Hematopathology Case Study: A 60 Year Old Man with Recurrent Bronchitis

Case History

60 year old man with recurrent bronchitis and extensive smoking history underwent CT scan. The CT scan showed an incidental finding of a 2.2 x 1.4 cm anterior mediastinal mass.

Excision

H&E4x
H&E 4x
H&E10x
H&E 10x
H&E20x
H&E 20x
cytokeratin cocktail
Cytokeratin cocktail
CD3
CD3
CD20
CD20
TdT
TdT

Diagnosis

The tissue shows nodules of epithelial cells in a lymphocyte-rich background. The epithelial cells have round to somewhat spindle shaped nuclei, vesicular chromatin and small mostly inconspicuous nucleoli. There is no high grade cytologic atypia, mitotic figures or necrosis seen. The nodules contain very few interspersed lymphocytes, but are surrounded by abundant lymphocytes which are small and mature appearing. A cytokeratin cocktail highlights the epithelial nodules and shows an absence of epithelial cells in the lymphocyte-rich areas. CD20 highlights stromal B-lymphocytes around the epithelial nodules which are arranged in follicles. CD3 highlights stromal T-lymphocytes, which surround the B-cell follicles and the epithelial nodules. TdT highlights only a very small subset of immature T-cells which are found scattered around the rim of the epithelial cell nodules. Overall, the findings are consistent with a micronodular thymoma with lymphoid stroma.

Discussion

The differential diagnosis for an anterior mediastinal mass includes thymoma, lymphoma, germ cell tumors, neurogenic tumors and benign cysts among other less common entities. Patients usually present with cough, chest pain, fever/chills or dyspnea and localizing symptoms are generally secondary to local tumor invasion. Typically, CT scans are the best modality to evaluate the mediastinum. Thymomas are the most common primary neoplasm of the anterior mediastinum, but are less than 1% of all adult malignancies. Patients are generally over 40 years old and between 30-50% of patients with a thymoma have myasthenia gravis, which occurs more frequently in women.1

The WHO has classified thymomas into 5 categories based on the morphology of the neoplastic epithelial cells along with the lymphocyte to epithelial cell ratio. Type A thymomas are composed of bland spindle/oval tumor cells with few or no admixed immature lymphocytes. Type B1 thymoma resembles normal thymus and has scattered epithelial cells in a dense background of immature T-cells. Type B2 thymoma is composed of epithelial cells in small clusters with a lymphocyte-rich background. Type B3 thymoma is primarily composed of mild to moderately atypical epithelial tumor cells in a solid growth pattern with few intermingled immature T-cells. Type AB thymomas are composed of lymphocyte-poor spindle cell (Type A) components as well as lymphocyte-rich (Type B) components.2

Micronodular thymoma with lymphoid stroma (MTWLS) is a rare type of thymoma and accounts for only 1% of all cases. Patients tend to be asymptomatic and the finding is usually incidental. The tumor tends to be well circumscribed and encapsulated with a tan cut surface. The histopathology is characterized by solid nests or nodules of epithelial tumor cells in a background of abundant lymphoid stroma. The tumor cells are bland spindle or oval cells without significant atypia or mitotic activity. The epithelial tumor cells are positive for pancytokeratins. The lymphoid stroma typically lacks keratin positive cells and consists of predominantly CD20 positive mature B-cells in follicles with admixed CD3 positive and TdT negative mature T-cells. There is typically a population of rare TdT positive immature T-cells that surrounds the epithelial nodules, as seen in this case. 2

Due to the rarity of MTWLS with only 74 cases reported since the first case described in 1999, there is limited data on its pathophysiology and prognosis. However, most cases are diagnosed as stage I/II disease according to the Masaoka-Koga staging criteria, involving only micro or macroscopic invasion into thymic or surrounding fatty tissue without invasion into neighboring organs.  Patients tend to have a very favorable prognosis with most patients alive without recurrence or metastasis many years after diagnosis.3

References

  1. Juanpere S, Cañete N, Ortuño P, Martínez S, Sanchez G, Bernado L. A diagnostic approach to the mediastinal masses. Insights Imaging. 2012;4(1):29-52.
  2. Travis WD, Brambilla E, Burke AP, et al. WHO Classification of Tumours of the Lung, Pleura, Thymus and Heart (Revised 4th edition). IARC: Lyon 2015.
  3. Qu L, Xiong Y, Yao Q, Zhang B, Li T. Micronodular thymoma with lymphoid stroma: Two cases, one in a multilocular thymic cyst, and literature review. Thorac Cancer. 2017;8(6):734-740.

Chelsea Marcus, MD is a Hematopathology Fellow at Beth Israel Deaconess Medical Center in Boston, MA. She has a particular interest in High-grade B-Cell lymphomas and the genetic alterations of these lymphomas.

Hematopathology Case Study: A 65 Year Old Male with a Skin Lesion on the Right Shoulder

Case History

A 65 year old Caucasian male presents with a skin lesion on his right shoulder. Physical examination reveals a 3 .0 cm  ×  1.5  cm hyperpigmented plaque with mild hyperkeratosis on his right shoulder and multiple scattered erythematous macules and plaques on the trunk and back Skin biopsy reveals involvement by Blastic Plasmacytoid Dendritic Cell Neoplasm (BPDCN). PET scan reveals no extracutaneous involvement.

The patient undergoes CHOP chemotherapy followed by autologous hematopoietic stem cell transplantation. He is subsequently treated systemically with lanolidomide, venetoclax, and idelalisib due to relapses of disease.

The patient returns to clinic three years later for follow-up. While his original cutaneous lesions are completely resolved, new lesions are noted on his back (representative lesion, Image 1). Hematologic evaluation is remarkable for pancytopenia with hemoglobin 8.7gm/dL, white blood cells 1.4 K/uL, and platelets 39 K/uL. A biopsy of the bone marrow is performed.

