Standardization of bone marrow specimen processing; immunohistochemistry and reporting

standardization and recommended protocols for processing; IHC and reporting systems in bone marrow aspirate and biopsies

1. Standardization Of Bone Marrow Specimens Processing, Immunohistochemistry And Reporting -Munira Saeed

2. 1. Introduction 2. The Bone Marrow Aspirate 3. Bone Marrow Trephine Biopsy 4. Reporting system 5. Verbal Reports 6. Turnaround Times 7. Storage 8. External Quality Assurance

3. Introduction Bone marrow (BM) aspirate and trephine biopsy examination is essential for the diagnosis & management of blood and BM disorders The lack of uniformity can lead to inconsistencies in disease diagnosis and classification, and thereby affect treatment and clinical outcomes In an attempt to standardize the indications for BM examination, the specimens required and report format, a set of consensus guidelines and recommendations have been made

4. Indications for BM examination

5. Prerequisites Clinical indications - specimens required known Thrombocytopenia / coagulopathy (platelet transfusion/reversal of anticoagulation) A blood count and smear obtained/ 2days Informed consent Adequate sedation and analgesia Either (aspirate or biopsy) may be performed first - 0.5–1 cm from first site using the respective needles- avoids hemorrhagic biopsy/ aspirate clotting

6. Bone marrow aspiration needle

7. Bone Marrow Core Biopsy Needle Types Jamshidi needle J- needle Moeller needle OnControl

8. Anatomic Site Posterior iliac crest- preferred Anterior iliac crest- immobile Medial tibial surface- infants Sternal aspirate- immobile, radiotherapy pelvis, ‘dry tap’ ,biopsy is not required X Cardiac tamponade X bone resorption

9. The Bone Marrow Aspirate 10- or 20-ml plastic syringe, to provide adequate negative pressure, attached to the aspiration needle To preserve morphology, the syringe should not contain anticoagulant

10. Second syringe - additional samples for supplementary tests, such as flow cytometry & cytogenetics It is suggested that these samples be collected with all BM aspirates ≈ 0.5 ml of the first draw of the aspirate- smears by the bedside; With increasing volumes drawn= progressive dilution of the aspirate with PB Additional aspirate into a EDTA tube to make smears, in case the sample clots rapidly amt of aspirate: amt of EDTA - minimize EDTA induced artefacts

11. A minimum of 6 smears Particle clot section 2 particle squash (‘crush’) slide The weight of the second slide on the first is sufficient to squash the marrow particles; no downward force should be applied Least possible amount of blood should be included in the clot sample

12. Staining of BM aspirate slides 2 smears and 1 squash slide - Romanowsky stain 1 smear and 1squash slide - Prussian Blue ( Perls ’ reaction) + Safranin-O/ Kernecht Red Coverslipped Additional slides - cytochemistry (e.g. MPO, NSE), IHC, FISH, or archived as unfixed, unstained smears, as required

13. Microscopy Low power(10X) - no & cellularity of particles, no. of megakaryocytes, abnormal cells Smear - higher power – morphology- cytological detail, parasites or cell inclusions- particularly cellular detail & differential counts Squash - cellularity, megakaryocyte numbers, focal disease, fibrotic marrows, abnormal cells No particles, megakaryocytes, precursors → ‘blood tap’ or peripheral blood No particles, megakaryocytes/precursor cells present → dilute sample → qualitative evaluation Particles with ↓ cellularity → qualitative description Blood count and peripheral smear stained - reviewed in conjunction with aspirate

14. Nucleated differential count Haemopoietic activity - compare proportions of different cell lineages & quantify abnormal cells Cell trails behind particles- minimally diluted with PB - well dispersed, least smudged cells ≥ 300 cells if not essential to diagnosis ≥ 500 cells ≥2 smears for precise %abnormal cell type for diagnosis and disease No. increased -count another smear, or second observer -abnormal cell count -critical threshold for disease stratification/ low threshold (e.g. 5%) /patchy involvement Total no. of cells counted - stated in the report

