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ORIGINAL ARTICLE
Year : 2016  |  Volume : 29  |  Issue : 2  |  Page : 360-366

Collagen I and collagen III expression in fibrotic bone marrow


1 Clinical Pathology Department, Faculty of Medicine, El-Menoufia University, Menoufia, Egypt
2 Clinical Pathology Department, Cairo University, Cairo, Egypt
3 Clinical Oncology Department, Faculty of Medicine, Cairo University, Cairo, Egypt

Date of Submission25-Feb-2015
Date of Acceptance07-Apr-2015
Date of Web Publication18-Oct-2016

Correspondence Address:
Amira M. F Shehata
Ahmed Abd El-Aziz Street, Tala, Menoufia, 32611
Egypt
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/1110-2098.192403

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  Abstract 

Objective:
The aim of this study was to assess the expression of collagen I and III in fibrotic bone marrow (BM) in different pathologies.
Background:
A wide variety of benign conditions and malignant disorders are associated with BM fibrosis. BM fibrosis can be demonstrated by histochemical staining using silver impregnation or by trichrome stains. Immunohistochemical stains, especially anticollagen I and III, are also used for more accurate assessment of BM fibrosis. This study demonstrates the expression of collagen I and III in fibrotic marrow in different pathologies. No clinical data, prognosis , survival were mentioned in this study and so the impact was not investigated.
Materials and methods:
In this study 50 formalin-fixed paraffin-embedded BM biopsy samples were selected from archived cases at the Clinical Pathology Department, Cairo University. These 50 cases included seven cases with myeloproliferative neoplasms, 14 with lymphoproliferative disorders, three with acute leukemia, four with metastasis, and 22 with reactive BM changes. Cases were divided into two groups according to the Masson's trichrome staining pattern: the fibrotic group, which included 13 cases, and the nonfibrotic group, which included 37 cases. The included cases were subjected to reticulin and trichrome stains, in addition to assessment of collagen I and III expression by immunohistochemistry.
Results:
As regards collagen I and III staining grades, grade 4 was significantly higher in the fibrotic group, whereas grade 0 was significantly higher in the nonfibrotic group.
Conclusion:
Collagen I and III are markedly expressed in fibrotic marrow and marrow fibrosis; in particular, collagen fibrosis appears to be more evident in myeloproliferative neoplasms and in tumor metastasis to the BM.

Keywords: bone marrow fibrosis, collagen I, collagen III, immunohistochemistry


How to cite this article:
Shehata AM, Kandel SH, Rizk SH, Khalifa KA, Fouad AS. Collagen I and collagen III expression in fibrotic bone marrow. Menoufia Med J 2016;29:360-6

How to cite this URL:
Shehata AM, Kandel SH, Rizk SH, Khalifa KA, Fouad AS. Collagen I and collagen III expression in fibrotic bone marrow. Menoufia Med J [serial online] 2016 [cited 2020 Feb 26];29:360-6. Available from: http://www.mmj.eg.net/text.asp?2016/29/2/360/192403


  Introduction Top


Bone marrow (BM) fibrosis is defined as an increase in BM stromal fiber content without specifying the exact amount or type (reticulin vs. collagen) of fiber increased [1].

BM fibrosis shown by silver (reticulin fibrosis) or trichrome (collagen fibrosis) stains can be associated with a number of hematologic and nonhematologic disorders [2].

Reticulin fibrosis may be evident in many benign conditions, including autoimmune and granulomatous diseases, and in different tumors, such as lymphoid neoplasms, as well as in myelodysplastic syndrome and acute myeloid leukemia. In contrast, collagen fibrosis is more characteristic of the advanced stages of myeloproliferative neoplasms (MPNs) or is associated with tumor metastasis to the BM [3].

Several grading schemes have been developed for reticulin deposition; the most commonly used is the Bauermeister scale. Few studies have evaluated reticulin fibrosis in healthy persons. Some degree of reticulin fibrosis was detected in a significant number of healthy persons. However, in a study involving 100 nonhematologic patients, neither grade 3 nor grade 4 reticulin fibrosis was observed on the Bauermeister scale (diffuse fiber deposition and areas of collagen deposition, respectively) [4].

