DISCLAIMER: This document was originally drafted in French by the Institut national d'excellence en santé et en services sociaux (INESSS), and that version can be consulted at www.inesss.qc.ca/index.php?id=49&L=1. It was translated into English by the Canadian Agency for Drugs and Technologies in Health (CADTH) with INESSS’s permission. INESSS assumes no responsibility with regard to the quality or accuracy of the translation.

While CADTH has taken care in the translation of the document to ensure it accurately represents the content of the original document, CADTH does not make any guarantee to that effect. CADTH is not responsible for any errors or omissions or injury, loss, or damage arising from or relating to the use (or misuse) of any information, statements, or conclusions contained in or implied by the information in this document, the original document, or in any of the source documentation.

Histomorphometric Analysis of a Non-decalcified Bone Sample

(for Non-complex Diseases and for Complex OM-, ROD-, and

HPTH-type Diseases)

April 2013

1

1 GENERAL INFORMATION

1.1 Submitting Company, Institution or Organization

Centre Hospitalier de l’Université de Montréal (CHUM), Hôpital Saint-Luc (Saint-Luc Hospital, University of Montreal Hospital Centre)

1.2 Application Submitted: July 16, 2012

1.3 Notice Issued: April 2, 2013

Note:

This notice is based on the scientific and commercial information (submitted by the requestor(s) and on a complementary review of the literature) according to the data available at the time that this test was assessed by INESSS.

2 TECHNOLOGY, COMPANY AND LICENCE(S)

2.1 Name of the Technology

Bone histomorphometry.

2.2 Brief Description of Technology

Bone histomorphometry is a histopathological analysis that is performed on non-decalcified bone specimens and that provides qualitative and quantitative information on bone structure (cortical and trabecular) and bone remodelling.

Histomorphometry of bone biopsy specimens is regarded as a reliable, well-established procedure for analyzing the pathogenesis of metabolic bone diseases, as well as for evaluating the mechanisms by which various drugs act on bone tissue (Kulak and Dempster, 2010; Hernandez et al., 2008; Lerma, 2007).

Histomorphometry is a diagnostic and therapeutic test.

2.3 Company or Developer

This analysis was developed in house at the Metabolic Bone Diseases Laboratory of the CHUM research centre. This laboratory is directed by Dr. Louis-Georges Ste-Marie, an endocrinologist. It was founded in 1984 and is the only Canadian laboratory that performs histomorphometric analysis on bone biopsy specimens.

2.4 Licence(s): N/A

2.5 Patent, if Applicable: N/A

2.6 Approval Status (Health Canada, FDA): Bone histomorphometry does not require the use of kits licensed by Health Canada.

2

2.7 Weighted Value

$1,359 (non-complex diseases)

$1,666 (complex diseases)

3 CLINICAL INDICATIONS, PRACTICE SETTINGS, AND TESTING PROCEDURES

3.1 Targeted Patients

Various categories of patients with bone disease whose etiology needs to be better defined: young people with unexplained bone fragility, fracture disease despite appropriate treatment of the bone, renal osteodystrophy, rare bone diseases (information taken from the form submitted by the requestor).

3.2 Clinical Indications

Histomorphometry of a bone biopsy specimen is generally used for the following purposes:

- to confirm or exclude a diagnosis of osteomalacia (Kulak and Dempster, 2010; Compston, 2005; Eriksen et al., 1994);

- to characterize the various forms of renal osteodystrophy (Kulak and Dempster, 2010; Hernandez et al., 2008; Compston, 2005; Eriksen et al., 1994);

- to diagnose certain rare metabolic bone diseases (Compston, 2005; Eriksen et al., 1994);

- to assess patients’ response to treatment of certain bone diseases, such as osteomalacia (Eriksen et al., 1994);

- to diagnose osteopenia in patients less than 50 years old and in patients with abnormal calcium metabolism (Eriksen et al., 1994);

- to investigate cases of unexplained bone fragility (Kulak and Dempster, 2010). In such cases, biopsy should be used when the diagnosis cannot be confirmed by non-invasive methods (Rauch, 2009).

Histomorphometry of a bone biopsy specimen is not recommended for diagnosing osteoporosis, in particular because of:

- the heterogeneity in the bone loss caused by osteoporosis (Kulak and Dempster, 2010; Compston, 2005);

- the low correlation between the bone mass present in biopsies of the iliac crest and that present in other bones, such as the head of the femur (Compston, 2005);

- the possibility of using other, less invasive methods, such as bone densitometry.

