Hemoglobin Electorphoresis in Children

This last month, I rotated through our Children’s hospital, which included reviewing hemoglobin electrophoresis tests. I’d learned about them before in residency, but they can be quite more interesting (complicated) than I expected.

Hemoglobin electrophoresis is a blood test to look at different types of hemoglobin to determine if there are any abnormalities. In a children’s hospital it is frequently ordered as a reflex for an abnormal newborn screen or when a child is incidentally found to be anemic. The test is performed in 2 stages. 1st lysed blood samples are run on gel electrophoresis and different types of hemoglobin are separated as they move at different speeds. Several types of hemoglobin will run within the same region, so a secondary method of separation is always employed.

Below, you can see how some bands in the same area of an acidic gel (agarose) are actually very different on the alkaline gel (cellulose acetate) and vice versa.

At our hospital, we use HPLC and measure retention times of the hemolysate to quantify and identify different hemoglobin types present. As a basic primer you should recall that hemoglobin is a tetramer with a pair of alpha globin + a pair of either beta, delta or gamma globin (each separate genes).

Alternative hemoglobins are enriched in populations where malaria is endemic as these variants may provide improved fitness by promoting resistance to the malarial parasite that reproduces inside red blood cells. Thus, many people of African or south east Asian descent may carry these variants.

Our case is that of a 2 year old girl with anemia who had testing sent by her primary care doctor for the following CBC:

This is indicative of microcytic anemia, but unlike some Thalessemias the RBC isn’t very high. More on this later.

Looking at the gel result, there is a large band in the area coinciding with Hgb C. We also see the normal Hgb A2 and a small amount of Hgb F. We know Hgb F can be increased in Hgb SS and thus could also be present if she had Hgb C trait or disease.

InkedBlog 1B_LI

Looking at the next HPLC result, we see there is a similar very high level of Hgb C (68%) with corresponding levels of Hgb F and Hgb A2 (note: acetylated Hgb F and Hgb F are added together). Thus, this fits with a homozygous C with some compensatory A1 and F, right?

Remember Hgb C is a β -globin variant and you only have 2 β -globin genes, so if you are homozygous for the C variant on the β-globin gene (HBB), then Hgb A1, which is made of normal β-globin would be impossible to produce. Also you might be bothered by all of these small peaks. However, there are often small peaks that can’t be definitively identified and are likely post-translationally modified hemoglobin. But in the context of an abnormal Hgb A1 that shouldn’t be there, we dug deeper.

One of the most common hemoglobinopathies is Beta Thalassemia (β-Thal), which clinically manifests when less of the beta hemoglobin protein is produced. Heterozygous mutations lead to Beta Thalassemia minor with minimal symptoms, while homozygous mutations lead to β-thal major with symptoms of anemia. Mutations in the β -globin gene, HBB, can lead to complete loss of β-globin (β0 variant) or partial of β-globin (β+ variant).

As this patient has less than 50% of Hgb A present (expected amount), they could also have a β+ variant as well. This would make them compound heterozygous for C and β+.

One of the hallmarks of Thalassemia is an increase in Hgb A2 (normal 2.5-3.5%). Hemoglobin A2 is a normal variant of A that is composed of two alpha and two delta chains (δ2α2). We see in our case that the Hgb A2 is normal at 2.5%. So it seems the patient doesn’t display a typical Thalassemia picture.

One condition that could create this scenario is if there is a variant in the delta chain of A2 that causes it to elute differently. Indeed, there is a delta variant that creates hemoglobin A2 prime (A2’) that moves near the S region of the HPLC. And when we look back at our unknown hemoglobins, Hgb X is marked at 1.03 of the S region and has an abundance of 3.9%. This supports it being the Hgb A2’ and if we add this together with the Hgb A2 we get an elevated 6.6% A2 total, which would be consistent with Beta Thalassemia. Lastly, one would wonder if we could find this third hemoglobin variant A2’ on the alkaline gel. Previous studies have shown the A2’ variant is more negatively charged, so on a basic gel, it should move further from the negative anode than the other hemoglobins. We don’t see anything to the left of the HgbC, but if we flip the gel over and look under the patient label, you can see a faint band that is likely the A2’!