Image 1. Skin lesion on back.

Biopsy Findings

Core biopsy
CD56
Aspirate

H&E stained sections demonstrate a normocellular bone marrow with diminished trilineage hematopoiesis and sheets of amphophilic, blastoid cells with irregular borders occupying most of the marrow cells. Immunohistochemistry demonstrates a cellular population with CD56. The aspirate smears show similar findings with numerous clustered blastoid cells (92%) with a monocytoid appearance, often with basophilic vacuolated cytoplasm. There is also a decrease in myeloid and erythroid precursors.

Flow cytometric analysis performed on the bone marrow aspirate reveals a dim CD45 population with expression of CD4, CD56, partial CD7, dim and partial CD5, and CD38. The same population lacks expression of immaturity markers such as CD34, MPO, and TdT. The morphologic and phenotypic findings found in the marrow specimen are diagnostic of extensive involvement of the marrow by BPDCN.

Discussion

BPDCN is a rare and highly aggressive malignancy derived from precursors of plasmacytoid dendritic cells. Its nomenclature has constantly changed over years as the understanding of this entity has been improved. It has been variously known as blastic natural killer cell lymphoma/leukemia, agranular CD4+ natural killer cell leukemia, and CD4+CD56+haematodermic neoplasm. It is currently classified under acute myeloid leukemia and related precursor neoplasms in the most recent WHO classification of tumours of haematopoietic and lymphoid organs.

Limited data exist regarding the incidence of BPDCN; however, it is estimated to account for 0.7% of primary cutaneous skin lymphomas and 0.44% of all hematological malignancies. This hematodermic malignancy predominantly affects elderly male patients with mean age ranging from 60 to 70; however, a few cases have also been reported in childhood and infancy. As demonstrated in our case, the patients typically present with multiple violaceous skin lesions, which may be associate with erythema, hyperpigmentation, purpura, or ulceration. Extracutaneous involvement is reported to occur in the bone marrow, peripheral blood, and lymph nodes.

Diagnosis of BPDCN relies on histological and immunophenotypic findings. Histologically, BPDCN may show a monomorphic infiltrate of medium-sized immature blastoid cells with round nuclei, finely dispersed chromatin, and cytoplasmic vacuoles. They typically display immunophenotypic expression of markers CD4, CD56, CD123, and T-cell leukemia/lymphoma 1 (TCL1) without any lineage-specific markers of T cells or B cells. Chromosomal abnormalities involving 5q, 12p,13q, 6q, 15q, and 9p have been reported. The differential diagnosis entails, but is not limited to, mature T-cell lymphoma, nasal-type NK/T-cell lymphoma, myeloid sarcoma/acute myeloid leukemia and T-cell lymphoblastic lymphoma/leukemia

The clinical course of BPDCN is aggressive, with a median survival of 9 to 16 months. The patients with disease limited to the skin may have a better prognosis, while advanced age and advanced clinical stage are indicators of poor prognosis.  There is currently no consensus on optimal management and treatment because of low incidence of BPDCN; however, most patients are treated with regimens used for other hematopoietic malignancies (i.e. CHOP and hyperCVAD) followed by allogeneic stem cell transplantation for eligible patients. They often respond well to chemotherapy with complete resolution of skin lesions; however, relapse of disease can occur due to resistance to chemotherapeutic agents, which may have happened in our case.

References

  1. Lim MS, Lemmert K, Enjeti A. Blastic plasmacytoid dendritic cell neoplasm (BPDCN): a rare entity. BMJ Case Rep. 2016;2016:bcr2015214093.
  2. Grushchak S, Joy C, Gray A, Opel D, Speiser J, Reserva, Tung R, Smith SE. Novel treatment of blastic plasmactoid dendritic cell neoplasm: a case report. Medicine (Baltimore). 2017 Dec;96(51):e9452.
  3. Dhariwal S, Gupta M. A case of blastic plasmacytoid dendritic cell neoplasm with unusual presentation. Turk J Haematol. 2018 Jul 24. doi: 10.4274/th.2018.0181.
  4. Shi Y, Wang E. Blastic plasmacytoid dendritic cell neoplasm: a clinicopathologic review. Arch Pathol Lab Med. 2014 Apr;138(4):564-9.
  5. Bulbul H, Ozsan N, Hekimgil M, Saydam G, Tobu M. Report on three patients with blastic plasmactoid dendritic cell neoplasm. Turk J Haematol. 2018 Sep;35(3):211-212.
  6. Kerr D 2nd, Sokol L. The advances in therapy of blastic plasmacytoid dendritic cell neoplasm. Expert Opin Investig Drugs. 2018 Sep;27(9):733-739.
  7. Pagano L, Valentini CG, Pulsoi A, Fisogni S, Carluccio P, Mannelli F, et al. Blastic plasmactoid dendritic cell neoplasm with leukemic presentation: an Italian multicenter study. Haematologic. 2013 Feb;98(2):239-246.

-Jasmine Saleh, MD MPH is a pathology resident at Loyola University Medical Center with an interest in dermatopathology and hematopathology. Follow Dr. Saleh on Twitter @JasmineSaleh.

-Kamran M. Mirza, MD PhD is an Assistant Professor of Pathology and Medical Director of Molecular Pathology at Loyola University Medical Center. He was a top 5 honoree in ASCP’s Forty Under 40 2017. Follow Dr. Mirza on twitter @kmirza.

