Sampling Methods
Urine/Blood Analysis

DIETER Y. KOLLER

University Children's Hospital Vienna, Vienna, Austria

	INTRODUCTION

TOP INTRODUCTION WHAT DO WE KNOW? WHAT DO WE NEED... HOW CAN WE ACHIEVE... REFERENCES

Asthma and other recurrent wheezing disorders, cystic fibrosis, acute respiratory distress syndrome, and chronic lung disease of prematurity are the most frequent pediatric disorders in which inflammatory processes within the airways play a crucial role (1). Although airway inflammation is of crucial pathogenetic importance in these diseases, because direct access to the airways is rarely possible, alternative markers of airway inflammation are needed for diagnostic and therapeutic purposes (Table 1).

This article explores the use of biomarkers in peripheral blood and in urine (4, 5) as possible indirect measures to study airway inflammation (Table 2). In particular, the article focuses on the use of biomarkers, especially the use of eosinophil granule proteins in the blood and urine of children with asthma/wheezy bronchitis and cystic fibrosis, in comparison with healthy subjects.

	WHAT DO WE KNOW?

TOP INTRODUCTION WHAT DO WE KNOW? WHAT DO WE NEED... HOW CAN WE ACHIEVE... REFERENCES

Peripheral Blood Biomarkers

Eosinophil granule proteins. The eosinophil contains four cationic proteins: eosinophil cationic protein (ECP), major basic protein (MBP), eosinophil peroxidase (EPO), and eosinophil protein X (EPX is also known as eosinophil-derived neurotoxin [EDN]). Of these proteins, MBP is also produced by basophils and the placenta (6), whereas ECP, although sometimes present in neutrophils, is not produced by the neutrophil. These proteins are released during activation of eosinophils.

Eosinophil granule proteins can be detected in both serum and plasma. However, in serum the levels of, for example, ECP are much higher than in ethylenediamine tetraacetic acid (EDTA)-plasma. Because the turnover of ECP in the circulation is fast (half-life approximately 45 min [7]) it is not surprising that in EDTA-plasma, levels of ECP are frequently low and cannot be used to distinguish between healthy and diseased children. In addition, this can also be explained by the fact that in peripheral blood without additives, eosinophils can release proteins, when activated or primed, during the clotting process. In blood samples containing EDTA as an additive, eosinophils are inactivated and are therefore unable to release their granule proteins ex vivo (8). It appears that ECP levels in EDTA-plasma reflect the concentrations in the circulation whereas serum concentrations reflect the releasability of eosinophil granule proteins from eosinophils.

Blood should thus be collected under tightly controlled and standardized conditions (i.e., temperature, type of container, duration of clotting process) to give repeatable results (8). Despite the use of blood-collecting tubes, and processing of blood samples, diurnal variation in eosinophil granule proteins in serum should be considered (7).

A wide variation in reference values has been cited in the literature, depending on the protocol used for the collection and processing of blood samples. When using comparable standardized protocols, serum ECP levels between 2 and 20 µg/L are considered normal for adults (11). Comparable results were observed in school-age children with a mean age of 10.2 yr (3.2- 19.7 µg/L), toddlers with a mean age of 4.1 yr (3.8-22.6 µg/L), and infants with a mean age of 8 mo (5.1-16.7 µg/L) (4, 11).

ECP and EPX levels in serum samples from healthy children, children with stable asthma, and children with cystic fibrosis have been shown to be repeatable (11). In contrast, in asthmatic children with an acute exacerbation the variability of repeated ECP determinations was increased (D. Y. Koller and colleagues, unpublished manuscript).

Eosinophil markers in peripheral blood are not disease specific. It seems that increased concentrations of serum ECP, as well as of the other eosinophil granule proteins, indicate the participation of the eosinophil in any (chronic) inflammatory process (12, 13).

Therefore, what do serum levels of eosinophil granule proteins actually reflect? Serum ECP levels correlate with ECP levels in bronchoalveolar lavage (BAL) fluid from patients with asthma (14), as well as with ECP concentrations in the sputum of patients with cystic fibrosis (12). This indicates that eosinophil activity in the blood reflects eosinophilic inflammation in the lung, in children primarily with lung disease. In addition, atopy and infections of the respiratory tract enhance eosinophil activity measured by serum ECP in children with asthma (4).

For epidemiological purposes, especially in field studies, the use of eosinophil granule measurements in serum is limited by strict and time-consuming sampling procedures and the need for venipuncture.