In summary this case arose from 3 separate mutations in a single patient. She was compound heterozygous for a Hgb C and β+ variants in the β-globin gene and she was heterozygous for an A2’ variant on the delta-globin gene.  This was certainly a case where paying close attention mattered.

References:

  1. Abdel-Gadir D, Phelan L, and Bain BJ. Haemoglobin A2′ and its significance in beta thalassaemia diagnosis. Int J Lab Hematol. 2009 Jun;31(3):315-9. doi: 10.1111/j.1751-553X.2008.01038.x. Epub 2008 Feb 21.
  2. https://ghr.nlm.nih.gov/condition/beta-thalassemia

-Dr. Charles Timmons MD PhD is a pediatric pathologist at Children’s Medical Center in Dallas, TX. His responsibilities include signing out hemoglobin electrophoresis, HPLC and globin sequencing, and has been residency director for 17 years.

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

Hematopathology Case Study: An 80 Year Old Man with Rapid Onset Cervical Adenopathy

Case History

An 80 year old man presented with rapid onset of cervical adenopathy over a period of few months. The largest lymph node measuring 6 cm was biopsied and sent for histopathological evaluation.

Biopsy Findings

Sections from the lymph node showed effacement of the lymph node architecture by a fairly monotonous population of medium to large sized lymphoid cells arranged in vague nodular pattern. Focally, a starry sky pattern was observed. The cells were 1.5-2 times the size of an RBC, with high N:C ratio, irregular angulated nuclei and small nucleoli. A high mitotic rate of 2-3 mitoses/hpf was seen.

Immunohistochemistry

Immunohistochemical stains showed that the lymphoma cells were positive for CD20, CD5, SOX-11, and negative for Cyclin D1, CD10, CD23, CD30, BCL-1, and BCL-6. Ki67 index was about 70%.

Diagnosis

A diagnosis of Mantle cell lymphoma, pleomorphic variant was made.

Discussion

Mantle cell lymphoma is a peripheral B cell lymphoma, occurring in middle aged or older adults, with a male: female ratio of 7:1. Although Cyclin D1 expression is considered a hallmark of mantle cell lymphoma, yet about 7% cases are known to be Cyclin D1 negative. In these cases, morphological features and SOX-11 positivity helps in establishing a definitive diagnosis.

Differential Diagnosis

In the assessment of morphological features of lymphoma, the cell size is an important starting point. In this case, the lymphoma cells ranged from medium to large sized. The following differential diagnoses were considered:

  • Burkitt lymphoma

This case showed a “starry sky” pattern focally. A medium sized population of cells, high mitotic rate and a high Ki67 index (70%) favoured a Burkitt lymphoma. However, although commonly seen in Burkitt lymphoma, a “starry sky” pattern is not specific for this type of lymphoma. Also, the lack of typical “squaring off” of nuclei, basophilic cytoplasmic rim were against the diagnosis of Burkitt lymphoma. The nuclei in this case showed 0-1 small nucleoli, unlike the typical basophilic 2-3 prominent nucleoli of Burkitt lymphoma. Moreover, Ki67 index, even though high was not enough for Burkitt lymphoma where it approaches 100%. The cells were negative for CD10 and Bcl-6, which are almost always found in a Burkitt lymphoma. Hence, a diagnosis of Burkitt lymphoma was ruled out.

  • Diffuse Large B cell Lymphoma

The presence of interspersed large cells with nucleoli, irregular nuclei, high mitotic rate, and a high Ki67 index with a history of very rapid enlargement of lymph node suggested a diagnosis of Diffuse Large B cell lymphoma. However, the scant cytoplasm, lack of bizarre cells, and absence of CD10, BCl-2, BCl-6 were against a diagnosis of DLBCL.

