Gram Stain Examination – Beyond Infectious Organisms

Case History

A 72 year old female with past medical history of stage IV ovarian adenocarcinoma treated with chemotherapy and interval debulking surgery, presented to emergency room with a one week history of confusion and worsening balance.

CT scan of the head showed new communicating hydrocephalus.  A magnetic resonance imaging couldn’t be performed initially because of patient’s uncontrolled agitation.  Lumbar puncture (LP) was performed.  Following this procedure the patient’s mental status showed some improvement and therefore neurosurgery team decided to insert ventriculoperitoneal (VP) shunt to treat her hydrocephalus and prevent recurrence of seizures.

It was Friday afternoon when a microbiology technologist brought the patient’s cerebrospinal fluid (CSF) gram stain to be reviewed.  It was confirmed that no inflammatory cells and organisms were present.  However, cells in the background looked very atypical (Image 1a, b).

CSF1
Image 1:  Gram stain of CSF showing atypical epithelial cells at (a) 40x and (b) 100x with oil.
CSF2
Image 1b.

Discussion

The gram stain is used to provide preliminary information about the microorganism present in the specimen.  Gram stain differentiates bacteria into two fundamental varieties of cells.  Bacteria that retain the initial crystal violet stain (purple) are said to be “Gram-positive,” whereas those that are decolorized and stain red with carbol fuchsin (or safranin) are said to be “Gram-negative” (1).  An adequate examination of a gram-stained smear includes observing numerous representative fields and the fields containing neutrophils yield the most information (2).  Gram stain provides information about number of bacteria present, gram reaction and shape of the bacteria.  In background we can also see epithelial cells and inflammatory cells.  However, it’s a good practice to also appreciate and investigate any odd looking findings.

To investigate further, we visited the hematology laboratory to view their CSF slide to determine if these cells were a processing artifact.  After it was confirmed that hematopathology saw the same atypical cells, a cytopathologist was requested to review the gram stain since patient’s CSF cytology specimen was to be processed after the weekend.  Cytopathologist favored our suspicion and decided to process the cytology specimen late in the day on Friday and it was confirmed that those atypical cells were consistent with the metastatic adenocarcinoma.

Neurosurgery team was immediately contacted to reconsider insertion of the VP shunt as the shunt would drain fluid from the CSF into the peritoneal cavity and thus there was concern for transferring of malignant cells from central nervous system into abdomen/pelvis. However, after consulting oncology team it was later decided to proceed with the procedure since patient’s primary tumor originated in abdomen/pelvis and current intraabdominal tumor burden was not significant as compared to the symptoms driven by CNS involvement. Proceeding with this procedure was considered to be palliative and the best course of action to improve the patient’s quality of life.

References

  1. Beveridge TJ. Use of the gram stain in microbiology. Biotech Histochem.2001 May;76(3):111-8.
  2. Barenfanger J, Drake C. Interpretation of gram stains for the nonmicrobiologist. 2001 July;32(7):368–375.

KM

-Kiran Manjee, MD, is a 1st year anatomic and clinical pathology resident at University of Chicago (NorthShore).

-Erin McElvania, PhD, D(ABMM), is the Director of Clinical Microbiology NorthShore University Health System in Evanston, Illinois. Follow Dr. McElvania on twitter @E-McElvania. 

 

Hematopathology Case Study: A 56 Year Old Man with Sinus Congestion and Axillary Adenopathy

Case History

A 56 year old male presented to his PCP complaining of sinus congestion, rhinorrhea, night sweats, decreased appetite and fevers of up to 101-102 every evening. Hematologic evaluation revealed a neutropenia and a lymphopenia. An infectious disease work up was negative. His LDH was elevated. Physical examination reveals an enlarged left axillary lymph node. An excisional biopsy was performed.

Biopsy Findings

Figure 1.jpg

Figure 2.jpg

H&E stained sections demonstrate an enlarged node with effaced architecture and scattered residual follicles with small, mature cells. There is a proliferation of intermediate to large, to very large, atypical and highly pleomorphic cells many of which demonstrate bizarre forms, irregular nuclear morphology and acidophilic nucleoli. The lymphoma cells are noted to focally traverse through adipose tissue. Occasional hallmark cells are appreciated.

To further characterize the infiltrate, immunohistochemical stains were performed and interpreted with appropriate controls. The lymphoma cells were diffusely positive for CD45 (LCA), CD43, and CD30 (membranous and Golgi) with a Ki-67 of 80-90%. These cells were negative for CD20, PAX-5, CD3, CD4, CD8 (mostly), CD5, D10, BCl-2, BCl-6 and ALK1.

