Patients with chronic kidney disease (CKD) have complex

Transcription

1 Histomorphometric Measurements of Bone Turnover, Mineralization, and Volume Susan M. Ott Department of Medicine, University of Washington, Seattle, Washington A recent Kidney Disease: Improving Global Outcomes report suggested that bone biopsies in patients with chronic kidney disease should be characterized by determining bone turnover, mineralization, and volume. This article focuses on the calculations and interpretation of these measurements. In most cases of renal osteodystrophy, the bone formation rate is roughly similar to the bone resorption rate; therefore, the bone formation indices can be used to describe turnover. It is important to remember that these conventions will not apply in some situations. Activation frequency should not be confused with bone formation rate or bone metabolic unit birth rate. Abnormal mineralization can be described using the osteoid volume, increased osteoid maturation time, or increased mineralization lag time. The concept of bone volume is the easiest to understand, but there is a large error from one biopsy to the next in the same person. There are some difficulties with each of the measurements, and further research in patients with chronic kidney must be done to enable a consensus to be reached about cut points to define categories within the spectrum of renal osteodystrophy and how to evaluate treatment responses. Clin J Am Soc Nephrol 3: S151 S156, doi: /CJN Patients with chronic kidney disease (CKD) have complex abnormalities in their bones (1 6). Some of the pathologic factors and the resulting histologic changes are shown in Figure 1. Many more factors affect bone metabolism in patients with CKD than in patients with other types of metabolic bone disease, in which one major pathogenic disturbance usually predominates. In addition to abnormalities shown here, patients may have had bone disease before developing renal failure (e.g., osteoporosis, vitamin D deficiency, steroid-bone disease), which will contribute to the pathologic findings of the bones. Bone biopsies are performed to understand the pathophysiology and course of bone disease, to relate histologic findings to clinical symptoms of pain and fracture, and to determine whether treatments are effective. A recent Kidney Disease: Improving Global Outcomes (KDIGO) report (7) suggested that bone biopsies in patients with CKD should be characterized by determining bone turnover, mineralization, and volume (TMV). The exact methods of measuring these parameters, however, were not specified. This article focuses on the calculations and interpretation of these measurements. The three parameters (TMV) for defining categories of renal osteodystrophy were selected by the KDIGO committee on the basis of their experience with examining bone biopsies from patients with CKD and our current understanding of bone physiology. It has been recognized that these patients display a spectrum of bone formation rates (BFR) from abnormally low to very high. Other measurements that help to define low or high turnover (e.g., eroded surfaces, number of osteoclasts, fibrosis, woven bone) tend to be associated with the BFR as measured by tetracycline labeling. This is the most definite dynamic measurement, so it was chosen to represent bone turnover. Note Correspondence: Dr. Susan M. Ott, Box , University of Washington, Seattle, WA Phone: ; smott@u.washington.edu that a simple change in the BFR will not reveal whether a patient has improved, because restoration of normal physiology may require either increase or decrease in bone turnover, depending on the starting point. The second parameter was mineralization, to distinguish those with osteomalacia (who can form matrix but not form mineralized bone) from those with adynamic disease (who do not even form the matrix). The final parameter was bone volume, which was not usually included in previous schemes of describing renal osteodystrophy. The committee believed that bone volume would be likely to contribute to bone fragility and is separate from the other parameters. The bone volume is the end result of changes in bone formation and resorption rates: If overall BFR is greater than overall bone resorption rate, then the bone is in positive balance and the bone volume will increase. Some histologic findings in bone document causes of bone disease, such as aluminum, amyloid, or iron deposition; these are independent of the TMV classification. There are many other measurements that are not considered here because either they are too new, there is too little information about their meaning, or they seem to be secondary. It should be stressed that renal osteodystrophy is complicated and further research may indicate that some of these measurements will provide necessary information about treatment of patients: The amount of woven bone, the number and condition of osteocytes, the number of osteoblasts and osteoclasts, the number of apoptotic cells, the structural microarchitecture, the cortical porosity, the mineralization density, the mechanical stiffness of the bone material, the volume of fibrosis, the resorption depth, the volume of the fat cells, the thickness of the lamella, and the characteristics of the tetracycline labels. Immunohistochemical measurements of bone proteins may be used in the future but are beyond the scope of this discussion. In 1987, the American Society for Bone and Mineral Research Copyright 2008 by the American Society of Nephrology ISSN: /

2 S152 Clinical Journal of the American Society of Nephrology Clin J Am Soc Nephrol 3: S151 S156, 2008 Figure 1. Pathophysiologic mechanisms of renal osteodystrophy. The dashed arrows show feedback loops that are frustrated by the renal dysfunction. Histomorphometry Nomenclature Committee published an article on standardization of nomenclature, symbols, and units (8). This was widely accepted in the scientific community, and the KDIGO bone histomorphometry committee unanimously agreed that these conventions should also be used for renal osteodystrophy. The American Society for Bone and Mineral Research Histomorphometry Nomenclature Committee article provided valuable rationale for the measurements, the importance of the referents, the descriptions of the core samples (whether transiliac or vertical), the methods of measuring the primary measurements, and the equations for deriving the common indices of bone physiology. Although the bone journals all require articles to use this standard nomenclature, some of the recommendations are not always followed. Common omissions are the referent for BFR, specification of the minimum width of osteoid that is measured, and reporting derived indices without the primary data. The bone biopsies are taken from the anterior iliac crest because that site has normative data and because it is safe to take a biopsy; there are no major nerves, organs, or blood vessels, and the bone is large enough that the biopsy will not compromise bone strength. The size of the biopsy should be at least 4 mm in diameter; in many research studies, the size is 8 mm to allow more accurate measurements. It is critical that the sample not be decalcified. The vast majority of bone biopsies are done for possible cancer, which requires decalcification and very thin sections, so pathology laboratories automatically place the bone samples into decalcifying solution and it is then impossible to tell anything about mineralization. A bone biopsy that is done for diagnosis of any metabolic bone disease requires double tetracycline labeling. Turnover Healthy bone is a dynamic tissue, continually resorbing bone and replacing it with new bone in discrete areas known as bone metabolic units (BMU). The BMU in solid cortical bone drills a tunnel and then refills it. On the cancellous bone surface, the BMU can channel like a river flowing or can spread over the surface like fudge flowing over ice cream; therefore, the turnover of bone is different from the turnover on the skin, where the entire surface is continuously forming and shedding. At each BMU, the volume of new bone could completely fill the space that was resorbed, in which case there would be a neutral bone balance at that BMU. More common, the new bone does not quite fill in the resorbed space and the bone balance is negative at that location. On cancellous surfaces, the newly formed bone could occupy a larger volume than the bone resorbed, which would lead to a positive bone balance. The overall bone balance for the skeleton represents the sum of balances at all of the BMU. If each BMU yields a slight loss of bone, then the overall bone loss will depend on the number of BMU. The overall bone balance is also the difference between the average bone resorption rate and the average BFR. A dynamic review of the bone remodeling is available at edu/bonephys. If the bone is in balance, then turnover is either the rate of bone formation or the rate of bone resorption, because they are the same. The term turnover can be ambiguous if the bone is not in balance. Some people equate turnover to bone resorption, others with bone formation. A condition such as steroidbone disease, with high resorption and low formation rates, will then be designated high turnover by some and low turnover by others. The opposite happens during growth, recovery from lactation, or treatment with anabolic drugs, when the bone formation is higher than resorption. When bone is not in balance, both bone formation and bone resorption rates are needed to define the physiologic status. Postmenopausal osteoporosis is a bone disease in which the bone resorption rate is increased as a result of estrogen deficiency and the BFR are also increased to compensate for the high bone resorption (by a process called coupling, whose mechanisms are still uncertain), but the formation rates do not quite match the resorption rates and the net effect is loss of bone. This common form of high turnover is deleterious to the skeleton, because there is net loss of bone. Also, more subtle effects include increased chance of perforation of the trabecular walls, more cement lines that may be weak areas, and a greater proportion of new bone that is not as mineralized and lacks stiffness of mature bone. Nevertheless, the high BFR itself is not the problem, and anabolic medications hold more promise for reversing osteoporosis than antiresorbing ones, which merely prevent the disease from getting worse. In most cases of renal osteodystrophy, the bone is roughly in balance, so those with high BFR also have high resorption rates and those with adynamic bone disease have low bone resorption rates. The bone formation indices are used to describe turnover, because they can be measured with much more accuracy than the bone resorption rates. It is important to remember that these conventions will not apply in some situations. BFR can be measured directly. The length of the tetracycline labels (mineralizing surface per bone surface [MS/BS]) multiplied by the distance between labels (mineral apposition rate

3 Clin J Am Soc Nephrol 3: S151 S156, 2008 Bone Turnover, Mineralization, and Volume S153 [MAR]) is the area of new bone formed during the label interval. In other words, the BFR depends both on the rate of apposition at the surface and the total surface involved in forming bone: BFR MS/BS MAR This can be expressed in reference to the bone surface (as in this equation), bone volume, or tissue volume (after adjustment for bone surface per bone or tissue volume). The appropriate referent depends on the situation. The most easily understood concept of turnover is the BFR as a percentage per year of the bone volume. The absolute amount of bone formed (BFR/ TV) would theoretically provide the best correlation with a serum marker of bone formation. The tetracycline labels usually are seen clearly, and measurements are straightforward. Sometimes the edges tapir gradually into the inactive bone surface, making the exact end of the label indefinite, but this causes only minor measurement difficulties. More common, when there is very rapid bone formation, the labels become blurry and diffuse, making them difficult to measure. Conversely, when the bone is forming very slowly, the labels do not show separation and it is difficult to tell whether a label is a double label or a single label. Use of two different kinds of tetracycline (demeclocycline and tetracycline) is helpful in these cases because they have different colors when viewed using fluorescence microscopy. Because bone formation occurs in discrete BMU, an area of bone surface is not continuously active. There are intervals when a spot on the surface is actively forming bone; the duration of one of these intervals is called the formation period (FP). The rest of the time, that bone surface is either resorbing or quiescent. The total period (TP) is the duration between the beginning of one FP and the beginning of the next FP. The number of times per year that this spot begins the FP is the activation frequency (Acf). The Acf is related to the BFR, but it is not a BFR, because the Acf also depends on the amount of bone formed during each remodeling cycle. This amount of bone formed during an average remodeling cycle is represented by the wall thickness (WTh), which is the thickness of new bone made in one cycle. The FP is calculated as the WTh divided by the MAR: FP WTh/MAR For example, if the WTh is 40 m and the apposition rate is 0.5 m/d, then that spot on the surface was forming bone for 80 d. The WTh is a straightforward concept that is easy to demonstrate on an ideal trabecula that has clear lamella with distinct orientations (Figure 2). In practice, however, this can be a difficult and subjective measurement. Often the orientation of lamellae on the old bone is the same as on the new bone, and it is difficult to tell where the cement line is. On thin trabecula, there may be only one cement line, and it is hard to tell which BMU was most recent. Measurements must be strictly randomized to avoid bias of measuring only the thickest and clearest parts of the wall. Walls cannot be measured if the surface is eroded or covered with osteoid, because the calculations assume complete walls. In patients with CKD, this can be a problem because so much of the surface is actively forming or Figure 2. Trabecular surface showing an osteoid surface, an eroded surface, and the wall width. The section is viewed under polarized light to show the lamellar structure. resorbing bone. When the bone is undergoing changes, such as with therapy or worsening disease, some of the existing BMU will be old and the WTh will not represent the current dynamic state of the bone. There is an alternative method of estimating the WTh on the basis of extrapolation of walls on forming surfaces, but that requires many tedious measurements on multiple sections (9). An alternative method of calculating the FP is based on the phenomenon of label escape (10). Between the first and the last tetracycline labels, some BMU have stopped forming bone and others have started to form bone. These BMU will have only a single label. The longer the interval between labels, the more labels will be only single labels; therefore, the ratio of single to double labels is related to the FP and the label intervals. By extension, three labels can be used to give more precise measurements, and the FP is calculated as the label interval divided by the ratio of double labels to double triple labels. This method of calculating FP is not generally used because the labels can be infrequent enough to make the ratio unreliable unless a very large surface is measured. Once the FP has been calculated, the TP can be calculated by using a surface-interval transformation. The ratio of mineralizing surface (MS) to bone surface (BS) corresponds to the ratio of FP to TP. MS FP BS TP Therefore, TP is the FP divided by the mineralizing surface per bone surface. TP FP MS/BS For example, if the FP is 80 d and 10% of the surface is forming, then the TP is 800 d. Frequencies are the inverse of periods, so the Acf is once every 800 d, or 0.45 per year: Acf 1/TP

4 S154 Clinical Journal of the American Society of Nephrology Clin J Am Soc Nephrol 3: S151 S156, 2008 Note that the mineralizing surface is used to calculate both the Acf and the BFR, which is why the two are related. By combining the previous equations, it can be shown that the Acf is the BFR divided by the WTh: Acf 1/TP TP FP MS/BS therefore Acf MS/BS FP MS/BS BFR/MAR and FP WTh/MAR therefore Acf BFR/MAR WTh/MAR The MAR cancel each other out, so the final equation is Acf BFR WTh It is important to understand that Acf can increase even though the BFR is unchanged. As seen from the previous equation, the Acf can increase merely from a decrease in WTh. With aging, the Acf increases; this is due to a combination of increased bone formation and decreased WTh. Some people think that an increased Acf causes bone loss. They confuse bone balance with bone turnover. It is the imbalance between resorption and formation that causes osteoporosis, not the increased Acf. If the BFR is greater than the bone resorption rate, then an increased Acf is associated with increasing bone volume (e.g., after therapy with intermittent teriparatide). In a subset of patients with CKD, the cancellous bone volume is increased, probably resulting from increased parathyroid hormone that has stimulated bone formation even more than bone resorption. The Acf is also confused with the frequency of originating new BMU (BMU birth rate). One must remember that Acf refers to just one spot on the bone surface. When a traveling BMU reaches a spot, that surface becomes active. The birth rate of new BMU depends on how long they live. A bone with many short-lived BMU can have the same Acf, WTh, and BFR as a bone with a few long-lived BMU. The concept of BMU lifespan is simple to understand but cannot be measured on a twodimensional section of bone, because BMU wander in and out of the plane of the section. Other measurements that relate to the formation aspects of bone turnover include the osteoblastic surface, the number of active osteoblasts, and the osteoid surface. None of these gives the same accurate information as the tetracycline labeling. Bone resorption is related to the number of osteoclasts and the depth of resorption cavities, but there is no direct way to calculate the bone resorption rate. Some formulas that have been proposed require an assumption that the bone is in balance. Acf and BFR have been used as indices of bone turnover in patients with CKD. There is definitely a spectrum from very low to very high. Further research is necessary to determine the best place for cut points that will place patients into categories that make sense in terms of pathophysiology or response to therapy. Serum biochemical markers of bone formation and resorption could potentially be very helpful in determining these rates (11 13). They have not yet been validated in patients with CKD. In patients with osteoporosis, the markers relate to BFR determined by tetracycline in some situations but not in others, and there is considerable variability. This is where more research is needed. Mineralization After the osteoblasts lay down new collagen, they direct mineralization of the matrix. This is normally a regulated and orderly process, but in patients with CKD, the mineralization can be delayed or disorganized. This results in thickened osteoid. Rapid bone formation also can result in thick osteoid, but in that case, the tetracycline labels are also more widely separated. The osteoid maturation time is the osteoid width divided by the distance between labels per day. The mineralization lag time is the osteoid maturation time adjusted for the percentage of osteoid surface that has a tetracycline label. This adjustment, however, assumes that the osteoid surface without a label is in a resting phase, but there is no evidence to support a resting phase. Osteomalacia has been defined by various investigators as increased osteoid volume, increased osteoid maturation time, or increased mineralization lag time. Parfitt (14) defined osteomalacia in patients with malabsorption when they had a combination of osteoid volume/bone volume 10%, osteoid thickness 12.5 m (note that the thickness is a measurement that requires a correction for section obliqueness, which is the width/1.2), and mineralization lag time 100 d. There is no consensus about the exact definition of abnormal mineralization in patients with CKD. Volume The concept of bone volume is the easiest to understand. It is direct and reproducible to measure this within a sample. There is a large error, however, from one biopsy to the next in the same person. From right to left iliac crest, there is an average of 29% difference (15). Bone volume is related to bone strength, but the same volume can have different microarchitecture. The trabecular thickness can be calculated from measurements of the bone surface per volume relationship, and this is an index that relates to bone strength. Other methods of measuring the architecture include the strut length, the star volume, and the number of nodes connecting trabeculae. These two-dimensional measurements are inferior to newer, three-dimensional measurements of connectivity that are made with microcomputed tomography. There is consensus that bone volume is expressed as bone volume per tissue volume, which is the same as bone area per tissue area when expressed in two dimensions. This can be measured in cortical or in cancellous bone. Although there is agreement about the measurement, it has not been related to other categories of renal osteodystrophy. Each category of