Hematopathology Case Study: A 66 Year Old Man with Abdominal Pain and Diarrhea

Case History

66 year old man with a past medical history of Sjogren syndrome, hypertension and hypothyroidism presented to an outside hospital with abdominal pain and diarrhea. CT imaging showed extensive lymphadenopathy, splenomegaly, mesenteric thickening and ureteral dilatation, all highly concerning for lymphoma or other malignancy. A needle core biopsy of “mesenteric mass” showed reactive lymphoplasmacytic infiltrate in a sclerotic background. A second needle core biopsy of a retroperitoneal lymph node was performed which showed reactive features and numerous plasma cells. An excisional biopsy of an inguinal lymph node was ultimately performed due to worsening lymphadenopathy with increased PET FDG avidity.

Excisional Biopsy

H&E 10x
H&E 20x
H&E 20x
H&E 40x
IgG
IgG4

Diagnosis

At low power view, there is intact follicular architecture with reactive appearing germinal centers that are polarized and show tingible body macrophages. The higher power view shows a feature that can be seen in Castleman disease called a “lollipop” which has a thickened vessel with hypertrophied endothelial cells penetrating the germinal center. In addition, there are areas with sclerotic remnants of germinal centers, a feature that can be seen in infection or other reactive conditions. Notably, the interfollicular space is expanded by numerous plasma cells and rare scattered eosinophils. Overall, the lymph node shows reactive follicular and paracortical hyperplasia. Stains for IgG and IgG4 showed an elevated IgG:IgG4 ratio with 54% of IgG plasma cells positive for IgG4. These findings raise the possibility of IgG4-related lymphadenopathy (IgG4LAD). In addition to histopathologic findings, the patient was tested for a serum IgG4 level, which is an important diagnostic criterion for the diagnosis of IgG4 disease. Seen above, serum testing on 10/25/18 showed a low/normal IgG subclass 4 of 17 mg/dl. After the excisional biopsy was performed, suspicion of involvement by IgG4-related disease increased. A sample was re-sent on 11/27/18 with instructions to perform serial dilutions in case of the prozone effect (see discussion below). The test came back with an elevated serum IgG subclass 4 of 1239 mg/dl, further supporting the diagnosis of IgG4-related disease.

Discussion

IgG4-related disease is a condition that was originally described in patients with autoimmune pancreatitis. These patients were found to have elevated serum IgG4 concentrations and have large numbers of IgG4-positive plasma cells. IgG4-related disease is now recognized as a fibroinflammatory condition and has been described in almost every organ system. Similar histopathological characteristics can be seen regardless of location and include tumefactive lesions, dense lymphoplasmacytic infiltrate, increased IgG4-positive plasma cells and storiform fibrosis.1

IgG4 normally accounts for less than 5% of the total IgG in healthy people and is the least abundant IgG subclass. IgG4 is also seen to be involved in other immune-mediated conditions such as pemphigus vulgaris, idiopathic membranous glomerulonephritis, and thrombotic thrombocytopenic purpura. The majority of patients with IgG4-related disease have elevated serum IgG4 concentrations, but 30% of patients may have normal IgG4 concentrations.1 When testing IgG4 serum levels, it is important to be aware of the prozone effect. This occurs when very high concentrations of the antibody that is being measured are present. This will prevent appropriate antibody-antigen binding and agglutination from occurring. This will result in a falsely low level of antibody being detected. In a report by Khosroshahi et. al., after identifying the prozone effect in one patient with IgG4-related disease, 38 patients who had previously been tested for serum IgG4 levels were re-tested. The prozone effect was found to affect 26% of patients with IgG4-related disease. The samples were re-tested with serial dilutions and the mean serum concentration rose from 26 mg/dl to 2,008 mg/dl. This could have a large impact on patient care, as elevated serum IgG4 concentrations are very important in making the diagnosis of IgG4-related disease. 2

IgG4-related lymphadenopathy (IgG4LAD) is somewhat distinct from tissue based IgG4-related disease and presents with solitary or multifocal lymph node enlargement. Lymph nodes are involved by a lymphoplasmacytic infiltrate with increased IgG4-positive plasma cells and tissue eosinophils and are not typically involved by storiform fibrosis. Five microscopic subtypes have been described and include multicentric Castleman disease-like changes, follicular hyperplasia, interfollicular lymphoplasmacytic proliferation, progressive transformation of germinal centers, and a variant with the formation of inflammatory pseudotumor-like lesions. The differential diagnosis of this entity is broad and purely reactive lymph nodes as well as multicentric Castleman disease (MCD), in particular needs to be ruled out. The presence of HHV8 infection and elevated IL-6 and CRP can favor MCD, while tissue eosinophilia favors IgG4LAD.3    

The diagnostic criteria for the diagnosis of IgG4LAD includes lymph node involvement,  a serum IgG4 level greater than 135 mg/dl, and histologic findings of lymphoplasmacytic infiltrates with either >10 IgG4-positive plasma cells per hpf or a ratio of IgG4-positive to IgG-positive plasma cells to be greater than 40%. As seen in this case, patients may undergo multiple biopsies, often with extensive work-ups before a diagnosis is made. Treatment depends on the organ involved and extent of damage/dysfunction. While some cases may only need clinical follow-up, others will require urgent aggressive treatment. Glucocorticoids are typically the first line of therapy and have been shown to be effective in a majority of patients with IgG4-related disease. 3

References

  1. Stone, J, Zen, Y, Deshpande, V. IgG4-Related Disease. N Engl J Med 2012; 366:359-551.
  2. Khoroshahi, A, Cheryk, LA, Carruthers, MN, et. al. Brief Report: spuriously low serum IgG4 concentrations caused by the prozone phenomenon in patients with IgG4-related disease. Arthritis Rheumatol 2014; 66(1):213-7.
  3. Wick, M, O’Malley, D. Lymphadenopathy associated with IgG4-related disease: Diagnosis and differential diagnosis. Seminars in Diagnostic Pathology 2018; 35(1)61-66.