Neutrophil-derived proteins. In bronchial asthma, the assessment of neutrophil-derived products in peripheral blood has been shown to be less useful (15). However, in cystic fibrosis high serum levels of neutrophil-derived proteins, such as myeloperoxidase (MPO), lactoferrin, and human neutrophil lipocalin (HNL), have been found (16). Furthermore, serum levels of MPO are significantly correlated with MPO concentrations in the sputum (12), leading to the assumption that measurement of neutrophil activity in serum reflects endobronchial neutrophilic inflammation in cystic fibrosis.

The newly discovered protein HNL has been shown to distinguish between chronic bacterial colonization or infection and acute bacterial pulmonary exacerbation in cystic fibrosis, with higher specificity and sensitivity than other neutrophil markers such as MPO or lactoferrin (16). The protocol for processing blood samples for neutrophil markers is not as demanding as for eosinophil granule proteins. Therefore, neutrophil parameters may be more easily applied in routine clinical practice. However, because neutrophil-derived proteins may increase during bacterial infection outside the airways they cannot be used solely for the assessment of neutrophil participation within the airways (4).

Cytokines and adhesion molecules. The determination of cytokines and adhesion molecules in peripheral blood to assess or monitor airway inflammation is currently limited by a lack of knowledge of which factors influence their passage from the lung into the circulation, degradation, and clearance from the blood. In addition, as cytokines and adhesion molecules are primarily local messengers, we may not know what peripheral blood levels reflect and whether the concentrations are functionally relevant. To date, interleukin 1 (IL-1), IL-4, IL-5, IL-6, IL-8, interferon , granulocyte-macrophage colony-stimulating factor (GM-CSF), and tumor necrosis factor  (TNF-) have been measured in peripheral blood in patients with asthma, cystic fibrosis, and other respiratory disorders as well as in healthy control subjects (17). However, investigations are limited by the poor sensitivity of most of the assays (e.g., IL-5 was detectable in only approximately 50% of patients with acute bronchial asthma), although assays are continuously being improved.

Increased serum concentrations of adhesion molecules such as intracellular adhesion molecule 1 (ICAM-1), endothelial leukocyte adhesion molecule 1 (ELAM-1), or E-selectin, or soluble cytokine receptors, such as the soluble IL-2 receptor, have been described in patients with asthma, cystic fibrosis, or other pulmonary or atopic disorders. However, there is a large overlap between healthy and ill subjects. Moreover, the role of circulating adhesion molecules is still unclear.

Mast cell-derived mediators. Determination of histamine levels in plasma requires sensitive methods. In addition, histamine is rapidly cleared from the circulation. Tryptase is another mast cell marker, but when assessed in peripheral blood, it gives no important information on airway inflammation (18). Therefore, measurement of established mast cell markers in peripheral blood does not contribute to the evaluation of airway inflammation (18).

Biomarkers in Urine

Eosinophil granule proteins. Urinary EPX can be detected not only in children with asthma and cystic fibrosis but also in healthy subjects. In pilot studies of children with asthma initial results were encouraging (19).

Urine should be collected as a spot sample. However, a urinary tract infection or the presence of leukocytes must be excluded before urine is processed because if infection is present urinary EPX levels are increased 10-fold. Before assessment of urinary EPX, urine samples should be diluted 11 times with a phosphate-buffered saline containing bovine serum albumin, Tween 20, EDTA, and N-cetyltrimethylammonium bromide (19, 20). Urinary EPX levels must be corrected for urinary creatinine concentrations. The reproducibility of urinary EPX measurements is good in healthy subjects and in children with inflammatory disorders. However, urinary EPX levels show a diurnal variation, with the lowest levels in the evening (D. Y. Koller and colleagues, unpublished manuscript). Therefore, the time of collection is also important.

Urinary EPX determination is feasible for use in epidemiological field studies (22). In one study it was possible to establish normal values in healthy school-age children for urinary EPX (median, 63.6 µg/mmol; 5th percentile, 28.8 µg/mmol; 90th percentile, 141 µg/mmol creatinine). Urinary EPX levels appear to be more strongly correlated with atopy than with asthma (22).

Others. To date, methylhistamine, neopterin, and some other markers in urine have not been found to be of great value.

	WHAT DO WE NEED TO KNOW?

TOP INTRODUCTION WHAT DO WE KNOW? WHAT DO WE NEED... HOW CAN WE ACHIEVE... REFERENCES

1. What determines the level of inflammatory biomarkers in serum/urine in children with inflammatory respiratory disease?

2. Do we have a marker in peripheral blood/urine whose concentration is not highly dependent on the sampling procedure? If not, can we identify others?

3. How many blood/urine markers do we need to assess the complex inflammatory process within the lung in certain diseases? Is it ever possible to assess inflammatory processes with a single marker?

4. Can we exclude confounding factors such as atopy, extrapulmonary inflammation, or infection?

5. Are high levels of eosinophil granule proteins in children with acute-phase asthma really related to asthma or are they a by-product of the atopic status of the patient? What role does viral or bacterial infection play in eosinophilic activation?