5 Clin J Am Soc Nephrol 3: S151 S156, 2008 Bone Turnover, Mineralization, and Volume S155 renal osteodystrophy may have patients with high or low bone volume (16). Some investigators have noted that the average bone volume is lower in those with adynamic bone disease, but there is still wide overlap. With the new TMV system of classification, the bone volume will be included in the descriptions of renal osteodystrophy. It is expected that the patients with low bone volume will be at higher risk for clinical disease and fractures, but more research is required to prove the theory. Bone volume is related to the bone density measured by dual-energy x-ray absorptiometry (DEXA). The radiographic methods, however, cannot tell how dense bone material is, so a bone with high volume and low mineralization will have the same DEXA value as one with lower volume and higher mineralization density. This property is important, because DEXA cannot tell whether a bone has osteomalacia. Also, bisphosphonates do not increase bone volume, but they increase the DEXA values because the bone becomes more densely mineralized (harder, as in a piece of petrified wood). Despite these difficulties, DEXA has the advantage of measuring a larger and more representative area of bone, is noninvasive, and can be done repeatedly on the same location. In the general population, each SD decrease in DEXA predicts approximately a two-fold increase in osteoporotic fractures. Quantitative computed tomography (QCT) is another noninvasive method of measuring bone mass. QCT also cannot differentiate between osteomalacia and other kinds of bone disease or between bones with dense mineralization. It has the advantage of providing measurements in three dimensions and of separating the cortical from the cancellous bone. In the general population, QCT and DEXA give similar predictions for fractures, but neither of these techniques seems to be as predictive in patients with severe CKD (10). The emerging technique of micromagnetic resonance imaging can determine bone volume in a virtual biopsy that also can be analyzed for trabecular structure and connectivity. This noninvasive technique can image the same small area in bone over time and can document trabecular perforations that increase (in women going through menopause) or fill in (in men who are treated with androgens [17]). In the future, this technique may become the method of choice for determining bone volume. Conclusions The results of bone biopsies for patients with CKD should include description of the turnover (using BFR/TV or Acf), mineralization (using osteoid width or osteoid maturation time), and volume (bone volume/tissue volume). These are calculated using standardized methods, but there are some difficulties with each of the measurements, and further research in patients with CKD must be done to enable us to reach a consensus about cut points to define categories of these diseases and how to evaluate treatment responses. Disclosures None. References 1. Elder G: Pathophysiology and recent advances in the management of renal osteodystrophy. J Bone Miner Res 17: , Ferreira A: Development of renal bone disease. Eur J Clin Invest 36[Suppl 2]: 2 12, Hruska KA, Teitelbaum SL: Renal osteodystrophy. N Engl J Med 333: , Sherrard DJ, Hercz G, Pei Y, Greenwood C, Manuel A, Saiphoo C, Fenton SS, Segre GV: The spectrum of bone disease in end-stage renal failure: An evolving disorder. Kidney Int 43: , Cunningham J, Sprague SM, Cannata-Andia J, Coco M, Cohen-Solal M, Fitzpatrick L, Goltzmann D, Lafage-Proust MH, Leonard M, Ott S, Rodriguez M, Stehman-Breen C, Stern P, Weisinger J, Osteoporosis Work Group: Osteoporosis in chronic kidney disease. Am J Kidney Dis 43: , Freemont T, Malluche HH: Utilization of bone histomorphometry in renal osteodystrophy: Demonstration of a new approach using data from a prospective study of lanthanum carbonate. Clin Nephrol 63: , Moe S, Drueke T, Cunningham J, Goodman W, Martin K, Olgaard K, Ott S, Sprague S, Lameire N, Eknoyan G, Kidney Disease: Improving Global Outcomes (KDIGO): Definition, evaluation, and classification of renal osteodystrophy: A position statement from Kidney Disease: Improving Global Outcomes (KDIGO). Kidney Int 69: , Parfitt AM, Drezner MK, Glorieux FH, Kanis JA, Malluche H, Meunier PJ, Ott SM, Recker RR: Bone histomorphometry: Standardization of nomenclature, symbols, and units. Report of the ASBMR Histomorphometry Nomenclature Committee. J Bone Miner Res 2: , Paddock C, Youngs T, Eriksen E, Boyce R: Validation of wall thickness estimates obtained with polarized light microscopy using multiple fluorochrome labels: Correlation with erosion depth estimates obtained by lamellar counting. Bone 16: , Ott SM: Calculation of active formation period using label escape and three labels. Bone 14: , Fletcher S, Jones RG, Rayner HC, Harnden P, Hordon LD, Aaron JE, Oldroyd B, Brownjohn AM, Turney JH, Smith MA: Assessment of renal osteodystrophy in dialysis patients: Use of bone alkaline phosphatase, bone mineral density and parathyroid ultrasound in comparison with bone histology. Nephron 75: , Haas M, Leko-Mohr Z, Roschger P, Kletzmayr J, Schwarz C, Domenig C, Zsontsich T, Klaushofer K, Delling G, Oberbauer R: Osteoprotegerin and parathyroid hormone as markers of high-turnover osteodystrophy and decreased bone mineralization in hemodialysis patients. Am J Kidney Dis 39: , Martin KJ, Olgaard K, Coburn JW, Coen GM, Fukagawa M, Langman C, Malluche HH, McCarthy JT, Massry SG, Mehls O, Salusky IB, Silver JM, Smogorzewski MT, Slatopolsky EM, McCann L, Bone Turnover Work Group: Diagnosis, assessment, and treatment of bone turnover abnormalities in renal osteodystrophy. Am J Kidney Dis 43: , 2004

6 S156 Clinical Journal of the American Society of Nephrology Clin J Am Soc Nephrol 3: S151 S156, Parfitt A: Osteomalacia and related disorders. In: Metabolic Bone Disease, edited by Aviolo LV, Krane SM, Philadelphia, Saunders, 1990, pp Chavassieux PM, Arlot ME, Meunier PJ: Intersample variation in bone histomorphometry: Comparison between parameter values measured on two contiguous transiliac bone biopsies. Calcif Tissue Int 37: , Schober HC, Han ZH, Foldes AJ, Shih MS, Rao DS, Balena R, Parfitt AM: Mineralized bone loss at different sites in dialysis patients: Implications for prevention. J Am Soc Nephrol 9: , Benito M, Vasilic B, Wehrli FW, Bunker B, Wald M, Gomberg B, Wright AC, Zemel B, Cucchiara A, Snyder PJ: Effect of testosterone replacement on trabecular architecture in hypogonadal men. J Bone Miner Res 20: , Jamal SA, Hayden JA, Beyene J: Low bone mineral density and fractures in long-term hemodialysis patients: A metaanalysis. Am J Kidney Dis 49: , 2007

7 Downloaded from jcp.bmj.com on August 17, Published by group.bmj.com Review article Histomorphometry of bone J Clin Pathol 1983;36: PA REVELL From the Department of Morbid Anatomy, The London Hospital Medical College, Whitechapel, London El 2AD SUMMARY This review of the histomorphometry of bone outlines methods of biopsy and processing of specimens in the laboratory, the basic principles of morphometry, and the measurements made in order to obtain estimates of the proportional volumes and surfaces occupied by different components of bone. Variability such as that between methods, observers and laboratories is discussed and a brief outline of automatic and semiautomatic methods of image analysis also given. Histopathology is a subject normally considered in purely descriptive terms but a quantitative approach is sometimes of value. This is true of the pathology of bone. Bone morphometry has a role to play in the study of particular metabolic disturbances and their treatment. Group means and standard deviations are best used as a method of comparison under these circumstances. Morphometry may also be helpful in the evaluation of an individual biopsy. Care is needed over the reproducibility of techniques and the methods may be time consuming, but these aspects will almost certainly change with the development of relatively low cost semiautomatic computer-linked image analysis systems. Bone biopsy Several factors determine the site selected for bone biopsy, namely: (a) ease of clinical availability, (b) an area where there is active bone turnover and (c) adequate amounts of trabecular bone. The iliac crest is usually chosen.' 2 Modern techniques of biopsy involve obtaining a cortex to cortex core of bone from the iliac crest with a wide bore trephine.3 Ideally the biopsy should be obtained from a standard site.4 An alternative method is to take a vertical core downwards from the iliac crest. A comparison by Visser et a15 was unable to demonstrate a systematic difference between the two techniques with respect to the measurement of volume densities. Wedges of iliac crest obtained at necropsy should include the area normally examined in biopsy specimens. The complications of iliac crest biopsy have Accepted for publication 29 June 1983 recently been reported by Duncan et a16 who presented data for nearly biopsies, three fifths of which were transiliac, the remainder obtained by a superior approach through the iliac crest. Morbidity was low with both methods, the most common problem being haematoma in patients with primary haematological diseases or those receiving heparin during haemodialysis. Other complications included neuropathy affecting the lateral cutaneous nerve of the thigh, wound infection, pain, fracture and osteomyelitis. The overall incidence of complications in this large number of biopsies was 0-5%. Duncan and colleagues6 found that the large majority of patients experience little pain. A similar finding was obtained by Johnson et al.7 The experience of the person performing the biopsy plays a part in determining the suitability of the specimen for histological examination. When bone biopsy is performed as an occasional procedure, the specimen is often fragmented and inadequate. Laboratory processing of the biopsy The biopsy may be fixed in formalin, 70% ethanol or methanol. The latter two fixatives give better preservation of tetracycline fluorescence.8'-0 It is essential to prepare undecalcified plastic embedded sections and our own preference is for methyl methacrylate sectioned at 6-7 um on a Jung K microtome. Thicker sections (15,um) may be used for ultraviolet microscopy of tetracycline fluorescence. Changes in the amount of total bone and osteoid together with the activity of the cells at trabecular surfaces are the important features in metabolic bone disease. All the necessary details are detect- 1323

8 Downloaded from jcp.bmj.com on August 17, Published by group.bmj.com 1324 able in haematoxylin-eosin stained sections but it is preferable to use several other techniques to highlight particular features. The differentiation of osteoid from mineralised bone is easily achieved using von Kossa counterstained with van Gieson, eosin, safranin or almost any other similar stain. The Goldner trichrome method gives good contrast between mineralised and unmineralised bone, as do other trichrome methods. Solochrome cyanin is used in some centres. A close correlation has been shown between results for measurements of osteoid volume when solochrome and von Kossa methods are compared." 12 Comparability was less good between solochrome and trichrome, the latter tending to underestimate the amount of osteoid."' 13 Features at trabecular surfaces are reasonably well seen in haematoxylin-eosin and Goldner trichrome methods. Toluidine blue or thionin staining give good definition of cellular details. Osteoclasts are easily visualised by these methods, but some workers are now using cold formalin fixation, equal parts methyl/glycol methacrylate embedding medium and acid phosphatase staining for the demonstration of these cells.'4 The width of the osteoid seams depends on: (a) the osteoblastic apposition rate, that is the rate of production of osteoid by osteoblasts, (b) the rate of mineralisation of the osteoid so produced by the osteoblasts.'5 Clearly, an increase in the amount of osteoid relative to total bone tissue (hyperosteoidosis) is not necessarily due to osteomalacia and it is important to decide whether there is a calcification defect under these circumstances. Staining methods which demonstrate the mineralisation front include solochrome cyanin, Sudan black, cobalt salts and toluidine blue at ph 2.8.2'5-18 The mineralisation front appears as a granular purple line at the junction of osteoid and mineralised bone in the toluidine blue method. None of these methods is particularly reliable and the mineralisation front is best demonstrated by incorporation of a tetracycline label into the bone before biopsy. The fluorescence of tetracyclines in bone was described by Milch et al'9 and adapted as a means of labelling the mineralisation front by various workers Tetracyclines are bound at sites of active calcification, as shown by the close anatomical relationship between tetracycline and 45Ca deposition in bone.2223 Further information and references relating to use of tetracycline labelling of bone may be obtained from the literature A single label gives information about any defect in calcification, while double-labelling enables measurement of bone mineralisation rate. The time Revell required between administration of label and biopsy before reproducible results are obtained has been found to be 48 to 72 h.2y Tetracycline given for two or three days followed by an interval of three days before biopsy provides a suitable regimen for single labelling, while double labelling is obtained by the use of two three day courses of tetracycline separated by 10 days. Basic principles of the morphometry of bone It is not proposed to give a detailed account of the theory of morphometry, which is available from other sources.3032 Basically, the methods involve the application of probability theory to geometry by the use of estimates, rather than exact measurements. Repeated counting is used in order to make the estimates as accurate as possible. Although the measurements are made on two dimensional images (allowing for section thickness), the information derived may be interpreted on a three dimensional basis. Results are usually expressed in percentages though some workers use ratios. Bone quantification may be performed using one of the following: (a) inexpensive simple eye-piece graticules, (b) semiautomatic instruments in which a digitising tablet is linked to a desk-top microcomputer, (c) fully automatic computer-linked image analysis equipment. The following account will deal mainly with point counting and so-called linear intercept methods using eye-piece graticules. Point counting is performed by the superimposition of a series of points on the microscope field with an eye-piece graticule and enables the estimation of areas. The principle of point counting is extremely simple. The number of points or hits occurring on a particular feature, for example, bone trabeculae is counted and expressed as a percentage of the total number of possible hits (Fig. 1). The adjacent field is counted, and so on, until a sufficient number of measurements has been made to obtain an accurate estimate of the true amount of trabecular bone as a percentage of total tissue. Thus if n, n2, n3 etc. are the numbers of points falling on trabeculae and N1, N2,N3 etc. are the total possible numbers of points in each field, the area A, expressed as a percentage, can be calculated as: nl + n2 + n3... nx N, +N2 +N3. NXlOO where x is the number of fields necessary to obtain a reproducible result.

9 . s~~~~~~~~~~~~ Histomorphometry of bone Fig. 1 Diagram to show the principle ofpoint counting using the Zeiss Integration Plate II eye-piece graticule. Ifthe islands are considered to be bone trabeculae, then in the small central square there are 8 out of 25 points falling on bone-that is 32 % of total tissue is bone. Alternatively, 36 points fall on bone in the large square which contains 100 points-that is, 36% oftotal tissue is bone. (With permission of Carl Zeiss (Oberkochen) Ltd.) S Nominal value 9- * Test positions I, I I Total No ot points Check of measured values (test object) Fig. 2 Diagram to show the effect ofincreasing the number offields examined and points counted on the accuracy ofthe estimate obtained for a volume measurement. There is considerable variation in the results obtained when small numbers offields are measured, but the values gradually settle to a "nominal" value, shown as a horizontal line. Obviously the number offields could be increased further, in theory until a constant value were obtained. (With permission of Carl Zeiss (Oberkochen) Ltd.) Downloaded from jcp.bmj.com on August 17, Published by group.bmj.com 1325 The simplest way to overcome the problem of how much to count is to calculate the mean value after a given number of fields, count more fields and recalculate the mean value, continuing until the mean value settles to a more or less constant level. This value is described as the "nominal value" and is best understood by reference to Fig. 2. Since measurements are made on a basically two dimensional object, the value obtained is an area, or area fraction (Aa), of the whole area of tissue counted. The Delesse principle, described by a French geologist in the middle of the last century,33 states that area is an unbiased estimator of volume so that it is usual to express values as volumes even though areas have been measured. The linear intercept method is used for measurements of surface area or simple length measurements. An eye-piece graticule enables the superimposition of a series of parallel lines upon the microscope field to be examined. The method is easily understood by reference to Fig. 3, which illustrates the measurement of osteoid surface using five lines. The number of intercepts, or more accurately intersections, that osteoid and total trabecular surface make with these lines is counted. Further measurements are performed on successive adjacent fields until an accurate estimate of the percentage of surface occupied by a particular feature, in this case osteoid, is obtained. Repetition until a nominal value is achieved applies in the same way as for 7tn 4LR \~~C Fig. 3 Diagram to show the use of the linear intercept method for measuring surface lengths. The broad black borders represent areas ofosteoid. There are S places where the lines intersect with osteoid-covered surface and 20 places where lines intersect trabecular surface-that is, osteoid surface is 25 % oftrabecular surface. This diagram is a simplified representation of the superimposition of lines in the Integration Plate II, shown in Fig 1.

10 Downloaded from jcp.bmj.com on August 17, Published by group.bmj.com 1326 point counting. Measurements of osteoid, resorption, osteoblastic surfaces may be made by this method. The mineralisation front, as seen by ultraviolet light microscopy after tetracycline labelling of the biopsy, is also measured in this way. If double labelling of the biopsy with tetracycline is available, then it is possible to measure the distance between the two labels using a calibrated micrometer eye-piece. This measurement is performed at four equidistant points along each surface showing two fluorescent lines, as illustrated in Fig. 4, and the measurement repeated a sufficient number of times, say twenty The appositional rate is calculated by dividing the mean distance between the labels (d), by the time interval between administration of labels (t). In order to obtain a true appositional rate, it is necessary to apply a correction factor since the two lines of tetracycline label are actually sectioned in random planes varying between perpendicular and horizontal. The mathematical method for the derivation of this correction factor is available in papers by Frost28 and Teitelbaum and Nichols.25 Frost28 has recommended a correction factor of 0 74-that is, the true appositional rate may be calculated as 0 74 times the value obtained from actual measurement. Measurements on bone biopsies There is considerable variation in the literature with respect to the terms used for the various values obtained in bone morphometry. The volume of trabecular bone as a proportion of total tissue (bone and bone marrow) is termed the trabecular bone volume, absolute volume of trabecular bone, fractional bone volume, fractional trabecular bone volume, volume density of bone or relative trabecular volume. Surface density is strictly a measure of surface area related to volume and expressed in mm2/ cm but "surface density" and the abbreviation S, are often used more loosely. It is vital therefore to have a clear idea of what is being expressed when examining particular results. A list of parameters and how they are measured is shown in Table 1. The osteoid index provides a useful guide to the thickness of osteoid seams, and is derived mathematically from the osteoid volume and osteoid surface, using the following formula: Osteoid volume Osteoid index = x 100. Osteoid surface It has been shown to correlate well with the actual measurement of osteoid seam width."5 A rapid and fairly reliable method of assessing osteoid seams is Revell Fig. 4 Diagram to illustrate the measurement of appositional rate in bone with a double tetracycline label. The distance (d) between two fluorescent lines of incorporated tetracycline is measured with a calibrated eye-piece graticule at four equidistant points. The appositional rate is obtained with knowledge ofthe time interval (t) between administration ofthe labels-that is, appositional rate = dlt. (See text for references to correction factor required for measurement ofthe appositional rate.) the use of polarised light microscopy, when the number of birefringent lamellae (bright lines) may be counted. Up to four such lamellae are present in normal bone3"38 so that a greater number than this is an indicator of hyperosteoidosis. NORMAL VALUES Definitive normal values for the measurements made in bone morphometry are impossible to give, since they vary from laboratory to laboratory. It is essential that each centre obtains a number of normal bone samples at necropsy from the iliac crest of previously ambulant cases dying suddenly with cardiovascular disease and having no other known pathology. Measurement of these specimens enables a normal range to be obtained. Examples of typical normal values are given in Table 2. Several workers have demonstrated a gradual decrease in the trabecular bone volume of normal bone with increasing age2 39`41 The mineralisation front should normally occupy more than 80% of the osteoid surface, but this decreases slightly in older subjects.2 A mineralisation front of less than 20% represents a definite calcification defect. The mineralisation rate (appositional rate) as measured by double-labelling with tetracycline is normally about 1,um/day or slightly less. Active osteoid formation by osteoblasts usually comprises around 5 % of the total trabecular surface

11 Downloaded from jcp.bmj.com on August 17, Published by group.bmj.com Histomorphometry of bone 1327 Table 1 Trabecular bone volume Osteoid volume Osteoid surface (Active) osteoblastic surface Resorption surface Osteoclastic resorption surface Mineralisation front Osteoid index Appositional rate (see text, correction factor) Osteoclastic index Volume of trabeculae Volume of trabeculae and marrow Volume of osteoid Volume of osteoid and mineralised bone Length of surface occupied by osteoid Total length of trabecular surface Length of trabeculae occupied by active osteoblasts Total length of trabecular surface Length of trabecular surface occupied by resorption lacunae Total length of trabecular surface Length of surface occupied by osteoclasts Length of resorption lacunae Line length of mineralisation (Tetracycline fluorescence) Total length of trabecular surface Osteoid volume Osteoid surface x 100 x 100 x 100 x Distance between labels gm/day Time = Estimate of numbers of osteoclasts as either osteoclasts per high power field, osteoclasts per sq mm or sq cm of tissue or osteoclasts per mm trabecular surface x too x 100 x 100 x 100 Table 2 Normal values for iliac crest bone expressed as percentages (from Melsen et all') Age (yr) Male Female Trabecular bone volume ± ± ± ± ± ± ± ± ± ± ± ± ±2-0 > ± ±3-2 Osteoid volume ± ± ± ± ± ± ± ± ± ± ± ± t-5 > ± ±0-2 Osteoid surface ± ± ± ± t ± ± ± ± ± ± ± ± ±3-4 > ± t0-8 Resorption surface ± ± ± ± ± ± ± ± ± ± ± ± ± ±0-5 > ± ±0-6 and active resorption less than 1 % of total trabecular surface.2 VARIABILITY Important aspects of variability in bone quantification include differences between (a) sites in the same bone (b) bones in the same patient (c) observers (d) laboratories, as well as (e) variations in methods, such as staining techniques and magnifications used in microscopy. There have been numerous studies in which different sites in the iliac crest were compared. Minimal differences have been demonstrated between biopsies at adjacent sites in the same iliac crest although differences do occur when the bone is more than 2 cm posterior or inferior to the standard site of biopsy.4 Comparison of results obtained from the left and right iliac crests have been performed by several workers and no systematic differences have been demonstrated. The influence of staining methods on bone histomorphometry has already been mentioned, comparability being good between solochrome and von Kossa for measurements of osteoid volume, and less good between solochrome and trichrome methods. Melsen el al4 found higher values for osteoid volume and osteoid surface using Masson trichrome compared with toluidine blue. There are several questions which arise with respect to the use of tetracycline labelling of bone. The timing of administration of label and biopsy is

