructokinase, enzymes
that are active early in the Embden–Meyerhof
pathway. Glycolysis is inhibited instantly in eryth-
rocytes, leukocytes and platelets when the blood
ph of 5.3 and 5.9 is obtained by adding citrate
buffer. This inhibition can last up to 10 hours at
 14th EFLM Continuous Postgraduate Course in Clinical Chemistry and Laboratory Medicine
room temperature. Their patented additive con-
tained a mixture of citric acid, trisodium citrate,
disodium EDTA (to chelate magnesium), and NaF
(for inhibition over a longer period of time) in a
gravimetric ratio of 3.4:1.6:4.8:0.2 (10 mg/mL
blood). This was incorporated into Venosafe® Gly-
caemia test tubes from the Terumo Corporation.
Recently, test tubes containing citrate buffer have
been available on the market and several studies
of their performance have been done. Gambino
has performed a stability study of the this test
tube, and showed that without the separation
from the cells, glucose concentration dropped
only 0.3% after 2 hours, while after 24 hours the
decrease was only 1.2%. In comparison, in fluoride
test tube, decrease after two hours was 4.5% and
after 24 hours 7% (13,14). Because of the piling evi-
dence on superiority of the citrate tube in compar-
ison with the fluoride tubes, these findings were
incorporated into guidelines. In the document is-
sued by the American Association for Clinical
Chemistry and American Diabetes Association in
the 2011, the authors propose that sample should
be put immediately in icy-water slurry, and plasma
separated from the cells within 30 minutes. If this
cannot be obtained, a tube containing rapid inhib-
itor of glucose such as citrate buffer should be
used. Tubes with only NaF are not enough to pre-
vent glycolysis (15).
Since then this tube was widely implemented, and
several studies of the clinical impact of the tube
change have been reported. del Pino et al. have
found an increase of 6% in prevalence of positive
results in screening test for gestational diabetes,
3.9% increase in confirmatory test for gestational
diabetes and 4.5% increase in positive oral glucose
S43
tolerance test for non-pregnant adults, however
two latter were not statistically significant (16).
Norman M. et al. have also investigated clinical im-
pact of change in glucose testing tube. They have
compared mean glucose values obtained from the
laboratory information system prior, during and
after change from fluoride tubes to fluoride/citrate
tubes. They have discovered a difference in mean
values is +0.80 mmol/L, which represents a 14% in-
crease. Because there were no other changes in
laboratory analyzers, reagents or equipment, this
difference is attributed solely on inhibition of loss
of glucose by glycolysis in the first two hours in
the fluoride tube that is now prevented. Based on
the distribution of glucose concentration, they
have calculated that approximately 18% of pa-
tients were wrongly diagnosed based on the glu-
cose fasting concentration (17).
This problem could be expected since all estab-
lished cut-off values for diabetes diagnosing are
determined using sodium fluoride tubes. Even if it
is proven that the citrate tube is superior and the
concentration of glucose is measured more accu-
rately, the change from fluoride to the citrate tube
has a significant clinical impact. All laboratories
that introduce the new tube into routine practise
should communicate this change with the clini-
cians and inform them of the expected effect on
the laboratory result. It is now necessary to rede-
fine cut-off values for diagnosis and monitoring
diabetes patients using the new citrate tube.
Preanalytical variability can significantly influence
laboratory results. All factors contributing to the
variability have to be carefully monitored to pre-
vent preanalytical errors that can cause harm to
the patient.
Biochemia Medica 2014;24(Suppl 1):S1-S78
14th EFLM Continuous Postgraduate Course in Clinical Chemistry and Laboratory Medicine
References
1. Kuwa K, Nakayama T, Hoshino T, Tominaga M. Relationshi-
ps of glucose concentrations in capillary whole blood, ve-
nous whole blood and venous plasma. Clin Chim Acta
2001;307:187-92.
2. Kruijshoop M, Feskens EJ, Blaak EE, de Bruin TW. Validation
of capillary glucose measurements to detect glucose into-
lerance or type 2 diabetes mellitus in the general populati-
on. Clin Chim Acta 2004;341:33-40.
3. Simundic AM, Nikolac N, Dukic K, Zoricic M. Quality of the
capillary sample in non-fasting healthy volunteers. (unpu-
blished results)
4. American diabetes Association. Standards of Medical Care
in Diabetes 2014. Diabetes Care 2014;37(Suppl 1):S14-80.
5. Frank EA, Shubha MC, D’Souza CJ. Blood glucose determi-
nation: plasma or serum? J Clin Lab Anal 2012;26:317-20.
6. Shi RZ, Seeley ES, Bowen R, Faix JD. Rapid blood separati-
on is superior to fluoride for preventing in vitro reductions
in measured blood glucose concentration. J Clin Pathol
2009;62:752-3.
7. Waring WS, Evans LE, Kirkpatrick CT. Glycolysis inhibitors
negatively bias blood glucose measurements: potential im-
pact on the reported prevalence of diabetes mellitus. J Clin
Pathol 2007;60:820-3.
8. Gambino R, Bruns DE. Stabilization of glucose in blood
samples: out with the old, in with the new. Clin Chem Lab
Med 2013;51:1883-5.
9. Gambino R. Sodium fluoride: an ineffective inhibitor of
glycolysis. Ann Clin Biochem 2013;50:3-5.
Biochemia Medica 2014;24(Suppl 1):S1-S78
S44
10. Fernandez L, Jee P, Klein MJ, Fischer P, Perkins SL, Brooks
SP. A comparison of glucose concentration in paired speci-
mens collected in serum separator and fluoride/potassium
oxalate blood collection tubes under survey ‘field’ conditi-
ons. Clin Biochem 2013;46:285-8.
11. Mikesh LM, Bruns DE. Stabilization of glucose in blood
specimens: mechanism of delay in fluoride inhibition of
glycolysis. Clin Chem 2008;54:930-2.
12. Mikesh LM, Bruns DE. Stabilization of glucose in blood
specimens: mechanism of delay in fluoride inhibition of
glycolysis. Erratum. Clin Chem 2008;54:1261.
13. Gambino R, Piscitelli J, Ackattupathil TA, Theriault JL, An-
drin RD, Sanfilippo ML, Etienne M. Acidification of blo-
od is superior to sodium fluoride alone as an inhibitor of
glycolysis. Clin Chem 2009;55:1019-21.
14. Bruns DE. Are fluoride-containing blood tubes still needed
for glucose testing? Clin Biochem 2013;46:289-90.
15. Sacks DB, Arnold M, Bakris GL, Bruns DE, Horvath AR, Kir-
kman MS, et al. Executive summary: guidelines and re-
commendations for laboratory analysis in the diagno-
sis and management of diabetes mellitus. Clin Chem
2011;57:793-8.
16. del Pino IG, Constanso I, Mourín LV, Safont CB, Vázquez PR.
Citric/citrate buffer: an effective antiglycolytic agent. Clin
Chem Lab Med 2013;51:1943-9.
17. Norman M, Jones I. The shift from fluoride/oxalate to acid
citrate/fluoride blood collection tubes for glucose testing -
the impact upon patient results. Clin Biochem 2014;47:683-
5.
 14th EFLM Continuous Postgraduate Course in Clinical Chemistry and Laboratory Medicine
Self measurement of glucose – how
useful is it and how can it be done
Sverre Sandberg
The Norwegian Quality Improvement of Primary Care
Laboratories (NOKLUS), University of Bergen, Bergen, Norway
Corresponding author: sverre.sandberg@isf.uib.no
Abstract
Large amounts of money are used for self moni-
toring of blood glucose (SMBG) instruments al-
though several systematic reviews have shown
conflicting evidence when the effect of SMBG on
long term complications of diabetes mellitus or
decrease of HbA1c have been evaluated, especially
concerning the effect in type II DM not using insu-
lin. Whereas in persons with diabetes using insulin
more than three times a day, the evidence of use-
fulness of SMBG is high (1), the evidence in diabet-
ic persons not using insulin is dubious and de-
pendent on the conditions. The International Dia-
betes Federation recommends that in persons
with non-insulin treated type2 DM “SMBG should
be used only when individuals with diabetes (and/
or their care-givers) and/or their healthcare pro-
viders have the knowledge, skills and willingness
to incorporate SMBG monitoring and therapy ad-
justment into their diabetes care plan in order to
attain agreed treatment goals” (2). Another impor-
tant point in this recommendation is that “The
purpose(s) of performing SMBG and using SMBG
data should be agreed between the person with
diabetes and the healthcare provider. These
agreed-upon purposes/goals and actual review of
SMBG data should be documented”. And then to
link clinical use to analytical performance, the last
recommendation reads: “SMBG use requires an
easy procedure for patients to regularly monitor
the performance and accuracy of their glucose
meter”.
Thus to summarize, there are some presupposi-
tions that should be present for successful SMBG
monitoring
S45
a) Instruments should have good enough quality.
b) Patients should be able to use the instruments.
c) Patients should be able to interpret the results
and take actions when necessary.
Quality specifications should be set for these in-
struments and the users should definitely be in-
volved. In theory such quality specifications can
vary from situation to situation. Quality specifica-
tions, based on how results from the instruments
are interpreted by the diabetic patients, showed
that imprecision should be less than 5% and bias
less than 5% (3). The new ISO 15197:2013 dealing
with self-measurement of glucose states that 95%
of the results must be within ±0.83 mmol/L at glu-
cose concentrations <5.55 mmol/L and within
±15% at glucose concentrations ≥5.55 mmol/L.
This is slightly stricter compared to the limits in
ISO 15197 from 2003. Although such criteria can be
met when well-trained technicians are using the
instruments, they are often not fulfilled by pa-
tients for all instruments although the instruments
seems to improve as a function of time.
We have developed a standardized protocol
where evaluations of glucometers were carried
out both by a technicians and patients (with train-
ing and without training) and with three different
lots of strips. In 2008 we evaluated nine different
instruments (4) have been by this method and in
2014 13 systems were evaluated. The results from
these evaluations will be presented.
Users of SMBG can benefit from participation in
external quality assurance systems (5) and by com-
paring their results with larger instruments at the
GP offices or pharmacies (6).
Self-monitoring of blood glucose (SMBG) has been
available for patients with diabetes for more than
30 years. Today, SMBG is important components in
diabetes management, helping patients achieve
and maintain normal blood glucose concentra-
tions. Implementation of SMBG as an effective gly-
caemic control tool requires that instruments have
acceptable analytical quality, that the patients are
educated in using them, and that actions are taken
upon the results. This presentation will give an
overview of the use and usefulness of SMBG.
Biochemia Medica 2014;24(Suppl 1):S1-S78
14th EFLM Continuous Postgraduate Course in Clinical Chemistry and Laboratory Medicine
References
1. American Diabetes Association. Standards of medical care
in diabetes – 2007. Diabetes Care 2007;30: S4–S41.
2. Self-Monitoring of Blood Glucose in Non-Insulin-Treated
Type 2 Diabetes. Recommendations based on a Works-
hop of the International Diabetes Federation Clinical Gu-
idelines. Available at: http://www.idf.org/webdata/docs/
SMBG_EN2.pdf, Accessed on 2nd July 2014.
3. Kristensen GBB, Sandberg S. Self-monitoring of blood
glucose with a focus on analytical quality: an overview. Cli-
nical Chemistry and Laboratory Medicine. 2010;48:963–72.
Biochemia Medica 2014;24(Suppl 1):S1-S78
S46
4. Kristensen GB. Monsen G. Skeie S. Sandberg S. Standardi-
zed evaluation of nine instruments for self-monitoring of
blood glucose. Diabetes Technol Ther 2008;10:467–77.
5. Kristensen GB, Nerhus K, Thue G, Sandberg S. Results and
feasibility of an external quality assessment scheme for se-
lf-monitoring of blood glucose. Clin Chem. 2006;52:1311–
17.
6. Kjome RLS, Nerhus K, Sandberg S. Implementation of a
method for glucose measurements in community pharma-
cies. Int J Pharm Pract. England; 2010;18:13–9.
 14th EFLM Continuous Postgraduate Course in Clinical Chemistry and Laboratory Medicine
Can glycated albumin assist in
management of diabetes mellitus?
Jasna Lenicek Krleza
Department of Laboratory Diagnostics, Children’s Hospital
Zagreb, Zagreb, Croatia
Corresponding author: jlenicek@gmail.com
Introduction
Diabetes mellitus can be described as a group of
disorders in carbohydrate metabolism in which
glucose is produced in excess amounts, leading to
hyperglycemia. Diabetes usually occurs in three
forms: type 1, type 2 and gestational. There are
other forms of the syndrome, but they are quite
rare.
Diabetes is prevalent around the world. According
to the International Diabetes Federation, approxi-
mately 382 million people were estimated to have
the disease in 2013. This prevalence is rapidly in-
creasing, with the total number of affected indi-
viduals around the world expected to double by
2030 (1,2).
High blood glucose concentrations cause numer-
ous types of negative effects, which can lead over
time to tissue dysfunction, organ failure, and life-
threatening health complications. For example, di-
abetes increases the risk of heart and kidney dis-
ease, stroke, blindness, peripheral blood vessel
disease, peripheral neuropathy, foot ulcers and
complications that require amputation of the low-
er extremities.
The concept of “prediabetes” has been coined to
indicate fasting glucose levels that are higher than
normal but not high enough to warrant a diagno-
sis of diabetes. Type 2 prediabetes increases the
risk of cardiovascular disease, but its symptoms
have not been clearly defined (3).
Many long-term effects of diabetes are due to pro-
tein glycation associated with the disease. Glyca-
tion, a non-enzymatic Maillard reaction, occurs
when glucose molecules spontaneously react with
the amine group of proteins, giving rise to stable
S47
ketoamines. The fraction of proteins that are gly-
cated is elevated under hyperglycemic conditions,
and these modified proteins cause chronic diseas-
es as diabetes complications. Table 1 highlights
several important proteins that can become ex-
tensively glycated under hyperglycemic condi-
tions (4).
Some of these glycated proteins can be used as
biomarkers to determine the degree of glycemia
in individuals with diabetes or prediabetes. Glycat-
ed hemoglobin (HbA1c) is the recommended and
most often used biomarker for assessing hypergly-
cemia in the clinic. Measuring levels of HbA1c pro-
vides a measure of glycemia valid for 2-3 months,
the same as the lifespan of erythrocytes. Today,
HbA1c is the “gold standard” for assessing glyce-
mia during diabetes management, and since 2009
it has been recommended by both the American
Diabetes Association (5) and the World Health Or-
ganization (6) as a diagnostic criterion for diabetes,
with a diagnostic cut-off of >6.5% (48 mmol/mol).
Levels of HbA1c correlate linearly with average glu-
cose concentration in most patients with diabetes
(7). In some patients, however, levels of HbA1c are
inadequate for determining the average glucose
concentration and therefore for assessing glycemic
state; in such patients, this biomarker is useless for
predicting diabetes complications likely to devel-
op. Lack of linear correlation between HbA1c levels
and average glucose concentration arises because
numerous factors affect HbA1c levels, including
genetics, hematological factors and the presence
of certain comorbidities, such as hemoglobinopa-
thy, certain anemias, and disorders associated with
shorter erythrocyte lifespan (Table 2)(6).
Diabetes management, especially in early phases,
requires frequent monitoring because significant
changes can occur within 2-3 months. Such moni-
toring is particularly important for individuals on
therapy to treat prediabetes, patients undergoing
new therapy or a change in their current therapy,
individuals on intensive insulin therapy during ear-
ly stages of diabetes, pregnant women and pa-
tients on hemodialysis (4,6).
For such patients for whom HbA1c levels cannot di-
rectly be used to assess glycemic state, Cohen et al.
Biochemia Medica 2014;24(Suppl 1):S1-S78
14th EFLM Continuous Postgraduate Course in Clinical Chemistry and Laboratory Medicine

Table 1. Proteins susceptible to glycation, particularly under hyperglycemic conditions.*

Matrix proteins Enzymes Plasma proteins

Collagen Cathepsin B Albumin
Myelin Lysozyme Immunoglobulin
Fibronectin Pancreatic ribose Apo A-I, II
Fibrin Copper/zinc SOD Apo B
Carbonate dehydratase Apo C-I
Membrane proteins β-N-acetyl hexominase Apo E
Red cell Glu transport protein Alcohol dehydrogenase Haptoglobin
Red cell spectrin Aldose reductase Ferritin
Red cell membrane protein Aldehyde reductase Transferrin
Endothelial plasma membrane protein Sorbitol dehydrogenase α1-antitrypsin
Na+/K+-ATPase Plasminogen
Intracellular proteins Plasminogen activator
Hemoglobin Hormones Fibrinogen
Crystallin Thyroid hormone Fibrin
Tubulin Insulin Antithrombin III
Calmodulin β2-microglobulin
Ceruloplasmin
*Adapted from ref. (4).

