Crystal induced Arthropathies A Comparative Study of 186 Patients with Gout, Apatite Rheumatism, Chondrocalcinosis and Primary Synovial Chondromatosis

Objective

The authors demonstrate the histological characteristics of gout (G), apatite rheumatism (AR), chondrocalcinosis (Ch-C), and primary synovial chondromatosis (prSynCh) in conventionally processed tissue samples, furthermore illustrate the sensitivity of their non-staining technique.

Microscopic Characteristics of Gout, Apatite Rheumatism, Chondrocalcinosis and Primary Synovial Chondromatosis

Microscopic characteristics of MSU (monosodium urate monohydrate [NaC5H3N4O3·H2O] – monosodium salt of uric acid [C5H4N4O3]) crystal deposits in patients with the clinical diagnosis of gout: Deposits of MSU crystals of gouty tophi were irregular intra- or extraarticular eosinophilic formations, stained with HE, which were surrounded by inflammatory cellular infiltrates and/or by fibrosis depending of the stage of the pathological process (Figure 1a-f).

In most cases the MSU crystals dissolved in 8% formaldehyde solution and in aqueous dyes, and were not present in traditionally fixed HE stained sections. Occasionally, especially in deeper layers of tissue samples or in case of large amounts of MSU deposits, the MSU crystals were preserved and remained demonstrable in aqueous formaldehyde solution fixed tissues, stained with HE.

In unstained sections viewed with polarized light the great amounts of MSU crystals of swart natural color retained (Figure 1g-i), and were arranged in characteristic bundles, or sporadically as globules (Figure 1j-o).

The size of needle-shaped MSU crystals ranged from smaller to 40 µm with the polarization microscope. Under polarized light using Red I compensator the needle shaped crystals showed an intensive negative birefringence according to the long axis (Figure 1j-o).

Typically, the MSU deposits in gouty tophi were not accompanied by amorphous calcium phosphate [Ca3(PO4)2] and/or calcium carbonate [CaCO3] deposits, in contrast to HA and/or CPPD crystal depositions. Chondroid-, osteoid- or new bone formation was not detected in association with MSU deposits.

In our patient cohort calcium pyrophosphate dihydrate (CPPD – [Ca2P2O7.2H2O]) crystal deposits were found in 1 patient with gout; histologically co-existent gout and chondrocalcinosis was diagnosed. Gout was associated with rheumatoid arthritis (rheumatoid nodules) in 3 patients.

MSU and calcium hydroxyapatite (HA – [Ca5(PO4)3(OH)]) crystal combination was not detected; co-existent metabolic diseases such as apatite rheumatism or primary synovial chondromatosis were not seen in combination with tophaceous gout.

Comparative Analysis of Conventional Methods and Non-Staining Technique

Comparative analysis of conventional methods with the non-staining technique was performed on 105 of 211 serially sectioned tissue samples of 146 patients with gout; only the formaldehyde fixed tissue samples were examined, the alcohol fixed tissue samples were excluded. Regarding the location of the surgical excision the elbow was the most common site (n=50), followed by the small joints of the hand (n=12), the hallux (n=5), and the heel (n=3). In 35 cases of 105 tissue samples, the location of the tissue samples could not be determined.

In HE stained tissue sections MSU crystals were detected in 24 (22.86 % of 105), and were absent in 81 sections (77.14 % of 105).

In these 81 “urate negative” sections stained with HE, MSU crystals were demonstrable in 59 unstained tissue sections (72.84 % of 81).

With Gömöri’s methenamine silver method MSU crystals were found in 59 tissue sections (56.19 % of 105), and were not seen in 46 (43.81 % of 105).

In these 46 Gömöri negative tissue sections MSU crystals were seen in 24 with the non-staining technique (52.17 % of 46).

With the Schultz staining, MSU crystals were identified in 66 (62.86 % of 105), and were not in 39 (37.14 % of 105) tissue sections.