The morphologic features and immunophenotype of the cells was diagnostic of anaplastic large cell lymphoma, ALK negative.

Discussion

Anaplastic Large Cell Lymphoma (ALCL), ALK-negative (ALK-) is defined as a CD30+ T-cell neoplasm that morphologically resembles ALK-positive ALCL, but lacks ALK protein expression. It most commonly affects adults (aged 40-65 years), and has a slight male preponderance with a male-to-female ratio of 1.5:1. T. Most patients present with advanced disease (stage III-IV), lymphadenopathy and B symptoms. The most common differential diagnosis is ALK-positive ALCL.

The molecular deciphering of ALCL began in the 1990s with the discovery of a recurrent t(2;5) (p23;q35) translocation fusing the ALK gene and the nucleophosmin gene generating a NPM-ALK fusion protein, as well as other ALK translocations resulting in a high ALK kinase activity. This triggers the major oncogenic pathway in ALK-positive ALCL. Pharmacologic therapy has been developed to target ALK, and has shown efficacy. Thus, compared with ALK-negative cases, ALK-positive occurs in younger patients and has a better prognosis. ALK-negative ALCL also tends to involve both lymph nodes and extranodal tissues, although extranodal sites are less commonly involved than in ALK+ ALCL.

The other differential diagnoses of ALK- ALCL includes, primary cutaneous ALCL (C-ALCL), other subtypes of CD30+ T-cell or B-cell lymphoma with anaplastic features and classic Hodgkin Lymphoma. If a single lymph node or cutaneous cases are suggestive of ALK- ALCL, C-ALCL needs to be considered. Any cases that involve the gastrointestinal tract need to be distinguished from CD30+ enteropathy-associated and other intestinal T-cell lymphomas.

Molecular analysis of ALK- ALCL shows characteristic strong expression of CD30, in equal intensity in all the cells. Loss of T-cell markers is frequently seen, however, more than half of all cases express one or more T-cell markers. CD2 and CD3 are more commonly expressed than CD5, and CD43 is almost always expressed. CD4+ is frequently positive, while CD8+ is rare. Many cases also express cytotoxic markers TIA1, granzyme B, and/or perforin.

The genetic profile in ALK-negative ALCL has been found to be pretty heterogenous. Most notably, activating mutations of JAK1 and/or STAT3 have been shown to lead to activation of the JAK/STAT3 pathway. Chromosomal rearrangements of DUSP22 (i.e. chromosomal rearrangements in or near the DUSP22-IRF4 locus on 6p25.3) occur in 30% of the cases, and rearrangements of TP63 occur in about 8% of cases. Neither of the rearrangements have been reported in ALK+ ALCL.

From a prognostic standpoint, studies have shown that the rearrangements have effects on the survival rate. TP63-rearranged cases were shown to have an unfavorable prognosis worse than ALK- ALCL with neither rearrangement, while DUSP22-rearranged cases were shown to have favorable outcomes similar to ALK-positive ALCLs.

References

  1. Gaulard P, de Leval L. ALK-negative anaplastic large-cell lymphoma. 2016 Jan 14;127(2):175-7.
  1. Edgardo R. Parrilla Castellar et al., ALK-negative anaplastic large cell lymphoma is a genetically heterogeneous disease with widely disparate clinical outcomes Blood. 2014 Aug 28; 124(9): 1473–1480.

 

Bradon Zelman

-Brandon Zelman is 4th year medical student at the Philadelphia College of Osteopathic Medicine and an aspiring pathologist. You can follow Brandon on Twitter @ZelmanBrandon.

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 Newborn Infant with a High White Blood Cell Count

Case History

The patient is a 1 day old baby boy born at 39 weeks to a 44 year old woman. On physical examination, the baby had a mildly flattened occiput with thickened nuchal skin, downward slant of palpebral fissures with epichanthal folds and slightly low set ears. On imaging, he had a ventricular septal defect. A CBC was performed which revealed a white count of 34.2 K/uL with a differential that included 37 blasts.

Peripheral Blood Smear 

TAM1.jpg

TAM2

Cytogenetics

TAM3

TAM4.JPG

Diagnosis

The peripheral blood showed an increased white count, many nucleated red blood cells as well as a population of blasts. The cytogenetic analysis confirmed the suspicion of trisomy 21. Flow cytometry showed that the population of blasts expressed myeloid as well as erythroid and megakaryocytic lineage specific antigens. The patient was found to have a GATA1 mutation. Approximately one month after birth, the patient’s white count normalized to 7.2 K/uL with only 4 circulating blasts counted.