BM fibrosis involves deposition of collagen fibers in the marrow. There are over 16 types of collagen in the body, and BM collagen is mainly composed of type I (collagen) and type III collagen (reticulin) [5].

The connective tissue structure of the BM is not well appreciated on a routine hematoxylin and eosin stained-BM biopsy section and requires special histological stains such as Mallory's trichrome stain or Masson's trichrome stain, which can be used to identify collagen [6].

Assessment of collagen I and III expression in various tissues was carried out in many studies using various laboratory techniques; among these techniques is immunohistochemistry, which has the advantage of providing immunophenotypic information in association with preserved information regarding the spatial organization of labeled and unlabeled cells. Immunohistochemistry also permits assessment of the cytological features of cells expressing particular antigens [7].

The present study was designed to assess the expression of collagen I and III in fibrotic BM and demonstrate whether collagen I and III expression levels differ with the type of underlying BM disease.


  Materials and Methods Top


The current study included 50 formalin-fixed paraffin-embedded BM biopsy samples from archived coded cases at the Clinical Pathology Department, Faculty of Medicine, Cairo University. Cases were divided into two groups according to the BM fibrotic status based on Masson's trichrome stain: the fibrotic group and the nonfibrotic group. The fibrotic group included 13 cases and comprised five cases with MPNs, two cases with lymphoproliferative disorders (LPDs), four cases with BM metastasis, and two cases with reactive BM changes. The nonfibrotic group included 37 cases and comprised two cases with MPNs, 12 cases with LPDs, three cases with acute leukemia (AL), and 20 cases with reactive BM changes ([Table 1]).
Table 1: Underlying bone marrow pathology in fibrotic and nonfibrotic groups

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Cases were subjected to reticulin and trichrome staining to assess the grade and type of BM fibrosis, in addition to assessment of collagen I and collagen III expression using immunohistochemistry.


  Methods Top


Histochemical staining for fibrosis

Sliver impregnation for reticulin

Histochemical staining for reticulin was performed on paraffin sections using Gordon and Sweet's staining method [8].

Interpretation of reticulin staining: The defined reticulin fibers appeared black against a background of green tissue. Quantification of BM reticulin was based on a modified Bauermeister graduation [9].

Grade 0: No reticulin fibers demonstrable.

Grade 1: Occasional fine individual fibers and foci of a fine fiber network.

Grade 2: Fine fiber networks throughout most of the section; no coarse fibers.

Grade 3: Diffuse fiber networks with scattered thick coarse fibers but no mature collagen.

Grade 4: Diffuse, often coarse, fiber network with areas of collagenization.

Trichrome staining for collagen

Histochemical staining for collagen was performed on paraffin sections using Masson's trichrome stain [6].

Interpretation of trichrome staining: Collagen was stained blue, nuclei were stained black, and cytoplasm and erythrocytes were stained red.

The trichrome stain was graded as follows:

Grade 0: Negative for collagen.

Grade 1: Less than 10% of the section is positive for collagen.

Grade 2: 10–30% of the section is positive for collagen.

Grade 3: 30–50% of the section is positive for collagen.

Grade 4: More than 50% of the section is positive for collagen.

Immunohistochemical staining for collagen I and collagen III

Immunohistochemical staining for collagen I and III was performed on paraffin sections with immunoperoxidase visualization. After deparaffinization in xylene and rehydration in alcohol, sections were first preincubated in hydrogen peroxide for 10 min at room temperature to block endogenous peroxidase activity; then heat-mediated antigen retrieval (sodium citrate buffer at 95–98°C for 12 min) was performed, followed by application of protein block (incubated for 5 min at room temperature to block nonspecific background staining) before being incubated for 30 min at room temperature with a primary rabbit polyclonal antibody against collagen I or collagen III (1 : 600, Abcam, Cambridge, UK). Sections were washed four times in buffer and anti-mouse and rabbit, streptavidin peroxidase, and DAB chromogen were applied in this order. Each was applied for 10 min, and in between each application sections were washed four times in buffer. Adequate drops of hematoxylin were added to cover the sections; the sections were then rinsed 7–8 times in tap water, dehydrated, and mounted.