Chelsea Marcus, MD is a third year resident in anatomic and clinical pathology at Beth Israel Deaconess Medical Center in Boston, MA and will be starting her fellowship in Hematopathology at BIDMC in July. She has a particular interest in High-grade B-Cell lymphomas and the genetic alterations of these lymphomas.

A Year in the Life of a Hematology Laboratory

As this year comes to a close and we look forward to celebrating holidays with family and friends, we can also celebrate our accomplishments over the year. Our jobs in the clinical laboratory are vital in helping physicians make clinical decisions and we should celebrate the role we play in healthcare. In hematology, techs are busy doing daily tasks; QC,maintenance, and analyzing all the samples that come into the lab, 24 hours a day, 7 days a week. We work constantly to provide physicians with accurate and precise results in a timely fashion. But, what else goes on in hematology?

This past year has seen many changes and challenges in our hematology lab. In February, we switched our hematology analyzer to a new Sysmex system,and went to autoverification at the same time. This was a process that had begun months before with meetings with the Sysmex team, building rules for WAM,validations, training key operators, as well as site surveys and actual planning for the location of the instrument and water and electrical connections. Before we went live, new procedures had to be written and all techs had to be trained on the new system. Every tech in the department had to do competencies, everything had to be documented and the new procedures had to be signed. This doesn’t stop once the instrument is in use. There has been a continual learning process since then as techs become more familiar with the system.

During all the excitement and work involved with a new instrument, we, as have many labs, have had turnover in staff which has led to its own challenges. Techs have retired, moved out of state, been on maternity leave and have left us for other opportunities both in other areas of our lab and elsewhere. New staff needs to be hired and trained. Students need mentors during their rotations. It’s a cycle we go through every year, a never ending process. And, no sooner had we seemed to have everyone trained, then it was time for 6 month validations and competencies.

In September we moved to new Coagulation instruments, which, fortunately, was not as big a change as our Sysmex analyzers, because the coag instruments are newer versions of instruments we already used. Yet, there were validations to be done, training to be done on the new software, and procedures to write, all at a time when we were about to go live with a new LIS!

Perhaps our biggest project of the year came to fruition in September when we moved to Epic for a hospital wide software system. This was an undertaking which was well over a year in the making. Again, this transition involved many months of meetings, working with Epic and our IT department to create test codes and profiles and to build the system to our needs. We worked with Sysmex and WAM support to verify that there would be a smooth transition from the old system to the new. The month before go live, we did wet and dry testing of every possible scenario and tested every rule in WAM. And then we tested every rule in Epic. Many hours and late nights were spent entering test orders, creating spreadsheets, taking screen shots, and going back and forth to IT for changes and updates. An integral part of this Epic journey was more training for employees. Superusers were trained, training sessions were held for all, and then superusers helped to support other users at Go Live. And, of course, all of this this meant more procedures had to be written. The epic day arrived, and though things didn’t seem too smooth at first, the support teams were and continue to be available to help and make changes as necessary.

These are just a few of our particular challenges this year in the department. Even without these added projects, though, there is a lot that goes into operating a hematology laboratory. Every week, every month and every year, there are ‘extra’ or ongoing projects to be completed in every laboratory department. While we had some major changes this year, there were also many smaller ones. There are always new pieces of equipment that need to be validated, and new procedures or job aids to be written. Quality control has to be monitored, calibrations have to be performed, new lots need to be entered and tested, linearities have to be done. CAP surveys need to be assigned,reviewed and submitted. Inventory needs to be taken, vendors need to be met with or contacted and supplies need to be ordered. Equipment repairs ,troubleshooting and maintenance all need to be addressed. Training doesn’t stop at new hires and students. All techs have to complete annual competencies. Every year instruments have to be validated, new lot crossovers have to be done, and all procedures must be reviewed and updated. We need to get ready for inspections, or perform self-inspections. I’m sure I am leaving out a list of things, but this is a brief overview of all that goes into laboratory operations. It’s certainly more than just analyzing samples!

Who does all these ‘behind the scenes’ tasks? The department supervisor or technical specialist may be designated to make sure these are all completed, but often senior techs or career ladder techs can play an important role in meeting all these requirements. Many hospitals now have career ladders that allow techs to use the designation MLS II or III or MLT II or III. Our laboratory started such a program this year. For anyone interested in moving up the career ladder in their laboratory, there are many opportunities to be involved in lab operations and management. All the tasks that are required to run a lab cannot be done by one person alone. Tier program requirements differ from hospital to hospital but may ask candidates to submit a tier application and complete a list of achievements to show their commitment to the laboratory and their community before being designated a tier II or III.

Tier techs are generally required to meet education and certification requirements. They should be pro-active performers who are seen as leaders with excellent customer service skills. A tier level tech is a proficient performer with strong critical thinking and problem solving skills. They are mentors to coworkers and can train staff and perform competencies. Hospitals often look to these techs to contribute to the growth of the profession outside of the lab, as well. Being laboratory science community ambassadors, performing community service and upholding the mission and values of your facility all constitute qualities a hospital looks for in a tier tech.

Does this sound like you? We are constantly in need of techs to aspire to working in supervisory positions and management. With an increase in age of supervisors, managers and administrators, we are seeing an increase in retirements. We need more techs doing routine bench work to take the initiative and the steps to become tier techs and lead techs. We need aspiring supervisors and managers. Why wait? I encourage you to make a New Year’s resolution to seek out your facility’s tier or career ladder program. If one doesn’t exists, make it a project to see if one can be introduced!

Laboratories are often hidden in the basement, out of sight of visitors and out of mind of the general public. When we mention we work in a hospital, people ask “Are you a nurse?” Even though we may not be a well-known profession, we are a very important group of dedicated scientists and can be very proud of our accomplishments and contributions. I thank my supervisor and mentor, Gene Galligan, for her encouragement and support of the tier program and for all the things she has taught me in this very busy year.