In the 39 negative tissue section stained according to Schultz, MSU crystals were present in 17 unstained sections (43.59 % of 39).

According to Schultz’s staining the MSU crystals and/ or the not crystalline uric acid together were present n 81 tissue sections (77.14 % of 105), and were not in 24 (22.86 % of 105).

According to Schultz’s staining the 24 negative MSU and/ or not crystalline uric acid tissue sections (n=24) showed MSU positivity in 12 with non-staining technique (50.0 % of 24) (the non-crystalline uric acid without birefringence is not visible in unstained sections under polarized light).

In contrast with the classic stains and reactions the non- staining technic of Bély and Apáthy’s was more effective, MSU crystals were demonstrated in 83 (79.05 % of 105) tissue sections, and were absent only in 22 (20.95 % of 105).

In these negative unstained sections MSU crystals were not found by HE, according to Schultz staining or by Gömöri methenamine-silver method.

Table 3 summarizes the prevalence of MSU crystals in tissue sections of patients with gout, stained by conventional staining’s and reaction in comparison with Bély and Apáthy’s “non-staining” technique, furthermore the level of significance (“p” values) between different staining’s and techniques.

Remarks to Table 3 In the comparative study of patients with gout only 105 tissue samples was examined, and only the presence of crystals was registered; the amount of crystal deposits was not estimated, and was not compared.

All tissue samples were fixed in an 8% aqueous solution of formaldehyde at pH 7.6 for >24 hours at room temperature (20 Co) and embedded in paraffin, alcohol fixed tissue samples with known clinical diagnosis of gout were excluded).

Schultz’s staining is specific for MSU crystals [NaC5H3N4O3·H2O], non-crystalline uric acid [C5H4N4O3], and cholesterol crystals [C27H46O].

The Gömöri or Schultz stains were more effective in the detection of MSU than HE, and the non-staining technique of Bély and Apáthy’s was much more sensitive than all of these.

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Figure 1a-o: Demonstrates a characteristic gouty tophus with MSU crystals deposits.

Gouty arthritis (tophaceous gout), MSU [NaC5H3N4O3·H2O] – monosodium salt of uric acid [C5H4N4O3] crystal deposits, surrounded by fibrous connective tissue, without remarkable inflammatory reaction.

Tissue samples were fixed in an 8% aqueous formaldehyde solution, stained with HE, and viewed with the light microscope, and under polarized light, respectively.

MSU crystals are not demonstrable.in this characteristic tophus of conventionally processed tissue sections, the crystals dissolved in the aqueous formaldehyde solution and/or during the water-based HE staining. (a) HE, viewed with the light microscope, scale bar: 1000 [µm], magnification: ×40, (b) same as (a) scale bar: 1000 [µm], magnification: ×100; (c) same as (a) scale bar: 100 [µm], magnification: ×200 (d) HE, viewed under polarized light, same as (a), scale bar: 1000 [µm], magnification: ×40, (e) same as (d) scale bar: 1000 [µm], magnification: ×100; (f) same as (d) scale bar: 100 [µm], magnification: ×200 MSU crystals are retained in unstained sections, viewed with the light microscope, showing a natural swart color.

(g) Unstained section, viewed under polarized light, same tissue sample as (a), scale bar: 1000 [µm], magnification: ×40, (h) same as (g) scale bar: 1000 [µm], magnification: ×100; (i) same as (g) scale bar: 100 [µm], magnification: ×200 MSU crystals in unstained sections viewed under polarized light without Red I. compensator, and using Red I. compensator, respectively.

