*Piotr Glinicki, Wojciech Jeske, Wojciech Zgliczyński
The effect of the consumed meal and of serum sample freezing/thawing cycles on the chromogranin A (CgA) blood level**
Wpływ spożytego posiłku oraz cyklów zamrażanie/rozmrażanie próbek surowicy na stężenie chromograniny A (CgA) we krwi
Department of Endocrinology, Centre of Postgraduate Medical Education, Bielański Hospital, Warsaw
Head of Department: Professor Wojciech Zgliczyński, MD, PhD
Streszczenie
Wstęp. Chromogranina A (CgA) jest niespecyficznym biomarkerem guzów neuroendokrynnych (NEN). Jednym z ważnych zagadnień przygotowania pacjenta do badania jest ocena wpływu posiłku na stężenie chromograniny A. Inny ważny aspekt analityczny dotyczy stabilności cząsteczki CgA w warunkach in vitro.
Cel pracy. Celami badawczymi pracy była ocena wpływu posiłku na stężenie chromograniny A we krwi w teście poposiłkowym oraz ocena stabilności cząsteczki CgA w 3 cyklach zamrażanie/rozmrażanie.
Materiał i metody. Grupa badana liczyła 27 osób (7 mężczyzn, 20 kobiet) w wieku 22-76 lat. Wykonano test poposiłkowy według schematu: na czczo (0 minut) i 60 lub 120 minut po posiłku. Drugą grupę badaną stanowiło 10 próbek surowic pozyskanych od pacjentów w zakresie stężeń CgA wynoszącym 28,6-1115,1 ng/ml. Schemat testu: oznaczenie stężenia CgA w próbce pierwotnej, a następnie w 3 cyklach zamrożenie/rozmrożenie próbki surowicy. Stężenie chromograniny A oznaczono metodą immunoradiometryczną (IRMA).
Wyniki. Nie wykazano istotnych statystycznie różnic w stężeniu CgA oznaczanej w teście poposiłkowym: 0’-60’ (p = 0,0580) oraz 0’-120’ (p = 0,1024) oraz w 3 kolejno następujących po sobie cyklach zamrażanie/rozmrażanie próbek surowicy (p = 0,0624).
Wnioski. Spożycie posiłku u większości badanych nie miało wpływu na stężenie CgA we krwi, ale w pojedynczych przypadkach wpływ był znaczący (24-37%) i dlatego uzasadnione jest zalecenie, aby krew na to badanie pobierać na czczo. Trzykrotne zamrożenie/rozmrożenie próbek surowicy nie wpłynęło na wynik oznaczeń stężenia CgA we krwi.
Summary
Introduction. Chromogranin A (CgA) is a non-specific biomarker of neuroendocrine neoplasms (NEN). One of unsettled important question concerned with this marker is the effect of a meal on the level of chromogranin A. Yet another important analytical question is associated with in vitro stability of a CgA molecule.
Aim. The study was aimed at assessment of the effect of a meal on the blood level of chromogranin A in a postprandial test, and assessment of stability of a CgA molecule in serum samples undergoing three cycles of freezing/thawing.
Material and methods. The study group consisted of 27 persons (7 men, 20 women) age ranging between 22 and 76 years. A postprandial test was made, using the design a sample collected on fasting (0 minutes), and a second one collected 60 or 120 minutes after a meal. The second study group consisted of 10 serum samples collected from patients with CgA levels between 28.6 and 1115.1 ng/ml. Design of the test: CgA determination in a primary sample, and then after each of three serum sample freezing/thawing cycles. The CgA level was determined using the immunoradiometric method (IRMA).
Results. No statistically significant differences in CgA levels determined in the postprandial test were found: 0’-60’ (p = 0.0580) and 0’-120’ (p = 0.1024), nor after each of consecutive three freezing/thawing cycles (p = 0.0624).
Conclusions. In majority of subjects a meal had no effect on the blood CgA level, but in some single cases the effect was significant (24-37%), and therefore the recommendation that blood should be drawn on fasting seems valid. Three cycles of freezing/thawing of serum samples had no effect on the determined CgA blood levels.
Introduction
Chromogranin A (CgA) belongs to the family of secretory proteins called granins/secretogranins (1). CgA is a soluble, acidic glycoprotein, with molecular mass of 48 kDa, composed of a single polypeptide chain (2). It is present in human neuroendocrine cells (3, 4). It is stored and secreted along with catecholamines, peptide hormones and cell-specific neurotransmitters (5). CgA is released to blood where it may be determined as a, so called, circulating biomarker (6) and is classified as non-specific marker of neuroendocrine neoplasms (NEN) (7). Determination of blood CgA levels is useful in diagnostics of NEN, assessment of response to treatment and in detection of progression and recurrence at early stage of the disease (8, 9).
The knowledge of various pre-analytical and analytical factors that may influence the level of a tested analyte is an important question for laboratory research. The effect of a meal on the blood level of chromogranin A is one of important issues. Another analytical problem is associated with stability of a CgA molecule in in vitro conditions, in which the compound will be determined for clinical purposes.
Aim
The study was aimed at assessment of the effect of a meal on the blood level of chromogranin A in a postprandial test, and of stability of a CgA molecule in a test sample of serum undergoing three cycles of freezing/thawing.
Material and methods
Materials
The study group consisted of: 27 subjects (7 men, 20 women; age 22-76 years, mean age ± SD was 39 ± 17 years). They were healthy volunteers and patients in whom a postprandial test was performed. Design of the test: two blood drawings: on fasting (0 minutes) and 60 or 120 minutes after a meal. The meal consisted of: 2-3 sandwiches and water. Exclusion criteria: patients treated with proton pump inhibitors (PPI), H2 histamine receptor blockers, corticosteroids, serotonin reuptake inhibitors, and patients with chronic renal and hepatic diseases, inflammatory conditions of the alimentary tract, prostatic cancer, rheumatoid arthritis, severe arterial hypertension and neuroendocrine neoplasms.
The second study group consisted of: 10 samples of serum collected from patients with chromogranin A levels ranging between 28.6 and 1115.1 ng/ml. The study consisted of initial determination of the CgA level in a serum sample, and repeated determination after each three consecutive cycles of freezing/thawing.
The study was approved by the Bioethics Commission of Centre of Postgraduate Medical Education.
CgA level determination
Venous blood was drawn from the ulnar vein into a tube containing a clotting activator. 30 minutes later blood was centrifuged (10 minutes at 3500 rpm, in room temperature), and the obtained serum was frozen and stored at -30°C until the test (not longer than for a month).
Chromogranin A level was determined by the immunoradiometric method (IRMA) using CIS bio International kit (CGA-RIACT, France). The reference range for serum samples, according to test producer, was 10-100.0 ng/ml. Sensitivity of the method was 1.5 ng/ml. Intra-serial variability (CV) for samples at various concentrations: 29.9, 144 and 998 ng/ml was 6.0, 3.8 and 2.2%, respectively. Inter-serial variability for samples at various concentrations: 29.9, 144 and 998 ng/ml was: 8.5, 5.7 and 5.3%, respectively.
Statistics
Results are presented as median and range of concentration and as mean ± SD. Normality of distribution was analysed using the Shapiro-Wilk test. Differences of results between groups in the postprandial test were analysed using the Wilcoxon’s sign rank test, and differences of results between groups of frozen/thawed samples were analysed using the Friedman’s test.
Results
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Piśmiennictwo
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