PS: As I wrote this, The Joint Commission team arrived to start their accreditation process. There is never a dull moment in the laboratory!

Happy New Year!

-Becky Socha, MS, MLS(ASCP)CM BB CM graduated from Merrimack College in N. Andover, Massachusetts with a BS in Medical Technology and completed her MS in Clinical Laboratory Sciences at the University of Massachusetts, Lowell. She has worked as a Medical Technologist for over 30 years. She’s worked in all areas of the clinical laboratory, but has a special interest in Hematology and Blood Banking. When she’s not busy being a mad scientist, she can be found outside riding her bicycle.

Hematopathology Case Study: A 23 Year Old Man with Epistaxis, Fever and Pancytopenia

Case History

A 23 year old man presented to the hospital with recurrent fever up to 103F with associated nausea and vomiting, epistaxis, watery diarrhea, dyspnea, and decreased appetite for several days. Blood cultures from admission were positive for MSSA and a stool PCR was positive for Vibrio species. He was admitted and treated for sepsis. His CBC demonstrated a marked pancytopenia ( WBC count 0.6 K/μL) and the hematopathology team was consulted to review the peripheral blood film.

Peripheral blood smear.

Review of the peripheral blood confirmed a markedly pancytopenic picture with virtually no leukocytes in the region of best RBC “spread” (Image 1A). In the periphery of the smear (1B and C) clusters of leukocytes were noted where left-shifted granulocytes were seen. Many demonstrated nuclear irregularity and abnormal granulation (B) and some showed the presence of numerous Auer rods (Image 1C, arrows).

The presence of abnormally granulated immature neutrophilic precursors, and cells with numerous Auer rods was morphologically compatible with acute promyelocytic leukemia (APL) and a rush preliminary diagnosis was rendered. The patient was started on ATRA therapy and FISH for PML-RARA was expedited.

Discussion

Acute promyelocyticleukemia (APL) is characterized as an acute myeloid leukemia in which promyelocytes with the PML-RARA fusion predominate. The PML-RARA fusion is the result of a balanced translocation between chromosomes 15 and 17, designated ast (15;17)(q24.1;q21.2).  The promyelocyte progenitor cell is the cell of origin of APL. APL occurs most frequently in middle aged individuals, but can occur at any age.

The first account of APL was originally discussed in the late 1950s in which L. K.Hillestad, a hematologist from Norway, described a disorder as “a white blood cell picture dominated by promyelocytes and severe bleeding caused mainly by fibrinolysis.” The gene fusion was elucidated in the late 1970s at the University of Chicago demonstrating the balanced translocation between chromosomes 15 and 17. Cure rates at that time were still very low, until in the mid 1980s when researchers in China demonstrated the use of all-trans retinoic acid causing complete remission in APL patients.

Two distinct subtypes of APL exist: hypergranular (typical) or microgranular. The hypergranular variant is filled with large Auer rods and with dense cytoplasmic granules that can obstruct the nucleus. In contrast, the microgranular variant has a scantiness of cytoplasmic granules or small azurophilic granules.

The immunophenotype for APL is quite distinct and characterized by low or absent expression of CD34 and HLA-DR (in keeping with the cellular differentiation from blast to promyelocyte). APL cells are positive CD33 and CD13 with most cases showing expression of CD117 (sometimes weak). APL cells are usually negative for CD15, CD65, CD11a, CD11b, and CD18. The microgranular variant may display positive staining for CD34 and CD2. For both variants, IHC with antibodies to the PML gene demonstrates a nuclear multi granular pattern with nucleolar exclusion, a finding that is unique to APL and not seen in AML or normal promyelocyte morphology.

The main clinical symptom of APL is hemorrhagic, including gingival bleeding and ecchymosis but can progress to disseminated intravascular coagulopathy (DIC). Other symptoms of APL include those related to pancytopenia, including weakness, fatigue, and infections.

The prognosis for APL is considered to be excellent. Tretinoin (ATRA) interacts with the PML-RARA fusion product allowing for maturation and differentiation to occur along the granulocytic lineage, eliminating the promyelocyte population. Combination therapy with tretinoin and arsenic trioxide has become the gold standard of care leading to excellent remission rates.

References

  1. Kakizuka,A., et al. “Chromosomal translocation t (15; 17) in human acutepromyelocytic leukemia fuses RARα with a novel putative transcription factor,PML.” Cell 66.4 (1991): 663-674.
  2. Lo-Coco,Francesco, and Laura Cicconi. “History of acute promyelocytic leukemia: atale of endless revolution.” Mediterranean journal of hematologyand infectious diseases3.1 (2011).
  3. Rowley,JanetD, HarveyM Golomb, and Charlotte Dougherty. “15/17 translocation, aconsistent chromosomal change in acute promyelocytic leukaemia.” TheLancet 309.8010 (1977): 549-550.
  4. Swerdlow,Steven H. WHO Classification of Tumours of Haematopoietic and LymphoidTissues. International Agency for Research on Cancer, 2017.

-Christopher Felicelli is an M3 at Loyola University Chicago Stritch School of Medicine. Follow Chris on Twitter at @ChrisFelicelli

-Kamran M. Mirza, MD PhD is an Assistant Professor of Pathology and Medical Director of Molecular Pathology at Loyola University Medical Center. He was a top 5 honoree in ASCP’s Forty Under 40 2017. Follow Dr. Mirza on twitter @kmirza.

Hematopathology Case Study: A 54 Year Old Woman with a Bone Fracture

Case History

The patient is a 54 year old woman who presented to the hospital after a fall, which revealed a pathologic fracture of T1 and a spinal lesion from C6/C7 to T2. CT of the chest/abdomen and pelvis at the time showed a large mass in the anterior mediastinum with extensive lymphadenopathy and lytic lesions in the spine and ribs.

