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Technical Briefs |
1
Dipartimento di Chimica Inorganica ed Analitica, Università di Cagliari, Complesso Universitario di Monserrato, 09042 Monserrato-Cagliari, Italy
2
Dipartimento di Citomorfologia, Divisione di Anatomia Patologica, Università di Cagliari, Via Ospedale 60, 09124 Cagliari, Italy
a author for correspondence: fax 39-0706754478, e-mail crisponi{at}unica.it
ß-Thalassemia major is an autosomal recessive disease characterized by absent or decreased synthesis of the ß-globin gene (1). Thalassemic children, estimated at 100 000 worldwide, are affected by chronic anemia and need regular blood transfusion (2). Because of the limited capacity of iron excretion in humans, the iron in transfused red cells accumulates in the body. The liver, heart, and pancreas are the target organs of iron-induced injury; therefore, the major pathological manifestations observed in ß-thalassemia major are chronic liver disease, evolving to cirrhosis, and dilative cardiomyopathy, both characterized by severe iron deposition (3)(4). The dangerous effects of iron excess can be managed by administration of chelators capable of removing iron from transferrin, ferritin, and other iron stores (5)(6)(7). Determination of the hepatic iron concentration (HIC) is one of the most valid procedures in assessing real body iron burden (8)(9)(10), which is important for adjusting each patient’s chelation therapy over the years. HIC usually is measured on one part of a needle biopsy core, and the measured value is considered representative of the iron concentration in the whole liver (11). In 1995, a study by our group (3) first showed that iron is unevenly distributed in the livers of ß-thalassemic patients, and therefore, the iron content determined in a small liver fragment should be interpreted with caution because it cannot be considered a true representation of the mean HIC. These data were confirmed by us (12)(13)(14) and by other authors (15), who reported differences in HIC measurements on liver biopsy specimens.
Recently, a striking difference in HIC was found in biopsy samples from cirrhotic livers (range, 60–2851 µg/g dry weight) (16). It therefore seemed of interest to measure iron concentrations in a large number of needle biopsies with the following objectives: (a) to establish whether the determination of iron concentration in a needle biopsy is representative of the mean iron content of the whole liver; (b) to determine whether HIC measured in only a portion of the needle biopsy is indeed representative of the mean liver iron content; and (c) to determine the minimum weight of liver parenchyma needed to obtain a HIC value that would be useful to evaluate the body iron burden. To this end we performed 54 needle biopsies from an autopsy liver of a thalassemic patient and evaluated the effect of each factor by use of analysis of variance (17).
The clinical data of the 29-year-old male affected by homozygous ß-0-thalassemia major were reported previously (3).
At autopsy, the right lobe of the liver was divided into 18 areas with
diameters of ~1 cm (sampling sites), and three needle biopsies were
performed in each area. The three biopsies were subdivided into two,
three, and four parts (subsamples), respectively: the first subsample,
marked with index 1, corresponded to the subcapsular part, whereas the
indices for the other parts increased with depth. Fig. 1
A shows the sampling map for these 162 [18 x (2 + 3 +
4)] subsamples.
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The digestion procedure to obtain a solution suitable for inductively coupled plasma atomic emission spectroscopy (ICP-AES), the working calibration, and the ICP-AES conditions have been described previously (18).
All samples were weighed on a balance accurate to the sixth digit. It should be highlighted that weighing is fundamental to the precision of the entire procedure, e.g., a four-digit balance could lead to a 10% error when samples of ~1 mg are weighted. The sequence in analyzing the 162 samples was completely randomized to avoid any possible systematic error related to an ordered sequence. The same type of operation on all samples (e.g., weighing, diluting to the mark, spectral recording) was carried out by the same operator to avoid operator-generated errors. All procedures were tested by analyzing NIST bovine liver samples. During spectrophotometric analyses, one calibration out of five solutions was measured to check the reliability of the measurements.
HNO3 and the ICP calibrator for iron (10 g/L) were from Aldrich; Triton X was from Merck. NIST Standard Reference Material 1577b, Bovine Liver, was used to validate the measurements.
All data are represented as three 18 · p matrices, where
18 is the number of sampling sites, and p = 2, 3, or 4,
the number of subsamples into which each needle biopsy was subdivided.
The matrix elements xij represent iron
concentrations at sampling sites i (1–18) and depths
j (1 to p, where 1 is the index of the
superficial sample and p is the index of the deepest
sample). For each matrix, the total mean value over the
18 · p data points is defined as:
 |
j) could be defined among
samples at the same depth in different sampling sites, and 18 mean
values (i) could be defined
over the p subsamples in each needle biopsy:
 |
 |
j=1p i=118 (xij
- )2, with 18 · p -
1 degrees of freedom, can be divided into three contributions:
SSS = p · i=118(i - )2 has
17 degrees of freedom, and measures the part of the total sum
attributable to the differences among sampling sites;
SSD = 18 ·
j=1p(j -
)2 has p - 1 degrees of
freedom, and measures the variation attributable to the depth of
sampling;
SSR = j=1pi=118 (xij -
i - j +
)2 has (p - 1) x 17
degrees of freedom, and is the part that cannot be explained by a given
factor of variation and can be considered an estimate of experimental
error.
Analysis of variance can be performed, using the F-test (17), by comparing the variances attributable to the various factors with the variance attributable to the experimental error.
The results presented in Table 1
show some characteristics that need some comment:
).
.
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Our data clearly show that the iron concentration in a single liver needle biopsy, weighing ~5 mg and 2 cm long, may be considered representative of the mean HIC with a relative standard deviation of 15%. This value depends on the analytical procedure and the uneven iron distribution. Therefore, when only one portion of the needle biopsy core is used, the reduction in weight from 5 mg to 1 mg does not significantly increase the error attributable to the analytical procedure, although the measure is far less significant because the subcapsular portion of the needle biopsy contains much more iron than the inner part. In cases where the determination of HIC is important for monitoring iron chelation therapy, a random subdivision of the liver biopsy is to be avoided; if an entire liver biopsy is not available for chemical analyses, we suggest that the subcapsular and the deepest part of the biopsy be used as a unit to minimize the errors attributable to a casual choice of sample. Finally, we propose a consensus conference on the methods of trace element determination in needle biopsies. In our opinion, the standardization of these procedures may lead to measured HIC values more representative of the true HIC and much more useful for clinical purposes.
Acknowledgments
We thank the Assessorato alla Sanità della Regione Autonoma della Sardegna for financial support.
References
The following articles in journals at HighWire Press have cited this article:
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  J. C. Wood, C. Enriquez, N. Ghugre, J. M. Tyzka, S. Carson, M. D. Nelson, and T. D. Coates MRI R2 and R2* mapping accurately estimates hepatic iron concentration in transfusion-dependent thalassemia and sickle cell disease patients Blood, August 15, 2005; 106(4): 1460 - 1465. [Abstract] [Full Text] [PDF]
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, needle biopsies divided
into three parts; 
, needle biopsies divided into four parts.
d.w., dry weight.