Case Study Hematology: The Mouse Strikes again! Lymphocytes with Intracytoplasmic Inclusions

If you read my last blog, you heard the about the story “if You Give a Mouse a Cookie” by Laura Numeroff.4 The curious little mouse has a mind that never rests. As his mind wanders and hops from one thing to another, he keeps discovering more things to check out along the way. Medical laboratory lcientists are a lot like this. We’re a curious bunch, and, in investigations, one thing often leads to the next. Well folks, the mouse has struck again! We were given another cookie in the form of these beautiful cells.

Image 1. Lymphocytes with intracytoplasmic inclusions.

These cells were found by my coworker Liz Marr, MLS(ASCP), and the adventure began! First, we wanted to know what those were, and then we needed to find out more about them, and then, mostly, I wanted to know why in almost 40 years of working in and teaching hematology that I have never before seen this!

The story begins with our case history. We received a CBC from a 71 year old female with a 4 year history of untreated chronic lymphocytic leukemia/ small lymphocytic lymphoma(CLL/SLL). The patient’s recent history included a myocardial infarction(MI) 5 months prior. The patient was found to have leukocytosis (WBC 25.38 x 103/μL) and absolute lymphocytosis (18.25 x 103/μL) with normal hemoglobin and hematocrit (Hgb 13.4 g/dL, Hct 40.8%) and normal platelet count (272 x 103/μL). The differential had 71.9% lymphocytes with many abnormal forms noted. The slide was sent for a pathology review. The pathologist reported “Atypical lymphocytosis consistent with patient’s known chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL) Filament-like inclusions are present in the cytoplasm which has been previously reported in patients with CLL.”

Image 2. Lymphocytes with crystalline-like inclusions.

A curious tech can’t stop at just that description. If you tell me they are filament-like inclusions, I will have all kinds of questions. What are these filaments made of? Are they crystals, or something else? How common are these? Are these diagnostic of CLL? Are these only seen in CLL? What is their significance? And, of course, the most puzzling question, why have I never seen these before??

CLL is a form of non-Hodgkin lymphoma and is the most common leukemia in the Western world. It is generally a leukemia of older age with a median age at diagnosis around 67-72. The disease is widely variable, with some patients asymptomatic and requiring no treatment for many years, while others have a more rapidly progressive course of disease requiring treatment. About 60% of patients are diagnosed before they exhibit any symptoms. CLL and SLL are considered to be different manifestations of the same disease. In CLL, the abnormal B lymphocytes are found mostly in the peripheral blood and bone marrow, but in SLL, there is lymph node involvement, with abnormal cells mostly found in the lymph nodes. CLL is diagnosed based on absolute B lymphocyte counts ≥5 x 109/L. Flow cytometry typically reveals a distinctive cell immunophenotype with expression of CD19, CD5, CD23, and Κ/λ; and weak expression of CD20, CD79b, and surface immunoglobulin.1

The most recent flow cytometry report on our patient was from one year ago. An 8 color analysis with CD45/SSC gating was performed by LabCorp. The flow revealed an abnormal cell population representing 56% of total cells. Two monoclonal B cell populations were detected with identical phenotypes except for light chain expression. These cells expressed CD45, CD19, CD20, CD22, CD5, and CD23., CD38-. This phenotype was consistent with her previous diagnosis of CLL/SLL.

A literature search revealed only a few articles about intracytoplasmic inclusions in CLL. Cytoplasmic inclusions in lymphomas are uncommon, but have been noted as vacuoles, crystals, and pseudocrystals. These crystalline inclusions represent immunoglobulin(Ig) heavy and light chain that precipitate in the cytoplasm. Using electron microscopy it has been found that theses Ig deposits localize in the rough endoplasmic reticulum (RER).5 When surface Ig can be demonstrated on the B lymphocytes, it has been found to be same as Ig in the inclusions.6

In two published studies that describe these crystal like inclusions, photographs are very similar to the ones we found on our patient.3,5 It is interesting to note that, in these two studies, neither of the subjects was a known CLL patient. The inclusions were noted in the patients’ cells and the peripheral blood was subsequently sent for flow. Phenotypes reported confirmed monoclonal B-cells representing a large percentage of cells. Huang reported monoclonal B-cells which expressed CD45, CD19, CD20, CD22, CD79b, CD5, CD23, CD148 and CD200(hi), with partial expression lambda, and negative for FMC7, CD10, CD11c, CD49d, CD103, CD38, CD25, CD160, IgM, CD81, kappa and Ki67.3 In the Ramlal case study, phenotype was CD5, CD19, CD20, CD23, positive, CD10, FMC7 negative.5 On the basis of flow, along with the CBC results, the patients were diagnosed with CLL.

Of course, while researching this, the little mouse in me kept asking questions and finding more questions to ask. One question that I still had questions about was if these inclusions have any prognostic value. In three recent studies3,5,6 it was indicated that these inclusions can be used to help with diagnosis, but are not prognostic for course of disease. Rodriguez followed a patient with asymptomatic Rai stage 0 CLL. This patient consistently had inclusions noted in lymphocytes for 9 years before any progression of disease was noted.6 In the medical field even if one study reports no prognostic significance, this opinion could change in the future with more studies. Could these crystalline inclusions be used to forecast time to first treatment (TFT) or overall survival?(OS). So far, because of the rarity of these cytoplasmic inclusions, there is no evidence of prognostic value. As well, the mechanism related to their formation and their role in CLL is yet to be determined.

Our case study patient and the various reports found in literature had common flow cytometry immunophenotypes. Patients were all either previously diagnosed with CLL or lymphocytic lymphoma, or were diagnosed at the time of the findings of these inclusions. While these crystalline inclusions alone are not considered diagnostic for CLL, their recognition can be used to assist in a prompt diagnosis of a lymphoproliferative disease. And they are so pretty! What medical laboratory scientist doesn’t love pretty cells? Be like that mouse. Be curious, keep your eyes open, and be on the lookout for these interesting cells in CLL patients, but, more importantly, in patients with lymphocytosis without a known diagnosis of a lymphoproliferative disorder.

References

  1. AJMC, January 7, 2019
  2. Chronic Lymphocytic Leukemia: An Overview of Diagnosis, Prognosis, and Treatment
  3. Huang, Y., Zhang, L. Intracellular rod-like crystals in chronic lymphocyte leukemia. Int J Hematol 112, 267 (2020). https://doi.org/10.1007/s12185-020-02933-7
  4. Numeroff, Laura If You Give a Mouse a Cookie. 1986
  5. Ramlal, B, DiGiuseppe, JA. Intracytoplasmic crystalline inclusions in chronic lymphocytic leukemia. Clin Case Rep. 2019; 7: 1460– 1461. https://doi.org/10.1002/ccr3.2250
  6. Cecilia M. Rodríguez, Carmen Stanganelli, Claudio Bussi, Daniela Arroyo, Darío Sastre, Viviana Heller, Pablo Iribarren & Irma Slavutsky (2018)Intracytoplasmic filamentous inclusions and IGHV rearrangements in a patient with chronic lymphocytic leukemia, Leukemia & Lymphoma, 59:5,1239-1243, DOI: 10.1080/10428194.2017.1370549

-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.

The Utility of Flow Cytometry in Establishing the Correct Diagnosis of a Rare Aggressive Lymphoma

Case history

A 73 year old woman presented with shortness of breath and was found to have bilateral pleural effusions. She had a history of marginal zone B-Cell lymphoma involving the bone marrow, which was diagnosed 3 months before this presentation and was treated with Rituximab.

Thoracentesis revealed an atypical lymphoid population comprised of intermediate and large sized cells with eccentrically placed nuclei, multiple prominent nucleoli and scant to moderate amounts of basophilic cytoplasm (Image 1). Initial evaluation of the cytology material was concerning for large-cell transformation of the patient’s previously diagnosed marginal zone B cell lymphoma. A representative portion of the fine needle aspirate sample was sent for flow cytometric immunophenotyping.

Image 1. Cytology (Diff Quik, 400X). The atypical lymphoid population is comprised of intermediate and large sized cells with eccentrically placed nuclei, multiple prominent nucleoli and scant to moderate amounts of basophilic cytoplasm.

Flow cytometric immunophenotyping showed a distinct population of atypical cells with moderate CD45 expression and increased side scatter in keeping with cytoplasmic complexity (Figure 1, black arrows). On an initial screening B cell lymphoma panel these cells were negative for CD19 and positive for CD30 (partial), and CD44 (Figure 2).