3.3 Targeted Diseases: See Table 1.

3.4 Number of Patients to be Tested: See Table 1.

The application form states that the expected provincial volume for the coming three years is about 60 biopsies per year (about one per week).

3

3.5 Medical Specialists Involved

Nephrologists, endocrinologists, pathologists, orthopaedists, family physicians.

3.6 Testing Procedure

Histomorphometric analysis requires a non-decalcified bone sample, which involves taking a bone tissue biopsy. When the remodelling process is being analyzed, the patient receives two separate doses of tetracycline or a tetracycline derivative, 10 to 14 days apart, about three weeks prior to the biopsy. The biopsy is generally performed two to five days after the second dose of tetracycline is administered.

The biopsy is taken from the iliac crest. The anterior iliac crest is the preferred site, because it can provide a sample that contains both cortical and trabecular bone tissue (Dempster, 2008; Compston, 2005; Rao, 1983). The structure and cellular activity of the bone tissue at this site are similar to those observed at other locations in the skeleton, such as the hips and the vertebrae (Dempster, 2008). The procedure takes less than an hour, and the patient can go home the same day (Hernandez et al., 2008). Morbidity associated with this kind of biopsy is low. The main adverse effects associated with it are as follows:

- discomfort for 24 to 48 hours after the procedure (Rao, 1983);

- bruising (occurring mainly in people who are obese, or who are predisposed to hemorrhagic diseases, or are on hemodialysis) (Compston, 2005; Rao, 1983; Revell, 1983);

- infection, osteomyelitis, fracture of the iliac crest, temporary paralysis of the femoral nerve (Compston, 2005; Revell, 1983);

- the incidence of complications associated with transiliac bone biopsies is less than 1% (Compston, 2005; Revell, 1983). In this regard, an international, multi-centre study involving 9,131 transiliac biopsies showed complications in 64 patients (0.7%) (Dempster, 2008; Hernandez et al., 2008; Rao, 1983).

The sample is fixed with formalin, 70% ethanol, or methanol, and the non-decalcified bone tissue is then embedded in a plastic resin.

Thin sections (5 μm) are then sliced using an automated microtome with a tungsten carbonate blade.

The sections are then stained as required. In this regard, several different kinds of stain are available in order to:

- visualize the cells composing the bone tissue;

- differentiate mineralized bone from osteoid tissue (unmineralized bone matrix).

If appropriate, unstained sections can also be prepared, to view tetracycline labelling with a fluorescent microscope (Dempster, 2008).

The histological sections are then analyzed using a system that consists of a drafting table, a high-resolution camera mounted on an optical, polarized, or fluorescent microscope, and a computer equipped with OsteoMetrics software, specially designed for this purpose (Kulak and Dempster, 2010; Compston, 2005).

4

The histomorphometric variables are calculated from the measurements made with the microscope, such as the surface area, perimeter, and thickness of the bone structure. The nomenclature used in histomorphometry has been standardized by the American Society of Bone and Mineral Research (Parfitt et al., 1987).

This histomorphometric analysis can be used to assess several parameters, which are generally divided into two categories:

- ultrastructural parameters, which indicate the composition, mass, and structure of the bone;

- remodelling parameters, which provide information on the remodelling process. These parameters can be analyzed through the administration of labels (tetracycline) that are deposited at the sites of bone formation.

The results obtained are then analyzed by comparing them with bone histomorphometry values from normal patients. Histomorphometry allows for an in-depth analysis of several metabolic bone diseases, because these diseases present distinctive histomorphometric profiles (see Table 1).

The biopsy should be performed by Dr. Ste-Marie at the CHUM, and the analyses (handling and preparation of bone specimens) will be performed in the Metabolic Bone Disease Laboratory.

5

Table 1: Targeted Patients and Diseases

Targeted Pathology

Short Definition Number of Cases

Diagnosed in Quebec

17

Histomorphometric Parameters Altered by the Pathology

2010 2011 Relative Osteoid Volume

Osteoid Area

Osteoclastic Resorption

Area

Calcification Front

Mineralization Rate

Comments

Osteomalacia Deficient primary mineralization of the bone matrix.

Outcome: abnormal accumulation of unmineralized osteoid tissue, resulting in generalized softening of the bones and bone fragility.

