Hematopathology Case Study: A 16 Year Old Male with Fatigue, Fevers, and Weight Loss

Case History

16 year old male with a history of chronic pilonidal cyst presented with fatigue, fevers and weight loss. He was febrile and noted to have cervical and inguinal adenopathy. Labs were significant for a white count of 77,000 with 85% peripheral blasts, anemia and thrombocytopenia.

MPAL1
Bone marrow aspirate
MPAL2
Bone marrow core biopsy
MPAL3.png
Flow cytometry myeloid markers
MPAL4
Flow cytometry cytoplasmic markers
MPAL5
Flow cytometry T-cell markers

Diagnosis

The bone marrow aspirate shows cellular spicules with sheets of intermediate-to-large sized mononuclear cells with irregular nuclei, distinct nucleoli, dispersed chromatin, and scant to generous amphophilic cytoplasm, with occasional vacuoles, consistent with blasts.

The bone marrow core biopsy shows a greater than 95% cellular marrow, hypercellular for age with approximately 90% of the cellularity composed of an interstitial population of intermediate-to-large sized mononuclear cells with irregular nuclei, distinct nucleoli, dispersed chromatin, and scant to generous amphophilic cytoplasm, with occasional vacuoles, consistent with blasts.

Flow cytometry shows leukemic cells that express immaturity markers (TdT, CD34, CD117, HLA-DR), T cell lineage markers (CD2, CD7 cCD3), and multiple myeloid markers (CD13, CD117, and variable CD15 and CD11b as well as MPO in a small subset).

Bone marrow core biopsy staining (not shown) had similar findings with blasts showing dim-to-strong positivity for myeloperoxidase, lysozyme, CD34 and CD117, as well as strong positivity for TdT. CD7 was weakly positivity, as well as CD3. CD4 and CD5 were negative.

MPAL6
Genetics diagnostics
MPAL7
NGS panel

With the expression of MPO by flow cytometric analysis and immunohistochemistry, a final diagnosis of acute leukemia with myeloid and T lymphoid phenotypic features, most consistent with T/Myeloid Mixed Phenotype Acute Leukemia (MPAL) was rendered. 

Discussion

Most acute leukemias are definitively assigned to either myeloid, T or B lymphoid lineages. However, approximately 2-5% of patients diagnosed with acute leukemia display an ambiguous lineage after immunophenotyping. A portion of these cases are classified under the category of mixed phenotype acute leukemia (MPAL) by the current WHO nomenclature.1

In a study of 117 MPAL patients by Yan et al, 55% of the cases had combined B/Myeloid, while 33% had T/Myeloid, and 12% had B/T/Myeloid. CD34 was strongly positive in 82% of cases, which reinforces the idea that the cell of origin is a multi-potent stem cell capable of differentiating into both myeloid and lymphoid progenitors. Cytogenetic analysis revealed no chromosomal abnormality in 36% of the patients with MPAL, while 64% had complex karyotypes (>3 aberrations). Translocation (9;22) was the most common abnormality, found in 15% of patients. Monosomy 7, a common finding in myelodysplastic syndromes as well, was found in 7.6% of patients. Mutational analysis revealed IKZF1 deletions in 13% of patients, ASXL1 in 6.5% of patients and a variety of other mutations including ETV6, NOTCH1 and TET2.2

In 2016, Eckstein and colleagues demonstrated epigenetic regulatory genes such as DNMT3A, IDH2, TET3 and EZH2 are the most commonly mutated in MPAL. RAS mutations including NRAS and KRAS and tumor suppressors, such as TP53 and WT1, were frequently identified as well.3

Interestingly enough, the genetic features of MPAL often overlap with early T-cell precursor acute lymphoblastic leukemia (ETP-ALL). ETP-ALL is a high-risk subgroup, representing 10% of adult T-lineage acute lymphoblastic leukemia. It is defined by a characteristic immunophenotype (CD1a/CD8 negative with weak CD5) and distinct gene expression associated with early arrest in T-cell development. This subgroup, called the LYL1 group, expresses the early hematopoietic marker CD34 as well as myeloid antigens (CD13 or CD33), but lacks expression of both CD4 and CD8. These leukemias are associated with a poor prognosis, with a 10- year overall survival of 19% compared to 84% for all other T-ALLs.4

Zhang et al in 2012 performed whole genome sequencing on ETP-ALL cases and found a high frequency of mutations in factors mediating cytokine receptor, tyrosine kinase and RAS signaling. It also showed inactivating mutations in genes encoding transcription factors (GATA3, ETV6, RUNX1, IKZF1) as well as genes involved in histone modification, such as EZH2.5

Overall, the genetic features of both ETP-ALL and MPAL display an identical genomic pattern that involves multiple pathways, including tyrosine kinase signaling, cytokine receptor response, RAS pathway activation, and loss of function in tumor suppressors. These findings give credence to the hypothesis that the early T-cell precursor actually displays more of a pluripotent stem cell profile that is similar to myeloid neoplasms, thus confounding findings found during molecular profiling. With this paradigm in mind, molecular diagnostics cannot differentiate between ETP-ALL and in this case, MPAL.