15. Iron stain Prussian Blue - all initial BM aspirates ‘Dry tap’- core biopsy section -less reliable-decalcification removes iron; sideroblast – imprints Iron store - macrophages- several particles; graded subjectively- absent, reduced, normal, increased, or markedly increased Sideroblast - Total number(normal, reduced or increased) frequency location of granules (cytoplasmic/perinuclear) Ring sideroblasts - ≥5 granules – encircling ≥1/3nucleus - 100 erythroblasts

16. The Aspirate Report Name of institution Unique specimen identifier (laboratory accession number) Details of patient: Name of physician Name of requesting doctor Date Clinical history Indication Procedure performed site Ease/difficulty of aspiration Blood count, Blood smear Cellularity of particles and cell trails Nucleated differential cell count Total number of cells counted Myeloid : erythroid ratio Erythropoiesis, Myelopoiesis, Megakaryocytes, Lymphocytes, Plasma cells, Other haemopoietic cells, Abnormal cells (e.g. blast cells, metastatic infiltrates) Iron stain, Cytochemistry, Other investigations (e.g. cytogenetics, PCR, FISH, microbiology), Summary of flow cytometry findings, if available Conclusion WHO classification (if relevant), Disease code Signature and date The aspirate report should not be delayed whilst awaiting results of supplementary investigations and results that are pending should be noted in the initial report

17. Bone Marrow Trephine Biopsy

18. Imprint If no aspirate, imprint- examine cell composition and cytologic detail and a NDC can be performed Length- least 2 cm, shrinks by ≈ 20% after processing Longer in focal lesion - bilateral - increases the yield labelled -name, id, date and time of collection . Ischemic time to be monitored

19. Fixation and Decalcification Decalcification Shorter TAT methods not recommended for special studies Decalcification should be followed by careful rinsing of about 10 min to remove decalcification reagent Combined decalcifying & fixative solutions are not recommended Fixation Heating and stirring both improve fixation and should always be considered Selection depends on the desirable TAT Bouin’s solution contains picric acid – explosive, a recent study showed that Bouin’s did not provide IHC results comparable with formalin Fixatives containing mercury ( Zenker’s and B5) suitable for IHC but are toxic

20. Processing and Staining Staining Should be stained with H&E Giemsa staining may be carried out in addition to H&E stains- identifying plasma cells, mast cells, lymphoid cells, eosinophils & for distinguishing myeloblasts from proerythroblasts One section may be stained for reticulin by the silver impregnation method Embedded in paraffin wax / plastic Recommended thickness of 2 -3µ & even ≥ 6 sections at three levels into the cross-sectional diameter serial sections be mounted stepwise on glass slides

21. Microscopy 2-4 sections reviewed Overall marrow architecture and cellularity, focal lesions and patchy infiltrates The % cellularity- proportion of cells occupying the total marrow cavity, BM cellularity age adjusted Low power - adequacy, pattern, cellularity, presence of focal lesions, megakaryocyte number, abnormal cell clusters and location, bone structure , and osteoclastic and osteoblastic activity High power - haemopoietic activity and cytological detail. Higher power - fine cytological details, e.g. intracellular granules, Auer rods

22. Reticulin - quantified by grading from 0 to 3 (European consensus scoring system) or alternative system, the system used must be stated Focal increase in reticulin (e.g. seen after therapy) should be commented on if necessary for diagnosis Fiber density should be assessed only in hematopoietic areas. In grades MF-2 or MF-3 an additional trichrome stain is recommended by WHO

23. Immunohistochemistry 1) Pre analytical -time of procurement of the BM samples and ends by cutting the embedded tissue onto glass slides 2)Analytical -protocols used for IHC staining (including antigen retrieval, primary antibody incubation, incubation with detection system, color development for visualization of immunological reaction), counterstaining, and it ends with cover slipping 3)Post analytical - interpretation of the IHC results by the ( hemato )pathologist 4)Quality assurance - positive and negative controls, participation in PT provided by EQA programs, use of flow cytometry (FCM) to validate IHC, and training and education Lack of standardization of the pre-analytical component- prevents building EQA/PT programs for BM IHC