Osteomalacia in adults—the equivalent of rickets in children—can result from insufficient vitamin D in the diet or, more often, insufficient absorption of vitamin D (because of pancreatic disease or gluten intolerance).

Osteomalacia can be due to bone poisoning by certain substances (aluminum, fluorine, biphosphonates) or to a large increase in blood phosphorus levels (chronic kidney disease).

56 63 ↑ ↑ ↑ ↓ ↓

17

Source: RAMQ; fichier des services rendus à l’acte - médecins.

6

Targeted Pathology

Short Definition Number of Cases

Diagnosed in Quebec

17

Histomorphometric Parameters Altered by the Pathology

2010 2011 Relative Osteoid Volume

Osteoid Area

Osteoclastic Resorption

Area

Calcification Front

Mineralization Rate

Comments

Renal Osteodystrophy

A set of complications of the bones and internal organs related to the disruptions in phosphorus and calcium metabolism caused by kidney disease.

432 360 ↑ or ↓ ↑ or ↓ ↑ or ↓ ↑ or ↓ ↑ or ↓

Hyperpara- thyroidism

Condition characterized by hypersecretion of parathyroid hormone by one or more parathyroid glands. This hormone increases the level of calcium in the blood by promoting its gut uptake.

2 137 2 160 ↑ ↑ ↑ ↑ ↓ Cannot differentiate primary and secondary hyperpara-thyroidism

7

4 TECHNOLOGY BACKGROUND

4.1 Nature of the Diagnostic Technology

Unique for characterizing the various forms of renal osteodystrophy. Histomorphometry of a bone biopsy specimen is regarded as the gold standard for diagnosing renal osteodystrophy (Hernandez et al., 2008; Lerma, 2007).

Complementary for confirming or excluding a diagnosis of osteomalacia. Diagnosis of osteomalacia is essentially histological, but clinical, biochemical, and radiographic tests can sometimes allow a diagnosis without histology (Compston, 2005; Oprisiu et al., 2000).

4.2 Brief Description of the Current Technological Context

Renal osteodystrophy is manifested by pain and radiographic bone abnormalities revealing a resorption of the extremities of the short long bones. Clinical and X-ray results, and some biochemical tests, can guide differential diagnosis among the various types of bone diseases, but histological analysis of a bone biopsy is the only way to make a definite diagnosis of osteodystrophy (Oprisiu et al., 2000).

In osteomalacia, X-ray can reveal a demineralized, “blurry” appearance of the skeleton, distortions, and bone cracks. Biochemistry may confirm reduced levels of blood calcium and phosphorus, a drop in calcium levels in the urine and in the active form of vitamin D in the blood, and increased blood levels of bone alkaline phosphatases. When the diagnosis cannot be confirmed by X-ray and biochemistry, bone biopsy and histomorphometry are required (Compston, 2005).

4.3 Brief Description of the Advantages Cited for the New Technology

Provides information that the other diagnostic approaches (such as bone densitometry, biochemical markers, and bone remodelling) cannot (Rauch, 2009; Compston, 2005; Eriksen et al., 1994).

For example, administering tetracycline or a tetracycline derivative prior to bone biopsy is the only method that allows bone resorption to be calculated (Compston, 2005).

Enables the histological diagnosis of certain bone diseases to be made with certainty and their severity and therapeutic response to be characterized more precisely (Compston, 2005).

Makes it possible to describe bone remodelling activities and characteristics and to calculate the activity of the various cells involved in the bone remodelling process. As such, histomorphometry is the only method that can determine whether the results observed are caused by a change in cellular activity or by a change in the number of cells involved in the bone remodelling process (Eriksen et al., 1994).

Enables several metabolic bone diseases to be analyzed in detail, because they have distinctive histomorphometric profiles (Kulak and Dempster, 2010).

Makes it possible to define the diagnosis and inform the treatment (Oprisiu et al., 2000).

Compared with the other kinds of histopathological analyses available, this method preserves the integrity of the (non-decalcified) bone tissue.

8

4.4 Cost of the Technology and the Options

Information not available.