 

References

  1. Swerdlow, Steven H. WHO Classification of Tumours of Haematopoietic and Lymphoid Tissues. Revised 4th ed., International Agency for Research on Cancer, 2017.
  2. Yan et al. Clinical, immunophenotypic, cytogenetic, and molecular genetic features in 117 patients with mixed-phenotype acute leukemia defined by WHO-2008 classification. 2012 November;97(11):1708-12.
  3. Eckstein OS et al. Mixed Phenotype Acute Leukemia (MPAL) Exhibits Frequent Mutations in DNMT3A and Activated Signaling Genes. Exp Hematol. 2016 August; 44(8):740-744.
  4. Ferrando AA et al.  Gene expression signatures define novel oncogenic pathways in T cell acute lymphoblastic leukemia. Cancer Cell. 2002. 1:75–87.
  5. Zhang J et al. The genetic basis of early T-cell precursor acute lymphoblastic leukemia. Nature. 2012 Jan 11;481(7380):157-63.

 

Marcus, Chelsea_099-Edit

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

Not Your Grandmother’s Hematology

Last month we celebrated Lab Week, to recognize and show appreciation for Medical Laboratory Scientists and Technicians. Lab week is also a time to reminisce, and tell stories of the lab “in the old days.” I have worked with many technologists who have now been in the field for more than 50 years, and some who have worked in the same hospital all that time! Lab techs love to share stories about their experiences over the years, the days without computers, old methodologies, ancient lab equipment and manual testing. Listening to these stories always makes me think about just how far we have come in the field in the last 50- 60 years, and gives me a true appreciation for modern technology. It causes me to reflect on all the changes and developments that enable us to give physicians a wealth of knowledge that was previously unavailable.

During the first half of the 20th century, the complete blood count (CBC) was performed using exclusively manual techniques. Blood cell counts (erythrocytes, leukocytes, thrombocytes) were performed under the microscope using diluted blood samples and a hemocytometer. For each specimen, a technologist spent about 30 minutes at a microscope manually counting the cells and calculating the total count using a mathematical formula. A spectrophotometer was used to perform the hemoglobin by the cyanmethemoglobin method, and a spun hematocrit was performed. Indicies were calculated. A manual smear was made, stained, and cells were counted and differentiated under the microscope. To complete a CBC, all these procedures had to be performed individually, with duplicate testing and applying mathematical calculations, and could take over 2 hours. After all these tests were performed, results were reported on paper and sent to the patient’s doctor or the nursing floor.

In 1953 Wallace Coulter patented the Coulter Principle for counting and sizing microscopic particles. The Coulter Principle can be used for measuring any particles that can be suspended in an electrolyte solution, and has been used in the food and drug industry, in beer making, in the manufacture of construction materials and thousands of other applications. However, probably the most important application has been in the medical field where it has revolutionized the science of hematology. Coulter suspended red blood cells in a solution and, with an electrical current flowing, passed the solution through an aperture. As the cells pass through the current, the impedance between the terminals changes, and this change can be measured as a pulse. The first Coulter Counter measured the number of cells by counting the number of these pulses. The first Model A Coulter Counter was sold in 1956, manufactured in Coulter’s basement in Chicago. The Model A counted red blood cells in a sample in 10 minutes, a marked improvement over manual counting! The Coulter Counter was hailed for its speed, accuracy, and opportunities for reducing human error, tedium and eye strain.

grandma-heme-1
Image 1. Model A Coulter Counter, 1956. https://www.beckman.com/resources/discover/fundamentals/history-of-flow-cytometry/the-coulter-principle

During the 1960’s, an improved Model B Coulter Counter was developed and Model A and Model B were used to count both leukocytes and erythrocytes. Other Coulter Counter models soon followed, and competitors entered the market with their versions of cell counters. Within a decade, nearly every hospital in the United States had a Coulter Counter, and the new, advanced Coulter Model F was widely used. In 1968 the first fully automated hematology analyzer, The Coulter Counter Model S was introduced, and could perform a seven-parameter CBC. The Model S could perform not only WBC and RBC counts, but also reported Hemoglobin, Hematocrit, Mean Corpuscular Volume (MCV), Mean Corpuscular Hemoglobin (MCH) and Mean Corpuscular Hemoglobin Concentration (MCHC).  In 1955 it took one or several technologists 2 hours to perform a CBC, and in 1969 an automated hematology analyzer could analyze a sample in under 2 minutes.

