Smudge Cells: Artifacts or Clinically Significant?

In today’s hematology lab, when physicians order a CBC with differential, they typically request a CBC with automated differential. Thus, up to 85% of our CBCs are autovalidated because they are entirely within normal range, with no instrument flags. This leaves the technologist time to spend on those slides that do need a manual review. In reviewing a slide, we evaluate the WBCs, RBCs and platelets, and must pay attention to the counts as well as morphology.

But, what do we do when we have a cell we cannot identify? When we perform a manual differential under the microscope, technologists will joke or tell stories about the legendary “skipocyte”; that cell which, while it does not look malignant or clinically significant, we still can’t decide what it is, so it’s skipped. Perhaps the best way to deal with these cells would be to get consensus from other techs or the Hematology supervisor or to request a pathology review. However, despite the fact that we are taught that there is no such thing as a skipocyte, there are times when a tech will ignore the cell, hoping they don’t see another one. But, what do we do when we see smudge cells? Are they skipocytes? What exactly are they? Do we ignore these? Are they clinically significant? Do we count them as their own category of cells? Or something else?

Firstly, what is a smudge cell? Smudge cells, or basket cells, are remnants of leukocytes. They have no cytoplasm, and sometimes all that can be seen are smashed nuclei. Smudge cells are formed from leukocytes, typically lymphocytes, that are fragile, and are destroyed or smudged in the physical process of making a smear. But, what if the instrument makes the smear? In recent years, more labs are using automated analyzers that prepare and stain blood smears. Even though these have instrument settings based on the physical characteristics of each sample, we still tend to observe these traumatic injuries to leukocytes with automated slide making. Whether we make slides manually or the instrument makes them, these fragile cells appear on the stained slide as ruptured cells called smudge cells.

Image 1. Smudge cells seen on peripheral blood smear.

Smudge cells have also been called Gumprecht shadows, named after German scientists and researcher Ferdinand Adolph Gumprecht, who observed these on slides of patients with chronic lymphocytic leukemia (CLL). Smudge cells in patients with CLL are ruptured B-cells, but they can’t be distinguished morphologically from other disintegrated lymphocytes. We also see leukocytosis and smudge cells in viral conditions and chronic inflammatory diseases. However, the term Gumprecht shadows is reserved only for smudge cells in CLL cases.

Knowing what a smudge cell is, how do we handle them? Do we report the presence only? Do we count them? Or, do we ignore them entirely? Smudge cells are not skipocytes! For many years smudge cells were considered to be simply artifacts of slide making. More recently, studies have been conducted that show that there may be clinical significance to the number of smudge cells seen. While smudge cells are not diagnostic of CLL, it has been shown that, in newly diagnosed CLL, a larger percentage of smudge cells is a better prognostic factor. Patients with >30% smudge cells show longer times before requiring treatment and longer survival rates than patients with fewer smudge cells. These studies focused on vimentin, a protein that is important in lymphocyte cellular rigidity. Patients with low vimentin have more smudge cells and better survival rates.1,2

If we are performing a slide review, we are reviewing these slides because of some sort of instrument flag or rule trigger. There are several theories as to how smudge cells can be handled, and studies have been done to compare these theories.3 Laboratories have SOPs in place to guide technologist review and reporting, yet, I have noticed considerable variation in handling of smudge cells both within our lab and between labs. These pesky artefacts can be puzzling in both traditional (under the microscope) and digitized (CellaVision) microscopy and new technologists or unfamiliar operators can easily be misled. If we perform our manual differentials traditionally, under the microscope, we will no doubt notice the presence of smudge cells. It is important not to pass by these or consider them skipocytes. Some labs count these as their own category of cell and some labs merely report the presence of smudge cells. Other labs do not report smudge cells at all, with the exception being in known cases of CLL. In these CLL differentials, if the WBC count is very high, it may also be recommended to do a 200 cell differential. But, what happens when the manual diff doesn’t match the automated diff? The hematology analyzer will accurately count fragile cells, still intact in the specimen, and include them in the differential. If the cells then disintegrate on smear making, we see smudge cells on the slide. If we do not count these, this can affect the percentage of cell types in the differential, and potentially, in a patient with a low WBC, affect the absolute neutrophil count (ANC). If we are performing the manual differential (diff) in CellaVision, CellaVision identifies smudge cells and puts them in a separate category, but these are not reported as part of the diff. These are a ‘heads up’ to the technologist that further steps need to be taken to report out a differential. The importance of recognizing smudge cells is illustrated in Table 1 below for a patient sample with WBC 3.6 x 103/μL.

Table 1. Numbers of cells counted in three differentials on sample with WBC 3.6 x 103/μL.

The automated differential (auto diff) in this example, with 12% neutrophils counted, has an absolute neutrophil count of 432/ μL, which is considered critical (critical <500/μL). Fragile lymphocytes are intact in the blood sample and are counted by hematology analyzers.