Figure 1. The neoplastic population shows expression of CD30 and CD44.

The population of interest lacked expression of CD10, CD20, CD22 and surface immunoglobulin light chains and CD138 (Figure 2 and 3).

Figure 2. The neoplastic population lacks expression of CD10, CD19, CD20, and CD22.
Figure 3. The neoplastic population of cells are negative for surface immunoglobulin light chains and CD138.

CD30 expression prompted the investigation of additional T-cell markers to rule out a T cell lymphoma (Figure 4). This population showed dim expression of CD7 but was otherwise negative for pan T cell markers (CD2, CD3, CD5) as well as CD4 and CD8 (Figure 4).

Figure 4. The neoplastic population of cells are positive for CD7 (dim) and CD30 and negative for CD3, CD4, CD8, and CD26.

Given the unusual immunophenotype of the neoplasms, a diagnosis of diffuse large B cell lymphoma (transformation of the known marginal zone lymphoma) seemed less likely and other possibilities were considered.

The presence of CD30 expression and the plasmablastic morphologic features together with the clinical presentation with effusions raised the possibility of primary effusion lymphoma. IHC for anti-HHV8 was performed on the cell block sample (Image 2).

Image 2. Cytology (cell block 400X) A. Hematoxylin and eosin stain of the cell block reveals large, atypical lymphoid cells; Small and large atypical lymphoid cells are highlighted by CD30 (B), HHV8 (LANA-1) (C), and CD138 (focally) (D).

Final diagnosis  

Primary effusion lymphoma (HHV8 positive).

Discussion

Primary effusion lymphoma (PEL) is a large B-cell neoplasm usually presenting as serous effusions without a detectable tumor mass [1]. It is universally associated with the human herpesvirus 8 (HHV8). It usually occurs in the setting of immunodeficiency [2]. Some patients with PEL secondarily develop solid tumors in adjacent structures such as the pleura [3-5].

Immunophenotype of PEL:

POSITIVE: CD45, HLA-DR, CD30, CD38, VS38c, CD138, EMA, HHV8 (LANA1).

NEGATIVE: pan- B-cell markers (CD19, CD20, and CD79a), surface and cytoplasmic Ig, and BCL6.

PEL is usually negative for T/NK-cell antigens, although aberrant expression of T-cell markers may occur. PEL is usually positive for EBV-encoded small RNA (EBER) by in situ hybridization but negative for EBV latent membrane protein 1 (LMP1) by IHC.  This could be explained by EBV virus latency. It is ability of a pathogenic virus to lie dormant (latent) within a cell, denoted as the lysogenic part of the viral life cycle. EBV expresses its genes in one of three patterns, known as latency programs. EBV can exhibit one of three latency programs: Latency I, Latency II, or Latency III. Each latency program leads to the production of a limited, distinct set of viral proteins and viral RNAs. The Epstein-Barr virus encoded RNAs (EBERs): EBER1 and EBER2 are expressed during all latency forms [6], whereas LMP1 is expressed only in latency 2 and 3 rendering it a less sensitive marker for detection of EBV infection. EBV-negative PEL is common in elderly, HIV-negative patients from HHV8-endemic regions (Mediterranean) [7].

Differential Diagnosis

Most common cavities involved by PEL: pleural, pericardial, and peritoneal [8-10].

It was thought that PEL can involve an artificial cavity related to the capsule of a breast implant [11] although it was described only in one case report without appropriate HHV8 staining and before recognition of breast implant-associated anaplastic large cell lymphoma (BIA-ALCL), which this case probably was presenting [12].

Primary effusion lymphoma (PEL) Prognosis

The prognosis is very unfavorable. Median survival is < 6 months. Rare cases have been reported that responded to chemotherapy and/or immune modulation [13].

Flow Cytometry Utility

The importance of utility of flow cytometry in establishing a diagnosis of PEL has been previously shown by others [14]. In the series by Galan et al. the authors described a case of PEL in an 88-year-old HIV-negative female with right-sided pleural effusion without significant lymphadenopathies or other effusions. The cytological study of the pleural fluid revealed a dense proliferation of large plasmablastic cells. A six-color multiparametric flow cytometry immunophenotyping study revealed 45% of large in size and high cellular complexity cells positive for CD45 (dim), CD38, CD138, CD30 and HLA-DR; and negative for CD19, CD20, cytoplasmatic CD79a, surface and cytoplasmic light chains Kappa and Lambda, CD3, CD4, CD5, CD7, CD8, CD28, CD56, CD81, and CD117. In situ hybridization for EBV-encoded small RNA was negative and immunohistochemistry for Kaposi sarcoma herpesvirus (HHV8) confirmed the diagnosis of PEL. These results in addition to the current case highlight the utility of flow cytometry in the diagnosis of lymphomas involving body cavities.

In Summary

PEL is associated with a proliferation of large B-cells which are positive for HHV8, CD45 (dim), CD30, CD38, and CD138 and negative for lineage defining B cell markers (CD19, CD20, and CD79a). Although PEL is a very rare lymphoma, it is important to consider it in patients with pleural, pericardial, and peritoneal effusions by sending a sample for cytological examination and flow cytometric immunophenotyping. Due to the absence of a mass lesion, cytology and flow cytometry are essential for establishing the diagnosis of PEL.

References

  1. Said, J.a.C.E., Primary effusion lymphoma, in WHO Classification of Tumours of Haematopoietic and Lymphoid Tissues (Revised 4th edition), C.E. Swerdlow SH, Harris NL, Jaffe ES, Pileri SA, Stein H, Thiele J, Editor. 2017: Lyion. p. 323–324.
  2. Song, J.Y. and E.S. Jaffe, HHV-8-positive but EBV-negative primary effusion lymphoma. Blood, 2013. 122(23): p. 3712.
  3. Dotti, G., et al., Primary effusion lymphoma after heart transplantation: a new entity associated with human herpesvirus-8. Leukemia, 1999. 13(5): p. 664-70.
  4. Jones, D., et al., Primary-effusion lymphoma and Kaposi’s sarcoma in a cardiac-transplant recipient. N Engl J Med, 1998. 339(7): p. 444-9.
  5. Luppi, M., et al., Molecular evidence of organ-related transmission of Kaposi sarcoma-associated herpesvirus or human herpesvirus-8 in transplant patients. Blood, 2000. 96(9): p. 3279-81.
  6. Khan, G., et al., Epstein Barr virus (EBV) encoded small RNAs: targets for detection by in situ hybridisation with oligonucleotide probes. J Clin Pathol, 1992. 45(7): p. 616-20.
  7. Dupin, N., et al., Distribution of human herpesvirus-8 latently infected cells in Kaposi’s sarcoma, multicentric Castleman’s disease, and primary effusion lymphoma. Proc Natl Acad Sci U S A, 1999. 96(8): p. 4546-51.
  8. Otsuki, T., et al., Detection of HHV-8/KSHV DNA sequences in AIDS-associated extranodal lymphoid malignancies. Leukemia, 1996. 10(8): p. 1358-62.
  9. DePond, W., et al., Kaposi’s sarcoma-associated herpesvirus and human herpesvirus 8 (KSHV/HHV8)-associated lymphoma of the bowel. Report of two cases in HIV-positive men with secondary effusion lymphomas. Am J Surg Pathol, 1997. 21(6): p. 719-24.
  10. Beaty, M.W., et al., A biophenotypic human herpesvirus 8–associated primary bowel lymphoma. Am J Surg Pathol, 1999. 23(8): p. 992-4.
  11. Said, J.W., et al., Primary effusion lymphoma in women: report of two cases of Kaposi’s sarcoma herpes virus-associated effusion-based lymphoma in human immunodeficiency virus-negative women. Blood, 1996. 88(8): p. 3124-8.
  12. Lyapichev, K.A., et al., Reconsideration of the first recognition of breast implant-associated anaplastic large cell lymphoma: A critical review of the literature. Ann Diagn Pathol, 2020. 45: p. 151474.
  13. Ghosh, S.K., et al., Potentiation of TRAIL-induced apoptosis in primary effusion lymphoma through azidothymidine-mediated inhibition of NF-kappa B. Blood, 2003. 101(6): p. 2321-7.
  14. Galan, J., et al., The utility of multiparametric flow cytometry in the detection of primary effusion lymphoma (PEL). Cytometry B Clin Cytom, 2019. 96(5): p. 375-378.