C7-T1 Soft Tissue Excision

H&E 20X
H&E 50X
H&E 100X
CD30
BSAP/PAX5
KI-67

Diagnosis

Sections show sheets of large epithelioid-like cells with segmented nuclei with variably prominent nucleoli and ample amounts of eosinophilic cytoplasm.A majority of these larger cells have abundant cytoplasm and lobulated nucle iwith multiple nucleoli and a surrounding halo. They are consistent with Lacunar cells. These cells form large aggregates and are admixed with numerous neutrophils, histiocytes and scattered lymphocytes.

Occasional Hodgkin cells and multi-nucleated Reed-Sternberg cells are seen, as well as scattered medium size hyper chromatic cells with irregular nuclear contours and scant cytoplasm consistent with mummy cells.

Immunohistochemical staining revealed that the largea typical cells are immunoreactive for CD30, CD15 and PAX5/BSAP. CD45 highlighted background lymphocytes but showed infrequent dim staining in the large atypical cells. By Ki-67, the proliferation index is 50-60% in the large atypical cells. Taken together, the findings are consistent with Classic Hodgkin Lymphoma, nodular sclerosis, syncytial variant.

Discussion

Classic Hodgkin lymphoma (CHL) has four distinct subtypes including nodular sclerosis, lymphocyte-rich, mixed cellularity and lymphocyte-depleted. These subtypes differ based on characteristics of the background non-neopalastic reactive cells and the histomorphology of the Hodgkin/Reed-Sternberg cells (HRS). Nodular sclerosis Classic Hodgkin lymphoma accounts (NSCHL) for approximately 70% of all CHLs. The mediastinum is the most commonly involved site and it generally occurs in people between the ages of 15-34 years old. Generally, the histology shows nodules with surrounding fibrosis. There are a variable number of Hodgkin/Reed-Sternberg (HRS) cells mixed with other inflammatory cells. The characteristic HRS cell is called a lacunar cell. This is a type of HRS cell with more cytoplasm, less prominent nucleoli and can show retraction of the cytoplasm in formalin-fixed tissue that gives the cell a halo or “lacunae.”1

The syncytial variant (SV) of CHL, nodular sclerosis was first described in the 1980s. It presents in 5-15% of cases of NS CHL. It is characterized by sheets and clusters of “lacunar cells” typical of the type of HRS cell most commonly seen in NS CHL. Previous studies had determined the SV of CHL to have a worse prognosis and more aggressive course than other subgroups. In a more recent study by Sethi, et. al. the clinical features and response to treatment of patients with SV were compared to patients with typical NS CHL. Within the cohort, 43 patients with SV were compared to 124 patients with typical NS CHL. The study found that there was no significant difference in age, sex, performance status, stage, bulky disease, number of nodal sites and chemotherapy regimens used between the two groups.2

As far as treatment outcomes, the rate of complete response in the SV group was 74% vs. 87% in the NS group. This result approached statistical significance with a p=0.05. The medium progression-free survival in the SV group was significantly shorter compared with the NS group. The overall survival, however was not statistically different, indicating that salvage chemotherapy was ultimately able to match the clinical outcomes for patients with SV type to patients with NS type. 2

Currently, all CHLs are treated with adriamycin, bleomycin,vinblastine, decarbazine (ABVD) chemotherapy regimen plus or minus radiation therapy regardless of subtype. Patients with relapsed or refractory disease are treated with a “salvage” chemotherapy regimen followed by an autologous stem cell transplant. Emerging therapies including PD-1 inhibitor nivolumab have shown great effect in patients with CHL. PD-1 or programmed death ligand 1 is overexpressed on HRS cells. This ligand binds with receptorson T-cells to prevent the T-cell immune response and reduce cytokine activation and targeted  response against the proliferating HRS cells. By using an antibody against the PD-1 ligand in CHL,the ability of the tumor to suppress the immune response is decreased and patients have been shown to have better clinical response rates.3

Patients with SV do need to be recognized as a distinct subgroup that may have a higher risk of disease progression with first line chemotherapy agents.  Due to the high numbers of HRS cells seen in patients with SV and the increased failure rate of initial chemotherapy agents, antibody therapies such as PD-1 inhibitors may be even more successful in those patients.

References

  1. Swerdlow SH, Campo E, Harris NL, et al. WHO Classification of Tumours of Haematopoetic and Lymphoid Tissues (Revised 4thedition). IARC: Lyon 2017.
  2. Sethi, T.K.,  et al. Differences in Outcome of Patients with Syncytial Variant Hodgkin Lymphoma (HL) Compared with Typical Nodular Sclerosis HL. Blood. 2015;126(23),1441. Retrieved from http://www.bloodjournal.org/content/126/23/1441.
  3. Bond DA, Alinari L. Emerging treatment options for the management of Hodgkin’s lymphoma:clinical utility of nivolumab. J Blood Med. 2017;8:41-54. Published 2017 May 11. doi:10.2147/JBM.S117452.

Chelsea Marcus, MD is a third year resident in anatomic and clinical pathology at Beth Israel Deaconess Medical Center in Boston, MA and will be starting her fellowship in Hematopathology at BIDMC in July. She has a particular interest in High-grade B-Cell lymphomas and the genetic alterations of these lymphomas.

What’s “In” for Thrombocytopenia Diagnosis? Advanced Platelet Parameters: The Immature Platelet Fraction (IPF%) and the Immature Platelet Count (IPF#)

Platelets are our first line of defense in controlling bleeding. Abnormally low numbers of platelets can lead to easy bruising, tiny leaks from capillaries into the skin and mucous membranes, causing petechiae, and bleeding. The platelet count is a significant parameter in the CBC and it is therefore vital to be able to report accurate and precise platelet counts. Furthermore, physicians must be able to use this information to diagnose the cause of the thrombocytopenia in order to recommend treatment.