Interpretation of collagen I and collagen III staining

Quantification of collagen I and collagen III in BM biopsy sections was scored semiquantitatively (percentage of stained areas in the sections).

Grade 0: Negative.

Grade 1: Less than 10% of the section is positive.

Grade 2: 10–30% of the section is positive.

Grade 3: 30–50% of the section is positive.

Grade 4: More than 50% of the section is positive.

Statistical analysis

Data were collected, tabulated, and statistically analyzed using statistical package for the social science (SPSS, version 20; SPSS Inc., Chicago, Illinois, USA). Two types of statistics were determined:

  1. Descriptive statistics, expressed as number and percentage, and
  2. Analytic statistics, using the Z test, which was used to compare two prevalence percentages.


P values less than 0.05 were considered statistically significant.


  Results Top


The results of this study are presented in [Table 1],[Table 2],[Table 3],[Table 4],[Table 5],[Table 6],[Table 7] and [Figure 1],[Figure 2],[Figure 3],[Figure 4],[Figure 5].
Table 2: Reticulin staining grades in fibrotic and nonfibrotic groups

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Table 3: Collagen I staining grades in fibrotic and nonfibrotic groups

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Table 4: Collagen III staining grades in fibrotic and nonfibrotic groups

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Table 5: Comparison between pathological groups according to reticulin staining grades

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Table 6: Comparison between pathological groups according to trichrome staining grades

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Table 7: Comparison between pathological groups according to collagen I and collagen III staining grades

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Figure 1: Microphotograph of a bone marrow section with marked fibrosis and no hematopoietic elements stained with hematoxylin and eosin (×40)

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Figure 2: Microphotograph of a bone marrow section of a case of metastasis, stained with Gordon and Sweet's reticulin stain with light green counterstain showing extensive grade 3 reticulin fibrosis with focal accentuation (×40)

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Figure 3: Microphotograph of a bone marrow section of a case with marked fibrosis stained with trichrome stain showing wide positively stained area within the marrow space (×40)

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Figure 4: Microphotograph of a bone marrow section of a case of multiple myeloma stained with anticollagen I showing strong positivity (×40)

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Figure 5: Microphotograph of a bone marrow section of a case with myeloproliferative neoplasm stained with anticollagen III, showing strong positivity (×40)

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Reticulin staining grades

As regards reticulin staining grades, prevalence of grades 3 and 4 was significantly higher in the fibrotic group, whereas that of grade 0 was significantly higher in the nonfibrotic group ([Table 2]).

Collagen I expression

As regards collagen I staining grades, prevalence of grade 4 was significantly higher in the fibrotic group, whereas that of grade 0 was significantly higher in the nonfibrotic group ([Table 3]).

Collagen III expression

As regards collagen III staining grades, prevalence of grade 4 was significantly higher in the fibrotic group, whereas that of grade 0 was significantly higher in the nonfibrotic group ([Table 4]).

Results of different stains according to the underlying bone marrow pathology

According to the underlying BM pathology based on hematoxylin and eosin staining, cases were divided into MPNs, LPDs, AL, BM metastasis, and reactive BM changes.

The prevalence of MPNs and BM metastasis was significantly higher in the fibrotic group than in the nonfibrotic group, whereas the prevalence of reactive BM was significantly higher in the nonfibrotic group than in the fibrotic group ([Table 1]).

Comparison of different staining patterns between different pathological groups is shown in [Table 5],[Table 6],[Table 7].

Myeloproliferative neoplasms

Regarding the results of reticulin staining in MPNs, grade 3 and grade 4 were present in about 57.1% of the cases. As regards the trichrome staining pattern in MPNs, grade 4 was the most commonly represented grade, being seen in 42.8% of cases.