Discussion

In a patient with trisomy 21, this presentation is consistent with a diagnosis of transient abnormal myelopoiesis associated with Down syndrome (TAM). TAM occurs in 10% of newborns with Down syndrome. Patients typically present with cytopenias, leukocytosis, an increase in blasts and hepatosplenomegaly. Less commonly, patients can have respiratory distress, bleeding, skin rash or jaundice. The blasts are morphologically and immunophenotypically similar to those seen in acute myeloid leukemia. They often have basophilic cytoplasm, coarse basophilic granules and cytoplasmic blebbing, which suggests a megakaryocytic lineage. The immunophenotype generally includes expression of myeloid markers such as CD117, CD13 and CD33 plus erythroid and megakaryocytic markers like CD41, CD42b and CD61.1

In addition to trisomy 21, mutations in GATA1 are almost always seen in the blast cells of patients with TAM. GATA1 is a hematopoetic transcription factor. Bhatnagar, et al. (see diagram below) describe a three step model to explain the evolution of TAM. The initial event is abnormal hematopoesis in the fetal liver caused by trisomy 21. This causes an increase in megakaryocyte-erythroid progenitors in the hematopoetic stem cell compartment.  The second step is the acquisition of an N-terminal truncating GATA1 mutation before birth. GATA1 is a regulator of normal megakaryocyte and erythroid differentiation. The truncated mutation causes marked expansion of megakaryoblastic progenitors.

TAM5TAM has a high rate of spontaneous remission and typically resolves spontaneously in 90% of patients over several weeks to 6 months. This coincides with extinction of the GATA1 clone. However, in around 10% of these patients, myeloid leukemia of Down syndrome (ML-DS) develops within 5 years of the initial presentation. Additional mutations in cohesion component genes and epigenetic regulators occur in these patients that result in clonal expansion and non-transient leukemia. 2 Children who develop ML-DS generally have a good response to chemotherapy and a have a better prognosis than children without Down syndrome who develop AML.

References

  1. Swerdlow SH, Campo E, Harris NL, et al. WHO Classification of Tumours of Haematopoetic and Lymphoid Tissues (Revised 4th edition). IARC: Lyon 2017.
  2. Bhatnagar, Neha et al. “Transient Abnormal Myelopoiesis and AML in Down Syndrome: An Update.” Current Hematologic Malignancy Reports5 (2016): 333–341. PMC. Web. 21 Oct. 2018.

 

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.

 

 

Hematopathology Case Study: A 63 Year Old Man with Fatigue

Case history

A 63 year old male presented with extreme fatigue and weakness of unknown duration. Physical examination revealed scattered petechiae and mildly decreased muscle strength. His past medical history included a one year history of cough that had recently improved. Laboratory investigation demonstrated severe anemia and thrombocytopenia with a mild leukopenia.

Review of the peripheral blood smear showed smudge cells, circulating neutrophils with Döhle bodies and toxic granulation. CT scan of the chest showed upper/anterior mediastinal lymphadenopathy without hilar lymphadenopathy.

A biopsy of the bone marrow was performed.

Microscopic Findings

hod1.jpg

 

hod 2.jpg

The bone core biopsy revealed a hypercellular marrow for the patient’s age with a pronounced lymphohistiocytic infiltrate involving 30-40% of the biopsied marrow space. Interspersed along the infiltrate were large, atypical lymphoid cells with pleomorphic nuclei and prominent nucleoli. The marrow aspirate smear reveals progressive trilineage hematopoiesis with scattered hemophagocytic histiocytes.

Immunophenotype

hod3.jpg

The large atypical lymphoid cells were positive for CD30 and EBER, while being dimly positive for PAX5 and negative for CD20.

Diagnosis

The detection of mononuclear Hodgkin cells staining for CD30 along with a characteristic reactive infiltrate, together with dim PAX-5 staining, positive EBER, and negative CD20 is sufficient to diagnose involvement of a secondary site by Hodgkin lymphoma. The lymphoma was associated with a secondary hemophagocytic lymphohistiocytosis.

Discussion

Hodgkin lymphoma (HL) is a B-cell derived monoclonal lymphoid neoplasm. HL has a bimodal age distribution, with teenagers or patients in their early 20s and patients older than 55 years having the highest incidence. Although the typical presentation is with peripheral lymph node involvement, extranodal sites may be involved by either direct invasion or hematogenous dissemination. These sites include the spleen, liver, lung and bone marrow. About one third of patients have constitutional symptoms such as high fevers, night sweats, and weight loss.