Table 2. Factors influencing levels of HbA1c.*

Process Factors Effect on HbA1c level
iron, vitamin B12 deficiency, decreased erythropoiesis increase
Erythropoiesis erythropoietin administration, iron, vitamin B12, reticulocytosis, chronic liver
decrease
disease
Hemoglobin Genetic or chemical modifications of hemoglobin (hemoglobinopathies, HbF,
increase or decrease
modification methemoglobin)
alcoholism, chronic renal failure, decreased intra-erythrocyte pH increase
aspirin, vitamins C and E, certain hemoglobinopathies, increased intra-erythrocyte
Glycation decrease
pH
Genetic determinants increase or decrease
increased erythrocyte life span, e.g. due to splenectomy increase
Erythrocyte decreased erythrocyte life span, e.g. due to hemoglobinopathies, splenomegaly,
destruction rheumatoid arthritis or drugs such as antiretrovirals, decrease
ribavirin and dapsone.
hyperbilirubinemia, carbamylated hemoglobin,
increase
alcoholism, high-dose aspirin, chronic opiate use
Assays hemoglobinopathies increase or decrease
hypertriglyceridemia decrease
*Adapted from ref. (6).

Biochemia Medica 2014;24(Suppl 1):S1-S78

S48
 14th EFLM Continuous Postgraduate Course in Clinical Chemistry and Laboratory Medicine
have proposed calculating the “glycation gap”, de-
fined as the difference between the measured level
of HbA1c and the level predicted based on the
amount of glycated albumin (8). More recently,
Rodriguez-Segade et al. have shown that the com-
bination of glycation gap and glycated albumin lev-
el provide a better indication of nephropathy risk,
and may be more desirable for glycemic control (9).
The development of new biomarkers of hypergly-
cemia for cases when HbA1c levels are inadequate
has been the subject of intense investigation over
the last 5 years. One candidate biomarker is albu-
min, which accounts for approximately 60% of se-
rum proteins and is present in the blood at con-
centrations of 30-50 g/L. This protein is predicted
to be highly susceptible to glycation because it
contains numerous arginine and lysine residues
near its N- and C-termini. In addition, it persists for
2-3 weeks once released into the circulation, mak-
ing it potentially well-suited to be a biomarker that
can detect short and mid-term changes. In order
for glycated albumin to be measured routinely in
the clinic, the American Diabetes Association in
2011 called for studies to develop a standardized
method for its measurement as well as to clearly
establish its clinical usefulness and reliability for
predicting diabetes-related complications. Since
then, numerous studies have tried to follow these
recommendations and determine whether the
level of glycated albumin can be useful in manag-
ing diabetes. Here we examine those efforts (6,10).
Colorimetric determination of glycated
albumin
Recommended methods for determining glycated
albumin are based on affinity chromatography,
ion-exchange chromatography and high-perfor-
mance liquid chromatography (HPLC). Recent re-
search suggests that liquid chromatography-tan-
dem mass spectrometry (LC-MS/MS) may be the
“gold standard” method for quantitative determi-
nation of glycated proteins (11), including albumin
Usually glycated protein or glycated albumin are referred to as “fructosamine” if determined colorimetrically, or as “glycated albumin” or “glycated serum proteins” if
1
determined enzymatically (see next section).
S49
and all serum proteins, all of which are collectively
known as fructosamine1. These methods work well
with EDTA and heparin plasma samples, giving
them an advantage over the HbA1c assay, which
requires complete blood. However, all these tech-
niques are complicated and require sophisticated
equipment beyond the reach of many clinical lab-
oratories. A much simpler and less expensive alter-
native is a colorimetric method for fructosamine
determination known as the nitroblue-tetrazolium
(NBT) reduction method (12). The method was au-
tomated soon after it was first described.
Despite its advantages over more complicated
techniques, the NBT method does suffer from re-
duced specificity because the NBT reacts with vari-
ous endogenous reducing substances, including
thiol groups, ascorbate, and NADH--the levels of all
of which can vary from sample to sample. Some as-
say manufacturers attempt to reduce this interfer-
ence by incubating the reaction for slightly longer
periods (10-15 min). However, this approach is not
feasible with many laboratory analyzers, which lack
the flexibility to program longer incubations. Some
laboratories avoid the interference of the NBT assay
by using alternative colorimetric methods based
on 2‐thiobarbituric acid (TBA) or phenylhydrazine.
Although the NBT method has been widely auto-
mated, it is still vulnerable to interference, the
sources of which depend on the particular test.
The following have been widely reported to inter-
fere with this assay (13), though manufacturers’
test inserts should be always be consulted for spe-
cifics:
1. EDTA and heparin plasma samples give lower
fructosamine results than serum samples in
the NBT colorimetric assay, so the same type of
sample should always be used to monitor gly-
cemia in a given patient.
2. Urate, glutathione and vitamin C lead to artifi-
cially high fructosamine results.
3. Cysteine, methyldopa, dobesilate calcium, oxy-
tetracycline and hemolysis can cause artificial-
ly low fructosamine results.
Biochemia Medica 2014;24(Suppl 1):S1-S78
14th EFLM Continuous Postgraduate Course in Clinical Chemistry and Laboratory Medicine
4. Bilirubin has been shown to cause falsely ele-
vated fructosamine results.
5. The NBT assay, like other colorimetric assays, is
affected by changes in ambient temperature.
Enzymatic determination of glycated
albumin
Recently a quite precise and automated enzymatic
assay for determination of glycated albumin has
been commercialized by Diazyme Laboratories,
Asahi Kasei Pharma, and Randox Laboratories (14-
17). The principle of the enzymatic assay is shown
in Figure 1, and it can be performed with serum or
plasma on virtually all biochemical analyzers. The
commercial assays come supplied with ready-to-
use reagents, which simultaneously allow determi-
nation of not only glycated albumin but also sev-
eral other frequently used biomarkers, including
glucose, cholesterol and triglycerides. These multi-
ple determinations do not require multiple blood
samples or a total blood sample, as is required for
HbA1c determination. Stability tests indicate that
samples for the enzymatic assay can be stored for
up to 2 weeks at 2-8 ºC or up to 4 weeks frozen.
Although the tests from different manufacturers
rely on slightly different methods, they all show
good analytical characteristics and correlate well
with one another, as well as with HPLC-based
methods. The tests differ principally in what en-
Proteinase K
GSP/GA GPF
FructosaminaseTM
GPF PF or amino acids + H2O2
Peroxidase
H2O2 + TOOS + 4-AA Color + H2O2
Figure 1. Principle of the Diazyme enzymatic assay to deter-
mine glycated serum albumin. Proteinase K digests serum pro-
teins into low-molecular-weight glycated protein fragments
(GPF), then a specific fructosaminase™ (microbial amadoriase)
catalyzes the oxidative degradation of GPF Amadori product
to yield a protein fragment (PF) or amino acids and H2O2. The
H2O2 released is measured by a colorimetric Trinder end-point
reaction. The absorbance at 546 nm is proportional to the con-
centration of glycated serum proteins (GSP) or glycated albu-
min (14).
Biochemia Medica 2014;24(Suppl 1):S1-S78
S50
zyme is used and whether the results are ex-
pressed as a concentration (mmol/L or mmol/L) or
as glycated albumin fraction (%GA). Determina-
tion of %GA also involves determination of total al-
bumin.
While the Lucica GA-L kit determines %GA, the Di-
azyme GlycoGap kit determines the concentration
of glycated albumin in mmol/L, and the Randox kit
determines the concentration in mmol/L. The Lu-
cica GA-L kit determines albumin using a brom-
cresol purple (BCP) method that is more specific
than the bromocresol green (BCG) method most
often used to determine albumin in clinical labora-
tories. Each assay manufacturer provides reference
intervals for glycated albumin for diabetics and
non-diabetics in the appropriate concentration
units or %GA.
These enzymatic tests show extremely good re-
producibility and specificity, correlating closely
with glycated albumin levels determined by HPLC
(r > 0.98). Based on the performance of these en-
zymatic assays, which according to the manufac-
turers is evaluated in compliance with guideline
EP5-A of the Clinical and Laboratory Standards In-
stitute, the automated test shows the characteris-
tics of a reference method, even though it has yet
to be formally recognized as such.
Interference studies have reported various inter-
ferences, though these may vary from kit to kit,
and the manufacturer’s test insert should be con-
sulted in all cases.
1. Since EDTA plasma samples, but not serum
samples, have been internally validated to
show no matrix effects in enzymatic assays,
serum should be separated from cells immedi-
ately after blood collection.
2. As in the NBT colorimetric assay, cysteine,
methyldopa, dobesilate calcium, oxytetracy-
cline and hemolysis can cause artificially low
fructosamine results.
3. As in the NBT colorimetric assay, bilirubin has
been shown to cause falsely elevated fructos-
amine results.
Several common interfering substances in serum,
such as ascorbic acid, bilirubin, glucose, triglycer-
 14th EFLM Continuous Postgraduate Course in Clinical Chemistry and Laboratory Medicine
ide, uric acid and hemoglobin, usually show ≤ 10%
interference, though the manufacturer’s insert for
the particular test should be consulted.
Variability of glycated albumin and
limitations as a biomarker of glycemia
Disorders in albumin metabolism affect levels of
glycated albumin. Lower ratios of glycated albu-
min to blood glucose are observed in patients
with nephrotic syndrome or hyperthyroidism and
in patients on glucocorticoid therapy. These dis-
eases involve elevated albumin metabolism. Con-
versely, higher ratios of glycated albumin to blood
glucose are found in patients with liver cirrhosis
and hyperthyroidism--conditions associated with
reduced albumin metabolism.
Lower ratios of glycated albumin to blood glucose
are observed in obese people; these ratios appear
to reflect chronic micro-inflammation mediated
by adiponectin released from fat cells, which leads
to increased protein catabolism. Lower ratios of
glycated albumin to glucose are also observed in
smokers and in patients with hyperuricemia, hy-
pertriglyceridemia, or alcohol-induced fatty liver
disease associated with elevated levels of alanine
aminotransferase (ALT)(10).
Albumin metabolism changes rapidly in children,
and the levels of both albumin and glucose in in-
fants increase rapidly with age. While these effects
limit the reliability of glycated albumin as a bio-
marker of glycemia, they are still less severe than
the significant influence of changes in fetal hemo-
globin levels on HbA1c levels (10). As a result, gly-
cated albumin, not HbA1c, is used as an indicator
of glycemic control in newborns with diabetes.
Biological variability of glycated albumin
Determination of glycated albumin in serum and
plasma has become much easier since the devel-
opment of automated enzymatic tests. The analyt-
ical coefficient of variation of 1.7% is much lower
than the corresponding values of 2.8% for fruc-
tosamine and 2.4% for HbA1c published in 2013
S51
(18). That study reported a within-subject coeffi-
cient of variation (CVW) of 2.1% and between-sub-
ject coefficient of variation (CVG) of 10.6% for gly-
cated albumin; the corresponding values for albu-
min were 2.3% and 2.9%, and for fructosamine,
2.3% and 6.3%. This CVG of albumin (2.9%) is lower
than the 4.2% reported in the Westgard biodata-
base, while the CVGs for glycated albumin (10.6%)
and fructosamine (6.3%) are higher than the cor-
responding values of 10.3% and 5.9% in the West-
gard biodatabase. These comparisons indicate a
high degree of individuality for both albumin and
glycated albumin as biomarkers of glycemia. Some
authors have suggested that because of the sub-
stantial between-subject variation of glycated al-
bumin levels, the critical difference (CD) should be
used instead of target values for monitoring glyce-
mia (18). The estimated CD in this study was 7.5%
for GA, 9% for albumin and 10% for fructosamine.
This approach reduces between-subject variation,
allowing enzymatic determination of glycated al-
bumin to be recommended for clinical use in con-
junction with determination of HbA1c for monitor-
ing glycemic status (18).
A study in 2010 suggested that errors in determi-
nation of glycated albumin or HbA1c in patients
with type 2 diabetes are on the order of 18% and
can be attributed to between-subject variation
(19). While the causes of this large variation are
not entirely clear, variation in the erythrocyte
lifespan, especially in diabetics, is likely to contrib-
ute, as is variation in albumin half-life due to gly-
cation. The authors of that study strongly recom-
mended monitoring diabetes using a combina-
tion of two or more glycemia biomarkers, in order
to obtain more reliable information about glyce-
mic state.
Conclusion
The available evidence points to glycated albumin
levels as a useful marker for diabetes manage-
ment, although much still needs to be learned
about the mechanism of albumin glycation and
about how this marker compares with the much
better established HbA1c.
Biochemia Medica 2014;24(Suppl 1):S1-S78
14th EFLM Continuous Postgraduate Course in Clinical Chemistry and Laboratory Medicine
References
1. International Diabetes Federation: International Diabetes
Federation Diabetes Atlas, Editors: Guariguata L, Nolan T,
Beagley J, Linnenkamp U, Jacqmain O. 6th edition, 2013.
Available at: http://www.idf.org/diabetesatlas/download-
book , Accessed on on 16th August 2014.
2. Wild S, Roglic G, Green A, Sicree R, King H. Global prevalen-
ce of diabetes: Estimates for the year 2000 and projections
for 2030. Diabetes Care 2004;27:1047–53.
3. Sacks DB, Arnold M, Bakris GL, Bruns DE, Horvath AR, Kir-
kman MS, at al. Guidelines and Recommendations for La-
boratory Analysis in the Diagnosis and Management of
Diabetes Mellitus. Diabetes Care 2011;34:e61–e99.
4. Koga M, Kasayama S. Clinical impact of glycated albu-
min as another glycemic control marker.Endocrine Journal
2010;57:751-62.
5. The International Expert Committee. International com-
mittee report on the role of the A1c assay in the diagnosis
of diabetes. Diabetes Care 2009;32:1327–34.
6. WHO, Use of glycated haemoglobin (HbA1c) in the diagno-
sis of diabetes mellitus: abbreviated report of a WHO con-
sultation. Available at: http://www.who.int/cardiovascu-
lar_diseases/report-hba1c_2011_edited.pdf, Accessed on
20th August 2014.
7. Nathan DM, Kuenen J, Borg R, Zheng H, Schoenfeld D, He-
ine RJ for the A1c-Derived Average Glucose (ADAG) Study
Group: Translating the A1c assay into estimated average
glucose values. Diabetes Care 2008;31:1473–8.
8. Cohen RM, Holmes YR, Chenier TC, Joiner CH. Discordan-
ce between Hemoglobin A1c and Fructosamine: Evidence
for a Glycosylation Gap and its Relation to Nephropathy
in Longstanding Type 1 Diabetes. Diabetes Care 26:163-7,
2003.
9. Rodríguez-Segade S, Rodríguez J, Cabezas-Agricola JM,
Casanueva FF, Camiña F. Progression of nephropathy in
type 2 diabetes: the glycation gap is a significant predictor
Biochemia Medica 2014;24(Suppl 1):S1-S78
S52
after adjustment for glycohemoglobin (HbA1c) Clin Chem.
2011;57:264–71.
10. Furusyo N, Hayashi J. Glycated albumin and diabetes melli-
tus Biochimica et Biophysica Acta 2013;1830:5509–14.
11. Thornalley PJ, Rabbani N. Detection of oxidized and glyca-
ted proteins in clinical samples using mass spectrome-
try — A user’s perspective. Biochimica et Biophysica Acta
2014;1840:812-29.
12. Johnson R N, Metcalf P A, Baker J R. Fructosamine: a new
approach to the estimation of serum glycosyl-protein. An
index of diabetic control. Clin Chim Acta 1982;127:87-95.
13. Rigg C. Fructosamine (plasma, serum). Association for Cli-
nical Biochemistry 2012. Available at: http://www.acb.org.
uk/whatwedo/science/amalc.aspx , Accessed on 29th Au-
gust 2014.
14. Diazyme Laboratories and Abidin D, Liu L, Dou C, Datta A,
Yuan C. An improved enzymatic assay for glycated serum
protein. Anal Methods 2013;5:2461-9.
