Hematology Case Study: Thrombocytopenia in a 4 Year Old Child

A 4 year old child was brought to the pediatrician by her mother with a complaint of new onset of severe bruising on her legs. The mother could not recall any falls or bumps that would have caused the bruising. On exam, the physician also noted mucosal bleeding in the oral cavity. Questioning revealed that the patient had experienced flu like symptoms several weeks earlier. The physical exam was normal except for the bleeding. There was no family history of bleeding disorders. A CBC was ordered.

Reported CBC Results

WBC, RBC, Hgb, Hct, RBC indicies normal

Platelet count 26 x 103/μL

IPF 22% (reference range IPF% 1.0-7.0%) The physician evaluated the results, noting the normal CBC but decreased platelet count. The above results also show the immature platelet fraction (IPF), an additional Advanced Clinical Parameter reported from the Sysmex XN hematology analyzer. A low platelet count, as seen in this patient, will reflex a fluorescent platelet count (PLT-F). The impedance count (PLT-I) can be falsely increased if small RBCs or fragments are counted as platelets. On the other hand, in an optical platelet count, when measuring platelets by size (PLT-O), large platelets can be missed, giving a falsely low count. In this case there was a low platelet count and an instrument flag for an abnormal platelet scattergram. The PLT-F, on the other hand, uses a platelet specific dye which eliminates interference seen with other methods. The fluorescent dye labels the RNA, and forward scatter is used to determine size while side fluorescence is used to measure RNA content. With gating set based on cell volume and RNA content, the PLT-F can be measured. Therefore, the reflexed and more reliable PLT-F was the reported count.

Figure 1. PLT-F scattergram. The PLT-F channel measures forward scatter (FSC) on the Y axis and side fluorescence (SFL) on the X axis.1

Additionally, when there is an abnormal scattergram or a low platelet count, the IPF% and IPF# are also reported. The immature platelet fraction is a measure of the youngest platelets, or reticulated platelets. These are the first circulating platelets, right out of the bone marrow. An increased IPF indicates an increase in platelet production, yet this child’s platelet count was very low. This suggests that the thrombocytopenia may be due to excessive destruction of platelets; the bone marrow was actively making platelets, but they were being destroyed, causing the low platelet count.

Figure 2. 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 platelets.1

Diagnosis

Immune Thrombocytopenia- ITP.

Primary immune thrombocytopenia (ITP), formerly known as idiopathic thrombocytopenic purpura or immune thrombocytopenic purpura, is one of the most common bleeding disorders of children. In most cases, it presents with sudden onset of bruising and petechiae in an otherwise healthy child, with normal WBC and hemoglobin. ITP is an autoimmune bleeding disorder in which the immune system makes anti-platelet antibodies which bind to platelets and cause destruction. Even though the exact cause of ITP remains unknown, it is recognized that it can follow a viral infection or live vaccinations. While there are some similarities between pediatric ITP and ITP in adults, in children this tends to be an acute disease which is self-limiting and resolves itself in several weeks, with no treatment. However, in a small number of children, the disorder may progress to a chronic ITP. In contrast to ITP in children, a chronic form is more commonly seen in adults. It is usually a diagnosis of exclusion, does not follow a viral illness and requires treatment.

This patient recovered in a few weeks. One month after the initial episode, her PLT was 174 x 103/μL and her IPF% was 6.0%

Conclusion

An IPF reported with a CBC, in combination with a low platelet count, is fast, inexpensive, and can be extremely beneficial in aiding in a timely diagnosis. As the child’s platelet count recovered, the IPF% returned to normal range. ITP can therefore be monitored with a CBC. Thus, the IPF can be used not only to help diagnose but also as an indicator of remission.

References

  1. Sysmex America, 2019. www.sysmex.com/us. Used with permission
  2. Arshi Naz et al. Importance of Immature platelet Fraction as a predictor of immune thrombocytopenic purpura. Pak J Med Sci 2016 Vol 32 No 3:575-579
  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. D-Orazio, JA, Neely, J, Farhoudi,N. ITP in children: pathophysiology and current treatment approaches.J Pediatr Hematol Oncol.2013 Jan;35(1): 1-13

-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 69 Year Old Male with Weight Loss and Generalized Lymphadenopathy

Case History

The patient is a 69 year old male who presented to the hospital with a 3-month history of drenching night sweats, weight loss, fatigue, and generalized lymphadenopathy. He also endorsed a very itchy rash all over his body. He denied smoking. There was no other relevant social or family history.

Physical examination confirmed diffuse lymphadenopathy, hepatosplenomegaly and a mild diffuse skin rash. Notably, there was a 2.5 cm level-1 lymph node palpated in the left neck. This was subsequently biopsied.

Biopsy

Biopsy of the level-1 neck lymph node revealed a 2.3 x 1.5 x 1.2 cm mass pink-tan and firm mass. Sectioning revealed a glossy white-tan cut surface. H&E staining revealed a polymorphic lymphocytic infiltrate of in the interfollicular zones. The infiltrating lymphocytes ranged from small to large cells with abundant cytoplasm, eosinophils, and plasma cells. There was also a notable increase in the number of high endothelial vessels lined by lymphocytes with irregular nuclear borders and clear cytoplasmic zones.