MSU crystals show strong and negative birefringence. (j) MSU crystals, unstained section, viewed under polarized light, same field as (g) , scale bar: 1000 [µm], magnification: ×40, (k) same as (j) scale bar: 1000 [µm], magnification: ×100; (l) same as (j) scale bar: 100 [µm], magnification: ×200 (m) MSU crystals, unstained section, viewed under polarized light, using Red I compensator, same field as (g), scale bar: 1000 [µm], magnification: ×40, (n) same as (m) scale bar: 1000 [µm], magnification: ×100; (o) same as (m) scale bar: 100 [µm], magnification: ×200 The color of MSU crystals was green brown with the Schultz staining (Figure 2a-c), and were black (Figure 2g-i) with Gömöri’s methenamine-silver reaction.

In thin (2-3 µm) tissue sections the MSU crystals remained visible as birefringent needles under polarized light; the intensive staining did not cover completely the crystalline structure (Figure 2d-f and j-l).

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Tissue sample were fixed in an aqueous formaldehyde solution, and stained according to Schultz [8, 9]. (a) Schultz’s staining, viewed with the light microscope, scale bar: 1000 [µm], magnification: ×40, (b) same as (a) scale bar: 1000 [µm], magnification: ×100; (c) same as (a) scale bar: 100 [µm], magnification: ×200 (d) Schultz’s staining, viewed under polarized light, same as (a), scale bar: 1000 [µm], magnification: , scale bar: 1000 [µm], magnification: ×40, (e) same as (d) scale bar: 1000 [µm], magnification: ×100; (f) same as (d) scale bar: 100 [µm], magnification: ×200 Tissue sample fixed in an aqueous formaldehyde solution, and stained according to Gömöri [6, 7]. (g) Gömöri’s methenamine silver reaction, viewed with the light microscope, same field as (a), scale bar: 1000 [µm], magnification: ×40, (h) same as (g) scale bar: 1000 [µm], magnification: ×100; (i) same as (g) scale bar: 100 [µm], magnification: ×200 (j) Gömöri’s methenamine silver reaction, viewed under polarized light, same tissue sample as (g), scale bar: 1000 [µm], magnification: ×40, (k) same as (j) scale bar: 1000 [µm], magnification: ×100; (l) same as (j) scale bar: 100 [µm], magnification: ×200 Microscopic characteristics of HA (hydroxyapatite [Ca5(PO4)3(OH)]) crystal deposits in patients with clinically diagnosed apatite rheumatism (The chemical formula of HA is not uniform in the pertinent literature: [Ca5(PO4)3(OH)] or [Ca10(PO4)6OH2]) Nine (9) surgeries or joint interventions occurred in 4 patients with clinically diagnosed AR; the most commonly involved joint was the knee in 5 (55.55 %), followed by the shoulder in 3 (33.33 %) (including 2 punctions of the shoulder), and the hip in 1 (11.11 %) of the 9 cases.

During surgeries of these 4 patients 16 specimens were removed, the synovial membrane 7 of 16 samples (43.75), joint capsule 5 (31.25 %), bone and/or cartilage 2 (12.50 %), and bursa 2 (12.50 %) samples (Table 4).

The small and very soluble HA crystals with weak birefringence were not visible in HE stained tissue section (Figure 3).

HA crystals were accompanied typically with amorphous calcium phosphate [Ca3(PO4)2] and/or calcium carbonate [CaCO3] deposits, which were blue-violet with HE (Figure 3 a-c), orange with Alizarin red S staining (specific for calcium), and black with the von Kossa reaction (specific for phosphate and/or carbonate).

Evaluated by our semiobjective core system the average amount of amorphous mineral deposit was 1.063 per tissue samples.

Only minimal chondroid formation was detected (average 0.063 per tissue sample), osteoid or new bone was not found (Table 4).

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The readily soluble weakly birefringent small HA crystals are not detected in conventionally fixed tissue specimens stained with HE, only the accompanying amorphous calcium phosphate [Ca3(PO4)2] and/or calcium carbonate [CaCO3] deposits are visible as blue-violet deposits.