15. Diazyme GSP Assay (DZ112B) Performance Summary May,
2011. with premision Diazyme Laboratories.
16. Kohzuma T, Yamamoto T, Uematsu Y, Shihabi ZK, Freed-
man BI. Basic Performance of an Enzymatic Method for
Glycated Albumin and Reference Range Determination. J
Diabetes Sci Technol 2011;5:1455-62.
17. Randox Laboratories: Liquid Fructosamine:A medium
term indicator of diabetic control. Available at: http://
ow.ly/6H1DZ, Accessed on 29th August 2014.
18. Montagnana M, Paleari R, Danese E, Salvagno GL, Lippi
G, Guidi GC, Mosca A. Evaluation of biological variation
of glycated albumin (GA) and fructosamine in healthy su-
bjects. Clin Chim Acta 2013; 423:1–4.
19. Tahara Y, Shima K. Evaluation of error levels in hemoglo-
bin A1c and glycated albumin in type 2 diabetic patients
due to inter-individual variability. Diabetes Res Clin Pract
2010;89:115-20.
 14th EFLM Continuous Postgraduate Course in Clinical Chemistry and Laboratory Medicine
Pro and Contra of Incretin therapy in
Type 2 diabetes
Dario Rahelić
Department of Endocrinology, Diabetes and Metabolic
Disorders, Dubrava University Hospital, Zagreb, Croatia
Corresponding author: dario.rahelic@gmail.com
Abstract
Incretins are group of gastroinestinal hormones
involved in glucoregulation. Two main hormones
involved in glucoregulation are glucagon-like
peptide-1 (GLP-1) and gastric inhibitory peptide or
glucose-dependent insulinotropic polypeptide
(GIP). Both are rapidly inactivated by the enzime
dipeptidyl peptidase-4 (DPP-4). Incretin based
therapies include GLP-1 receptor agonists and
DPP-4 inhibitors. Their main mechanisms of ac-
tions are to increase insulin and inhibit glucagon
secretion, reduce gastric emptying and reduce
food intake. Positive effects on glycemia without
hypoglycemia and weight gain are their main ad-
vantages. Direct cardiovascular effects of GLP-1
receptor agonists are also documented. Concerns
have been raised that incretin based therapies
were associated with an increased risk of pancrea-
titis and pancreatic cancer. Recent trials do not
support the hypothesis. Preclinical studies in ani-
mals have suggested that incretin based therapies
could be associated with thyroid C-cell cancer.
However, it was not found in humans. Recent trial
of one DPP-4 inhibitor suggested increased risk
for hospitalization due to heart failure. Further
studies are required. Several large clinical trials are
in progress. In presentation pro et contra of incre-
tin based therapies will be presented.
S53
The practical issues in Type 2 diabetes
management - pharmacogenomic
consideration
Sabina Semiz
Department for Biochemistry and Clinical Analysis, Faculty of
Pharmacy, University of Sarajevo, Bosnia and Herzegovina
Corresponding author: sabinasemiz@hotmail.com
Type 2 diabetes (T2D) is a complex disease that
has a significant potential for stratification in its
management, including genetic etiology, treat-
ment outcomes, rate of progression, and develop-
ment of complications. In the light of exorbitant
and still increasing costs of treating T2D and its
complications, pharmacogenomics’ benefit of se-
lecting patients and adequate therapies offers a
considerable potential to improve cost-effective-
ness of drug therapy and avoid extra costs for side
- effects treatment. Several classes of oral antidia-
betic drugs (OAD) are currently available to treat
T2D patients, with sulphonylureas (SU), bi-
guanides, thiazolidinediones (TZD), and meglit-
nides being the most frequently used. Emerging
evidence has recently demonstrated that genetic
variation might be one of the key determinants of
an individual’s responses to OAD. A variety of ap-
proaches can be used to understand OAD’s phar-
macodynamic and pharmacokinetic mechanisms
related to interindividual differences in drug re-
sponse, with pharmacogenomics providing a
unique and powerful clinically relevant tool. Fur-
thermore, an enhanced understanding of genes
and pathways that determine OAD response has
also the potential to reveal new drug targets and
develop novel drugs for the treatment of diabetes.
Although benefits from a personalized diabetes
care are well established in patients with certain
monogenic forms of diabetes, pharmacogenom-
ics of common T2D is also anticipated and pro-
gressing rapidly. Here are summarized the results
of the several recent studies, which have analyzed
an association of genetic variations in drug - me-
tabolizing enzymes (DME), drug transporters (DT),
Biochemia Medica 2014;24(Suppl 1):S1-S78
14th EFLM Continuous Postgraduate Course in Clinical Chemistry and Laboratory Medicine
and specific drug targets with T2D treatment out-
comes in diverse population groups. The most re-
cent and promising advances appeared to be re-
lated to therapy with the biguanide drug met-
formin, a first-line drug used to treat newly diag-
nosed T2D. The glycemic response to metformin
appears to be highly variable, with about 35% of
patients failing to achieve acceptable control of
glucose levels on metformin monotherapy (1).
Variants in SLC47A1 (encoding the drug transport-
er, multidrug and toxin extrusion protein 1,
MATE1) and SLC47A2 (encoding MATE2 transport-
er) have also been associated with altered glucose
- lowering response to metformin in humans (2).
In the Diabetes Prevention Program (DPP), associ-
ations were also found for the STK11 (encoding
the drug target AMP kinase, AMPK) and SLC22A1
gene (encoding the drug transporter organic cati-
on transporter 1,OCT1). Although pharmacog-
enomics can also be a useful tool to point to a
novel biological mechanism of action of met-
formin, studies of pharmacodynamic genetics
have been limited. A recent genome-wide associ-
ation study (GWAS) found an association of ATM
(ataxia telangiectasia mutated) gene variation, in-
volved in AMPK activation, with treatment suc-
cess (3). Since the primary action of metformin
seems to be the inhibition of hepatic glucose pro-
duction through inhibition of gluconeogenesis,
interactions with loci associated within this path-
way (PCK1, phosphoenolpyruvate carboxykinase 1),
PPARA (peroxisome proliferator - activated receptor
alpha), and PARGC1A (peroxisome proliferator - acti-
vated receptor gamma, coactivator 1 alpha) were
also reported (4).
Single nucleotide polymorphisms (SNPs) of the
genes encoding potassium inwardly rectifier 6.2
subunit (Kir6.2) of pancreatic islet ATP- sensitive K+
(KATP) channel have been related to the efficacy
of secretagogue drugs, such as sulphonylureas.
This channel is essential for glucose - stimulated
insulin secretion from pancreatic β-cells, modu-
lates glucose uptake into skeletal muscle, glucose
production and release from the liver. KATP chan-
nels are heterooctamers assembled from Kir6.2
and the sulphonylurea receptor 1 (SUR1), encoded
by the KCNJ11 (potassium inwardly-rectifying
Biochemia Medica 2014;24(Suppl 1):S1-S78
S54
channel, subfamily J, member 11) and ABCC8 gene,
respectively. A common Glu23Lys polymorphism
(also known as E23K) in KCNJ11 is associated with
an increased risk of SU therapeutic failure. A recent
study found that KCNJ11 variations have been as-
sociated with altered response to gliclazide (5) and
glibenclamide (6). Interestingly, the most promis-
ing gene variants affecting the SU response are
those involved in drug pharmacodynamics, such
as the transcription factor 7-like 2 (TCF7L2) that en-
codes a transcription factor (Tcf-4), involved in the
regulation of cellular proliferation and differentia-
tion (7).
Meglitinides (glinides) represent a class of short -
acting insulin secretagogues that act by binding
to pancreatic - cells and inhibiting KATP channel
to stimulate insulin release. This is similar to the
mechanism of action of the sulphonylureas and
both, meglitinides and SU, bind to the SUR1 subu-
nit to inhibit channel activity. Due to their short
action, repaglinide and nateglinide have a lower
risk to induce hypoglycemia than SU. Further-
more, meglitinides offer an alternative OAD agent
of similar potency to metformin, and may be indi-
cated where side effects of metformin are intoler-
able or where metformin is contraindicated. A re-
cent study showed that SLCO1B1 gene, which en-
codes the organic anion-transporting polypep-
tide 1B1 (OATP1B1) that transports repaglinide
into hepatocytes, is a major factor that significant-
ly affects the repaglinide pharmacokinetics (8),
consistent with an enhanced hepatic uptake by
OATP1B1.
The thiazolidinediones activate their molecular
target PPARs (peroxisome proliferator - activated
receptors). TZD bind with greatest specificity for
PPARΥγ to promote adipogenesis and fatty acid
uptake. By reducing circulating fatty acid levels
and lipid availability in liver and muscle, these
drugs improve the patients’ sensitivity to insulin
and reduce hyperglycemia. Thus, variation in
PPARg would likely affect response to TZD and this
was suggested in a recent study that analyzed pi-
oglitazone response (9). Recently, several addition-
al gene variants have been also associated with
the TZD therapy outcomes (10), including adi-
ponectin, leptin, resistin, and tumor necrosis fac-
 14th EFLM Continuous Postgraduate Course in Clinical Chemistry and Laboratory Medicine
tor (TNF)-a that are of a particular interest due to
their important role in insulin resistance.
Variation in the cytochrome P450 (CYP) enzymes,
which metabolize oral antidiabetic drugs, ap-
pear also to impact their effects, including varia-
tion in CYP2C9 and CYP2C19 for SU metabolism,
CYP3A4 and CYP2C8 for repaglinide, CYP2C9 for
nateglinide, and CYP2C8 and CYP3A4 for pioglita-
zone.
Interestingly, a very recent systemic review re-
ported by Maruthur et al. (11), summarized the
major genetic variants that could predict response
to oral antidiabetic drugs. They performed a quali-
tative synthesis of results from twenty one stud-
ies, comprised from more than ten thousand sub-
jects, to determine if the effect of OAD treatment
on diabetes incidence, levels of glycosylated
hemoglobin (HbA1c), and fasting and postprandi-
al glucose is associated with genetic variations in
patients with impaired glucose tolerance or Type
2 diabetes. Based on this rigorous analysis, the au-
thors recommended as a priority further confir-
mation if variations of following selected genes
could be used to individualize the choice of diabe-
tes management: SLC22A1, SLC22A2, SLC47A1,
AMPK subunits (PRKAB2, PRKAA2, PRKAA1), and
STK11 for metformin; CYP2C9 and TCF7L2 for sulph-
onylureas; KCNJ11, SLC30A8 (solute carrier family 30
(zinc transporter), member 8), NEUROD1/BETA2 (neu-
rogenic differentiation 1 transcription factor), UCP2
(mitochondrial uncoupling protein 2), and PAX4
(paired box gene 4) for repaglinide; and PPARG2
and PTPRD (protein tyrosine phosphatase, receptor
type, D) for pioglitazone. Importantly, this study
(11) also indicated that although diabetes research
is extensively funded, the major limitation of the
pharmacogenomic research of Type 2 diabetes is
the lack of high - quality studies to identify and
confirm findings for specific interactions between
drug, genetic variation, and treatment outcome.
The most of pharmacogenomic studies on diabe-
tes treatment performed to date are small and in-
adequately replicated. The small size of many of
the studies does not exclude the possibility that
interactions exist, although they could not be
identified because of the lack of power. Thus,
since the pharmacogenomic associations in dia-
S55
betes that have been reported to date have had
limited impact on the individual treatments
choice, the value of genetic information in guid-
ing therapeutic decisions in T2D treatment must
be further tested in adequately designed and
carefully conducted clinical trials, controlling for
population stratification and relatedness. This im-
portant goal could only be achieved by a broad
transnational collaboration between numerous
research groups with large patient cohorts. Par-
ticularly, it would be pertinent to explore geno-
type - phenotype associations by using standard-
ized therapy outcomes (e.g., HbA1c at three
months) in order to reveal a number of genetic
variants that stand out as statistically significant
with high positive predictive value and may be
used as pharmacogenomic markers for an optimal
T2D treatment. With recent scientific and techno-
logical advances, as well as decreasing sequenc-
ing costs, pharmacogenomics has a great poten-
tial to yield therapeutic advances leading the way
towards personalized diabetes care. This stratified
approach to diabetes therapy should be also more
cost-effective than a classical ‘trial and error’ ap-
proach. Furthermore, analysis of the underlying
genetic factors related to OAD response may also
lead to the identification of novel targets and de-
velopment of improved, more effective antidia-
betic drugs.
In conclusion, the evidence has been accumulat-
ing to show that pharmacogenomics offers the
considerable potential to improve the manage-
ment of T2D and the effective prescribing of oral
antidiabetic drugs. As summarized here, signifi-
cant pharmacogenomic evidence has demonstrat-
ed an association between specific gene polymor-
phisms and interindividual variability in OAD ther-
apeutic and side effects. Thus, several variants re-
lated to drug-metabolizing enzymes, drug trans-
porters, drug targets, and diabetes risk genes that
were recently identified, could be employed to
predict treatment outcomes and treat Type 2 dia-
betes more efficiently. Further identification and
confirmation of drug - genotype interactions
would encourage a promotion of personalized
medicine in clinical settings, where genotype
would be used to guide diabetes therapy.
Biochemia Medica 2014;24(Suppl 1):S1-S78
14th EFLM Continuous Postgraduate Course in Clinical Chemistry and Laboratory Medicine
References
1. Cook MN, Girman CJ, Stein PP, Alexander CM. Initial mo-
notherapy with either metformin or sulphonylureas often
fails to achieve or maintain current glycaemic goals in pa-
tients with type 2 diabetes in UK primary care. Diabet Med
2007;24: 350–8.
2. Stocker SL, Morrissey KM, Yee SW, Castro RA, Xu L, Dahlin A,
et al. The effect of novel promoter variants in MATE1 and
MATE2 on the pharmacokinetics and pharmacodynamics
of metformin. Clin Pharmacol Ther 2013;93:186–94.
3. Zhou K, Bellenguez C, Spencer CC, Bennett AJ, Coleman RL,
Tavendale R, et al. Common variants near ATM are associa-
ted with glycemic response to metformin in type 2 diabe-
tes. Nat Genet 2011;43:117-20.
4. Jablonski KA, McAteer JB, de Bakker PIW, Franks PW, Pollin
TI, Hanson RL, et al.; Diabetes Prevention Program Resear-
ch Group. Common variants in 40 genes assessed for dia-
betes incidence and response to metformin and lifestyle in-
tervention in the diabetes prevention program. Diabetes
2010;59:2672–81.
5. Javorsky M, Klimcakova L, Schroner Z, Zidzik J, Babjako-
va E, Fabianova M, et al. KCNJ11 gene E23K variant and
therapeutic response to sulfonylureas. Eur J Intern Med
2012;23:245-9.
Biochemia Medica 2014;24(Suppl 1):S1-S78
S56
6. Pearson ER, Flechtner I, Njolstad PR, Malecki MT, Flanagan
SE, Larkin B, et al. Switching from insulin to oral sulfonylu-
reas in patients with diabetes due to Kir6.2 mutations. N
Engl J Med 2006;355:467-77.
7. Schroner Z, Javorsky M, Tkacova R, Klimcakova L, Do-
brikova M, Habalova V, et al. Effect of sulphonylurea tre-
atment on glycaemic control is related to TCF7L2 genoty-
pe in patients with type 2 diabetes. Diabetes Obes Metab
2011;13:89-91.
8. Kalliokoski A, Neuvonen M, Neuvonen PJ, Niemi M. The
effect of SLCO1B1 polymorphism on repaglinide pharma-
cokinetics persists over a wide dose range. Br J Clin Phar-
macol 2008;66:818-25.
9. Pei Q, Huang Q, Yang GP,   Zhao YC,  Yin JY,  Song M, et al.
PPAR-g2 and PTPRD gene polymorphisms influence type 2
diabetes patients’ response to pioglitazone in China. Acta
Pharmacol Sin 2013;34:255–61.
10. Schroner Z, Javorsky M, Kozarova M, Tkac I. Pharmaco-
genetics of oral antidiabetic treatment. Bratisl Lek Listy
2011;112:441-6.
11. Maruthur NM, Gribble MO, Bennett WL, Bolen S, Wilson LM,
Balakrishnan P, et al. The Pharmacogenetics of Type 2 Dia-
betes: A Systematic Review. Diabetes Care 2014;37:876–86.
