Chapter 4 Allgrove J, Shaw NJ (eds): Calcium and Bone Disorders in Children and Adolescents. Endocr Dev. Basel, Karger, 2009, vol 16, pp 49–57
Bone Biopsy: Indications and Methods Frank Rauch Genetics Unit, Shriners Hospital for Children and McGill University, Montréal, Qué., Canada
Abstract In the context of metabolic bone disorders, obtaining biopsies of iliac bone can be useful for establishing a diagnosis in an individual patient or for investigating pathomechanisms when a series of samples is examined. Although bone specimens are usually decalcified for routine pathology to facilitate sample processing, when investigating metabolic bone disorders it is usually much more informative to analyse undecalcified samples. Biopsy samples can be assessed qualitatively and quantitatively. Quantitative analysis by computerised histomorphometry of undecalcified bone biopsy samples is a key tool for studying bone metabolism and, to a lesser extent, bone mass and structure. Standard histomorphometric analyses focuses on trabecular bone and therefore mainly provides information on trabecular remodelling. Remodelling activity changes markedly with age during development. This has to be taken into account when histomorphometry is used in the paediatric setting. Children and adolescents with severe bone fragility should have a bone biopsy for diagnostic purposes unless the diagnosis is obvious from non-invasive examinations. Quantitative histomorphometric analysis of transiliac bone biopsy samples is especially valuable in clinical studies, as this method provides safety and efficacy data that can not be obtained in any other way. Copyright © 2009 S. Karger AG, Basel
In the context of metabolic bone disorders, obtaining biopsies of iliac bone can be useful for establishing a diagnosis in an individual patient or for investigating pathomechanisms when a series of samples is examined. Biopsy samples can be used for qualitative assessment – similar to the pathologist’s evaluation of other tissue specimens – and for quantitative analysis, called histomorphometry. Bone tissue is very hard and for that reason is more difficult to process than soft tissue. In routine pathology, bone tissue is therefore often decalcified and thus converted into a soft tissue. However, this leads to the loss of important information about bone mineralisation and bone cell activity. To assess metabolic bone disorders, it is therefore generally more informative to analyse samples undecalcified. Computerised quantitative histomorphometry of undecalcified bone biopsy samples is a method to obtain direct quantitative information on bone tissue. When tetracycline labelling is performed prior to biopsy, bone cell function can be studied in
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vivo. Important for paediatric use, bone histomorphometric results are not directly influenced by the growth process. In contrast to some currently popular indirect methods of bone analysis, histomorphometry yields results with a known meaning. Knowledge of bone tissue is also crucial for interpreting the findings of molecular and cellular studies. Despite these advantages, bone histomorphometry is underused in paediatrics. This may be partly due to the fact that histomorphometry requires an invasive procedure to obtain a bone sample, is labour intensive, and needs special equipment and expertise. Other reasons may include overestimation of the utility of non-invasive bone diagnostics and lack of information about what bone histomorphometry does. The present contribution tries to address this latter point. More detailed information on paediatric histomorphometry is available elsewhere [1].
Bone Biopsy Procedure
Bone histomorphometry was first developed to study rib bone samples. This was soon abandoned because the ilium proved to be a much more convenient site for obtaining bone samples. In principle, histomorphometric analysis can be performed in any bone. In clinical paediatrics, however, the utility of samples from nonstandard sites is limited because reference data are only available for the ilium. Quantitative bone histomorphometry requires an intact biopsy specimen of good quality. This implies that the transiliac sample must be obtained under standardised conditions and with appropriate tools. It is essential that the sample is not fractured or crushed and contains two cortices separated by a trabecular compartment. These requirements are often quite difficult to meet in small or very osteopaenic children. Bone specimens for histomorphometric evaluation are horizontal, full-thickness biopsy samples of the ilium from a site 2 cm posterior from the anterior superior iliac spine. This bone is easily accessible, does not require extensive surgery, and is associated with few postoperative complications. Also, this is the only site for which paediatric histomorphometric reference data have been published [2]. A correctly performed biopsy procedure should yield a sample containing two cortices that are separated by a trabecular compartment (fig. 1). Vertical samples (from the iliac crest downwards, also called Jamshidi approach) are of questionable utility because of the presence of the growth plate at the top of the iliac crest. Turnover is very high and cortical thickness is very low in bone tissue below the growth plate and results are therefore not representative. Thus, the often-used term ‘iliac crest biopsy’ is a misnomer, as the iliac crest actually should be avoided during the biopsy procedure. The more accurate term is ‘transiliac biopsy’. The most widely used bone biopsy instrument is the Bordier needle. The inner diameter of the needle should be 5 mm (which we use in patients up to the age of 12 years) or 6 mm (for patients older than 12 years). The size of the needle diameter is
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Fig. 1. Section of an entire iliac biopsy specimen of a 9-year-old boy without metabolic bone disease. In this section core width is 7.2 mm, cortical width (i.e. the average length of the arrows indicated in the two cortical compartments) is 894 μm and bone volume per tissue volume in the trabecular compartment is 24.5%. Osteoid and cellular structures can not be identified at this magnification.