	HOW CAN WE ACHIEVE THIS?

TOP INTRODUCTION WHAT DO WE KNOW? WHAT DO WE NEED... HOW CAN WE ACHIEVE... REFERENCES

1. To investigate what serum or urine measurements reflect, studies must be designed in which we compare biomarkers in various body fluids (serum, urine, nasal lavage fluid, sputum) including BAL in children, even in very young children with respiratory tract diseases.

2. Studies should be performed to investigate whether the measurement of specific mediators from different cell types known to be involved in pulmonary inflammation in certain lung diseases improves specificity in examining the inflammatory process.

3. To exclude any confounding factors such as atopy, or extrapulmonary inflammation or infection, studies including large groups of children with atopic and nonatopic asthma, nonasthmatic children with atopy, and subjects with acute infection (such as pneumonia) and chronic infection (such as cystic fibrosis) should be investigated for inflammatory markers in serum and in urine.

4. Although the results of many cross-sectional studies dealing with inflammatory markers in serum or urine appear to be encouraging, most are short-term and are underpowered. Therefore, long-term longitudinal studies with repeated measurements of inflammatory markers in an adequate number of patients are warranted to investigate the potential of biomarkers in monitoring the inflammatory process of a specific disease.

5. To examine the role of urinary markers in studying mechanisms, especially in atopic asthma, we should measure the levels of urinary biomarkers during and after intervention in an adequate number of patients, using different allergen or pharmacological challenges.

6. Because none of the known inflammatory biomarkers can be used solely to determine the complexity of the pulmonary inflammatory process in asthma or cystic fibrosis or in other chronic inflammatory lung diseases, the combination of cell products may give more detailed information on the degree or severity of inflammation.

	Footnotes

* Current affiliation: Department of Pediatrics, General Hospital, Amstetten, Austria.

Correspondence and requests for reprints should be addressed to Professor Dieter Y. Koller, M.D., Department of Pediatrics and Neonatology, General Hospital Amstetten, Krankenhausstrasse 21, A-3300 Amstetten, Austria.

	References

TOP INTRODUCTION WHAT DO WE KNOW? WHAT DO WE NEED... HOW CAN WE ACHIEVE... REFERENCES

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12. Koller, D. Y., M. Götz, I. Eichler, and R. Urbanek. 1994. Eosinophil activation in cystic fibrosis. Thorax 49: 496-499 [Abstract].

13. Halmerbauer, G., T. Frischer, and D. Y. Koller. 1997. Monitoring of disease activity by measurement of inflammatory markers in atopic dermatitis in childhood. Allergy 52: 756-769 .

14. Spry, C. F. J. 1988. Eosinophils: A Comprehensive Review and Guide to the Scientific and Medical Literature. Oxford University Press, Oxford.

15. Tauber, E., Y. Herouy, M. Götz, Urbanek, E. Hagel, and D. Y. Koller. 1999. Assessment of serum myeloperoxidase in children with bronchial asthma. Allergy 54: 177-182 [Medline].

16. Eichler, I., M. Nilsson, R. Rath, I. Enander, P. Venge, and D. Y. Koller. 1999. Human neutrophil lipocalin, a new highly specific marker for acute exacerbation in cystic fibrosis. Eur. Respir. J. 14: 1145-1149 [Abstract].

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18. Kristjansson, S., T. Shimizu, I. L. Strannegard, and G. Wennergren. 1994. Eosinophil cationic protein, myeloperoxidase and tryptase in children with asthma and atopic dermatitis. Pediatr. Allergy Immunol. 5: 223-229 [Medline].

19. Kristjansson, S., I. L. Strannegard, Ö. Strannegard, C. Peterson, I. Enander, and G. Wennergren. 1996. Urinary eosinophil protein X in children with atopic asthma: a useful marker of antiinflammatory treatment. J. Allergy Clin. Immunol. 97: 1179-1187 [Medline].

20. Lugosi, E., G. Halmerbauer, T. Frischer, and D. Y. Koller. 1997. Urinary eosinophil protein X in relation to disease activity in childhood asthma. Allergy 52: 584-588 [Medline].

21. Koller, D. Y., G. Halmerbauer, T. Frischer, and B. Roithner. 1999. Assessment of eosinophil granule proteins in various body fluids: is there a relation to clinical variables in childhood asthma. Clin. Exp. Allergy 29: 758-763 .

22. Tauber, E., G. Halmerbauer, T. Frischer, C. Gartner, F. Horak, Jr., A. Veiter, and D. Y. Koller. 2000. Urinary eosinophil protein X in children: the relationship to asthma and atopy and normal values. Allergy (In press)