The prevalence of grade 3 reticulin staining was significantly higher in MPNs than in reactive marrow (P = 0.01), and the prevalence of grade 4 trichrome staining was significantly higher in MPNs than in reactive marrow (P = 0.01).

Regarding collagen I and III expression in MPNs, grade 4 was the most evident grade for both collagen types, being seen in 66.6 and 57.1% of cases, respectively.

The prevalence of grade 4 collagen I staining was significantly higher in MPNs than in LPDs and reactive marrow (P = 0.01 and 0.0003, respectively), and the prevalence of grade 4 collagen III staining was significantly higher in MPNs than in reactive marrow (P = 0.04).

Lymphoproliferative disorders

Regarding the results of reticulin and trichrome staining in LPDs, grade 0 was the most evident grade, being seen in 71.5% of cases for reticulin and in 85.8% of cases for trichrome staining.

Regarding collagen I and III expression in LPDs, grades 0 and 1 were the most commonly encountered grades. Grade 0 was seen in 57.2% of cases for collagen I and in 23.1% of cases for collagen III, whereas grade 1 was seen in 35.7% for collagen I and in 53.8% for collagen III.

Acute leukemia

No reticulin-positive or trichrome-positive staining was detected in AL.

Regarding collagen I expression in the AL group, grade 0 was seen in two out of three cases, whereas grade 1 was seen in one case.

Regarding collagen III expression in AL, grades 0, 1, and 3 were seen in one case each.

Bone marrow metastasis

Regarding the results of reticulin staining, grade 4 was seen in three out of four cases and grade 3 was seen in one case. As regards trichrome staining, all of the studied cases showed grade 4 trichrome staining.

The prevalence of grade 4 reticulin staining was significantly higher in BM metastasis than in LPDs and reactive marrow (P = 0.005 and 0.0005, respectively), and the prevalence of grade 4 trichrome staining was significantly higher in BM metastasis than in LPDs and reactive marrow (P = 0.002 and 0.00001, respectively).

Regarding collagen I expression in metastasis, grade 4 was seen in three out of four cases and grade 2 was seen in one case. As regards collagen III expression in metastasis, grade 4 was seen in three out of four cases and grade 3 was seen in one case.

The prevalence of grade 4 collagen I staining was significantly higher in BM metastasis than in LPDs and reactive marrow (P = 0.02 and 0.0005, respectively), and the prevalence of grade 4 collagen III staining was significantly higher in BM metastasis than in LPDs and reactive marrow (P = 0.03 and 0.02, respectively).

Reactive group

Regarding the results of reticulin staining in reactive marrow, grade 0 was the most commonly presented grade, being seen in 59.1% of cases; grade 0 trichrome staining was the most encountered pattern of staining, being seen in 90.9% of cases.

Regarding collagen I and III expression in reactive marrow, grade 0 was the most commonly presented grade, being seen in 63.7% of cases for collagen I and in 61.1% of cases for collagen III.


  Discussion Top


Cases included in this study were classified into two groups according to trichrome staining: group 1 (the fibrotic group), which included 13 cases with positive trichrome stain, and group 2 (the nonfibrotic group), which included 37 cases with negative trichrome stain.

As regards reticulin staining grades, grades 3 and 4 were significantly higher in the fibrotic group, whereas grade 0 was significantly higher in the nonfibrotic group. In MPNs and metastatis, grades 3 and 4 were the most commonly found grades, whereas grade 0 was the most commonly found grade in AL, LPDs, and reactive BM changes. Similar results for reticulin fibrosis were reported by other authors in the same hematologic disorders [10],[11].

In the present study, immunohisochemical staining of collagen I and III showed that grade 4 was significantly higher in the fibrotic group, whereas grade 0 was significantly higher in the nonfibrotic group. Immunohistochemical staining for collagen fibers, especially for collagen I and III, has been used in other studies to assess the fibrotic pattern in many tissues. Zhao et al. [2] studied the expression of type I and type III collagen in case of gluteal muscle contracture. Cheng et al. [3] studied the immunohistochemical expression of collagen I and III in skin. Chen et al. [4] also studied the expression of type III collagen in case of liver fibrosis.