(a) HE, viewed with the light microscope, scale bar: 1000 [µm], magnification: ×40, (b) same as (a) scale bar: 1000 [µm], magnification: ×100; (c) same as (a) scale bar: 100 [µm], magnification: ×200 (d) HE, viewed under polarized light, same as (a), scale bar: 1000 [µm], magnification: ×40, (e) same as (d) scale bar: 1000 [µm], magnification: ×100; (f) same as (d) scale bar:

100 [µm], magnification: ×200 In unstained tissue sections, using a professional polarizing microscope with high, at least 100-Watt brightness, the small and very soluble HA prisms were visible in 10 of 16 (62.50 %) tissue samples, accompanied with CPPD crystals in 9 (56.25 %) cases; the HA crystals were present without CPPD crystals in the joint capsule of one patient (Table 4).

HA crystal prisms showed a weak positive, CPPD crystals a strong positive birefringence viewed by polarized light with Red I compensator (100x) (Figure 4).

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The HA crystals are colorless, translucent small (50-500 nm) rod-shaped prisms. Under polarized light the birefringence of HA crystals is weak.

(a) HA crystals, unstained sections, viewed under polarized light, scale bar: 1000 [µm], magnification: ×40, (b) same as (a) scale bar: 1000 [µm], magnification: ×100; (c) same as (a) scale bar: 100 [µm], magnification: ×200 (b) Under polarized light with Red I. compensator the birefringence of HA crystals is positive. (d) HE, viewed under polarized light, with Red I. compensator, same as (a), scale bar: 1000 [µm], magnification: ×40, (e) same as (d) scale bar: 1000 [µm], magnification: ×100; (f) same as (d) scale bar: 100 [µm], magnification: ×200 Apatite rheumatism, right shoulder, synovial membrane, HA (white ellipse) and CPPD (yellow ellipse) crystal deposits.

Under polarized light the birefringence of CPPD is stronger, compared to the HA crystals. (g) HA and CPPD crystals, unstained sections, viewed under polarized light, scale bar: 1000 [µm], magnification: ×40, (h) same as (g) scale bar: 1000 [µm], magnification: ×100; (i) same as (g) scale bar: 100 [µm], magnification: ×200 Under polarized light, with Red I. compensator, the direction of birefringence is positive according to the long axis of HA and CPPD crystals.

(j) HA and CPPD crystals, unstained sections, with Red I. compensator, same as (g), scale bar: 1000 [µm], magnification: ×40, (k) same as (j) scale bar: 1000 [µm], magnification: ×100; (l) same as (j) scale bar: 100 [µm], magnification: ×200 The individual small HA crystals formed clusters or aggregate of clusters. (in size of 100 µm or larger).

The 100 µm or larger clusters or aggregate of clusters were visible with an objective even of 40x magnification with non-staining techniques (Figure 5a-f).

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Figure 5a-f: Apatite rheumatism, right shoulder, subchondral bone.

The small prisms of HA crystals are arranged in larger spheroid microaggregates (white ellipses and yellow stars).

Under polarized light birefringence is positive according to the long axis of HA crystals, like that of collagen fibers.

(a) HA crystals, unstained sections, viewed under polarized light, scale bar: 1000 [µm], magnification: ×40, (b) same as (a) scale bar: 1000 [µm], magnification: ×100; (c) same as (a) scale bar: 100 [µm], magnification: ×200 Under polarized light with Red I. compensator the birefringence of HA crystals is positive. (d) HE, viewed under polarized light, with Red I. compensator, same as (a), scale bar: 1000 [µm], magnification: ×40, (e) same as (d) scale bar: 1000 [µm], magnification: ×100; (f) same as (d) scale bar: 100 [µm], magnification: ×200

Microscopic Characteristics of CPPD (calcium pyrophosphate dihydrate [Ca2P2O7.2H2O]) Crystal Deposits in Patients with Clinically Diagnosed Chondrocalcinosis

Sixteen (16) patients were operated with clinically diagnosed Ch-C; the most commonly involved joint was the knee in 8 (50.0 %), followed by the hip in 4 (25.0 %), wrist in 2 (12.50 %), elbow in 1 (6.25 %), and shoulder in 1 (6.25 %) of the 16 cases.