What a platelet count alone cannot tell us is the reason for thrombocytopenia. Just as there can be many reasons for a low hemoglobin, and many causes for an increased or decreased WBC, there are numerous causes for a decreased platelet count. After ordering a CBC, the next steps in determining etiology of thrombocytopenia have historically been a thorough physical with attention to any bleeding symptoms and organ enlargements, and a medical history. The medical history should include family history, and notation of recent viruses or drug therapies. After these tests, a bone marrow aspirate and biopsy may also be necessary to clarify etiology. While modern, automated hematology analyzers produce reliable platelet counts, measuring only the circulating platelet count does not give us any information as to the etiology, so there is a need for further testing. With thrombocytopenia, platelet counts can be less reliable than with normal counts.Platelet counts were originally performed by impedance methods, then better accuracy and precision was obtained with optical platelet counts. Physicians rely on precision with very low platelet counts to make informed decisions about when to transfuse patients. The problem with the impedance counts at the low end, is that RBC fragments, schistocytes and microcytic RBCs can be counted as platelets, giving a falsely high count. On the other hand, measuring platelets by size can miss large platelets leading to a falsely low count.

Historically, the mean platelet volume (MPV) has been used along with the platelet count to aid in making a differential diagnosis. The MPV is analogous to the red cell distribution width (RDW) for red cells, and can be used to as an indicator of the maturity of platelets. Young platelets are the largest, and as they age, the size decreases. The normal ranges for MPV are generally about 9-12 femtoliters (fl).The MPV will be higher if more platelets are being released from the bone marrow, and lower if fewer are being newly released and we are counting mature platelets. Thus, the MPV can be used as an indirect marker for platelet production. However, an inherent problem with the MPV is that, similarly to the impedance platelet count, this count can be unreliable because any RBC fragments or particles may interfere with the measurement.

So, what is a physician to do?And how can the lab provide information to help them make the best differential diagnosis and transfusion decisions? In an effort to provide a parameter that could help differentiate causes of thrombocytopenia, the concept of reticulated platelet counts (retPLT) was first introduced in research in the late 1960s. The term is used to describe immature, functional platelets in the peripheral blood.Reticulated platelets are to mature platelets as reticulocytes are to mature red blood cells. These are the youngest platelets, within 24 hours of being released from the bone marrow. Reticulated platelets are large, with increased amounts of RNA, and the number in the circulation can be used to provide an estimate of the rate of thrombopoiesis. Originally, these were stained with new methylene blue and manual counts were done, much like a manual reticulocyte count; tedious,and imprecise. It wasn’t until about 30 years later that a flow cytometry method was described for measuring retPLT. Using traditional flow cytometry, reticulated platelets can be stained with a Thiazole Orange dye and passed through a flow cytometer. This method, however, has been shown to have wide normal ranges from 1-15% because of the lack of analytical standardization. Variations in the concentration of the thiazole dye used, the timing, and the gate settings all make it difficult to compare results obtained from one laboratory to another. In addition, flow cytometry is time consuming, labor intensive and costly.

Newer flow cytometry methods are now available on select hematology analyzers. There are currently 2 companies that have analyzers that can report retPLT using routine CBC reagents and controls. Reticulated platelets can be measured with the same K2 EDTA tube used for the CBC. The test is automated, simple to perform, fast, and gives standardized results with tighter normal ranges. The Abbott CELL-DYN Sapphire measures the retPLT using a fluorescent dye and flow cytometry with 2 dimensional gating. Sysmex XE and XN analyzers offer several Advanced Clinical Parameters including measures of reticulated platelets, expressedas the Immature Platelet Fraction (IPF%) and the Absolute Immature Platelet Fraction Count (IPF#). Sysmex offers a fluorescent platelet count (PLT-F) as an addition to impedance counting (PLT-I) and optical counting (PLT-O). PLT-F is more reliable because it uses a platelet specific dye which eliminates noninterference seen with other methods. The fluorescent dye labels the RNA and forward scatter is used to determine size while fluorescence is used to measure RNA content. With gating set based on cell volume and RNA content, the PLT-Fcan be measured. When there is an abnormal scattergram or a low platelet count,the PLT-F is reflexed and the IPF% and IPF# are also reported.

What’s the clinical utility of the IPF? Thrombocytopenia can have many causes.Immature platelets are functioning platelets, and an increased IPF means that we have more newly formed immature platelets circulating. The IPF helps physicians to differentiate thrombocytopenia caused by platelet destruction or consumption versus thrombocytopenia caused by deficient platelet production in bone marrow failure. It is vital to know the pathogenesis of thrombocytopenia in making decisions about treatment. With these advanced parameters, these decisions can often be made without costly, time consuming flow cytometry,without an invasive bone marrow biopsy and without waiting for the results of such biopsy. This can often save a patient an unnecessary platelet transfusion.

The reference range for IPF% in healthy individuals is1.0-7.0%. Together with a low platelet count, an increased IPF indicates an increase in platelet production. This is seen in patients with excessive destruction of platelets. An example of the clinical utility of the IPF can be seen in the diagnosis of immune thrombocytopenic purpura (ITP). ITP is an autoimmune bleeding disorder in which the immune system makes anti-platelet antibodies which destroy platelets. Acute forms occur more often in children while adults can have chronic ITP. ITP can be diagnosed on clinical findings but laboratory confirmation is often necessary. This can be expensive with long turnaround times using traditional flow cytometry and/or bone marrow aspirates.An IPF reported with a CBC is fast, inexpensive, and be extremely beneficial in aiding a timely diagnosis. Patients with ITP have been shown to have the consistently highest IPF values with ranges from 7-28%.1 As their platelet counts recover, the IPF% returns to the normal range, without the need for transfusions. Thus, the IPF can be used not only to help diagnose but also as an indicator of remission.

