Beyond the CBC and Reticulocyte Count: Early Detection of Iron Deficiency Anemia

In my May 2018 post (Not your Grandmother’s Hematology), I discussed the history of hematology and chronicled how far we have come in the last 60 years. We have progressed from manual counting of cells to the first Coulter Counter in 1956, which revolutionized hematology by being able to automate the counting of red blood cells, to modern instruments that can report up to 30 parameters and perform up to 400 CBCs an hour. Among these parameters are what are termed advanced clinical parameters, new parameters which provide physicians with additional information about the state of blood cells. In this blog I will explore how one of these advanced clinical parameters, the Reticulocyte Hemoglobin content, can provide physicians with information that can assist them with earlier detection, differential diagnosis and better management of iron deficiency and iron deficiency anemia. 

Case Study 

A 29 year old female was seen by her gynecologist reporting a history of heavy menstrual bleeding with current bleeding lasting 15 days. The doctor discussed various treatment options with the patient and a CBC was performed. CBC results are shown below.

Test Result Flags Reference
WBC 7.23   4.5-10.5 K/CMM
RBC 4.38   3.70-5.30 M/CMM
HGB 12.0   12.0-15.5 GM/DL
HCT 36.2   36.0-46.0 %
MCV 82.6   80-100 FL
MCH 27.4   27.0-34.0 PG
MCHC 33.1   32.0-36.0 %
PLT 243   150-450 K/CMM
MPV 11.0   9.6-12.0 FL
RDW 12.5   0-15.1 %

This CBC shows no abnormal flags. Based on patient history and presentation, the physician questioned iron deficiency despite normal hemoglobin and hematocrit, MCV and MCHC. He ordered a reticulocyte profile on the same specimen with the following results:

Test Result Flags Reference Range
Retic 1.55   0.5-2.0 %
Abs Retic 0.0679 H 0.0391-0.057 M/CMM
Imm Retic Frac 14.9   2.3-15.9 %
Ret-Hgb 24.6 L 30-35 PG

Reticulocyte counts are the quantity of the youngest red blood cells released from the bone marrow into the peripheral blood. Reticulocytes are reported as a % and the absolute reticulocyte count is calculated by multiplying the Retic% by the RBC. The immature reticulocyte fraction (IRF) is the rate of production of reticulocytes and depends largely on the ability of the bone marrow to respond to erythropoietin. The reticulocyte hemoglobin (Ret-He) content is the amount of hemoglobin in newly formed red blood cells. (There are two different hematology systems that report reticulocyte hemoglobin content. The two nomenclatures used for reticulocyte hemoglobin are Ret-He and CHr and studies have been done that demonstrate their equivalence)

Note that the Ret-He reflects the quality of the newly formed reticulocytes. Ret-He is a direct measurement of the amount of hemoglobin in each reticulocyte, which indicates the amount of iron available for incorporation into the precursors of mature red cells. This patient’s retic% and IRF are within normal ranges, but her absolute reticulocyte count is high. A Ret-He less than 29 pg in an adult is indicative of iron deficiency. With a normal CBC and low Ret-He, this is an early indication that iron deficiency is indeed present. With the absence of sufficient iron, this patient would eventually develop a microcytic, hypochromic anemia. Therefore, Ret-He can measure and indicate inadequate hemoglobin production before the hemoglobin and hematocrit decrease.

In this case the importance of clinical awareness is illustrated. This physician remembered a recent laboratory technical bulletin announcing implementation of a new hematology analyzer system with the availability of new parameters for reticulocyte counts. When the CBC results came back from the laboratory, the patient had already gone home, and no serum had been drawn to perform a ferritin level. Rather than calling the patient back to have another sample drawn, the Ret-He could be done from the same blood sample already in the lab. Ret-He is a faster, easier and less expensive test than additional iron studies and bone marrow iron stains. Ret-He can easily be used at a very low cost to get that first piece of information to decide whether or not iron deficiency is a concern. A high or normal Ret-He would have ruled out an iron deficiency with a fairly high confidence level. In this case, the low Ret-He could be used to guide further workups. A subsequent blood drawn revealed a low ferritin and iron deficiency was confirmed. The patient was advised to take an iron supplement along with ongoing treatment for the bleeding.