During surgeries of these 16 patients 40 specimens has been removed, synovial membrane 15 of 40 samples (37.50 %), joint capsule 15 (37.50 %), bone and/or cartilage 5 (12.50 %), bursa 3 (7.50 %), and tendon 2 (5.0 %) (Table 4).

CPPD crystals were less soluble in the 8%aqueous formaldehyde solution or water containing dyes than HA crystals, and occasionally remained back and were demonstrable with polarized light in tissue sections stained with HE, Alizarin red S or with the von Kossa reaction (Figure 6a-f and Figure 7a-l, Table 4).

CPPD crystals appeared typically as planes of hexagonal, rhomboid, trapezoid, parallelogram-shape or fragments of these (Figure 6g-l). Their size varied less than 40 µm.

In unstained tissue section the CPPD crystals showed a relative strong positive birefringence viewed by polarized light using Red I compensator, compared to the weak positive birefringence of the HA crystals (Figure 6g-l versus 4a-l and Figure 7xxxxl).

CPPD crystals were accompanied in all cases by more or lesser amounts of amorphous calcium phosphate [Ca3(PO4)2] and/or calcium carbonate [CaCO3] deposits, appeared blue-violet with HE (Figure 6a-f), orange with Alizarin red S staining (Figure 8a-f) and black with the von Kossa reaction (Figure 8g-l).

According to our semi-objective score system in Ch-C patients the average mineral deposition was 1.054 per tissue sections (Table 4). In some cases, the massive amorphous (non-birefringent) minerals masked the crystals, which remained intact with Alizarin red S stain or with von Kossa’s reaction (Figure 8a-f and Figure 8g-l).

Only minimal chondroid formation was detected (average 0.054 per tissue sample), osteoid or new bone was not found (Figure 6a-f and Table 4).

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Figure 6a-l: Chondrocalcinosis (pseudogout, pyrophosphate arthropathy, calcium pyrophosphate dihydrate [Ca2P2O7.2H2O] (CPPD) crystal induced arthropathy, viewed with the light microscope and under polarized light, respectively.

CPPD crystals and crystal fragments are accompanied by amorphous calcium phosphate, or calcium carbonate deposits of blue-violet color of HE stained tissue sections. Minimal chondroid formation can be seen next to the amorphous mineral deposits (white ellipse) The less soluble CPPD crystals may remain back and are demonstrable with polarized light in tissue sections stained with HE (Figure 6a-f), Alizarin red S (see below Figure 7a-f) or with the von Kossa reaction (Figure 7g-l).

(a) HE, viewed with the light microscope, scale bar: 1000 [µm], magnification: ×40, (b) same as (a) scale bar: 1000 [µm], magnification: ×100; (c) same as (a) scale bar: 100 [µm], magnification: ×200 (d) HE, viewed under polarized light, same as (a), scale bar: 1000 [µm], magnification: ×40, (e) same as (d) scale bar: 1000 [µm], magnification: ×100; (f) same as (d) scale bar:

100 [µm], magnification: ×200 CPPD plane crystals are of hexagonal, rhomboid, trapezoid, parallelogram-shape or fragments of these, they range in size from smaller to 40μm, and show a relatively strong positive birefringence according to the long axis of the crystals with Red I. compensator.

(g) CPPD and HA (white ellipse) crystals, unstained sections, viewed under polarized light, same as (a), scale bar: 1000 [µm], magnification: ×40, (h) same as (g) scale bar: 1000 [µm], magnification: ×100; (i) same as (g) scale bar: 100 [µm], magnification: ×200 (j) CPPD and HA crystals, unstained sections, viewed under polarized light, with Red I. compensator, same as (g), scale bar: 1000 [µm], magnification: ×40, (k) same as (g) scale bar: 1000 [µm], magnification: ×100; (l) same as (g) scale bar: 100 [µm], magnification: ×200

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Figure 7a-l: Chondrocalcinosis, CPPD and HA crystal induced arthropathy, viewed with the light microscope and under polarized light, respectively.