Posters
P1
Quality improvement in point-of-care blood
glucose testing with Cobas IT 1000
Čeri A*, Baršić I, Rogić D
Department of Laboratory Diagnostics, University Hospital
Center Zagreb, Croatia
*Corresponding author: ceri.andrea@gmail.com
Background: The University Hospital Centre Za-
greb is the largest hospital in Croatia as well as a
unique institution regarding many medical proce-
dures, diagnostic methods and therapeutic proce-
dures. Our hospital has well-developed system of
point-of-care testing (POCT). Since POCT is per-
formed outside the central laboratory, it is very im-
portant to ensure supervision of each stage of per-
forming the point-of-care testing (quality control,
consumption of reagents, critical results, duplicate
samples).
Materials and Methods: More than 130 POCT in-
struments (blood gas analyzers, hematology ana-
lyzers, coagulation monitoring system analyzers
and glucometers) have been installed so far at the
University Hospital Center Zagreb in clinical de-
partments, where the emphasis was to reduce the
time span between collecting samples and get-
ting test results (i.e. turnaround time, TAT). 115 glu-
cometers are connected with central laboratory
via software Cobas IT 1000 (data management sys-
tem for hospital POCT). It connects glucometers,
collects and stores patient and control test results
centrally. Such integration allows thorough analy-
sis of results (reviewing Levey Jennings charts by
specific combination of test strip lot and control
lot number) and preparation of daily, monthly and
annual reports on results obtained from glucome-
ters. Also, it enables adding a new test strip lot to
all glucometers in just a few minutes.
Conclusions: POCT group devotes great attention
to improving quality of POCT. It is a time-consum-
ing job that requires a lot of patience and under-
standing. With Cobas IT 1000 we made step for-
ward to better quality of POCT glucometers. At
S57
our hospital, quality means providing safe and ap-
propriate care to our patients. Cobas IT 1000 al-
lows easier and faster control over the use of glu-
cometers and gives us more time for education of
non laboratory staff which is one of most impor-
tant components in quality improvement.
Key words: data management; Cobas IT 1000;
point-of-care testing
P2
Results of external quality control of HbA1c
in Croatian medical laboratories indicated
that POCT system differ from immunoassay
methods
Hrabric Vlah S*, Antoncic D, Grzunov A, Dobrijevic S,
Vlasic-Tanaskovic J, Bilic-Zulle L, Lenicek Krleza J
Croatian Center for Quality Assessment in Laboratory Medicine,
Croatian society of medical biochemistry and laboratory
medicine, Croatia
*Corresponding author: snjezana.hrabric.vlah@gmail.com
Background:  HbA1c  as valuable marker  in man-
agement of diabetes  is measured  in the most  of
medical laboratories in Croatia. Aim of this study is
to present the  results  of external quality control
assessment (EQA) in Croatian medical laboratories
for HbA1c.
Material and methods:  Results were ob-
tained  from  93  laboratories that participated  in cy-
cle of EQA scheme in 2013.The control sample was
commercial, lyophilized human blood specimen
with normal HbA1c value. The results were entered
on-line, as a dual system of reporting: DCCT (%) and
IFCC (mmol/mol), and DCCT results were analyzed.
Outliers (N=14) were excluded according to Tukey´s
model and mean (X), standard deviation (SD) and co-
efficient of variation (CV) were calculated.  According
to quality specifications established in CROQALM,
the allowable deviation from the mean was ±5%.
Results: The results were divided in two groups ac-
cording to the method (immunoassay and colori-
metric method).  For immunoassay methods (82
Biochemia Medica 2014;24(Suppl 1):S1-S78
14th EFLM Continuous Postgraduate Course in Clinical Chemistry and Laboratory Medicine
participants, six types of instruments), reported re-
sults were: X=5.4%,  SD=0.18, CV=3.3%. For colori-
metric method (11 participants, POCT instrument),
results were: X=9.8%, SD=1.94, CV=19.8%. Immuno-
assay group indicated acceptable CV (<5%) while
results (CV) obtained by colorimetric method (POCT
analyzer) were far outside of acceptable limits.
Conclusion: Immunoassay methods for HbA1c de-
termination  indicated  very good  inter-laborato-
ry  comparability. High CV in the group of POCT
analyzer revealed poor comparability within that
method group. Therefore,  it can be  concluded
that only a minority of participants that reported
results obtained on POCT analyzers demonstrated
low inter-laboratory agreement.   globin concentrations. The major hemolysis influ-
Key words: external quality control; HbA1c; inter-
laboratory comparability
P3
Hemolysis effect on coagulation test results
Coen Herak D, Dolčić M*, Vogrinc Ž
Department of Laboratory Diagnostics, University Hospital
Center Zagreb, Croatia
*Corresponding author: majadolcic@yahoo.com
Background: Due to the high risk of developing
thrombosis, the majority of diabetic patients are re-
ceiving antithrombotic therapy. During routine lab-
oratory monitoring of these agents a certain num-
ber of hemolyzed coagulation samples are usually
observed. As hemolysis influence on coagulation
test results hasn’t been extensively studied, our
aim was to investigate the effect of hemolysis on
routine coagulation tests: prothrombin time (PT),
activated partial thromboplastin time (aPTT) and fi-
brinogen by using serum hemolysis indices (HI)
and measuring free plasma hemoglobin concen-
trations as indicators of the degree of hemolysis.
Materials and methods: We measured PT, aPTT
and fibrinogen in 30 hemolyzed and non-
hemolyzed plasma samples from the same pa-
tients on the coagulation analyzer BCS® XP (Sie-
Biochemia Medica 2014;24(Suppl 1):S1-S78
S58
mens Healthcare Diagnostics) on two wavelengths
(405 and 570 nm). Free plasma hemoglobin con-
centrations were determined spectrophotometri-
cally and measurement of HI was performed on
biochemistry analyzer Cobas c6000 (Roche). In-vit-
ro hemolysis influence on coagulation test results
was studied by adding hemolysate in normal and
pathological plasma pool samples (final hemo-
globin concentrations: 0-10 g/L). Correlation be-
tween free plasma hemoglobin concentrations
and coagulation test results in hemolyzed samples
was assesed.
Results: Hemolysis affects all routine coagulation
tests independently of HI or free plasma hemo-
ence was observed for aPTT in a normal plasma
pool sample, whereas only minor influence was
observed for PT in a pathological plasma pool
sample. Linear correlation between free plasma
hemoglobin concentrations and HI was confirmed.
Conclusions: Although hemolysis affects all three
determined coagulation parameters, the influence
on each parameter is different, not always correlat-
ing with the intensity of hemolysis. Our results in-
dicate that HI is a reliable indicator of the degree
of hemolysis. Routine measurement of HI could
provide a better estimation of hemolysis influence
on coagulation test results and reduce the number
of unnecessary and repeated blood testing.
Key words: coagulation tests; hemolysis; hemoly-
sis indices; free hemoglobin plasma concentration
P4
OGTT - National survey
Gorenjak M*, Jevšnikar B
Department for Laboratory Diagnostics, University Clinical
Centre Maribor, Maribor, Slovenia
*Corresponding author: gormax@ukc-mb.si
Background: Slovenia has national guidelines for
screening and diabetes detection based on WHO
and ADA recommendations (Slovenske smernice
 14th EFLM Continuous Postgraduate Course in Clinical Chemistry and Laboratory Medicine
za klinično obravnavo sladkorne bolezni tipa 2 pri
odraslih osebah, 2011).
Materials and methods: In May 2013 we sent 120
questionnaires to all registered laboratories in Slo-
venia at primary, secondary and tertiary level of
health care. The questionnaires comprised of 38
questions regarding the type of tests, patients’ fa-
miliarity with the test procedure, procedure of the
OGTT test and methods of glucose determination.
Results: 80 solved questionnaires (67%) were re-
turned. 64 (71%) laboratories confirmed, that they
perform OGTT. Most of them perform between 1
and 20 OGTT per day. 60 (97%) laboratories do the
75g-OGTT. 54 (91%) of them also perform 75g-
OGTT during pregnancy. The answers in the sec-
ond part of the questionnaire showed some lack
of familiarity with the test procedure. Only 33% of
participants had written instructions for the pa-
tients. The results about a detailed procedure of
OGTT showed that in majority procedures com-
plied with national guidelines. 97% of laboratories
determined the first level of glucose from venous
blood. 76% of laboratories wouldn`t continue the
test if the level of glucose was 7 mmol/L for non
pregnant patients. For pregnant women the level
was 5,1 mmol/L in 92%. We also inquired about
the methods of glucose determinations, the re-
sults showed that 53% used HK, 30% GOD-PAP
and 12% some other amperometric methods. At
the end, we tried to find out how many test are
performed in our country per month. The conclu-
sion was that we do around 1400 tests, 900 tests
are done during pregnancy, which is half of all
pregnant women. In UKC Maribor the number of
pregnancy 75g-OGTT in 2012 was 405. 48 (11,6%)
had glucose levels beyond 5,1 mmol/L and were
diagnosed with gestational diabetes.
Conclusion: Slovenian laboratories in majority fol-
low recommendations and national guidelines
about OGTT.
Key words: OGTT; gestational diabetes; national
guidelines
S59
P5
Urinary protein analysis in diabetic patients
Kostovska I*, Cekovska S, Tosheska Trajkovska K, Labudovik D
Department of medical and experimental biochemistry,
Medical faculty, Skopje, Republic of Macedonia
*Corresponding author: irenakostovska22@yahoo.com
Background: Diabetic nephropathy is a major late
complication in type 2 diabetes mellitus and most
frequent cause of end stage renal disease. Protein-
uria, especially microalbuminuria is early sign of
renal involvement in diabetic patients. The aim of
this study was to investigate the relationship of to-
tal urinary proteins, microalbumin in urine and the
type of proteinuria with diabetes duration.
Material and Methods: This study included 62
patients with diabetes mellitus type 2, average age
of patients 45 ± 9 years, duration of disease from
4,6 years to 19,7 years. Microalbumin and total uri-
nary proteins were measured by turbidimetric
method in first morning urine sample. Urinary pro-
teins were separated by horizontal thinlayer SDS-
PAGE in two urinary samples: first morning urine
and urine excreted after routine daily activity.
Results: We found strong positive correlation be-
tween duration of disease and microalbuminuria
(r= 0,811; p< 0,05) and moderate positive correla-
tion between duration of disease and total urinary
proteins (r= 0,734; p<0,05). According to SDS-PAG
electrophoresis profiles the most common type of
proteinuria in patients with shorter duration of di-
abetes was selective glomerular proteinuria, while
in the patients with longer duration of diabetes
was detected non-selective glomerular with in-
complete tubular proteinuria.
Conclusion: There was positive correlation be-
tween microalbuminuria and total urinary pro-
teins with duration of diabetes. Presence of micro-
albuminuria alerts the physician to prevent further
renal damage by timely administration of ade-
quate therapy.
Key words: total urinary proteins; microalbumin;
diabetes mellitus; SDS-PAGE.
Biochemia Medica 2014;24(Suppl 1):S1-S78
14th EFLM Continuous Postgraduate Course in Clinical Chemistry and Laboratory Medicine
P6
Oxidized proteins and selenium in patients
with type 2 diabetes
Kozic I*, Bakula M, Grubic P, Rumenjak V
Department for Laboratory Diagnostics, Clinical Hospital Sveti
Duh, Zagreb, Croatia
*Corresponding author: irenakozic@yahoo.com
Background: Numerous studies suggest that the
diabetic condition is associated with oxidative
stress. Measurements of oxidized proteins are
used as unspecific indicators of oxidative stress.
The primary functions of selenium are defense
against oxidative stress through selenium-de-
pendent glutathione peroxidase and other seleno-
proteins.
The purpose of this study is to evaluate selenium
and oxidized protein levels in patients with type 2
diabetes compared to healthy controls.
Materials and methods: Subjects. The study
comprehended 54 patients with type 2 diabetes
with various complications, including hyperten-
sion, nephropathy, neuropathy and retinopathy.
Fifty eight age- and sex-matched subjects were in-
cluded for the control.
Selenium concentrations were determined using
Inductively Coupled Plasma-Mass Spectrometry,
ICP-MC (Perkin Elmer). Oxidized protein levels
were determined using ELISA method with 2,4-di-
nitrophenylhydrazine (DNP) (Biocell PC Test, Bi-
oCell Corporation, New Zealand).
Results: Mean values of selenium in type 2 diabet-
ic patients were 104.57±29,17 µg/L. Mean values of
selenium in control subjects were 124.09±25,15
µg/L (P=0,0002). Mean values of oxidized proteins
in type 2 diabetic patients were 0.2585±0,0511
nmol/mg. Mean values of oxidized proteins in con-
trol subjects were 0.1180±0.0623 nmol/mg
(P=<0,0001).
Conclusion: From the results it can be seen that
selenium concentrations in patients with type 2
diabetes were significantly lower compared to
Biochemia Medica 2014;24(Suppl 1):S1-S78
S60
healthy controls. At the same time concentrations
of oxidized proteins were significantly higher in
patients with type 2 diabetes compared to
healthy controls. Using that data we can assume
that lower selenium concentrations subsequently
contribute to reduced protection against oxida-
tive stress which results in higher levels of oxi-
dized proteins.
Key words: oxidized proteins; selenium; diabetes
mellitus
P7
Hyperglycaemia in children with cancer: two
case reports
Lenicek Krleza J1, Kozaj S1*, Grzunov A1, Rajcic A1, Stepan Giljevic J2,
Jakovljevic G2
1
Department od Laboratory Diagnostics, Children’s Hospital
Zagreb, Zagreb, Croatia
2
Haemato-Oncology Department, Children’s Hospital Zagreb,
Zagreb, Croatia
*Corresponding author: sanjakozaj@gmail.com
Background: Hyperglycaemia induced by stress
of disease or steroid therapy may indicate an early
defect in glucose regulation. Hyperglycaemia is
known to be a common adverse reaction of ster-
oid therapy in 20% to 50% of nondiabetic patients.
In cancer patients, corticosteroids are often pre-
scribed as part of treatment or for symptom man-
agement.
Aim: We present two cases with hyperglycaemia
during chemotherapy: one with corticosteroid-in-
duced hyperglycaemia and the other one with hy-
perglycaemia induced by stress of chemotherapy.
Patients: Case 1, a 12-year-old girl with acute lym-
phatic leukaemia; Case 2, a 2-year-old boy with
yolk sac testicular tumour. Both children, without a
history of diabetes, were treated with chemother-
apy at Haematology-Oncology Department, Za-
greb Children’s Hospital. Corticosteroid therapy
was introduced during chemotherapy (2nd cycle)
only in Case 1.
 14th EFLM Continuous Postgraduate Course in Clinical Chemistry and Laboratory Medicine
Results: Case 1: Before corticosteroid therapy,
blood glucose was normal (<5 mmol/L). Hypergly-
caemia developed after corticosteroid therapy in-
troduction, increasing glucose concentration to up
to 16.4 mmol/L, without acidosis. Hyperglycaemia
was present throughout the treatment. Treatment
without insulin maintained blood glucose at 11.0
mmol/L or less. Upon completion of corticosteroid
therapy and chemotherapy, glucose level returned
to the normal range.
Case 2: Before and during chemotherapy without
corticosteroids, glucose concentration was within
the reference range (<5.0 mmol/L). At the end of
the 4th chemotherapy cycle, glucose concentration
increased to 23.5 mmol/L (repeated after 12h: 22.8
mmol/L) with ketoacidosis (BE=-17.9). The boy was
referred to Endocrinology Department, where
type 1 diabetes was verified.
Conclusion: Our results point to the necessity of
glucose monitoring in cancer patients undergoing
chemotherapy, in particular when corticosteroids
are introduced in therapy. Corticosteroid induced-
hyperglycaemia is transient in most cases (Case 1).
In patients with prediabetes, stress and physical
effects of chemotherapy can lead to diabetes
(Case 2).
Glucose monitoring and continuous insulin thera-
py are recommended at glucose levels >7.8
mmol/L. This approach may prevent inflamma-
tion, immunosuppression, and symptoms of hy-
perglycaemia. Also, it may help recognize previ-
ously undiagnosed diabetes.