This case is just one example of the clinical utility of the Ret-He. Using the Ret-He, physicians can determine iron deficiency before iron deficiency IDA develops. A low Ret-He can alert a physician to iron deficiency without the presence of anemia, microcytosis or hypochromia. Ret-He can also be used to monitor and show early response to iron therapy before any other parameters change. A case example is that of a 5 month old who was brought to the emergency room with a Hgb of 7 g/dl and a Ret-He of 11.9 pg. In pediatric patients, a Ret-He less than 27.5 is an indicator of IDA. In this child, treatment with oral iron showed that the Ret-He had risen to 24.6 pg seven days after the onset of iron therapy, while the CBC remained virtually the same. This provided a very early indication that the iron therapy was effective.1 The Ret-He can also been used to minimize transfusions. The AABB Choosing Wisely Campaign lists 5 things that physicians and patients should questions before transfusion. One of the guidelines states “Don’t transfuse red blood cells for iron deficiency without hemodynamic instability.“2 Historically, physicians have used a ‘wait and see’ approach and watched Hgb levels drop before they start looking at iron. Using a Ret-He, iron deficiency could be determined, for example, in a patient with a Hgb of 11 g/dl. Oral or intravenous iron could be started before the Hgb drops below 7 g/dl and transfusion becomes necessary. The AABB Choosing Wisely Campaign emphasizes this by stating that patients with chronic iron deficiency or pre-operative patients with iron deficiency should be given iron therapy before transfusion is considered.2 Ret-He can give the earliest indication of iron deficiency and can be used to monitor the response to iron therapy. Another clinical utility of Ret-He has been to help diagnose or rule out iron deficiency in oncology patients. Additionally, Ret-He has been included in guidelines for anemia management in end stage renal disease patients on dialysis and who get erythropoietin.

The Ret-He parameter has proved clinically useful in early determination of functional iron deficiency. Traditionally ordered chemistry iron studies are indirect measures that have certain inherent inaccuracies due to the presence of inflammation and infection, or in patients on iron therapy. Ret-He is a direct and very effective screening tool and physicians can use Ret-He with other RBC indicies to improve anemia diagnosis and management in many patient populations. Ret-He can be used as a screening measure, and used to reflex for iron studies. Therefore, laboratories who have instruments that can report Ret-He and CHr should develop an education program to help clinicians effectively use Ret-He. Together physicians and laboratorians can develop their own guidelines for reflex testing and improvement for patient care.

References

  1. Case Studies Demonstrating the Clinical Application of the Advanced Clinical Parameters (1/20/2016) Chantale Pambrun, MD, FRCPC, Head of Division of Hematopathology and Assistant Professor of Pathology and Laboratory Medicine, IWK Children’s & Women’s Health Centre and Dalhousie University
  2. https://www.aabb.org/pbm/Documents/Choosing-Wisely-Five-Things-Physicians-and-Patients-Should-Question.PDF
  3. Advanced parameters offer faster, surer guidance to cancer care. Anne Paxton. CAP Today. Sept 2017
  4. The Value-driven Laboratory. Reticulocyte Hemoglobin Content (Ret-He): A Parameter Well-Established Clinical Value. Sysmex America White Paper.
  5. Sysmex Clinical Support Team. Utility of RET-He, August 10. 2015
  6. Brugnara C, Schiller B, Moran J. Reticulocyte hemoglobin equivalent (Ret-He) and assessment of iron-deficient states. Clinical Laboratory Hematology 2006;28:303 – 308.

 

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 67 Year Old Female with a Sore Throat

History 

A 67 year old female presents with a two-month history of sore throat. She endorses dysphagia and left-sided otalgia but denies voice changes, shortness of breath, hemoptysis, weight loss, fever or night sweats. She has smoked 1 pack/day for 41 years and occasionally drinks alcohol. Her past medical history is notable for systemic lupus erythematosus for which she takes Plaquenil.

Physical examination slightly elevated systolic blood pressure. She is afebrile. Pertinent neck exam findings include mild tonsillar asymmetry (left slightly larger than right), and a firm mass at left base of tongue, and a 3 cm lymph node in the neck (left level III). A biopsy sample was taken from the tongue mass. 