The 200 cell manual differential above merely notes the presence of smudge cells, but no quantifier is given. The ANC here is not critical (774/μL) and the lymph% is only 61, possibly leaving the physician to question how many smudge cells were present, and what the true lymph% may be.

In the CellavVision differential in Table 1, based on 100 WBCs counted, the total percentages of Neuts is 20%, lymphs 61% and monos 19%. If an unexperienced tech did not notice or investigate the 68 smudge cells, the manual differential (manual diff) reported from the CellaVision would be very different from the auto diff, and has an ANC of 720/μL, above the critical range.

If however, the smudge cells in CellaVision were reported as a separate category, our differential would now be based on 168 cells counted. 100 WBCs counted plus the 68 smudge cells counted = total of 168 cells counted. Our neut% is now 11.9 (20/168*100), lymph % 36.3(61/168*100), monos% 11.3 (19/168*100) and smudge cell % 40.5 (68/168*100). This ANC matches that from the auto diff. And, if we further consider that the smudge cells are lymphocytes, this brings the count to 11.9% neuts, 77% lymphs and 11.3% monos. (68 +61 = 129/168*100 = 77% lymphs) which closely matches our automated differential.

Lastly, the ‘something else’, is that we can make an albumin smear on these specimens. It has been a practice in labs to perform a manual differential on an albuminized blood smear when a certain number, defined by SOPs, of smudge cells are seen. If this is your lab procedure, it is important to recognize the presence of smudge cells on the manual differential or CellaVision differential and take the steps to make an albumin smear. Adding a drop of albumin to a few drops of the patient blood can add protein to the specimen and prevent the formation of smudge cells. Table 2 shows the manual diff on the sample in Table 1, performed on the albuminized slide. Note that this eliminates the smudge cells and corrects the diff results to match the original automated differential.

Table 2. Albumin smear results on sample from table 1.

It can be seen from these examples, that the method of counting differentials with smudge cells can alter the results reported to the physician. Any of the differential methods above that count smudge cells give essentially the same results. If smudge cells are not counted, the lymphs will be under reported and the neutrophils will be over represented compared to the auto diff. Excluding smudge cells from the manual differential count or merely reporting their presence without quantification will also yield unreliable results, and then necessitates performing an albumin differential.

If we are to choose between an albumin differential and an automated differential, which studies have shown to be equivalent3, making and staining an additional smear is time consuming and can affect turnaround times. Thus, guidelines have been suggested that the first choice for handling pesky smudge cells is to review the smear and report the automated diff with a morphology comment that smudge cells are present. If automated diffs are not available, smudge cells should be counted as lymphocytes, or in a separate category4, as illustrated in Table 1. Study findings indicate that this method is sufficient for reporting a reliable manual differential on known CLL patients3. By counting smudge cells separately, however, as discussed previously, these numbers can be used in newly diagnosed cases of CLL as a prognostic indicator.

There is still debate on the value of reporting smudge cells on routine CBC smears. In most routine cases, an auto diff without quantitating smudge cells is considered sufficient. Pathologists, however, differ on whether smudge cells should be reported.

The best course of action is always to consistently follow your own lab’s SOPs, to be aware of flags, rules triggered and operator alerts with regard to smears, and to always be on the lookout for smudge cells. They are not skipocytes!

References

  1. Nowakowski GS, Hoyer JD, Shanafelt TD, et al. Percentage of smudge cells on routine blood smear predicts survival in chronic lymphocytic leukemia. J Clin Oncol. 2009;27(11):1844-1849.
  2. Amal Abd El Hamid Mohamed, Nesma Ahmed Safwat. New insights into smudge cell percentage in chronic lymphocytic Leukemia: A novel prognostic indicator of disease burden. The Egyptian Journal of Medical Human Genetics, 19 (2018) 409–415
  3. Gene Gulati, Vandi Ly, Guldeep Uppal, Jerald Gong, Feasibility of Counting Smudge Cells as Lymphocytes in Differential Leukocyte Counts Performed on Blood Smears of Patients With Established or Suspected Chronic Lymphocytic Leukemia/Small Lymphocytic Lymphoma, Laboratory Medicine, Volume 48, Issue 2, May 2017, Pages 137–147, https://doi.org/10.1093/labmed/lmx002
  4. Denis Macdonald, MD, MBA, FRCPC, FCAP; Harold Richardson,et al. Practice Guidelines on the Reporting of Smudge Cells in the White Blood Cell Differential Count. Arch Pathol Lab Med—Vol 127, January 2003
  5. Luci Maria Sant’Ana Dusse; Tamiris Paula Silva, et al. Gumprecht shadows: when to use this terminology? J Bras Patol Med Lab, v. 49, n. 5, p. 320-323, 2013

-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: An 83 Year Old Man with an Elevated PTT

Case History

An 83 year old man with rapidly growing squamous cell carcinoma of the left temple and scalp underwent workup prior to surgery which showed an elevated PTT and a slightly elevated PT. The patient denied a history of abnormal coagulation tests or excessive bleeding or bruising. He also noted that he had previous surgeries including dental procedures without excessive bleeding. In addition, he did not have a history of clot formation.