12 Downloaded from jcp.bmj.com on August 17, Published by group.bmj.com m Eu 34- E E 32- (.4 0x 30- ' 28- a 26- u 24- ) 22- sv.l I vv I I X40 x63 x100 x160 x250 Magnitication IMean I SEM t I I v I I I I x25 x400 l I I I I l *8 Resolution ( n) I ce -21 X c- -20 '< -19 < Revell Fig. 5 Surface density (Sv) and volume density (Vv) of iliac crest cancellous bone at different magnifications, showing the systematic increase in the estimate of surface density with change in magnification and no difference in volume density. (From Olah,36 with the permission ofarmour-montagu, Levallois, France.) jn (I) * Student /pothologist r = 0 85 S= SEE= n= 20 y= 086x r = 0.95 s = 0* SEE = n=16 y= 082x Student lst / 2n o Student/pothol Fig. 6 Determination ofthe surface density (Sv) oftrabecular bone. Sixteen values obtained by a student at the beginning and end of a study of 150 normal cases, 20 values ofa student compared with a experienced pathologist on the same biopsies. (From Delling et al,45 with the permission of Armour-Montagu, Levallois, France.) a I I I I I I I important and biopsy should normally be two or three days after the last label. If the interval between two labels is much longer than three weeks then the percentage of osteoid bearing two labels will be decreased. The influence of microscopic resolution on the results obtained by bone morphometry has been described.3637 Changes in magnification between 25 and 400 times did not influence estimations of volume (volume density), but over the same range of magnification there was a systematic increase in the estimate of surface density.36 (Fig. 5) Delling and his colleagues45 have shown that there is greater variability in results obtained for surface measurements compared with volume measurements. This was particularly the case where cellular details at trabecular surfaces were being evaluted, and many more fields had to be measured to obtain reproducibility under these circumstances. The relatively small amount of cellular activity at trabecular surfaces and the irregular distribution of changes are contributing factors. The same authors also performed comparisons between observers and laboratories.45 The experience of the observer was found to be important, especially with respect to those surface measurements relating to cellular activity. The volume and surface densities of trabecular bone were not affected by observer experience in the sense that comparison of a student with himself and the student with an experienced pathologist both showed good statistical correlation (see Fig. 6). The shift in the line for the pathologist/

13 Downloaded from jcp.bmj.com on August 17, Published by group.bmj.com Histomorphometry of bone student comparison suggests that the pathologist was noting features which were not recognised by the student. Differences between centres looking at the same biopsies are also of interest. When four different morphometry groups were asked to assess 10 biopsies, there was considerable variation with respect to values obtained for osteoid surface measurements though trabecular bone volume showed much smaller variation.45 Semiautomatic and automatic methods in bone quantification The examination of large numbers of specimens is time-consuming and tedious using the simple eyepiece graticule. Automated and semiautomated computer-linked systems offer an alternative approach. Both types of equipment have the advantage that they enable a much greater throughput of material. Disadvantages include the amount of time required setting up the equipment before sections may be examined, and the need to stain sections in a way which is suitable for image analysis. Automatic image analysers may be divided into three main categories, according to whether they work by source-plane-scanning, specimen-plane-scanning or image-plane-scanning.32 Image-plane-scanning is the basis on which all television-linked systems operate. The best known system of this type is the Quantimet 720 and the following account is based on personal experience of its use Ċomputer-linked image analysis using the Quantimet may be considered in three stages. Input is by means of a closed circuit television linked to a light microscope. The signals resulting from the scanning of the microscope image by the television camera are used to produce an image on a television screen and at the same time for computer analysis of particular features. The results of the computer analysis are displayed numerically at the top of the television screen. It is also possible to superimpose the computer analysis display as an image on the television screen. The machine of which the author has experience has television display, standard function computer, a control system to set light sensitivity and shade correction, variable frame and scale settings, densitometer function and interfaces with a desk top microcomputer. Area, intercept, perimeter and size can be measured and a programmer module enables the machine to run automatically through a series of measurements on each field. The Quantimet is used in our own laboratories in two different ways for the measurement of bone his tology. Osteoid volume and trabecular bone volume are measured using the densitometer function. Signals from the television camera are passed to a detection module in which grey level thresholds are set in 64 steps from black to white. It is important that the image being analysed can be resolved into clearly distinguishable grey levels. Our preference is to use von Kossa stained sections counterstained with van Giesen and treated in such a way that coloration of the bone marrow features is deliberately leached out. The image is resolved into black, grey and white areas corresponding to mineralised bone, osteoid and bone marrow. Careful selection of the grey level settings enables accurate detection at the correct boundary point for the three features which are to be detected and measured. The areas over which there are particular grey levels are measured and values for "white, grey and black" obtained. Raw data for the areas of osteoid, mineralised bone and total tissue are then routed to the desk top computer (output terminal). The process is repeated for adjacent fields and all the raw data accumulated in the output computer. Osteoid volume and trabecular bone volume are calculated automatically by a programme in this computer, in just the same way as would be performed in conventional point counting. Although it is theoretically possible to obtain perimeter measurements using the same basic method, we have preferred to use the Quantimet as a semiautomatic image analyser. It is possible to outline trabecular surface, osteoid and resorption surfaces and lengths occupied by active osteoblasts and osteoclasts with a light pen (Fig. 7). The line lengths of each outlined feature are routed to the output computer for each field examined and the measurements calibrated so that it is possible to obtain an absolute value for the lengths measured. The output computer programme accumulates data for each measurement and after a previously specified number of fields has been examined, calculates osteoid surface, active osteoblastic surface, resorption surface, osteoclastic resorption surface and total length of trabecular surface. The numbers of osteoclasts seen is accumulated in the output computer as each field is examined, so that it is possible to obtain an osteoclast index, expressed as osteoclasts/mm trabecular surface. Semiautomatic systems for quantitative analysis of histology consist of a "digitising tablet", which is an electronic drawing board, linked to a microcomputer. The microscopic field may be projected onto this drawing board or a side-arm drawing tube used. Commercially available semiquantitative equipment designed for this purpose includes the Leitz ASM, Reichert-Jung MOP and Videoplan. The digitising tablet enables the outlining of features seen in the

14 Downloaded from jcp.bmj.com on August 17, Published by group.bmj.com 1330 Revell Fig. 7 Photograph of the Quantimet 720 television screen, showing the use of a light peni (white line) to outline the trabecular surface. The line is occupying 1941 picture points. microscope field and measurements of line length, perimeter, intercept or area, are then rapidly obtained. These measurements can be expressed in absolute terms bv previous calibration. Area and surface measurements for each field may be summed and the various bone morphometry parameters calculated by use of a suitable programme in the microcomputer. Semiautomatic methods are subjective, in that they rely on the drawing of features by the observer. The use of digitising tablets for bone quantification is described elsewhere COMPARISON OF BONE QUANTITATION METHODS A good correlation has been found between the actual volume of bone, measured by water displacement, and the point counting technique.3849 A difference of 1 5% was found between the use of the Zeiss eye-piece graticule and the Quantimet method by Giroux, Courpron and Meunier." No difference in accuracy has been found between point-counting and a semiautomatic method using a digitiser tablet.47 Smaller numbers of fields needed to be measured using the semiautomatic method to achieve the same coefficients of variance with respect to both non-cellular and cellular (bone formation and resorption) parameters. References Matrajt H, Bordier P, Martin J, Hioco D. Technique pour l'inclusion des biopsies osseuses nondecalcifies. J Microscopie 1967;6: Rasmussen H, Bordier P. The physiological and cellular basis of metabolic bone disease. Baltimore: Williams and Wilkins Bordier P. Matrajt H, Miravet B, Hioco D. Mesure histologique de la masse et de la resorption des travees osseuses. Pathol Biol (Paris) 1964;12: Melsen F. Melsen B, Mosekilde L. An evaluation of the quantitative parameters applied in bone histology. Acta Pathol Microbiol Scand 1978;86:63-9. ' Visser WJ, Niermans HJ, Roelofs JMM, Raymakers JA. Duursma SA. Comparative morphometry of bone biopsies obtained by two different methods from the right and left iliac crest. In: Meunier PJ, ed. Bone histomorphometry. 2nd International Workshop. Societe de la Nouvelle Imprimerie Fournie; Toulouse, France, 1976: Duncan H, Dao SD, Parfitt AM. Complications of bone biopsy. Metab Bone Dis et Rel Res 1980;2:suppl Johnson KA, Kelly PH, Jowsey J. Percutaneous biopsy of the iliac crest. Clin Orthop Rel Res 1977;123:34-6. Frost HM. Tetracycline-based histological analysis of bone remodelling. Calcif Tissue Res 1969;3: Parfitt AM, Villanueva AR, Crouch MM, Mathews CHE. Duncan H. Classification of osteoid seams by combined use of cell morphology and tetracycline labelling. Evidence for intermittency of mineralisation. In: Meunier PJ. ed. Bone morphometry. Societ& de la Nouvelle Imprimerie Fournie; Toulouse, France, 1976: 'Melsen F, Mosekilde L. Interpretation of single labels after in vivo double labelling. Metab Bone Dis et Rel Res 1980;2:suppl "Giroux JM, Courpron P, Meunier P. Histomorphometrie de l'osteopenie physiologique senile. Monographie du laboratoire de Researches sur I'Histodynamique osseuse. Lyon, Meunier P, Edouard C. Quantification of osteoid tissue in trabecular bone. Methodology and results in normal iliac bone. In: Jaworski ZFG, ed. Proceedings of the 1st Workshop on Bone Morphometry. Ottawa: University of Ottawa Press. 1976: Meunier P, Edouard C. Courpron P, Toussaint F. Morphometric analysis of osteoid in iliac trabecular bone. Methodology.

15 Downloaded from jcp.bmj.com on August 17, Published by group.bmj.com Histomorphometry of bone Dynamical significance of the osteoid parameters in vitamin D and problems related to uremic disease. Norman AW et al, eds. Berlin: Gruyter, 1975: Evans RA, Dunstan CR, Hills EE. Extent of resorbing surfaces based on histochemical identification of osteoclasts. In: Jee WSS, Parfitt AM, eds. Bone histomorphometry. 3rd International Workshop. Metab Bone Dis Rel Res 1980;2:suppl Meunier P, Edouard C, Richard D, Laurent J. Histomorphometry of osteoid tissue. The hyperosteoidoses. In: Meunier PJ, ed. Bone histomorphometry. 2nd International Workshop. Societe de la Nouvelle Imprimerie Fournie; Toulouse, France, 1976: Irving JT. A histological stain for newly calcified tissues. Nature 1958;181: '' Irving JT. The sudanophil material at sites of calcification. Arch Oral Biol 1963;8: Matrajt J, Hioco D. Solochrome cyanine R as an indicator dye of bone morphology. Stain Technol 1972;41: Milch RA, Hall DP, Tobie JE. Fluorescence of tetracycline antibiotics in bone. J Bone Jt Surg [Am] 1958;40: Baud CA, Dupont DH. Histologie intrastructurale sur la bifluorescence du tissu, osseux tonite par les t6tracyclines. C R Seances Acad Sci 1962;254: Frost HM. Tetracycline labelling of bone and the zone of demarcation. Can J Biochem 1962;40: Harris WH, Jackson RH, Jowsey J. The in vivo distribution of tetracycline in canine bone. J Bone Jt Surg [Am] 1962;44: Urist MR, Ibsen KH. Chemical reactivity of mineralised tissue with oxytetracycline. Arch Pathol 1963;76: Frost HM, Meunier P. Histomorphometry of trabecular bone. II. An empirical test for the theoretical correction for appositional rate measurements. In: Meunier PJ, ed. Bone histomorphometry. 2nd International Workshop. Societe de la Nouvelle Imprimerie Fournie; Toulouse, France, 1976: Teitelbaum SL, Nichols SH. Tetracycline-based morphometric analysis of trabecular bone kinetics. In: Meunier PJ, ed. Bone histomorphometry. Societe de la Nouvelle Imprimerie Fournie; Toulouse, France, 1976: Flora L. Idiosyncrasies of the measurement bone dynamics with fluorescent labels. In: Meunier PJ, ed. Bone histomorphometry. 2nd International Workshop. Societe de la Nouvelle Imprimerie Fourni6; Toulouse, France, 1976: Bordier PJ, Marie P, Miravet L, Ryckewaert A, Rasmussen H. Morphological and morphometrical characteristics of the mineralisation front. A vitamin D regulated sequence of the bone remodelling. In: Meunier PJ, ed. Bone histomorphometry. Societe de la Nouvelle Imprimerie Fournie; Toulouse, France, 1976: Frost HM. Histomorphometry of trabecular bone. I. Theoretical correction of appositional rate measurements. In: Meunier PJ, ed. Bone histomorphometry. 2nd International Workshop. Societe de la Nouvelle Imprimerie Fournie; Toulouse, France, 1976: Treharne RW, Brighton C(. The use and possible misuse of tetracycline as a vital stain. Clin Orthop Rel Res 1979;140: DeHoff RT, Rhines FN. Quantitative microscopy. New York: McGraw-Hill, Williams MA. Quantitative methods in biology. Amsterdam, New York, Oxford: North Holland Publishing Company, Aherne WA, Dunnill MS. Morphometry. London: Edward Arnold, Delesse MA. Proc6dd mechanique pour determiner la composi tion des roches. Annales des Mines 1848;13: Merz WA, Schenk RK. Quantitative structural analysis of human cancellous bone. Acta Anat 1970;75: Merz WA, Schenk RK. A quantitative histological study on bone formation in human cancellous bone. Acta Anat 1970;76: Olah AJ. Influence of microscopic resolution on the estimation of structural parameters in cancellous bone. In: Meunier PJ, ed. Bone histomorphometry. 2nd International Workshop. Societe de la Nouvelle Imprimerie Fournie: Toulouse, France, 1976: Woods CG, Morgan DB, Peterson CR, Gossman HH. Measurement of osteoid in bone biopsy. J Pathol Bacteriol 1968;95: Ellis HA. Peart KM. Quantitative observations on mineralised and non-mineralised bone in the iliac crest. J Clin Pathol 1972;25: Courpron P, Meunier P, Bressot C, Giroux JM. Amount of bone in iliac crest biopsy. Significance of the trabecular bone volume. Its values in normal and in pathological conditions. In: Meunier PJ, ed. Bone histomorphometry. 2nd International Workshop. Societ6 de la Nouvelle Imprimerie Fourni6; Toulouse, France, 1976: Beck JS, Nordin BEC. Histological assessment of osteoporosis by iliac crest biopsy. J Pathol Bacteriol 1960;80: Melsen F, Melsen B, Mosekilde L, Bergmann S. Histomorphometric analysis of normal bone from the iliac crest. Acta Pathol Microbiol Scand 1978;86: Garner A, Ball J. Quantitative observations on mineralised and unmineralised bone in chronic renal azotaemia and intestinal malabsorption syndrome. J Pathol Bacteriol 1966;91: Ritz E, Krempien B, Mehls 0, Malluche J. Skeletal abnormalities in chronic renal insufficiency before and during maintenance hemodialysis. Kidney Int 1973;4: Visser WJ, Roelofs JMM. Peters JPJ, Lentferink MHF, Duursma SA. Sampling variation in bone histomorphometry. In: Jee WSS, Parfit AM, eds. Bone histomorphometry. 3rd International Workshop. Metab Bone Dis Rel Res 1980;2: suppl Delling G, Luehmann H, Baron R, Mathews CHE, Olah A. Investigation of intra and inter-reader reproducibility. In: Jee WSS, Parfitt AM, eds. Bone histomorphometry. 3rd International Workshop. Metab Bone Dis Rel Resl980;2: suppl Birkenhager-Frenkel DH, Clermonts ECGM, Richter H. Histomorphometry by means of an x-y tabloid. Design of a computer programme; Disposition of Equipment. In: Jee WSS, Parfitt AM, eds. Bone histomorphometry. 3rd International Workshop. Metab Bone Dis Rel Resl980;2: suppl Malluche HH, Sherman D, Manaka R, Massey SG. Comparison between different histomorphometric methods. In: Jee WSS, Parfitt AM, eds. Bone histomorphometry. 3rd International Workshop. Metab Bone Dis Rel Res 1980;2: suppl Manaka RC, Malluche HH. A program package for quantitative analysis of histologic structure and remodelling dynamics of bone. Comput Programs Biomed 1981;13: Schwartz MP, Reeker RR. Direct and histomorphometric determinations of surface density and volume. In: Jee WSS, Parfitt AM, eds. Bone histomorphometry. 3rd International Workshop. Metab Bone Dis Rel Res 1980;2: suppl Requests for reprints to: Dr PA Revell, Department of Morbid Anatomy, The London Hospital Medical College, Whitechapel, London El 2AD.