Two broader forms of Hodgkin lymphoma exist: Classic Hodgkin lymphoma (CHL) and the less common nodular lymphocyte-predominant Hodgkin lymphoma (NLPHL). NLPHL tends to preserve the entire B-cell transcriptional phenotype, while the neoplastic cells in CHL fail to do so.

CHL is composed of mononuclear Hodgkin cells and multinucleated Reed-Sternberg cells surrounded by an infiltrate of non-neoplastic reactive cells that might encompass small lymphocytes, plasma cells, eosinophils, neutrophils, and histiocytes. Fibrosis may also be present in the form of bands or may be more diffusely spread. The four histological subtypes: nodular sclerosis CHL, lymphocyte-rich CHL, mixed cellularity CHL, and lymphocyte-depleted CHL are based on the composition and characteristic of the reactive infiltrate, and the cytological features of the neoplastic cells.

The classic Reed-Sternberg cell is binucleated, with prominent eosinophilic nucleoli, often referred to as having an “owl’s eye” appearance. However many neoplastic cells are not of the typical Reed-Sternberg variant, and can be mononuclear, termed Hodgkin cells, or cells with more condensed cytoplasm and pyknotic reddish nuclei known as mummified cells.

Hodgkin/Reed-Sternberg cells (HRS) in Classic Hodgkin Lymphoma fail to preserve their B-cell traits, and this is reflected by their immunophenotype. The majority of cases are negative for CD45, and although CD20 may be expressed, it is usually present only on a minority of the neoplastic cells and stain with varied intensity. The HRS cells stain with PAX5 with a lower intensity than the surrounding reactive cells, making them easily detectable. The HRS cell stains positive for CD30 and CD15 in nearly all cases. Both of them stain the membrane with accentuation around the Golgi apparatus. EBV associated Hodgkin Lymphoma will stain positive with EBER, detecting EBV-encoding small RNA.

Bone marrow involvement is rare, ~5-10% of cases, and suggest vascular dissemination of the disease. Bone marrow trephine biopsies are commonly performed in the staging of patients with newly diagnosed CHL which guides the further treatment and gives us information about prognosis. Involvement of the bone marrow represents stage IV disease (advanced stage) in the Ann Arbor staging classification and patients with advanced stage disease typically receive a more prolonged course of chemotherapy. The 5-year survival rate of stage IV Hodgkin lymphoma is ~65%,  a much worse prognosis when compared with stage I, stage II, and stage III with ~90%, ~90%, and ~80% 5-year survival rates respectively.

References

  1. Stein H, Pileri SA, Weiss LM, et al. Hodgkin Lymphomas. In Swerdlow SH, Campo E, Harris NL, Jaffe ES, Pileri SA, Stein H, Thiele J, editors: WHO classification of tumours of haematopoietic and lymphoid tissues, revised ed 4, Lyon, France, 2017, IARC Press, pp 423-464
  2. Ansell SM. Hodgkin Lymphoma: Diagnosis and Treatment. Mayo Clin Proc. 2015 Nov;90(11):1574-83.
  3. Howell SJ, Grey M, Chang J, Morgenstern GR, Cowan RA, Deakin DP, Radford JA. The value of bone marrow examination in the staging of Hodgkin’s lymphoma: a review of 955 cases seen in a regional cancer centre. Br J Haematol. 2002 Nov;119(2):408-11.
  4. Clarke C, O’Malley C, Glaser S. Hodgkin lymphoma. In: Ries LAG, Young JL, Keel GE, Eisner MP, Lin YD, Horner M-J, eds. SEER Survival Monograph: Cancer Survival Among Adults: U.S. SEER Program, 1988-2001, Patient and Tumor Characteristics. National Cancer Institute, SEER Program, NIH Pub. No. 07-6215, Bethesda, MD, 2007.

 

Hans.jpg

-Hans Magne is a 6th- year medical student at Poznan University of Medical Sciences. Follow Hans on Twitter @HHamnvag

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: The Case of Lymphocytosis

Case History

53 year old female was found to have leukocytosis upon a wellness examination. A CBC was performed and found a WBC of 34.0, HgB 13.2, and Plt of 263,000. The WBC differential consisted of 80% Lymphocytes and 12% neutrophils. The patient states that she is feeling well, no fever, chills, or night sweats. She denies any adenopathy. Flow Cytometry was recommended as well as morphologic review along with Cytogenetics and FISH (fluorescence in-situ hybridization).