Lab Values

Differential Diagnosis

At this point, the differential diagnosis for a prolonged PTT included the presence of an inhibitor (specific factor inhibiter vs. non-specific lupus anticoagulant) vs. reduced levels/activity of intrinsic pathway factors that would prolong the PTT, but would not significantly affect clot formation. This would include factors XI and XII. 

Additional Testing

An inhibitor screen/mixing study was performed and was positive. An inhibitor screen is performed by mixing the patient’s plasma with pooled normal plasma and running a PT or PTT.  If the PT/PTT corrects than the screen is negative. This means that a factor or factors were deficient in the patient’s plasma and were replaced with the pooled normal plasma resulting in a correction of the PT/PTT. In this case, a PTT at time 0 of 68 seconds and a PTT at 2 hours of 66 seconds was a failure to correct and indicated that an inhibitor was present, thus a positive result was entered.

The dilute Russell’s viper venom time (dRVVT) was used to test for a lupus anticoagulant. The screening test is performed by adding Russell viper venom, which directly activates coagulation factor X in the presence of calcium and a phospholipid poor reagent to the patient’s plasma and calculating time to clot. The confirmation test is the same assay with added excess phospholipid. In the presence of phospholipid dependent antibodies, the time to clot will be shorter for the confirmation test. The screen and confirmation ratios are normalized ratios (NR) of the patient sample result in seconds divided by the mean of the normal range in seconds. If the screen is <1.20, the confirmation test will not be run. If the screen is greater than 1.20 as seen here, the confirmation test will be run. The end result is reported as a normalized ratio of the screening test over the confirmation test. If the NR is greater than 1.20, than a lupus anticoagulant is reported as present.

Specific factor assays are performed by mixing the patient’s plasma with substrate plasma that is severely deficient in the factor being measured. Factor deficient plasma would be expected to give a prolonged clotting time. When patient plasma is mixed with factor deficient plasma, the clotting time will shorten and the degree of correction is proportional to the factor level in the patient’s plasma. The clotting times for the patient sample are compared to a reference curve. The reference curve is made with dilutions of normal plasma (containing 100% factor) added to factor deficient substrate plasma. All tests are run with 3 dilutions at 25%, 50% and 100% and curves are checked for parallelism errors, which might indicate the presence of an inhibitor. For this patient, factor XI was initially resulted as 1%, which would indicate a factor deficiency.

This is an example of a factor assay that shows parallelism. The reference plasma calibration curve and the patient plasma are parallel lines. 1

Analysis

From the results, it initially appeared that there was both a lupus anticoagulant and a factor XI deficiency. However, it would be odd for a patient with no reported coagulation abnormalities to suddenly have both a lupus anticoagulant and a factor XI deficiency. The raw data from the factor XI assay was obtained.

Upon review, the factor XI assay did show parallelism errors. Parallelism is tested by performing serial dilutions of a standard with known normal concentrations of factor and recording the time to clot. This line is shown with the red arrow. In contrast, the patient sample appears to be a flat line that is not parallel to the calibration curve. Parallelism errors were flagged because from the 50% to 25% dilution, the corrected results more than doubled. If there is a >20% change between dilutions, this indicates possible interference and additional dilutions should be run to dilute out the inhibitor. The 25% dilution had a corrected result of 2.9, which was greater than a 20% increase from the 50% dilution result of 1.3. Once more dilutions were performed; the Factor XI level was ultimately close to 100%.

Additional factors were checked to see if they also increased with dilutions. This would add support to the theory of a non-specific inhibitor (lupus anticoagulant) that was affecting all of the factor levels, rather than a specific factor XI inhibitor or a concurrent factor XI deficiency. The curve from factor IX (below) showed a similar phenomenon. As the sample underwent additional dilutions, the corrected result increased significantly (from 12.8 at 50% to 26.8 at 25%). Ultimately, the factor level was close to 82%.

The curve from factor VIII also showed low results to begin with and ultimately normal levels with additional dilutions. Altogether, this supported the presence of a strong lupus anticoagulant that was non-specifically interfering with all of the factor levels and prolonging the PTT.

Discussion

A prolonged PTT can be caused by many factors. In a patient without a bleeding history, lupus anticoagulant and certain factor deficiencies are high on the differential. The most common specific factor inhibitors are to FVIII and FIX. These generally arise in hemophilia patients treated with factor concentrates. It is very rare for a patient to develop an inhibitor to factor XI or XII.

Factor XI acts in the intrinsic pathway of the clotting cascade and is important for hemostasis. Deficiency of factor XI is rare and mainly occurs in Ashkenazi Jews. Generally, it does not cause spontaneous bleeding; however excessive blood loss can occur during surgical procedures.