Image 1. Polymorphic infiltrate of small, mature appearing lymphocytes (A), with prominent blood vessels and clear cytoplasm (B). Most of these cells were CD3 positive T cells (C) with expanded CD21 positive FDC meshworks (D) and scattered CD30 positive immunoblasts (E)

Further characterization by immunohistochemical staining showed the majority of the interfollicular cells to be CD3 and CD5 expressing T cells. These were a mix of CD4 and CD8 positive cells but with marked CD4 predominance. CD7 appeared positive in a smaller population of T-cells compared to CD3 (consistent with loss of this pan-T-cell marker). Varying numbers of the interfollicular cells were positive for CD10, BCL-6, CXCL-13, and PD-1 with a strong positivity for ICOS, phenotypically consistent with an expansion of Tfh (T-follicular helper cell) cells.

Interspersed between the T cells were numerous CD20 positive cells with prominent nucleoli that also revealed CD30 positivity. CD21 staining revealed expanded follicular dendritic cell meshworks. EBER ISH was positive in a rare subset of cells. Kappa and lambda ISH showed an increased number of polytypic plasma cells.

Flow Cytometry showed the presence of a small population of T-cells that were CD4 positive but CD3 negative. There was no evidence of B-cell clonality. TCR-G PCR was positive.

A final diagnosis of Angioimmunoblastic T-cell lymphoma (AITL) was rendered.

Discussion

AITL is a relatively rare neoplasm of mature T follicular helper cells, representing about 1-2% of all non-Hodgkin lymphomas. It is; however, one of the more common subtypes of peripheral T-cell lymphomas, accounting for 15-30% of this subgroup. The condition was first reported in 1974 in Lancet as a non-neoplastic abnormal immune reaction1. It was first recognized as a distinct clinical entity in in 1994 in the Revised European American Lymphoma Classification2. The disease shows a geological preference to Europe (28.7%) over Asia (17.9%) and North America (16%). AITL occurs primarily in middle aged and elderly individuals and shows a slight predominance of males over females.

The disease has a strong association with EBV infection, but the neoplastic T-cells are almost always EBV negative, creating an interesting question of EBV’s function in the etiology of AITL. AITL most often presents late in the disease course with diffuse systemic involvement, including hepatosplenomegaly, lymphadenopathy and other symptoms such as rash with pruritis and arthritis. Lab findings include cold agglutinins, rheumatoid factor and anti-smooth muscle antibodies. There also tends to be immunodeficiency secondary to the neoplastic process. The clinical course of AITL is variable, but the prognosis is poor, with the average survival time after diagnosis being < 3 years. The histological features and genetic findings have not been found to impact clinical course.

Microscopically, AITL presents with either partial or total effacement of the normal lymph node architecture with perinodal infiltration. The cells of AITL are small to medium-sized lymphocytes with clear to pale cytoplasm, distinct cell membranes and very minimal cytological atypia. These cells often congregate around the high endothelial venules. The T-lymphocytes are present in a largely polymorphous inflammatory background of other lymphocytes, histiocytes, plasma cells and eosinophils. There are 3 overlapping sub-patterns of AITL. The first of these is similar to a reactive follicular hyperplasia, and can only be distinguished from normal hyperplasia by use of immunohistochemical stains to differentiate the neoplastic cells from normal reactive cells. The second pattern has retained follicles, but they show regressive changes. The third pattern has completely or sub totally effaced. These three patterns seem to be on a spectrum with one another, given that progression from the first to the third pattern has been seen on consecutive biopsies in the same patient.

Cytologically, AITL cells express pan-T-cell markers including CD2, CD3 and CD5 and the vast majority are CD4 positive. CD3 may be quantitatively decreased or absent by flow cytometry. There are a variable number of CD8 positive T-cells. The tumor cells also show the immunophenotyping of normal T follicular helper cells including CD10, CXCL13, ICOS, BCL6 and PD1 in 60-100% of cases. CXCL13 and CD10 are the most specific, whereas PD1 and ICOS are the most sensitive.

References

  1. Horne, C., Fraser, R., & Petrie, J. (1974). Angio-Immunoblastic Lymphadenopathy With Dysproteinemia. The Lancet, 304(7875), 291. doi:10.1016/s0140-6736(74)91455-x
  2. Harris, N.l. “A Revised European-American Classification of Lymphoid Neoplasms: a Proposal from the International Lymphoma Study Group.” Current Diagnostic Pathology, vol. 2, no. 1, 1994, pp. 58–59., doi:10.1016/s0968-6053(00)80051-4.
  3. Swerdlow, Steven H. WHO Classification of Tumours of Haematopoietic and Lymphoid Tissues. International Agency for Research on Cancer, 2017.
  4. “Angioimmunoblastic T Cell Lymphoma.” Pathology Outlines – PathologyOutlines.com, http://www.pathologyoutlines.com/topic/lymphomanonBAITL.html.

-Zachary Fattal is a 4th year medical student at the Central Michigan University College of Medicine. He is pursuing a career in pathology and has a special interest in hematopathology, cytopathology and blood bank/transfusion medicine. You can follow him on Twitter @Paraparacelsus.

