Paraffin embedded marrow clot sections as an adjuvant procedure in the diagnosis of bone marrow diseases.

Objectives: This study was conducted to evaluate the role of histopathological examination of paraffin embedded marrow clot sections in the diagnosis of different neoplastic and non-neoplastic marrow diseases, and whether these sections can replace the need for performing bone marrow trephine biopsy. Methods: Patients included in the study are those with clinical manifestations of hematological disorders who were subjected to peripheral blood examination, marrow aspirate cytology with complimentary bone marrow trephine and marrow clot biopsy. Along with marrow aspiration, the blood left behind after preparing marrow smears, was used to prepare paraffin embedded histological clot sections. The results for 114 patients were analyzed simultaneously for their concordance to highlight the usefulness of marrow clot sections in the diagnosis of different diseases affecting the bone marrow. Immunohistochemical stains were used whenever indicated to obtain additional diagnostic information. Results: Marrow clot sections were diagnostic and concordant with the complete blood count, marrow aspirates and trephines in 63% of the cases. While trephines were unconvincing in another 15.7% of the cases, the clot sections were diagnostic. In 1.7% of the cases, the clot sections and trephines were diagnostic while marrow aspirates were inconclusive, thus in 81% of the cases, marrow clot sections provided the diagnosis and replacing the need for performing trephine biopsy. However, trephine biopsy is still considered necessary in 19 % of the cases in whom the clot sections are of poor quality and unconvincing Conclusion: In most of the cases clot sections can replace the need for trephine biopsy, but trephine biopsy is still required in other cases. The clot sections are useful as an adjuvant procedure to increase the diagnostic yield in marrow studies. Because it is difficult to expect which patients will be diagnosed by clot sections and which will need trephine biopsy, it is advisable to perform both trephine and clot biopsy together with the marrow aspirates.


INTRODUCTION
one marrow examination is useful in the diagnosis of various hematological diseases. Both neoplastic and nonneoplastic hematological diseases can be diagnosed with a maximum diagnostic yield by the simultaneous examination of peripheral blood, marrow aspirate smears, marrow trephine and clot biopsy sections. [1][2][3] The blood clot collected during marrow aspiration can be used to prepare paraffin embedded histological sections. With the use of immunohistochemical stains, marrow clot sections may provide an adjuvant procedure to improve the diagnostic yield in different neoplastic and non-neoplastic marrow diseases. Studies, worldwide, have been done to evaluate the role of marrow clot sections as an adjuvant diagnostic method in different marrow diseases. [2][3][4][5][6] However, this is the first study on marrow clot sections to be conducted in our locality to evaluate the role of paraffin embedded marrow clot sections in the diagnosis of different marrow diseases, and whether these sections can replace the need for performing bone marrow trephine biopsy.

MATERIALS AND METHODS
This is a prospective study, performed in Basrah governorate-Iraq, during the period from January 2013 through April 2015. A 114 patients, sixty five males and forty nine females, all are those with clinical manifestations of hematological disorders who were subjected to peripheral blood examination, marrow aspirate cytology with complimentary bone marrow trephine and marrow clot biopsy. Along with marrow aspiration, 0.5-2 ml of the aspirated blood (the blood left behind after preparing marrow smears) was fixed in 10% formal saline overnight. Trephine biopsy was fixed in Bouin's solution overnight. If more decalcification is required, trephine biopsy is further decalcified with 20% HCL for 1-3 hours. Unnecessary prolonged decalcification of trephine biopsies with acid was avoided to insure good staining B quality. Over fixation of marrow clot biopsies was avoided as this may make the clot brittle with subsequent difficult sectioning. The trephine and clot biopsies are then processed through alcohol, xylene, impregnation in paraffin wax and blocking. Sections (3-5 µm thickness) were stained by Hematoxylin and Eosin stains. Immunohistochemical stains were used as indicated. Clinical data and laboratory findings, including complete blood count, marrow aspirate cytology and results of bone marrow trephine and clot sections for each patient were recorded in an Excel sheet for subsequent analysis.

RESULTS
A total of 114 patients were included in this study. Sixty five (57.1%) were males and forty nine (42.9%) females. The mean age was 51.7 year. The commonest indication for marrow study was anemia (51.7%). Backache due to vertebral destructive lesion was the indication for marrow study in 15.7% of the cases, ( The final diagnosis was made by the concordance of the results of complete blood count (CBC), marrow aspirate smears, trephine biopsy and marrow clot biopsy (Table-2).  (+) = conclusive (-) = inconclusive Asp=marrow aspirate smears, Clot = clot sections, Treph = trephine biopsy.

DISCUSSION
The role of histopathological examination of paraffin embedded marrow clot sections has been evaluated as an additional diagnostic method in different neoplastic and nonneoplastic diseases affecting the bone marrow. However, limitations of marrow clot sections have been noted in these works. [2][3][4][5][6] In this study, marrow clot sections are proved to be diagnostic in 80.7% of cases. In 63% of the cases, clot sections yielded results that are concordant with that of complete blood count, trephine biopsy and marrow aspirate smears. In 17.5% of the cases the trephine biopsy was inconclusive, while the clot sections yielded results that are concordant with those of marrow aspirate smears and complete blood count. Tkachuk and Hirschmann concluded that marrow clot sections can be conclusive while trephine biopsy and marrow aspirate cytology failed to show any pathology. [4] Dee et al found that , in some cases, marrow clot sections and smears are diagnostic of lymphoma whereas the trephine biopsy appeared negative. [9] Similarly, Mathur and Soutar in their work on the role of marrow clot sections in confirming marrow involvement by multiple myeloma, reported that "the clot sections provided the diagnosis in 75% of the patients (19/28) and in five of these 19(26%) the trephine biopsy was negative". [7] This highlights the necessity for preparing marrow clot sections whenever bone marrow study is performed, particularly when trephine biopsy is obtained with difficulty. Furthermore, in immunohistochemical staining, antigen retrieval is better in clot sections than trephine biopsy since decalcification is not required. [5] Decalcification has the disadvantage of damaging nucleic acids and proteins reducing the staining quality. [8] As compared with trephine biopsy, the clot biopsy saves time, avoids discomfort, reduces cost with less incidence of complications. [7] This study showed that in most of the cases (80.7%) the clot sections can replace the need for trephine biopsy. However trephine biopsy is still required in other cases, in whom the clot biopsies are of poor quality and inconclusive. This is in agreement with other studies [5,7] Ong et al has concluded that "a bone marrow section comprised of sinusoidal blood is inadequate for morphological interpretation", the authors recommend the "cell block technique to ensure maximum capture of material needed to establish a diagnosis". [6] Because it is difficult to anticipate which patients will be diagnosed by the clot sections, and which will need trephine biopsy, it is advisable to perform both trephine and clot biopsy together with the marrow aspirate. The clot biopsy is prepared as an adjuvant procedure to increase the diagnostic yield.

CONCLUSION
Examination of marrow clot sections in conjunction with the complete blood count, marrow aspirate smears and trephine biopsy is necessary to improve the diagnostic yield of these procedures. In most of the cases clot sections can replace the need for trephine biopsy, but trephine biopsy is still required in other cases.