24. IHC Class I (lesser risk) : IHC both interpreted and used by pathologists: Qualitative - for diagnostic purposes- determination of cell lineage Validation -medical director of laboratory. Test performance characteristics- descriptive- ‘positive’ or ‘negative’. Class II (greater risk) : IHC prognostic/predictive nature-therapies / decision-making used by treating physician Qualitative and quantitative - prognostic and/or predictive markers. Validation - published guidelines for each marker. Test performance characteristics- descriptive/qualitative (positive vs. negative) or (semi) quantitative (% positivity or H-score or other results of a specific scoring system). At present, only few Class II- relevant to BM IHC- metastatic breast cancer, metastatic lung cancer, or NPM-1 when molecular studies may not be available

25. IHC Recommendations 1. CAP-ACP classification is recommended to properly design and follow QA requirements, Class II IHC tests need higher level of QA 2. All Class II IHC tests follow national and/or international guidelines for recommended protocol for validation 3. If not available, medical director prepares design for Class II markers, according to published guidelines for validation 4. Class II IHC tests need to be run with calibrated positive control and reagent negative controls, used. It is recommended that (calibrated) controls are placed on the same slide as the patient’s sample (so-called ‘on-slide’ controls) 5. Reagent negative controls are generally not recommended for Class I IHC tests

26. IHC Analytical Standards Recommendations 1.IHC protocols need to be validated 2.Monitoring of protocol performance characteristics using appropriate calibrated controls is recommended. 3. SOPs for each IHC test that is performed in the laboratory need to be developed

27. IHC Post Analytical Standards Recommendations 1. All BM aspirate results should be available to ( hemato )pathologists directly or indirectly smears 2. Interpretation of IHC results should be performed in consideration of the sample adequacy. 3. It is recommended that the interpretation starts with the evaluation of the external control(s). 4. Evaluation of the patient sample starts with detection and evaluation of an internal positive control if such is present

28. IHC Quality Assurance Recommendations Positive and negative controls - Published guidelines should be followed whenever possible Participation in proficiency testing (PT), provided by external quality assurance programs - recommended that laboratories participate only if the EQA program provides samples with identical or nearly identical BM tissue processing Use of flow cytometry to validate IHC- If FCM is available and the disease state is such that it enables straight forward interpretation of IHC results, corresponding IHC testing can be evaluated to compare the number of cells and the levels of expression between the two methods

29. The bone marrow trephine report Name of institution ID Details of patient Name of responsible physician Name of requesting doctor Date Significant clinical history lab results Indication Procedure performed Anatomic site length of core Adequacy and macroscopic appearance of core Percentage and pattern of cellularity Bone architecture Location, number, morphology and pattern of differentiation :- erythroid, myeloid, megakaryocytic lineages, lymphoid cells, plasma cells and macrophages Abnormal cells and/or infiltrates Reticulin stain Immunohistochemistry Histochemistry Other investigations (e.g. FISH, PCR) Conclusion Disease code Signature and date of report

30. Reporting System

31. Conclusion. — A framework for bone marrow synoptic reporting will improve completeness of the final report in a manner that is clear, concise, and consistent among institutions

32. BONE MARROW: Final Integrated Diagnosis BONE MARROW: Histologic Assessment Clinical context ___ New diagnosis, untreated ___ New diagnosis, treatment status unknown ___ Follow up sample Procedure ___ Bone marrow aspiration ___ Bone marrow aspirate clot ___ Bone marrow core biopsy ___ Bone marrow core touch preparation (imprint) Peripheral Blood Complete Blood Cell Count Bone Marrow Morphology Bone Marrow Cellularity: ___% Bone Marrow Blasts: ___ % Bone Marrow Lymphocytes (report for lymphoid malignancies): ___ % Dysplasia (report for myeloid malignancies) ___ Absent___ Present (select all that apply)___ Granulocytic lineage___ Erythroid lineage___ Megakaryocytic lineage Iron stain (report for myeloid malignancies) Reticulin/Trichrome stains (fibrosis grade) Histologic Group Biomarker Studies Immunohistochemistry Flow Cytometry Cytogenetics Fluorescence in situ Hybridization Molecular Diagnostics CAP Protocol