Image 2. “Woman Using a Model F Coulter Counter Cell Counter,” 1969. Beckman Historical Collection, Box 58, Folder 94. Science History Institute. Philadelphia. https://digital.sciencehistory.org/works/736664585.

grandma-heme-2
Image 2. “Woman Using a Model F Coulter Counter Cell Counter,” 1969. Beckman Historical Collection, Box 58, Folder 94. Science History Institute. Philadelphia. https://digital.sciencehistory.org/works/736664585.

As these improvements and advancements continued, and Coulter patents expired, new manufacturers entered the field. Technicon Instruments Corporation, Ortho Diagnostics, Instrumentation Laboratories and Toa Medical Electronics, (presently Sysmex Corporation) were among the first Coulter competitors. From a simple automated blood cell count, to the first seven-parameter CBC, we saw hematology changing before our eyes. More reliable automated platelet counts were added in the 1970s. In the 1980s we saw the first hematology analyzers that could perform automated differentials and the first automated reticulocyte analyzers. In the late 1990’s, we saw the advent of digital cell images and automated manual differentials.

Today, modern automated cell counters sample blood, and quantify, classify, and describe cell populations. These instruments use optical light scatter, impedance methods based on the Coulter principle or a combination of both optical and impedance methods. Progressive improvement in these instruments has allowed the enumeration and evaluation of blood cells with great accuracy, precision, and speed, at a very low cost per test. The latest descendant of the Model A Coulter Counter, the LH 750, can determine 26 reportable hematological parameters. The Sysmex XN-9100 with four XN analyzers reports 30 parameters and has a throughput of up to 400 CBCs and 75 smears per hour. Today’s analyzers can accomplish more and more routine diagnostics, and the role of the hematology technologist continues to evolve and expand.

grandma-heme-3.tif
Image 3. Sysmex XN-9100™ Automated Hematology System
https://www.sysmex.com/us/en/Brochures/XN9100ScalableAutomationBrochure_mkt-10-1177_10252017.pdf

This is not your grandmother’s hematology! We’ve truly come a very long way in 60 years. Modern hematology instruments not only perform a CBC, but they give us next generation diagnostics as well. Many give us advanced clinical parameters and other new parameters which provide physicians with additional information about the state of blood cells. We can report out immature granulocytes with every differential, automated nucleated red blood cell counts, immature platelet fractions and fluorescent platelet counts, and report the amount of hemoglobin in reticulocytes and the immature reticulocyte fraction. Future directions of hematology instrumentation include the addition of even more new parameters. In upcoming Hematology blogs I will be presenting case studies that highlight each of these advanced clinical parameters and discuss how physicians can use this new information in making diagnoses.

 

References

  1. Beckman Coulter, Inc. History http://www.fundinguniverse.com/company-histories/beckman-coulter-inc-history/
  2. https://www.beckman.com/resources/discover/fundamentals/history-of-flow-cytometry/the-coulter-principle 
  3. Clinics in laboratory Medicine. Development, history, and future of automated cell counters Green RWachsmann-Hogiu S. Clin Lab Med. 2015 Mar;35(1):1-10. doi: 10.1016/j.cll.2014.11.003. Epub 2015 Jan 5. March 25, Vol 35, Issue 1, p1-10
  4. Cytometry: Journal of Quantitative Cell Science. Wallace H. Coulter: Decades of invention and discovery Paul Robinson First published: 17 April 2013 https://onlinelibrary.wiley.com/doi/epdf/10.1002/cyto.a.22296
  5. J.clin.Path. An assessment of the Coulter counter model S P.H.Pinkerton,I.Spence, J.C. Ogilvie, W.A Ronald, Patricia Marchant, and P.K, Ray. 1970,23,68-76 http://jcp.bmj.com/content/jclinpath/23/1/68.full.pdf
  6. SLAS TECHNOLOGY: Translating Life Sciences Innovation. The Coulter Principle: Foundation of an Industry. Marshall Don, Ph.D., Beckman Coulter, Inc.. Volume: 8 issue: 6, page(s): 72-81. Issue published: December 1, 2003 https://doi.org/10.1016/s1535-5535(03)00023-6
  7. Medical Electronic Laboratory Equipment 1967-1968. G.W.A Dummer and J. MacKenzie Robertson. 1967 Pergamon Press

 

Socha-small

-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 68 Year Old Man with Epidural Mass

Case History

A 68 year old man with no significant past medical history presented with 3 weeks of upper back pain and bilateral leg weakness. He denied numbness, tingling, leg pain or urinary or fecal incontinence. MRI showed severe cord compression at the upper thoracic spine with a T2-T5 epidural mass. Due to the patient’s decline, an urgent decompression was scheduled and the patient underwent T2-T5 thoracic laminectamies with resection of extramedullary epidural tumor.