Lupus anticoagulants are directed against proteins that complex with phospholipids. Although they prolong the PTT, they are associated with an increase in thrombosis rather than bleeding. In addition to interfering with the PTT assay, lupus anticoagulants may interfere with individual factor assays and result in non-parallelism (patient curve is not parallel to calibration curve) as seen in this patient. With increasing dilutions, the lupus activity will be disproportionately neutralized and the coagulation factor activity will increase in a non-parallel manner. 1

In a letter to the editor by Ruinemans-Koerts et al., they performed a set of experiments to investigate whether lupus anticoagulants vs. individual FVIII and FIX inhibitors can cause non-parallelism in the one-stage factor assay.  Non-parallelism was only detected using lupus sensitive reagents in plasma with high titers of lupus anticoagulants. The FVIII and FIX inhibitor containing samples both resulted in curves that were parallel to reference sample.

This curve shows that the factor IX inhibitor line is parallel to the reference plasma, while the lupus anticoagulant line is not. 1

Ultimately, this demonstrates the importance of running dilutions and being aware of parallelism errors when performing factor assays. This is especially important in patients with known or suspected lupus anticoagulants. In this case, the unlikely presence of a FXI deficiency with no previously reported coagulation testing abnormalities or bleeding history raised the suspicion of an inhibitor interfering with the factor assay. With a concurrent positive inhibitor screen and lupus anticoagulant test, as well as interference demonstrated with multiple factor assays, the best unified conclusion was a strong lupus anticoagulant. 1

References

  1. Ruinesman-Koerts, J., Peterse-Stienissen, I, and Verbruggen, B. ”Non-parallelism in the one-stage coagulation factor assay is a phenomenon of lupus anticoagulants and not of individual factor inhibitors. “ Letter. Thrombosis and Hemostasis, 2010, p.104.5.

Chelsea Marcus, MD is a Hematopathology Fellow at Beth Israel Deaconess Medical Center in Boston, MA. She has a particular interest in High-grade B-Cell lymphomas and the genetic alterations of these lymphomas.

Hematopathology Case Study: A 39 Year Old Woman Presenting with Persistent Cough and Pericardial Effusion

Case history

The patient is a 39 year old woman presenting with a persistent cough. Upon work up, a pericardial effusion is noted. Pericardiocentesis is performed and a smear made from the pericardial fluid reveals atypical lymphoid cells.

Cytology of the Pericardial Fluid

Image 1. Pericardial fluid cytology showing reactive mesothelial cells surrounded by benign small lymphocytes and atypical large lymphocytes.

Additional imaging reveals an anterior mediastinal mass measuring 12.6 cm. Excision of the mediastinal mass is performed. Sections of mediastinal mass show a variable population of lymphoid cells ranging from small to medium lymphocytes and some atypical large lymphocytes. These atypical large lymphocytes have irregular nuclear contours with abundant cytoplasm, vesicular chromatin and prominent nucleoli. These atypical large lymphoid cells are consistent with Hodgkin Reed-Sternberg cells. Abundant eosinophilic and scattered neutrophilic infiltration are noted within the nodules. These nodules are surrounded by dense collagen bands.

Image 2. H&E sections showing small to medium sized lymphoid cells with scattered large Hodgkin Reed-Sternberg cells infiltrating through fibrosis (frozen section A) and inflammatory cells predominantly eosinophilic infiltration (B) Fascin (C) and CD30 (D) are positive for atypical lymphoid cells.

Immunohistochemistry studies are performed, atypical large lymphoid cells are positive for CD30, Fascin and PAX5, while rare small to medium sized lymphocytes are positive for CD20, however, large atypical lymphoma cells are negative for CD20. Tumor cells are negative for CD3, CD5, CD15, LCA, ALK and EBER ISH. CD3 and CD5 highlight the reactive T cells in the background.

Image 3. PAX5 is positive in some tumor cells.

Overall, the case is consistent with nodular sclerosis classic Hodgkin lymphoma.  The presence of sheets of large lymphoma cells is suggestive of the syncytial variant.

Discussion

Nodular sclerosis classic Hodgkin’s lymphoma (NSCHL) subtype has a distinct epidemiology, clinical presentation and histology. NSCHL is more common in females with peak aged between 15 and 34 years. The risk is higher in high socioeconomic status. The patients are presenting with particularly mediastinal mass and 40% B symptoms.

NSCHL can be distinguished from the other subtypes of Hodgkin’s lymphoma (HL) with characteristic histologic features. There is a nodular growth pattern and the nodules are surrounded by collagen bands representing nodular sclerosis.  The lymphoma is composed of variable number of Hodgkin Reed-Sternberg (HRS) cells, small to medium sized lymphoid cells and non-neoplastic inflammatory cells, predominantly eosinophils, neutrophils and histiocytes. HRS cells have multinucleated or binucleated with irregular nuclear contours and prominent nucleoli. HRS cells induce fibroblastic activity by expressing IL-13 and the fibrosis begins in the lymph node by invaginating into the lymph node along vascular septa.

