jbm > Volume 31(2); 2024 > Article
Sellman, Tong, Burton, and Kröger: Retrospective Characterization of Bone Histomorphometric Findings in Clinical Patient Specimens

Abstract

Background

Bone histomorphometry provides comprehensive information on bone metabolism and microstructure. In this retrospective study, we aimed to obtain an overview of the typical indications, referring hospitals, and histomorphometric quantification-based diagnoses of the bone tissue in our histomorphometry laboratory, the only laboratory in Finland carrying out histomorphometric examination of clinical bone biopsies.

Methods

Between January 1, 2005 and December 31, 2020, 553 clinical bone biopsies were sent to our histomorphometry laboratory for histomorphometric examination. The median age of the patients was 55 years (range, 0.2-89.9 years), 51% of them were males, and 18% comprised pediatric patients. We received bone biopsy specimens from 23 hospitals or healthcare units. The majority of the samples we sent by nephrologists.

Results

The most common bone biopsy indications were suspicion of renal osteodystrophy (ROD), unknown bone turnover status in osteoporosis, and several or untypical fractures. The most common quantitative bone histomorphometry-based diagnosis was ROD.

Conclusions

This study provides information on the clinical application of bone histomorphometry in Finland. Precise and quantitative ROD evaluation is the most common indication for bone histomorphometry, being crucial in clinical decision-making and targeted treatment of this patient group.

Graphical Abstract

INTRODUCTION

Histomorphometric analyses of bone tissue provide reliable quantification of bone metabolism and microarchitecture. It is a valuable tool for studying the etiology and pathogenesis of metabolic bone diseases (MBDs).[1] Histomorphometric analysis should always be interpreted along with patient history and laboratory data. Bone histomorphometry can provide critical information when suspicion of a mineralizing defect or a rare MBD is raised and assessing the response to their treatment.[2]
As one of the complications of chronic kidney disease (CKD), related bone abnormalities (CKD-MBD) can be referred to as renal osteodystrophy (ROD) which contributes to the elevated fracture risk in these patients.[3] In diagnostics and classification of ROD, bone histomorphometry has been considered the gold standard. Yet the challenge is the paucity of nephrologists trained for the procedure.[4] As an invasive procedure, iliac bone biopsy-related discomfort and risk should be proportionate to the gained information.[2] It is important that each bone histomorphometry laboratory uses a normative database for quantitative parameters.[4] Recently, a need for harmonization of reference values has been raised.[5]
In this study, we present typical indications of patients referred to bone histomorphometry examination in Finland and diagnoses based on quantitative bone histomorphometry of bone biopsy specimens.

METHODS

1. Bone biopsies

Iliac crest bone biopsies were taken from patients in 21 healthcare units/hospitals in Finland and 2 healthcare units in Sweden. The data include referral indications for bone biopsy and parameters, based on reports in our histomorphometry laboratory during January 1, 2005 to December 31, 2020.
For fluorochrome double labeling, the patients had 2 short tetracycline courses (tetracycline 500 mg x3 p.o. for 2 days) with an interval of 10 days, and the biopsy was taken 4 to 5 days after discontinuation of medication. However, in case of unscheduled surgeries, tetracycline labeling could not be implemented. Bone biopsies were obtained using either vertical or horizontal techniques. In vertical technique, a biopsy was obtained from a standardized site 2 cm posterior to the anterior superior iliac spine using an 8 to 11 G needle (T-Lok; Angiotech, Reading, PA, USA) and in horizontal technique 2 cm below and posterior to the anterior superior iliac spine in horizontal technique using a 7.5 mm bone biopsy trephine (Rochester Bone Biopsy Trephine; Medical Innovations International Inc., Rochester, MN, USA).[6,7]
Iliac crest specimens were stored in 70% to 80% ethanol and sent to our histomorphometry laboratory. The specimens were dehydrated in ethanol (70%) before being embedded in methylmethacrylate (MMA) according to standard protocols.[8] After embedding, 5 μm thick sections were cut using a microtome (Reichert-Jung; Cambridge Instruments, Heidelberg, Germany) and stained with Masson Goldner trichrome stain by light microscopy. 10 μm sections were cut for ultraviolet microscopy.