Kamran M. Mirza, MD, PhD, MLS(ASCP)CM is an Assistant Professor of Pathology and Medical Education at Loyola University Health System. A past top 5 honoree in ASCP’s Forty Under 40, Dr. Mirza was named to The Pathologist’s Power List of 2018. Follow him on twitter @kmirza.

Hematopathology Case Study: A 36 Year Old Woman with an Incidental Neck Mass

Case History

A 36 year old female underwent thyroidectomy for multinodular goitre that led to the fortuitous discovery of a neck mass. The neck mass specimen submitted comprised two lymph nodes measuring 2.2 cm and 1.3 cm in the greatest dimensions, with a fleshy tan cut surface.

Biopsy Findings

H&E stained sections revealed numerous non-necrotizing granulomas effacing and replacing normal lymph node architecture. These consisted of pale epithelioid histiocytes and Langhans type of giant cells. The granulomas lacked a peripheral rim of lymphocytes. AFB and GMS stains were negative for microorganisms

Diagnosis

A diagnosis of non-necrotizing granulomatous lymphadenitis was rendered noting that in the correct clinical context the findings could represent sarcoidosis.

Discussion

Granulomatous inflammation is a special type of chronic inflammatory response characterised by the formation of discrete collections of histiocytes called granulomas. Activated histiocytes appear as epithelioid cells with round to oval nuclei, often with irregular contours and abundant granular eosinophilic cytoplasm with indistinct cell borders. They may coalesce to form multinucleated giant cells. When found in the lymph node, the reaction pattern is called granulomatous lymphadenitis. It can be caused by a variety of different conditions, and therefore, requires thorough workup to come to a conclusive diagnosis.

On the basis of presence or absence of necrosis, granulomatous lymphadenitis can be classified as necrotizing or non-necrotizing. Additionally, the presence of an abscess, usually central, indicates a suppurative lymphadenitis.

Non-necrotizing granulomatous lymphadenitis:

Sarcoidosis lymphadenitis is the prototype of non-necrotizing granulomatous lymphadenitis. It shows the presence of discrete granulomas without a peripheral rim of lymphocytes, called “naked granulomas”. The early phase shows follicular hyperplasia and sinus histiocytosis, followed by appearance of epithelioid cell nodules toward the end of this phase. The peak phase shows well-demarcated granulomas composed of epithelioid cells with scattered multinucleated giant cells observed throughout the lymph node. Granulomas may occasionally coalesce. In the late phase, increased collagen fibers result in fibrosis and hyalinization. There are no neutrophils and it is uncommon to find small foci of central necrosis. Numerous inclusions such as asteroid, Schaumann, or Hamazaki-Wesenberg bodies can be seen. In this case, we observed well-demarcated granulomas throughout the lymph node, typical of the peak phase without any caseous necrosis or suppuration.

Other causes of granulomatous lymphadenitis can be ruled out as follows.

Sarcoid-like lymphadenitis: It shows a similar pattern of non-necrotizing lymphadenitis like sarcoidosis. However, classically sarcoid like reaction shows scattered small epithelioid granulomas with sparsely arranged epithelioid cells. The border of the granulomas is usually obscure. The CD4:CD8 ratio ranges from 0.8 to 2.25 while in sarcoidosis, it is >3.5. These findings help distinguish sarcoid-like lymphadenitis from sarcoidosis.

Sarcoid-like adenitis may be seen in numerous conditions such as carcinoma, Toxoplasmosis, fungal infections, tuberculosis, immunocompromised states, pneumoconiosis etc. The fact that tuberculosis and fungal infections can present with a non-necrotizing granulomatous lymphadenitis highlights the importance of performing fungal (PAS & GMS) and AFB (Ziehl Neelson) stains in non-necrotizing lymphadenitis as well. In this case, the granulomas had distinct borders, numerous epithelioid cells, no organisms were identified on special stains, nor was there any history of immune compromise; ruling out a sarcoid-like reaction.

Berylliosis: The lymph node picture in Berylliosis is identical to that of sarcoidosis. We may even see asteroid bodies or Schaumann bodies. A diagnosis can be established by eliciting a history of chronic exposure to Beryllium. Beryllium lymphocyte proliferation test (BeLPT) is a test that measures Beryllium sensitization and is very specific for Beryllium exposure. There was no known history of exposure to Beryllium in this case.

Toxoplasmosis: A classic triad of follicular hyperplasia, small granulomas composed of epithelioid cells within and around hyperplastic follicles and, monocytoid B cell hyperplasia, is observed in toxoplasmosis lymphadenitis. This case did not show follicular hyperplasia, ruling out toxoplasmosis.

Necrotizing granulomatous lymphadenitis

Even though we did not find any necrosis in this case, yet, it is worthwhile to review briefly the various causes of necrotizing lymphadenitis.

  • Non-suppurative

Tuberculosis: Histology of a tuberculous lymph node is characterised by central caseous necrosis surrounded by an epithelioid cell layer. The outermost layer is comprised of lymphocytes and fibrosis. Plasma cells are not observed. Diagnosis can be established by performing an AFB stain that demonstrates acid fast rod shaped bacteria in the areas of necrosis. Organisms can also be detected by PCR.