Key words: hyperglycaemia; chemotherapy; corti-
costeroid therapy; child; diabetes
S61
P8
Urinary patterns analysis of type 2 diabetes
mellitus patients by nuclear magnetic
resonance spectroscopy method (1H-NMR)
Stefan LI1*, Nicolescu A2, Sandu M3, Popa GS3,4, Moţa M3,4, Deleanu C5
1
Craiova County Clinical Emergency Hospital, Department of
Clinical Chemistry and Laboratory Medicine, Craiova, Romania 
2
Petru Poni Institute of Macromolecular Chemistry, Group of
Biospectroscopy, Iasi, Romania 
3
Craiova County Clinical Emergency Hospital, Department of
Diabetes Nutrition and Metabolic Diseases, Craiova, Romania 
4
University of Medicine and Pharmacy, Department of Diabetes
Nutrition and Metabolic Diseases, Craiova, Romania 
5
Costin D. Nenitescu Institute of Organic Chemistry, Group of
Biospectroscopy, Bucharest, Romania
*Corresponding author: lorenaivona@yahoo.com
Background: Proton nuclear magnetic resonance
spectroscopy method (1H-NMR) was applied to in-
vestigate the urinary patterns of type 2 diabetes
mellitus (T2DM) patients to identify possible dis-
turbances that may accompany T2DM. We investi-
gate the potential relationship between diabetic
retinopathy (DR), diabetic neuropathy (DN), esti-
mated glomerular filtration rate (eGFR), anthropo-
metric indicators (body mass index (BMI), waist cir-
cumference (WC), waist to hip ratio (WHR) and
waist to stature ratio (WSR)), duration of T2DM and
urinary metabolites in T2DM.
Materials and Methods: Serial urine samples of
167 healthy subjects and 185 T2DM patients were
investigated by 1H-NMR. The 1H-NMR spectra have
been recorded on a Bruker Avance DRX 400 MHz
spectrometer. The results are evaluated in mmol/
mol of creatinine. p<0.05 was taken as significant.
A significant difference between the urinary excre-
tion of valine, 3-hydroxyisolateric acid, alanine,
gamma-aminobuthyrate, betaine, citric acid, tri-
methylamine-N-oxide and glycine at the healthy
individuals and T2DM patients was found. The val-
ues for 3-hydroxyisolateric acid and gamma-amin-
obuthyrate increase in T2DM patients with retin-
opathy vs. without retinopathy. There was no cor-
relation between DN and urinary metabolite pic-
ture in T2DM patients.
Biochemia Medica 2014;24(Suppl 1):S1-S78
14th EFLM Continuous Postgraduate Course in Clinical Chemistry and Laboratory Medicine
Results: We found significant correlations be-
tween eGFR and dimethylamine (r=0.194, p=0.031),
gamma-aminobuthyrate (r=0.239, p=0.049), ace-
tate (r=0.29, p=0.035) and pyruvate (r=0.275,
p=0.014) in T2DM patients. Our analysis revealed
significant decreased concentrations for citrate, di-
methylamine and glycine in T2DM patients with
the increase of BMI. WC were positively correlated
with gamma-aminobuthyrate (r=0.42, p=0.01) and
dimethylamine (r=0.39, p=0.03) and, no correlation
were observed between WHR, WSR and urinary
metabolites in T2DM patients. There are higher uri-
nary concentrations for alanine, 3-hydroxyisovaler-
ic acid, citrate and dimethylamine in newly diag-
nosed type 2 DM patients, while the hippurate in-
creased with the increase of duration of type 2 DM.
Conclusions: 1H-NMR spectroscopy can be a
method to explore urinary metabolite as markers
for early detection of associated diseases and com-
plications in diabetes.
Key words: nuclear magnetic resonance spectros-
copy method; type 2 diabetes mellitus; urine
P9
Urinary biochemistry of type 1 diabetes
mellitus patients using Proton Nuclear
Magnetic Resonance Spectroscopy Method
(1H-NMR)
Stefan LI1*, Nicolescu A2, Sandu M3, Popa GS3,4, Moţa M3,4, Deleanu C5
1
Craiova County Clinical Emergency Hospital, Department of
Clinical Chemistry and Laboratory Medicine, Craiova, Romania  N-oxide and gamma-aminobuthyrate are higher
2
Petru Poni Institute of Macromolecular Chemistry, Group of
Biospectroscopy, Iasi, Romania 
3
Craiova County Clinical Emergency Hospital, Department of
Diabetes Nutrition and Metabolic Diseases, Craiova, Romania  tabolites are interesting in various aspects, such as
4
University of Medicine and Pharmacy, Department of Diabetes
Nutrition and Metabolic Diseases, Craiova, Romania  nisms of the disease or potential early diagnostic
5
Costin D. Nenitescu Institute of Organic Chemistry, Group of
Biospectroscopy, Bucharest, Romania
*Corresponding author: lorenaivona@yahoo.com
Background: In recent years, Proton Nuclear Mag-
netic Resonance Spectroscopy Method (1H-NMR)
Biochemia Medica 2014;24(Suppl 1):S1-S78
S62
had emerged as a premier research method for
the analysis of biological samples.
In the first section of this study we compared the
value of the concentrations of different metabo-
lites between normal and type 1 diabetes mellitus
(T1DM) patients to obtain basic knowledge of pos-
sible differences in the urinary excretion or con-
centrations of a series of metabolites between pa-
tients with type 1 diabetes and nondiabetic indi-
viduals and to assess the NMR potential as a diag-
nostic tool. In the next setion of the study T1DM
patients were evaluated according to age and du-
ration of T1DM and the NMR profile of metabolites
concentrations established.
Materials and Methods: Serial urine samples of
167 control subjects and 132 T1DM patients were
investigated by 1H-NMR method. The patients had
a history of T1DM less than 5 years. The NMR spec-
tra were recorded on a Bruker Avance DRX 400
MHz spectrometer. To 0.9 ml urine, 0.1 ml of stock
solution of 5 mM sodium 3-(trimethylsilyl)-[2, 2, 3,
3-d4]-1-propionate in D2O has been added. The re-
sults are evaluated in mmol/mol of creatinine.
p<0.05 was taken as significant.
Results: A significant difference between the uri-
nary excretion of lactate, citrate, hippurate and
gamma-aminobuthyrate at the healthy individuals
and T1DM patients was found.
The T1DM patients below 35 years old tended to
have higher urinary values of lactate, alanine,
pyruvate, citrate, choline and hippurate than T1DM
patients above 35 years old. The urinary excretion
of valine, lactate, citrate, glycine, , trimethylamine-
in patients with duration of T1DM less than 1 year.
Conclusions: Type 1 diabetes mellitus urinary me-
providing clues for the biochemistry and mecha-
markers in diabetes renal involvement.
Key words: nuclear magnetic resonance spectros-
copy method; type 1 diabetes mellitus; urine
 14th EFLM Continuous Postgraduate Course in Clinical Chemistry and Laboratory Medicine
P10
Plasma cholinesterase method comparison
Finderle P*, Snoj N
Clinical Institute of Clinical Chemistry and Biochemistry,
University Medical Centre Ljubljana, Slovenia
*Corresponding author: petra.finderle@kclj.si
Background: Cholinesterases are enzymes capa-
ble of hydrolyzing choline esters. There are two
types of cholinesterases: Acetilcholinesterase
(E.C.3.1.1.7) present in red blood cells and nerve tis-
sues, and Cholinesterase (E.C.3.1.1.8) found in plas-
ma, liver, heart, and other tissues. The biological
role of plasma cholinesterase (CHE) has not been
clearly established, however, it has been reported
that CHE may be involved in lipoprotein metabo-
lism. It is known, that Diabetes mellitus is a low
grade systematic inflammation disease. Persistent
hyperglycemia causes increased production of
free radicals, especially reactive oxygen species,
which affect lipoproteins metabolism. For this rea-
son the uses of plasma CHE measurements in dia-
betes are nowadays increasing.
Materials and methods: We evaluated two meth-
ods for plasma CHE determination with different
detection principles: dry chemistry test with re-
flectance measurement (Vitros Chemistry Cho-
linesterase), and liquid chemistry test with absorb-
ance measurement (Simens Advia Chemistry Cho-
linesterase). Both methods are based on the cata-
lytical activity of CHE to hydrolyse butyrylthiocho-
line to thiocholine, which afterwards reacts with a
dying compound. The rate of change in the meas-
ured signal is proportional to CHE activity in the
sample.
We collected 58 heparin plasma samples with rou-
tinely ordered CHE determination. The data was
statistically evaluated with least-square analysis
method.
Results: Both methods showed a very good
agreement (r=0,996), although dry chemistry
method results are significantly lower than liquid
chemistry results (p<0.0001). The difference can be
S63
overcome by taking into account the correlation
equation (intercept=8,753 and slope=0,721) and
different method linearity limits.
Conclusions: We have shown that both methods
meet appropriate expectations for plasma cho-
linesterase determination in diabetes patients.
Key words: analysis comparison; cholinesterase; li-
poprotein metabolism; diabetes
P11
Is there a role for HbA1c in diagnosing
gestational diabetes in high-risk
pregnancies?
Krhač M 1*, Radišić Biljak V 1, Radeljak A 1, Božičević S 1, Prašek M 2,
Vučić Lovrenčić M 1
1
Department of Medical Biochemistry and Laboratory Medicine
2
Vuk Vrhovac University Clinic for Diabetes, Endocrinology and
Metabolic Diseases, Merkur University Hospital, Zagreb, Croatia
*Corresponding author: mkrhac@kb-merkur.hr
Background: The use of HbA1c had been recently
proposed for the diagnosis of diabetes melitus,
but it’s diagnostic use for gestational diabetes
(GDM) had not been recommended. Thus, a cum-
bersome and poorly reproducible 75 g oral glu-
cose tolerance test (oGTT) remains the only rec-
ommended diagnostic tool for GDM.
The aim of this study was to assess the diagnostic
accuracy of HbA1c in pregnant women with high
risk for developing GDM.
Materials and Methods: 785 pregnant women,
referred to our clinic for the diagnosis of GDM un-
derwent a standard 75 g oGTT procedure with ve-
nous plasma glucose measured with an automat-
ed hexokinase procedure (Beckman Coulter
AU680, USA) at fasting, 1h and 2h after glucose
load (FPG,1h-PG and 2h-PG, respectively). Mean
gestational age was 28 ± 5,2 weeks.The classifica-
tion of glycaemic status was carried out according
to the IADPSG/WHO criteria. HbA1c was sampled
at fasting and measured with an automated im-
Biochemia Medica 2014;24(Suppl 1):S1-S78
14th EFLM Continuous Postgraduate Course in Clinical Chemistry and Laboratory Medicine
munoturbidimetric procedure (TinaQuant-Integra
400Plus, Roche Diagnostics, USA).
Results: Women with GDM (N=367) were older
(P=0,006), and had higher both pre-gestational
BMI and gestational body-weight (P<0,001) than
healthy women (N=418). HbA1c was significantly
higher in the GDM-group (P<0,001), as were FPG,
1h-PG and 2h-PG, respectively (P<0,001). ROC-
curve analysis for HbA1c revealed an AUC of 0,647
(P<0,001), and a cutoff at 5,1%/32 mmol/mol (69,5%
sensitivity, 50% specificity). Stepwise linear regres-
sion analysis identified FPG, 1h-PG and gestational
age as the most significant determinants of HbA1c
level (P<0,001), followed by the body weight
(P=0,0303) and age (P=0,0327), whereas 2hPG and
pre-gestational BMI had no influence on HbA1c
level.
Conclusions: Despite significantly higher HbA1c in
GDM, there is a substantial overlap of HbA1c val-
ues between the healthy and GDM population,
which might compromise its diagnostic use in a
high-risk population. However, our results suggest
that assessment of HbA1c-utility for screening of
GDM in general population merits further investi-
gation.
Key words: gestational diabetes; oral glucose tol-
erance test; HbA1c
P12
Antioxidative Effect of Naringenin on the
Activity of Superoxide Dismutase and
Glutathione Peroxidase in HepG2 Cells
under Hyperglycaemic Conditions
Knežević T1*, Petlevski R2
1
Primary health centre Zagreb Centar, Zagreb, Croatia
2
Faculty of Pharmacy and Biochemistry, University of Zagreb,
Zagreb, Croatia
*Corresponding author: tami.knezevic@gmail.com
Background: Oxidative stress is believed to play a
pivotal role in the etiology and pathogenesis of di-
abetes mellitus and its late complications. Hyper-
Biochemia Medica 2014;24(Suppl 1):S1-S78
S64
glycaemia-induced oxidative stress in diabetes is a
consequence of both increased production of free
radicals and reduced capacity of antioxidative de-
fence. The citrus-derived flavonoid, naringenin,
possesses antioxidative, free radical scavenging
and metal chelating properties. The aim of this
work was to investigate the antioxidative effect of
naringenin on the activities of superoxide dis-
mutase (SOD) and glutathione peroxidase (GPx) in
HepG2 cells under hyperglycaemic conditions.
Materials and methods: Activities of SOD and
GPx were measured in lysates of HepG2 cells (ATCC
HB­8065, Manassas, USA). HepG2 cells tested were
divided in six groups: control group, diabetes
group (30 mM glucose) and four diabetes-narin-
genin groups (30 mM glucose + naringenin in one
of the following concentrations: 1000, 100, 10 and
1 µg/mL). Activities of both enzymes were meas-
ured spectrophotometrically and expressed by
protein concentrations.
Results: The results were reported as specific ac-
tivities of SOD and GPx. All groups tested were
compared to diabetes group. Student t-test was
used to compare tested groups. P-value <0.05 was
adopted as statistically significant. Diabetes-narin-
genin group (30 mM glucose + 1 µg/mL narin-
genin) showed significantly increased activities of
SOD (P<0.001) and GPx (P=0.007). On the other
hand, in diabetes-naringenin group (30mM glu-
cose + 10 µg/mL naringenin) only SOD showed in-
creased activity (P=0.002). The other two diabetes-
naringenin groups did not show significantly high-
er activities of SOD and GPx.
Conclusion: The results obtained from this work
indicate that naringenin exhibits its antioxidative
effect on the activity of SOD and GPx in HepG2
cells under hyperglycaemic conditions when ap-
plied in lower concentrations, 1 µg/mL and 10 µg/
mL.
Key words: naringenin; oxidative stress; hypergly-
caemia; antioxidative enzymes
 14th EFLM Continuous Postgraduate Course in Clinical Chemistry and Laboratory Medicine
P13
Performance of equations for estimating
glomerular filtration rate in diabetic patients
Šimetić L*, Drmić S, Zibar L, Begić I, Šerić V
Clinical Hospital Center and University Medical School Osijek,
Osijek, Croatia
*Corresponding author: lusimetic@gmail.com
Background: Acetoacetate, glucose and hydroxy-
butyrate have been reported as interference in the
Jaffé method for creatinine determination. They
can be found in urine of diabetics. They could be
responsible for overestimation of GFR by creati-
nine clearance (CrCl), or be a source for variability
in CrCl when compared with eGFR.
Materials and methods: Study enrolled 235 males
(ages 18 to 83 years, mean 56.00 ± 14.34) and 265
females (ages 19 to 86 years, mean 58.38 ± 13.93).
There were 267 diabetics and 233 non-diabetics. We
tested performance of Cockroft-Gault, MDRD and
CKD-EPI equations. Serum creatinine was analyzed
by enzymatic method (with creatininase, creatinase,
sarcosine oxidase and phenol-aminophenazone
peroxidase), and urine creatinine by Jaffé method
on Beckman Coulter AU680. Calibration material
was Beckman Coulter system calibrator (traceable
to NIST SRM 967) and Beckman Coulter urine cali-
brator (traceable to ID-MS reference method). Sta-
tistical analysis was done by Kolmogorov-Smirnov
test, bivariate correlation and ROC (MedCalc).
Results: In diabetics, as well as non-diabetics,
MDRD showed the highest correlation with CrCl.
In the group of diabetics CKD-EPI equation
showed the highest AUC at CrCl cut off 90 ml/min,
which would make this formula the best predictor
of CrCl when it comes to discriminating normal
and slightly decreased GFR. In identifying diabet-
ics with moderately decreased GFR (cut off of 60
ml/min) MDRD had higher AUC than CKD-EPI. Ob-
served differences between AUC were not signifi-
cant (only in non-diabetics CKD-EPI was signifi-
cantly better at predicting CrCl than CG). Results
were considered significant at p<0.05.
S65
Conclusions: All equations had better correlation
with CrCl in non-diabetics than in diabetics. Possi-
bly the reason is hyperfiltration of the glomeruli in
the initial stage of diabetes that affects CrCl (in-
creasing urine creatinine and volume), but not the
eGFR or ketone interfering with Jaffé method.