In the present study, we tried to demonstrate the relationship between reticulin (sliver impregnation method) collagen (Masson's trichrome method) and collagen I and III (immunohistochemical staining) staining patterns and the underlying BM pathology.

A comparison of their different staining patterns in different BM pathologies was performed. MPNs and marrow metastasis were associated with marked BM fibrosis, as grade 4 was the most evident grade in trichrome staining for collagen I and III expression in both disease entities. Also, reticulin staining grades 3 and 4 were the most common grades in MPNs and marrow metastasis, respectively.

In accordance with our results in MPNs, a study by Jesus et al. [5], who assessed fibrosis of BM stroma in 22 patients with chronic myeloid leukemia in chronic phase before and after treatment using Gomori staining for reticulin, found on evaluation of reticulin grades of the first BM biopsy before treatment that 59.09% of the samples showed significant fibrosis and were graded between 2+ and 3+, 13.64% revealed myelofibrosis grade 4+, only 9.09% of samples were grade 0, and 22.73% were grade 1+.

In accordance with our results, many studies documented the presence of high grades of fibrosis in BM metastasis. Boman et al. [6] reported reticulin and/or collagen fibrosis in 32/35 BM biopsies of patients with neuroblastoma. Another study performed by Kaur et al. [1] on nine patients with BM metastasis found BM fibrosis in 66.6% of cases and osteosclerosis in 22.2% of cases, as detected by reticulin and trichrome staining.

In LPDs in this study, grade 0 was reported as the most evident grade for both reticulin and trichrome staining; the same was seen for collagen I expression. As regards collagen III expression, grade 1 was the most evident grade. Concordant results were obtained by Etienne et al. [7], who assessed BM fibrosis in 16 cases with LPDs using Masson's trichrome and Gordon–Sweet reaction and stated that BM fibrosis was mild (MF1) in nine patients, moderate (MF2) in six patients, and severe (MF3) in one patient.

Tadmor et al. [8] studied BM fibrosis by Gomori or Gordon–Sweet staining methods in 173 cases diagnosed as chronic lymphocytic leukemia (CLL) and reported that a total of 22 (12.5%) patients had reticulin fibrosis grade 0, 96 (55%) patients had grade 1, 33 (20%) patients had grade 2, and 22 (12.5%) patients had grade 3.

As regards cases with AL (three cases of acute myeloid leukemia (AML)) in the present study, all were negative for reticulin and trichrome staining, two cases were negative for collagen I, and one case was negative for collagen III. However, variable results have been reported in different studies according to the type of leukemia. In a study by Kazi et al. [0] to detect fibrosis by reticulin and trichrome staining on trephine biopsies of 14 cases of acute myeloid leukemia, 71.4% of cases showed grade 1 to grade 3 fibrosis.

In another study by Bharos et al. [9] to detect BM fibrosis in childhood acute lymphoblastic leukemia (ALL), the percentages of patients with different grades of fibrosis were as follows: grade 0, 2.5%; grade 1, 33.3%; grade 2, 37%; grade 3, 19.7%; and grade 4, 7.5%.

In the present study, 44% of cases had reactive BM changes. Most of these cases were negative for both trichrome and reticulin staining, in addition to collagen I and III.

Similarly, negative staining for trichrome and reticulin was observed in the majority of reactive BM cases studied by Kazi et al. [0], which included 30 cases of erythroid hyperplasia and 12 cases of myeloid hyperplasia.


  Conclusion Top


Collagen I and III are markedly expressed in fibrotic marrow and marrow fibrosis; in particular, collagen fibrosis appears to be more evident in cases with MPNs and tumor metastasis to the BM.

Conflicts of interest

There are no conflicts of interest.[19]

 
  References Top

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    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5]
 
 
    Tables

  [Table 1], [Table 2], [Table 3], [Table 4], [Table 5], [Table 6], [Table 7]



 

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