16 Downloaded from jcp.bmj.com on August 17, Published by group.bmj.com Histomorphometry of bone. P A Revell J Clin Pathol : doi: /jcp Updated information and services can be found at: alerting service These include: Receive free alerts when new articles cite this article. Sign up in the box at the top right corner of the online article. Notes To request permissions go to: To order reprints go to: To subscribe to BMJ go to:

17

18

19

20

21

22

23

24

25

26

27

28

29

30

31

32

33 REVIEW JBMR Standardized Nomenclature, Symbols, and Units for Bone Histomorphometry: A 2012 Update of the Report of the ASBMR Histomorphometry Nomenclature Committee David W Dempster, 1,2 Juliet E Compston, 3 Marc K Drezner, 4 Francis H Glorieux, 5 John A Kanis, 6 Hartmut Malluche, 7 Pierre J Meunier, 8 Susan M Ott, 9 Robert R Recker, 10 and A Michael Parfitt 11 1 Department of Pathology, College of Physicians and Surgeons of Columbia University, New York, NY, USA 2 Regional Bone Center, Helen Hayes Hospital, West Haverstraw, NY, USA 3 Department of Medicine, Cambridge University Hospitals NHS Foundation Trust, Cambridge, United Kingdom 4 University of Wisconsin School of Medicine and Public Health, Madison, WI, USA 5 Genetics Unit, Shriners Hospital for Crippled Children and McGill University, Montreal, Canada 6 Centre for Metabolic Bone Diseases, University of Sheffield Medical School, Sheffield, United Kingdom 7 Department of Medicine, Division of Nephrology, Bone and Mineral Metabolism, University of Kentucky College of Medicine, Lexington, KY, USA 8 INSERM Unit 1033, Faculte Alexis Carrel, Lyon, France 9 Department of Medicine, Harborview Medical Center, University of Washington, Seattle, WA, USA 10 Osteoporosis Research Center, Creighton University School of Medicine, Omaha, NE, USA 11 University of Arkansas for Medical Sciences, Little Rock, AR, USA Introduction Before publication of the original version of this report in 1987, practitioners of bone histomorphometry communicated with each other in a variety of arcane languages, which in general were unintelligible to those outside the field. The need for standardization of nomenclature had been recognized for many years, (1) during which there had been much talk but no action. To satisfy this need, B Lawrence Riggs (ASBMR President, 1985 to 1986) asked A Michael Parfitt to convene an ASBMR committee to develop a new and unified system of terminology, suitable for adoption by the Journal of Bone and Mineral Research (JBMR) as part of its Instructions to Authors. The resulting recommendations were published in 1987 (2) and were quickly adopted not only by JBMR but also by all respected journals in the bone field. The recommendations improved markedly the ability of histomorphometrists to communicate with each other and with nonhistomorphometrists, leading to a broader understanding and appreciation of histomorphometric data. In 2012, 25 years after the development of the standardized nomenclature system, Thomas L Clemens (Editor in Chief of JBMR) felt that it was time to revise and update the recommendations. The original committee was reconvened by David W Dempster, who appointed one new member, Juliet E Compston. The original document was circulated to the committee members and was extensively revised according to their current recommendations. The key revisions include omission of terminology used before 1987, recommendations regarding the parameters and technical information that should be included in all histomorphometry articles, recommendations on how to handle dynamic parameters of bone formation in settings of low bone turnover, and updating of references. Preliminary Definitions It is generally agreed that a bone is an individual organ of the skeletal system, but the term bone has at least three meanings. The first is mineralized bone matrix excluding osteoid; this usage conforms rigorously to the definition of bone as a hard tissue. Osteoid is bone matrix that will be (but is not yet) mineralized, and is sometimes referred to as pre-bone. The second meaning of bone, and the one we have adopted, is bone matrix, whether mineralized or not, ie, including both mineralized bone and osteoid. The third meaning of bone is a tissue including bone marrow and other soft tissue, as well as bone as just defined. We Received in original form August 15, 2012; revised form September 26, 2012; accepted October 8, 2012; accepted manuscript online xxxx xx, Address correspondence to: David W Dempster, PhD, Regional Bone Center, Helen Hayes Hospital, Route 9W, West Haverstraw, NY 10993, USA. ddempster9@aol.com Journal of Bone and Mineral Research, Vol. 28, No. 1, January 2013, pp 1 16 DOI: /jbmr.1805 ß 2013 American Society for Bone and Mineral Research 1

34 refer to the combination of bone and associated soft tissue or marrow as bone tissue. Tissue is defined (3) as an aggregation of similarly specialized cells united in the performance of a particular function. In this sense, bone, bone marrow, and the contents of osteonal canals are certainly not the same tissue, but in a more general sense, most textbooks of histology recognize only four fundamental tissues epithelium, nerve, muscle, and connective tissue (4) of which the last-named includes bone and all its accompanying nonmineralized tissue. In current clinical and radiologic parlance, trabecular and cortical refer to contrasting structural types of bone. But trabecular does not appear in any standard textbook of anatomy or histology as a name for a type of bone; rather, spongy or cancellous is used. Spongiosa (primary or secondary) is best restricted to the stages of endochondral ossification; cancellous is most commonly used in textbooks (4,5) and is the term we have chosen. We retain the noun trabecula and its associated adjective trabecular to refer to an individual structural element of cancellous bone, in accordance with current practice in histology, (4) pathology, (6) and biomechanics. (7) Etymologically, a trabecula is a beam or rod, and in young people plates rather than rods are the predominant structural elements, both in the spine (8) and in the ilium, (9) but no convenient alternative is available. The size, shape, and orientation of trabeculae (as just defined) vary considerably between different types of cancellous bone. (9,10) Density is a frequent source of confusion in discussions about bone. We propose that the term should be restricted as far as possible to its primary meaning in physics of mass per unit volume, (11,12) with a subsidiary meaning analogous to population density, which is applied mainly to cells. This precludes the use of density in its stereologic sense, as will be discussed later. Corresponding to the definitions given earlier, the volume to which mass is referred can be of mineralized bone, bone, bone tissue (cortical or cancellous), or a whole bone. Mineralized bone density is slightly less than true bone density, which excludes the volume of osteocyte lacunae and canaliculi. (11) This volume is small and generally ignored; lacunar volume can be readily measured, (13) but canalicular volume is inaccessible to light microscopy. Bone density reflects the volumetric proportion of osteoid; bone matrix volume, excluding lacunar and canalicular volume, has been referred to as absolute bone volume. (14) Bone tissue density reflects the volumetric proportion of soft tissue, or porosity. Whole bone density, often referred to as apparent bone density, reflects the volumetric proportions of cortical bone tissue, cancellous bone tissue, and diaphyseal marrow within a bone, the organ volume of which is usually measured by Archimedes principle. (15) Osteoblast is defined differently in the clinical and experimental literature. In young, rapidly growing small animals, most bone surfaces are undergoing either resorption or formation and virtually all cells on the surface are either osteoclasts or osteoblasts, (16) but in the adult human, most bone surfaces are quiescent with respect to bone remodeling. We refer to the flat cells that cover quiescent internal (nonperiosteal) bone surfaces as lining cells and restrict the term osteoblast to cells that are making bone matrix currently or with only temporary interruption, rather than including all surface cells that are not osteoclasts. (16) Lining cells are of osteoblast lineage and are thought to have osteogenic potential. (17) The term osteoclast is restricted to bone-resorbing cells containing lysosomes and tartrate-resistant acid phosphatase; they are usually multinucleated, although some osteoclast profiles may have only one or no nucleus. Criteria for identification of osteoblasts and osteoclasts, whether morphologic or histochemical, (18,19) should always be stated or referenced. Dimensional Extrapolation and Stereology A two-dimensional histological section displays profiles of threedimensional structures. Four types of primary measurement can be made on these profiles area, length (usually of a perimeter or boundary), distance between points or between lines, and number. (20) Some histomorphometrists report all results only in these two-dimensional terms because the assumptions needed for extrapolation to three dimensions may be difficult to justify and because the diagnostic significance of the measurements or the statistical significance of an experimental result are not affected. For these limited objectives, this is a reasonable view, but bone cannot be fully understood unless conceived in threedimensional terms. In every other branch of science that uses microscopy as an investigative tool, the ultimate goal is to understand three-dimensional reality by the application of stereology, which is the relevant mathematical discipline. (20 22) We believe that this also should be the goal of bone histomorphometry. Accurate three-dimensional data are necessary for proper comparison between species, between bones, and between different types of bone, for input into finite element models of bone strength, for realistic estimation of radiation burdens, and for many aspects of bone physiology, such as the calculation of diffusion distances and the measurement of individual cell work. But as a practical matter, it is unrealistic to insist on universal adoption of a three-dimensional format. All stereologic theorems require that sampling be random and unbiased, a condition only rarely fulfilled in bone histomorphometry; the closest feasible approach is to rotate the cylindrical bone sample randomly around its longitudinal axis before embedding. (20,23) In the past, the use of a hemispherical grid (20 22) in the ocular lens was a convenient way of ensuring randomness of test line orientation, but even this cannot compensate for sampling bias introduced at an earlier stage. With the exception of the conversion of area fractions to volume fractions, most stereologic theorems also require that the structure be isotropic, meaning that a perpendicular to any element of surface has an equal likelihood of pointing in any direction in space. (20,24) Although not true for all cancellous bone, in the ilium there is only moderate deviation from isotropy, and stereologic theorems may be used with acceptable error. (24,25) But it is more accurate to apply the theory of vertical sections; a cycloid test grid is required, which is incompatible with the use of a digitizer, (23,26) but there is no other way of obtaining truly unbiased estimates. Because Haversian canals generally do not deviate from the long axis by more than 108, stereologic problems in diaphyseal cortical bone 2 DEMPSTER ET AL. Journal of Bone and Mineral Research

35 are minimal, but investigation of the correct stereologic approach to iliac cortical bone has not been done. Accordingly, we recommend that everyone reporting histomorphometric data should select one of two options: either present all results strictly and consistently in two dimensions, using the terms perimeter (for length), area, and width (for distance), or (as favored by the committee) present only the corresponding three-dimensional results using the terms surface, volume, and thickness; with the latter option, an explanation is needed for each type of measurement of exactly how it was derived from the primary two-dimensional measurement, as described later. A mixture of two- and threedimensional terms should not be used in the same article. The only exception is number, the fourth type of primary measurement, for which there is no convenient way of extrapolating to three dimensions without making assumptions concerning the three-dimensional shape of the objects counted. (21,22) Direct enumeration of number in three dimensions is possible if the same object can be identified in serial sections of known thickness and separation, (27) but this method has not yet been applied to bone. Topological properties such as connectivity also cannot be determined from two-dimensional sections. (28) The original committee chose not to adopt the terminology of the International Society of Stereology, as was suggested at the First International Workshop on Bone Morphometry. (29) Stereologists use the term density in a very general sense to identify any measurement referred to some defined containing volume, (21,22) so that fractional volume is volume density (V v ) and surface area per unit volume is surface density (S v ). Although the unification of scientific terminology is desirable in the long term, the practical disadvantage of using density in two different senses outweighs the theoretical advantage. Nevertheless, all investigators wishing to remain at the cutting edge of bone histomorphometry will need to be thoroughly familiar with the terminologic conventions of stereology because many important methodologic articles applicable to bone are published in the Journal of Microscopy, which is the official journal of the International Society of Stereology. (26 28) The Importance of Referents Primary two-dimensional measurements of perimeter, area, and number are indices of the amount of tissue examined and can be compared between subjects only when related to a common referent, which will be some clearly defined area or perimeter within the section. Absolute perimeter length and absolute area in two dimensions have no corresponding absolute surface area and absolute volume in three dimensions, but it is convenient to refer to perimeters as surfaces and to areas as volumes if the appropriate referent is clear from the context. Primary twodimensional measurements of width (and corresponding threedimensional thicknesses) and mean profile areas of individual structures have meaning in isolation and are the only type that do not require a referent. Different referents serve different purposes and lead to different interpretations, so that use of multiple referents is unavoidable, and it is important to clearly distinguish between them. (30) Commonly used referents include tissue volume (TV), bone volume (BV), bone surface (BS), and osteoid surface (OS) and their corresponding two-dimensional areas or perimeters. With explicit identification of the referent, the use of relative as a qualifying term becomes redundant. The volume of the cylindrical biopsy core is not commonly used as a referent at present but is needed for comparison with physical methods of measuring bone density, (31) for comparing the absolute amounts of cortical and cancellous bone lost because of aging or disease, (31) for determining the contributions of different types of bone and different surfaces to various histological indices, such as amount of osteoid and surface extent of osteoblasts, (32) and for examining in detail the relationships between histological and biochemical indices of whole-body bone remodeling. (32) Use of the core volume (CV) as a referent provides the closest approach possible from an iliac biopsy to the in vivo level of organization corresponding to bone as an organ. An intact, full-thickness transiliac biopsy can be regarded as representative of the entire bone (18,33) because the length of the cylindrical biopsy core perpendicular to the external surface depends mainly on the width of the iliac bone at the site of sampling. Cortical thickness can be measured with a vertical biopsy through the iliac crest, (5) but the proportions of cortical and cancellous tissue in the bone cannot be measured. However, with either type of biopsy, the results can be weighted by the proportions of cortical and cancellous bone tissue in the entire skeleton. (34) The same principle can be applied to rib biopsies and to long bone cross sections by using the whole area enclosed by the periosteum as the referent. Lexicon of Bone Histomorphometry The recommended individual terms are listed in Table 1 in alphabetical order of their abbreviations or symbols. Several general comments are in order. First, like a dictionary, the lexicon is intended to be consulted, rather than memorized. Second, the use of abbreviations is always discretionary, never compulsory. Although designed mainly to save time or space, there is a more subtle reason for abbreviations, as for other symbols. Words frequently carry unwanted implications from their use in other contexts, but confusion is less likely with symbols that can be approached with fewer preconceptions. (1) Nevertheless, our purpose is not to encourage or discourage the use of abbreviations and symbols but to ensure that the same ones are used by everybody. To this end, we have made the lexicon comprehensive to anticipate future needs and forestall the introduction of new abbreviations with different meanings. We have included metals frequently identified in bone (with their usual elemental abbreviations) and terms commonly used in quantitative microscopy and stereology, as well as terms for all the major structural features of bone and of bones and for some important concepts of bone physiology. Terms with unfamiliar meanings are explained and defined in relation to their use. With one exception, the abbreviations and symbols in Table 1 consist of only two letters; BMU (basic multicellular unit) is retained because it is important and widely used and lacks a suitable alternative. The most commonly used descriptive terms are given a single capital letter. Other terms have an additional Journal of Bone and Mineral Research BONE HISTOMORPHOMETRY STANDARDIZED NOMENCLATURE 3

36 Table 1. Abbreviations and Symbols of Terms Used in Bone Histomorphometry A Apposition(al) G Grow(th)(ing) Ot Osteocyt(e)(ic) Ad Adipocyte a H Haversian P Period Ab Absolute Hm Hematopoietic Pf Profile Ac Activation Hp Hypertrophic Pl Plate Aj Adjusted Ht Height Pm Perimeter (2D) b Al Aluminum Hz Horizontal Po Por(e)(ous)(osity) Ar Area (2D) b H Hit Ps Periost(eal)(eum) a Activ(e)(ity) I Interface f (3D) b Pt Point B Bone Ia Intra Q Quiescent BMU Basic multicellular unit Ic Intercept R Rate Bd Boundary (2D) b Il Initial Rd Radi(al)(us) C Core In Internal Rf Referen(ce)(t) Ca Canal(icula)(r) Ir Inter Rm Remodeling Cd Corrected Is Instantaneous Rs Resorption e Ce Cell It Interstitial Rv Reversal Cg Cartilage I Intersection S Surface (3D) b Cm Cement L Label(led) Sa Sample Cn Cancellous Lc Lacuna(r) g Se Section Cp Cytoplasm(ic) Le Length Sg Sigma Ct Cort(ex)(ical) Li Lining Sm Seam Cy Cycle Lm Lamella(r) Sn Spongiosa D Dimension(al) Ln Line Sp Separation De Depth Lo Longitudinal St Structur(e)(al) Dg Degenera(tive)(tion) L Lag S Single Dm Diameter M Mineral(iz)(ing)(ation) T Tissue Dn Density Ma Marrow Tb Trabecula(r) i Do Domain Md Mineralized Th Thickness (3D) b Dp Diaphys(is)(eal) Me Medullary Tm Termin(al)(us) Dt Delta Ml Modeling Tr Transitional d Double c Mo Mononucle(ar)(ated) Tt Total E Ero(ded)(sion) Mp Metaphys(is)(eal) T Time Ec Endocortical Mu Multinucle(ar)(ated) U Unit En Envelope Mx Matrix V Volume (3D) b Ep Epiphys(is)(eal) M Maturation Vd Void Es Endost(eal) d (eum) N Number of profiles or structures Vk Volkmann Ex External N Number of sampling units h Vt Vertical F Formation e Nc Nucle(us)(ar) W Wall Fa Fat(ty) Nd Node Wi Width (2D) b Fb Fibro(sis)(us) O Osteoid Wo Woven Fe Iron Ob Osteoblast(ic) y Year Fr Front Oc Osteoclast(ic) Z Zone f Frequency On Osteon(al) Note: For further definitions and explanations, see text. a Included here and in Table 3 because of the rekindled interest in assessing adipocyte parameters in the marrow space of iliac crest bone biopsies and the shared progenitor cell with osteoblasts. b 2D or 3D refers to the format in which data are reported, not the dimensions of an individual quantity. c Also day, but context should eliminate ambiguity. d Endocortical þ cancellous. e As a process, not as a morphologic feature. f Between osteoid and mineralized bone. g If unqualified, osteocytic, not Howship s. h For example, subjects, sites, sections, etc. i An individual structure, not a type of tissue. 4 DEMPSTER ET AL. Journal of Bone and Mineral Research

37 lowercase letter, chosen in many cases to emphasize the second or later syllable and usually avoiding the second letter of the word abbreviated by the single capital letter. Single lowercase letters are used for terms that are in some sense related to time, for the primary data of classical grid counting (hit and intersection), and for n in its usual statistical sense. When used in combination, double-letter abbreviations should be demarcated by a period; in the absence of periods, each letter is to be construed as an individual abbreviation. In this way, any combination of abbreviations can be unambiguously deciphered without having to determine which terms are included in the lexicon. The Nomenclature System Bone histomorphometry can be applied to many types of material, but the most common are sections of cylindrical biopsy samples of iliac bone obtained from human subjects and sections of long bones obtained from experimental animals. For orientation, we first present the terminology for describing these sections. Description of section Core (C) refers to the entire biopsy specimen (Fig. 1). For transiliac biopsies, the distance between external (Ex) and internal (In) periosteum is termed width (Wi) because it is related to the thickness of the iliac bone at the biopsy site; for vertical biopsies through the iliac crest, the term length (Le) is more appropriate. Core width is subdivided into cortical (Ct) widths and cancellous (Cn) width; for transiliac biopsies, measurements on the two cortices (including their width) are usually pooled, but it is possible to keep track of their identity and examine them separately. In this case, the two cortices are generally distinguished by their width (thick versus thin). Identification of the inner and outer cortex would require that one be marked in some way (eg, by ink or cotton thread) at the time of the biopsy, but this is seldom done. The outer cortex generally has more attached fibrous and muscle tissue than the inner cortex. The other dimension of the core is referred to as diameter (Dm), although only sections through the central axis of the cylinder have the same diameter as the trephine; the more accurate term chord length is too cumbersome. If the axis of the transiliac core is oblique to the plane of the ilium, its dimensions are apparently changed (Fig. 2). It is convenient to define core diameter as mean periosteal length (external and internal) regardless of obliquity because true values for cortical and cancellous width corrected for obliquity are then given by the relationships between length and area set out in the legend to Fig. 2. (31,35) For long bone cross sections (Fig. 3), bone diameter (B.Dm) is similarly subdivided into two cortical widths and either cancellous diameter (Cn.Dm) for metaphyseal (Mp) cross sections, or marrow diameter (Ma.Dm) for diaphyseal (Dp) cross sections. The relationships between these diameters and bone area, cortical area, and cancellous or marrow area depends on the precise geometry of the cross section. For biomechanical purposes, such measurements may be needed at multiple locations in relation to the in vivo orientation. For both iliac and long bone sections, it is necessary for certain purposes to recognize a transitional zone (Tr.Z) lying between cortical and cancellous bone tissue and intermediate in geometrical and topological features. (36) This zone is not indicated in Figs. 2 or 3 because methods of defining its boundaries are not yet fully developed. A threshold-based algorithm has recently been used to address this problem in high-resolution peripheral quantitative computed tomography (pqct) images. (37,38) This may be applicable to iliac crest bone biopsy samples, but this has not yet been tested. For all bones, all interior surfaces in contact with bone marrow are referred to as endosteal (Es) and are subdivided into cancellous bone surface and endocortical (Ec) surface; the latter is the inner boundary of the cortex. Demarcation between these components is subject to large observer error (39) unless made in accordance with some well-defined rule (40) and will also Fig. 1. Sections of representative bone biopsies from different sites. Upper: transiliac (outer cortex on left). Lower: vertical (iliac crest on left). Supplied by H Malluche; transiliac biopsy reproduced from Malluche and Faugere (5) with permission. Journal of Bone and Mineral Research BONE HISTOMORPHOMETRY STANDARDIZED NOMENCLATURE 5