However, this didn’t have an effect on the eGFR
performance in diabetics in this study. All eGFR
had great performance and no one was statistical-
ly better than the others.
Key words: Diabetes mellitus; creatinine clear-
ance; estimeted glomerular filtration rate
P14
Serum Omentin-1 Level in Diabetic Patients
on Haemodialysis: a pilot study
Kocijancic M1*, Vujicic B2, Racki S2, Zaputovic L3, Dvornik S4
1
Medical biochemistry laboratory of Primorsko-goranska
county health care Rijeka, Croatia
2
Department of Nephrology, Dialysis and Transplantation,
Clinical Hospital Center Rijeka, Croatia
3
Department of Cardiovascular Disease, Clinical Hospital Center
Rijeka, Croatia
4
Department of Laboratory Diagnostics, Clinical Hospital
Center Rijeka, Croatia
*Corresponding author: marija.percan@gmail.com
Background: The most common cause in the inci-
dence of end stage renal disease (ESRD) is diabe-
tes mellitus. The main cause of mortality in diabe-
tes positive dialysis patients is cardiovascular dis-
ease (CVD). It’s important to identify and prevent
cardiovascular risk factors in dialysis patients. The
serum omentin-1 level was found to be associated
with cardio- metabolic disorders such as insulin re-
sistance, diabetes, dyslipidemia and CVD. Omen-
tin-1 may play an important role in the pathogen-
esis of atherosclerosis and it may be predictive pa-
rameter of co morbidities associated with obesity
and glucose metabolism.
The aim of this study was to examine possible clin-
ical significance of serum omentin-1 level in hae-
modialysis (HD) patients.
Biochemia Medica 2014;24(Suppl 1):S1-S78
14th EFLM Continuous Postgraduate Course in Clinical Chemistry and Laboratory Medicine
Methods: A total of 60 prevalent HD patients at
Clinical Hospital Center Rijeka were included in the
cross-sectional study. Patients were divided into
two groups according to presence of diabetes. Ve-
nous blood sample was withdrawn after an over-
night fasting before midweek HD session. Serum
omentin-1 level was assessed by enzyme-linked
immunosorbent assay.
Results: Diabetes negative group was comprised
26 subjects: 16 males and 10 females of average
ages 67 (range 33-88) and diabetes positive group
was comprised 34 subjects: 25 males and 9 fe-
males of average ages 70 (range 31-87). Omentin-1
levels of diabetic HD patients were found to be
lower than of non-diabetic HD patients (8,4±3,8
µg/l vs. 12,7±2,9 µg/l, respectively; P<0,001).
Conclusions: Serum omentin-1 levels were signifi-
cantly lower in diabetic HD patients. We believe
that decreased omentin-1 levels could play an im-
portant role in progression of atherosclerosis
through its action on the vascular endothelial in-
flammatory state in this patients group. Further in-
vestigation in prospective clinical study with
greater number of patients is needed.
Key words: dialysis; diabetes; omentin-1 protein
P15
How well are the patients in Croatia
informed about the OGTT procedure?
Banković Radovanović P1*, Kocijančić M2
1
Medical biochemistry laboratory, General Hospital Pula,
Croatia
2
Medical biochemistry laboratory of Primorsko-goranska
county health care Rijeka, Croatia
*Corresponding author: patricija@hi.htnet.hr
Background: Pre-analytical errors still constitute
the largest source of errors in laboratory work. The
accuracy and reliability of any diagnostic proce-
dure is strongly dependent on the proper patient
preparation and patient’s knowledge about the
certain procedure. We hypothesized that most of
Biochemia Medica 2014;24(Suppl 1):S1-S78
S66
the patients are not informed well enough about
the proper OGTT procedure for blood sampling.
The aims of this study were to investigate: i) how
well patients are informed about the OGTT proce-
dure for laboratory blood testing; ii) the most com-
monly used way to inform the patients and iii)
whether the particular ways of informing differ an-
yhow.
Materials and methods: The anonymous ques-
tionnaire was conducted across the country in 23
Croatian primary and secondary healthcare cen-
tres. All 329 patients were instructed by laboratory
staff on how to answer the questionnaire, but not
about the OGTT procedure itself. The participants
filled the questionnaire before the first blood
draw. Data analysis was performed by using non-
parametric tests were appropriate, with the P val-
ue less than 0.05, as the level of significance.
Results: The results showed that: (i) a greater pro-
portion of participants (90%) had enough informa-
tion about adequate preparation for the OGTT,
but only a minority of the patients (42%) were
completely familiar with the entire OGTT proce-
dure, (ii) their major source of instructions was
their gynaecologist (66%), and (iv) the ways of in-
forming patients made a difference in the patients’
awareness (P = 0.030).
Conclusion: In general patients are not familiar
enough with the whole OGTT procedure. In order
to improve the pre-analytical phase of the labora-
tory work it is very important that the laboratory
provides the doctors with clear and understanda-
ble written instructions for the preparation of the
patients as well as that all changes and updates
are available to all users of laboratory services in
an adequate timeframe.
Key words: pre-analytical errors; survey; patient
education
 14th EFLM Continuous Postgraduate Course in Clinical Chemistry and Laboratory Medicine
P16
Hb1Ac comparıson and evaluatıon ın
patıents wıth cardıovascular dıseases
Isiksacan N*, Koser M
Central Laboratory, Bakirkoy Dr Sadi Konuk Training and
Research Hospital, Bakirkoy, Istanbul, Turkey
*Corresponding author: nisiksacan@gmail.com
Background: Type II Diabetes Mellitus(T2DM) is
the most frequent endocrin disease. Elevated
HbA1c level is reported as a risk factor in cardiovas-
cular diseases whereas cardiovascular diseases
cause death and disability among diabetic pa-
tients.
The aim of the study is to compare the immuno-
turbidimetry and high performance liquid
chromatography(HPLC) methods and determine
the HbA1c status among patients who were ad-
mitted to our hospital and policlinics to select the
best method for determining HbA1c in order to
meet the needs of the patients.
Materials-Methods: HbA1c levels from 20.503 pa-
tients (12288 females and 8215 males) were col-
lected from the Laboratory Information
system(LIS) retrospectively. Patients were admit-
ted to our Cardiology and Cardiovascular Surgery
Hospital for cardiovascular reasons and to our In-
ternal Medicine policlinics for non-cardiovascular
reasons between 2011 and 2012, whereas HbA1c
levels were analyzed with high performance liquid
chromatography (HPLC). Also venous blood sam-
ples obtained in EDTA containing tubes from 237
in-and-out patients of our hospital (155 females
and 82 males) were analyzed in Premier Hb
9210(Trinity Biotech –USA) and Cobas c501(Roche
–Germany) using HPLC and immunoturbidimetry
respectively. This two methods were compared.
Results: Patients with HbA1c level ≤6 % were 47,5
%, ≤7 were 74,5 %, ≤8 were 85,2% and ≥8 were
14,8 % of all patients. After the comparison of the
two methods there was found a strong correlation
from all patients (n= 237)(r2=0.90) and from pa-
tients with HbA1c level ≥6.0 % (n=125) (r2=0.88).
S67
Conclusion: The importance of HbA1c in patients
with cardiovascular diseases and the HbA1c levels
among our patients indicate that HPLC represents
a better choice to determine HbA1c in our hospi-
tal. But immunoturbidimetry can be the choice of
the method in laboratories with low number of
patients. Each laboratory should consider which
method is the best.
Keywords: HbA1c; HPLC; immunoturbidimetry;
cardiovascular disease; comparison
P17
Evaluation of the analytical quality of
B-ANALYST as a POCT analyzer for HbA1c
determination
Menéndez-Valladares P*, Fernández-Riejos P, Sánchez-Mora C,
Pérez-Pérez A, Sánchez-Margalet V, González-Rodríguez C
Clinical Biochemistry Department, Virgen Macarena University
Hospital, Seville, Spain
*Corresponding author: palomamenval@hotmail.com
Background: Type 2 diabetes mellitus (DM2) rep-
resents a major public health issue. The incidence
and prevalence of DM2 continue to grow world-
wide. The risk of DM2 is associated with morbidity
and mortality, which increase healthcare costs.
Glaciated hemoglobin (HbA1c) is a good marker
for glycemic control. Point-of-care testing technol-
ogy (POCT) for HbA1c determination allows a bet-
ter glycemic monitoring of diabetic patients and
avoids complications of poorly controlled diabe-
tes.
Materials and Methods: we randomly selected
samples received in our routine laboratory for
HbA1c determination. Samples were collected by
venipuncture into EDTA tubes and analyzed in du-
plicate. We use the HA-8180 (Menarini Diagnos-
tics®) as a reference method which employs cation-
ic exchange High Performance Liquid Chromatog-
raphy (HPLC). The B-Analyst (Menarini Diagnos-
tics®) is based on the principle of latex agglutina-
tion immuno-turbidimetric method. For the com-
Biochemia Medica 2014;24(Suppl 1):S1-S78
14th EFLM Continuous Postgraduate Course in Clinical Chemistry and Laboratory Medicine
parison study we selected 120 samples divided in
4 ranges. For inter-assay variability, we repeated
the same sample during 12 consecutive days. For
intra-assay study we repeated each sample ten
consecutive times during the same day.
Results: regression analysis of the data for the
method comparison between HA-8180 and B-An-
alyst showed a slope of 1,0097 and an intercept of
0,1287. The Pearson’s correlation coefficient was
0,9894 (p<0,0001). Bias study of HbA1c measure-
ments for B-Analyst showed a mean difference re-
spect to HA-8180 of 0,205 with a 95% confidence
interval. The concordance correlation coefficient
to asses accuracy was 0,9904 (0,9866-0,9932). The
CV for the inter-assay study was 1,4%. For the in-
tra-assay study we analyzed 3 samples with differ-
ent HbA1c%, whose CV were 1,03% (4,7% HbA1c),
0,46% (6,4% HbA1c) and 0,78% (8,1% HbA1c).
Conclusions: the evaluated B-Analyst showed
good linear correlation with the reference meth-
od. It also showed good accuracy both in the inter-
assay and in the intra-assay. The B-Analyst carried
out with quality specifications required for moni-
toring of diabetic patients.
Key words: POCT; diabetes; HbA1c
P18
Application of the guidelines for the request
of microalbuminuria in diabetic patients
Lukic V*
Department of Clinical Chemistry, Railway Healthcare Institute,
Belgrade, Serbia
*Corresponding author: veralukic.lab@gmail.com
Background: Microalbumiuria is defined as excre-
tion of 30–300 mg of albumin/24 h. American Dia-
betes Association and National Kidney Foundation
guidelines, but also Serbian clinical guidelines for
diabetes, emphasise significance of microalbumi-
nuria determination in diabetic patients because
of early diagnosis of incipient diabetic nephropa-
thy. It is recommended that annual testing for mi-
Biochemia Medica 2014;24(Suppl 1):S1-S78
S68
croalbuminuria in patients without clinical pro-
teinuria should begin 5 years after diagnosis of
type 1 diabetes and at the time of diagnosis of
type 2 diabetes. The diagnosis of microalbuminu-
ria requires the demonstration of increased albu-
min excretion on 2 of 3 tests performed within 6
months.
Aim of this paper is to show frequency of microal-
buminuria testing among population of diabetic
patients in Railway Healthcare Institute, Belgrade.
Materials and methods: Data were extracted
from laboratory information system about num-
ber of patients with diagnosis of diabetes mellitus
and laboratory tests which were ordered for them
in 2013.
Results: In 2013. 2269 patients with diabetes mel-
litus made 3915 visits in our laboratory. 61051 labo-
ratory analyses were done for them. But among all
those tests only 111 were microalbuminuria test-
ing, which means that only 4.89% of diabetic pa-
tients in our institution had their annually microal-
buminuria testing done.
Conclusions: Microalbuminuria is under request-
ed test, potentially affecting longer-term health
outcomes. Causes for this could be that doctors
don’t apply national and international recommen-
dations in their everyday practice, but also the fact
that health insurance in Serbia doesn’t accept
costs of this test at the primary health care level, so
patients have to pay for it. Corrective measures
could be: education of primary health care doctors
(we have organized two lectures about microalbu-
minuria clinical significance in 2013), education of
diabetic patients and putting microalbuminuria
on the list of tests which are covered by health in-
surance.
Key words: microalbuminuria; diabetes; guide-
lines
 14th EFLM Continuous Postgraduate Course in Clinical Chemistry and Laboratory Medicine
P19
Plasma glucose concentration, HbA1c
together in assessing stability of diabetes
Nake A1*, Hatibi S2
1
Medical University of Tirana, Faculty of Tech Medical Sciences;
Tirana
2
Regional Health Authority, Tirana
*Corresponding author: a_mfshm@yahoo.com
Introduction: Diagnosis and monitoring of dia-
betic patients performs with two basic examina-
tions, measurement of plasma glucose concentra-
tions and HbA1c values as a condition for stability
of average plasma glucose values and delaying of
clinical complications by endocrinologist.
Aim: Argumentation of necessity of evaluation of
plasma glucose concentrations and HbA1c values
together for stability of plasma glucose concentra-
tion.
Material and methods: We’ve studied plasma
glucose values (mg/dL) and HbA1c (%) determined
by reference standard methods in 37 patients with
diabetes, with average age 67.2 ± 10.1. We’ve cal-
culated and compared average values of plasma
glucose concentrations between two groups of
patients those with HbA1c = 4-8 (first group) and
those with HbA1c> 8(second group). We’ve calcu-
lated Pearson correlation coefficient between
plasma glucose and HbA1c, respectively.
Results: In our diabetic patients: HbA1c = 8.79 ±
1.74; plasma glucose 191 ± 61.5; Pearson correla-
tion coefficient between plasma glucose concen-
trations and HbA1c levels, resulted :r = 0.52; Calcu-
lation and comparison of average values of plasma
glucose concentrations between two groups of
patients resulted respectively: 132 ± 54 and 231 ±
70.3. It was observed statistically significant differ-
ence between these values (p <0.0001).
Conclusion: HbA1c monitoring is an indispensa-
ble examination to evaluate the degree of instabil-
ity of diabetes together with plasma glucose con-
centration. The value of Pearson correlation coeffi-
cient tells us the necessity of performing these
S69
two examinations, especially for monitoring pur-
poses.
Key words: blood glucose; glycated hemoglobin
HbA1c; diabetes
P20
Dried Blood Spot: Could potassium solve
hematocrit issue?
Bilandžija I*, Fumić K
Department of Laboratory Diagnostics, University Hospital
Centre Zagreb, Zagreb, Croatia
*Corresponding author: ivabilandzija@yahoo.com
Background: Dried blood spots (DBS) on filter pa-
per is one of the easiest and noninvasive way to col-
lect samples from different population, with several
advantages over using conventional sampling. It
brings lot of diagnostics possibilities in different dis-
eases including diagnostics and monitoring diabe-
tes. However, it is still associated with some issues.
Hematocrit (Hct) is the most widely discussed chal-
lenge, as deviation of Hct values may significantly
impact DBS-based quantitation and different dif-
fuse on filter paper which results in sample concen-
tration differences per punch area. It is particularly
important in newborn samples where Hct values
tend to change hourly. To solve this issue we meas-
ured potassium (K+) concentration, endogenous in-
tracellular electrolyte that also varies with Hct.
Materials and Methods: We used 85 DBS sam-
ples from newborns, children and adults. To evalu-
ate K+ extraction from DBS several procedures
were tested. After extraction, K+ was measured by
indirect potentiometric using the ISE module on
Cobas 6000 Clinical Chemistry Analyzer. Hct was
measured on Sysmex XE-5000.
Results: 0.25% Tritona X-100 with freezing and de-
frosting showed as the most effective way to re-
lease as much as K+ from 3-mm DBS punches in
the least possible volume. Potassium concentra-
tion was from 0.36 to 1.89 mmol/L, with hemato-
crit values from 0.158 to 0.697 L/L. We noticed a
Biochemia Medica 2014;24(Suppl 1):S1-S78
14th EFLM Continuous Postgraduate Course in Clinical Chemistry and Laboratory Medicine
significant dispersion of results when potassium
concentration was greater than 1.3 mmol/L or he-
matocrit was greater than 0.6 L/L.
Conclusion: We developed a procedure to predict
the approximate Hct of DBS, based upon K+ meas-
urement. Comparing measured and calculated he-
matocrit showed good correlation between their
values. From measured potassium concentration
calculated hematocrit values can be used for correc-
tion of the measured DBS analyst values. For K+ val-
ues above 1.3 mmol/L and hematocrit above 0.6 L/L
(neonatal population) a special curve relationship
with concentrations of potassium has to be made.