MRI-t2
MRI T2 SAG T-Spine
frozhe2x
Frozen Section H&E, 2x 
frozhe20x
Frozen Section H&E, 20x

Imaging

Frozen Section Diagnosis

“Round blue cell tumor.  Await permanents for final diagnosis.”

Differential Diagnosis

Small round blue cell tumor is a term generally used for a group of neoplasms characterized by small, round, basophilic, relatively undifferentiated cells on H & E staining. The differential diagnosis is wide, but includes Ewing’s sarcoma/peripheral neuroectodermal tumor, mesenchymal chondrosarcoma, small cell osteosarcoma, desmoplastic small round cell tumor and Non-Hodgkin Lymphoma. 1

 he2x

H&E, 2x

he4x
CD20, 4x

 

cd20-4x
CD20, 4x
bcl2-4x
BCL2, 4x
cd10-4x
CD10, 4x
cd21-4x
CD21, 4x
ki-67
Ki-67, 4x
IGH
IGH/BCL2 double fusion FISH probe. White arrows: IGH/BCL2 fusion

Diagnosis

Sections show fragments of fibrous tissue and focal bone with extensive crush artifact. There is an abnormal lymphoid infiltrate with areas showing a vaguely nodular architecture. The lymphocytes are small to medium in size with irregular cleaved nuclei, inconspicuous nucleoli and small amounts of cytoplasm. Scattered centroblastic cells are seen but are <15 per high power field. Between the nodules, the cells are centrocytic appearing. Rare mitotic figures are identified.

By immunohistochemistry, the neoplastic cells are immunoreactive for CD20 and BCL2. BCL2 is brighter in the vague nodular areas which are also highlighted by CD10 and BCL6. CD23 is variably positive in a large subset of cells. MUM1 is negative. CD21 highlights the enlarged and irregularly shaped follicular dendritic cell meshwork present in the areas with nodules. CD3 and CD5 highlights admixed T-cells. The proliferation index by Ki-67 is low and approximately 10%.

Cytogenetic analysis using fluorescent in-situ hybridization performed on paraffin embedded sections revealed numerous cells with an IGH/BCL double fusion probe signal pattern consistent with IGH/BCL2 gene rearrangement.

Overall, the morphologic and immunophenotyipic findings in conjunction with the cytogenetic results are in keeping with involvement by a B-cell lymphoma most consistent with a follicular lymphoma. The follicles present contain <15 centroblasts per hpf and the low proliferation fraction makes it most compatible with a low grade (WHO morphologic grade 1-2/3) follicular lymphoma.

Discussion

The differential diagnosis for an extramedullary epidural tumor is wide and can include anything from an epidural abscess to a metastasis. Although rare, lymphoma must be considered, especially when initial pathology shows “Round blue cells.”

Making the diagnosis of follicular lymphoma involves assessing the H & E slides for follicular architecture, characteristic immunostains including positivity for BCL2 within follicles and the typical t(14;18) IGH/BCL2 translocation, which occurs in 90% of cases. 2

Primary spinal epidural lymphoma (PSEL) includes extramedullary/extranodal lymphomas of the epidural space for which there are no other sites of disease at the time of diagnosis. As demonstrated in Figure 1 below, the lymphoma is seen entirely within the epidural space. 3

fig1
Figure 1. Primary spinal epidural lymphomas. Journal of Craniovertebral Junction and Spine (2011).

 

An epidural location for lymphoma is observed in 0.1-6.5% of cases. Patients tend to present in the fifth to seventh decade of life with a higher proportion of male to female cases. Presenting symptoms include weakness in the upper or lower limbs and back pain corresponding to the site of involvement of tumor. The most common tumor site is the thoracic spine (75%) followed by lumbar and cervical. Most epidural spinal tumors are B-cell lymphomas of intermediate and high grade, but low grade lymphomas have been reported. 3

Although rare, lymphoma is an important consideration in the differential diagnosis for tumors involving the spine. Surgical intervention is often necessary to relieve spinal cord compression and to make a histologic diagnosis. Treatment includes radiation and chemotherapy. Patients with primary spinal epidural lymphoma tend to have a better prognosis than patients with systemic lymphoma involving the epidural space, as well as patients with metastatic carcinoma. 3

References

  1. Hameed, Meera: Small Round Cell Tumors of Bone. Arch Pathol Lab Med (2007) 131: 192-204.
  2. Louis D.N., Ohgaki H., Wiestler O.D., Cavenee W.K. (Eds.): WHO Classification of Tumors of the Central Nervous System. IARC: Lyon 2007.
  3. Cugati G, Singh M, Pande A, et al. Primary spinal epidural lymphomas. Journal of Craniovertebral Junction and Spine (2011) 2(1): 3-11.