  • Lymphoblastic lymphoma

A diagnosis of lymphoblastic lymphoma was favoured by the irregularly angulated nuclei, and presence of nucleoli. However, the cells of lymphoblastic lymphoma have a more delicate nuclear chromatin, higher mitotic rate as against the relatively condensed chromatin and the low to high variable mitotic rate of Mantle cell lymphoma. Also, lymphoblastic lymphomas are more commonly of the T cell subtype and occur commonly in younger individuals. In this case, B cell markers were positive (CD 20), and the patient was 80 year old, disfavouring a lymphoblastic lymphoma. The blastoid variant of mantle cell lymphoma is practically indistinguishable from lymphoblastic lymphoma, except that it is Tdt negative.

Cyclin D1 negativity in Mantle cell lymphoma

In the cases of Cyclin D1 negative mantle cell lymphomas, morphology plays a critical role in coming to a diagnosis of mantle cell lymphomas. In this case, points that favoured the diagnosis of mantle cell lymphoma were clinical features such as older age (80 years), and male gender, and morphological features such as a vaguely nodular pattern of growth, irregular nuclei, and 0-1 small nucleoli. Due to the presence of variably sized cells with distinct nucleoli, a pleomorphic variant was considered. Even though Cyclin D1 was found to be negative, the cells were positive for SOX-11.

SOX-11 is a transcription factor that is not normally expressed in B cells, but is sensitive and fairly specific for mantle cell lymphomas. It is important to note that SOX-11 is also positive in 25% Burkitt lymphoma, 100% lymphoblastic lymphoma, and 66% T-prolymphocytic leukemia. Herein lies the importance of recognising morphological features, as all of these lymphomas that may express SOX-11 were ruled on the basis of morphology. A more specific antibody, MRQ-58 may be used for greater specificity. The presence of SOX-11 is considered a specific biomarker for Cyclin-D1 negative mantle cell lymphomas. In these cases, there is upregulation of Cyclin D2 or D3 that may substitute for Cyclin D1 upregulation. But, immunohistochemical detection of Cyclin D2 or D3 is not helpful for establishing a diagnosis, as other lymphomas are commonly positive for these markers. Hence, it is important to perform SOX-11 immunohistochemistry to diagnose the Cyclin D1 negative variant of mantle cell lymphoma.

SOX-11 can be used not just for the diagnosis, but also for determining prognosis of mantle cell lymphoma. Indolent MCL usually lack SOX-11 expression. The pattern of SOX-11 staining has also been used a marker of prognosis. Cytoplasmic expression of MCl, seen in only a few cases was associated with a shorter survival as compared to the more common nuclear staining of SOX-11.

Conclusion

In this age, lymphoma diagnosis relies heavily on the use of immunohistochemical markers. However, this case highlights the importance of morphological features in diagnosing lymphomas with unusual immunohistochemical marker profile. Although, this case was negative for Cyclin D1, considered a hallmark of Mantle cell lymphoma, yet, the combination of morphological features with SOX-11 staining helped in clinching the diagnosis. To avoid a misdiagnosis, it would be prudent to perform SOX-11 staining in all lymphoma cases morphologically resembling MCL, but lacking Cyclin-D1.

-Swati Bhardwaj, MD has a special interest in surgical pathology and hematopathology. Follow her on Twitter at @Bhardwaj_swat.

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

Hematology Case Study: Thrombocytopenia in a 4 Year Old Child

A 4 year old child was brought to the pediatrician by her mother with a complaint of new onset of severe bruising on her legs. The mother could not recall any falls or bumps that would have caused the bruising. On exam, the physician also noted mucosal bleeding in the oral cavity. Questioning revealed that the patient had experienced flu like symptoms several weeks earlier. The physical exam was normal except for the bleeding. There was no family history of bleeding disorders. A CBC was ordered.

Reported CBC Results

WBC, RBC, Hgb, Hct, RBC indicies normal

Platelet count 26 x 103/μL

IPF 22% (reference range IPF% 1.0-7.0%) The physician evaluated the results, noting the normal CBC but decreased platelet count. The above results also show the immature platelet fraction (IPF), an additional Advanced Clinical Parameter reported from the Sysmex XN hematology analyzer. A low platelet count, as seen in this patient, will reflex a fluorescent platelet count (PLT-F). The impedance count (PLT-I) can be falsely increased if small RBCs or fragments are counted as platelets. On the other hand, in an optical platelet count, when measuring platelets by size (PLT-O), large platelets can be missed, giving a falsely low count. In this case there was a low platelet count and an instrument flag for an abnormal platelet scattergram. The PLT-F, on the other hand, uses a platelet specific dye which eliminates interference seen with other methods. The fluorescent dye labels the RNA, and forward scatter is used to determine size while side fluorescence is used to measure RNA content. With gating set based on cell volume and RNA content, the PLT-F can be measured. Therefore, the reflexed and more reliable PLT-F was the reported count.