Biopsy

EBV-1

H&E stained sections reveal sheets of large lymphocytes. The lymphoid cells are medium to large in size with irregular nuclear contours and prominent nuclei. Areas of necrosis are prominent. No specific areas of epithelial ulceration are noted. Immunophenotypic characterization of the larger cells reveals positivity for CD20, CD30, CD79a, PAX5, MUM1, Epstein Barr virus encoded RNA (EBER) and a variable Ki-67 proliferation index, which is up to 60-70% in the larger cells, but around 20-30% overall. Only rare cells are positive for BCL-2 and BCL-6. The lymphoma cells are negative for keratin AE1/AE3, CD10, CD4, CD8, CD21, CD23, CD7, CD5, Cyclin D1, CD68, CD56, and CD43. The background T cells express CD5 and CD7 and are a mixture of CD4 and CD8 with CD4 predominance.

We considered the diagnosis of EBV-positive mucocutaneous ulcer (a more indolent entity); however, the lack of history of an ulcer/ulceration and the presence of a mass-lesion (with additional adenopathy) does not support this diagnosis.

The findings are most consistent with EBV-positive DLBCL, NOS (WHO 2017), previously known as EBV positive DLBCL of the elderly (WHO 2008). 

Discussion 

Epstein Barr Virus, a member of the Herpesviridae family is mostly known for causing Infectious Mononucleosis. However, the ubiquitous virus which is present in about 90% of adults but often asymptomatic1, has a predilection for epithelial cells including B-cells.2 Incorporation of the viral genome and viral takeover of the cells proliferative machinery underlies the pathogenesis of any EBV-related disease/malignancy. It has been associated with a gastric carcinoma, fulminant hepatitis, undifferentiated nasopharyngeal carcinoma, and B cell, T cell and NK cell lymphomas3, including EBV+ diffuse large B-cell lymphoma, not otherwise specified (DLBCL-NOS).

EBV-positive diffuse large B-cell lymphoma, not otherwise specified (EBV+ DLBCL-NOS) was formerly known as EBV-positive diffuse large B-cell lymphoma (DLBCL) of the elderly. The WHO classification substituted “not otherwise specified” in place of “for the elderly” to reflect two things: 1) EBV is associated with other specific neoplastic Large B-Cell diseases such as lymphomatoid granulomatosis, and 2) EBV+DLBCL can affect younger individuals as well as the elderly. 2

EBV+DLBCL-NOS patients may occur in nodal or extranodal sites, with up to 40% presenting with extranodal sites at least in the early stages. Patients may be asymptomatic with or without B symptoms but usually, patients present with rapidly enlarging tumors at single or multinodal sites, as well as at extranodal sites. 4

The patient’s presentation with sore throat and the finding of neck mass with EBV-positive large B-cells associated with ulcer-like necrosis raises a differential diagnosis that ranges from reactive to malignant. Table 1 shows a comparison between three differential diagnoses: EBV+DLBCL-NOS; EBV-positive mucocutaneous ulcer; and infectious mononucleosis.

EVB-t1
Table 1. Comparison of 3 EBV-positive differentials in the head and neck

Unfortunately, there is currently no uniformly agreed standard of treatment for EBV+DLBCL which has a worse prognosis than EBV negative DLBCL.2 The standard treatment for DLBCL (rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone- R-CHOP) is used but it responds poorly to treatment, with a median survival of 2 years.

Therefore, early detection by clinical suspicion and testing all DLBCL patients for EBV is very important.2 

 References

  1. Tsuchiya S. Diagnosis of Epstein–Barr virus-associated diseases. Critical Reviews in Oncology and Hematology. 2002;44(3):227-238. https://www.sciencedirect.com/science/article/pii/S1040842802001142. doi: 10.1016/S1040-8428(02)00114-2.
  2. Murthy SL, Hitchcock MA, Endicott-Yazdani T, Watson JT, Krause JR. Epstein-barr virus–positive diffuse large B-cell lymphoma. Proceedings (Baylor University.Medical Center). 2017;30(4):443-444. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5595389/.
  3. Okano, Motohiko, MD, PhD|Gross, Thomas G., MD, PhD. Acute or chronic life-threatening diseases associated with epstein-barr virus infection. American Journal of the Medical Sciences, The. 2012;343(6):483-489. https://www.clinicalkey.es/playcontent/1-s2.0-S0002962915309435. doi: 10.1097/MAJ.0b013e318236e02d.
  4. Swerdlow S, Campo E, Harris NL, Jaffe ES, Pileri SA, Stein H, Thiele J, Arber D, Hasserjian R, Le Beau M. WHO classification of tumours of haematopoietic and lymphoid tissues. 2017.
  5. Dunmire SK, Hogquist KA, Balfour HH. Infectious Mononucleosis. Current topics in microbiology and immunology. 2015;390:211-240. doi:10.1007/978-3-319-22822-8_9.