This case was previously presented by authors as eCSI Case on International Clinical Cytometry Society website.  For more information please follow: https://www.cytometry.org/public/newsletters/eICCS-10-1/article7.php

-Dr. Loghavi is an Assistant Professor of hematopathology and molecular pathology MD Anderson Cancer Center in Houston, TX. She received her MD degree from the Azad University in Tehran, Iran. Shen then completed an Anatomic and Clinical Pathology residency training at Cedars Sinai Medical Center in Los Angeles, CA, followed by Surgical pathology, Hematopathology and Molecular pathology fellowship training at the University of Texas, MD Anderson Cancer Center. Dr. Loghavi is passionate about medical education. Her clinical and research interests are focused on hematologic malignancies, with particular focus on myeloid neoplasm and the applications of flow cytometric immunophenotyping and molecular methods in detection of minimal/measurable residual disease. She has authored 100 peer-reviewed articles, 5 book chapters, and numerous abstracts in the fields of hematopathology and molecular pathology. 

-Kirill Lyapichev, MD, FASCP, is a board-certified anatomical and clinical pathologist who completed 2 years of hematopathology fellowship at MD Anderson Cancer Center. He is currently a molecular genetic pathology fellow at MD Anderson Cancer Center. Additionally, he is interested and involved in other research projects including neoplastic as well as non-neoplastic entities: MALT lymphoma, Castleman Disease, Kikuchi-Fujimoto Disease, and others. In 2020 he was selected as one of ASCP’s 2020 Top 40 Under Forty. Follow him on Twitter: @KirillLyapichev.

The Story of the Mott Cell, COVID-19 and the Cute Little Mouse

I have worked in hematology for many years, and there are certain things that never fail to excite technologists. Working in New Hampshire, it was always exciting to sickle cells or malaria, something common to some, but not common in our patient population. I now work in Baltimore, and see sickle cells nearly every day, and we come across malaria not too infrequently, but we still share good examples and save them for training. When we see something different or unusual, we always share the finding. Cells may need to be sent to the pathologists for a pathology review, and we always check back to see the pathologist’s identification and comments. Medical Technologists by nature are a curious bunch, and we always want to see ‘cool’ things. I wrote a blog two years ago about the only patient I have ever seen with Trypanosoma (Hematology Case Study: The Race to Save a 48 Year Old Man from a Rare Disease). Last month I wrote about Blue-green cytoplasmic inclusions (COVID-19 Patients with “Green Crystals of …” STOP! Please Don’t Call Them That). So, when I saw something else ‘cool’ and different on a peripheral smear, and then saw it AGAIN, on another patient, and saw other techs here in the US and in other countries were also mentioning these, because it’s my nature, I got curious.

When I write these blogs, I often feel a little bit like the mouse in the children’s story “If You Give a Mouse a Cookie”, by Laura Joffe Numeroff. It’s about an adorable little mouse who asks for a cookie, and then decides he needs a glass of milk to go with it, and then he needs a straw, and it goes on and on, in a circle, back to the beginning. Maybe it’s that the mouse is a little ADD, but I like to believe that he’s just creative and curious. I start with an idea, and often go off on many tangents before a blog is finished and comes back to where I started.. When I started writing this, it was because I saw an interesting cell, and I started exploring, and found that others had seen them, too. Then I started looking through my textbooks for references and information, and searched for recent research or studies, and then I wanted to find out more… just like that mouse.

There are some things that we learn about in school and we may see on CAP surveys, but no matter where you work, they are still rarely seen, so are a novelty. Mott cells are one of these things. I have a collection of Hematology texts from grad school and years of teaching Hematology. Several of these don’t even mention Mott cells, but, when they do, it’s barely a sentence in a discussion of plasma cells. I happen to have a very old copy of Abbott Laboratories “The Morphology of Human Blood Cells” . The one with the red cover, from 1975. The term Mott cell does not appear in this manual, but they do show pictures and describe “Plasma cells with globular bodies (Grape, Berry or Morula cells)”, and describe these globules as “Russell bodies”.1 So some of us who have been working in the field for many years may remember Russell bodies and Morula cells, or Grape cells, even if the term Mott cell is not familiar. Regardless of what we or textbooks call them, they tend to trigger a memory because the images are so unique.

So, again, I’m a bit like that mouse and getting distracted with the background. Why am I writing this blog? In recent months I have seen cells identified as plasmacytoid lymphocytes and Mott cells in several hospitalized patients. I have heard reports of these cells in other facilities as well. So, like a good medical technologist, I got curious about Mott cells. What are they, and what is their significance? And why are we seeing more of these now?

Mott Cells are named after surgeon F.W. Mott. In the 1890’s, William Russell first observed these cells with grape like globular inclusions, but did not recognize what the inclusions were or their significance. Russell examined the cytoplasmic globular inclusions and assumed that these cells were fungi. Ten years later, Mott described cells he called morular cells. He recognized that these cells were plasma cells and the inclusions were indicative of chronic inflammation. Thus, today we refer to these cells as Mott cells, morular cells or grape cells, and the inclusions as Russell bodies.2

Hematology texts describe Mott cells as morphologic variations of plasma cells packed with globules called Russell bodies. We know that plasma cells produce immunoglobulin. When the plasma cells produce excessive amounts of immunoglobulin, and there is defective immunoglobulin secretion, it accumulates in the endoplasmic reticulum and golgi complex of the cells, forming Russell bodies. Russell bodies are eosinophilic, but in the staining process the globulin may dissolve and they therefore appear to be clear vacuoles in the cell under the microscope. Thus, a plasma cell with cytoplasm packed with these Ig inclusions is called a Mott cell.

Mott recognized that these atypical plasma cells were present in inflammation. Plasma cells are not typically seen on peripheral blood smears and constitute less than 4% of the cells in a normal bone marrow. Yet, on occasion, we can see plasma cells, including Mott cells, on peripheral blood smears in both malignant and non-malignant conditions. Mott cells are associated with stress conditions occurring in a number of conditions including chronic inflammation, autoimmune diseases, lymphomas, multiple myeloma, and Wiskott–Aldrich syndrome.3

So, why are we seeing an increased mention of Mott cells now? We seem to be seeing these on patients testing positive for SARS-CoV-2. I have seen cells on patients at my facility that resemble Mott cells. I belong to a Hematology Interest group and over the past few months I have seen several people post pictures of Mott cells, cells with Russell bodies, and plasmacytoid lymphocytes identified on peripheral blood smears of COVID-19 patients. Other techs chimed in with comments that they have also seen these cells recently. I have even seen a comment propose that these cells are indicative of COVID-19 infection.

SARS-CoV-2 definitely causes inflammatory processes and stress conditions in the body, so it makes sense that we may see these cells in COVID-19 positive patients.

Figure 1 shows a Mott cell on an image from Parkland Medical Center Laboratory, Derry, NH. A Mott cell was identified by pathologist in a male patient who tested negative for COVID-19 at the time the sample was drawn, and subsequently tested positive. Mariana Garza, a Medical Technologist working at Las Palmas Medical Center in El Paso, TX shared a case of a 59 year old diabetic male, diagnosed with COVID-19. The patient’s WBC was 31 x 103/μL. Two Mott cells were identified by pathologist on his differential. So, the curious little mouse in me researched some more.

Image 1. Mott cell. Photo courtesy Parkland Medical Center, Laboratory, Derry, NH.