BCG lymphadenitis: About 0.7 to 2.3% of BCG vaccinated children may develop BCG lymphadenitis that is smaller than tuberculous lymphadenitis. Early phase shows follicular hyperplasia and sinus histiocytosis. Later, there is development of micronodules of epithelioid granulomas without necrosis and epithelioid cell granulomas with central caseous necrosis. Langhans giant cells are rare.

Fungal infections: Fungal infections by Histoplasma, Cryptococcus, coccidiodomycosis, pneumocystis may also cause a necrotizing granulomatous inflammation. There are numerous neutrophils, and fungal structures can be seen. GMS and PAS can be used in cases where it is difficult to the find the fungal elements on H&E.

  • Suppurative

Tularemia: There are three forms of histological changes, Abscess form, showing abscess with central necrosis and mononuclear cells, Abscess-granulomatous form with granulomas with central necrosis, which form large lesions with central abscesses, and granulomatous form with caseating necrosis at the centre of the granulomas.

Cat Scratch disease: Similar to tularemia, there are three phases of histologic presentation, an early phase of follicular hyperplasia, intermediate phase of microabscess, and a late phase of granulomatous inflammation. Monocytoid B cell clusters are observed close to the abscess.

Conclusion

Sarcoidosis is usually diagnosed by excluding other causes of granulomatous inflammation, as we did in this case. Characteristic non-necrotizing, discrete granulomas were seen throughout the lymph node. The age of the patient and female gender epidemiologically support the diagnosis. This case reflects an example work up of a granulomatous lymphadenitis that is a morphologic presentation of myriad diseases.

-Swati Bhardwaj, MD has a special interest in surgical pathology and hematopathology. Follow her on Twitter at @Bhardwaj_swat.

Kamran M. Mirza, MD, PhD, MLS(ASCP)CM is an Assistant Professor of Pathology and Medical Education at Loyola University Health System. A past top 5 honoree in ASCP’s Forty Under 40, Dr. Mirza was named to The Pathologist’s Power List of 2018. Follow him on twitter @kmirza.

Hematology Case Study: A 69 Year Old Female with Breast Implants

Case History

A sixty nine year old female who underwent right breast reconstruction about 13 years ago due to breast cancer presents to the doctor office with right breast pain and right breast enlargement over the last two months. She has lost some weight and does not recall any trauma to this area. She had a textured saline implant. Examination reveals no definite palpable masses. MRI of right breast showed intact saline implant with moderate amount of fluid surrounding the implant within the intact external capsule. No adenopathy was noted. Right breast implant was removed and complete capsulectomy was performed.

Image 1. A. Section of breast capsule with rare atypical hyperchromatic cells (arrow). B. Cytospin preparation of the fluid surrounding the implant with numerous atypical lymphocytes. C. Cell block of the fluid with large atypical lymphocytes. D, E. Lymphocytes are positive for CD30 (image D) and negative for ALK-1 (image E). F. CD30 positive cells in the section of the implant.

Diagnosis

Breast implant-associated anaplastic large cell lymphoma.

Discussion

Breast implant associated anaplastic large cell lymphoma is a provisional entity that is morphologically and immunophenotypically similar to ALK-negative anaplastic large cell lymphoma. It arises primarily in association with a breast implant. It is a very rare entity with an incidence of 1 in 500,000 to 3 million women with implants. Tumor cells may be localized to the seroma cavity or may involve pericapsular fibrous tissue. Sometimes it can form a mass lesion. Locoregional lymph node may be involved. The mean patient age is 50 years. Most patient presents with stage 1 disease, usually with peri-implant effusion. The mean interval from implant placement to lymphoma diagnosis is 10.9 years. There is no association with the type of implant. Histologic examination shows two different types of proliferations. In patients with seroma, the proliferation is confined to the fibrous capsule (“in situ” iALCL). However, the distribution of neoplastic lymphocytes could be heterogeneous with some cellular areas with numerous large pleomorphic cells of varying size and some fibrotic areas with rare atypical lymphocytes. It is beneficial to look at the seroma fluid in addition to capsule sections, because sometimes the neoplastic lymphocytes are predominantly present in fluid (as in our case). Patients presenting with tumor mass show more heterogeneous proliferations infiltrating surrounding tissues (“infiltrative” iALCL). They consists of either sheets are clusters of large neoplastic cells accompanied by a large number of eosinophils. By immunohistochemistry, the tumor cells are strongly positive for CD30. CD2 and CD3 are more often positive than CD5. CD43 is almost always expressed. Most cases are CD4 positive. The prognosis is very good in patients with disease confined to the capsule. The median overall survival is 12 years. However, patients with a tumor mass could have a more aggressive clinical outcome.

References

1. Swerdlow SH, Campo E, Harris NL, et al. WHO Classification of Tumours of Haematopoetic and Lymphoid Tissues (Revised 4th edition). IARC: Lyon 2017.