Lab Identification

CBC

WBC               33.68  [103/uL] NEUT             4.17 [103/uL]      12.3 [%]
RBC                4.54  [106/uL] LYMPH          26.91 [103/uL]    79.9 [%]
HGB               13.2   [g/dL] MONO           2.04 [103/uL]      6.1  [%]
HCT               40.0   [%] EO                  0.39 [103/uL]      1.2  [%]
MCV               88.1   [fL] BASO             0.11 [103/uL]      0.2  [%]
MCH               29.1   [pg] IG                    0.06 [103/uL]      0.2  [%]
MCHC            33.0   [g/dL] NRBC             0.00 [103/uL]      0.0  [%]
RDW-CV         14.3   [%]
PLT                263   [103/uL]
MPV                9.4   [fL]

Morphologic Review

CLL1.jpg
Image 1. 40x magnification showing lymphocytosis.
CLL2.jpg
Image 2. 60x magnification showing lymphocytosis with occasional smudge cells.

Flow Cytometry: Population of Interest – Abnormal Lymphocytes

cll3.JPG

cll4.JPG

cll5.JPG

Cytogenetics: 46,XX[20] Normal Female Karyotype

It is not unusual to observe a normal karyotype in CLL due to the limited number of abnormal cells and/or low spontaneous proliferative activity of the malignant cells. Fluorescence in situ hybridization studies may identify cytogenetic abnormalities of prognostic significance in interphase nuclei not observed in the metaphase cells analyzed. In cases of CLL molecular profiling may be performed to aid in predicting course of the disease. If clinically indicated these studies may be considered. Standard cytogenetic analysis may not detect subtle submicroscopic rearrangements and may not include metaphases from abnormal cell populations with low mitotic rates or present in low levels.

Metaphases Counted: 20 Metaphases Analyzed: 20
Metaphases Karyotyped: 2 Culture Type: 48EB, 72IL2/DSP30
Banding Technique: GTG Banding Resolution: 400

Fluorescence in-situ Hybridization: Abnormal – 13q14 deletion present

Fluorescence in situ hybridization (FISH) analysis was performed using a specific set of probes for Chronic Lymphocytic Leukemia (CLL). This study revealed a 13q14 deletion. Counts for all other probes were within the normal reference range. This finding represents an ABNORMAL result. Deletion of 13q14 is the most common deletion in CLL being reported in 10-20% of cases by conventional cytogenetics and up to 64% of cases by FISH analysis. When present as a sole abnormality this deletion is associated with a good prognosis and a median survival longer than CLL patients with a normal karyotype.

Del(6q) Not Detected
Del(11q)(ATM) Not Detected
Trisomy 12 Not Detected
Del(13q)/-13 DETECTED
t(11;14) Not Detected
Del(17p)(TP53) Not Detected

Discussion

Chronic Lymphocytic Leukemia is a neoplasm of about 5 cases per 100,000 people with a median age of around 70 years old. The neoplasm is composed of monotypic mature B-cells that typical express CD5. Other immunophenotypic characteristics of the leukemic B-cells include CD19, CD20, CD22, and CD79b with dim surface expression of one of the immunoglobulin light chains, Kappa or Lambda. These cells typically express CD200 which helps differentiate the leukemia from Mantle Cell Lymphoma/Leukemia. Patients found to have a mutated IGHV genes typically have a better prognosis than those with an unmutated genes. Expression of ZAP70, CD38, or CD49d is also associated with an adverse prognosis. Complex karyotypes also trend towards a poor outcome. Adverse predictive factors include rapid lymphocyte proliferations in the blood, typically doubling in < 12 months.

Monoclonal B-cell Lymphocytosis is typically characterized as a monoclonal
BCell count of <5 X109/L in the peripheral blood. Monoclonal B-cell Lymphocytosis with a Chronic Lymphocytic Leukemia-type phenotype is the most common which accounts for about 75% of all cases. It has been reported that virtually all Chronic Lymphocytic Leukemias are preceded by Monoclonal B-cell Lymphocytosis, although not all MBLs progress to CLL.