38 Fig. 2. Diagram of sections through cylindrical biopsy core of ilium. Direction of trephine perpendicular on left, oblique on right. C.Wi ¼ core width; C.Dm ¼ core diameter; Ct.Wi ¼ cortical width; Cn.Wi ¼ cancellous width. Relationships to areas: C.Ar ¼ core (or section) area ¼ C.Dm C.Wi; Ct.Ar ¼ cortical area ¼ C.Dm Ct.Wi; Cn.Ar ¼ cancellous area ¼ C.Dm Cn.Wi. Provided the inner and outer periosteum do not depart seriously from parallelism and their mean length is used for C.Dm, these relationships remain true for the oblique section because the areas enclosed by the interrupted and solid lines are equal. (35) Consequently, the relationships can be used to estimate C.Wi, Ct.Wi, and Cn.Wi without measuring the angle of obliquity. depend on whether the transitional zone is measured separately. Interior surfaces not in contact with bone marrow are generally referred to as cortical (Ct), with optional qualification as intra (In); the cortical surface can also be referred to as the Haversian canal (H.Ca) or osteonal canal (On.Ca) surface. Standard format The following standard and universally applicable method for reporting all data should be used: Source Measurement/ Referent. Note that the complete elimination of ambiguity applies to punctuation as well as to terminology; the dash ( ) and slash (/) are used only as illustrated and periods are used only as described earlier. Source refers to the structure on which the measurement was made, whether this was a particular surface or a particular type of tissue. Most of the commonly used sources have already been defined (Table 2); many others are definable by using the lexicon (Table 1). If measurements are restricted to some subdivision of a source, such as the outer portion of a cortex (41) or the central zone of cancellous tissue, (33) the same symbol can be used, but the appropriate qualification should be made in the description of methods. For measurements made on the entire section, the source is identified as total (Tt). Usually it will not be necessary to specify the source each time a particular quantity is referred to if only one source is used in an article, it need only be mentioned once. If several sources are included, their names can be used as subheadings for presentation of results in tables or text, and in most cases will need to be repeated only if measurements from several sources are discussed together, such that confusion between them is possible. For some measurements, such as trabecular thickness, only one source is possible and its specification is redundant. The need for referents was described earlier. The most commonly used referents have already been defined and are listed in Table 2, but the relationships between them need further explanation, as follows (OV is used here as an example of Fig. 3. Diagram of cross sections through the shaft of a long bone; metaphyseal region is on the left, and diaphyseal region is on the right. For clarity, the cancellous bone of the metaphysis is not shown. B.Dm ¼ bone diameter; Ct.Wi ¼ cortical width; Cn.Dm ¼ cancellous diameter; Ma.Dm ¼ marrow diameter. 6 DEMPSTER ET AL. Journal of Bone and Mineral Research

39 Table 2. Sources and Referents in Bone Histomorphometry Sources the variable in the numerator; the asterisk is the most typographically convenient symbol for multiplication): OV/BS BS/BV ¼ OV/BV OV/BS BS/TV ¼ OV/TV ¼ OV/BV BV/TV OV/BS BS/CV ¼ OV/CV ¼ OV/BV BV/CV Referents Name Abb. Name Abb. Total core Tt Bone surface BS Cortical bone tissue Ct Bone volume BV Cancellous bone tissue Cn Tissue volume TV Endocortical surface Ec Core volume CV Periosteal surface Ps Osteoid surface OS Transitional zone Tr.Z Bone interface BI Diaphyseal bone Dp Eroded surface ES Metaphyseal bone Mp Mineralized surface Md.S Epiphyseal bone Ep Osteoblast surface Ob.S Medullary bone Me Osteoclast surface Oc.S Abb. ¼ abbreviation. Those listed will cover most situations in both human and nonhuman studies, but neither list is exhaustive. Combinations of source terms may be needed, such as Dp.Ec for diaphyseal bone, endocortical surface. The three surface/volume ratios and the two volume/volume ratios are the key quantities needed to convert from one referent to another. (30) BS/BV is equivalent to S/V in stereologic terminology, and BS/TV and BS/CV are equivalent to S v (surface density) in stereologic terminology. These ratios are derived from the corresponding two-dimensional perimeter/area ratios B.Pm/B.Ar, B.Pm/T.Ar, and B.Pm/C.Ar by multiplying either by 4/p (1.273), which is correct for isotropic structures, (20 22) or by 1.2, which has been experimentally determined for human iliac cancellous bone. (25) The ratios increase with microscopic resolution, so that the magnification must always be stated and preferably standardized. (42) BV/TV and BV/CV correspond to V v (volume density) in stereologic terminology and are numerically identical with the corresponding area/area ratios B.Ar/T.Ar and B.Ar/C.Ar. (20 22) For some purposes, a subdivision of the bone surface is needed as a referent (Table 2). Osteoblast surface (Ob.S) and mineralizing surface (MS) are often related to osteoid surface (/OS). Osteoclasts usually avoid osteoid, and it can be useful to relate osteoclasts to the mineralized surface (/Md.S), previously called nonosteoid surface, (43) as an alternative to the more usual referents bone surface and eroded surface (/ES). Various kinetic indices of bone formation can be related to the osteoblast surface (/Ob.S) or to the number of osteoblast profiles (/N.Ob), as well as to osteoid surface or bone surface. (30) Finally, it may be appropriate to use the interface between mineralized bone and osteoid, or bone interface, as a referent (/Bl) for the length of tetracycline label or of positive aluminum staining because the interface is where these features are located. In many cases, as when only one referent is used for each measurement, the referent need only be specified once and not repeated each time the measurement is mentioned. If more than one referent is used, measurements with the same referent can be grouped together to avoid repetition. Primary measurements These are listed together with abbreviations in both 3D and 2D form in Table 3. Many have already been defined but some need additional explanation. Area measurements Mineralized volume is used for simplicity instead of mineralized bone volume and is given by (bone volume osteoid volume). Osteoid may need to be qualified as lamellar, OV(Lm), or as woven, OV(Wo). Note the distinction in the lexicon between M, which refers to a process, and Md, which refers to a state: for convenience, all tetracycline-based measurements are considered with the kinetic indices discussed earlier. Void is a general term applicable to all tissue that is not bone (44) and includes marrow in cancellous bone and Haversian and Volkmann canals in cortical bone. For both types of tissue, porosity (Po) ¼ void volume/tissue volume. Problems can arise with area measurements on individual profiles, such as cells or cortical canals. The profiles can be treated as an aggregate of tissue, indicated by use of the appropriate referent. For example, Ce.V/TV is the total area of all cell profiles referred to the total area of tissue and expressed in 3D terms. The profiles can also be treated as individual structures, indicated by absence of a referent; eg, Ca.Ar is the mean area of individual canal profiles. If confusion is still possible, the term could be qualified as total (Tt) or mean (x). Mean areas in 2D cannot be extrapolated to mean volumes in 3D unless the structures are counted in 3D. (27) Assuming cylindrical geometry, mean canal area can be used to estimate canal radius (Ca.Rd), but it is preferable to measure this directly, as described later. Perimeter measurements Osteoid seams do not end abruptly so that some minimum width should be specified for measurement of osteoid surface (OS). We avoid the terms formation (or forming) surface and resorption (or resorbing) surface because the implications of current activity may be erroneous, and for the same reason we avoid the qualification active. Eroded surface (ES) is synonymous with crenated or lacunar surface and comprises the osteoclast surface (Oc.S) and the reversal surface (Rv.S); individual erosions can also be classified as osteoclast positive, ES(Ocþ), or osteoclast negative, ES(Oc ). Some mononuclear cells probably resorb bone, (45) and better methods are needed for identifying and classifying the nonosteoclast cells on the eroded surface or reversal cells. Quiescent surface (QS) is synonymous with resting or inactive surface; the term implies that remodeling activity will return at some future time. The thin layer of unmineralized connective tissue lying beneath the flat lining cells on quiescent surfaces should not be referred to as osteoid. (46) It is possible that some eroded surface covered by flat lining cells should be counted as quiescent surface rather than as reversal surface. Journal of Bone and Mineral Research BONE HISTOMORPHOMETRY STANDARDIZED NOMENCLATURE 7

40 Table 3. Primary Measurements in Bone Histomorphometry Type of measurement Name of measurement Abbreviations Area Bone volume a BV B.Ar Osteoid volume OV O.Ar Mineralized volume Md.V Md.Ar Void volume Vd.V Vd.Ar Marrow volume Ma.V Ma.Ar Fibrosis volume Fb.V Fb.Ar Canal volume b Ca.V Ca.Ar Cell volume b,c Ce.V Ce.Ar Cytoplasmic volume b,d Cy.V Cy.Ar Nuclear volume b,d Nc.V Nc.Ar Length Bone interface e BI B.Bd Bone surface f BS B.Pm Osteoid surface OS O.Pm Eroded surface ES E.Pm Quiescent surface g QS Q.Pm Mineralized surface h Md.S Md.Pm Osteoblast surface Ob.S Ob.Pm Single-labeled surface i sls sl.pm Double-labeled surface i dls dl.pm Osteoclast surface Oc.S Oc.Pm Reversal surface j Rv.S Rv.Pm Distance k Cortical thickness l Ct.Th Ct.Wi Wall thickness W.Th W.Wi Mineralized thickness Md.Th Md.Wi Osteoid thickness O.Th O.Wi Label thickness L.Th L.Wi Trabecular thickness m Tb.Th Tb.Wi Interstitial thickness It.Th It.Wi Trabecular diameter n Tb.Dm o 3D 2D Canal radius Ca.Rd o Cell height c Ce.Ht o Nuclear height d Nc.Ht o Erosion depth E.De o Number p Osteoblast number N.Ob Osteoclast number N.Oc Osteocyte number N.Ot Adipocyte number N.Ad Nuclear number d N.Nc Canal number N.Ca Seam number N.Sm Erosion number NE Profile number N.Pf a Area in 2D. b Potential confusion between tissue aggregates and individual structures; see text. c Specify cell type if needed, eg, Oc.V or Oc.Ar. d Qualify by cell type if needed, eg, Oc.Nc.V. e Boundary in 2D. f Perimeter in 2D. g BS (OS þ ES). h ES þ QS. i Alternative terms are single- (or double-) labeled interface (sli, dli). j ES Oc.S. k Between points or lines. Table 3. (Continued ) l Width in 2D; for the cortex, width and thickness are numerically equal, but for other measurements, thickness ¼ width divided by 4/p or by 1.2. m Assumes that trabeculae are thin plates;(54) ¼ 2/(BS/BV). n Assumes that trabeculae are cylindrical rods;(58) ¼ 4/(BS/BV). o No unique corresponding term in 2D. p No 3D equivalent by standard methods; with appropriate referent could be referred to as density. For further details, see text. Distance measurements In principle, all distance measurements can be obtained in two ways either by direct measurement at multiple locations or by indirect calculation from measurements of area and perimeter. The direct method is more precise and can provide a frequency distribution and a standard deviation as well as a mean value but requires that measurement sites be randomly selected. (47) The indirect method is less laborious and less subject to sampling bias. The direct method is usually used for wall thickness, distance between labels, and cell and nuclear dimensions, and the indirect method is usually used for trabecular thickness (plate model), diameter (rod model), and separation. Both methods are widely used for osteoid thickness and cortical thickness. The direct method is essential for reconstructing the remodeling sequence from the relationships between individual measurement values at particular locations and instantaneous values at particular times during the remodeling cycle. (45,48) The mean value determined by either method in an individual must be distinguished from the mean value in a group of subjects. Mineralized thickness is the distance from the cement line to the interface between bone and osteoid. (48) It is used in remodeling sequence reconstruction (45) and in characterizing different types of abnormal osteoid seam, and defining different stages of severity in osteomalacia; (49) the mean value should be close to the difference between wall thickness and osteoid thickness. Label thickness is measured on an individual label; it has been used in the rat for calculation of the rate of initial mineral accumulation (50) and in human subjects as an index of treatment response in renal osteodystrophy. (51) Interstitial thickness (It.Th) is the mean distance between cement lines on opposite sides of a trabecula, usually calculated as Tb.Th- 2 W.Th for the plate model. (52) Canal radius is an index of bone loss from the cortical surface, but too little is known of the internal geometry of iliac cortical bone to decide the most stereologically correct method of measurement. On the reasonable but unproven assumption that elliptical profiles are the result of oblique sections through cylindrical canals, direct measurements can be restricted to the short axes of the ellipses. (53) Number measurements Most of these are self-explanatory, but restriction to 2D and invariable need for a referent must be reemphasized. In most cases, the referent will be an area or perimeter, but number of nuclei can also be expressed per cell; eg, N.Nc/Oc is the mean number of nuclear profiles per osteoclast profile. Profile number without qualification refers to isolated bone profiles in cancellous bone tissue, a quantity that increases with age as 8 DEMPSTER ET AL. Journal of Bone and Mineral Research

41 connectivity declines and then decreases as some remaining structures are completely removed. Nodes are branch points and termini are endpoints in a trabecular network that has been skeletonized to facilitate examination of its topological properties. (55) The ratio of nodes to termini (Nd/Tm) in a section is an index of spatial connectivity. (56) Derived indices These can be either structural or kinetic (Table 4). Many of the calculations are based on assumptions that are reasonable but not rigorously established, and individual investigators may decide to use all, some, or none of the indices that we have selected. Structural indices Trabecular number (or density) is usually calculated with dimensions Length 1 (in specifying dimensions, length and time are usually abbreviated L and T, but these have other meanings in the lexicon) according to the parallel plate model as (BV/TV)/Tb.Th, which is numerically equal to one-half of BS/TV for cancellous bone. (57) With the alternative cylindrical rod model, (58) Tb.N is given with dimensions Length 1 by (4/p BV/TV) 0.5 /Tb.Dm. To maintain consistency between the alternative models, this is preferred to the corresponding squared value with dimensions Length 2. It should be noted that there is ambiguity in the term trabecular number, which has been measured using a different method by others. (59) By the ASBMR definition, trabecular number goes down with estrogen deficiency, whereas with the alternate definition, (59) it goes up. Trabecular separation, defined as the distance between edges rather than between midpoints, is calculated according to the parallel plate model as Tb.Th (TV/ BV l), or as (1/Tb.N) Tb.Th. This quantity when multiplied by p/2 is an estimate of the mean distance across marrow cavities. (20,24) According to the cylindrical rod model, and assuming a parallel rectangular lattice, trabecular separation is given by Tb.Dm ((p/4 TV/BV) 0.5 1) but cannot be related in any simple way to the size of the marrow cavities. Trabecular spacing, defined as the distance between midpoints, is given by 1/Tb.N, and can also be measured directly. (60) Mineralizing surface The extent of surface active in mineralization at a particular time is given by the total extent of the labeled surface resulting from label administration at that time. The total extent of double label Table 4. Derived Indices in Bone Histomorphometry Type of index Name of index a Abbreviation a Formula b Structural Trabecular number Tb.N (BV/TV)/Tb.Th c Trabecular separation Tb.Sp (1/Tb.N) Tb.Th c Trabecular width Tb.Wi (BV/TV)/Tb.N Kinetic Mineralizing surface d MS (dls þ sls/2)/bs e Mineral apposition rate MAR Ir.L.Th/Ir.L.t Adjusted apposition rate f Aj.AR MAR (MS/OS) Osteoid apposition rate OAR same g Mineral formation rate d MFR MAR (MS/BS) Bone formation rate d BFR same g Bone resorption rate d BRs.R see text Mineralization lag time Mlt O.Th/Aj. AR Osteoid maturation time Omt O.Th/MAR h Formation period FP W.Th/Aj.AR Resorption period Rs.P FP (Oc.S/OS) h Reversal period Rv.P FP (ES Oc.S)/OS Remodeling period i Rm.P FP (ES þ OS)/OS BMU life span (sigma) Sg (or s) see text Quiescent period QP FP (QS/OS) Total period j Tt.P FP (BS/OS) Activation frequency k Ac.f (1/Tt.P) a Name and abbreviation are the same whether 2D or 3D expression used, except for mineralizing surface. b For 3D expression, in applying these formulae, it is essential to keep track of units throughout the calculations. c For parallel plate model, see text for rod model. d Referent must be specified; /BS is used in formula. e Other methods of measurement and calculation can be used (see text). f Time averaged over osteoid seam life span. g Mean value given by preceding quantity in steady state and in absence of osteomalacia. h For a more accurate method, see Eriksen.(45) i Rs.P þ Rv.P þ FP. j Rm.P þ QP. k l/tt.p. Journal of Bone and Mineral Research BONE HISTOMORPHOMETRY STANDARDIZED NOMENCLATURE 9