2. Jaffe, E , Arber, D, et al. Hematopathology (second edition) 2017.

-Junaid Baqai, MD, was born in Chicago, IL but spent most of his life in Karachi, Pakistan. He graduated from DOW Medical College in Pakistan and did his residency in anatomic and clinical pathology at Danbury Hospital, CT followed by hematopathology fellowship from William Beaumont Hospital, Michigan and oncologic-surgical pathology fellowship from Roswell Park Cancer Institute, New York. He currently serves as Medical Director of hematology, coagulation and flow cytometry at Memorial Medical Center and Medical Director of Laboratory at Taylorville Memorial Hospital.

Hematopathology Case Study: A 76 Year Old Man with Lymphadenopathy

Case History

76 year old man with a history of chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL) with new anterior mediastinal mass and increasing lymphadenopathy.

Lymph Node Biopsy

H&E

Diagnosis

Tissue sections show a diffuse atypical lymphoid infiltrate that completely effaces the normal nodal architecture. The infiltrate is composed of numerous small lymphocytes with round to mildly irregular nuclei, clumped chromatin, inconspicuous nucleoli and scant cytoplasm. There are also expanded pale areas that contain intermediate sized cells with more open chromatin and distinct single to multiple nucleoli. These cells are most consistent with prolymphocytes/paraimmunoblasts and form the proliferation centers characteristic of CLL/SLL. Occasional centroblastic-type B-cells are noted within these proliferation centers. In addition, there are scattered single to multinucleated cells that have irregular nuclear membranes with pale, vesicular chromatin and prominent inclusion-like, eosinophilic nucleoli. These cells morphologically resemble Hodgkin cells, Reed-Sternberg cells, mummified forms and other variants. These large cells are more evident in areas with a histiocyte rich background and around foci of necrosis. Occasionally, apoptotic bodies and mitotic figures are seen.

 Immunohistochemical studies show that the vast majority of the small-intermediate lymphocytes express B-cell markers CD20 (dim) and PAX5 and co-express CD5 and CD23 (subset). This is consistent with a background of CLL/SLL. The large atypical cells are positive for CD30, PAX5 and CD20 (variable). CD3 highlights numerous scattered background small T-cells, which are increased in the areas with the large cells. In situ hybridization for Epstein Barr viral RNA (EBER ISH) is mainly staining the large atypical cells. By Ki-67, the proliferation fraction is overall increased (40%) with increased uptake by the large atypical cells.

The morphologic and immunophenotypic findings are consistent with involvement by the patient’s known small lymphocytic lymphoma/chronic lymphocytic leukemia (SLL/CLL) with aggressive morphological features. The aggressive features include expanded proliferation centers and an elevated Ki-67 proliferative index (40%). Additionally there are histiocyte/T-cell rich areas composed of multiple EBV positive large atypical cells with morphologic and immunophenotypic features compatible with Hodgkin/ Reed-Sternberg cells. These areas are most in keeping with evolving classic Hodgkin lymphoma. Sheets of large cells indicative of large cell transformation are not seen, although increased scattered large centroblastic-type B cells are present.

Discussion

Lymph node involvement by CLL/SLL will typically show a diffuse proliferation of small lymphocytes with effacement of the normal nodal architecture.  The small lymphocytes have round nuclei, clumped chromatin and scant cytoplasm. Scattered paler areas known as proliferation centers are characteristic of this entity. The proliferation centers are composed of a mixture of cell types including small lymphocytes, prolymphocytes and paraimmunoblasts. Prolymphocytes are small to medium in size with relatively clumped chromatin, whereas paraimmunoblasts are larger cells with round to oval nuclei, dispersed chromatin, eosinophilic nucleoli and slightly basophilic cytoplasm. Some cases show increased and enlarged proliferation centers with a higher proliferation rate. This must be distinguished from large cell transformation.1

Aggressive features of CLL/SLL include proliferation centers that are broader than a 20x field or becoming confluent. An increased Ki-67 proliferation >40% or >2.4 mitoses in the proliferation centers can also portend a more aggressive course. These cases tend to have worse outcomes than typical CLL/SLL and better outcomes than cases that have undergone Richter transformation to diffuse large B-cell lymphoma (DLBCL). Transformation to DLBCL occurs in 2-8% of patients with CLL/SLL. Less than 1% of patients with CLL/SLL develop classic Hodgkin lymphoma (CHL). In order to diagnose CHL in the setting of CLL/SLL, classic Reed-Sternberg cells need to be found in a background appropriate for CHL, which includes a mixed inflammatory background. The majority of these CHL cases will be positive for EBV.1

Richter’s transformation is defined as an aggressive evolution of CLL. While the most common type of transformation is to a high-grade B-cell Non-Hodgkin lymphoma, other histological transformations have been described. This includes CHL, lymphoblastic lymphoma, hairy cell leukemia and high-grade T-cell lymphomas. The prognosis for patients who present with transformation to CHL is poor compared to de novo CHL.2 A large study from the M.D. Anderson Cancer Center described 4121 patients with CLL/SLL and found that only 18 patients or 0.4% developed CHL. The median time from CLL to CHL diagnosis was 4.6 years. Fourteen of the patients received chemotherapy. The overall response rate was 44% with a complete response rate of 19%. The median overall survival was 0.8 years and all patients eventually died from disease recurrence or progressive disease.3 This dismal prognosis is similar to patients with Richter transformation to DLBCL and much worse than patients with de novo CHL, which is curable in >85% of cases.1

References

  1. Swerdlow SH, Campo E, Harris NL, et al. WHO Classification of Tumours of Haematopoetic and Lymphoid Tissues (Revised 4th edition). IARC: Lyon 2017.
  2. Janjetovic S, Bernd HW, Bokemeyer C, Fiedler W. Hodgkin’s lymphoma as a rare variant of Richter’s transformation in chronic lymphocytic leukemia: A case report and review of the literature. Mol Clin Oncol. 2016;4(3):390–392.doi:10.3892/mco.2016.727.
  3. Tsimberidou, AM, O’Brien, S and Kantarjian, HM, et. al. Hodgkin transformation of chronic lymphocytic leukemia. Cancer. 2006;107(6).doi.org/10.1002/cncr.22121.