Immunophenotypically, the lymphoma cells are mostly positive for CD30 and 75-85% positive for CD15. Association with EBV can be demonstrated with EBER in-situ hybridization.  The malignant lymphocytes in NSCHL are variably expressing CD20, PAX5 and CD79a, however, T cell antigen markers, particularly CD4 and CD2 are aberrantly expressed in NSCHL.

NSCHL is classified mostly as grade 2 and the prognosis is better than the other subtypes of HL.  Doxorubicin, bleomycin, vinblastine and dacarbazine (ABVD) is the most frequent induction regimen for NSCHL patients with over 70% response rate.

Patients with Syncytial Variant Nodular Sclerosis Classic Hodgkin Lymphoma experience a lower than expected rate of complete therapeutic response with shorter progression-free than non-SV NSCHL treated with standard therapy. Syncytial Variant NSCHL should therefore be recognized as a high-risk subgroup within the otherwise traditionally docile NSCHL classification. This case fits the classic presentation for syncytial variant with presentation as bulky (mediastinal) disease.

References

  1. Eberle FC, Mani H, Jaffe ES. Histopathology of Hodgkin’s Lymphoma. Cancer J. 2009 Mar-Apr;15(2):129-37.
  2. Swerdlow SH, Campo E, Harris NL et al. WHO Classification of Tumors of Haematopoietic and Lymphoid Tissues (Revised 4th Edition). IARC: Lyon 2017.
  3. Sethi T, Nguyen V, Li S, Morgan D, Greer J. Differences in outcome of patients with syncytial variant Hodgkin Lymphoma compared with typical nodular sclerosis Hodgkin Lymphoma. Ther Adv Hematol 2017, Vol. 8(1):13-20.

Ayse Irem Kilic is a 2nd year AP/CP pathology resident at Loyola University Medical Center. Follow Dr. Kilic on twitter @iremessa.

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

Hematopathology Case Study: A 43 Year Old Man with Difficulty Breathing

Case History

43 year old man presented with symptoms of superior vena cava syndrome including swelling of the head and neck and difficulty breathing. He was found to have a 9 cm anterior mediastinal mass on imaging.

Excisional Biopsy

Top: H&E morphology of diffuse large cells infiltrating through fibrotic tissue.
Bottom: Small lymphocytes with scattered large multinucleated Hodgkin and Reed-Sternberg (HRS) cells.
Left: CD30 showing dim/variable staining in the diffuse large cell component.
Right: CD30 highlighting Hodgkin and Reed-Sternberg cells with a golgi and membranous staining pattern.
Left: CD15 showing golgi staining in the diffuse large cell component.
Right: CD15 highlighting Hodgkin and Reed-Sternberg cells with a golgi and membranous staining pattern.
Left: CD20 diffusely highlighting the large cell infiltrate.
Right: CD20 highlighting small B-cells surrounding a negative HRS cell.
Left: PAX5 diffusely highlighting the large cell infiltrate.
Right: PAX5 showing bright staining in small B-cells surrounding a dimly stained HRS cell.
Left: Ki-67 showing a high proliferation index (90%) in the diffuse large cell component.
Right: Ki-67 showing increased staining in the HRS cells.

Diagnosis

Sections show fragments of fibrotic tissue with crush artifact. Two distinct morphologies are seen in different tissue fragments. Some tissue fragments show infiltration by cords and aggregates of abnormal large lymphoid cells with irregular nuclear contours, somewhat vesicular chromatin, small nucleoli and small to medium amounts of cytoplasm. Frequent apoptotic cells and mitotic figures are seen. In other tissue fragments, the large cell component is absent and there are focally vague nodules. The nodules are composed of small mature appearing lymphocytes, rare eosinophils and scattered medium and large mononuclear and multinucleated cells with prominent nucleoli consistent with Hodgkin cells and Reed-Sternberg cells, respectively. Admixed histiocytes are also seen.

By immunohistochemistry, the areas with different morphologies also show different staining patterns. The areas with the large cell infiltrate are immunoreactive for CD20, BCL6, and MUM1, dimly positive or negative for CD45 and negative for CD10. CD30 is variably positive in the large cell population and CD23 is largely negative. CD15 shows a golgi staining pattern. The Hodgkin and Reed-Sternberg (HRS) cells present in the areas without the large cell infiltrate are brightly immunoreactive for CD30 and CD15 (membranous and golgi pattern), dim positive for PAX5 and are negative for CD20. CD20 and PAX5 highlight small B-cells present in aggregates surrounding the HRS cells. By Ki-67 staining, the proliferation index is high (90%) within the diffuse large cell component and also highlights the HRS component.

Overall, the findings are of a composite lymphoma composed of both a diffuse large B-cell lymphoma (DLBCL) and a classic Hodgkin lymphoma (CHL).  