 

Marcus, Chelsea_099-Edit

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

Blood Bank Case Study: Transfusion Transmitted Malaria

Case Study

A 26 year old African American female with sickle cell anemia presented to a New York emergency room with cough, chest pain, fever and shortness of breath. Laboratory results showed an increased white blood cell count, slightly decreased platelet count and a hemoglobin of 6.2 g/dl. Her reticulocyte count was 7%, considerably below her baseline of 13%. Consulting the patient’s medical records revealed history of stroke as a child and subsequent treatment with chronic blood transfusions. She was admitted to the hospital for acute chest syndrome and aplastic crisis and care was transferred to her hematologist. Two units of RBCs were ordered for transfusion.

The blood bank technologists checked the patient’s blood bank history and noted her blood type was A, Rh(D) positive, with a history of a warm autoantibody and anti-E. The current blood bank sample confirmed the patient was blood type A, RH(D) positive with a negative DAT but the antibody screen was positive. Anti-E was identified. Per request of the hematologist, phenotypically similar units were found and the patient was transfused with 2 units of A RH(negative), C/E/K negative, HgS negative, irradiated blood. The patient’s hemoglobin rose to 8g/dl and she was discharged from the hospital 3 days after transfusion.

Ten days after discharge the patient returned to the emergency room with symptoms including aching muscles, fever and chills. A delayed transfusion reaction was suspected. A type and screen was immediately sent to the blood bank. The post transfusion type and screen remained positive for anti-E, DAT was negative. No additional antibodies were identified. However, a CBC sent to the lab at the same time revealed malarial parasites on the peripheral smear. The patient was consulted for a more complete medical history and reported that she had never traveled outside of the country. A pathology review was ordered and the patient was started on treatment for Plasmodium falciparum.

plasfal1

Discussion

Red Blood cell transfusions can be life saving for patients with sickle cells anemia. These patients are frequently transfused by either simple transfusion of red cell units or by exchange transfusion. Because of this, alloimmunization is reported to occur in 20% to 40% of sickle cell patients.1 Blood bank technologists are very diligent in adhering to strict procedures and follow a standard of practice aimed to prevent transfusion reactions. While preventing immune transfusion reactions may be the most forefront in our minds when transfusing the alloimmunized patient, it is important to consider transfusion transmitted diseases as a potential complication of blood transfusions.

Malaria is caused by a red blood cell parasite of any of the Plasmodium species. Mosquito transmitted infection is transmitted to humans through the bite of an infected mosquito. Transfusion-transmitted malaria is an accidental Plasmodium infection caused by a blood transfusion from a malaria infected donor to a recipient.

Donors, especially those from malarial endemic countries who may have partial immunity, may have very low subclinical levels of Plasmodium in their blood for years. Even these very low levels of parasites are sufficient to transmit malaria to a recipient of a blood donation. Though very rare, transfusion-transmitted malaria remains a serious concern for transfusion recipients. These transfusion-transmitted malaria cases can cause high percent parisitemia because the transfused blood releases malarial parasites directly into the recipient’s blood stream.

Blood is considered a medication in the United States, and, as such, is closely regulated by the FDA. Blood banks test a sample of blood from each donation to identify any potential infectious agents. Blood donations in the US are carefully screened for 8 infectious diseases, but malaria remains one infectious disease for which there is no FDA-approved screening test available. For this reason, screening is accomplished solely by donor questioning.2 A donor is deferred from donating if they have had possible exposure to malaria or have had a malarial infection. Deferral is 12 months after travel to an endemic region, and 3 years after living in an endemic region. In addition, a donor is deferred from donating for 3 years after recovering from malaria. It is important, therefore, for careful screening to take place by questionnaire and in person, to make sure that the potential donor understands and responds appropriately to questions concerning travel and past infection.

Malaria was eliminated from the United States in the early 1950’s. Currently, about 1700 cases of malaria are reported in the US each year, almost all of them in recent travelers to endemic areas. From 1963-2015, there have been 97 cases of accidental transfusion-transmitted malaria reported in the United States. The estimated incidence of transfusion-transmitted malaria is less than 1 case in 1 million units.4 Approximately two thirds of these cases could have been prevented if the implicated donors had been deferred according to the above established guidelines.3 While the risk of catching a virus or any other blood-borne infection from a blood transfusion is very low, a blood supply with zero risk of transmitting infectious disease may be unattainable. With that being said, the blood supply in the United Sates today is the safest it has ever been and continues to become safer as screening tests are added and improved. Careful screening of donors according to the recommended exclusion guidelines remains the best way to prevent transfusion-transmitted malaria.