5 EVIDENCE

5.1 Clinical Relevance (Utility and Validity) and Analytical Validity

5.1.1 Other Tests Replaced: N/A

5.1.2 Diagnostic or Prognostic Value: N/A

5.1.3 Therapeutic Value

Histomorphometry of a bone biopsy can be used to characterize the various forms of renal osteodystrophy and decide on a course of treatment (Kulak and Dempster, 2010; Hernandez et al., 2008; Compston, 2005; Eriksen et al., 1994). For example, some presentations may be associated with hyperparathyroidism, or with beta-2 microglobulin amyloidosis, or with aluminic bone disease. The treatments for these three conditions are totally different, so it is necessary to distinguish them with certainty, because a poor treatment decision can have serious consequences for the patient (for example, parathyroidectomy exacerbates aluminic bone diseases) (Oprisiu et al., 2000).

5.2 Clinical Validity

The reliability and security of the results are ensured by:

- the use of positive and negative control specimens in the histological staining procedures;

- the use of a micro-ruler to verify the histomorphometric analysis system used with the microscope.

Summary Table

Component Presence Absence Not Applicable

Sensitivity X

Specificity X

Positive predictive value (PPV) X

Negative predictive value (NPV) X

Likelihood ratio (LR) X

ROC curve X

Accuracy X

9

5.3 Analytical (Technical) Validity

Limitations associated with bone biopsy

The biopsy is an invasive procedure requiring special technical expertise that few clinicians have (Hernandez et al., 2008).

Only a small number of specialized centres have the expertise needed to handle and prepare the bone samples (Hernandez et al., 2008).

Obtaining a suitable bone specimen requires an experienced operator who has mastered the biopsy method (Hernandez et al., 2008).

The biopsy cannot be performed on all people with bone diseases (Kulak and Dempster, 2010).

Lesions observed in the iliac crest are not necessarily representative of lesions present in other bones (heterogeneity of pathologies) (Compston, 2005).

Differences in histomorphometric parameters have been observed in bone specimens taken from adjacent sites in the same iliac crest (Chavassieux et al., 1985a; Revell, 1983; Visser W.J., 1980).

No significant difference was observed between the specimens taken from the left and right iliac crests (Revell, 1983; Visser W.J., 1980).

Variation within the same bone specimen: various histomorphometric indices were measured in two sections that came from the same biopsy but were separated by a distance of 200 μm. A correlation of 0.87 was measured for surface density (Sv) and 0.85 for volume density (Vv) (Delling G., 1980).

Intra-observer variation Intra-observer variations ranging from 1% to 6% have been seen, depending on the histomorphometric index measured (Chavassieux et al., 1985a; Chavassieux et al., 1985b; Delling G., 1980). These variations have now been now minimized by the use of automated, computer-assisted methods.

Inter-observer variation Some significant differences have been observed in the measurement of certain histomorphometric indices (Wright et al., 1992; Chavassieux et al., 1985b; Delling G., 1980).

Subjectivity in the identification of certain histological parameters (for example, difficulty in delineating the corticomedullary junction of the bone) (Wright et al., 1992).

Certain histomorphometric indices (surface density (Sv) and volume density (Vv)) have shown an inter-observer correlation of 0.85 (Delling G., 1980).

A comparison between experienced observers (pathologists with experience in histomorphometry) and inexperienced observers (students) showed differences in the identification of certain histological parameters (for example, difficulties in distinguishing mineralized bone tissue from osteoid tissue) (Wright et al., 1992; Delling G., 1980).

All of these limitations have now been minimized by the use of automated, computer-assisted methods and standardization of methodologies.

10

Limitations associated with the methodology used

There is a significant correlation between the values obtained by the manual method and those obtained by the automated methods (0.98 < r < 0.90) (Chavassieux et al., 1985b).

Use of a higher resolution for the microscope is associated with a higher estimate of surface density (Sv) (Revell, 1983).

It is difficult to establish comparative values, because the histomorphometric values for normal subjects vary (Kulak and Dempster, 2010).

5.4 Recommendations for Listing in Other Jurisdictions

Information not available (the Metabolic Bone Diseases Laboratory at the CHUM Research Centre is the only Canadian laboratory that performs histomorphometry on bone biopsy specimens).

6 ANTICIPATED OUTCOMES OF INTRODUCING THIS TEST

6.1 Impact on Human and Material Resources

The required human and material resources are already in place at the CHUM Research Centre Metabolic Bone Diseases Laboratory.

6.2 Economic Impacts of Including This Test in the Health and Social Services System

The cost for the laboratory to introduce histomorphometry of bone biopsy specimens into the health care system is about $100,000 per year (60 analyses x $1,666).