Discussion

Composite lymphomas occur when two morphologically and immunophenotypically distinct lymphomas occur at the same anatomical site. They are most commonly composed of two Non-Hodgkin B-cell lymphomas (NHL), however rare cases of composite CHL with NHL have been reported. In a review of the literature, Goyal et. al. documented 20 previously reported cases of composite lymphoma with CHL and DLBCL components. The median age at presentation was 51 years with 12 men and 9 women. Fifteen of the cases presented with nodal involvement and of those, three had mediastinal disease. The most common subtype of CHL was nodular sclerosis. Evaluation for IGH gene rearrangements was performed on both components of 6 cases, with either a complete or partial clonal relationship between the components seen in all of the cases tested. This suggests a shared origin from a common B-cell precursor.1

A review of literature by Wang et. al. documented 10 previously described composite lymphomas consisting of DLBCL and CHL. The most common site of occurrence was in lymph nodes, followed by three cases seen in the stomach, one case in the small intestine and one case in the anterior mediastinum. CHL is more commonly associated with EBV infection than NHL In the reviewed cases, 6 showed positivity for EBV infection in both the DLBCL and CHL components. This suggests that the lymphomas shared a common EBV-infected progenitor cell, and are also clonally related as seen in the Goyal review. 2

Composite lymphomas must be distinguished from another WHO defined entity called B-cell lymphoma, unclassifiable, with features intermediate between diffuse large B-cell lymphoma and classic Hodgkin lymphoma. This entity has previously been referred to as “grey-zone lymphoma.” These lymphomas tend to present as mediastinal masses and can cause superior vena cava syndrome. They show a wide spectrum of histologic appearances within a single tumor and often show sheet-like growth of pleomorphic cells. Some areas may resemble CHL while others resemble DLBCL. The neoplastic cells typically do not show the characteristic immunophenotype of either CHL or DLBCL. Areas that may resemble CHL will show preservation of B-cell markers, while areas more characteristic of DLBCL might lose B-cell markers and express CD30 and CD15. These tumors will show clonal rearrangement of the immunoglobulin genes. They tend to have a more aggressive clinical course and worse outcome than either CHL or DLBCL. 3

This case was ultimately diagnosed as a composite lymphoma (CL) because it consisted of separate areas with the morphologic and immunophenotypic features of both classic Hodgkin lymphoma and diffuse large B-cell lymphoma. Patients tend to have a poor prognosis with short survival. There is no standardized treatment for composite lymphomas due to their rare occurrence; however cases with a component of DLBCL are generally treated with aggressive chemotherapy such as R-CHOP.

References

  1. Goyal, G. et al. “Composite Lymphoma with Diffuse Large B-Cell Lymphoma and Classical Hodgkin Lymphoma Components: A Case Report and Review of the Literature.” Pathology – Research and Practice vol. 212,12(2016):1179-1190. http://www.ncbi.nlm.nih.gov/pubmed/27887763.
  2. Wang, Hong-Wei et al. “Composite diffuse large B-cell lymphoma and classical Hodgkin’s lymphoma of the stomach: case report and literature review” World journal of gastroenterology vol. 19,37(2013):6304-9.
  3. Swerdlow SH, Campo E, Harris NL, et al. WHO Classification of Tumours of Haematopoetic and Lymphoid Tissues (Revised 4th edition). IARC: Lyon 2017.

Chelsea Marcus, MD is a Hematopathology Fellow at Beth Israel Deaconess Medical Center in Boston, MA. She has a particular interest in High-grade B-Cell lymphomas and the genetic alterations of these lymphomas.

Hematology Case Study: Symptomatic Anemia in Myelodysplastic Syndrome with Progression to Acute Myelogenous Leukemia

The patient is a 77 year old woman who presented in late Jan 2019 with severe anemia. In Feb 2017 she was diagnosed with myelodysplastic syndrome with no evidence of transformation to acute myelogenous leukemia. A bone marrow biopsy at the time showed 5-7% blasts in the bone marrow. She went through 5 rounds of chemotherapy with Vidaza (azacytidine) over the course of 9 months, with no significant response. She received one unit of RBCs with her 4th round of chemo and was given Aranesp (darbepoetin alfa) injections for anemia support. Aranesp is a man-made erythropoiesis stimulating protein which can be used to treat symptomatic anemia associated with myelodysplastic syndromes (MDS). After the 5th cycle of chemo, because of the lack of response, Vidaza was discontinued. Since then she has received several RBC transfusions to treat anemia and the Aranesp injections have continued.

In Oct 2018, the patient’s CBC showed leukocytosis, anemia, thrombocytopenia and neutrophilia.  See results below:

Patient results 10/2018       reference ranges

WBC  31.6                         4.5-10.5 x 103/μL

RBC  3.0                           3.7-5.3 x 106/μL

Hgb  7.0                            12.0-15.5 g/dl

Hct  23.6                            36.0-46.0 %

MCV  78.4                         80-100 fl

Plt  82                                150-450 x 103/μL

The CBC with automated differential performed at this visit flagged for a smear review. The technologist suspected blasts and the slide was sent for a pathologist’s review. The pathologist’s interpretation was that the differential showed “an aberrant myeloblast population, representing 6% of leukocytes along with an immature appearing monocytic population with phenotypic aberrancies representing 21% of leukocytes.” A leukemia/lymphoma flow cytometry was ordered. Results of the flow cytometry commented that an acute myeloid leukemia could not be excluded, however the differential diagnosis could also include chronic myelomonocytic leukemia. 