Figure1. Platelet scattergrams from a healthy individual with a normal IPF (a) and a patient with a high IPF (b). Mature platelets appear as blue dots, green dots represent the IPF with increased cell volume and higher fluorescence intensity compared to mature platelets1

In contrast to what we see with ITP, thrombocytopenia with alow normal or decreased IPF indicates decreased bone marrow production of platelets. Patients with bone marrow failure are more likely to have bleeding episodes with low platelet counts and may need transfusion. Rapid differential diagnosis using the IPF can help physicians help these patients get early treatment.

IPF may also be a reliable indicator of bone marrow recovery. Traditionally, neutrophil counts have been used as an indicator of recovery after a bone marrow transplant. IPF can be used as an indicator of imminent platelet recovery. It has been shown that,post-transplant, the IPF% increases before the platelet count. In a study done with stem cell transplant patients, it was shown that the absolute neutrophil count took an average of 13 days to recover, compared to 9 days for the IPF. The IPF was shown to recover before the Immature reticulocyte count, platelet count and absolute neutrophil count, giving physicians earlier indication that the transplant was successful.2 This is significant because it can eliminate the need for bone marrow biopsies and platelet transfusions.  

Thrombocytopenia is not an uncommon finding in neonates, particularly in the neonatal intensive care unit (NICU). There are various causes for this, including sepsis, placental insufficiency and immune thrombocytopenia. The IPF% and IPF# can be used to diagnose and distinguish the cause of thrombocytopenia in neonates, and direct the treatment. When platelet count platelet count drops below 50 x 103/Lin an otherwise healthy appearing infant in the first 72 hours of life, neonatal alloimmune thrombocytopenia (NAIT) can be suspected. This condition is similar in pathogenesis to hemolytic disease of the fetus and newborn (HDFN), and is caused by an incompatibility in human platelet antigens between mother and baby. This occurs most often when the mother is HPA-1b and the father and baby are HPA-1a. The mother forms anti-HPA-1a which crosses the placenta and destroys the fetus’ platelets.This is a thrombocytopenia caused by platelet destruction, and the IPF% is high. The condition is self-limiting and resolves in 1-4 weeks. Neonatal sepsis can also present with a high IPF, but typically is found in very sick or premature babies and the degree of thrombocytopenia is not as severe as with NAIT. In contrast, neonatal thrombocytopenia due to placental insufficiency would exhibit a decreased IPF due to a deficiency in platelet production. Using the IPF% and IPF# to help differentiate the causes of neonatal thrombocytopenia can help steer the treatment and save infants from unnecessary invasive procedures and transfusions.

TheIPF has proved to be very valuable in the clinical setting. It has been used in the investigation of etiology in secondary thrombocytopenias due to chronichepatitis C, liver disease and HIV. It has been used to guide treatment in thrombocytopenias such as thrombotic thrombocytopenic purpura (TTP). IPF can also be useful in evaluation of hereditary platelet thrombocytopenias. The IPF% and IPF# can be compared after transfusion to support the theory that, after platelet transfusion, theIPF% will decrease due to the newly increased platelet count, but the IPF#remains the same. This validates that the IPF is a reflection of continual platelet production by the bone marrow.4

IPF%and IPF# are expanded CBC parameters that physicians can use to aid in differentiation of various thrombocytopenic states. Treatment for the different classes of thrombocytopenia can differ drastically, and knowing the class of thrombocytopenia helps direct the management. The IPF parameters are automated,easy to perform at the same time as the CBC, and provide standardized results that are inexpensive and available 24 hours a day in the hospital setting. Using the IPF can also reduce diagnostic costs for the patient. Many studies have been conducted on the varied applications of the IPF and research continues investigating possible further uses of this advanced clinical parameter. This is the new hematology, constantly providing the clinician with better tools for making diagnoses and treating patients. Platelet counts alone and MPVs are out. Make room for the new kid on the block; the IPF is in.

References

  1. Arshi Naz et al. Importance of Immatureplatelet Fraction as a predictor of immune thrombocytopenic purpura. Pak J MedSci 2016 Vol 32 No 3:575-579
  2. Zucker ML et al. Immature Platelet fraction asa predictor of platelet recovery following hematopoietic progenitor celltransplanatation. Lab Hematol 2006 12(3):125-30
  3. Briggs,C. Assessment of an immature plateletfraction (IPF) in peripheral thrombocytopenia. Br J Haematol 2004Jul;126(1):93-9
  4. Sysmex White Paper. The role of the ImmaturePlatelet Fraction(IPF) in the differential diagnosis of thrombocytopenia. www.sysmex.com/us
  5. Fujii,T et al.. A new approach to detectreticulated platelets stained with thiazole orange in thrombocytopenicpatients. Thromb Res. 2000 Mar 15;97(6):431-40
  6. Cremer Malte The immature platelet fraction(IPF) in neonates. Diagnostic Perspectives 2011 Vol1:36-42
  7. Cremer M. et al. Immature platelet values indicateimpaired megakaryopoietic activity in neonatal early-onset thrombocytopenia.Thrombosis and Haemostasis 2010; May;103(5):1016-21

-Becky Socha, MS, MLS(ASCP)CM BB CM graduated from Merrimack College in N. Andover, Massachusetts with a BS in Medical Technology and completed her MS in Clinical Laboratory Sciences at the University of Massachusetts, Lowell. She has worked as a Medical Technologist for over 30 years. She’s worked in all areas of the clinical laboratory, but has a special interest in Hematology and Blood Banking. When she’s not busy being a mad scientist, she can be found outside riding her bicycle.