2. Bone histomorphometry

The histomorphometric analyses were conducted using Bioquant Osteo I and II (Bioquant Image Analysis, Nashville, TN, USA) August 19, 2009 to May 7, 2018 and OsteoMeasure system (OsteoMetrics, Atlanta, GA, USA) May 14, 2018 to December 31, 2020. The nomenclature, abbreviations, and parameters follow the recommendations of the American Society for Bone and Mineral Research (ASBMR).[9] For each biopsy specimen 1 or 2 sections were randomly selected and analysed with bone histomorphometry. Biopsy specimens were analysed by one of 3 experts (Heikki Kröger, Inari Burton, or Xiaoyu Tong). The histomorphometric diagnosis was set by Heikki Kröger. Regions of interest were carefully selected to ensure that only trabecular bone was measured, i.e., no cortical or subcortical bone. Biopsy specimens were measured by using a magnification of 200x.
Bone volume over total volume (BV/TV, %) was determined using the whole field of view including bone and TV. Osteoid (unmineralized bone tissue) volume/TV (OV/TV, %) and OV/BV ratios were determined similarly. Eroded bone surface (ES/BS, %) and OS/BS (%), were measured as well. Osteoblasts and osteoclasts were counted (Ob.S/BS, Oc.S/BS, %) along the related bone surface. Osteoid thickness (O.Th, μm) was determined by the average thickness of osteoid seams. Dynamic parameters, mineralizing surfaces (MS/BS, %), and mineral apposition rate (MAR, μm/d), were defined using ultraviolet microscopy on unstained sections. Bone formation rate per unit of BS (BFR/BS, μm3/μm2/d) and activation frequency (Ac.F, N/y) were calculated accordingly. Mineralization lag time (d) was determined using adjusted MAR for MS and O.Th. The histomorphometric diagnosis was based on the parameters of trabecular BV, bone turnover, and mineralization in combination with the patient history. Our histomorphometry laboratory uses published normative values to set bone histomorphometric diagnosis.[10-13]
We classified the bone biopsies in groups by referring healthcare unit, biopsy indications, tetracycline labeling, diagnosis, quality of biopsy specimen, and histomorphometric parameters. Biopsies could have up to 4 indications and up to 3 diagnoses.

3. Statistical analysis

The data was analysed after collecting and pooling it into Microsoft Excel. Biopsies were classified by referring healthcare unit, indications, and diagnoses. Histomorphometric parameters were compared between diagnostic groups which were defined by the BV (osteoporosis, normal bone) or based on the bone metabolism (accelerated, decreased, or normal bone metabolism). Mean values and standard deviations (SD) were calculated for age and histomorphometric parameters. Shapiro-Wilk test was used to determine whether the data was normally distributed. Variations of histomorphometric parameters between different diagnostic groups were evaluated by Kolmogorov-Smirnov Z tests due to the non-normal data distribution and narrow as well as unequal group size.[14] The Bonferroni correction was applied. All analyses were performed using SPSS Statistical software (version 22; SPSS Inc., Chicago, IL, USA). All P-values less than or equal to 0.05 were considered statistically significant.

RESULTS

From January 1, 2005 to December 31, 2020, 553 patients were referred for bone histomorphometry to our histomorphometry laboratory (Fig. 1). Median age 55 years, range 0.2 to 89.9 years, 51% males. A total of 97 of the biopsies (18%) were from pediatric patients (under 18 years old). All biopsy specimens had written referrals and report sheets. In this study, the histomorphometric parameters are reported only for 311 cases, whose data were available in electronic format.
Bone biopsies were sent from 21 hospitals/healthcare units in Finland and 2 in Sweden. The majority of the biopsies were sent from Helsinki University Hospital, the division of nephrology. Referring healthcare units and the number of bone biopsies sent from them are shown in Supplementary Table 1.
Table 1 demonstrates indications for bone biopsy. There could be more than one indication, in which case all the indications were listed. The most common indications for bone histomorphometry were suspicion of ROD or osteoporosis (e.g., unknown origin or turnover state). In addition, biopsies were taken from fracture cases (e.g., untypical, several) and from pediatric patients (e.g., several fractures).
Histomorphometric findings and diagnoses are shown in Table 2. The most common findings were osteoporosis, ROD, and normal bone turnover. Biopsies could have up to 3 separate findings/diagnoses.
ROD diagnoses are shown in Table 3. ROD diagnoses were further divided into subgroups i.e., hyperparathyroidism, mild hyperparathyroidism, mixed uremic osteodystrophy, mineralization defect, osteomalacia, and osteitis fibrosa. Biopsy specimens were also divided into groups by BV and bone turnover. There were 2 groups related to the amount of bone: normal bone (N=117), osteoporosis (N=166), and 3 groups related to bone metabolism: accelerated (N=38), normal (N=14), and decreased (N=33) bone metabolism.
Figure 2 demonstrates the differences (mean and SD) in histomorphometric parameters between normal, decreased and accelerated bone turnover. Evidently, the significant differences were between accelerated and decreased bone metabolism in OV/BV, OS/BS, ES/BS, O.Th, Ob.S/BS, Oc.S/BS, MAR, MS/BS, and Ac.F.