Key words: dried blood spot; hematocrit; potassi-
um concentration; newborns
P21
Association of serum nesfatin-1/NUCB2
with metabolic risk factors in non-obese,
normoglycemic subjects
Bergmann K1*, Olender K1, Kretowicz M2, Manitius J2,
Sypniewska G1
1
Department of Laboratory Medicine, Nicolaus Copernicus
University Collegium Medicum in Bydgoszcz, Poland
2
Department of Nephrology, Hypertension and Internal
Diseases, Nicolaus Copernicus University Collegium Medicum
in Bydgoszcz, Poland
*Corresponding author: bergmann@vp.pl
Background: Nesfatin-1 is a polypeptide encoded
in the N-terminal region of Nucleobindin2 (NUCB2),
expressed in the hypothalamus, pancreatic islets,
gastric endocrine cells and adipocytes. Recent stud-
ies indicate its role in regulation of satiety and stimu-
lation of insulin secretion. We assessed the relation-
ship between serum nesfatin-1/NUCB2 and selected
metabolic risk factors in normoglycemic individuals.
Materials and Methods: Study included 80 nor-
moglycemic, non-obese (BMI <30 kg/m2) subjects
aged 25-40 years (32 women, 48 men). Basic an-
thropometric parameters (weight, BMI, WHR) and
blood pressure measurements were performed.
Laboratory tests: fasting plasma glucose, glycated
Biochemia Medica 2014;24(Suppl 1):S1-S78
S70
hemoglobin (HbA1c), lipid profile, insulin, CRP,
apolipoproteins AI and B were measured on auto-
matic analyzers (Abbott Architect ci8200, Roche
Cobas e411). Adiponectin and nesfatin-1/NUCB2
were assayed by commercially available ELISA kits
(BioVendor R&D, Phoenix Pharmaceuticals Inc.).
Results: Nesfatin-1/NUCB2 levels ranged 0.53-
14.38 ng/mL and were significantly higher in wom-
en compared to men (1.28 vs. 0.82 ng/mL; p=0.02).
In men nesfatin-1/NUCB2 correlated negatively
with glucose (R= -0.51; p=0.009), insulin (R= -0.33;
p=0.038) and HOMA-IR (R= -0.42; p=0.027), while
inverse relationship was observed in women. Mul-
tivariable regression analysis with glucose, insulin
and HOMA-IR in females and with glucose, HOMA-
IR and adiponectin in males explained 87% and
32% of nesfatin-1/NUCB2 variability.
Conclusions: Association of serum nesfatin-1/
NUCB2 with metabolic risk factors differs essential-
ly by gender, however this issue requires further
investigation in large, population-based study.
Key words: nesfatin-1/NUCB2; adipocytokines;
metabolic risk; insulin resistance; type 2 diabetes
P22
Importance of the analysis of the
fructosamine 3-kinase gene promoter
region: experience in an Italian cohort of
diabetic patients
Avemaria F1*, Carrera P2, Lapolla A3, Ferrari M 2,4, Mosca A1
1
Department of Pathophysiology and Transplantation, The
University of Milano, Milano, Italy
2
Division of Genetics and Cell Biology, San Raffaele Research
Hospital, Milano, Italy
3
University Medical School, Padova University, Padova, Italy
4
Faculty of Medicine and San Raffaele Research Hospital,
Milano, Italy
*Corresponding author: francesca.avemaria@unimi.it
Background: Nonenzymatic glycation is one of
the most important factors in the pathogenesis of
diabetic complications. Fructosamine 3-kinase
(FN3K) appears to be responsible for the removal
 14th EFLM Continuous Postgraduate Course in Clinical Chemistry and Laboratory Medicine
of fructosamine from proteins, suggesting a pro-
tective role in nonenzymatic glycation. Recently,
genetic variants in the FN3K gene have been
found in diabetic patients.
The aim of the present study is the conclusion of
the molecular investigation of the FN3K gene in an
Italian cohort of diabetic patients and healthy sub-
jects, by analyzing its promoter region. We have
evaluated the presence of two polymorphisms,
the c.-385A/G (rs3859206) and the c.-232A/T
(rs2256339), known to be associated with FN3K en-
zymatic activity in erythrocytes.
Materials and methods: Seventy diabetic sub-
jects, 35 T1DM and 35 T2DM and 33 healthy blood
donors with no history of diabetes were evaluated
using PCR and direct sequencing of the FN3K gene
promoter region. The allelic frequencies for each
polymorphism were calculated and the Hardy-
Weinberg equilibrium was estimated using the χ2-
test.
Results: Among the diabetic patients for the poly-
morphism c.-385A/G we detected 40 patients GA,
15 AA and 15 GG; for c.-233A/T,we observed 46 pa-
tients AT, 11 TT and 13 AA. Regarding the control
subjects, we observed for the c.-385A/G 18 individ-
uals GA, 11 AA and 4 GG, and for the c.-233A/T 16
individuals AT, 8 TT and 9 AA. Moreover, our
screening identified two new variants (c.-421C/T,
c.-429delATCGGAG). Furthermore, combining
these results with our previous investigation, we
observed that T2DM patients with GG genotype
for the polymorphism c.-385A/G and CC for
c.900C/G presented a lower concentration of
HbA1c.
Conclusions: The analysis of FN3K promoter re-
gion allowed us to complete the screening of the
gene in our cases, reinforcing the idea that some
variants could act together and influence the FN3K
enzymatic activity as reflected on the levels of
HbA1c.
Keywords: Diabetes; Fructosamine 3-kinase;
FN3K; HbA1c.
S71
P23
Evaluation of the performance of an
immunoturbidimetric HbA1c reagent
applied to the Siemens ADVIA 2400
automatic analyzer
Barbaro M1*, Carobene A1, Ku-Chulim C2, Cochrane R3, Passerini
G1, Ceriotti F1
1
Department of Laboratory diagnostics, Research Hospital San
Raffaelo, Milano, Italy
2
Hospital Centro Médico de las Américas, Mèrida, Yucatán,
México. Supported by IFCC Professional Scientific Exchange
Programme
3
Axis-Shield Laboratories, Technology Park, Dundee, United
Kingdom
*Corresponding author: barbaro.mose@hsr.it
Introduction: Glycated Hemoglobin (HbA1c) is
recognized as the gold standard for long-term gly-
cemic control. The use of HbA1c to screen and di-
agnose diabetes mellitus has increased, leading to
the suggestion of a HbA1c level of 48 mmol/mol
(6.5%) as a diagnostic cut-off. Our aim was to eval-
uate a new immunochemical reagent based on la-
tex particles (Axis Shield), using Siemens ADVIA
2400.
Material and Methods: Intra-assay and total im-
precision, interferences studies (bilirubin ~850
µmol/L, triglycerides ~16.9 mmol/L, total protein
~140 g/L, sodium cyanate ~50 mg/dL, ascorbic
acid ~50 mg/dL, urea ~24.99 mmol/L, glucose
~105.46 mmol/L, rheumatoid factor ~700 U/mL),
method comparison vs Sebia Capillary Electropho-
resis, lot to lot reproducibility, linearity and carry
over were conducted on ADVIA 2400 according to
CLSI protocols. Additionally, 40 samples with tar-
get values by NGSP were measured by the two
methods.
Results: CVs % obtained by intra-assay impreci-
sion, on 3 human specimens at different concen-
trations (<48 between 48-64, and >64 mmol/mol)
in 20 replicates, were <4%. CVs% by total impreci-
sion, performed in 20 days with 4 calibrations on 5
materials [control low, control high and 3 human
samples], resulted <4%. Interferences were stud-
Biochemia Medica 2014;24(Suppl 1):S1-S78
14th EFLM Continuous Postgraduate Course in Clinical Chemistry and Laboratory Medicine
ied on two human samples without obtaining sig-
nificant biases (<10%). Methods comparison, per-
formed on 120 samples ranging 23–137 mmol/
mol, obtaining r=0.9809 as regression coefficient
and a mean bias at decisional level (48 mmol/mol)
<2.0%. The results obtained with the 40 NGSP
samples has allowed the certification of the new
reagent.
Conclusions: The availability of fully automated
method for the determination of HbA1c will not be
only desirable but will become an important clini-
cal need. The ADVIA 2400 is able to perform the
analysis in 10 minutes. Furthermore, this method
showed good performance in our evaluation, ro-
bustness with respect to endogenous interference,
and a good correlation when compared with rou-
tinely used CE especially against NGSP materials.
Keywords: HbA1c; Glycated Hemoglobin; NGSP
P24
Validation of five point-of-care glucometers
Ćelap I*, Milevoj Kopčinović L, Kosovec V, Grabusin P, Begčević I,
Vrkić N
Clinical Institute of Chemistry, Medical School University
Hospital Sestre milosrdnice, Zagreb, Croatia
*Corresponding author: ivana.celap@gmail.com
Background: Self-monitoring of blood glucose is
a convenient way in the management of patients
with type 2 diabetes mellitus (T2DM). Although
numerous glucometers are available in the mar-
ket, only rare can achieve requested performance
characteristics given by International Federation in
Clinical Chemistry and Laboratory Medicine (IFCC)
and, especially, American Diabetes Association
(ADA). ADA sets analytical error at <5% while IFCC
allowable error is <20%. The aim of the study was
to investigate analytical performance characteris-
tics of five glucometers versus reference laborato-
ry method.
Materials and methods: Glucometers of five dif-
ferent manufacturers [Conture (Bayer Vital GmbH,
Biochemia Medica 2014;24(Suppl 1):S1-S78
S72
Leverkusen, Germany), Xceed (Abbott Diabetes
Care Inc., Almeda, CA, USA), AccuCheck Active
(Roche Diagnostics, Mannheim, Germany), Bioni-
me GM550 (Bionime GmbH, Switzerland) and X-
meter (Glucocard, Arkray Factory Inc, Shiga, Ja-
pan)] were validated. Laboratory glucose was
measured with the IFCC reference method with
the hexokinase on AU 2700 (Beckman Coulter,
Brea, CA, USA). Venous blood was drawn from 30
T2DM patients and healthy volunteers in sodium
fluoride/potassium oxalate vacutainers (Greiner
Bio-One, Kramsmünster, Austria). Immediately af-
ter venous blood sampling, capillary blood glu-
cose was determined on all glucometers for each
patient.
Results: All glucometers have shown satisfactory
imprecision (Conture 5%; AccuCheck 4.2%; Xceed
5.1%; X-meter 5.2%, Bionime 3.3%). Relative mean
biases between glucometer and reference meth-
od were: Conture (4.02%), Bionime GM550 (14.31%),
AccuCheck Active (3.16%), Xceed (6.08%), X-meter
(12.46%). Passing-Bablok regression analysis have
shown systematic shift for AccuCheck Active (y=-
0,2906 [-0.6162 – (-0.1500)]+1,0156 (1,0000 - 1,0541))
x and proportional difference for Bionime GM550
(y=0,06113 (-0,3079 - 0,5261) + 0,8473 (0.7826 –
0.8947))x. Error grid analysis showed 100 % results
in A zone for all glucometers except Bionime with
only 4 % results in A zone.
Conclusion: Results of the study showed that glu-
cometers Conture and Xceed satisfy analytical
performance according to IFCC recommendations
while only Conture fulfill ADA criteria.
Keywords: glucose; point-of-care; glucometer;
validation
 14th EFLM Continuous Postgraduate Course in Clinical Chemistry and Laboratory Medicine
P25
Estimation of the detection of gestational
diabetes mellitus - do we need to resign
from 1h50g load?
Grudzień U*, Panek A, Kozłowska D, Swadźba J
Diagnostyka Sp z o.o., Cracow, Poland
*Corresponding author: urszula.grudzien@diag.pl
Background: The aim of the study was to estimate
and compare detection of Gestational Diabetes
Mellitus (GDM) using criteria from Polish Diabetes
Association (PDA) recommendation 2013 and 2014,
and verify sensitivity and specificity of 1h50g load.
Materials and methods: Tests were performed on
group of 23440 pregnant women between 2008-
2013 at Diagnostyka Company, Cracow, Poland.
The cutoff values for the OGTT and 1h50g load
were estimated according to criteria from PDA.
Results: OGTT was made on 16155 women (one
step model). GDM were diagnosed in 1511 (9.4%)
using 2013 criteria and in 1523 (9.4%) using 2014cri-
teria respectively. 1h50g test were performed in
7285 women. Glucose intolerance was observed in
1388 women, GDM had been confirmed with OGTT
(two step model) in 404(2013yr) and in 312 patients
(2014yr). Detection of GDM were 5.8% (2013yr) and
4.5% (2014yr). For evaluated sensitivity and specific-
ity of 1h50g loud according to new and old cutoffs,
2719 women were chosen, and divided into two
groups: first with glucose intolerance and diabetes
(1405 women) and second with negative test re-
sults (1314 women). In first group GDM was diag-
nosed in 421 (2013yr) and 329 (2014yr). In second
group detection was 102 (3.8%) and 40 (1.5%) pa-
tients. Sensitivity and specificity were compared us-
ing both diagnostic criteria: 2013 recommendation-
sensitivity: 80.5%,specificity: 55.1%, negative pre-
dictive value (NPV): 92.2%, positive predictive value
(PPV): 29.9%, negative likelihood ratio (LR): 0.35,
positive LR 1.8 and odds ratio (OR): 5.084; 2014 rec-
ommendation-sensitivity: 89.2%, specificity: 54.2%,
NPV: 97.0%, PPV: 23.4%, negative LR: 0.2; positive
LR: 1.9; and OR 9.7.
S73
Conclusions: In two step model using old cutoffs
3.8% of patients were undiagnosed, detection of
GDM was lower than using one step model. The
numbers of undiagnosed patients using two step
model and new criteria decreased, sensitivity of
1h50g loud elevated, but detection was still lower
than using one-step model
Key words: gestational diabetes; pregnancy; rec-
ommendation
P26
User verification for HbA1c on AU 480
Beckman Coulter
Sablek J1*, Todorić Z2, Sablek Lj1
¹ Medical biochemisty laboratory Ljerka Sablek, Požega, Croatia
2
Medical biochemisty laboratory, Labplus Polyclinics, Split,
Croatia
*Corresponding author: jette0106@yahoo.com
Background. Glycated hemoglobin (HbA1c) is
formed by non-enzymatically binding of glucose
to the free amino group of the N-terminal end of
the β-chain of hemoglobin A. HbA1c is used to
measure average glucose blood levels over the
lifespan of the erythrocytes (120 days), and is a
longlasting indicator of blood glucose levels, which
makes it good indicator of diabetic disease control.
With this study, we wanted to evaluate new meth-
od in laboratory by defining precision and true-
ness for the determination of HbA1c at the AU 480
(Beckman Coulter, USA), by immunoturbidimetric
method.
Materials and methods: We determined the con-
centrations of total hemoglobin and HbA1c. HbA1c
is measured in a latex agglutination inhibition test.
If the sample does not contain HbA1c, there will be
a microparticle agglutination marked with anti-
bodies and aglutinators. The presence of HbA1c in
the sample results in reduced levels of agglutina-
tion. The increase in absorption is inversely pro-
portional to the concentration of HbA1c in the
sample.
Biochemia Medica 2014;24(Suppl 1):S1-S78
14th EFLM Continuous Postgraduate Course in Clinical Chemistry and Laboratory Medicine
Results: We determined concentrations of HbA1c,
during five days, in triplicate, in commercially avail-
able control samples for two concentration levels
declared by the manufacturer. From the obtained
values of concentrations of HbA1c, we ​​ calculated
repeatability, intermediate precision, intra-labora-
tory precision and systematic error (bias).
To evaluate precision, we compared our variation
coefficient of intra-laboratory precision (for level 1
it’s 1.3 %, for level 2 it’s 1.5 %) with the desirable
specification for imprecision from Westgard bio-
logical variation database (0.9 %).
To evaluate trueness, we compared our bias (at
level 1 is 0.54 %, at level 2 is 4.06 %) with desirable
specification for inaccuracy from Westgard biolog-
ical variation database (1.5 %).
Conclusion: Based on the obtained values​​ , we
concluded that our method fot HbA1c determina-
tion is not ready to use in routine yet, we have to do
corrections in all preanalitical and analitical steps.