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.

Slide Review and You

Welcome back everybody!

Last month, I wrote about some projects I did while rotating through the pathology program at Danbury Hospital in Connecticut. This month I’m in a more clinical setting with a hematology/oncology clerkship at Northwell’s Staten Island University Hospital. But, over the past few months of rotations (and arguably a lot longer before medical school) I’ve been noticing a part of laboratory medicine which often intersects with our clinical colleagues at the bedside. I’ve told you about the pitfalls and successes in the relationships between surgeons and anatomic pathologists before, where frozen sections are critical and time is of the essence. And we’ve all seen collaboration between the bench and bedside before—think microbiology and infectious disease, blood bank and literally everyone, etc. Still, one collaborative effort sort of happens behind the shadows, behind phone calls and lab reports, and sometimes with no communication at all! So, what kind of vigilante medicine am I talking about? Who is this Batman of medicine? It’s just our friends in hematology.

When you’re working the hematology bench in the lab, it’s pretty commonplace for a physician on a hematology service to call and ask for a peripheral smear to review. Many times, it’s for the purpose of teaching residents, fellows, or medical students but more often than not it’s a confirmatory exercise. See, when that hematologist asks to review a slide, she’s probably coming down to the lab to look at the morphology of red cells and white cells to help in their differential diagnosis. They might have a patient with a suspected thalassemia or hemoglobinopathy and, before starting the full work up of lab tests, just want to see if there are any RBC morphology traits or target cells that stand out. Thrombocytopenia? Let’s make sure there’s no platelet clumping. Maybe they’ve got a patient with some kind of liver or kidney pathology and are on the hunt for acantho- or echinocytes. Or better yet, someone went hiking, there’s an infectious etiology on their differential—let’s go hunting for babesia, malaria, oh or even erlichia!

Image 1. Here’s a few examples of three parts of a patient’s smear that are contributory to a particular pathology in vivo. Think you know what it is? I bet you’d be surprised…not all that hyper-segments is a B12/Folate deficiency. But technically it is; read about cobalamin and homocysteine pathology in a neonatal patient here: http://www.bloodjournal.org/content/128/21/2584 (Source: Blood 2016)

I know what you’re thinking. Wait—that’s our job as medical laboratory scientists; our literal job. Our instruments, that we validate, and correlate, and make sure work fantastically give us flags. We investigate those flags and look at smears ourselves! We collaborate with other lab techs, and with our pathologist colleagues and send out final lab results with all kinds of helpful information: including platelet clumping, microorganisms, RBC and WBC morphology, and loads more. What gives?

Hold on to your lab coats. I’ll get there in a minute.

Slide review and differential training in medical school and residency

[This section intentionally left blank]

Image 2. There is nothing wrong with your television set. Do not attempt to adjust the picture. You are about to experience the awe and mystery which reaches from the inner mind to… the bench tech working in hematology. The one who went to school for this? Medical school and residency are starkly devoid of any in-depth, comprehensive learning for differentials.

A Differential, Differential

So let me address the issue I brought up: why do hematologists come down to the lab to look at the slides themselves, when perfectly capable BOC certified, degree-holding medical laboratory scientists and pathologists sign out validated differentials? It might not happen this way at all hospitals, but I think the answer is a simple two-part problem.

First, as with the many things I’ve learned in medical school, one of the lab-centric pieces of information that is well understood is that, well, no one really knows what the lab does and how it operates. Virtually nobody knows the depth and breadth of the testing that pathologists manage, let alone the scientific precision and accuracy that instrument validation requires. Learning that MLS techs are certified, can hold graduate degrees, and even do their own research is often surprising to most of our clinical colleagues. And—I will tell you for a fact—that pathology and laboratory testing methodology is not covered in medical school the way you might think. Pathology is more of a class of distinguishing the identifying details of a disease, not understanding the interdisciplinary diagnostic teamwork that goes into those CBC index results on a computer screen on the clinical floors.