(a) CPPD and HA (white ellipse) crystals, unstained sections, viewed under polarized light, scale bar: 1000 [µm], magnification: ×40, (b) same as (a) scale bar: 1000 [µm], magnification: ×100; (c) same as (a) scale bar: 100 [µm], magnification: ×200 (d) CPPD and HA crystals, unstained sections, viewed under polarized light, with Red I. compensator, same as (a), scale bar: 1000 [µm], magnification: ×40, (e) same as (d) scale bar: 1000 [µm], magnification: ×100; (f) same as (d) scale bar: 100 [µm], magnification: ×200

(g) HA crystals, unstained sections, viewed under polarized light, scale bar: 1000 [µm], magnification: ×40, (h) same as (g) scale bar: 1000 [µm], magnification: ×100; (i) same as (g) scale bar: 100 [µm], magnification: ×200

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Figure 8a-l: Chondrocalcinosis (pseudogout, pyrophosphate arthropathy, calcium pyrophosphate dihydrate [Ca2P2O7.2H2O] (CPPD) crystal induced arthropathy), viewed with the light microscope and under polarized light, respectively.

Non-crystalline calcium containing mineral deposits are staining orange with calcium specific Alizarin red S. Alizarin red S does not stain the CPPD crystals, but staining of the massive deposits of amorphous minerals may mask the crystals, without detectable birefringence or only with weak intensity. (g) Alizarin red S, viewed with the light microscope, same as (a), scale bar: 1000 [µm], magnification: ×40, (h) same as (g) scale bar: 1000 [µm], magnification: ×100; (i) same as (g) scale bar: 100 [µm], magnification: ×200 (j) Alizarin red S, viewed under polarized light, same as (g), scale bar: 1000 [µm], magnification: ×40, (k) same as (j) scale bar: 1000 [µm], magnification: ×100; (l) same as (j) scale bar: 100 [µm], magnification: ×200 The non-crystalline amorphous mineral deposition starts with calcium, and the phosphate and/or carbonate appear later. In demonstrated (illustrated) stage the intensive orange color of calcium stained with the Alizarin red S is accompanied by negative or with minimal black staining with the von Kossa reaction.

(m) von Kossa reaction, viewed with the light microscope, same as (a), scale bar: 1000 [µm], magnification: ×40, (n) same as (m) scale bar: 1000 [µm], magnification: ×100; (o) same as (m) scale bar: 100 [µm], magnification: ×200 (p) Alizarin red S, viewed under polarized light, same as (m), scale bar: 1000 [µm], magnification: ×40, (q) same as (p) scale bar: 1000 [µm], magnification: ×100; (r) same as (p) scale bar: 100 [µm], magnification: ×200 In unstained tissue section, the large and less soluble CPPD crystals were visible in 22 of 40 (62.50 %) tissue samples, and were accompanied by HA crystals in all cases.

The HA crystals were present in 26 of 40 (56.25 %) cases; 4 of them without demonstrable CPPD crystals.

In 2 patients the HA crystals were not accompanied by CPPD crystals; in 2 joint capsules and in 2 bone and/or cartilage specimens CPPD crystals were not detected (Table 4).

Microscopic Characteristics of Clinically Diagnosed Primary Synovial Chondromatosis

Twenty-one (21) joints of 20 patients with clinically diagnosed and histologically confirmed prSynCh were operated; the most commonly involved joint was the knee in 14 (66.67 %), followed by the hip in 5 (23.81 %), and elbow in 2 (9.52 %) of the 21 cases (Table 4).

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Figure 9a-l: Synovial osteo-chondromatosis, knee joint (2569-2008), synovial osteomatosis, elbow (1864-89), and synovial osteo-chondromatosis with minimal amorphous mineral deposits (1727-2016).