 

Adesola picture-small

-Adesola Akinyemi, M.D., MPH, recently earned his MPH-Health Policy and Management from New York Medical College. He plans on pursuing residency training in pathology. His interests include cytopathology, neuropathology, and health outcomes improvement through systems thinking and design.

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: Two Cases with Surprising Hematopoetic Elements

Case 1 History

Sixty-one year old man with new diagnosis of Bud-Chiari syndrome and extensive peripheral, splenic and hepatic venous thrombosis with increasing fatigue, abdominal discomfort and abnormal liver function tests. A liver biopsy was performed and a hypercoagulability work-up, including JAK2 mutation analysis was initiated.

2-cases-liver2x
Liver core biopsy 2X
2-cases-liver10x
Liver core biopsy 10X

Diagnosis

The liver biopsy showed extensive hemorrhage, hepatocellular necrosis and collapse with mild portal and lobular mixed inflammation. Occasional megakaryocytes and nucleated red blood cell precursors were noted. The case was sent to hematopathology for further review.

2-cases-liver40x
Liver core biopsy 40X
2-cases-liver40x2
Liver core biopsy 40X
2-cases-jak2analysis
JAK2 Mutations Analysis

Hematopathology Diagnosis

Sections show liver parenchyma with changes of the patient’s known history of venous outflow obstruction, as well as extramedullary hematopoiesis, including scattered megakaryocytes (arrows) and erythroid precursors (circle). In the setting of a positive JAK2 V617F mutation, this constellation of findings is consistent with a myeloproliferative neoplasm.

Case 2 History

Fifty-nine year old man with a history of hypertension and alcohol abuse with posterior mediastinal lymphadenopathy. Recent bone marrow biopsy showed mildly hypercellular bone marrow with megakaryocytic and myeloid hyperplasia, and increased stromal reticulin with concern for primary myelofibrosis. A lymph node biopsy was performed.

2-cases-lymph10x.jpg
Lymph node biopsy 10X
2-cases-lymph40x
Lymph node biopsy 40X
2-cases-myelo.jpg
Myeloperoxidase
2-cases-CD71
CD71
2-cases-CD61
CD61
2-cases-CD34
CD34
2-cases-CD3
CD3
2-cases-CD20
CD20

Diagnosis

The lymph node biopsy shows fragments composed of adipocytes and maturing trilineage hematopoiesis. Multiple small to medium sized lymphoid aggregates are also seen, composed of small and mature appearing lymphocytes. The lymphocytes are a mixture of CD3 positive T cells and CD20 positive B cells with focal B cell predominance. Myeloperoxidase highlights myeloid precursors, which comprise 70-80% of the cellularity. CD71 highlights erythroid precursors, which comprise 20-30% of the cellularity. CD61 highlights megakaryocytes. CD34 highlights vessels and only rare CD34-positive cells are seen. Taken together, the findings are consistent with extramedullary hematopoiesis.