Second, hematologists are specialists just like any other practicing clinician. They know their stuff! They manage patient diagnosis, treatment, and follow-up with the most up to date literature, national cancer guidelines, and anything else available to better their patients’ outcomes. Despite the notes in the CBC results that there are numerous macrocytes with hypersegmented neutrophils, or 3+ schistocytes reported in a manual differential—seeing is believing. It helps to see the slide yourself and get a feel for the disease “state” with your own eyes. Moreso, it could be a learning opportunity. It’s well within a clinicians’ scope to come down and look at a peripheral smear, I actually encourage it. But it should come with a few caveats…I’ll get to those too…

I-CARE

One of the places I was proud to hang my lab coat was actually my first job as an assistant lab technician in the blood bank at Rush University Medical Center in Chicago. Before I got my MLS and way before grad school or med school, I was a blood bank “expediter.” Super fancy title, but all I did was make sure specimens were logged in and blood products were up to par with labels on their way out. Clerical but critical! (Let me have this, please…haha) Anyway, part of the culture at that hospital has stayed with me all these years. I’ve talked before about culture and the way it permeates an institution’s practice like at the Mayo Clinic, but for my first foray into clinical work their acronym was clutch: I CARE.

  • I for innovation
  • C for collaboration
  • A for accountability
  • R for respect and
  • E for excellence

Why am I telling you this? No, there are no royalties. I just think it’s an easy way to remind ourselves about the meaning of interdisciplinary medicine and they way we should work together across specialties, and from bench to bedside. When we incorporate those values into our work for the purpose of improving patient care and outcomes, everyone wins. In this case, effective utilization of resources tells us that peripheral slide review means different things to different people. In the setting of hematologic work-ups, flags and review at the bench can signal something to the clinician which could spark a conversation with the pathologist. All parts contributing to a whole of patient care. Vigilante medicine is bad news. Collaboration is key.

One place I was lucky enough to be a part of this interdisciplinary collaboration was Swedish Covenant Hospital. One of the hematology physicians would routinely call me and ask to look as peripheral smears down in the lab, often as a group with med students, residents, and fellows. I’d throw the image of his patients’ slides on a large flat screen and go over what certain traits meant with regard to morphology and identification from the lab setting. Dr. Cilley would add what this all meant clinically and discuss treatment algorithms and next steps. That was collaboration at it’s finest: lab tech working with pathologists, clinicians working with the lab, and patient’s benefiting from all of it.

Video 1. ASCP’s 2015 Membership Video. I was super thrilled to be part of this video back in 2015 after winning the Midwest regional ASCP member of the year. If you’re bored enough to make it about 40 seconds into the video, that was my actual desk where Dr. Cilley and his residents would come to discuss patient slides. I would talk to them about morphology and hematologic clues with digital hardware and software to make it clear in group settings, rather than taking turns at the scope. Good times. (Source: https://www.youtube.com/watch?v=86fBRXGrZFo)
Video 2. Dr. Jeffrey Cilley talks about treating cancer as a “team approach” and he’s right. Hematology/Oncology to patient. Lab to clinician. Bench to bedside. (Source: https://www.youtube.com/watch?v=q0waKLyT1Dg)

Teamwork makes the dream work

About those caveats for collaboration I mentioned earlier… Let me put it briefly: it’s well within the scope of a clinician to come over to the laboratory and get some information on their patient’s lab results/testing. But why not consider the following:

  • If a physician calls to review a smear, offer to go over it with them. Likewise, to our clinical friends: if you go to the lab for a slide don’t be batman—ask the tech what they think!

Experienced techs are one of the hospital’s most valuable resources. Some folks I’ve worked with have been looking at slides longer than I’ve been using my eyes at all! They’ll save you and your residents the time when those terrifying intracellular microorganisms are really just overlying platelets. I mean, they’ve got a cute halo.

  • If you need help, just ask. This applies to everyone.

Talking with the tech about the slide is great start, but there’s more resources in the lab than most people know what to do with! Clinical physicians: check the shelves around the hematology microscope. Stuck on something? Find a CAP atlas or a proficiency survey booklet guide. Easy to read. Techs and pathologists: have someone who constantly comes down for slide review despite your immaculate and detailed SOPs on CBC results reporting? Have a quick chat about the work that goes into resulting those diffs—you might even improve your heme TAT, who knows?

  • If it’s well within the right of a physician to leave the unit and see a patient’s slide, logic says that maybe, just maybe, it should be okay for a pathologist to leave the lab and see a patient at the bedside!

Hospitals are full of never-ending rounding white coats, all asking patients questions, and all contributing specialty notes to their charts. But its not only to prevent patients from getting a decent nap. We’re all parts of a large interdisciplinary patient team. A recent Medscape survey found that somewhere around 3% of pathologists see patients, routinely! Got an interesting case in the lab, someone who’s part of lots of tumor boards, someone with an interesting case to write up, or even someone who nobody knows exactly what’s going on with? Try walking over to 4 south and have a conversation with Mr. Jones; it might help. At least he’ll know how many people are working on his care team!

The bottom line: we’re in this together, and like the flag on the ASCP ship says, we’re Stronger Together. Innovation, collaboration, accountability, respect, and excellence are—and should be—simple cornerstones of clinical medicine that translate across every discipline. When we share information and expertise, everyone gets better at what they do.

Bonus Image. This was a hard picture to take. Usually, a quick hematologist just comes down to see if there are any real schistocytes. But, after reading a draft of this post, BatDoc’s cool with chatting about red cell indices and automated flow cytometry methods in auto-diff validation. That’s the hero we deserve, and the one healthcare needs! (Source: https://gunaxin.com/batman-doesnt-police-stop-visiting-children-hospital)

Thanks for reading!