Several published research papers have studied morphologic changes in peripheral blood cells in COVID-19 patients. As we now know, SARS-CoV-2 affects many organs including the hematopoietic and immune systems. A study in Germany showed that COVID-19 patients exhibited abnormalities in all cell lines; white blood cells, red blood cells and platelets. Increased WBC counts were seen in 41% of samples in their study. Differentials performed on study patients showed lymphocytopenia in 83%, and monocytopenia in 88%. Red blood cell morphology changes were noted. Platelet counts ranged from thrombocytopenia to thrombocytosis, but giant platelets were noted across the board.4

Mott cells are indicative of chronic inflammation and may have significance in association with COVID-1. In the above mentioned study, aberrant lymphocytes were noted in 81% of patients who were SARS-CoV-2 positive, and observable in 86% of the same patients after they tested negative. The paper shows plasmacytoid lymphocytes and Mott cells amongst these aberrant lymphocytes. Moreover, morphologic changes in neutrophils, such as a left shift and pseudo‐Pelger‐Huët anomaly, decreased after virus elimination but changes in lymphocytes, indicators of chronic infection, remained.4

Another study also reported reactive or plasmacytoid lymphocytes and Mott cells observed in peripheral blood.4,5 Researchers at Northwick Park Hospital, UK, presented a case study of a 59 year old male with COVID-19 with a normal WBC and thrombocytosis. His differential revealed lymphocytopenia. His differential also showed lymphoplasmacytoid lymphocytes and Mott cells. In their conclusions it is stated that “In our experience, the lymphocyte features illustrated above are common in blood films of patients presenting to hospital with clinically significant Covid‐19. The observation of plasmacytoid lymphocytes supports a provisional clinical diagnosis of this condition.”5

Can these variant plasma cells, along with other commonly seen morphological changes, be used as part of a diagnostic algorithm for SARS-Cov-2 infection? As we see more COVID-19 patients there will be more, larger studies done and more Mott cells identified. Some disorders, such as Epstein Barr Virus and Dengue Fever are characterized by distinct viral changes in cells. However, since Mott cells can be seen in many conditions, these alone could not be considered diagnostic, but the indications are that these cells, along with the entire differential and morphological patterns, could prove to be a straightforward and easy to perform supplementary diagnostic tool. More, larger studies need to be done. It was concluded in the German study, that this pattern of morphologic changes in cells could be further investigated and validated with a larger blinded study, and that this information could lead to the development of a morphologic COVID‐19 scoring system.4 In the meantime, keep an eye out for Mott cells. These should not be ignored and should be in some way noted because they may be of future diagnostic use. That’s all or now, folks! Something to dig deeper into in another blog! The mouse strikes again!

Many thanks to Nikki O’Donnell, MLT, Parkland Medical Center, Derry, NH and Mariana Garza, MT, Las Palmas Medical Center in El Paso, TX for sharing their case studies and photos.

Becky Socha MS, MLS(ASCP)CMBB

References

  1. Diggs, LAW, Sturm, D, Bell,A. The Morphology of Human Blood Cells, Third edition. Abbott Laboratories. 1975.
  2. ManasaRavath CJ, Noopur Kulkarni, et al. Mott cells- at a glance. International Journal of Contemporary Mudeical Research 2017;4(1):43-44.
  3. Bavle RM. Bizzare plasma cell – mott cell. J Oral Maxillofac Pathol. 2013;17(1):2-3.doi: 10.4103/0973-029X.110682.
  4. Luke, F, Orso, E, et al. Coronavirus disease 2019 induces multi‐lineage, morphologic changes in peripheral blood cells:eJHaem. 2020;1–8.
  5. Foldes D, Hinton R, Arami S, Bain BJ. Plasmacytoid lymphocytes in SARS-CoV-2 infection (Covid-19). Am J Hematol. 2020;1–2. https://doi.org/10.1002/ajh.
  6. Numeroff, Laura. If You Give a Mouse a Cookie, 1985.

-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.

COVID-19 Patients with “Green Crystals of …” STOP! Please Don’t Call Them That

Blue-green cytoplasmic inclusions in neutrophils and monocytes are a novelty in hematology. It is rare to see these inclusions on peripheral smears, and when we do, there is excitement, but sadness too, because, when noted, they usually indicate a poor prognosis and impending death. Thus, we have heard them called “green crystals of death” or “death crystals.” I know I would not want to read a family member’s medical chart and see reference to “death crystals.” It’s an insensitive term, and one the medical community is trying to discourage. And, in fact, though it typically does indicate a poor prognosis, not all cases lead to death. In published reports, it has been shown that short term mortality in patients with these crystals is about 60%.1

These rare inclusions are refractile and irregular in shape, and are found in neutrophils, and occasionally in monocytes. Color seems to be subjective here. They call them green when inclusions in photos or cells I am looking at look very blue to me. The color perceived may depend on the type of stain (Giemsa, Wright or Wright-Giemsa) used and how fancy we get in color names and descriptions. Or, maybe I’m just color blind! Some people (like my husband) are “lumpers” and call anything blue-green, blue, or green, but don’t recognize subtleties of colors. Thus, I guess to make everyone happy, or to compromise, the blue-green description may fit them best.

Image 1. Blue-green inclusions seen in neutrophils. Photos courtesy of Alana D. Swanson. UMMC

These blue-green inclusions were originally reported in patients with hepatic injury and failure. Laboratory results include elevations in AST, ALT and LDH. More recently, there have been cases with no evidence of hepatic injury. Researchers are now finding that these crystals can occur in patients with tissue injury other than liver, and in patients with multiorgan failure. In patients with no liver injury, what is a common factor is that LDH is elevated, indicating tissue injury. Additionally, along with these crystals, lactic acid levels can be used as a predictor of survival. Higher levels of lactic acidosis at the time crystals are noted is a negative predictor of survival.2

In trying to determine the clinical significance of these crystals, they have been subject to a number of different stains to determine their content. The association with hepatic failure led researchers to hypothesize that the crystals were a bile product in circulation. Since then, the crystals have been found to be negative in bile stains. When stained with other stains, Oil Red O showed positive in neutrophils, indicating high lipid content. The inclusions did not stain positive with iron stain or myeloperoxidase. Acid fast stains showed the inclusions to be acid fast positive.3 These crystals also show an interesting similarity to sea-blue histiocytes, which further associates them with tissue injury. After analysis, it is now thought that these crystals contain lipofuscin-like deposits representing lysosomal degradation products, and may be present in multiple types of tissue injury.2

With the current pandemic, I have seen reports of these crystals in COVID-19 patients. I have heard of fellow technologists seeing these, and a recent paper described the first reported cases in patients with COVID-19. These recent incidences may lead to new information about exactly what clinical significance they hold. About one third of COVID-19 patients have elevated ALT and AST, though it is not yet clear whether the liver dysfunction is directly caused by the virus, due to sepsis, or other complications of patient comorbidities. Many COVID-19 patients have mild disease, yet some develop severe pneumonia, respiratory complications, and multiorgan failure. Mortality is increased in these severely affected patients. To better understand and manage treatment for COVID-19, physicians seek to identify biological indicators associated with adverse outcomes.1

In a New York City study, Cantu and colleagues reported on six COVID-19 patients who presented with blue-green crystals in neutrophils and/or monocytes. All six patients had an initial lymphocytopenia, and significantly elevated AST, ALT, LDH and lactic acid at the time the crystals were noted. All of the patients had comorbidities, yet only two of the six presented with acute liver disease. Interestingly, in the six cases reported on in the study, only one had blue-green inclusions reported from the original manual differential. The others were found retrospectively when correlating the cases with patients known to have elevated ALT and AST. All patients died within 20 days of initial diagnosis.1

The consensus of several papers in the last few years is that these crystals are being underreported. As seen in the above study, the crystals were originally seen in just one of the six patients. A look back revealed the other cases. With an increase in COVID-19 cases in our facilities, these blue-green crystal inclusions may be a novelty that is wearing off. We may see a rise in their presence, and need to be able to recognize and report them. This information is important to report if clinicians are to use these crystal inclusions along with acute transaminase and lactic acid elevations to predict poor patient outcomes.

Clinicians, hematologists, and laboratory technologists should be educated and have a high level of awareness of these inclusions. The University of Rochester conducted a study a few years ago that noted that, because these crystals are rare, techs may not be on the lookout for them. Once techs see them, they seem to be on the alert and more are reported. The hospital instituted an “increased awareness” campaign, which resulted in an increase in detection. This revealed cases that were not related to liver injury, including patients with metastatic cancer and sepsis. However, an important correlating factor was that all of the patients had mild to severe elevations in liver enzymes. With more awareness, we are starting to see them in patients without hepatic injury, but with other inflammation and tissue injury.4

Image 2. Blue- green crystal inclusions seen in a patient diagnosed with sepsis and multiorgan failure. Photo courtesy of Karen Cable, YRMC.

Let’s raise our level of awareness of these maybe-not-so-rare crystal inclusions. And, please be sure to call them by their preferred name, blue-green neutrophil inclusions! Let’s not talk about death crystals or crystals of death.

Many thanks to my colleague Alana D. Swanson, MLS(ASCP)CM , University of Maryland Medical Center and Karen Cable, Hematology Section Lead, Yavapai Regional Medical Center, Arizona, for the photos used in this blog. 