Figure 1. PLT-F scattergram. The PLT-F channel measures forward scatter (FSC) on the Y axis and side fluorescence (SFL) on the X axis.1

Additionally, when there is an abnormal scattergram or a low platelet count, the IPF% and IPF# are also reported. The immature platelet fraction is a measure of the youngest platelets, or reticulated platelets. These are the first circulating platelets, right out of the bone marrow. An increased IPF indicates an increase in platelet production, yet this child’s platelet count was very low. This suggests that the thrombocytopenia may be due to excessive destruction of platelets; the bone marrow was actively making platelets, but they were being destroyed, causing the low platelet count.

Figure 2. Platelet scattergrams from a healthy individual with a normal IPF (a) and a patient with a high IPF (b). Mature platelets appear as blue dots, green dots represent the IPF with increased cell volume and higher fluorescence intensity compared to mature platelets.1

Diagnosis

Immune Thrombocytopenia- ITP.

Primary immune thrombocytopenia (ITP), formerly known as idiopathic thrombocytopenic purpura or immune thrombocytopenic purpura, is one of the most common bleeding disorders of children. In most cases, it presents with sudden onset of bruising and petechiae in an otherwise healthy child, with normal WBC and hemoglobin. ITP is an autoimmune bleeding disorder in which the immune system makes anti-platelet antibodies which bind to platelets and cause destruction. Even though the exact cause of ITP remains unknown, it is recognized that it can follow a viral infection or live vaccinations. While there are some similarities between pediatric ITP and ITP in adults, in children this tends to be an acute disease which is self-limiting and resolves itself in several weeks, with no treatment. However, in a small number of children, the disorder may progress to a chronic ITP. In contrast to ITP in children, a chronic form is more commonly seen in adults. It is usually a diagnosis of exclusion, does not follow a viral illness and requires treatment.

This patient recovered in a few weeks. One month after the initial episode, her PLT was 174 x 103/μL and her IPF% was 6.0%

Conclusion

An IPF reported with a CBC, in combination with a low platelet count, is fast, inexpensive, and can be extremely beneficial in aiding in a timely diagnosis. As the child’s platelet count recovered, the IPF% returned to normal range. ITP can therefore be monitored with a CBC. Thus, the IPF can be used not only to help diagnose but also as an indicator of remission.

References

  1. Sysmex America, 2019. www.sysmex.com/us. Used with permission
  2. Arshi Naz et al. Importance of Immature platelet Fraction as a predictor of immune thrombocytopenic purpura. Pak J Med Sci 2016 Vol 32 No 3:575-579
  3. Briggs,C. Assessment of an immature plateletfraction (IPF) in peripheral thrombocytopenia. Br J Haematol 2004Jul;126(1):93-9
  4. Sysmex White Paper. The role of the ImmaturePlatelet Fraction(IPF) in the differential diagnosis of thrombocytopenia. www.sysmex.com/us
  5. D-Orazio, JA, Neely, J, Farhoudi,N. ITP in children: pathophysiology and current treatment approaches.J Pediatr Hematol Oncol.2013 Jan;35(1): 1-13

-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 69 Year Old Male with Weight Loss and Generalized Lymphadenopathy

Case History

The patient is a 69 year old male who presented to the hospital with a 3-month history of drenching night sweats, weight loss, fatigue, and generalized lymphadenopathy. He also endorsed a very itchy rash all over his body. He denied smoking. There was no other relevant social or family history.

Physical examination confirmed diffuse lymphadenopathy, hepatosplenomegaly and a mild diffuse skin rash. Notably, there was a 2.5 cm level-1 lymph node palpated in the left neck. This was subsequently biopsied.