The histological diagnosis in 9 of 21 operated joints was chondromatosis (characterized by chondroid formation without osteoid or bone formation), in 11 osteo-chondromatosis (characterized by chondroid with osteoid and/or bone formation), and in 1 osteomatosis (characterized exclusively by bone formation) (Figure 9a-l).

True medullary spaces accompanied bone formation in 8 of 12 cases (with osteo-chondromatosis 11 or osteomatosis 1).

Different stages of chondroid, osteoid and/or bone formation existed side by side in the same histologic section.

During surgeries of these 21 joint 40 specimens has been removed, synovial membrane 15 of 40 samples (37.50 %), joint capsule 15 (37.50 %), bone and/or cartilage 5 (12.50 %), bursa 3 (7.50 %), and tendon 2 (5.0 %) (Table 4).

Chondroid and/or bone formation (with or without true medullary spaces) was abundant; calculated by our semi- objective score system the average chondroid and/or bone formation was 1.975 per tissue sections.

Chondroid and/or bone formation was accompanied by minimal amorphous mineral deposition (0.270 per tissue sections) Figure 9m-o.

In unstained sections of surgical specimens HA [Ca5(PO4)3(OH)] (Figure 10a-l), and CPPD [Ca2P2O7.2H2O] crystals were detected in all cases.

Synovial chondromatosis (a-c) osteo-chondromatosis with early stage of true medullary cavity formation (d-f), osteo-chondromatosis with advanced stage of true medullary cavity formation (g-i), and synovial osteomatosis, elbow (j-l).

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Figure 10a-l: Synovial osteo-chondromatosis, knee joint, joint capsule with HA crystal prisms, and pericapsular bone with HA crystal clusters.

(a) Synovial chondromatosis, HE, viewed with the light microscope, scale bar: 1000 [µm], magnification: ×40, (b) same as (a) scale bar: 1000 [µm], magnification: ×100; (c) same as (a) scale bar: 100 [µm], magnification: ×200 (d) Synovial osteo-chondromatosis with early stage of true medullary cavity formation HE, viewed with the light microscope, scale bar: 1000 [µm], magnification: ×40, (e) same as (d) scale bar: 1000 [µm], magnification: ×100; (f) same as (d) scale bar: 100 [µm], magnification: ×200 (g), Synovial osteo-chondromatosis with advanced stage of true medullary cavity formation HE, viewed with the light microscope, scale bar: 1000 [µm], magnification: ×40, (h) same as (g) scale bar: 1000 [µm], magnification: ×100; (i) same as (g) scale bar: 100 [µm], magnification: ×200 (j) Synovial osteomatosis, HE, viewed with the light microscope, scale bar: 1000 [µm], magnification: ×40, (k) same as (j) scale bar: 1000 [µm], magnification: ×100; (l) same as (j) scale bar: 100 [µm], magnification: ×200 Synovial osteo-chondromatosis with minimal amorphous mineral deposition, viewed with the light microscope. (m) HE, the section is practically negative, scale bar: 1000 [µm], magnification: ×20, (n) Alizarin red S, same as (m) scale bar: 1000 [µm], magnification: ×20; (o) von Kossa reaction, same as (m) scale bar: 100 [µm], magnification: ×20 The small prisms of HA crystals are arranged in larger spheroid microaggregates. Under polarized light the direction of birefringence is positive according to the long axis of HA crystals, like that of collagen fibers.