33. VERBAL REPORTS When a verbal report is given to a clinician, a comment should be added to indicate the name of the pathologist providing the information, to whom the report was given, and the time and date

34. TURNAROUND TIMES BM Aspirate Processing TAT- collection of the aspirate -slides available for microscopy : 2-6working hours Reporting TAT , or the time from when the slides are available for microscopy to the time when a verbal or written report is issued: Urgent: 3 working hours- verbal/ 24 h- written Less urgent : 48 hours- written Trephine biopsy specimens Processing TAT - fixation and decalcification regimens, laboratory procedures, usually 24–72 hr Reporting TAT : Urgent: 3 working hours- verbal/ 24 h- written Less urgent: 5 days- written If IHC or other stains performed: additional 24–48 h

35. STORAGE Spare BM aspirate slides may be wrapped tightly in aluminum foil for storage at -20 Celsius to preserve cellular antigens. Unfixed and unstained aspirate smears stored at room temp for long periods may give variable results on Giemsa staining Aspirate slides fixed in absolute methanol preserve DNA for future FISH, or DNA extraction and subsequent PCR amplification. The duration of storage of BM specimens and reports should comply with national regulatory guidelines Where these are not available, BM slides should be stored for at least 20 years, or indefinitely, if possible Digital images and electronic reports may be stored indefinitely.

36. EXTERNAL QUALITY ASSURANCE Participation in external quality assurance (EQA) schemes for both technical and interpretative elements of BM examination are encouraged and recommended to ensure accuracy, reproducibility and standardization.

37. References ICSH guidelines for the standardization of bone marrow specimens and reports, LEE ET AL, Int. Jnl . Lab. Hem. 2008, 30, 349–364 ICSH guidelines for the standardization of bone marrow immunohistochemistry, TORLAKOVIC ET AL, Int. Jnl . Lab. Hem. 2015, 37, 431–449 Protocol for the Examination of Hematologic Malignancies in Bone Marrow, Version: Bone Marrow 4.0.0.0 Protocol Posting Date: February 2019 Combined Pathology-Driven Algorithmic Testing and Reporting for Bone Marrow Examination, Pearson et al, Arch Pathol Lab Med, ahead of print Bone Marrow Synoptic Reporting for Hematologic Neoplasms, Cordelia Sever et al, Arch Pathol Lab Med—Vol 140, September 2016 Data-Driven Iterative Refinement of Bone Marrow Testing Protocols Leads to Progressive Improvement in Cytogenetic and Molecular Test Utilization, Seegmiller et al, Am J Clin Pathol 2016;146:585-593 Diagnostic testing managed by hematopathology specialty and other laboratories: costs and patient diagnostic outcomes, Engel-Nitz et al, BMC Clinical Pathology 2014

38. Making the most of bone marrow trephine biopsy, Wilkins et al, (2009) Histopathology 55, 631–640 Optimal processing of bone marrow trephine biopsy: the Hammersmith Protocol, Naresh et al, J Clin Pathol 2006;59:903–911 Bone Marrow Biopsy Needle Type Affects Core BiopsySpecimen Length and Quality and Aspirate Hemodilution,Brestoff et al, Am J Clin Pathol February 2019;151:185-193 Comparison of the bone marrow trephine sample quality between OnControl drill system and the Jamshidi needle, Forwood et al, Int J Lab Hematol . 2019;1–7 Bone marrow solid core biopsy needle: a critical assessment of the utility, benefits and limitations of the instruments employed in current day haematology and oncology, Islam A. J Clin Pathol 2017 Immunohistochemistry of Bone-Marrow Biopsy, STEFAN0 ET AL, (1997) Immunohistochemistry of Bone-Marrow Biopsy, Leukemia & Lymphoma, 26:sup1, 69-75