References

  1. Cantu, M, Towne, W, Emmons, F et al. Clinical Significance of blue-green neutrophil and monocyte cytoplasmic inclusions in SARS-CoV-2 positive critically ill patients. Br J Haematol. May 26, 2020.
  2. Hodgkins, SR, Jones, J. A Case of Blue-Green neutrophil inclusions. ASCLS Today. 2019;32:431.
  3. Hodgson, T.O., Ruskova, A., Shugg, C.J., McCallum, V.J. and Morison, I.M. Green neutrophil and monocyte inclusions – time to acknowledge and report. Br J Haematol, 2015;170: 229-235.
  4. Patel,N, Hoffman,CM, Goldman,BJ et al. Green Inclusions in Neutrophils and Monocytes are an Indicator of Acute Liver Injury and High Mortality. Acta Haematol. 2017;138:85-90

-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.

Patient Advocacy: A Laboratory Professional at the Bedside

Before I became an MLS Program Director, I worked for nearly 20 years in Hematology. I was particularly interested in Coagulation and was excited to work as the Coordinator of the Special Hematology lab, overseeing coagulation and special RBC testing. Our Pathology Department offered a consultation service for these cases and I was included along with a team of pathologists, residents, fellows, and clinicians that worked with patients and their families to diagnose patients and manage their treatment plans.

One of my most memorable moments was when we had a patient with a previously diagnosed platelet disorder who became pregnant and sought advice regarding the delivery of her child. Her doctors worked with our pathologists to weigh the risk of bleeding complications associated with different modes of delivery, while also considering the welfare of the child who may have inherited the platelet disorder. It was decided that they would take a non-surgical approach to minimize risk for the mother, but would monitor the baby closely. That’s where I came in!  I was asked to be on call for the child’s delivery in order to be available to collect samples to monitor the baby’s progress and perform the necessary testing to inform her doctor’s decisions. At the time, on-call meant carrying a pager. When my pager went off, I met the obstetrical team at the hospital and accompanied them into the delivery suite. Labor progressed as expected and when the baby’s head was visible, I assisted the doctor in collecting a tiny amount of blood from the baby’s head, enough to look quickly under a microscope to determine if the baby’s platelets showed any similarity to the mom’s. I was delighted to say that the platelets appeared normal in number and size, minimizing the bleeding risk for the baby. The patient continued to deliver a healthy baby girl without complications.

Once the delivery was complete, I was able to collect enough blood from the placenta to perform definitive testing to rule out any evidence of the platelet disorder in the baby. This was an opportune time as the testing required a large volume which would have been difficult to collect from an infant. Once again, the testing ruled out any evidence of the bleeding disorder in the baby. Mom not only had a beautiful baby, but enjoyed the peace of mind associated with the results of her laboratory testing. As was often the case with our patients, we would see them from time to time in the management of their bleeding disorder. It was always a joy to see our patient visit with her daughter.

-Susan Graham, MS, MT(ASCP)SHCM is the Chair and MLS Program Director in the Department of Clinical Laboratory Science at SUNY Upstate Medical University. Ms. Graham is a current volunteer for ASCP, serving on the BOC Board of Governors, the Hematology and Joint Generalist Exam Committees and the Patient Champions Board. 

A Day in the Life

Who are medical laboratory scientists? We call ourselves clinical laboratory scientists, medical technologists, med techs, medical laboratory technicians, MLTs, or simply “techs.” Around the clock each day we provide vital information to physicians. We perform a variety of laboratory procedures from identifying microorganisms to providing blood for emergency transfusions. We’re trained in clinical chemistry, hematology, microbiology, and transfusion medicine. We are dedicated to delivering accurate and precise, high quality results to physicians. These providers rely on us for the diagnosis and monitoring of patients. I’ve heard it said that “without the lab, you’re just guessing.” We are a somewhat unknown but very important part of the medical field.

Many of us joined this profession because we are organized, have a strong attention to detail, are intrigued by science, and want to help others. We want to work in the medical field, but may not really want patient contact. In my case, I knew I loved biology, chemistry and math, had an analytical mind, and pay a great deal of attention to detail, but I didn’t really want to deal with “people,” so I thought I had found the perfect profession. Working in a lab, in the basement, I wouldn’t have any patient contact. Little did I know that for many years I’d be looked up to as an “expert phlebotomist;” the tech the phlebotomists would come to when they missed a “tough stick.” I was often called to the floors and the outpatient lab to draw patients. I worked 3rd shift where we were our own phlebotomists. And little did I know that I’d discover a love of teaching, and actually enjoy standing in front of a group of students, teaching them. I never thought I’d enjoy public speaking, but now I speak at conferences and symposiums and love sharing my love and knowledge of Hematology and Transfusions Medicine with my audiences.

I’ve been teaching for years, but continue working in the laboratory as well, because I feel the best teachers are the ones with first hand, current experiences to share. When I work with my students, I like to coach them to think problems through and to solve puzzles instead of simply memorizing facts. Med techs often choose the profession because they have a strong ability in science, but also keen investigative instincts, and enjoy the challenge of solving puzzles. We graduate with a plethora of knowledge, but it doesn’t stop there. We need to take this with us to our jobs, build on it, and use it every day to learn to think through and solve these puzzles and problems quickly and accurately. It’s a profession where you never stop learning.

So, where is this going? Graduation is coming, and a new set of med techs will be set forth into the labs of the world, armed with knowledge and ready to learn yet even more. So, what is it really like working in a hospital lab? Here’s a little glimpse of a typical day in the Hematology lab.

It starts a lot like the Beatles tune: “Woke up, fell out of bed, dragged a comb across my head. Found my way downstairs and drank a cup, and looking up I noticed I was late…” Which reminds me, I remember reading somewhere that medical technologists are the profession that drinks the most coffee. But, so much for being side tracked. Waking up at the crack of dawn, rushing in the door, clocking in before 7 am, on a typical morning we all check the schedule to see where we are scheduled for the day and to see who called out sick. On this day, there was only one sick call, which necessitated a little juggling of the schedule because we were already short staffed. (We can’t wait for you new grads to start!) That was our first problem of the day solved. And then we got a call that the 2nd heme tech was stuck in traffic. Techs are very adaptable, and can think on their feet. Looking around, I suddenly noticed I was alone in Hematology, and our CellaVision was down. On top of sick and late calls, the overnight tech had left early. I jumped right in. I took inventory of the situation, and saw messages about 2 pathology review fluid slides that were left from the previous shift. I took out QC to warm up, started finishing up the morning run and worked on the CellaVision. Soon my partner for the day arrived, just in time to hear the XN analyzer start beeping. Did I mention that techs are really good at multi-tasking?

I got the CellaVision up and running again: second problem of the day fixed. After shutting off the alarm on the XN, we began investigating, reran the specimen, called the floor, and discovered it was a contaminated sample: third problem of the day solved. We had a morning of calling critical labs to the providers, trekking across to the other building to bring the pathology reviews to the pathologists, and handling sample barcode issues. I took a quick look at the clock and realized it was 9:30 am, and we had just finished the morning QC and maintenance. Time for that coffee! (I actually am apparently one of the few med techs who doesn’t drink coffee, but I managed a quick break and a cup of tea.) Our hematology techs assist with bone marrow collections, making the slides, processing them and bringing the slides to the pathologists, then to surgical pathology and cytology. The whole process can take 1 ½ – 2 or more hours, and this day was our lucky day. We had two scheduled bone marrows, and another one that was a surprise. Three bone marrow and only two techs in the department!

While we were up in oncology and interventional radiology and processing bone marrows, the CellaVision acted up again, and I had to call service. I left a message for evening shift that service would be coming in that afternoon. A reagent ran out and I had to fill out the reagent replacement log. One other things that med techs do very well, is documenting what we did. There is a saying in the lab that “if it’s not documented, it didn’t happen.” We had a couple racks of unreceived specimens delivered to the department, and had to resolve the unregistered samples. Stats kept coming in, we had a T4T8 to run, and lunch time came and went, with neither of us getting a real lunch. Body fluids started coming in, three in a row. And guess what? One of them needed a pathology review! Med techs also get plenty of exercise when the pathologists are in a different building than the lab. The next phone call I got was from a second-shift tech who was running late. It seemed like the start of the day all over again! Before we knew it, it was 3:30 and time to go home.

We had a full day, a great day. It makes me feel good to know that we are doing such vital work. I feel proud that our team works well together. Not every day is quite this busy, but the busy ones are when we learn the most.