By Jan 2018, the patient was receiving blood transfusions every 6-8 weeks. CBC results from this visit shown below:

Patient results 1/2019         reference ranges

WBC  36.5                         4.5-10.5 x 103/μL

RBC  2.7                           3.7-5.3 x 106/μL

Hgb  6.2                            12.0-15.5 g/dL

Plt  65                                150-450 x 103/μL

Unfortunately the differential on this visit showed over 25% myeloblasts, confirmed by pathologist’s review. This sample was sent out for a second leukemia/lymphoma panel. A myeloblast phenotype was detected representing 27% of the leukocytes.

Diagnosis: Acute monoblastic/monocytic leukemia, no remission.

Image 1. Blasts, RBC morphology consistent with severe anemia
Image 2. Blasts seen on slide.

Myelodysplastic syndrome is a disorder of hematopoietic cell production involving clonal proliferation of an abnormal hematopoietic stem cell. It is most commonly diagnosed in patients in their 70s. Failure of the bone marrow to produce mature healthy cells is a gradual process, and therefore MDS is not necessarily a terminal disease. However, pancytopenia is a hallmark of MDS, and when pancytopenia is accompanied by the loss of the body’s ability to fight infections and control bleeding, MDS can be fatal. In addition, patients with MDS have a high risk of conversion to AML. About 30% of patients diagnosed with MDS will progress to acute myeloid leukemia (AML).

This patient was exhibiting pancytopenia, with accompanying anemia and infections, until her WBC began climbing several months ago. This was accompanied by the left shift and blasts seen on the peripheral smear, and prompted the flow cytometry studies.

Acute monoblastic/monocytic leukemia is considered a type of acute myeloid leukemia. In order to fulfill World Health Organization (WHO) criteria for AML-M5, a patient must have greater than 20% blasts in the bone marrow, and of these, greater than 80% must be of the monocytic lineage. AML-M5 can further be classified as M5a or M5b depending on whether the monocytic cells are predominantly monoblasts (>80%) or a mixture of monoblasts and promonocytes (<80% blasts).

The patient’s situation was discussed with the patient and her family. The patient chose more conservative and palliative treatment options over further chemotherapy.

References

https://www.merckmanuals.com/professional/hematology-and-oncology/leukemias/myelodysplastic-syndrome-mds

http://wiki.clinicalflow.com/amol-acute-monoblasticmonocytic-leukemia-m5

-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 33 Year Old Man with a Mass Behind the Ear

Case History

A 33 year old man of Japanese ethnicity presents with a 2 month history of a mass behind the right ear. Examination reveals a non-tender local with no other local or generalized adenopathy or hepatosplenomegaly. Laboratory investigations reveal an elevated ESR, serum IgE and peripheral blood eosinophilia. The lesion is excised.

Biopsy Findings

H&E stained sections demonstrate a follicular hyperplasia. The germinal centers demonstrate polarity and tingible body macrophages (A). Focally, follicular centers reveal eosinophilic microabscesses (B, C). Immunohistochemical analysis with an IgE stain reveals deposition in germinal centers (D). A diagnosis of Kimura disease is rendered.

Discussion

Kimura disease, also known as eosinophilic lymphoid follicular hyperplasia is a rare, chronic inflammatory disorder of unknown etiology. While an infectious etiology has been suggested, no pathogen has been identified to be causal, to date. Historically, Kimura disease was considered to be the same as Angiolymphoid Hyperplasia with Eosinophilia (ALHE); however, these entities are not the same.

Generally occurring in Asian males, Kimura disease is most common in the 3rd decade of life and in a head/neck site. It presents as painless, slow-growing adenopathy. An association with nephrotic syndrome has been reported. Peripheral blood eosinophilia, elevated ESR, and serum IgE are common findings. Histologically, nodes reveal hyperplastic follicles with well-formed germinal centers and mantle zones with deposition of IgE and eosinophilic microabscesses, as seen in this case. Perinodal soft tissue may be involved. Necrosis may be present, but is not extensive. Cytologically, FNA material may reveal polymorphous cell population with many eosinophils.

Prognosis is indolent; however, most cases recur after excision and radiation therapy usually yields best outcome.

References:

  1. Zhou P. et al. Kimura disease. Dermatol Online J. 2017 Oct 15;23(10).
  2. García Carretero R et al. Eosinophilia and multiple lymphadenopathy: Kimura disease, a rare, but benign condition. BMJ Case Rep. 2016 Aug 31;2016. pii: bcr2015214211. doi: 10.1136/bcr-2015-214211.
  3. Sun QF et al. Kimura disease: review of the literature. Intern Med J 2008;38:668–72.  