References

  1. LabQ, Clinical laboratory 2014 No.8, Transfusion Medicine. Jeanne E. Hendrickson, MD, Christopher Tormey, MD, Department of Laboratory Medicine, Yale University School of Medicine
  2. Technical Manual, editor Mark K. Fung-18th edition, AABB. 2014. P 201-202
  3. https://www.cdc.gov/malaria/about/facts.html. Accessed April 2018
  4. The New England Journal of Medicine. Transfusion-Transmitted Malaria in the United States from 1963 through 1999. Mary Mungai, MD, Gary Tegtmeier, Ph.D., Mary Chamberland, M.D., M.P.H., June 28, 2001. Accessed April 2018
  5. Malaria Journal. A systematic review of transfusion-transmitted malaria in non-endemic areas. 2018; 17: 36. Published online 2018 Jan 16. doi: 1186/s12936-018-2181-0. Accessed April 2018
  6. http://www.aabb.org/advocacy/regulatorygovernment/donoreligibility/malaria/Pages/default.aspx

 

Socha-small

-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.

To Be (MDS) or Not To Be? The Conundrum of Cytoplasmic Vacuolation in Hematopoietic Precursors

Every hematopathologist and pathology trainee knows to be wary of the myriad of causes that could mimic the dysplastic changes seen in marrows involved by MDS. Many times morphology alone, without genetic or cytogenetic evidence of clonality can be tricky. The list of things that can recapitulate changes seen in MDS seems to grow longer every day – and with it the length of our ‘canned comments’ on ruling out reactive causes of dysplasia. Within the recent past, two bone marrow biopsies crossed my microscope, both sent to ‘rule out’ MDS. Both had almost identical morphologic findings, but very different diagnoses. Here are some representative images from the marrow aspirates and iron stains:

mds
Figure 1. Representative Wright-Giemsa stained cells from Case 1 (A and B) with accompanying iron stain (C) showing numerous ring sideroblasts.  Representative Wright-Giemsa stained cells from Case 2 (D) with accompanying iron stain (E) showing some ring sideroblasts. 

Discussion

Images A through C come from case 1, a 67-year-old woman with a past medical history of non-alcoholic steatohepatitis (NASH) complicated by hepatic encephalopathy and recurrent ascites who underwent bone marrow biopsy for new onset pancytopenia with transfusion-dependent anemia. The marrow was slightly hypercellular for age and showed progressive trilineage maturation. Granulocytic and erythroid progenitors did not reveal quantitatively significant dysplasia. The one dysplastic megakaryocyte identified is pictured here (panel A). Interestingly many erythroid and granulocytic precursors showed cytoplasmic vacuolation (panel B showing granulocytic vacuolation). An iron stain (panel C) revealed 44% ring sideroblasts. Case 2 is represented in images D and E and was from a 64-year-old man with no significant past medical history who presented with lethargy and anemia. This marrow was also slightly hypercellular for his age and showed borderline-significant dysplasia in megakaryocytic maturation. Granulopoiesis and erythropoiesis were unremarkable except for cytoplasmic vacuolations in many cells (panel D). An iron stain showed 8% ring sideroblasts (panel E).

Both cases were signed out descriptively, urging the clinician that we needed to rule out reactive causes of dysplasia before a definitive diagnosis of MDS could be rendered. In both cases we suggested waiting for the cytogenetics results for a more comprehensive analysis. Additionally, we recommended testing for serum copper since copper deficiency can be the cause of dysplastic morphology, cytoplasmic vacuolation, and ring sideroblasts.

Case 1 revealed markedly diminished copper and normal cytogenetics. Copper replenishment was curative. Case 2 revealed normal copper levels and a complex karyotype that contained numerous MDS-associated abnormalities confirming the clonal, and therefore malignant nature of these changes. Despite being almost identical morphologically, these case were diagnostically and prognostically poles apart.

Copper is an element that serves as a micronutrient required for hematopoiesis. It’s presence in many readily available foods including meat, fish, nuts, and seeds renders diet-related copper deficiency a rare phenomenon. Zinc-supplementation is one of the causes of copper deficiency in published reports. Copper deficiency has been well documented to mimic dysplastic changes seen in MDS; but these morphologic findings and cytopenia are reversible. Characteristically, cytoplasmic vacuolation is an important morphologic clue that there could be an underlying paucity of serum copper.  Another aspect of copper deficiency is the presence of ring sideroblasts which also can mean MDS. It is very important to consider this differential diagnosis when dealing with marrow specimens sent to rule out MDS. This Lablogatory post highlights the significant overlap between presentation and morphologic findings between MDS and copper deficiency supporting the notion that a high index of suspicion, good communication, stat copper levels, and cytogenetics or MDS FISH studies are very helpful in delineating benign from malignant.