To all the students I have worked with this year, and all students everywhere, welcome to the lab! We need curious minds, and new techs who are ready to unravel the puzzles and solve the problems we see every day. We need new “diagnostic detectives.” I am very proud every year to see or new graduates accept the challenge and become medical laboratory professionals. 2020 Graduates, welcome to our world!

-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.

Hematology Case Study: Thrombocytopenia in a 50 Year Old Male

A 50 year old male patient receiving chemotherapy for treatment of gastric cancer presented to ER. Labs reported: 

WBC = 5.4 x 103/μL

Hgb = 8.9 g/dL

PLT (impedance) = 26 x 103/μL

PLT-F (fluorescent) = 9 x 103/μL

IPF = 21%

The hemoglobin was consistent with the patient history. Flags on the original impedance platelet count included thrombocytopenia, platelet clumps and platelet abnormal distribution. The sample was checked for clots, with no clots found. A fluorescent platelet count (PLT-F) was reflexed and the critical platelet count was called to the ER physician. The high immature platelet fraction (IPF%) indicates increased platelet production. Despite the increased production, the patient still had a severe thrombocytopenia. This would suggest thrombocytopenia caused by platelet destruction or consumption. Examination of the blood smear showed the presence of moderate numbers of schistocytes.

Image 1. Schistocytes seen on peripheral blood smear

Additional labs were ordered. BUN and Creatinine were slightly elevated. PTINR and APTT were within normal range. LDH was markedly increased. The physician was able to use this information, along with the clinical presentation and history, to diagnose Thrombotic Thrombocytopenic Purpura (TTP). Plasma exchanges were initiated. The patient expired 3 days later.

The difference between the impedance platelet count and the fluorescent platelet count in this patient is actually related to the presence of schistocytes. With thrombocytopenia, platelet counts can be less reliable than with normal counts. Automated 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 treatment. 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, as we see in this case. On the other hand, measuring platelets by size (optical) can miss large platelets leading to a falsely low count. The PLT-F is more reliable because it uses a platelet specific dye which eliminates these interferences. The fluorescent dye labels the RNA. 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-F can be measured. When there is an abnormal scattergram or a low platelet count, the PLT-F is reflexed and the IPF% is also reported.

The Immature platelet fraction (IPF) can also be used to help understand the etiology and aid in diagnosis. Historically, the MPV has been 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. Reticulated or immature platelets are the youngest platelets, within 24 hours of being released from the bone marrow. Measurement of these is a concept that first emerged in the late 1960s, before automated hematology analyzers performed platelet counts. Thus, the original method was staining with new methylene blue and manually counting, much like a manual reticulocyte count. These manual methods tend to be tedious and imprecise. In the last 20 yeas we have developed flow cytometry methods for performing a reticulated platelet count. Reticulocytes are stained with Thiazole Orange and passed through a flow cytometer. Unfortunately, there is no standardization for the procedure as there are variations in dye concertation, timing and gate settings. As well, this method is also time consuming, labor intensive, costly, and requires highly trained technologists to perform.

Newer flow cytometry methods to count these youngest platelets are available on Sysmex and Abbott CELL-DYN analyzers. The IPF (Sysmex) or RetPLT(Abbott) can be performed along with the routine CBC with no additional sample or time required. Knowing the reticulated or immature platelet fraction can help physicians to differentiate pathogenesis. A decreased percent of newly formed platelets may indicate that thrombocytopenia is caused by deficient platelet production, as seen in bone marrow failure. Increased circulating immature platelets with a low platelet count may suggest that the bone marrow is making adequate platelets and the thrombocytopenia is caused by platelet destruction or consumption. Treatment for these scenarios is different, and the physician must determine the etiology in order to determine treatment

Thrombotic thrombocytopenic purpura (TTP) is a microangiopathic hemolytic anemia with thrombocytopenia and organ failure caused by microvascular thrombosis. Platelets clump in the small blood vessels and cause the low platelet count. The hemolytic anemia causes schistocytes which can be seen on the peripheral blood smear. In this case, the low platelet count and high IPF, schistocytes on the smear and the patient presentation were all important factors that led to a speedy diagnosis and start of therapy.

Plasma exchange is the treatment of choice for TTP. With the advent of therapeutic plasma exchange, mortality from TTP has decreased from about 90% to 10-20%. In patients who have relapses or become refractory, vincristine has been used successfully as an adjunct to plasma exchange.4 The exact etiology of TTP is unknown. It can be secondary TTP, often triggered by chemotherapy drugs, or can be sporadic. Sporadic, or idiopathic, TTP is now thought to be associated with an acquired autoimmune deficiency of a plasma metalloprotease named ADAMTS13. The ADAMTS13 gene controls this enzyme, which is involved in blood clotting. In acquired TTP, the ADAMTS13 gene isn’t faulty. Instead, the body makes antibodies that block the activity of the ADAMTS13 enzyme. In these cases, a lack of activity in the ADAMTS13 leads to TTP. Almost all cases of recurrent TTP have severe ADAMTS13 deficiency. These patients benefit from immunosuppressive therapy with vincristine along with plasma exchange.

However, despite the decreased mortality seen with plasma exchange, patients with cancer, infections, transplant patients, or those receiving certain drug therapy have a much worse prognosis.4 In this case study, this was this patient’s first episode of TTP and he was undergoing chemotherapy for gastric cancer. The patient’s unfortunate outcome is most likely linked to this finding.

References

  1. 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
  2. Johannes J. M. L. Hoffmann, Nicole M. A. van den Broek, and Joyce Curvers (2013) Reference Intervals of Reticulated Platelets and Other Platelet Parameters and Their Associations. Archives of Pathology & Laboratory Medicine: November 2013, Vol. 137, No. 11, pp. 1635-1640.
  3. M Meintker, Lisa & Haimerl, Maria & Ringwald, Juergen & Krause, Stefan. (2013). Measurement of immature platelets with Abbott CD-Sapphire and Sysmex XE-5000 in haematology and oncology patients.
  4. J. Evan Sadler, Joel L. Moake, Toshiyuki Miyata, James N. George Clinical chemistry and laboratory medicine : CCLM / FESCC. 51. 1-7. 10.1515/cclm-2013-0252.; Recent Advances in Thrombotic Thrombocytopenic Purpura. Hematology Am Soc Hematol Educ Program 2004; 2004 (1): 407–423. doi: https://doi.org/10.1182/asheducation-2004.1.407
  5. Sysmex White Paper. The role of the Immature Platelet Fraction(IPF) in the differential diagnosis of thrombocytopenia. www.sysmex.com/us

-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.

Is it Christmas? Hematology Case Study: Coagulopathy

A 2 year old male was brought into the pediatrician’s office by his mother after tripping over a toy truck 2 days earlier. The mother stated that the child cut the inside of his lip in the fall, and the lip had been oozing blood for the past 2 days. The child had also experienced a bloody nose several times since the fall. Upon examination, the child appeared in general good health with no other bruising or bleeding. Examination of the joints revealed swelling in the right knee. The physician took a family history, and the mother reported that her younger brother has ‘some sort of bleeding problem’ and experienced prolonged bleeding after a tonsillectomy as a child, and after several surgeries as a young adult. The physician ordered blood work on the child.

  • Hgb 9.5 g/dl
  • Hct 30%
  • Platelet  185 x 103/ uL
  • INR  1.1
  • aPTT 57 sec
  • Mixing Test: corrected
  • Thrombin Time: normal

Based on these results, the prolonged aPTT warranted further investigation. A differential diagnosis involved ruling out other causes for the prolonged aPTT. The physician ordered mixing studies, factor VIII and factor IX assays and vWF. Mixing studies are used to determine if etiology of prolonged PT or PTT is due to a factor deficiency or an inhibitor. If the aPTT remains prolonged after mixing with normal plasma, this indicates an inhibitor. If the prolonged PTT becomes normal after the mixing studies, this would indicate a factor deficiency. The factor VIII and vWF were normal, but factor IX activity was 25%. Diagnosis: Factor IX deficiency. (It was also confirmed, after speaking with the child’s uncle, that he also had a factor IX deficiency)

So, you may ask, what does this have to do with Christmas? In the spirit of the season, I chose to present a Case Study on Factor IX deficiency, aka Christmas Disease. But, alas, this really has nothing to do with the holiday. Maybe it has something to do with the fact that the first article about this disorder was published in the British Medical Journal on Dec 27, 1954 (just 2 days after Christmas)? But, not so. Actually, Factor IX deficiency is also called Christmas Disease because it is named after Stephen Christmas, the first patient described to have Factor IX deficiency. Stephen Christmas was diagnosed with hemophilia in Toronto in 1949, at the age of 2. The family was visiting relatives in London in 1952 and it was there that doctors discovered that he was not deficient in Factor VIII, the cause of Classic Hemophilia as it was known at the time. It was discovered that he was deficient in another coagulation protein. This new protein was named Christmas protein and later became known as Factor IX.