Key words: glycated hemoglobin; automation;
verification
P27
Low-grade inflammatory state in pregnancy
complicated by diabetes
Panek A*, Grudzień U, Kozłowska D, Swadźba J.
Diagnostyka Sp. z.o.o. Cracow, Poland
*Corresponding author: andrzej.panek@diag.pl
Background: A low-grade systemic inflammation
is concomitant in diabetes. The aim of the study
was to investigated the relation between low-
grade systemic inflammation expressed as C-reac-
tive protein in gestational diabetes.
Material and methods: The CRP concentrations
in pregnant women with normal glucose toler-
ance (NGT) and those with gestational diabetes
mellitus (GDM) were measured at the same time as
the oral glucose tolerance test (OGTT).Tests were
performed on group of 603 pregnant women in
Biochemia Medica 2014;24(Suppl 1):S1-S78
S74
2013 at Diagnostyka Company, Cracow, Poland, 64
patients were diagnosed as gestational diabetes.
Results: CRP level were significantly higher in
GDM patients compared to NGT: GDM median 3.9
(quartile 1.4-4.8) mg/l; NGT median 2.3 (quartile
1.1-3.3) mg/L;p =0.019. GDM patients were signifi-
cantly older: median 33 (quartile 29-38) yr; median
31 (quartile 28-35) yr p=0.003. 584 women with
CRP between 0and 5 mg/L were chosen and divid-
ed into two group: first with CRP level 0-3 mg/L
(400 women: 362 NGT and 38 GDM patients), and
second with CRP 3-5mg/L (184 women: 172 NGT
and 12 GDM). CRP were significantly higher in
GDM patients in second group: GDM median 4.3
(quartile 4.1-4.7) mg/L; NGT median 3.8 (quartile
3.3-4.3) mg/L; p=0.014; whereas, no change was
found in CRP in first group: GDM median 1.5 (quar-
tile 1.0-2.0) mg/L; NGT median 1.5 (quartile 0.8-2.1)
mg/L; p= 0.875. Using ROC-analysis the cutoff
point for CRP were estimated as 4.9 mg/L for sec-
ond group of CRP, and 5.8 mg/L for whole popula-
tion. There were no significantly correlation be-
tween CRP level and fasting plasma glucose nor
glucose level after 2 hour 75 gram loud.
Conclusions: CRP as a marker of low-grade in-
flammation state was associated with gestational
diabetes mellitus in statistically significant group
number two.
Key words: gestational diabetes; C-reactive pro-
tein; pregnancy
P28
Correlation between two analytical method
of HbA1c measuring in aligned and
misaligned diabetic patients
Stec K*, Grudzień U, Panek A, Kozłowska D, Swadźba J
Diagnostyka Sp z o.o., Cracow, Poland
*Corresponding author: urszula.grudzien@diag.pl
Backgrounds: Glycated hemoglobin (HbA1c) is
representative of the mean blood glucose level
over three months. The aim of the study was to
 14th EFLM Continuous Postgraduate Course in Clinical Chemistry and Laboratory Medicine
compared the HbA1c value using both HPLC and
turbidymetric (T) assay in diabetic patients with
aligned diabetes (AD) and with misaligned diabe-
tes (MD).
Materials and Method: HbA1c (T), HbA1c (HPLC),
glucose level and hemoglobin (Hb) were per-
formed on group of 53 men (21AD, 22MD) and 79
women (46AD, 26MD), between 2011-2013 in Diag-
nostyka Company, Cracow, Poland.
Results: Significant differences were observed be-
tween both HbA1c (T) ,HbA1c HPLC and glucose
leveling women: median [quartile] for: HbA1c (T)
5.2 (4.9-5.4)[%];5.8 (5.4-6.2)[%] p<0,001; HbA1c
(HPLC): 33.1 (29.9-35.3) [mmol/mol]; 39.7 (35.3-
44.0) [mmol/mol], p<0.001; glucose 13.3 (12.7-14.4)
[mmol/l]; 13.4 (12.7-14.2) p<0.001 (AD,MD respec-
tively). In men: HbA1c (T) 5.3 (5.2-5.6) [%]; 5.8 (5.4-
6.5) [%] p=0.004; HbA1c HPLC: 34.2 (33.1-37.5)
[mmol/mol], 39,11 (36.4-47.3) [mmol/mol] p<0.001;
glucose: 4.9 (4.5-5.2) [mmol/l]; 6.0 (5.6-7.2) [mmol/l]
p<0.001 (AD,MD respectively) Significant correla-
tion were found between HbA1c (T) and HbA1c
(HPLC) in men: R=0.7112, p=0.003; R=0.7108,
p=0.003 (AD,MD respectively) and in women:
R=0.6452, p=0.003; R=0.6331; p=0.011 (AD,MD re-
spectively). No significant correlation were found
between both HbA1c (T) nor HbA1c (HPLC) and
glucose level in AD or MD group however, in com-
bined group of women and men, the significant
correlation were found: HbA1c (T)
r=0.4786,p=p=,001; HbA1c HPLC r=0,4338, p=,003;
HbA1c (T) ,6115,p<0,001; HbA1c HPLC 0,8731,
p<0,001 (women, men respectively).
Conclusions: Significant correlation were found
between both analytical methods of HbA1c meas-
uring in aligned and misaligned diabetes (women
and men). No significant correlation were found
between both HbA1c (T) nor HbA1c (HPLC) and
glucose level in AD and MD patients (women and
men). Although the significant correlation were
found in combined group of women and men
(AD+MD)
Key words: HbA1c; HPLC; diabetes mellitus
S75
P29
Testing paracetamol interference with
glucose analysis in serum
Trifunović J
Medical biochemisty laboratory, Special hospital for medical
rehabilitation Varaždinske Toplice, Varaždinske Toplice, Croatia
*Corresponding author: jasenka.trifunovic@yahoo.com
Background: Paracetamol (acetaminophen) is
widely used over-the-counter pain reliever and fe-
ver reducer. Studies have shown that paracetamol
interferes with some home blood-glucose-meas-
urement systems. The aim of this study was to in-
vestigate if the paracetamol interferes with glu-
cose analysis in serum.
Materials and Methods: Testing was performed
in accordance with IFCC Drug Effects in Clinical
Chemistry. Part 2. Guidelines for Evaluation of Ana-
lytical interferences, 1984. Two paracetamol con-
centrations have been tested to cover the concen-
tration spectrum (200 µg/mL and 1000 µg/mL).
Two serum pools (normal and high glucose con-
centration) have been used as samples. Determi-
nation of glucose (Siemens, UK) was done on Di-
mension Xpand Plus analyser (Siemens, UK). All
measurements were performed in duplicate. Bias
exceeding 10% is considered „interference“.
Results: There were no difference between mean
glucose concentration in test samples (5.0 mmol/L)
and control samples (5.0 mmol/L) after adding 200
µg/mL and 1000 µg/mL of paracetamol in serum
pool with normal glucose concentration. After
adding 200 µg/mL and 1000 µg/mL of paraceta-
mol in serum pool with high glucose concentra-
tion there were no difference present between
mean glucose concentration in test samples (11.1
mmol/L) and control samples (11.1 mmol/L).
Conclusion: Paracetamol has no significant effect
(bias less then 10%) on glucose analysis when pre-
sent in serum at concentration of 200 µg/mL or
1000 µg/mL.
Key words: drug interference; glucose; paraceta-
mol
Biochemia Medica 2014;24(Suppl 1):S1-S78
14th EFLM Continuous Postgraduate Course in Clinical Chemistry and Laboratory Medicine
P30
Determination of potential urine biomarkers
of kidney disease by LC-MS / MS
Vojtova L1*, Přikryl P2, Hrušková Z3, Vokurka M2,Tesař V3, Zima T1
1 Institute of Clinical Biochemistry and Laboratory Diagnostics,
1st Faculty of medicine, Charles University and General Teaching
Hospital in Prague
2 Institute of Pathological Physiology, 1st Faculty of medicine,
Charles University in Prague
3 Department of Nephrology, 1st Faculty of medicine, Charles
University in Prague
*Corresponding author: lucie.vojtova@vfn.cz
Background: Proteinuria may be caused by either
increased glomerular basement membrane per-
meability, or by impairment of secretion-reabsorp-
tion tubular processes. It is often used as a surro-
gate marker in monitoring and predicting out-
come in patients with chronic kidney disease but it
is non-specific. We established a proteomics ap-
proach for identification of nephropathy-related
biomarkers in urine, including Diabetic nephropa-
thy (DN), which is a well-known complication of
long-standing diabetes. The main aim of this work
was to assess differences in urine proteins in pa-
tients with nephropathies and healthy controls
and to identify abnormal proteins as potential bio-
markers of the renal disease.
Materials and Methods: In our pilot project, we
have analyzed protein composition of urine in 20
patients with IgA nephropathy and an appropriate
healthy control group. The proteins were separat-
ed by the isoelectric focusing method (IEF) in the
first step. The second dimensional separation and
protein characterization was performed using iT-
RAQ quantitative analysis followed by mass spec-
trometry LC-MS/MS.
Results: We have identified 46 urinary proteins sig-
nificantly up- or down regulated in the urine of IgA
nephropathy patients. We have found changes in
proteins of complement system, Regulation of actin
cytoskeleton or retinol biosynthesis. Whilst these
preliminary data require further studies that will
correlate the results with clinical, laboratory and
Biochemia Medica 2014;24(Suppl 1):S1-S78
S76
histopathological data, some of the proteins, name-
ly Cathepsin, Retinol-binding protein, Protein AMBP
were also represented in urine of patients with dia-
betes mellitus in previous studies made by others.
Conclusion: We have optimized proteomic meth-
odology that is able to identify potential urine bio-
markers of kidney disease. Study of changes of uri-
nary proteins could help to establish the diagno-
sis, assess the disease activity and/or predict the
prognosis of different renal diseases, including dif-
ferent glomerulonephritis or DN.
Key words: kidney disease; LC-MS/MS; urine pro-
teins
P31
Demand management of hemoglobin A1c
testing according to the American Diabetes
Association’s Standards of Care 2014
Varo Sánchez GM*, Muñoz Calero M, Salas Herrero E, Montilla
López C
Clinical Laboratory Analysis. Hospital San Juan de Dios del
Aljarafe, Sevilla, Spain
*Corresponding author: gemavaro@hotmail.es
Background: Recently, American Diabetes Asso-
ciation (ADA) has considered the A1c test to diag-
nose diabetes (≥6.5%). Furthermore, ADA has de-
fined a category of increased risk of diabetes (pre-
diabetes) whose values of HbA1c are between
5.7%-6.4%. The low difference of the HbA1c levels
between the categories defined requires a strictly
control of testing interval. ADA recommends
measurement of HbA1c at least two times a year
whether a patient’s glycemic targets have been
reached and maintained. Unstable patients may
require four analyses a year.
The aim is the evaluation of time interval of the
HbA1c during the last year in Aljarafe´s area.
Materials and Methods: This study of manage-
ment of the HbA1c test is based on the consensus
among primary care and Aljarafe’s Hospital to im-
 14th EFLM Continuous Postgraduate Course in Clinical Chemistry and Laboratory Medicine
prove diabetes outcomes (Plan Integral Diabetes
de la Junta de Andalucía). Intervals testing condi-
tions were: 180 days for stable patients and 90 days
for unstable and non-maintained subjects. The A1c
test was performed using a method that is NGSP
certified and standardized to the DCCT assay.
Results: The total of HbA1c tests solicited was
23017, with 1174 (5.10%) were rejected for being
out of the time intervals considered. The number
of tests was: 18157 (72.2%) with 1 test/year, 5638
(22.4%) with 2 tests/year, 1020 (4.1%) with 3 tests/
year and with 327 (1.3%) 4 tests/year. The average
of HbA1c tests for each group was: 6.28%, 6.96%,
7.62% and 7.9%, respectively. The prevalence of di-
abetes in the sample studied was 5.1%.
Conclusions: The consensus approved in our Hos-
pital about the time interval of the HbA1c has con-
tributed to improve the diabetes management
with reasonable criteria for demanding.
The strategies used for demanding of A1c suppose
the reduction of the number of duplications. Un-
necessary tests do not make a positive contribu-
tion in the appropriate diabetes diagnose.
Key words: Diabetes; HbA1c test; demand man-
agement; primary care
P32
Activities of superoxide dismutase and
glutathione peroxidase in plasma of patients
with Balkan endemic nephropathy
Gnjidic T1*, Pavlovic D², Radic T³, Coric V³, Simic T³, Savic-Radojevic A³
¹ General Hospital “Blazo Orlandic”, Bar, Montenegro
² University Hospital, Foca, Serbian Republic
³ Institute of Medical and Clinical Biochemistry, Faculty of
Medicine, University of Belgrade, Belgrade, Serbia
*Corresponding author: tasajoja@yahoo.com
Beckground: Balkan endemic nephropathy (BEN)
is a familial chronic tubulointerstitial disease with
insidious onset and slow progression to terminal
renal failure. The aim of this study was to investi-
gate the influence of oxidative stress on BEN by
S77
measuring the activities of antioxidative enzymes,
superoxide dismutase (SOD) and glutathione per-
oxidase (GPX).
Material and methods: Activities of plasma GPX
and SOD were determined spectrophotometrical-
ly in 30 BEN patients and 31 controls with nephro-
lithiasis.
Results: The activity of GPX was significantly lower
in patients with BEN compared to the control group
(368.1±133.8, 425.7±82.5, respectively, p<0.05).
When the BEN patients were divided according to
eGFR, in patients with eGFR>90ml/min/1,73m² GPX
activity was significantly higher compared to those
with eGFR<60ml/min/1,73m². Moreover, significant
negative correlation was observed between GPX
activity and creatinine, urea and proteinuria (r=-
0.530, p<0.05; r=-0.704, p<0.001; r=-0.475, p<0.05;
respectively). However, SOD activity was about 10%
higher in plasma of BEN patients in comparison
with controls, but without statistical significance
(48.7±9.6, 44.2±10.8, respectively, p>0.05).
Conclusions: The reduced GPX activity in patients
with BEN might be a consequence of the atrophy
of the kidneys present in these patients. As a result
of the reduced enzyme antioxidant capacity, it
may be assumed that the increased production of
free radicals may be an important factor in the
progression of BEN.
Key words: Balkan endemic nephropathy; oxida-
tive stress; superoxide dismutase; glutathione per-
oxidase.
P33
Pharmacovigilance - case report
Romić M
National Blood Transfusion Institute of Serbia
*Corresponding author: mfrak@beotel.net
Background: a brief introduction.National Blood
Transfusion Institute is producing medicines
which are obtained from human plasma. Medi-
Biochemia Medica 2014;24(Suppl 1):S1-S78
14th EFLM Continuous Postgraduate Course in Clinical Chemistry and Laboratory Medicine

cines which we are producing human is albumin,
specific immunoglobulin - tetanus and rabies im-
munoglobulin. Our Directive for medicines and
medical devices (Sl.Glasnik 84/2004) define that is
necessary to use pharmacoviglance for all medi-
cines.
The term pharmacovigilance comprises all scien-
tific and data gathering activities relating to the
detection, assessment, and understanding of ad-
verse events. This includes the use of pharma-
coepidemiologcal studies. Pharmacovigilance ac-
tivities are undertaken with the goal of identifying
adverse events and understanding, to the extent
possible, their nature, frequency, and potential risk
factors.
Materials and methods: The little girl, after dogs
bit her received usual anti rabies prophylaxis in
the health center The little girl, after dogs bit her
received usual anti rabies prophylaxis in the health
center She react with severe reaction. After we re-
ceived sample from patient to analyze it. We de-
cided to analyze in her sample subclass of IgA. We
reanalyzed sample of drug (Human rabies immu-
noglobulin) from the same batch.

Results:
IgA IgM IgG Subcleses Subclases
Name of sample
g/L g/L g/L IgA1 IgA2
Normal values 0.9-4.5 0.6-2.5 8-19 0.61-3.04 0.10-1.13
Patient M. J. 2.42 0.65 9.0 2.15 0.18
Humani rabies imunoglobulin 2.26 0.78 130 1.90 0.21

Conclusion: man rabies immunoglobulin. Any reaction could

After comparison of these results with normal val- possible to manifest for all population with or
ues we concluded that, this reaction is not con- without diabetes.
nected with IgA immunodeficiency, or with Hu- Key words: pharmacovigilance; rabies; diabetes

Biochemia Medica 2014;24(Suppl 1):S1-S78

S78