Cortical compartment
Trabecular compartment
Core width
important, because an appropriately large sample area must be available for histomorphometric analyses to obtain representative measures. A smaller needle diameter means that a smaller bone sample is obtained and that the margin of error of the histomorphometric analysis is wider. Most children younger than 14 years of age require general anaesthesia for the procedure. Local anaesthesia can be sufficient for older adolescents. Patients are allowed to get out of bed after 3 h and can usually be discharged on the same day. Another prerequisite for histomorphometric evaluation is bone labelling. Dynamic parameters of bone cell function can only be measured when the patient has received two courses of tetracycline label prior to biopsy. Tetracycline compounds form calcium chelate complexes that bind to bone surfaces. These complexes are buried within the bone at sites of active bone formation, whereas they redissociate from the other bone surfaces once serum tetracycline levels decrease. The tetracycline trapped at formation sites can then be visualised under fluorescent light (fig. 2). The most widely used tetracycline compound is demeclocycline hydrochloride (DeclomycinT, Ledermycin®). Two labelling cycles are given before the biopsy procedure, each one lasting for 2 days. Declomycin is given orally in two doses per day with a daily dose of 15–20 mg/kg body weight (maximum dosage: 900 mg/day). The first labelling course is given on days 17 and 16 before the biopsy procedure, the second course is given on days 5 and 4 before the procedure. The two courses are thus separated by an interlabel time of 10 days. Although children and adolescents generally tolerate tetracycline double labelling well, some side effects, such as allergic reactions, vomiting, and photosensitivity, might be observed. Administering the drug after meals can diminish gastrointestinal side effects. It is important that these meals do not include milk or other dairy products because tetracycline complexes with calcium contained in the food and is not absorbed adequately. Sun exposure must be
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Fig. 2. Trabecular remodelling. Multiple short tetracycline double-labels are present. Dynamic bone formation parameters are derived from the length of the tetracycline labels and the distance between the labels.
avoided while taking tetracyclines. Tetracycline use is generally not recommended for children younger than nine years of age because discolouration of teeth may occur. However, the previously mentioned schedule appears to be safe in this respect. At the Montreal Shriners Hospital, it has been used for more than 350 biopsies in children younger than 9 years of age and tooth discolouration has never been observed.
Sample Processing
The biopsy sample should be placed in a fixative solution as soon as possible after the procedure. The fixation process aims at the preservation of bone tissue constituents by inactivating lysosomal enzymes. The choice of fixative and temperature at which the sample should be kept depends on the planned staining techniques. For routine histomorphometry, 70% ethanol or 10% buffered formalin at room temperature can be used. The duration of fixation should be at least 48 h but should not exceed 10 days because the tetracycline labels are washed out when fixation is too long. Once in fixative, the sample can be sent to the laboratory where samples are cut and stained and where the histomorphometric analyses are performed.
Histomorphometric Parameters
Histomorphometrists use standardised terminology and clear definitions that were established by a working group of the American Society for Bone and Mineral Research [3]. According to these definitions, ‘bone’ is bone matrix, whether it is mineralised or not. Unmineralised bone matrix is called osteoid. The term ‘tissue’ refers to both bone and associated soft tissue, such as bone marrow. Histomorphometric measurements
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are performed in two-dimensional sections. Nevertheless, in order to stress the threedimensional nature of bone, the terminology committee favoured a three-dimensional nomenclature for reporting histomorphometric results [3]. Thus, what appears as a line in a microscopic bone section is called a surface, whereas what is visible as an area under the microscope is referred to as a volume. This is done simply by convention, and should not be mistaken as actual three-dimensional measurements. Histomorphometric parameters can be classified into four categories (table 1): Structural parameters, static bone formation parameters, dynamic formation parameters, and bone resorption parameters. Structural parameters describe the size and the amount of bone. The outer size of a transiliac biopsy specimen is called core width, a measure which reflects the thickness of the ilium. Cortical width is the average width of the two cortices. Bone volume per tissue volume of trabecular bone represents the proportion of the marrow cavity which is occupied by bone. In trabecular bone, bone volume per tissue volume can be schematically separated into two components, trabecular thickness and trabecular number. The group of static formation parameters comprises the surface extent, thickness and relative amount of osteoid, as well as the surface extent of osteoblasts (called osteoid surface per bone surface, osteoid thickness, osteoid volume per bone volume and osteoblasts surface per bone surface, respectively; table 1). Wall thickness reflects the amount of bone that is created by an osteoblast team during a remodelling event. Wall thickness should not be confused with cortical thickness, with which is does not have any relationship. Dynamic bone formation parameters yield information on in vivo bone cell function and can only be measured when patients have received two courses of tetracycline label prior to biopsy (table 1). The two basic parameters are the surface extent of mineralisation activity (mineralising surface per bone surface) and the speed of mineralisation in a direction perpendicular to the bone surface (mineral apposition rate). From these primary measures, mineralisation lag time and bone formation rate per bone surface are derived mathematically. It