A little bit more about the history of Factor IX deficiency. Before the discovery of the Christmas protein, it was thought that Hemophilia was a single disorder, caused by a deficiency of Factor VIII. With the discovery of this new protein, Classic Hemophilia (Factor VIII deficiency), was given the name Hemophilia A, and this new Factor IX deficiency became known as Hemophilia B. Yet another nickname for this disorder is the Royal Disease. Hemophilia was prominent in the European royal families in the 19rth and 20th centuries. Queen Victoria of Britain was a carrier of hemophilia and passed the gene on to three of her children. Her children and descendants married into the royal families of Germany, Russia and Spain, giving her the nickname the Grandmother of Europe. But, these marriages also served to spread the disease to these other royal houses, giving hemophilia the nickname Queen Victoria’s curse. The last known member of the royal families of Europe to carry the gene passed away in 1945, 9 years before that article in the British Medical Journal (December 27, 1954). So, how do we know that Hemophilia B is the hemophilia responsible for the Royal Disease? In 2009, DNA testing on bones identified as  Anastasia and Alexei Romanov, the last Russian royal family descendants of Queen Victoria, determined that the Royal Disease was Hemophilia B.

I remember teaching Hematology and Genetics before 2009 using a pedigree chart of Queen Victoria’s family to teach students about Hemophilia as an X linked recessive disorder. We created Punnett squares that showed the inheritance from Queen Victoria to her family members and descendants across Europe. I always enjoyed this lecture, because it was a fun piece of historical trivia paired with a good science lesson. After 2009, the science of the inheritance did not change, but we now knew that this Royal Disease was Hemophilia B. Hemophilia B is caused by mutations in the F9 gene which is responsible for making the factor IX protein.  The F9 gene is on the X chromosome. Hemophilia B, like Hemophilia A, is X linked, carried by the mother. 50% of males born to a carrier mother will have the disease and 50% of daughters will be carriers. All daughters of affected males will be carriers, but their sons will not be affected. Hemophilia A is more common than Hemophilia B, affecting about one in 5,000 males. Hemophilia B affects about one in 25,000 males. It has been though that up to about 30% of Hemophilia B cases occur as a spontaneous mutation and are not inherited. This has been thought to be the case with Queen Victoria. She has been believed to be ‘case zero’, the first hemophilia case in her family. However, some newer articles that have researched her family history suggest that she may have had a half-brother who had the disease.1 There are also other related disorders including a rare autoimmune acquired hemophilia B and another rare form of Hemophilia B called Hemophilia B Leyden.

The coagulation process involves many chemical reactions, from the initial event that triggers bleeding, to the formation of a clot. The sequence of events are generally depicted as a coagulation cascade to illustrate and simplify understanding of the process. The coagulation cascade is divided into 2 pathways, the intrinsic and extrinsic system, and a common pathway. This segregation of sections is not physiological, but allows for the grouping of factor defects and the interpretation of laboratory testing. Most problems with coagulation factors fall into one of three categories: a factor is not produced, there is a decreased production, or the factor is produced but not functioning properly. Hemophilia B is a factor IX deficiency. It is classified as mild, moderate or severe based upon the activity level of factor IX. In mild cases, bleeding symptoms may occur only after surgery or trauma and may not be diagnosed until later in life. In moderate and severe cases, bleeding symptoms may occur after a minor injury or even spontaneously. These moderate to severe cases are usually diagnosed at a younger age.

This child was diagnosed with Hemophilia B, based on coagulation studies, Factor IX assay results and family history. Treatment involves replacement of Factor IX to promote adequate blood clotting and prevent bleeding episodes.

References

  1. Turgeon, Mary Louise, Clinical Hematology: Theory & Procedures, 6th ed.  Lippincott Williams and Wilkins, Philadelphia, 2017.
  2. https://www.hog.org/publications/detail/the-royal-disease-a-family-history-update-on-queen-victoria
  3. https://rarediseases.org/rare-diseases/hemophilia-b/
  4. https://pediatriceducation.org/2015/12/14/what-is-it-called-christmas-disease/
  5. https://www.stago-us.com/hemostasis/tests-clinical-applications/hemophilia-b/

-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 and Molecular Diagnostics Case Study: A 63 Year Old Man with Fatigue

The following case is an interesting overlap of Hematopathology and Molecular Diagnostics, and shows the utility of sequencing to detect a cancer before biopsy could.

A 63 year old gentleman presented to a heme/onc physician with six months of intractable anasarca, fatigue, and a recent mild thrombocytopenia (Table 1). They were otherwise in healthy condition. The physician initiated a lymphoma work-up that included a bone marrow biopsy. The tests were negative for M-protein.

Table 1. Summary of symptoms and relevant abnormal labs.

The bone marrow biopsy was somewhat limited, but the core contained multiple marrow elements. After a thorough review by a Hematopathologist, no evidence of dysplasia or other irregularities could be detected (Image 1). Flow cytometry detected no aberrant blast population. Cytogenetics detected 20del [16/20] and 5del [3/20]. These findings did not clearly indicate a specific diagnosis.

Image 1. 40x view of the bone marrow specimen at the initial presentation. No evidence of dysplasia was found.

As the clinical suspicion for a malignancy was high, the bone marrow specimen was sent for sequencing on a 1385-gene panel test. The test included tumor-normal matched DNA sequencing (“tumor” sample: bone marrow, normal: saliva), RNA whole transcriptome sequencing on the bone marrow, and Copy Number Variant (CNV) analysis. Tumor-normal matched sequencing helps rule out variants that are normal and present in the patient.

Somatic mutations were determined as those that were present in the “tumor” sample and not in the matched normal sample. The somatic variants found are listed below with their variant allele frequency (VAF) in parenthesis. Recall that a VAF of 40% means that a mutation is present in the heterozygous state in 80% of cells.

  • IDH2 (p.R140Q, 46%)
  • SRSF2 (p.P95T, 51%)
  • CBL (p.R499*, 47%)
  • KRAS (p.K117N, 12%)
Figure 1. View of IGV, which displays the NGS reads for IDH1 along with the variant allele highlighted in red. The color of the bars indicates the direction of the reads (forward in red and reverse in blue). This reflects the allele frequency of approximately 50%.

The mutations in these genes are commonly found in myeloid cancers including myselodysplastic syndrome. Activating mutation in IDH2 (isocitrate dehydrogenase 2) increase the production of the oncometabolite 2-HG, which alters methylation in cells taking them to an undiffereitiated state. SRSF2 (Serine And Arginine Rich Splicing Factor 2) is a part of the spliceosome complex, which regulates how sister chromatids separate from each other. Failures in the proper function of the complex creates genomic instability. CBL (Casitas B-lineage Lymphoma) is a negative regulator of multiple signaling pathways, and loss of function mutations (as seen here) lead to increased growth signals through several tyrosine kinase receptors. KRAS (Kirsten RAt Sarcoma virus) is an upstream mediator of the RAS pathway, which acquires mutations that lead to constitutive activation and sends growth signals to cells causing them to proliferate.

Furthermore the CNV analysis also found the heterozygous loss of chromosome 20 as reported in cytogenetics. CNV analysis did not detect chromosome 5 deletion, as it was below the limit of detection (20% for CNV analysis).

Figure 2. This plot shows the normalized read frequency of genes across each of the chromosomes is shown here. The drop at chromosome 20 is shown in a pale brown color on the right side of the graph. This is consistent with the cytogenetic findings. The loss of 5q isn’t seen as it is below the limit of detection of 30%.

These mutations are all individually common in MDS, but the co-occurance of each gives very strong evidence that MDS is the diagnosis (Figure 3). There have also been studies that provide prognostic implications for several of the genetic mutations present. Some mutations like SRSF2 or CBL at high VAF (>10%) indicate a poor prognosis, but mutations in IDH2 or TP53 at any frequency have not only a high chance of progression, but also a faster time to onset of disease. Another non-genetic risk factor for developing MDS is an elevated RDW, which we saw in our patient.

Figure 3. From Becker et al 2016.

