Summary
Chromogranin A (CgA) as a main, nonspecific marker of
neuroendocrine tumors (NET) has been applied for diagnostic
purposes and for monitoring the treatment of NET. Currently few
commercial immunoassays (IRMA, RIA, ELISA, CLIA, TRACE)
are available allowing measurement of CgA concentration in serum
or plasma. These methods have various sensivity and specivity and
differ in many analitycal parameters.
CgA blood concentration, however, may be influenced by various
factors or coexisting pathological conditions. Among factors
causing a substantial increase of blood CgA concentration are:
treatment with proton-pump inhibitors or H₂ -receptor blockers,
chronic atrophic gastritis (type A), impaired renal function, prostate
cancer, rheumatoid arthritis with high level of RF IgM.
In addition there are many conditions which may have a
moderate or little influence on the concentration of CgA, among
them are: inflammatory bowel disease, deteriorating liver function,
untreated essential hypertension, heart failure, hypercortisolism,
pregnancy.
Proper assessment of the CgA results requires detailed
knowledge about various preanalytical conditions influencing it's
concentration in blood.
The aims of the study were as follow:
1. To compare directly the results of serum and plasma CgA
concentrations by IRMA and ELISA method in patients with
neuroendocrine tumours.
2. To confirm the noted ealier differences in CgA levels measured
in serum and plasma, and to establishe respective reference
ranges in a group of healthy males.
3. To investigate the effect of postprandial test on the concentration
of CgA.
4. To investigate the stability of CgA in serum samples submitted
to three freeze-thaw cycles.
5. To investigate plasma CgA concentration in patients with
various adrenal tumours concentrating mainly on the value of
this determination as a screening test in cases suspected of
pheochromocytoma.
6. To evaluate the usefulness of CgA measurements in patients
with multiple endocrine neoplasia (MEN).
Material and methods
150 blood donors and 122 patients with various NET 207
patients with adrenal tumours including 20 patients with
pheochromocytoma, 28 patients with MEN syndromes and 27
patients with indication for the postprandial test.
Results
In patients with NET CgA concentrations were markedly higher
in plasma than serum. Using IRMA method these difference in
the range between 10-100 ng/ml approached 20% - 70%
(median 61 vs. 42, p<0,0001), in the range 101-300 ng/ml – 12%
- 60% (median 147 vs. 101, p<0,0001), and in the range 301 1076 ng/ml – 14% - 40% (median 486 vs. 356, p<0,0033).
Highly significant (p<0,0001) positive correlation (r > 0.9)
between the results of serum and plasma (K₂EDTA) CgA levels
was found. The differences between CgA results in serum and
plasma using ELISA were similar only a bit smaller. No
significant differences between CgA levels in EDTA-plasma and
heparinized-plasma samples in ELISA method (p=0,2531), and
only very little difference in IRMA method (p<0,0021) were
notted.
In healthy males-blood donors, the median (and the range) of CgA
concentration were as follow: for serum samples - 42,0 ng/ml (16108 ng/ml) and for plasma (EDTA₂K) samples - 58,0 ng/ml (23-153
ng/ml). The differences between serum and plasma ranged 15% –
79% (median 26%). Plasma CgA levels were significantly higher in
relation to serum CgA levels (p<0,0001). Correlation of CgA in
serum and plasma was r = 0,8493; p<0,01. The reference ranges for
CgA measured in serum and plasma in males, expressed as 2,5 to
97,5 percentiles were: 21,0 – 108,0 ng/ml and 31,0 – 153,0 ng/ml
respectively.
The concentrations of CgA in 0’-60’ postprandial test in healthy
subjects, were as follow: at fasting state the median of CgA was 26
ng/ml (19 - 46 ng/ml) and after 60 minutes - 34 ng/ml (22 – 44
ng/ml), the difference was 0% - 24% (p = 0,0580), whereas in
patients – before meal (0’), the median of CgA concentration was
33,0 ng/ml (12 – 89 ng/ml), after 120 minutes - 33,5 ng/ml (19 – 91
ng/ml), the difference was 0% - 37% (p = 0,1024).
Testing the effect of 3 consecutive cycles of freeze / thaw the notted
differences in
CgA concentration were not significant, 0.1% -
12.5%, median 5.2% (p = 0.0624).
In the majority of patients with adrenal tumours not derived from
neuroendocrine cells (chromaffin cells), except those with marked
hypercortisolemia plasma CgA concentrations was below the cutoff value. In few patients with marked hypercortisolemia CgA
levels were higher and ranged 150 – 319 ng/ml
(median 225
ng/ml). In 17 of 20 of patients with pheochromocytoma CgA levels
were markedly elevated and ranged 180 – 1971 ng/ml (median 425
ng/ml), but in 3 subclinical cases CgA levels were not elevated.
In patients with MEN-1 (n = 12), the median CgA concentration
in serum was 158 ng / ml (34 - 1800 ng / ml). The median CgA
concentration in NET tumors forming part of the MEN 1
(including gastrinoma and carcinoid) was 974 ng / ml (216 1800 ng/ml), which resulted with 58% sensitivity and 100%
specificity for the cut-off point of 100 ng / ml. In patients with
MEN-2A (n = 10) median CgA concentration in serum was 61
ng / ml (42 - 268 ng / ml). In this group of patients there were
eight patients after surgery (pheochromocytoma and / or
medullary thyroid carcinoma), in whom the median CgA
concentration after surgery was 65 ng / ml (42-76 ng / ml). In
two
patients
with
pheochromocytoma,
serum
concentration amounted to 237 ng / mL and 268 ng / ml.
CgA
Conclusions
1. Concentration of chromogranin A in plasma appears to be
significantly higher than in serum.
2. Due to
significant difference in CgA concentration measured in
plasma and serum, the obtained results should be referred to the
separate reference ranges corresponding to the type of material used.
3. Food intake in most subjects had no effect on serum CgA , but in
some cases the impact was significant.
4. Three times freezing and thawing of serum samples had no influence
on measured CgA concentration.
5. In preliminary differential diagnosis of adrenal tumours a markedly
increased CgA level might be a useful additional marker of
pheochromocytoma, however because in some subclinical cases CgA
level may not be raised, therefore a non increased CgA level does not
exclude pheochromocytoma, only suggest that such diagnosis is less
likely.
6. In the majority of patients with adrenal tumours not derived
from neuroendocrine cells and in some with adrenal carcinoma,
except those with significant hypercortisolemia, the plasma CgA
concentrations were below the cut-off value.
7. In patients with Multiple Endocrine Neoplasia investigation of CgA
level may be used in monitoring eventual coexistence or appearance
of
carcinoid,
pheochromocytoma.
pancreatic
neuroendocrine
tumours
or