should be noted that a high bone formation rate does not necessarily lead to a net gain of bone. If the remodelling balance is zero, the amount of bone will remain unchanged even if bone formation rate is very high. The combination of a negative remodelling balance and high bone formation rate will even lead to rapid bone loss. Thus, bone formation rate per bone surface in trabecular bone indicates the activity of bone turnover rather than bone gain [4]. Bone resorption can only be quantified with static parameters, which makes evaluation of bone resorption the least informative aspect of histomorphometric analysis. It is possible to quantify the extent of bone surface that is covered by osteoclasts or which looks eroded (osteoclast surface per bone surface and eroded surface per bone surface, respectively), but it is not possible to tell from these measures how much bone resorption is actually going on. This may be an issue in the evaluation of renal bone disease. In chronic renal failure, osteoclasts resorb bone more slowly than normal, so that the extent of osteoclast and eroded surfaces overestimates the rate of bone resorption [5].
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Table 1. The most commonly used histomorphometric parameters Parameter Structural parameters Core width, mm Cortical width, μm Bone volume/tissue volume, %
Trabecular thickness, μm Trabecular number, /mm
Static formation parameters Osteoid thickness, μm Osteoid surface/bone surface, % Osteoid volume/bone volume, % Osteoblast surface/bone surface, % Wall thickness, μm Dynamic formation parameters Mineralising surface/bone surface, % Mineral apposition rate, μm/day Mineralisation lag time, days Bone formation rate/bone surface, μm3*μm-2*y-1 Static resorption parameters Eroded surface/bone surface, % Osteoclast surface/bone surface, %
Significance
overall size of the biopsy specimen distance between periosteal and endocortical surfaces space taken up by mineralised and unmineralised bone relative to the total size of a bone compartment self-explanatory number of trabeculae that a line through trabecular compartment would hit per millimetre of its length distance between the surface of the osteoid seam and mineralised bone percentage of bone surface covered by osteoid percentage of bone volume consisting of unmineralised osteoid percentage of bone surface covered by osteoblasts mean thickness of bone tissue that has been deposited at a remodelling site percentage of bone surface showing mineralising activity distance between two tetracycline labels divided by the length of the labelling interval time interval between the deposition and mineralisation of matrix amount of bone formed per year on a given bone surface percentage of bone surface presenting a scalloped appearance percentage of bone surface covered by osteoclasts
Bone Metabolism in Children and Adolescents
The volume of trabecular iliac bone increases markedly between 2 and 20 years of age [2]. This increase is entirely explained by trabecular thickening, whereas there is no change in trabecular number. Iliac trabeculae probably become thicker during development
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because bone remodels with a positive balance [6]. It has been estimated that, during a remodelling cycle, osteoblasts lay down about 5% more bone than osteoclasts resorb. In other words, 95% of the bone formation activity is required just to replace the bone that has been previously removed by the osteoclasts. Since the difference between resorption and formation is very small, a high remodelling activity is necessary to make trabeculae noticeably thicker. Remodelling activity is indeed elevated in young children, decreases until the age of 8 or 9 years, and increases again during puberty. After the age of puberty, remodelling activity declines into the much lower adult range [6].
Indications for Bone Biopsy in Paediatrics
The main use of iliac bone biopsies is to provide diagnostic clues in unclear bone fragility disorders. For example, some forms of osteogenesis imperfecta can be diagnosed on the basis of a characteristic histologic pattern [7]. Polyostotic fibrous dysplasia is sometimes difficult to distinguish from osteogenesis imperfecta on clinical grounds, but the diagnosis is usually quite obvious on bone histology. This has therapeutic implications, as children with osteogenesis imperfecta usually respond much better to bisphosphonate treatment than patients with fibrous dysplasia [7, 8]. Thus, children with multiple long-bone fractures or vertebral body compressions without adequate trauma should have a bone biopsy unless the diagnosis is obvious from non-invasive examinations. Another indication for bone biopsy is progressive bone deformity, which may sometimes arise without clear history of fractures. A bone biopsy sample allows evaluation of trabecular and cortical bone structure, the mineralisation process, bone lamellation (woven bone vs. lamellar bone, the appearance of lamellae), the presence of calcified cartilage, the activity of bone metabolism and the appearance of bone cells. All of this information is important in the assessment of skeletal disease processes, but none of it is reflected in ‘bone density’, whatever technique is used to measure it. These considerations are particularly relevant in the context of renal bone disease, which is a frequent but understudied problem after juvenile renal failure [9]. When the only aim is to assess an individual patient, it is not absolutely necessary to analyse the sample with quantitative histomorphometry. A qualitative evaluation of the histological appearance may be sufficient in such cases. However, a quantitative analysis is necessary in clinical research settings, when numbers are needed to describe the average effect of a disease or a treatment in a group of patients. Histomorphometric evaluation of bone biopsy samples should be a standard feature of studies that evaluate experimental drugs to treat bone disorders in children and adolescents. Current noninvasive methods for studying the amount, distribution, and metabolism of bone are fraught with technical limitations and uncertainties regarding the interpretation of results. The availability of histomorphometric data allows judging treatment effects in a rational way.
