Standardized nomenclature, symbols, and units for bone histomorphometry: A 2012 update of the report of the ASBMR Histomorphometry Nomenclature Committee

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 19872 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. 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 refer to the combination of bone and associated soft tissue or marrow as “bone tissue.” “Tissue” is defined3 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 tissue4—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 textbooks4, 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 spine8 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. A two-dimensional histological section displays profiles of three-dimensional 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 three-dimensional 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 grid20-22 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 10°, stereologic problems in diaphyseal cortical bone 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 three-dimensional 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” (Vv) and surface area per unit volume is “surface density” (Sv). 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 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 two-dimensional measurements of width (and corresponding three-dimensional 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 bone18, 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. 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 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. 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. “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 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 Faugere5 with permission. Diagram of sections through cylindrical biopsy core of ilium. Direction of trephine perpendicular on left, oblique on right. C.Wi = core width; C.Dm = core diameter; Ct.Wi = cortical width; Cn.Wi = cancellous width. Relationships to areas: C.Ar = core (or section) area = C.Dm*C.Wi; Ct.Ar = cortical area = C.Dm*Ct.Wi; Cn.Ar = cancellous area = 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. 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 error39 unless made in accordance with some well-defined rule40 and will also 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. 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 = bone diameter; Ct.Wi = cortical width; Cn.Dm = cancellous diameter; Ma.Dm = marrow diameter. 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 cortex41 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. OV/BS*BS/BV = OV/BV OV/BS*BS/TV = OV/TV = OV/BV*BV/TV OV/BS*BS/CV = OV/CV = OV/BV*BV/CV 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 Sv (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/π (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 Vv (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. 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. “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 bone44 and includes marrow in cancellous bone and Haversian and Volkmann canals in cortical bone. For both types of tissue, porosity (Po) = 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 (). 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. 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(Oc+), 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. 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 reconstruction45 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 accumulation50 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 deci