42 plus half the extent of single label is equivalent to the mean of the separately measured first label length (L1) and second label length (L2), thus following the normal scientific procedure of taking the mean of two separate observations when they are available. Use of the neutral term mineralizing surface (MS) or mineralizing interface (MI) allows a choice between the mean of the two labels, the second label alone (because it is closer in time to the biopsy), the total label (if only one label was given), in vitro tetracycline staining, (61) histochemical identification of the mineralization front, (43) or autoradiography after radiocalcium administration. Whatever the choice, the specification and validation of the method and of the exact conditions of measurement are the responsibility of the investigator and should be clearly stated. MS can be expressed in relation to a variety of referents (Table 2); MS/OS is equivalent to the fraction of osteoid seam life span during which mineralization occurs. It should be noted that length of individual labels varies depending on the fluorochrome. Parfitt and colleagues (62) showed that demethylchlortetracycline labels were significantly longer than oxytetracycline labels, regardless of the order in which they were administered. This should be taken into account in the calculation of MS/BS and BFR, and it is particularly important to do so when a quadruple labeling protocol is used to assess longitudinal changes in bone formation rate in a single biopsy. (63,64) Apposition rates Mineral apposition rate (MAR) is the distance between the midpoints (30) or between the corresponding edges (65) of two consecutive labels, divided by the time between the midpoints of the labeling periods. Both the number of sites available for measurement and the mean value of the measurement may vary with the length of the labeling interval, (30,65) which must always be stated and preferably standardized. We avoid the terms calcification rate and mineralization rate because they may lead to confusion between mineral apposition and mineral accumulation (66) and are often used in radiocalcium kinetics to refer to the whole body bone formation rate. There is no convenient way of distinguishing between the two-dimensional and three-dimensional quantities by different names, so that if the latter is chosen, it is important that the dimensional extrapolation factor be used consistently. Adjusted apposition rate (Aj.AR) is calculated as MAR MS/OS, and represents either the mineral apposition rate or the bone formation rate averaged over the entire osteoid surface. (66,67) It is analogous to the osteon radial closure rate (68) and is synonymous with effective apposition rate, (69) corrected apposition rate, (70) formation velocity, (71) and bone formation rate BMU level surface referent, (67) but none of these alternative names is satisfactory. The concept is important because in a steady state and in the absence of osteomalacia the adjusted apposition rate is the best estimate available from a biopsy of the mean rate of osteoid (or matrix) apposition. Under these conditions, the rates of formation of mineralized bone and of bone matrix, timeaveraged over the osteoid seam life span, including periods of activity and inactivity, are identical even though their instantaneous values are systematically out of step, (66) and the term osteoid apposition rate (OAR) may be used. We refer to these quantities (Aj.Ar and OAR) as apposition rates rather than as formation rates to maintain the distinction that an apposition rate has meaning at a point on the surface, whereas a formation rate has meaning only in relation to some aggregate of tissue, either surface or volume. An apposition rate represents in some sense the activity of a team of osteoblasts, but a formation rate is influenced by the rate of remodeling activation and so depends on the number of osteoblast teams as well as on their activity. The team rather than the single cell is emphasized as the conceptual unit because the activity of the team depends on the number of its members as well as on their individual productivity. Formation and resorption rates Mineral formation rate (MFR) is the volume of mineralized bone formed per unit time, calculated as the product of mineral apposition rate and mineralizing surface as defined earlier. If this term could be misinterpreted as relating to the physical chemistry of mineralization, the more precise term mineralized bone formation rate (Md.BFR) can be used. In a steady state and in the absence of osteomalacia the mineral formation rate is identical with the bone formation rate (BFR), and except when the distinction is important, the latter and more familiar term should be used. There is a bone formation rate corresponding to each possible referent for mineralizing surface: /OS, /BS, /BV, /TV, and /CV. Bone formation rate calculated using the osteoid surface referent is numerically identical to the adjusted apposition rate, as explained earlier. Expressing bone formation rate per unit of bone surface (BFR/BS) seems most logical when considering hormonal effects on bone remodeling. (32) Bone formation rate per unit of bone volume (BFR/BV) is equivalent to the bone turnover rate, which determines bone age and various age-dependent properties of bone. (66) Bone formation rate per unit of tissue volume (BFR/TV) seems most logical when considering biochemical markers of bone remodeling because the entire tissue is perfused and contributes its products to the circulation. (32) The significance of the core volume referent was discussed earlier. Bone resorption rate (BRs.R) cannot be measured directly by histomorphometry but can be estimated indirectly as the bone formation rate increased or decreased by an assumed or measured rate of change of bone volume, provided that all terms are expressed in relation to the same referent. (30,72,73) Previous gains or losses of bone from a surface can be estimated by comparing trabecular thickness and number, cortical thickness, and osteonal canal radius with mean values in age-matched control subjects, but it cannot be assumed that bone formation persisted at the current rate throughout the time over which these changes occurred. Because the rate of bone loss rarely exceeds 10% of the rate of bone turnover, under most circumstances the error from assuming that resorption and formation rates are equal is less than the error of measurement, but it is more accurate to assume that mineralized volume changes in proportion to some local or whole body measurement of bone mineral. (73) An alternative is to use sequential biopsies to estimate the change in bone volume, (30) which is satisfactory for a group of adequate size but subject to 10 DEMPSTER ET AL. Journal of Bone and Mineral Research

43 substantial error from sampling variation in a single subject. However it is estimated, BRs.R can be expressed in relation to a variety of different referents, including osteoclast number. (73) Timing of mineralization Mineralization lag time (Mlt) is the mean time interval between deposition and mineralization of any infinitesimal volume of matrix, averaged over the entire life span of the osteoid seam, and is given by O.Th/Aj.AR. The concept is important in the understanding of osteomalacia and the control of osteoid volume because it can be demonstrated that OV/BV ¼ BFR/ BV Mlt, (49) corresponding respectively to the birth rate and life span of individual moieties of osteoid. (66) Osteomalacia has been defined as O.Th >12.5 mcm (corrected for obliquity) and Mlt >100 days. (5,49) Mlt must be distinguished from osteoid maturation time (Omt), which is the mean time interval between the onset of matrix deposition and the onset of mineralization at each bone-forming site. The name implies that the delay results from extracellular modification of the matrix, such as collagen cross-linking. (66) In the growing rat, Mlt and Omt are identical, but in human subjects Omt is usually shorter and never longer than Mlt. Omt can be estimated as O.Th/MAR, and has also been referred to as direct rather than indirect Mlt, (74) but it is more accurate to measure Omt by remodeling sequence reconstruction. (45) Omt provides less insight into the mechanisms of osteoid accumulation than Mlt, but it may be more convenient for diagnostic use because, unlike Mlt, it is always normal in osteoporosis. (49) Techniques that can be readily applied to embedded biopsy samples, such as microradiography, backscattered electron imaging, and fourier transform infrared spectroscopy, yield important information about the degree of secondary mineralization, which can be correlated with static and dynamic indices of bone formation. (75 77) Remodeling cycle duration and its subdivisions Formation period (FP) is the mean time required to rebuild a new bone structural unit (B.St.U) or osteon from the cement line back to the bone surface at a single location, and is given by W.Th/ Aj.AR. It includes so-called downtime or offtime (68) or whatever other mechanism contributes to the difference between osteoid surface and mineralizing surface that cannot be attributed to label escape, (62) and so can be qualified as active, FP(aþ), given by W.Th/MAR, or inactive, FP(a ), given by W.Th/Aj.AR (OS/MS-l), or FP-FP(aþ). One example of a mechanism that could contribute to a difference between osteoid surface and mineralizing surface is the presence of thin osteoid seams during the terminal period of bone formation when MAR is too low to allow detectable separation of labels or deposition of sufficient amount of tetracycline to allow its visualization. (78) FP(aþ) has also been referred to as osteoblast life span. (79) FP is the key quantity needed for calculation of all other temporal subdivisions of the remodeling sequence. In a steady state, fractions of space are equivalent to fractions of time, (66) so that xp ¼ xs/os FP, where x is any remodeling state other than formation, including osteoclastic resorption, reversal, and quiescence (Table 4), but these calculations will reflect the uncertainty in classifying reversal cells. (45,65) Osteoclasts are motile and their area of activity probably extends beyond their current contact area (66) and in principle the osteoclast domain (Oc.Dm) determined by scanning electron microscopy (80) could be used to calculate RP. The sum of the resorption, reversal, and formation periods is the remodeling period (Rm.P), which is the average total duration of a single cycle of bone remodeling at any point on a bone surface. Rm.P is substantially shorter (by a factor of 2 or 3) than the total duration of bone remodeling activity that follows a single event of activation, because once initiated, the remodeling process moves for a variable distance across the bone surface or through the bone. (66) For example, many cortical osteons are much longer than a single cortical BMU, including both cutting and closing cones, (66) and the three-dimensional extent of many trabecular osteons is much larger than the extent of a single erosion or a single osteoid seam. (8) Although not commonly recognized, it is this extended period (66) that is the true BMU life span (or sigma) needed for attainment of a new steady state after any pathogenic process or therapeutic intervention. (68) As it is still used and appears frequently in key reference material, s remains an acceptable symbol for this crucially important concept; however, Sg is an alternative that avoids the inconvenience and outmoded use of Greek letters. Activation interval and frequency The sum of the remodeling period and the quiescent period (QP) is the total period (Tt.P), which is the average time interval between the initiation of two successive remodeling cycles at the same point on the surface. (45,66) The reciprocal of Tt.P is the activation frequency (Ac.f), which is the probability that a new cycle of remodeling will occur at any point on the surface by the event of activation. (45,66) Ac.f can also theoretically be calculated in the more traditional manner as the birth rate of remodeling sites of assumed or measured mean area, (66) and expressed in relation to the various volume referents in Table 2. However, the caveat here is that current technology does not permit measurement of the remodeling site area, particularly in cancellous bone. It can be shown that Ac.f W.Th ¼ BFR/BS, which is reasonable because W.Th can be regarded as the average amount of bone formed per activation event. Assessment of dynamic parameters when remodeling rates are low When methods were first developed for assessment of tetracycline-based parameters of bone formation, states of low remodeling rate were rarely encountered in human iliac crest bone biopsies, except in specific disease states. However, with the advent and widespread use of potent antiresorptive agents, biopsies are now frequently encountered in which turnover rates are so low that there are no labels or only single labels in cancellous and/or cortical bone. (81) One potential reason for lack of labels is that the tetracycline was not taken or properly or efficiently absorbed. This can sometimes be ruled out by the presence of labels in another biopsy compartment, eg, in the cortex or on the endocortical surface. The suspicion that a paucity of labels truly represents a low turnover rate can be supported by low values for static parameters of bone formation, such as osteoid surface or osteoblast number. Reduced Journal of Bone and Mineral Research BONE HISTOMORPHOMETRY STANDARDIZED NOMENCLATURE 11

44 tetracycline uptake presents a problem for the reporting and interpretation of the data and requires a uniform approach. In situations where there are no labels in an adequate sampling area, we recommend that MAR be recorded as a missing datum and that the number of such samples in a treatment group be clearly stated in the results section of the article. In this situation, it is appropriate to record a value of zero for MS/BS and to include these samples in the calculation of group means for MS/ BS. In biopsies where only single labels or too few double labels are present to measure MAR reliably, MS/BS can be measured and reported in the usual way. MAR can be recorded as a missing value or one has the option of assigning (imputing) a minimum value to MAR. Two such values have been determined empirically: 0.3 mcm/d, (82) or 0.1 mcm/d, (83) based on either the lowest measurable average value for MAR in the first case or the lowest measured value in the second. The lowest measured value for MAR in the laboratory where the analysis is performed could also be used. Alternatively, if double labels are present in another envelope of the biopsy, say within the cortex, the measured value for MAR in that envelope can be used or one could use the average value for MAR for the cohort to which the subject belongs. In any of these approaches, if MAR is expressed in three dimensions, the appropriate correction factor should be applied (see above). The advantage of assigning a value to MAR when only single labels are present is that a larger number of samples can be used to calculate group means for MAR and parameters derived from it, such as BFR, with the caveat that group means for MAR and the derived parameters may be biased upwards, whereas exclusion of such samples will have the opposite effect. The key recommendation here is that all articles clearly state the numbers of samples in a group with double labels, the number with only single labels, and the number without labels and the method of dealing with single labels. Another option is to present the results using both methods. (84,85) Some authors have applied extended search protocols to hunt for labels throughout the biopsy. (84,86) Although this allows a statement to be made on the proportion of biopsies in a treatment group that have labels, it does not change the quantitative data and the extra effort may be disproportional to the additional information obtained. If labels are low or absent in an adequate sampling area, it is likely that they will be low or absent in the rest of the biopsy. The presence or absence of single and double labels should be described separately for cortical and cancellous bone. (87) The above recommendations for estimating and reporting MAR would also apply in the rare situations in which only one tetracycline label is administered. Units and dimensions Two primary units of length, micrometer (mcm) and millimeter (mm), and two primary units of time, day (d) and year (y), should be used, with the choice depending on convenience, consistency, and the principle of providing the most important information in front of rather than after the decimal point. Dimensions are useful for checking equations and derivations (88) and for indicating the similarities between some quantities expressed in different units. For surface/surface and volume/ volume ratios, we prefer percentages rather than decimal fractions; in this case, the percent sign can be used to combine the referent and unit (eg, OS%BS instead of OS/BS(%)). If abbreviations are not used for these ratios, the names can be simplified by writing the type of measurement only once (eg, osteoid/bone [surface]). We avoid units such as mm 2 /cm 2 because their magnitude changes with transition from two to three dimensions (eg, 1 mm 2 /cm 2 ¼ 10 mm 3 /cm 3 ). Such units also do not conform to the SI (89) and make it more difficult to perceive that the quantity is dimensionless. All section dimensions should be expressed in mm, all primary perimeter and area measurements in mm or mm 2, and all surface/volume ratios in mm 2 /mm 3 (Length 1 ). Thickness measurements should be expressed in mcm, with mm as an alternative for cortical thickness. Apposition rates should be expressed as mcm/d (Length Time 1 ) and formation rates with volume referent as %/ y (Time 1 ). Times and periods should be expressed in days or years as most appropriate and activation frequency in /y (Time 1 ). Summary of Nomenclature System and Recommended Parameters We recognize that many who perform bone histomorphometry or interpret its results will on most occasions need to use only a small proportion of the foregoing material. Accordingly, we provide here a summary of its most important features, but this is not intended to stand on its own without reference to the main body of the article. We also provide a list of parameters that preferentially should be included in all histomorphometry studies. Definitions All acceptable terms are listed in Table 1; only the most basic are discussed here. The term bone refers to bone matrix whether mineralized or not and bone tissue refers to bone as defined with its associated marrow or other soft tissue. Bone tissue is usually either cortical or cancellous; the junction between them, which is the inner border of the cortex, is referred to as endocortical surface. A trabecula is an individual structural element of cancellous bone tissue, whether plate-like or rod-like in form. The term osteoid refers to unmineralized bone matrix that in the normal course of events will become fully mineralized, and does not include the thin layer of permanently unmineralized collagen-containing connective tissue that lies beneath bone lining cells on all quiescent surfaces. The junction between osteoid and mineralized bone is referred to as the bone interface or, more precisely, osteoid-bone interface. The term osteoblast is restricted to cells that are assumed to be currently making bone and does not refer to all cells with osteogenic potential. The qualifications active and inactive are not used; inactive osteoblasts are called lining cells. Terms that embody assumptions, such as formation (or forming) surface and resorption (or resorbing) surface, are avoided. Instead, the purely descriptive terms osteoid surface and eroded surface are used. The extent of currently active mineralization is referred to as mineralizing surface (or 12 DEMPSTER ET AL. Journal of Bone and Mineral Research

45 interface) regardless of how it is estimated. The method used for its determination must be specified and justified. A cylindrical biopsy specimen from the ilium, whether transverse or vertical, is referred to as a core, and the term total is generally used only when measurements are made on the entire core. General principles Dimensional expression There must be consistent use of only two-dimensional or only three-dimensional terminology and units throughout the same article or the same report. Primary measurements are referred to as area, perimeter, and width if expressed in two dimensions and as volume, surface (or interface), and thickness if expressed in three dimensions (Table 1). Number, the fourth type of primary measurement, can be expressed three-dimensionally only if serial sections are examined. If three-dimensional expression is used, the method of calculation should be exactly specified and its underlying assumptions carefully considered. Stereology The terminology and symbols of the International Society of Stereology will not be used. Consequently, the term density retains its primary meaning in physics of mass per unit volume. However, this in no way diminishes the importance of stereologic theory for proper sampling, measurement, and dimensional extrapolation. Referents An absolute area, perimeter, or number measurement is useful only as an index of the amount of tissue examined, for which acceptable minimum values should be specified (see below); the term absolute is not used in any other sense. Of the four types of primary measurement, only width (or thickness) can be interpreted without a referent, which will normally be some defined and measured area (or volume) or perimeter (or surface) in the section. Because several referents are possible for virtually all measurements, the chosen referent must always be specified consistently and explicitly; when this is done, terms such as ratio and relative are redundant and should not be used. If only one referent is used, or if measurements with the same referent are grouped together, the referent may need to be mentioned only once, but it must be repeated each time if there is any possibility of confusion. Abbreviations These consist of the first letters in the same order as the words in the name, without superscripts or subscripts. Each symbol component has only one meaning, as specified in Table 1, and no latitude in the choice of abbreviations is allowed. Single capital letters are used for the most frequent terms, a capital letter and an additional lowercase letter for less frequent terms, and a single lowercase letter for terms that are in some sense related to time. Double letter abbreviations must be demarcated by periods; in the absence of periods, each letter is to be construed as a separate abbreviation. Standard format The same format is used for all measurements: Source Measurement/Referent. The source is the type of structure or region within a sample on which the measurement was made and will most commonly be cortical bone tissue (Ct), cancellous bone tissue (Cn), endocortical surface (Ec), or total biopsy core (Tt), but many other sources are in occasional use (Table 2) or can be defined using the lexicon (Table 1). Circumstances in which the source can be omitted from the name are detailed in the body of the text. Current practice is inconsistent in this respect; even when measurements have only been made on cancellous bone tissue, the source is almost always mentioned for some measurements (eg, trabecular bone volume) and frequently omitted for others (eg, osteoid volume and surface). The need for and the rules pertaining to referents were given earlier. The most commonly used referents are tissue volume (TV), bone volume (BV), bone surface (BS), osteoid surface (OS), and bone interface (BI), but many other referents can be defined for particular purposes (Table 2). The principal referents are related by the surface to volume ratios BS/BV (S/V in stereologic terminology) and BS/TV (S v in stereologic terminology). Adequate tissue sampling and recommended measurements For human iliac crest biopsies, we recommend a Bordier/ Rochester type trephine with an internal diameter of at least 7.5 mm (5 mm for pediatric samples). Useful qualitative and quantitative information can be obtained with smaller bore trephines and with vertical rather than horizontal biopsies, but the number of variables that can be reliably quantified is more limited. (5,90) The minimum acceptable tissue area to be sampled is 30 mm 2 and the minimum acceptable bone perimeter is 60 mm. We recommend that sections be collected from at least two, and, preferably, three regions within the biopsy starting at about halfway through the core and separated by approximately 300 mcm. Short core widths (Fig. 2) may require additional sampling to achieve the minimum acceptable tissue area and bone perimeter. The number of sections and the tissue area and bone perimeter measured should be reported in all publications. The committee recommends that a minimum of five double labels be used to measure MAR. If necessary MAR can be estimated with fewer double labels, but in that case the lowest number of double labels used to estimate MAR in a group of subjects should be stated. Table 5 gives a list of measurements that, where practical and appropriate, should be performed and reported in all histomorphometry studies, (81) together with their abbreviations and units. Note that the recommended units are based on two units for length (mcm and mm) and two units for time (day and year), and that percent is preferred for dimensionless ratios. It is conventional to distinguish between static and dynamic measurements, the former not requiring tetracycline labeling, but it is perhaps more important to distinguish between primary Journal of Bone and Mineral Research BONE HISTOMORPHOMETRY STANDARDIZED NOMENCLATURE 13