Discussion

Extramedullary hematopoiesis (EMH) is defined as hematopoiesis that occurs outside of the bone marrow. It can occur in both normal and pathologic states and has been seen in several hematologic disorders including chronic myeloproliferative neoplasms. Myeloproliferative neoplasms (MPN) are a group of clonal hematopoetic stem cell disorders that include polycythemia vera (PV), essential thrombocythemia (ET) and primary myelofibrosis (PMF).1 JAK2V617F mutation is the most frequent mutation associated with MPNs, found in roughly 96% of patients with PV and 65% of patients with ET and PMF. This mutation leads to constitutive activation of the JAK/STAT pathway and is a driver of myeloproliferation.2  The patient in case 1 was found to have a JAK2 mutation during the work-up for hypercoagulability. This suggests that he may have an underlying MPN, however JAK2 mutations have been found in patients with venous thrombosis, but without overt evidence of MPNs.3 The patient in case 2 had a bone marrow biopsy with features concerning for primary myelofibrosis. In PMF, there is generally a proliferation of myeloid cells in addition to marrow fibrosis. Increasing fibrosis can eventually result in pancytopenia as the fibrosis takes over the marrow space in addition to altering the bone marrow environment so that it is unable to support normal hematopoiesis. Ultimately, this can lead to extramedullary hematopoesis. EMH most commonly occurs in the spleen and liver, but has been described in many other sites including the mediastinum and lymph nodes. In addition to being a driver of proliferation, it is thought that JAK2 mutations make hematopoetic stem and progenitor cells more sensitive to growth factors and can cause the cells to mobilize to the liver and spleen.4  Patients with EMH can have symptoms related to the site of involvement. Depending on the extent of involvement and location, EMH may require treatment with low dose radiation. While EMH is a rare finding, it should prompt an investigation for an underlying MPN.

References

  1. Imai K, Aoi T, Kitai H,et al. A case of perirenal extramedullary hematopoiesis in a patient with primary myelofibrosis. CEN Case Reports. 2017;6(2):194-199. doi:10.1007/s13730-017-0274-1.
  2. Kim CH. Homeostatic and pathogenic extramedullary hematopoiesis. Journal of blood medicine. 2010;1:13-19.3https://www.ncbi.nlm.nih.gov/pubmed/17263783.
  3. De Stefano, V, Fiorini, A, Rossi, E, et al. Incidence of JAK2 V617F mutation among patients with splanchnic or cerebral venous thrombosis and without overt chronic myeloproliferative disorders. Journal of Thrombosis and Haemostasis. 2007;5(4):708-14. https://www.ncbi.nlm.nih.gov/pubmed/17263783.
  4. Passamonti F, Maffioli M, Caramazza D, et al. Myeloproliferative neoplasms: From JAK2 mutations discovery to JAK2 inhibitor therapies. Oncotarget. 2011;2(6):485-490.

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.

Hematology Case Study: A 51 Year Old Woman with Fever and Chills

A 51 year old patient presented to the emergency room with abdominal pain and fever. Fever was associated with diaphoresis, chills and headaches. Patient was in Tanzania for 3 months. She was admitted to the hospital while she was there for some unknown infection, details of which are not available.

CBC done revealed normocytic normochromic anemia with a hemoglobin of 9.2 g/dl and thrombocytopenia. Platelet count was 100 K/uL. On review of peripheral blood revealed presence of malarial parasite (ring forms).

mal1.PNG

mal2

Malaria is an infectious disease caused by Plasmodium parasites. These parasites are primarily spread by the bite of infected female Anopheles mosquitos. There are four main types of Plasmodium (P) species that infect humans:

  • Plasmodium vivax and Plasmodium ovale, which cause a relapsing form of the disease, and
  • Plasmodium malariae and Plasmodium falciparum, which do not cause relapses.

mal3

Malaria must be recognized promptly in order to treat the patient in time.

Microscopy (morphologic analysis) continues to be the “gold standard” for malaria diagnosis. Parasites may be visualized on both thick and thin blood smears stained with Giemsa, Wright, or Wright-Giemsa stains. Giemsa is the preferred stain, as it allows for detection of certain morphologic features (e.g. Schüffner’s dots, Maurer’s clefts, etc.) that may not be seen with the other two. Ideally, the thick smears are used to detect the presence of parasites while the thin smears are used for species-level identification. Quantification may be done on both thick and thin smears.

Various antigen kits are available to detect antigens derived from malarial parasites. These rapid diagnostic tests (RDT) offer a useful alternative to microscopy in situations where reliable microscopic diagnosis is not available.

Vajpayee,Neerja2014_small

-Neerja Vajpayee, MD, is the director of Clinical Pathology at Oneida Health Center in Oneida, New York and is actively involved in signing out surgical pathology and cytology cases in a community setting. Previously, she was on the faculty at SUNY Upstate for several years ( 2002-2016) where she was involved in diagnostic work and medical student/resident teaching.

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.

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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.

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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
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Frozen Section H&E, 2x 
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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.