References

  1. Dohner H, et al. N Engl J Med 2000; 343:1910-6.
  2. Hamblin TJ.Best Practice & Research Clinical Haematology. 2007; 20(3):455 – 68.
  3. Swerdlow SH, Campo E, Harris NL, Jaffe ES, Pileri SA, Stein H. WHO classification of tumours of haematopoietic and lymphoid tissues, fourth edition. Lyon, France: IARC; 2017
  4. Nowakowski GS, et al. Br J Hematol. 2005; 130:36 – 42.
  5. Atlas of Genetics and Cytogenetics in Oncology and Hematology http://atlasgeneticsoncology.org/

 

Troy-Krieger-small

Troy G. Krieger, MS, MLS(ASCP)CMSCYMCMQLSCMCLS(MT) graduated from Montana State University Billings with a BS in Biology, Medical Laboratory Science option. He received a NAACLS Certificate and clinical training from the University of North Dakota in Grand Forks, ND, where he also received his Master’s degree. He is a Medical Laboratory Scientist / Flow Cytometrist at Yellowstone Pathology Institute, Inc in Billings, MT and his interests include Hematology, Immunopathology, and Flow Cytometry.

Hematopathology Case Study: A 43 Year Old Man with History of Latent TB

Case History

43 year old Vietnamese speaking man with a history of treated latent TB who presented with one month of fevers, night sweats, weight loss, and acute left facial swelling with associated pain, nasal congestion and 2 nose bleeds. The patient was found to have a polypoid mass within the left interior nasal cavity.

Biopsy Left Nasal Mass

nkt-he10x
H&E 10x
nkt-he40x
H&E 40x
nkt-cd3
CD3
nkt-cd5
CD5
nkt-cd56
CD56
nkt-perforin
Perforin
nkt-67
KI-67
nkt-eber.jpg
EBER

Diagnosis

The biopsy shows nasal mucosa with a dense submucosal lymphoid infiltrate and large areas of necrosis. The lymphocytes are somewhat pleomorphic, medium to large in size with irregular nuclear contours, vesicular chromatin and inconspicuous nucleoli. There are scattered mitoses and apoptotic cells.

By immunohistochemistry, CD3 highlights the lymphoma cells, which comprise the majority of the lymphoid infiltrate. The lymphoma cells co-express CD56 and CD7 (dim) and are negative for CD2 and CD5. The lymphoma cells also express cytotoxic markers perforin and granzyme (major subset). CD20 highlights only rare small clusters of B-cells. The lymphoma cells are also positive for EBER (Epstein-Barr Virus encoded RNA) in situ hybridization.  The Ki67 (MIB1) proliferation index is 60% with focal areas exhibiting up to 80%.

Taken together, the morphologic and immunophenotypic findings are consistent with an extranodal NK/T cell lymphoma, nasal type.

Discussion

Extranodal NK/T cell lymphoma, nasal type is an aggressive lymphoma that is more prevalent in Asian and South American populations. It occurs most often in adults and is more common in men than women. It is generally located in the upper aerodigstive tract, with the nasal cavity being the prototypical site. Patients tend to present in a manner similar to the patient described in this case, with rhinorrhea, pain, nasal obstruction and epistaxis due to a mass lesion. 1 The term “lethal midline granuloma” was once used to describe this entity because patients can present with locally destructive mid-facial necrotizing lesions. The early non-specific symptoms can pose a diagnostic challenge, and often result in treatment delays, which makes the aggressive disease more lethal. 2

The entity is described as NK/T cell lymphoma because although most cases are of NK-cell origin, some cases are comprised of cytotoxic T-cells. Natural killer (NK) cells are non-T and non-B lymphocytes that are part of the innate immune system. They respond immediately to antigenic challenge and are able to directly kill virally infected cells without the help of antigen presenting cells. They also secrete cytokines to increase the innate immune response. NK cells are classically positive for cytoplasmic CD3 and CD56, as well as cytotoxic molecules granzyme and perforin. Of note, NK-cells lack recombination activating gene enzymes and therefore have no clonal molecular marker for gene rearrangement such as the T-cell receptor or Immunoglobulin heavy chain.  3

Microscopically, the involved sites generally have widespread mucosal destruction. There is an angioentric and angiodestructive growth pattern that results in extensive necrosis. Another important diagnostic consideration is the very strong association with EBV. EBV is present in a clonal episomal form. This means that the infection occurs prior to and likely plays a pathogenic role in the development of NK/T cell lymphomas. 3

Following diagnosis, staging and management of the disease involves quantification of circulating EBV DNA. This can be used as a laboratory marker for disease status and progression or remission of disease. PET/CT is performed for accurate staging and patients are most commonly treated with a combination of radiotherapy and the SMILE regimen, which includes dexamethasone, methotrexate, ifosfamide, L-asparaginase and etoposide. NK/T cell lymphomas are aggressive and patients tend to have a short survival and poor overall response to therapy. 3

A recent study by Kwong, et al. showed the potential use of PD1 (programmed death ligand 1) blockade drug pembrolizumab in the treatment of relapsed or refractory NK/T-cell lymphoma. As mentioned above, the lymphocytes in this entity are invariably infected with EBV. PD1 is known to be up regulated in cells infected with EBV. In the study, seven patients who had failed treatment with the SMILE regimen were treated with pembrolizumab. After a medium follow-up of 5 months, 5 patients remained in complete remission and all patients had objective responses to treatment. 4 This shows promise as a potential new treatment for patients with this uncommon, but deadly disease.