References

  1. Dalal N. et al. Copper deficiency mimicking myelodysplastic syndrome. Clin Case Rep. 2015 May; 3(5): 325–327.
  2. Willis M.S. Zinc-Induced Copper Deficiency: A Report of Three Cases Initially Recognized on Bone Marrow Examination. AJCP. 2005 Jan; 123(1): 125–131
  3. D’Angelo G. Copper deficiency mimicking myelodysplastic syndrome. Blood Res. 2016 Dec; 51(4): 217–219.
  4. Karris S and Doshi V. Hematological Abnormalities in Copper Deficiency. Blood 2007 110:2677

 

Mirza-small

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

Hematopathology Case Study: A 45 Year Old Male with Mediastinal Mass

Case History

A 45 year old male underwent a chest MRA for aortic dilation due to his history of an aneurysmal aortic root. Upon imaging, an incidental anterior mediastinal mass was seen that measured 4.0 cm. In preparation for an upcoming cardiac surgery, the patient underwent a thymectomy with resection of the mass. The sample is a section from the mediastinal mass.

Diagnosis

HVCD-HE-2x
H&E, 2x
HVCD-HE-4x
H&E, 4x
HVCD-HE-lollipop
H&E, 10x. Green Arrows: “lollipop” germinal centers
HVCD-HE-twinning
H&E, 10x. Red arrow: focal “twinning” of germinal centers

Sections show an enlarged lymph node with several follicles demonstrating atrophic-appearing germinal centers which are primarily composed of follicular dendritic cells. These areas are surrounded by expanded concentrically arranged mantle zones. Focal “twinning” of germinal centers is present. Additionally, prominent centrally placed hyalinized vessels are seen within the atrophic germinal centers giving rise to the “lollipop” appearance.

By immunohistochemistry, CD20 highlights B-cell rich follicles while CD3 and CD5 highlight abundant T-cells in the paracortical areas. CD10 is positive in the germinal centers while BCL2 is negative. CD21 highlights expanded follicular dendritic meshwork. CD138 is positive in a small population of plasma cells and are polytypic by kappa and lambda immunostaining. HHV8 is negative. MIB1 proliferation index is low while appropriately high in the reactive germinal centers.

Overall, taking the histologic and immunophenotypic findings together, the findings are in keeping with Castleman’s disease, hyaline vascular type. The reported clinical and radiographic reports suggest a unicentric variant.

Discussion

Castleman’s disease comes primarily in two varieties: localized or multicentric. The localized type is often classified as the hyaline vascular type (HVCD). Demographically, it’s a disease of young adults but can be found in many ages. The most common sites for involvement are the mediastinal and cervical lymph nodes.

The classic histologic findings of HVCD involve numerous regressed germinal centers with expanded mantle zones and a hypervascular interfollicular region. The germinal centers are predominantly follicular dendritic cells and endothelial cells. The mantle zone gives a concentric appearance, often being likened to an “onion skin” pattern. Blood vessels from the interfollicular area penetrate into the germinal center at right angles, giving rise to another food related identifier, “lollipop” follicles. A useful diagnostic tool is the presence of more than one germinal center within a single mantle zone.

The differential diagnosis of HVCD includes late stage HIV-associated lymphadenopathy, early stages AITL, follicular lymphoma, mantle cell lymphoma, and a nonspecific reactive lymphadenopathy. A history of HIV or diagnostic laboratory testing for HIV would exclude the first diagnosis. AITL usually presents histologically as a diffuse process but atypia in T-cells with clear cytoplasm that co-express CD10 and PD-1 outside of the germinal center are invariably present. EBER staining may reveal EBV positive B immunoblasts in early AITL, which would be absent in HVCD. The most challenging differential would include the mantle zone pattern of mantle cell lymphoma. Flow cytometry revealing a monotypic process with co-expression of cyclin D1 on IHC would further clarify the diagnosis.1

Overall, unicentric Castleman’s disease is usually of the hyaline vascular type. Surgical resection is usually curative in these cases with an excellent prognosis.2

 

References

  1. Jaffe, ES, Harris, NL, Vardiman, J, Campo, E, Arber, D. Hematopathology. Philadelphia: Elsevier Saunders, 2011. 1st ed.
  2. Ye, B, Gao, SG, Li, W et al. A retrospective study of unicentric and multicentric Castleman’s disease: a report of 52 patients. Med Oncol (2010) 27: 1171.

 

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-Phillip Michaels, MD is a board certified anatomic and clinical pathologist who is a current hematopathology fellow at Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA. His research interests include molecular profiling of diffuse large B-cell lymphoma as well as pathology resident education, especially in hematopathology and molecular genetic pathology.

Hematology Case Study: The Race to Save a 48 Year Old Man from a Rare Disease

A 48-year-old Caucasian male presented to a Baltimore Emergency Room complaining of fever, chills, and aches. He stated he had not been feeling well for the past week. His symptoms had progressed rapidly over the last 3 days to include night sweats, nausea and excessive somnolence. History taken in the ER revealed the patient had returned 10 days prior from a Safari in Botswana and Zambia. The patient was admitted to the ICU, in shock, with a BP of 75/50. Even though the patient had taken anti-malarial medication, the doctors suspected malaria. Blood was sent to the lab for a blood parasite exam and treatment for malaria was started while the doctors waited for the confirmation.