DISCUSSION

This study is a retrospective analysis of the typical clinical indications, referring medical specialties and healthcare units, and most common histomorphometric findings and diagnoses in Finland from years 2005 to 2020.
The median age of the patients was 55 years and 51% of the patients were males. Eighteen percent were pediatric patients. The indications for pediatric bone histomorphometry included for example suspicion of genetic osteoporosis in presence of the family history of severe osteoporosis, low-energy fractures, short stature, renal disease, or Turner’s syndrome. It is interesting to note that half of the bone biopsies were obtained from men. Since osteoporosis is a more common condition in women,[15] the majority of bone densitometry scans are performed in women. However, secondary osteoporosis is proportionally more common in men.[15] This might explain a high number of male bone biopsies in our study. In cases of severe osteoporosis with fractures, it is important to differentiate between low and high bone turnover when planning osteoporosis treatment, and exclude mineralization defects.
Bone biopsies were sent to our histomorphometry laboratory from several medical specialties and 23 different hospitals/healthcare units. This indicates that bone tissue may be affected by several medical conditions. Our histomorphometry laboratory is the only laboratory that examines clinical bone biopsies in Finland. Although we report our data from the years 2005-2020, bone histomorphometry laboratory in Kuopio started in early 1980,s and since that several thousand bone histomorphometric analyses have been performed in our laboratory.
The annual number of biopsy specimens analyzed in our histomorphometry laboratory is relatively low, under 65 specimens annually between 2005 to 2020. This suggests that non-invasive diagnostics may have partly replaced bone biopsy. Further, the recognition and familiarity of bone histomorphometry have weakened. However, bone histomorphometry is a precise method used particularly in unclear clinical situations. For instance, when the treatment requires confirming the actual bone status such as in case of many atypical fractures, it is necessary to exclude MBDs as an underlying cause for the fracture.
Most bone diseases can be diagnosed with clinical, radiological, and biochemical examinations which are non-invasive methods. These methods are developing and already provide three-dimensional structural parameters from hip and spine that correlate with the ones measured by histomorphometry, although the latter has been proven to provide much better spatial resolution and represents much more accuracy in evaluating microarchitecture.[16] It has been suggested that high-resolution peripheral quantitative computed tomography (HR-pQCT) could be used to evaluate the effects of kidney disease on cortical, trabecular microarchitecture and to predict the risk of fracture.[17] However, it is still debated whether the addition of HR-pQCT parameters to dual energy X-ray absorptiometry effectively improves fracture discrimination.[18] On the other hand, only mineralized tissue can be detected by non-invasive approaches and none of these approaches demonstrated an adequate diagnostic accuracy for CKD.[19] Bone histomorphometry is the only method used to ensure bone status at the tissue and cellular level. Even though non-invasive biochemical markers are part of the diagnostics in many bone diseases, bone histomorphometry remains the gold standard for the diagnosis and specific classification of ROD.[20] Further development of image analyzers and techniques will promote and fasten the analysis of bone biopsy in the future.[21]
Bone histomorphometry has limitations as an invasive procedure, and therefore, it is not as practical as a first-line clinical tool. Moreover, the rarity of bone histomorphometry laboratories also limits its wider use. Also, the variation and shortage of reference data could limit its widespread use in clinical settings. Reference data is at risk of selection bias. Due to its invasive procedure, the reference data is usually based on a small group of biopsy specimens. Many factors such as age, sex, and ethnicity have an impact on bone turnover and there is no consensus on how these factors should be considered in reference data.[5]
Tetracycline labeling is necessary for complete histomorphometric analysis. However, it was unsuccessful for various reasons in some cases, and because of that, dynamic parameters could not be defined for all biopsy specimens in this study. In total, 20% of the biopsy specimens were either not labeled accordingly, not labeled at all, or there was no mention of the labeling at all (likely acute cases during surgery).
Because of the 16-year retrospective study design, histomorphometry equipment and laboratory technicians have changed over the years. We have also updated the reference data according to new research published. In terms of methodology, the variance comes mainly from sampling, staining procedures, and measuring methods.[21] However, these changes have not affected the establishment of a histomorphometric diagnosis, that is based on the same histomorphometry specialist during the whole study period (HK). Despite the limitations, histomorphometric analysis of bone biopsy remains the best clinical approach to describe the static and dynamic parameters of bone turnover in patients with MBD. Our study agrees that precise quantitative evaluation of ROD is the most common indication for bone histomorphometry.[22]
This study contributes to understanding the utilization of bone histomorphometric examination and its various applications within clinical settings. By investigating the usage patterns and distribution of bone histomorphometry indications across different medical specialties, our study provides valuable insights into its role in diagnosing and managing various bone-related conditions. Provided information can inform healthcare professionals about the scenarios where bone histomorphometry may be particularly beneficial, facilitating more targeted and efficient use of this diagnostic tool.