 

References

  1. Swerdlow SH, Campo E, Harris NL, et al. WHO Classification of Tumours of Haematopoetic and Lymphoid Tissues (Revised 4th edition). IARC: Lyon 2017.
  2. Mallya V, Singh A, Pahwa M. Lethal midline granuloma. Indian Dermatology Online Journal. 2013;4(1):37-39. doi:10.4103/2229-5178.105469.
  3. Tse, E, Kwong, Yok-Lam. The diagnosis and management of NK/T-cell lymphomas. Journal of Hematology and Oncology. 2017:10:85. Doi: 10.1186/s13045-017-0452-9.
  4. Yok-Lam, K, Thomas, S.Y. Chan, Daryl Tan, et. al. PD1 blockade with pembrolizumab is highly effective in relapsed or refractory NK/T-cell lymphoma failing L-asparaignase. Blood. 2017:129:2437-2422. Doi: 10/1182/blood-2016-12-758641.

 

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.

Beyond Bands: The Immature Granulocyte Count

In today’s clinical laboratories there are an increasing number of tests available for physicians to order. In a constant effort to provide the best tools for patient care, laboratories typically issue a Laboratory Technical Bulletin when new platforms or new testing is available. These help introduce physicians to or update them on the ever changing array of test availability. In Hematology, Advanced Clinical Parameters are an extension of the traditional CBC and Automated Differential. Used appropriately, these new tests can provide a wealth of knowledge that can be available to physicians in making diagnoses. In my July post, (Beyond the CBC and Reticulocyte Count: Early Detection of Iron Deficiency Anemia), I discussed the Reticulocyte Hemoglobin (RetHe) and it’s uses in early detection and better management of iron deficiency anemia. In this blog I will explore the Immature Granulocyte Count (IG%), another of the Advanced Clinical Parameters.

Historically, the 100 cell manual differential has been used to enumerate percentages of each cell type present and to detect the presence of abnormal cells, including immature granulocytes. An increase in band count >10 %, was, and is still often used with other screening tests as an indicator of infection or sepsis. The first microscopic image analyzer, the Perkin-Elmer Cellscan in 1966, could recognize neutrophils, lymphocytes and monocytes with 90% accuracy, and a 100 cell differential could be completed overnight. With improved, faster computers this instrument was marketed in the mid 1970’s and then purchased by Coulter Electronics in 1977 to become the Coulter Diff 3. These first hematology analyzers to provide an automated differential yielded a 3 part differential, and then more sophisticated instrumentation gave us the 5 part diff. In the beginning, an automated differential only counted 100-200 cells and manual diffs performed by a trained technologist were still considered superior. Today’s hematology analyzers count over 30,000 cells using impedance and flow cytometry to give us a statistically superior automated differential.

An automated 5 part differential will flag a ‘Left Shift’ when bands are seen. Many laboratories then perform a scan or manual diff. However, bands are very subjective and therefore it has been questioned if the band count is truly useful as a clinical indicator of sepsis. If a band count >10% is an indicator of sepsis, what happens when 2 or 3 technologists perform diffs on the same slide and get a band % range between 5% and 19%? 5% would not indicate sepsis, whereas 19% exceeds the 10% cutoff almost twofold. Two experienced technologists can perform a manual diff on the same slide and get band counts in a fairly wide range depending on their training and which definition of bands they use and which 100 cells they see in their count. On the other hand, a 6 part automated differential counts and differentiates over 30,000 neutrophils, lymphocytes, monocytes, eosinophils, basophils and immature granulocytes. The auto diff separates bands and immature granulocytes. Band cells are considered mature and included in the neutrophil count. The Advanced Clinical Parameter, Immature granulocytes (IG%) include metamyelocytes, myelocytes and promyelocytes. In the peripheral blood these immature granulocytes are an indicator of leukopoiesis, cells just coming out of the bone marrow and represent the earliest information possible and an earlier indication of inflammation and infection than a band count. The example below illustrates this concept.
Case Study 1

