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. It is convenient to core diameter as mean length" and of because true for cortical and cancellous width for are given by the relationships between length and area in the to of representative bone biopsies from different transiliac cortex on vertical on by transiliac biopsy from and with of sections through cylindrical biopsy core of of perpendicular on oblique on core width; core cortical width; cancellous to core area cortical area cancellous area the inner and outer periosteum do not from and their mean length is used for these relationships remain true for the oblique section because the areas enclosed by the and are the relationships can be used to and without measuring the of For long bone cross sections (Fig. bone diameter is similarly subdivided into two cortical widths and either cancellous diameter for cross or marrow diameter for diaphyseal cross sections. The relationships between these and bone area, cortical area, and cancellous or marrow area depends on the of the cross section. For such measurements may be needed at multiple in relation to the in vivo For both iliac and long bone it is necessary for purposes to recognize a between cortical and cancellous bone tissue and in and This is not in or because methods of its are not yet fully A has been used to this in quantitative This may be applicable to iliac bone biopsy but this has not yet been For all bones, all surfaces in with bone marrow are referred to as and are subdivided into cancellous bone surface and the latter is the inner of the cortex. between these is to unless made in accordance with some and will also on whether the is measured separately. surfaces not in with bone marrow are generally referred to as cortical with as the cortical surface can also be referred to as the Haversian or osteonal of cross sections through the of a long bone; is on the and diaphyseal is on the For the cancellous bone of the is not bone cortical width; cancellous marrow The standard and applicable method for reporting all data should be that the of to as well as to the and are used only as and are used only as described refers to the structure on which the measurement was whether this was a particular surface or a particular type of tissue. of the commonly used have been defined many are by using the lexicon 1). If measurements are restricted to some of a such as the outer of a or the central of cancellous the same can be but the appropriate should be made in the of For measurements made on the entire the source is identified as it will not be necessary to the source each time a particular is referred only one source is used in an it need only be If are their can be used as for of results in or and in most cases will need to be only if measurements from are discussed such that confusion between them is For some such as only one source is possible and its is redundant. The three and the two are the key needed to from one referent to is to in stereologic terminology, and and are to in stereologic are derived from the corresponding two-dimensional and either by which is correct for or by which has been determined for human iliac cancellous The with so that the always be stated and and to in stereologic terminology and are with the corresponding and For some a of the bone surface is needed as a referent surface and surface are often related to osteoid surface usually and it can be to osteoclasts to the mineralized surface as an alternative to the more usual referents bone surface and surface indices of bone formation can be related to the osteoblast surface or to the number of osteoblast profiles as well as to osteoid surface or bone it may be appropriate to use the between mineralized bone and or bone as a referent for the length of or of because the is these features are In many as when only one referent is used for each measurement, the referent need only be and not each time the measurement is If more than one referent is measurements with the same referent can be to are listed with abbreviations in both and in Table have been defined but some need additional is used for of mineralized bone volume and is given by volume osteoid Osteoid may need to be as or as the in the lexicon between which refers to a and which refers to a for all measurements are with the indices discussed is a general term applicable to all tissue that is not and includes marrow in cancellous bone and Haversian and canals in cortical bone. For both types of tissue, can with area measurements on individual such as cells or cortical The profiles can be as an of tissue, by use of the appropriate referent. For is the area of all cell profiles referred to the area of tissue and in terms. The profiles can also be as individual by absence of a is the mean area of individual If confusion is the term be as or mean areas in cannot be to mean volumes in unless the structures are in cylindrical mean area can be used to but it is to this as described later. Osteoid do not so that some width should be for measurement of osteoid surface We the terms formation surface and resorption surface because the implications of current may be and for the same reason we the surface is with or lacunar surface and the osteoclast surface and the surface individual can also be as osteoclast or osteoclast Some cells and methods are needed for and the cells on the surface or cells. surface is with or the term that will at some future The of connective tissue the flat lining cells on quiescent surfaces should not be referred to as It is possible that some surface by flat lining cells should be as quiescent surface rather than as In all distance measurements can be obtained in two by measurement at multiple or by calculation from measurements of area and The method is more and can a and a standard deviation as well as a mean but that measurement be randomly The method is less and less to sampling The method is usually used for distance between and cell and dimensions, and the method is usually used for thickness diameter and methods are widely used for osteoid thickness and cortical The method is for the from the relationships between individual measurement at particular and at particular during the The mean determined by either method in an individual be distinguished from the mean in a of Mineralized thickness is the distance from the line to the between bone and It is used in and in different types of osteoid and different stages of in the mean should be to the between thickness and osteoid thickness is measured on an individual it has been used in the for calculation of the of and in human subjects as an of in thickness is the mean distance between on of a usually as for the is an of bone from the cortical surface, but too is known of the internal of iliac cortical bone to