See you next time!

–Constantine E. Kanakis MSc, MLS (ASCP)CM graduated from Loyola University Chicago with a BS in Molecular Biology and Bioethics and then Rush University with an MS in Medical Laboratory Science. He is currently a medical student actively involved in public health and laboratory medicine, conducting clinicals at Bronx-Care Hospital Center in New York City.

What’s in a Differential?

When a complete blood count (CBC) and differential is ordered by a physician, most labs today have instrumentation capable of performing an automated differential. Depending on the instrument results and flags, we may need to perform a scan, review of the slide, or a manual differential. However, the definition of a manual differential today may be a bit different than the historical definition. A typical manual differential, when I first started working as a technologist, consisted of counting and differentiating 100 white blood cells under a microscope, and performing a red blood cell morphology along with a platelet estimate. Today, the 3 components of the manual differential have not changed, but more and more  labs are using an automated digital counting device, such as CellaVision. Whether counting cells under the microscope or scanning and verifying or reclassifying cells in CellaVision, it is important to always address all 3 parts of the manual review.

When an automated CBC has flagged that abnormal RBC morphology may be present, a peripheral blood smear should be reviewed. Reporting the red blood cell (RBC) morphology is an important component of a differential. Evaluation and interpretation of RBC morphology may provide the physician with important diagnostic information regarding the underlying cause of a variety of disorders, including anemia and systemic disease. Therefore, it is important to be able to accurately recognize and identify RBC morphologic abnormalities.

Red blood cell morphology can be subjective, and therefore inconsistent. Therefore, Laboratories must have training and competency programs as well as  procedures which dictate how they will report RBC morphology. Some labs use a numbering system, 1+, 2+, 3+, and others report, ‘rare’, ‘few’, moderate’ or ‘many’. Some morphological, such as rouleaux, can just be reported as present, with no quantified. Any method is acceptable, as long as there is consistency in reporting.

When performing RBC morphology,  these semi-quantitative report formats for should be based on clinical significance. Some RBC morphologies and inclusions are clinically significant,even when they are present in very low numbers. Sickle cells are one of these abnormalities that are significant even if only seen in very small numbers. Malaria or other parasites are clinically significant in any number. Fragmented cells such as schistocytes and helmet cells should also be noted if seen in any number. Other abnormalities which can be clinically significant in very low numbers are polychromasia, spherocytes and teardrop cells.

There are many other abnormal RBC morphologies which are only clinically significant if seen in larger numbers. Laboratories may choose to only report the presence of ovalocytes, target cells, burr cells, macrocytes, microcytes or hypochromia when greater than a defined percentage of cells exhibit these morphologies. Other laboratories choose to not report macrocytes, microcytes and hypochromia at all, instead relying on the physician to use the RBC indicies for their indication. The 2 most important things to remember, whatever your procedures are, is to be consistent, and not to ignore the:RBC morphology.

In addition to performing RBC morphology, a manual differential also requires platelet examination. A smear should be examined for a platelet estimate and abnormalities. This is particularly important when platelet clumps or an abnormal platelet scattergram are flagged on the CBC.  If an instrument uses optical platelet counts, large platelets can be missed. A fluorescent platelet count (PLT-F) , performed on Sysmex analyzers, will stain only platelets and give an accurate platelet count. The fluorescent count eliminates interferences seen with other methods. However, even when reporting a PLT-F, it may still required to review the smear for a platelet estimate, particularly with a very low count, or with clumped platelet flags. Clumped platelets are not an uncommon phenomenon, and an accurate platelet count can not be reported if significant clumping is present. The presence of giant platelets or hypogranular platelets, seen on the slide,  can also aid the physician in diagnosis or patient management.

CellaVision users have the added benefit of automation which simplifies the process of performing manual differentials. The system automatically locates and takes digital images of cells, including white blood cells, red blood cells and platelets.This simplifies the process of performing a manual differential. White  blood cells are pre-classified, RBC images are provided, and platelet images allow platelet estimates to be performed easily. The new advanced RBC application software can pre-classify RBCs.  This makes it even easier than before to perform reliable, standardized RBC morphology. (Watch for my next Hematology blog about the new RBC software!)

Particular disorders or abnormalities often involve characteristic changes to RBC morphology “Assessment of RBC morphology can be the best tool for laboratory hematology professionals to recommend clinical and laboratory follow‐up in a patient with anemia and to select the right tests for definitive diagnosis.”1 Too often, I have seen technologists perform a manual differential and either superficially skim over the RBC and platelet components, or totally forget them. Don’t forget your RBC morphology and platelet estimate and morphology! With today’s automated differential and autovalidation, 75-85% of CBCs are autovalidated. This allows us to spend quality time on those manual reviews that need to be done. Be sure to spend your time thoroughly reviewing the slides. A scan, slide review or manual differential, whether done under the scope of with CellaVision,tells the physician that we have looked at the slide or cells, which must include all 3 parts of manual review… WBCs , RBCs and platelets. Don’t sign it out until it’s complete!

References

  1. J. Ford, Red Blood Cell Morphology. International Journal of Laboratory Hematology. 2013
  2. https://www.labce.com/red-cell-morphology.aspx

-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.