Joint capsule, the small prisms of HA crystals prisms are located on, and between collagen fibers. (a) HA crystals, unstained sections, viewed under polarized light, the ratio of scale bars: 100:1000 [µm], magnification: x100, (b) same as (a) scale bar: 100 [µm], magnification: x200, (c) same as (a), scale bar: 100 [µm], magnification: x400. (d) HA crystals, unstained sections, viewed under polarized light, using Red I compensator, same as (a), the ratio of scale bars: 100:1000 [µm], magnification: x100, (e) same as (d) scale bar: 100 [µm], magnification: x200, (f) same as (d), scale bar: 100 [µm], magnification: x400. (g) Pericapsular bone, HA crystal clusters, unstained sections, viewed with the light microscope, the ratio of scale bars: 100:1000 [µm], magnification: ×100; (h) same as (g) scale bar: 100 [µm], magnification: ×200, (i) same as (g), scale bar: 100 [µm], magnification: x400. (j) HA crystals, unstained sections, viewed under polarized light, using Red I compensator, same as (g), the ratio of scale bars: 100:1000 [µm], magnification: x100, (k) same as (j) scale bar: 100 [µm], magnification: x200, (l) same as (j), scale bar: 100 [µm], magnification: x400.

Comparative Analysis of Apatite Rheumatism, Chondrocalcinosis and Primary Synovial Chondromatosis with Non-Staining Technique and with Conventional Methods

The comparative analysis was performed on 73 unstained tissue samples of 28 patients with the clinical diagnosis of apatite rheumatism, chondrocalcinosis or primary synovial chondromatosis (Table 4) Amorphous calcium phosphate [Ca3(PO4)2], and/or calcium carbonate [CaCO3] deposits were appraised in 90 tissue sections of 40 patients (Table 4) The chondroid, osteoid and/or bone formation was evaluated in 96 tissue sections of 40 patients (Table 4).

Table 4 summarizes the prevalence of HA and CPPD crystals in 73 unstained sections, the amount of amorphous mineral deposits in 90 tissue sections stained by HE, Alizarin red S and/or von Kossa reaction furthermore the extent of chondroid, osteoid and/or bone formation in 96 HE stained tissue section of patients.

Table 4 Distribution of HA and CPPD crystals, amount of amorphous mineral deposits, furthermore extent of chondroid, osteoid and/or bone formation in patients with clinically diagnosed AR, Ch-C or prSynCh

The prevalence of HA and/or CPPD was determined with polarized light in 73 unstained tissue sections of 28 patients.

16 tissue sections of 4 patients with clinical diagnoses of AR, 40 tissue sections of 16 patients with clinical diagnoses of Ch-C, and 17 tissue sections of 8 patients with clinical diagnoses of prSynCh were analyzed in unstained sections with polarized light.

Patients with gout are not tabulated; HA and/or CPPD crystals were not associated with MSU (except CPPD in one patient).

MSU crystals were not accompanied by amorphous mineral deposits, and chondroid, osteoid or bone formation.

The most commonly involved joint structures of patients with AR, Ch-C, and prSynCh were the synovial membranes in 30 (41.10 %), followed by the capsules in 28 (38.36 %), bone and/or cartilage in 8 (10.96 %), bursae in 5 (6.85 %), and tendons in 2 (2.74 %) of 73 cases.

1* Calcification was determined in 37 of 40 HE stained tissue sections of 16 patients with Ch-C; 3 of 40 tissue samples were not available for serial sections (the specimens were too small for further serial sections).

2* Chondroid, osteoid and/or bone formation was determined in 40 HE stained tissue sections of 16 patients with Ch-C.

3* Prevalence of HA and CPPD crystals was determined only in 17 unstained tissue sections of 8 patients with prSynCh.

4* Calcification was evaluated in 37 HE of 40 HE stained tissue sections of 20 patients with prSynCh (3 tissue samples were not available for histological analysis).

5* Chondroid, osteoid and/or bone formation was evaluated in 40 HE stained tissue sections of 20 patients with prSynCh.

prSynCh – primary Synovialis Chondromatosis
Ts – Tissue sample
Calc – Calcification (amount of calcium phosphate [Ca3(PO4)2] and/or calcium carbonate [CaCO4])
Ch/Bone – Chondroid, osteoid or Bone formation