A patient admitted to the ER with high fever and chills has a CBC and auto diff ordered. His WBC is elevated and percent neutrophils are high. The physician suspects sepsis despite the normal WBC and orders a CRP and blood cultures. CRP results are within normal limits. The immature granulocyte counts are shown below on admission and in 4 subsequent phlebotomies. Antibiotics were started 3 hours after initial triage. The patient was admitted to the hospital and Streptococcus pneumoniae was isolated from the blood cultures at 48 hours.

left shift 1

The IG% and absolute counts are increased in this patient when other markers of infection are not. An IG% of >1% is indicative of a true left shift and >3% may predict positive blood cultures. The ED can also use the IG to determine that the infection is community acquired and not nosocomial if the IG% is high on admission. Note the band counts here that could be high or low depending on who performed the diff. This demonstrates the inherent subjectivity and imprecision of band counts and manual differential counts because they are done on only 100 cells. The immature granulocyte count is automated, not subjective. In checking the slide for correlation, we see the presence of these immature granulocytes. The IG% is thought to be better as a predictor of sepsis than the WBC and band count. For this reason, with the availability of a 6 part diff, labs have encouraged physicians to order and use the automated differential.
Another application of the utility of the IG% is seen in comparing IG% and ITR, immature to total neutrophil ratio. ITR is used by neonatologists to determine infection. The ITR is a calculation based on a 100 cell manual differential. In this formula, the immature neutrophils are bands, myelos, metas and pros. An ITR <0.2% is a negative predictive value for sepsis.
Immature neutrophils/Total neutrophils=bands+myelos+metas+pros/total neutrophils= ITR

Case Study 2

left shift 2

In 2 babies less than 30 days old, we see again here the phenomenon of a wide variety of bands counted by 2 techs. In the ITR, bands are counted as immature cells. Despite the imprecision of the band count, Baby A has >10% bands counted by both techs and the ITR range is >0.2% in both calculations. However, in Baby B, we see that the band count from one differential was 5% and the second tech counted 17% bands. Since the bands are used to calculate the ITR, one calculation gives an ITR of 0.06%, a negative predictor of infection, and the second count gives an ITR of 0.22%, above the 0.2% threshold. At the same time, if physicians are looking at an absolute neutrophil count(ANC), the number of neutrophils available to fight off infection, this number is calculated using bands and neutrophils. In one formula, bands are lumped with immature cells, and in the other, bands are included with mature cells. This can be confusing information. If we were instead to rely on the automated differential, the IG% gives us a more clear and precise measure of sepsis. Using the previously stated criteria for infection, the IG% of Baby A is clearly above 3% and in Baby B is well below 1%.

How do band counts compare to the IG%? Should bands be eliminated as an indicator of infection? Studies have been done that suggest that the WBC and band counts are not as reliable in predicting infection as the IG% and ANC. Suggestions have been made that the left shift should be redefined with IG% rather than bands. Bands may be too subjective to be the best indicator of infection and they lead to an imprecision in the ITR. The IG count can highlight potential acute infection or inflammation at its earliest stages, even when other parameters are still within normal ranges. IG counts (% and #), reported with the automated differential, reduce turnaround time and are valuable for clinicians to use in conjunction with other current indicators for the diagnosis of infection and inflammation.

References

  1. Ansari-Lari, M.Ali et al. Immature Granulocyte Measurement Using the Sysmex XE-2100 Relationship to Infection and Sepsis Am. J Clin Pathol. 2003,Nov;120(5):795-9.
  2. Balamurugan Senthilnayagam, et al. Automated Measurement of Immature Granulocytes: Performance Characteristics and Utility in Routine Clinical Practice. Patholog Res Int. 2012: 483670. Published online 2012 Feb 15. doi: 10.1155/2012/483670
  3. Cavallazzi, R et al. Is the band count useful in the diagnosis of infection? An accuracy study in critically ill patients. J Intensive Care Med. 2010, Nov-Dec;25(6):353-7.
    Dutcher, Thomas F. Automated Leukocyte Differentials: A Review and Prospectus. Laboratory Medicine, Volume 14, Issue 8, 1 August 1983, Pages 483–487, https://doi.org/10.1093/labmed/14.8.483
  4. McDaniel, Holly. Ban the Bands. Sysmex News, 2013. https://www.sysmex.com/crc/ACP/IG_Ban_the_Bands.pdf
  5. Sysmex America White Paper. Getting Beyond the Flags: Quantitative assessment of immature granulocyte (IG) populations may improve the assessment of sepsis and inflammation. http://www.sysmex.com/us

 

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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.