DECLARATIONS

Funding

The authors received no financial support for this article.

Ethics approval and consent to participate

This registry study used only anonymized data and did not require ethical committee approval. The study has been registered approved at The Science Service Centre of Kuopio University Hospital, Finland (no. 5203117, 21.9.2019, and 23.2.2021).

Conflict of interest

No potential conflict of interest relevant to this article was reported.

Fig. 1
The annual number of patients undergoing bone histomorphometric analysis in our histomorphometry laboratory.
jbm-2024-31-2-132f1.jpg
Fig. 2
Mineral apposition rate (MAR), mineralizing/bone surface (MS/BS), and activation frequency (Ac.F) compared between accelerated bone turnover (N=19), decreased bone turnover (N=7), and normal bone turnover (N=41). Osteoid/bone volume (OV/BV), osteoid surface (OS/BS), eroded surface (ES/BS), osteoid thickness (O.Th), osteoblast surface (Ob.S/BS), osteoclast surface (Oc.S/BS), and BV over total volume (BV/TV) compared between accelerated bone turnover (N=24), decreased bone turnover (N=19), and normal bone turnover (N=53). The error bars indicate the standard deviation of the mean. The asterisk (*) highlights the distinction between the groups in which the significant difference exists. All P-values less than or equal to 0.05 were considered statistically significant.
jbm-2024-31-2-132f2.jpg
jbm-2024-31-2-132f3.jpg
Table 1
Indications for bone histomorphometric examination in our histomorphometry laboratory from 2005 to 2020 (N=553)
Indication for bone histomorphometry N (%)
Suspicion of renal osteodystrophy 338 (61.1)
Osteoporosis 195 (35.3)
Fractures 138 (25.0)
Secondary/tertiary hyperparathyroidism 118 (21.3)
Pediatric specimen 95 (17.2)
Research specimen 39 (7.1)
Osteomalacia 23 (4.2)
Organ transplant 23 (4.2)
Adynamic bone disease 18 (3.3)
Atypical fracture 13 (2.4)
Hyperparathyroidism 12 (2.2)
Hip/Pelvic fracture 7 (1.3)
Suspicion of unknown bone syndrome 6 (1.1)
Mineralization defect 6 (1.1)
Suspicion of metabolic bone disease 4 (0.7)
Paget’s disease 4 (0.7)
Alport syndrome 4 (0.7)
Celiac disease/Crohn’s disease 3 (0.5)
No indication 3 (0.5)
Turner variant 1 (0.2)
Down syndrome 1 (0.2)
Hypoparathyroidism 1 (0.2)
Table 2
Histomorphometric diagnoses and findings (N=553)
Histomorphometry diagnoses N (%)
Osteoporosis 166 (30.0)
Renal osteodystrophy 150 (27.1)
Normal bone 117 (21.2)
Hyperparathyroidism 114 (20.6)
Mild hyperparathyroidism 46 (8.3)
Non-diagnostic specimen 44 (8.0)
Hyperosteoidosis 40 (7.2)
Accelerated bone metabolism 38 (6.9)
Mixed uremic osteodystrophy 35 (6.3)
Decreased bone metabolism 33 (6.0)
Peritrabecular fibrosis 16 (2.9)
Normal bone metabolism 14 (2.5)
Mineralization defect 13 (2.4)
Osteomalacia 10 (1.8)
Osteitis fibrosa 2 (0.4)
Table 3
Renal osteodystrophy subgroups based on bone histomorphometric analysis (N=553)
Renal osteodystrophy subgroups N (%)
Hyperparathyroidism 114 (20.6)
Mild hyperparathyroidism 46 (8.3)
Mixed uremic osteodystrophy 35 (6.3)
Mineralization defect 13 (2.4)
Osteomalacia 10 (1.8)
Osteitis fibrosa 2 (0.4)

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ORCID iDs

Linnea Sellman
https://orcid.org/0000-0001-5389-1108

Xiaoyu Tong
https://orcid.org/0000-0002-7868-0478

Inari S Burton
https://orcid.org/0009-0004-3496-4067

Heikki Kröger
https://orcid.org/0000-0003-4245-8186

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