46 Table 5. Terminology, Abbreviations, and Units for Recommended Primary Measurements and Derived Indices in Cancellous Bone Tissue Parameter a,b Abbreviation Units Tissue area c T.Ar mm 2 Bone area c B.Ar mm 2 Bone perimeter c B.Pm mcm Bone volume BV/TV d % Wall thickness W.Th mcm Osteoid surface OS/BS % Osteoid volume OV/BV % Osteoblast surface Ob.S/BS e % Osteoblast number N.Ob/BS /mm Osteoid thickness O.Th mcm Eroded surface ES/BS % Osteoclast surface Oc.S/BS f % Osteoclast number N.Oc/T.A g /mm 2 Bone surface BS/TV mm 2 /mm 3 Double-labeled surface dls/bs % Single-labeled surface sls/bs % Mineralizing surface MS/BS % Mineralizing surface MS/OS % Mineral apposition rate MAR mcm/d Adjusted apposition rate Aj.AR mcm/d Mineralization lag time Mlt D Osteoid maturation time Omt D Activation frequency Ac.F N/y Cortical thickness Ct.Th mcm Cortical porosity Ct.Po % Bone formation rate BFR/BS mcm 3 /mcm 2 /d Bone formation rate h BFR/BV %/y a Measurement name only; need for inclusion of source and/or referent in name varies with context, as discussed in text. b Three-dimensional expression except where otherwise stated. c Should always be included to allow the reader to assess adequacy of tissue sampling. Should be expressed as a range (minimum maximum) for the samples analyzed. d The full name and abbreviation would be cancellous bone volume/ tissue volume (Cn-BV/TV). e OS is another frequently used referent. f ES sometimes used as an additional referent. g Bone perimeter is an alternative referent; note that expression must be 2D, not 3D. h Equivalent to rate of bone turnover. measurements (Table 3) and derived indices (Table 4). By primary measurement is meant not the absolute raw data, but the use of no more manipulation of the raw data than is needed to express them in terms of a referent or to divide by a constant such as the time interval between labels. Derived indices require more complex arithmetical manipulation and usually rest on one or more assumptions that should always be made clear. Derived indices should not be reported without the primary measurements from which they are derived. Disclosures All authors state that they have no conflicts of interest. Acknowledgments Authors roles: DWD produced the first draft of the updated report. All other authors reviewed and revised this and subsequent drafts. References 1. Parfitt AM. Terminology and symbols in bone morphometry. In: Jaworski ZFG, editor Proceedings of the First International Workshop on Bone Morphometry. Ottawa: Ottawa University Press; p Parfitt AM, Drezner MC, Glorieux FH, Kanis JA, Malluche H, Meunier PJ, Ott SM, Recker RR., Bone histomorphometry: standardization of nomenclature, symbols, and units. Report of the ASBMR Histomorphometry Nomenclature Committee. J Bone Miner Res. 1987; 2: Dorland s illustrated medical dictionary. 26th ed. Philadelphia: W.B. Saunders; Fawcett DW. A textbook of histology II ed. Philadelphia: W.B. Saunders; Malluche HH, Faugere M-C. Atlas of mineralized bone histology. New York: S. Karger AG; Revell PA. Pathology of bone. Berlin: Springer-Verlag; Cowin SC. Wolff s Law of trabecular architecture at remodeling equilibrium. J Biomech Eng. 1986;108: Arnold JS, Wei LT. Quantitative morphology of vertebral trabecular bone. In: Stover B, Jee WSS, editors Radiobiology of plutonium. Salt Lake City: The J.W. Press; p Whitehouse WJ., Cancellous bone in the anterior part of the iliac crest. Calc Tiss Res. 1977;23: Singh I. The architecture of cancellous bone. J Anat. 1978;127: Robinson RA. Chemical analysis and electron microscopy of bone. In: Rodahl K, Nicholson JT, Brown EM, editors Bone as a tissue. New York: McGraw-Hill; p Courpron P, Meunier P, Bressot C, Giroux JM., Amount of bone in iliac crest biopsy. Significance of the trabecular bone volume. In: Meunier PJ, editor Bone histomorphometry. Second international workshop. Paris: Armour Montagu, p Malluche HH, Sherman DS, Meyer W, Massry SG. A new semiautomatic method for quantitative static and dynamic bone histology. Calcif Tissue Int. 1982;34: Frost HM. Bone remodelling dynamics. Springfield: C. C. Thomas; Carter DR, Spengler DM. Mechanical properties and composition of cortical bone. Clin Orthop. 1978;135: Vaughan JM. The physiology of bone 2nd ed. Oxford: The Clarendon Press; Dobnig H, Turner RT. Evidence that intermittent treatment with parathyroid hormone increases bone formation in adult rats by activation of bone lining cells. Endocrinology. 1995;136: Dunstan CR, Evans RA. Quantitative bone histology: a new method. Pathology. 1980;12: Chappard D, Alexandre C, Riffat G. Histochemical identification of osteoclasts. Review of current methods and reappraisal of a simple procedure for routine diagnosis on undecalcified iliac bone biopsies. Bas Appl Histochem. 1983;27: Parfitt AM. The stereologic basis of bone histomorphometry. Theory of quantitative microscopy and reconstruction of the third dimension. In: Recker R, editor Bone histomorphometry. Techniques and interpretations. Boca Raton (FL): CRC Press; p DEMPSTER ET AL. Journal of Bone and Mineral Research

47 21. Aherne WA, Dunnill MS., Morphometry. London: Edward Arnold; Elias H, Hyde DM. A guide to practical stereology. Basel: Karger; Vesterby A, Kragstrup J, Gundersen HJG, Melsen F. Unbiased stereological estimation of surface density in bone using vertical sections. Bone. 1987;8: Whitehouse WJ. The quantitative morphology of anisotropic trabecular bone. J Microsc. 1974;101: Schwartz MP, Recker RR. Comparison of surface density and volume of human iliac trabecular bone measured directly and by applied stereology. Calcif Tissue Int. 1981;33: Baddeley AJ, Gundersen HJG, Cruz-Orive LM. Estimation of surface area from vertical sections. J Microsc. 1986;142: Gundersen HJG. Stereology of arbitrary particles. J Microsc. 1986; 143: DeHoff RT. Quantitative serial sectioning analysis: preview. J Microsc. 1983;131: Schenk RK. Comments on terminology symbols. In: Jaworski ZFG, editor Proceedings of the First Workshop on Bone Morphometry. Ottawa: University of Ottawa Press; p Frost HM. Bone histomorphometry: analysis of trabecular bone dynamics. In: Recker R, editor Bone histomorphometry. Techniques and interpretations. Boca Raton (FL): CRC Press; p Parfitt AM, Rao DS, Stanciu J, Villanueva AR, Kleerekoper M, Frame B. Irreversible bone loss in osteomalacia: comparison of radial photon absorptiometry with iliac bone histomorphometry during treatment. J Clin Invest. 1985;76: Parfitt AM, Simon LS, Villanueva AR, Krane SM. Pro-collagen type 1 carboxy-terminal extension peptide in serum as a marker of collagen biosynthesis in bone. Correlation with iliac bone formation rates and comparison with total alkaline phosphatase. J Bone Min Res. 1987;2: Boyce BF, Courpon P, Meunier PJ. Amount of bone in osteoporosis and physiological senile osteopenia. Comparison of two histomorphometric parameters. Metab Bone Dis Rel Res. 1978;1: Horsman A. Bone mass. In: Nordin BEC, editor Calcium, phosphate and magnesium metabolism. Edinburgh: Churchill-Livingstone; Euclid. Euclid s Elements, Book I, Proposition Keshawarz NM, Recker RR. Expansion of the medullary cavity at the expense of cortex in postmenopausal osteoporosis. Metab Bone Dis Rel Res. 1984;5: Hamilton EJ, Ghasem-Zadeh A, Gianatti E, Lim-Joon D, Bolton D, Zebaze R, Seeman E, Zajac JD, Grossmann M. Structural decay of bone microarchitecture in men with prostate cancer treated with androgen deprivation therapy. J Clin Endocrinol Metab. 2010;95: E Zebaze RM, Ghasem-Zadeh A, Bohte A, Iuliano-Burns S, Mirams M, Price RI, Mackie EJ, Seeman E. Intracortical remodelling and porosity in the distal radius and post-mortem femurs of women: a crosssectional study. Lancet. 2010;15; (375): Compston JE, Vedi S, Stellon AJ. Inter-observer and intra-observer variation in bone histomorphometry. Calcif Tissue Int. 1986;38: Duncan H. Cortical porosis; a morphological evaluation. In: Jaworski ZFG, editor First Workshop on Bone Histomorphometry. Ottawa: University of Ottawa Press; p Arnold JS, Bartley MH, Tont SA, Jenkins DP., Skeletal changes in aging and disease. Clin Orthop Relat Res. 1966;49: Olah AJ. Effects of microscopic resolution on histomorphometrical estimates of structural and remodeling parameters in cancellous bone. Path Res Pract. 1980;166: Rasmussen H, Bordier PJ. The physiological and cellular basis of metabolic bone disease. Baltimore: Williams and Wilkins; Martin RB. Porosity and specific surface of bone. CRC Crit Rev Biomed Eng. 1984;10: Eriksen EF. Normal and pathological remodeling of human trabecular bone: three dimensional reconstruction of the remodeling sequence in normals and in metabolic bone disease. Endocr Rev. 1986;7: Parfitt AM. The cellular basis of bone remodeling. The quantum concept re-examined in light of recent advances in cell biology of bone. Calc Tissue Int. 1984;36:S37 S Kragstrup J, Gundersen HJG, Melsen F, Mosekildc L. Estimation of the three-dimensional wall thickness of completed remodeling sites in iliac trabecular bone. Metab Bone Dis Rel Res. 1982;4: Parfitt AM, Villanueva AR, Mathews CHE, Aswani JA. Kinetics of matrix and mineral apposition in osteoporosis and renal osteodystrophy: relationship to rate of turnover and to cell morphology. In: Jee WSS, Parfitt AM, editors Bone histomorphometry. Third International Workshop. Paris: Armour-Montagu; p Parfitt AM. Osteomalacia related disorders. In: Avioli LV, Krane SM, editors Metabolic bone disease 2nd ed. Philadelphia: W. B. Saunders; p Baylink D, Stauffer M, Wergcdal J, Rich C. Formation, mineralization and resorption of bone in vitamin D deficient rats. J Clin Invest. 1970;49: Teitelbaum S, Hruska KA, Shieber W, Debnam JW, Nichols SH. Tetracycline fluorescence in uremic and primary hyper-parathyroid bone. Kidney Int. 1977;12: Courpron P. Bone tissue mechanisms underlying osteoporoses. Orthop Clin North Am. 1981;12: Broulik P, Kragstrup J, Mosekilde L, Melsen F. Osteon cross-sectional size in the iliac crest. Acta Path Microbiol Immunol Scand Sect A. 1982;90: Merz WA, Schenk RK. Quantitative structural analysis of human cancellous bone. Acta Anat (Basel). 1970;75: Garrahan NJ, Mellish RW, Compston JE. A new method for the two dimensional analysis of bone structure in human iliac crest biopsies. J Microsc. 1986;142: Compston JE, Mellish RWE, Garrahan NJ. Age-related changes in iliac crest trabecular macroanatomic bone structure in man. Bone. 1987; 8: Parfitt AM, Mathews CHE, Villanueva AR, Kleerekoper M, Frame B, Rao DS. Relationship between surface, volume and thickness of iliac trabecular bone in aging and in osteoporosis: implications for the microanatomic and cellular mechanisms of bone loss. J Clin Invest. 1983;72: Malluche HH, Meyer W, Sherman D, Massry SG. Quantitative bone histology in 84 normal American subjects. Calcif Tissue Int. 1982;34: Boyce RW, Ebert DC, Youngs TA, Paddock CL, Mosekilde L, Stevens ML, Gundersen HJ. Unbiased estimation of vertebral trabecular connectivity in calcium-restricted ovariectomized minipigs. Bone. 1995;16: Weinstein RS, Hutson MS. Decreased trabecular width and increased trabecular spacing contribute to bone loss with aging. Bone. 1987; 8: Aaron JE, Makins NB, Francis RM, Peacock M. Staining of the calcification front in human bone using contrasting fluoro-chromes in vitro. J Histochem Cytochem. 1984;32: Parfitt AM, Foldes J, Villanueva AR, Shish MS. Difference in label length between demethylchlortetracycline and oxytetracycline: implications for the interpretation of bone histomorphometric data. Calcif Tissue Int. 1991;48: Lindsay R, Cosman F, Zhou H, Bostrom MP, Shen VW, Cruz JD, Nieves JW, Dempster DW. A novel tetracycline labeling schedule for longitudinal evaluation of the short-term effects of anabolic therapy with a Journal of Bone and Mineral Research BONE HISTOMORPHOMETRY STANDARDIZED NOMENCLATURE 15

48 single iliac crest bone biopsy: early actions of teriparatide. J Bone Miner Res. 2006;21: Lindsay R, Cosman F, Zhou H, Nieves J, Dempster DW. Letter to the editor: double and quadruple tetracycline labeling of bone. J Bone Miner Res. (in press). 65. Tarn CS, Anderson W. Tetracycline labelling of bone in vivo. Calcif Tissue Int. 1980;30: Parfitt AM. The physiologic and clinical significance of bone histomorphometric data. In: Recker R, editor Bone histomorphometry. Techniques and interpretations. Boca Raton (FL): CRC Press; p Melsen F, Mosekilde L., Dynamic studies of trabecular bone formation and osteoid maturation in normal and certain pathological conditions. Metab Bone Dis Rel Res. 1978;1: Frost HM. Tetracycline-based histological analysis of bone remodeling. Editorial. Calc Tiss Res. 1969;3: Jaworski ZFG. Bone histomorphometry: outline of theory and practice. In: Simmons OJ, Kunin AS, editors Skeletal research. Vol 2. New York: Academic Press; p Parfitt AM, Mathews C, Rao D, Frame B, Kleerekoper M, Villanueva AR., Impaired osteoblast function in metabolic bone disease. In: DeLuca HF, Frost H, Jee W, Johnston C, Parfitt AM, editors Osteoporosis: recent advances in pathogenesis and treatment. Baltimore: University Park Press; p Parfitt AM, Villanueva AR. Hypophosphatemia and osteoblast function in human bone disease. In: Massry SG, Letteri JM, Ritz E, editors Proceedings of the 5th International Workshop in Phosphate and Other Minerals. Regulation of phosphate and mineral metabolism. Adv Exp Med Biol 151: Wu K, Jett S, Frost HM. Bone resorption rates in rib in physiological, senile, and postmenopausal osteoporoses. J Lab Clin Med. 1967; 69: Gruber HE, Ivey JL, Thompson ER, Chestnut CH III, Baylink DJ. Osteoblast and osteoclast cell number and cell activity in postmenopausal osteoporosis. Miner Electrolyte Metab. 1986;12: Evans RA, Flynn J, Dunstan CR, George CRP, McDonnell GD. Bone metabolism in chronic renal failure. Miner Electrolyte Metab. 1982; 7: Roschger P, Paschalis EP, Fratzl P, Klaushofer K. Bone mineralization density distribution in health and disease. Bone. 2008;42: Boivin G, Farlay D, Bala Y, Doublier A, Meunier PJ, Delmas PD. Influence of remodeling on the mineralization of bone tissue. Osteoporos Int. 2009;20: Paschalis EP, Mendelsohn R, Boskey AL. Infrared assessment of bone quality: a review. Clin Orthop Relat Res. 2011;469: Frost HM. Bone histomorphometry: correction of the labeling escape error. In: Recker RR, editor Bone histomorphometry. Techniques and interpretations. Boca Raton (FL): CRC Press; p Arlot M, Edouard C, Meunier PJ, Neer RM, Reeve J. Impaired osteoblast function in osteoporosis: comparison between calcium balance dynamic histomorphometry. Br Med J. 1984;289: Jones SJ, Boyde A, Ali NN, Maconnachie E. A review of bone cell and substratum interactions. An illustration of the role of scanning electron microscopy. Scanning. 1985;7: Recker RR, Kimmel DB, Dempster D, Weinstein RS, Wronski TJ, Burr DB. Issues in modern bone histomorphometry. Bone. 2011;49: Foldes J, Shih MS, Parfitt AM. Frequency distributions of tetracyclinebased measurements: implications for the interpretation of bone formation indices in the absence of double-labeled surfaces. J Bone Miner Res. 1990;5: Hauge E, Mosekilde L, Melsen F. Missing observations in bone histomorphometry on osteoporosis: implications and suggestions for an approach. Bone. 1999;25: Reid IR, Miller PD, Brown JP, Kendler DL, Fahrleitner-Pammer A, Valter I, Maasalu K, Bolognese MA, Woodson G, Bone H, Ding B, Wagman RB, San Martin J, Ominsky MS, Dempster DW. Denosumab Phase 3 Bone Histology Study Group. Effects of denosumab on bone histomorphometry: the FREEDOM and STAND studies. J Bone Miner Res. 2010;25: Dempster DW, Zhou H, Recker RR, Brown JP, Bolognese MA, Recknor CP, Kendler DL, Lewiecki EM, Hanley DA, Rao DS, Miller PD. Woodson GC 3rd, Lindsay R, Binkley N, Wan X, Ruff VA, Janos B, Taylor KA. Skeletal Histomorphometry in Subjects on Teriparatide or Zoledronic Acid Therapy (SHOTZ) study: a randomized controlled trial. J Clin Endocrinol Metab. 2012;97: Recker RR, Delmas PD, Halse J, Reid IR, Boonen S, García-Hernandez PA, Supronik J, Lewiecki EM, Ochoa L, Miller P, Hu H, Mesenbrink P, Hartl F, Gasser J, Eriksen EF. Effects of intravenous zoledronic acid once yearly on bone remodeling and bone structure. J Bone Miner Res. 2008;23: Compston JE. Histomorphometric interpretation of bone biopsies for the evaluation of osteoporosis treatment. BoneKEy Rep [Internet] Apr 4;1; DOI: /bonekey Riggs DS. The mathematical approach to physiologic problems. Baltimore: Williams and Wilkins; Young DS. Implementation of SI units for clinical laboratory data. Style specifications and conversion tables. Ann Intern Med. 1987;10: Faugere MC, Malluche HH. Comparison of different bone-biopsy techniques for qualitative and quantitative diagnosis of metabolic bone diseases. J Bone Joint Surg Am. 1983;65: DEMPSTER ET AL. Journal of Bone and Mineral Research