All of these high-risk factors together led us to push for a diagnosis of MDS based off of molecular findings, and the patient was started on treatment with Azacitadine. Our assessment was confirmed 3 months later when, the patient’s follow up bone marrow biopsy showed significant progression with megakaryocytic and erythroid dysplasia and hyperplasia and reticulin fibrosis MF2 (Image 2). Aberrant blasts were detected (1-2%), but not elevated. This demonstrates how molecular findings predicted and predated the patient’s rapid progression to morphologic disease.

Image 2. Dysplastic, hyperplastic megakaryocytes and erythroid lineage.

In summary, multiple molecular mutations indicative of MDS were found in a symptomatic patient’s unremarkable bone marrow biopsy months before a rapid progression to MDS.

References

  1. Steensma DP, Bejar R, Jaiswal S et al. Blood 2015;126(1):9-16.
  2. Sellar RS, Jaiswal S, and Ebert BL. Predicting progression to AML. Nature Medicine 2018; 24:904-6.
  3. Abelson S, Collord G et al. Prediction of acute myeloid leukemia risk in healthy individuals. Nature 2018; 559:400-404.
  4. Desai P, Mencia-Trinchant N, Savenkov O et al. Nature Medicine 2018; 24:1015-23.
  5. Becker PM. Clonal Hematopoiesis: The Seeds of Leukemia or Innocuous Bystander? Blood.2016 13(1)

-Jeff SoRelle, MD is a Chief Resident of Pathology at the University of Texas Southwestern Medical Center in Dallas, TX. His clinical research interests include understanding how the lab intersects with transgender healthcare and improving genetic variant interpretation.

A 66 Year Old Male with Diarrhea, Weight Loss, and Night Sweats

Case History

A 66 year old man with past medical history of recently diagnosed Clostridioides difficile colitis presented to emergency department with diarrhea, weight loss of 52 pounds in 4 months, and occasional night sweats. CT imaging revealed dilation of small bowel with thickened mucosal folds. The duodenum was subsequently biopsied to reveal diffuse intestinal lymphangiectasia containing PAS positive and Congo red negative eosinophilic material and lamina propria foamy macrophages. Laboratory investigations revealed normocytic anemia, proteinuria, and peripheral IgM kappa monoclonal gammopathy.

Biopsy Findings

Image 1. Aspirate.
Image 2. Core biopsy.
Image 3. CD138.
Image 4. Kappa ISH.
Image 5. Lambda ISH.

Bone marrow aspirate shows increased plasma cells and mast cells. H&E stained sections demonstrate a normocellular bone marrow with trilineage hematopoiesis and involvement by 35% plasma cells. By immunohistochemistry, CD138 highlights clusters of plasma cells that predominantly express kappa light chain restriction.

FISH and Mutation Analysis

FISH demonstrated loss of chromosome 11 and gain of chromosome 15, which was consistent with plasma cell dyscrasia. MYD88 mutation analysis did not detect the mutation.

Diagnosis

The findings of the patient’s normocytic anemia, IgM monoclonal gammopathy, and intestinal lymphangectasia with an associated plasma cell dyscrasia involving the bone marrow favor a lymphoplasmacytic lymphoma/Waldenström macroglobulinemia.

Discussion

Waldenstrom macroglobulinemia (WM) is a malignant B-cell lymphoproliferative disorder characterized by lymphoplasmacytic infiltration of the bone marrow and peripheral IgM monoclonal gammopathy.1 It is rare with an overall incidence of 3 per million persons per year, accounting for 1-2% of hematologic cancers.1 It occurs predominantly in Caucasian males, with a median age of 63-68 years old at diagnosis.1-3

Patient may be asymptomatic for years and require observation or experience a broad spectrum of signs and symptoms. These symptoms may be attributable to the tumor infiltration of the bone marrow and lymphoid tissues, IgM circulating in the blood, and IgM depositing into tissues. The most common clinical presentation of WM is fatigue and nonspecific constitutional symptoms, such as fever, night sweats, and weight loss, due to normochromic, normocytic anemia. 20-30% of patients may exhibit lymphadenopathy and hepatosplenomegaly due to infiltration of peripheral tissues. High concentration of IgM in the circulation may lead to hyperviscosity, resulting in oronasal bleeding, gingival bleeding, blurred vision due to retinal hemorrhages, and neurological symptoms, including headache, ataxia, light-headedness, dizziness, and rarely, stroke.2-3 The gastrointestinal manifestations are rare; however, IgM monoclonal protein may deposit into the lamina propria of the GI tract, causing diarrhea, steatorrhea, and GI bleeding.4 Other IgM-related manifestations include cold agglutinin hemolytic anemia, cryoglobulin, and amyloid deposition in tissues.3

Diagnosis of WM includes evidence of IgM monoclonal gammopathy and at least 10% of bone marrow infiltration by lymphoplasmacytic cells.5 Monoclonal gammopathy can be detected by the monoclonal spike, or M-spike, on serum protein electrophoresis.3 Serum immunofixation may be performed to identify the type of monoclonal protein and the type of light chain involved.3 In terms of immunophenotype, neoplastic cells express surface IgM, cytoplasmic Igs, CD38, CD79a, and pan B-cell markers (CD19, CD20, and CD22). CD10 and CD23 are absent. Expression of CD5 occurs in approximately 5-20% of cases.6 Recent studies have reported two most common somatic mutations in WM, which are MYD88 L265P mutations (90-95% of cases) and CXCR4 (30–40% of cases).7 Absence of these mutations, however, do not completely exclude the diagnosis of WM.

The International Staging System for WM identifies five factors associated with adverse prognosis, including age older than 65, hemoglobin < 11.5g/dL, platelet count < 100K/μL, beta-2-microglobulin > 3mg/L, and monoclonal IgM concentration > 7g/L.3 Patients younger than the age of 65 years with 0 or 1 of these factors are in the low-risk category with a median survival of 12 years.3 In contrast, patients with 2 or more risk factors are in the intermediate- and high-risk categories and have a median survival of almost 4 years. 3

Management of WM depends on the patient’s clinical manifestations.Furthermore, patients with minimal symptoms should be managed with rituximab, whereas patients with severe symptoms related to WM should receive more aggressive treatment, including dexamethasone, rituximab and cyclophosphamide. Hyperviscosity syndrome is an oncologic emergency that requires removal of excess IgM from the circulation via plasmapheresis.8

References

  1. Neparidze N, Dhodapkar MV. Waldenstrom’s Macroglobulinemia: Recent advances in biology and therapy. Clin Adv Hematol Onco. 2009 Oct;7(10): 677-690.
  2. Leleu X, Roccaro AM, Moreau AS, Dupire S, Robu D, et al. Waldenstrom Macroglobulinemia. Cancer Lett. 2008 Oct;270(1):095-107.
  3. Tran T. Waldenstrom’s macroglobulinemia: a review of laboratory findings and clinical aspects. Laboratory Medicine. 2013 May;44(2):e19-e21.
  4. Kantamaneni V, Gurram K, Khehra R, Koneru G, Kulkarni A. Distal illeal ulcers as gastrointestinal manifestation of Waldenstrom Macroglbulinemia. 2019 Apr; 6(4):pe00058.
  5. Grunenberg A, Buske C. Monoclonal IgM gammopathy and Waldenstrom’s macroglobulinemia. Dtsch Arztebl Int. 2017 Nov;114(44):745-751.
  6. Bhawna S, Butola KS, Kumar Y. A diagnostic dilemma: Waldenstrom’s macroglobulinemia/plasma cell leukemia. Case Rep Pathol. 2012;2012:271407.
  7. Varettoni M, Zibellini S, Defrancesco I, Ferretti VV, Rizzo E, et all. Pattern of somatic mutations in patients with Waldenstrom macroglobulinemia or IgM monoclonal gammopathy of undetermined significance.
  8. Oza A, Rajkumar SV. Waldenstrom macroglobulinemia: prognosis and management. Blood Cancer Journal. 2015;5:e394.

-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, MLS(ASCP)CM is an Assistant Professor of Pathology and Laboratory Medicine, Medical Education and Applied Health Sciences at Loyola University Chicago Stritch School of Medicine and Parkinson School for Health Sciences and Public Health. A past top 5 honoree in ASCP’s Forty Under 40, Dr. Mirza was named to The Pathologist’s Power List of 2018 and placed #5 in the #PathPower List 2019. Follow him on twitter @kmirza.