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

Hematopathology Case Study: A 60 Year Old Man with Recurrent Bronchitis

Case History

60 year old man with recurrent bronchitis and extensive smoking history underwent CT scan. The CT scan showed an incidental finding of a 2.2 x 1.4 cm anterior mediastinal mass.

Excision

H&E4x
H&E 4x
H&E10x
H&E 10x
H&E20x
H&E 20x
cytokeratin cocktail
Cytokeratin cocktail
CD3
CD3
CD20
CD20
TdT
TdT

Diagnosis

The tissue shows nodules of epithelial cells in a lymphocyte-rich background. The epithelial cells have round to somewhat spindle shaped nuclei, vesicular chromatin and small mostly inconspicuous nucleoli. There is no high grade cytologic atypia, mitotic figures or necrosis seen. The nodules contain very few interspersed lymphocytes, but are surrounded by abundant lymphocytes which are small and mature appearing. A cytokeratin cocktail highlights the epithelial nodules and shows an absence of epithelial cells in the lymphocyte-rich areas. CD20 highlights stromal B-lymphocytes around the epithelial nodules which are arranged in follicles. CD3 highlights stromal T-lymphocytes, which surround the B-cell follicles and the epithelial nodules. TdT highlights only a very small subset of immature T-cells which are found scattered around the rim of the epithelial cell nodules. Overall, the findings are consistent with a micronodular thymoma with lymphoid stroma.

Discussion

The differential diagnosis for an anterior mediastinal mass includes thymoma, lymphoma, germ cell tumors, neurogenic tumors and benign cysts among other less common entities. Patients usually present with cough, chest pain, fever/chills or dyspnea and localizing symptoms are generally secondary to local tumor invasion. Typically, CT scans are the best modality to evaluate the mediastinum. Thymomas are the most common primary neoplasm of the anterior mediastinum, but are less than 1% of all adult malignancies. Patients are generally over 40 years old and between 30-50% of patients with a thymoma have myasthenia gravis, which occurs more frequently in women.1

The WHO has classified thymomas into 5 categories based on the morphology of the neoplastic epithelial cells along with the lymphocyte to epithelial cell ratio. Type A thymomas are composed of bland spindle/oval tumor cells with few or no admixed immature lymphocytes. Type B1 thymoma resembles normal thymus and has scattered epithelial cells in a dense background of immature T-cells. Type B2 thymoma is composed of epithelial cells in small clusters with a lymphocyte-rich background. Type B3 thymoma is primarily composed of mild to moderately atypical epithelial tumor cells in a solid growth pattern with few intermingled immature T-cells. Type AB thymomas are composed of lymphocyte-poor spindle cell (Type A) components as well as lymphocyte-rich (Type B) components.2

Micronodular thymoma with lymphoid stroma (MTWLS) is a rare type of thymoma and accounts for only 1% of all cases. Patients tend to be asymptomatic and the finding is usually incidental. The tumor tends to be well circumscribed and encapsulated with a tan cut surface. The histopathology is characterized by solid nests or nodules of epithelial tumor cells in a background of abundant lymphoid stroma. The tumor cells are bland spindle or oval cells without significant atypia or mitotic activity. The epithelial tumor cells are positive for pancytokeratins. The lymphoid stroma typically lacks keratin positive cells and consists of predominantly CD20 positive mature B-cells in follicles with admixed CD3 positive and TdT negative mature T-cells. There is typically a population of rare TdT positive immature T-cells that surrounds the epithelial nodules, as seen in this case. 2

Due to the rarity of MTWLS with only 74 cases reported since the first case described in 1999, there is limited data on its pathophysiology and prognosis. However, most cases are diagnosed as stage I/II disease according to the Masaoka-Koga staging criteria, involving only micro or macroscopic invasion into thymic or surrounding fatty tissue without invasion into neighboring organs.  Patients tend to have a very favorable prognosis with most patients alive without recurrence or metastasis many years after diagnosis.3

References

  1. Juanpere S, Cañete N, Ortuño P, Martínez S, Sanchez G, Bernado L. A diagnostic approach to the mediastinal masses. Insights Imaging. 2012;4(1):29-52.
  2. Travis WD, Brambilla E, Burke AP, et al. WHO Classification of Tumours of the Lung, Pleura, Thymus and Heart (Revised 4th edition). IARC: Lyon 2015.
  3. Qu L, Xiong Y, Yao Q, Zhang B, Li T. Micronodular thymoma with lymphoid stroma: Two cases, one in a multilocular thymic cyst, and literature review. Thorac Cancer. 2017;8(6):734-740.

Chelsea Marcus, MD is a Hematopathology Fellow at Beth Israel Deaconess Medical Center in Boston, MA. She has a particular interest in High-grade B-Cell lymphomas and the genetic alterations of these lymphomas.