Bone Biopsy
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The most important argument for performing bone biopsies in paediatric studies probably concerns patient safety. This is especially true when children and adolescents are treated with long-acting drugs, such as bisphosphonates. Analysis of bone samples provides safety measures that can not be obtained in any other way. For example, bone histologic studies have demonstrated that bisphosphonate treatment in children can lead to accumulation of calcified cartilage material in bone tissue, a disquieting finding that calls for caution in the use of these drugs in growing patients with minor skeletal symptoms [10, 11]. Thus, including bone biopsies in study protocols is crucial for documenting the efficacy of therapy as well as its safety.
Conclusions
Standard histomorphometric analysis of transiliac bone biopsies mainly provides information on trabecular remodelling. Assessment of cortical modelling processes is feasible but has rarely been used until now. When histomorphometric studies are performed in children and adolescents, it is important to take the age dependency of many histomorphometric parameters into account. When new treatments of bone disorders are studied, analysis of transiliac bone biopsy samples provides safety and efficacy data that can not be obtained in any other way. Children with multiple longbone fractures or vertebral body compressions that are not explained by adequate trauma should have a bone biopsy unless the diagnosis is obvious from non-invasive examinations.
Acknowledgements Thanks go to Mark Lepik for preparing the figures. The author is a Chercheur-Boursier Clinicien of the Fonds de la Recherche en Santé du Québec. This work was supported by the Shriners of North America.
References 1 Rauch F: Bone histomorphometry; in Glorieux FH, Pettifor J, Jueppner H (eds): Pediatric Bone. San Diego, Academic Press, 2003, pp 359–374. 2 Glorieux FH, Travers R, Taylor A, et al: Normative data for iliac bone histomorphometry in growing children. Bone 2000;26:103–109. 3 Parfitt AM, Drezner MK, Glorieux FH, et al: Bone histomorphometry: standardization of nomenclature, symbols, and units: report of the ASBMR Histomorphometry Nomenclature Committee. J Bone Miner Res 1987;2:595–610.
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4 Parfitt AM: Renal bone disease: a new conceptual framework for the interpretation of bone histomorphometry. Curr Opin Nephrol Hypertens 2003;12: 387–403. 5 Jaworski ZF, Lok E, Wellington JL: Impaired osteoclastic function and linear bone erosion rate in secondary hyperparathyroidism associated with chronic renal failure. Clin Orthop Relat Res 1975; 107:298– 310.
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6 Parfitt AM, Travers R, Rauch F, Glorieux FH: Structural and cellular changes during bone growth in healthy children. Bone 2000;27:487–494. 7 Rauch F, Glorieux FH: Osteogenesis imperfecta. Lancet 2004;363:1377–1385. 8 Plotkin H, Rauch F, Zeitlin L, Munns C, Travers R, Glorieux FH: Effect of pamidronate treatment in children with polyostotic fibrous dysplasia of bone. J Clin Endocrinol Metab 2003;88:4569–4575.
9 Groothoff JW, Offringa M, Eck-Smit BLF, et al: Severe bone disease and low bone mineral density after juvenile renal failure. Kidney Int 2003;63:266– 275. 10 Rauch F, Travers R, Plotkin H, Glorieux FH: The effects of intravenous pamidronate on the bone tissue of children and adolescents with osteogenesis imperfecta. J Clin Invest 2002;110:1293–1299. 11 Whyte MP, Wenkert D, Clements KL, McAlister WH, Mumm S: Bisphosphonate-induced osteopetrosis. N Engl J Med 2003;349:457–463.
Frank Rauch Genetics Unit, Shriners Hospital for Children 529 Cedar Avenue Montréal, Qué., H3G 1A6 (Canada) Tel. +1 514 842 5964, Fax +1 514 842 5581, E-Mail
[email protected]
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