49 Technical Approach to Iliac Crest Biopsy Joel D. Hernandez, Katherine Wesseling, Renata Pereira, Barbara Gales, Rick Harrison, and Isidro B. Salusky Department of Pediatrics, David Geffen School of Medicine at University of California, Los Angeles, Los Angeles, California Bone histomorphometry has been the gold standard in the evaluation and diagnosis of renal osteodystrophy. The recent new definition of renal osteodystrophy as chronic kidney disease mineral and bone disorder has once again highlighted the use of bone biopsy as a powerful and diagnostic tool to determine skeletal abnormalities in chronic kidney disease. The procedure of iliac crest bone biopsy has been proved safe and associated with very minimal morbidity. In this review, the clinical indications, preparation, instrumentation, and potential complications are discussed. Because current biochemical markers are poor predictors of bone turnover, volume, and mineralization, a wider use of bone biopsy and histomorphometry will lead to a better understanding of the bone and mineral disorders that are associated with chronic kidney disease. Clin J Am Soc Nephrol 3: S164 S169, doi: /CJN Renal osteodystrophy represents a spectrum of skeletal lesions that range from high-turnover disorders (osteitis fibrosa and mild lesions of secondary hyperparathyroidism) to low-turnover bone diseases (osteomalacia and adynamic lesion) (1). Mixed lesions of renal osteodystrophy have histologic evidence of both osteomalacia and hyperparathyroidism, with the rate of bone formation depending on the predominant lesion. Bone biopsy is the gold standard to establish the precise diagnosis of renal osteodystrophy, determine the severity of the bone disease, and assess the skeletal response to different therapeutic interventions (2,3). The use of this procedure for histomorphometric analysis has provided the basis of our current understanding of the different subtypes of renal bone disease and their pathogenesis in patients with chronic kidney disease (CKD). In the past few years, various studies have linked the abnormalities of bone and mineral metabolism with the process of vascular calcification (4 6). The magnitude of vascular and skeletal complications that are associated with CKD and the recognition that these complications are integrally linked have led to the recent new definition of renal osteodystrophy as a broader clinical syndrome of abnormalities of mineral and bone metabolism secondary to CKD and/or extraskeletal calcifications. The new consensus on the definition of renal osteodystrophy highlights the use of bone biopsy as a powerful and diagnostic tool to determine precise skeletal abnormalities (7). In describing these abnormalities, it has now been recommended to include three important elements obtained from bone histomorphometric analysis: Bone turnover, mineralization, and volume. These three histologic variables provide clinically relevant information on bone pathology that can be used Correspondence: Dr. Isidro B. Salusky, Department of Pediatrics, David Geffen School of Medicine at UCLA, A2-331, MDCC, Le Conte Avenue, Los Angeles, CA Phone: ; Fax: ; isalusky@mednet.ucla.edu to define pathophysiology and guide clinicians with therapy (7). Although bone histomorphometry is the gold standard for diagnosis of bone turnover state in patients with CKD, the use of bone biopsy has been limited by several factors, such as the invasiveness of the procedure, the lack of technical training, and the limited number of specialized centers with expertise in tissue handling and preparation (3). Serum parathyroid hormone (PTH), however, has been used as a noninvasive biomarker of bone turnover. Indeed, in cross-sectional studies, the introduction of first-generation immunometric PTH assay (1st PTH-IMA) has allowed reasonable prediction of the different subtypes of bone lesions in CKD (8 10); however, this predictive value of serum PTH is diminished when patients are treated with intermittent calcitriol therapy such that the development of adynamic bone has been observed in patients with PTH levels that are consistent with secondary hyperparathyroidism (11). In addition, it has been shown that 1st PTH-IMA detect not only the full-length and biologically active PTH (1-84) but also PTH fragments that accumulate in renal failure (12,13). Cross-reaction with PTH amino-truncated fragments [ntpth(1-84)] have resulted in the overestimation of the true concentration of PTH (1-84). For addressing this shortcoming, second-generation PTH-IMA (2nd PTH-IMA) have been developed. These assays use detection antibody that is raised against the first four amino acids at the amino-terminal end and recognizes exclusively the biologically active full-length PTH(1-84) with no cross-reaction with ntpth(1-84) fragments (14,15); however, its value in predicting the types of bone lesions is yet to be fully established. Likewise, other serum biomarkers and imaging techniques that are used to assess bone health have not been carefully studied in the context of CKD. Thus, bone biopsy remains the most accurate diagnostic tool in the evaluation of renal osteodystrophy. The current clinical indications for performing the procedure are summarized in Table 1 (16). The applications of this procedure are not limited to clinical evalu- Copyright 2008 by the American Society of Nephrology ISSN: /

50 Clin J Am Soc Nephrol 3: S164 S169, 2008 Iliac Crest Biopsy for Evaluation of Renal Osteodystrophy S165 Table 1. Clinical indications for bone biopsy in patients with CKD based on KDOQI (16) and KDIGO (7) a 1. Fractures with minimal or no trauma (pathologic fractures) 2. Suspected aluminum bone disease on the basis of clinical symptoms or history of aluminum exposure 3. Intact plasma PTH levels between 100 and 500 pg/ ml (11.0 to 55.0 pmol/l; in stage 5 CKD) with coexisting conditions such as unexplained hypercalcemia, severe bone pain, or unexplained increases in bone alkaline phosphatase activity 4. Severe progressive vascular calcification 5. Before parathyroidectomy if there has been significant exposure to aluminum in the past or if the results of biochemical determinations are not consistent with secondary or tertiary hyperparathyroidism 6. Before beginning treatment with bisphosphonates a CKD, chronic kidney disease; KDIGO, Kidney Disease: Improving Global Outcomes; KDOQI, Kidney Disease Outcomes Quality Initiative; PTH, parathyroid hormone. ation and diagnosis but extend to basic and clinical research. Future studies have been recommended to evaluate how bone biopsy can be best used in clinical practice and how more clinicians can be trained to perform such a procedure (7). Figure 1. Tetracycline double labeling on bone-osteoid surface as seen in fluorescent light. Clinical Procedure Tetracycline Labeling The value of bone biopsy in the diagnosis of renal osteodystrophy is enhanced when dynamic aspects of bone turnover are characterized (17). For determination of the level of bone turnover, bone formation rates, and mineralization defects, double labeling of the bone with tetracycline compounds is done before performance of the bone biopsy. Tetracycline compounds chelate calcium on bone surfaces and are deposited within the bone at sites of active mineralization. The labels can then be visualized in fluorescent light during histomorphometric evaluation (Figure 1). Double bands of tetracycline fluorescence can be seen circumscribing the amount of new bone formed during the labeling interval. Tetracycline and demeclocycline HCl are the most commonly used compounds. The intensity of the label would depend on the drug concentration and dosage. In children, dosage for oral tetracycline is 10 to 15 mg/kg per d and for demeclocycline HCl is 15 to 20 mg/kg per d divided three times a day. Adult dosage for tetracycline HCl is 500 mg by mouth two times a day. Demeclocycline HCl can be given on a similar schedule as tetracycline but at a lower dosage of 300 mg. There are different protocols for double-tetracycline labeling, but the basic and optimal setup would involve allowing a certain amount of time to elapse between two courses of antibiotics. In our center, antibiotics are given during two 2-d periods with each period separated by 10 to 12 d free of tetracycline. Iliac crest bone biopsy will take place 2 to 5 d after double labeling with tetracycline. The drug is usually administered after meals to avoid gastrointestinal discomfort. The patient should avoid milk and dairy products, which contain calcium, as well as calcium-containing binders because they can bind to tetracycline and prevent adequate absorption of the drug. It is imperative that the patient adhere strictly to tetracycline dosing and schedule so as not to encounter any problems in interpretation and computation of the dynamic histomorphometric parameters. Biopsy Sites, Instruments, and Technique Quantitative bone histomorphometry requires a good-quality bone specimen. Obtaining good samples would depend on the instrument used, biopsy technique, and a skillful and experienced operator (3,18). Iliac crest has been the preferred site when doing bone biopsy, because it is easily accessible and associated with fewer complications. Either the right or left iliac crest can be biopsied. Iliac crest biopsies can be obtained in a horizontal and vertical direction with associated advantages and disadvantages (3,19 21). The vertical approach would have specimen that is mostly trabecular bone. In the horizontal approach, intact two cortices separated by trabecular bone can be obtained (19). In either approach, a manual trochar or electric drill can be used (Figures 2 and 3). In our center, modified Bordier trephine (Lepine à Lyon, Lyon, France) with trochar core diameter of 5 to 7 mm is used. This needle consists of four parts: A pointed trochar; guide sleeve with sharp, serrated edges; trephine biopsy needle; and blunt extractor (Figure 3) (17). Regardless of the instrument and technique used, the expertise of the operator plays a significant role in obtaining an intact bone core. Care should be taken in performing the procedure so that the bone sample is not fractured or crushed. It should contain two cortices separated by trabecular bone if the transiliac approach is used. In children, the vertical approach has not been widely used because the presence of the growth plate at the top of the iliac crest may be a confounding factor (22). Bone specimen below the growth plate is normally char-

51 S166 Clinical Journal of the American Society of Nephrology Clin J Am Soc Nephrol 3: S164 S169, 2008 Figure 2.Electric drill for horizontal and vertical iliac crest biopsies. Figure 3. Modified Bordier trephine, from top to bottom, includes a pointed trochar, an outer guide or sleeve, a trephine, and a blunt extractor. might be problematic, especially in the relatively dehydrated postdialysis patient, and PD patients should be advised to decrease the dialysate dextrose concentration the night before biopsy. Combinations of narcotic, typically fentanyl, and benzodiazepine, often midazolam, have been used successfully as well. Local anesthetic should be used to decrease procedural stimulation and provide some postprocedure anesthesia. The patient is placed in the supine position with the ilium and umbilicus exposed; the anterior ilium is cleaned with chlorhexidine or povidone-iodine solution and draped. The biopsy site is located 2 cm posterior to the anterior-superior iliac spine (Figure 4) (20). This site is easily accessible and associated with minimal complications (23). Approximately 10 ml of 1% lidocaine is used to anesthetize the skin, subcutaneous tissue, and periosteum of the iliac crest (Figure 5). The skin and the subcutaneous tissue are anesthetized with lidocaine using a 25-G needle. The periosteum is anesthetized with lidocaine using a 1.5-inch 20-G needle and injecting while moving over the surface of the lateral ilium on an area of 1 to 2 cm. A vertical skin incision of 0.5 to 1.0 cm is then made at the previously identified site using a scalpel with a No. 11 blade. The underlying muscle and fascia are separated by blunt dissection until the lateral iliac periosteum is exposed (Figure 6). Before doing the biopsy, the trephine biopsy needle is filled with bone wax (using a 1- to 2-cm block of wax), which will help to secure the bone specimen within the cutting trephine while it is being withdrawn. The pointed trochar is inserted through the outer guide sleeve and then inserted through the skin incision. The outer guide and pointed trochar are then applied firmly to the exposed bone, pointing toward the umbilicus. The outer guide is then rotated until it is firmly implanted and anchored to the lateral ilium (Figure 7). Making sure that the outer guide is firmly seated on the bone surface prevents slippage of the biopsy trephine along the surface of the bone when it is advancing through the sleeve during the biopsy. At this time, the pointed trochar is then withdrawn and the trephine is inserted through the outer guide. An assistant stands on the other side acterized with high bone turnover and has very low cortical thickness. Biopsy Procedure The biopsy should be performed in a procedural area that is equipped to monitor a sedated patient and provide for a monitored recovery. In hemodialysis patients, we avoid doing the procedure during dialysis day to avoid hematoma and bleeding from heparin exposure. Peritoneal dialysis (PD) patients are instructed not to have a day dwell of PD fluid on the day of the biopsy. The patient should be instructed to fast before the procedure. Moderate to deep sedation is required and should be administered by personnel who are trained and credentialed to do so. In our center, a combination of fentanyl for analgesia combined with propofol infusion for sedation has resulted in excellent biopsy conditions and rapid recovery. Hypotension Figure 4. The biopsy site is identified 2 cm posterior to anterior iliac crest (dotted line outlines the iliac crest).

52 Clin J Am Soc Nephrol 3: S164 S169, 2008 Iliac Crest Biopsy for Evaluation of Renal Osteodystrophy S167 Figure 5. Lidocaine (10%) is used to anesthetize skin, subcutaneous tissue, and periosteum. Figure 7. The pointed obturator together with the outer guide is inserted and applied firmly to the exposed bone. The guide is rotated and implanted on the lateral ilium. Figure 6. After an incision is made, the muscle and fascia are separated by blunt dissection until the periosteum is exposed. of the patient and holds the hips down. The trephine is rotated clockwise with a steady moderate pressure and gradually increased until the cutting action on the bone is felt and the trephine has advanced through the full depth of the iliac crest (Figures 8 and 9). For obtaining an intact bone core, it is essential to use a gentle, steady pressure to advance the trephine, particularly in patients with fragile osteoporotic or soft bones (20). Heavy pressure should be avoided. After the inner cortical bone is penetrated, the trephine is rotated 360, first in a clockwise direction and then counterclockwise. This step would help to free the biopsy specimen from the connective tissue at the inner periosteal surface. The trephine is then removed using a slow, rotating, counterclockwise motion, and once free, the outer guide is removed while gauze is placed over the incision site. The blunt extractor is inserted through the top of the trephine, gently pushing out the bone core specimen. If the specimen does not come out easily, then a few Figure 8. The trephine is inserted into the outer guide and rotated counterclockwise with steady pressure until the cutting action of the trephine on the bone is felt. light taps with the handle of the large trochar would suffice to expel the bone core. Bone biopsy can also be done using a new electric drill (Straumann, Cambridge, MA; Figure 2) with a one-step drilling and extraction procedure. This drill is now widely used and has the advantage of providing easier and shorter surgical time for performing bone biopsies. The use of disposable drill bits in this new instrument decreases infectious complications, avoids problems with dull drill bits, and eliminates the need for frequent resharpening (21). After incision of the skin is made and blunt dissection of the subcutaneous tissue is done, a funnelshaped winged positioner is introduced through the incision and placed over the iliac crest surface. The axis of the hand-held funnel is aligned by an assistant with the axis of the underlying bone to stabilize and prevent the trephine from exiting through

53 S168 Clinical Journal of the American Society of Nephrology Clin J Am Soc Nephrol 3: S164 S169, 2008 water then covering it with a Band Aid. The sutures are then removed 7 to 10 d after bone biopsy. Figure 9. Trephine advancing through the full length of the iliac crest. the pelvic bone during drilling. The trephine consists of a precutter drill bit, which can be placed first to remove subcutaneous tissue and then cut through the periosteum, and a core drill or actual trephine, which drills and separates the bone biopsy core from the surrounding bone. The precutter on the drill is placed in the center of the funnel, and the electric drill is engaged by pressing the first trigger or button (21). The actual trephine then replaces the precutter and is inserted into the funnel in the same manner. Drilling is done with minimal pressure and continues until the base of the drill just reaches the bottom of the funnel without touching it. Within the trephine, internal forceps fracture the core of bone graft away from the iliac crest. As the sleeve of the drill is pulled slowly upward while continuing to drill, extraction forceps move over the bone core and capture the sample. The entire trephine is now removed slowly upward while the drill is still rotating. An internal plunger is used to push out the core of bone from the trephine. The operator ejects the bone core sample while pressing the second button on the drill. Sterile medical wax is then used to plug the cavity created by the trephine. It is important that care and prevention of exerting undue pressure while using the drill be observed because bone powder can accumulate in the periphery of the trabecular bone. Avoidance of using high speed in drilling can prevent creation of heat artifacts on bone cells (3). The bone specimen after the biopsy is placed in a container filled with 10% phosphate-buffered formalin. The incision is approximated and closed with 3-0 nylon sutures. Povidoneiodine ointment is applied, and the site is covered with a small adhesive pad (Band Aid; Johnson & Johnson, New Brunswick, NJ) and elastic pressure dressing. The procedure takes approximately 15 to 20 min, and blood loss is less than 1 ml. When fully awake after sedation wears off, the patient can get out of bed in 3 h and go home the same day. The pressure dressing is removed after 24 h and replaced with a Band Aid. The patient can shower after 48 h, cleaning the incision site with soap and Specimen Handling and Processing In our center, biopsy specimens are fixed in commercially prepared 10% phosphate-buffered formalin and kept at room temperature for 24 h. The specimen is then transferred into 70% ethanol alcohol and sent at room temperature to the laboratory. In other centers, however, the bone specimen is immediately placed in 70% ethanol after the bone biopsy procedure. Regardless of the initial fixative used, specimen should be placed right away into the solution to preserve the bone tissue. Duration should not exceed 48 h because tetracycline labeling can be washed out with prolonged fixation. Concentrated formalin should not be used because it has the tendency to leach out calcium, aluminum, and tetracycline from bone (3). After the initial placement into the fixative solution, the specimen is dehydrated in alcohol and later embedded in methylmethacrylate. Cutting of the specimen is done using a special microtome. The sections are later stained with either toluidine blue or Masson-Goldner trichome. Two computer-based software applications are commercially available to facilitate bone histomorphometric measurements: OsteoMeasure (Osteometrics, Decatur, GA) and Bioquant Osteo II (Bioquant Image Analysis Corp., Nashville, TN). The software applications are userfriendly and automatically calculate more than 100 bone parameters based on American Society for Bone and Mineral Research nomenclature. Complications Complications as a result of bone biopsies can include pain, hematoma, wound infection, and, rarely, neuropathy. Studies have shown that horizontal and transiliac bone biopsies are associated with very small morbidity and no mortality (24). The operator s experience is an important factor in minimizing morbidity and ensuring specimen adequacy. Our group has performed more than 700 bone biopsies in pediatric patients in the past several years with excellent results and with minimal morbidity. Allergic reactions, gastrointestinal disturbances, and photosensitivity secondary to tetracycline intake can occur. Giving the drugs after meals can diminish gastrointestinal upset, and avoiding sun exposure while taking tetracycline prevents skin photosensitivity. The bone procedure described here is generally well tolerated with minimal pain and discomfort. Pain is the most feared complication, which can be easily prevented by use of generous amounts of local anesthetic over the skin and periosteum (20). Osteomyelitis and skin incision site infection can be avoided by adherence to strict aseptic techniques during the procedure (19). Duncan et al. (25) surveyed complications in 9131 transiliac biopsies as reported by physicians who performed the procedure. The total incidence of complications was 0.6%: 22 (0.24%) patients had hematoma, 17 (0.20%) had pain, 11 (0.12%) had transient neuropathy, and six (0.07%) had wound infection. Two (0.02%) patients had hip fractures, and one (0.01%) had osteomyelitis. No deaths or permanent disability was reported.