In the Hematology laboratory, technologists perform microscopy of thick and thin blood smears to look for malarial parasites. The thin smear is a typical Wright Giemsa stained wedge smear, and the thick smears are prepared and stained so that the red blood cells are lysed, and the sample is concentrated, making examination easier. Thorough, careful examination of the thick smear is aimed to identify whether a particular parasite is present, but they require a long drying period and take several hours to prepare and read. Thin smears can detect the parasites but also permit identification of particular species of malaria. While the thick smears were drying the technologist examined the thin smear.

The technologist who examined this patient’s thin smears saw parasites (image 1) under her microscope. She consulted with a supervisor and pathologist to confirm, and the patient’s doctor was notified that the patient did not have malaria, but instead, had Trypanosoma! This was an exciting find in the laboratory, as there have been only 40 cases seen in the US in the past 50 years.

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Image 1. This slide shows the parasite, in dark blue. The parasite causes
African trypanosomiasis, also known as sleeping sickness
(Courtesy of Greater Baltimore Medical Center).

The race for diagnosis and treatment did not stop there, as there are 2 types of African trypanosomiasis, or African sleeping sickness, and effective and appropriate treatment must be started in a timely fashion. Both types look identical on a blood smear and both are considered universally fatal, if not treated. West African trypanosomiasis and East African trypanosomiasis are caused by the tsetse fly, which only lives in rural Africa. The patient stated he did remember being bitten by tsetse flies, and because there had been such a short span of time between being bitten and the onset of symptoms, doctors concluded that the patient had the rarer and fast-acting East African trypanosomiasis, which can kill within months.

Epidemiologists at CDC were contacted, who then consulted other infectious disease specialists at CDC. There are 2 treatments depending the stage of the disease. Surinam is the first line of defense, but melarsoprol, which is arsenic-like and very toxic, must be used if the parasites have reached the central nervous system. Because of the urgent need to start treatment, emergency shipments of both drugs were flown to Baltimore. The patient was started on Surinam to reduce the number of parasites in his blood to a level low enough to allow a spinal tap to be performed. After confirming that the CSF showed no signs of the parasite, treatment with surinam was continued and the patient was discharged a week later and has made a full recovery.

Because of the excellent work done by the medical technologists who made the first discovery, the speed with which the critical calls were made, the actions of the doctors involved, and the cooperation of the CDC, this patient received his first dose of Surinam a little over 24 hours after his blood was sent to the lab. This case shows the importance of a thorough medical and travel history in differential diagnosis. It also illustrates the importance of the competency evaluations and surveys in which all laboratory professionals are required to participate. None of the technologists, doctors or scientists involved had ever actually seen a case of African Trypanosomiasis, they had only read about it in books and seen it on competency assessments.

This case is based on an actual case from 2016. My coworker, Gail Wilson, was the technologist who first saw the Trypanosoma on the slides. Many thanks to Gail for her keen eye and attention to detail!

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Image 2: L&R: Trypanosoma brucei in thin blood smears stained with Giemsa. Center: A close up of a tsetse fly. Credit: DPDx

 

References 

  1. Jon E. Rosenblatt Barth Reller Melvin P. Weinstein.pages 1103-1108, Laboratory Diagnosis of Infections Due to Blood and Tissue Parasites Clinical Infectious Diseases, Volume 49, Issue 7, 1 October 2009; retrieved March 2018 from https://academic.oup.com/cid/article/49/7/1103/316703
  1. Ivo Elliott, Trupti PatelJagrit Shah, and Pradhib Venkatesan. West-African trypanosomiasis in a returned traveller from Ghana: an unusual cause of progressive neurological decline BMJ Case Rep. 2014; 2014: bcr2014204451. Published online 2014 Aug 14.doi: 1136/bcr-2014-204451; retrieved March 2018 from https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4139564/
  1. Lena H. Sun. Medical Detectives raced to save a man from a rare, ‘universally lethal’ disease; retrieved March 2018 from https://www.washingtonpost.com/news/to-your-health/wp/2016/12/22/medical-detectives-raced-to-save-a-man-from-a-rare-universally-lethal-disease/?utm_term=.16d7b136bc47
  1. Parasites – African Trypanosomiasis (also known as Sleeping Sickness). Retrieved March 2018 from https://www.cdc.gov/parasites/sleepingsickness/
  1. DPDx- Laboratory Identification of parasites of Public Health Concern; retrieved March 2018 from https://www.cdc.gov/dpdx/

 

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-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.