6.3 Main Organizational, Ethical, Social, Legal, and Political Issues

Organizational issues: the biopsy has to be done in Montreal by Dr. Ste-Marie personally, so arranging for patients from outside Montreal to receive this service is an issue.

11

7 INESSS NOTICE IN BRIEF

Histomorphometric Analysis of a Non-Decalcified Bone Sample (for Non-Complex Diseases and for Complex OM-, ROD-, and HPTH-Type Diseases)

Status of the diagnostic technology

X Established

Innovative

Experimental (for research only)

Replacement for technology:________________, which is becoming obsolete

INESSS recommendation

Include in the Index

Do not include in the Index

X Reassess or include conditionally:

The test must comply with the ministerial directive:

- The analyses must be conducted by clinical staff in a clinical laboratory (not in a research laboratory).

- The analysis must be supervised by the department of medical biology.

Additional information is needed about the accessibility of the test and the clinical indications for it.

INESSS decision regarding any required work

Draw connection with listing of drugs, if it is a companion test.

Produce an optimal user manual.

Produce indicators, if close monitoring is required.

X Verify the weighted value. Although no economic study was performed, lower costs have been seen in some laboratories in the United States.

NOTE: Unique expertise; it is important not to lose it.

12

REFERENCES

Chavassieux PM, Arlot ME, Meunier PJ. Intersample variation in bone histomorphometry: comparison between parameter values measured on two contiguous transiliac bone biopsies. Calcified tissue international 1985a;37(4):345-50.

Chavassieux PM, Arlot ME, Meunier PJ. Intermethod variation in bone histomorphometry: comparison between manual and computerized methods applied to iliac bone biopsies. Bone 1985b;6(4):221-9.

Compston JE. Bone Histomorphometry׃ Elsevier, Inc.; 2005.

Delling G. LH, Baron R., Mathews C.H.E., Olah A. Investigation of Intra- and Inter-Reader Reproductibility. Jee WSS Parfit AM Eds Bone histomorphometry Third International Workshop, Metabolic Bone Disease and Related Research 1980:419-27.

Dempster DW. Histomorphometric Analysis of Bone Remodeling ׃ Academic Press; 2008.

Eriksen EF, Axelrod DW, F. M. Bone Histomorphometry ׃ Raven Press Ltd, 1994.

Hernandez JD, Wesseling K, Pereira R, Gales B, Harrison R, Salusky IB. Technical approach to iliac crest biopsy. Clin J Am Soc Nephrol 2008;3(Suppl 3):S164-9.

Kulak CA et Dempster DW. Bone histomorphometry: a concise review for endocrinologists and clinicians. Arq Bras Endocrinol Metabol 2010;54(2):87-98.

Lerma EV. Diagnosis of renal osteodystrophy. Clinical Reviews in Bone and Mineral Metabolism 2007;5 (1):21-6.

Oprisiu R, Hottelart C, Ghitsu S, Said S, Westeel PF, Moriniere P, et al. [Renal osteodystrophy (1): invasive and non-invasive diagnosis of its pathologic varieties]. Nephrologie 2000;21(5):229-37.

Parfitt AM, Drezner MK, Glorieux FH, Kanis JA, Malluche H, Meunier PJ, et al. Bone histomorphometry: standardization of nomenclature, symbols, and units. Report of the ASBMR Histomorphometry Nomenclature Committee. J Bone Mineral Research 1987;2(6):595-610.

Rao DS. Practical approach to Bone Biopsy. Dans ׃ R.R. R, réd. Bone Histomorphometry: Techniques and Interpretation. CRC Press; 19833-11 ׃.

Rauch F. Bone biopsy: indications and methods. Endocrine development 2009;16:49-57.

Revell PA. Histomorphometry of bone. J Clin Pathol 1983;36(12):1323-31.

Visser W.J. RJMM, Peters J.P.J., Lentferink M.H.F., Duursma S.A. Sampling Variation In Bone Histomorphometry. Jee WSS Parfit AM Eds Bone histomorphometry Third International Workshop, Metabolic Bone Disease and Related Research 1980:429-34.

Wright CD, Vedi S, Garrahan NJ, Stanton M, Duffy SW, Compston JE. Combined inter-observer and inter-method variation in bone histomorphometry. Bone 1992;13(3):205-8.