Biopsy

Biopsy of the level-1 neck lymph node revealed a 2.3 x 1.5 x 1.2 cm mass pink-tan and firm mass. Sectioning revealed a glossy white-tan cut surface. H&E staining revealed a polymorphic lymphocytic infiltrate of in the interfollicular zones. The infiltrating lymphocytes ranged from small to large cells with abundant cytoplasm, eosinophils, and plasma cells. There was also a notable increase in the number of high endothelial vessels lined by lymphocytes with irregular nuclear borders and clear cytoplasmic zones.

Image 1. Polymorphic infiltrate of small, mature appearing lymphocytes (A), with prominent blood vessels and clear cytoplasm (B). Most of these cells were CD3 positive T cells (C) with expanded CD21 positive FDC meshworks (D) and scattered CD30 positive immunoblasts (E)

Further characterization by immunohistochemical staining showed the majority of the interfollicular cells to be CD3 and CD5 expressing T cells. These were a mix of CD4 and CD8 positive cells but with marked CD4 predominance. CD7 appeared positive in a smaller population of T-cells compared to CD3 (consistent with loss of this pan-T-cell marker). Varying numbers of the interfollicular cells were positive for CD10, BCL-6, CXCL-13, and PD-1 with a strong positivity for ICOS, phenotypically consistent with an expansion of Tfh (T-follicular helper cell) cells.

Interspersed between the T cells were numerous CD20 positive cells with prominent nucleoli that also revealed CD30 positivity. CD21 staining revealed expanded follicular dendritic cell meshworks. EBER ISH was positive in a rare subset of cells. Kappa and lambda ISH showed an increased number of polytypic plasma cells.

Flow Cytometry showed the presence of a small population of T-cells that were CD4 positive but CD3 negative. There was no evidence of B-cell clonality. TCR-G PCR was positive.

A final diagnosis of Angioimmunoblastic T-cell lymphoma (AITL) was rendered.

Discussion

AITL is a relatively rare neoplasm of mature T follicular helper cells, representing about 1-2% of all non-Hodgkin lymphomas. It is; however, one of the more common subtypes of peripheral T-cell lymphomas, accounting for 15-30% of this subgroup. The condition was first reported in 1974 in Lancet as a non-neoplastic abnormal immune reaction1. It was first recognized as a distinct clinical entity in in 1994 in the Revised European American Lymphoma Classification2. The disease shows a geological preference to Europe (28.7%) over Asia (17.9%) and North America (16%). AITL occurs primarily in middle aged and elderly individuals and shows a slight predominance of males over females.

The disease has a strong association with EBV infection, but the neoplastic T-cells are almost always EBV negative, creating an interesting question of EBV’s function in the etiology of AITL. AITL most often presents late in the disease course with diffuse systemic involvement, including hepatosplenomegaly, lymphadenopathy and other symptoms such as rash with pruritis and arthritis. Lab findings include cold agglutinins, rheumatoid factor and anti-smooth muscle antibodies. There also tends to be immunodeficiency secondary to the neoplastic process. The clinical course of AITL is variable, but the prognosis is poor, with the average survival time after diagnosis being < 3 years. The histological features and genetic findings have not been found to impact clinical course.

Microscopically, AITL presents with either partial or total effacement of the normal lymph node architecture with perinodal infiltration. The cells of AITL are small to medium-sized lymphocytes with clear to pale cytoplasm, distinct cell membranes and very minimal cytological atypia. These cells often congregate around the high endothelial venules. The T-lymphocytes are present in a largely polymorphous inflammatory background of other lymphocytes, histiocytes, plasma cells and eosinophils. There are 3 overlapping sub-patterns of AITL. The first of these is similar to a reactive follicular hyperplasia, and can only be distinguished from normal hyperplasia by use of immunohistochemical stains to differentiate the neoplastic cells from normal reactive cells. The second pattern has retained follicles, but they show regressive changes. The third pattern has completely or sub totally effaced. These three patterns seem to be on a spectrum with one another, given that progression from the first to the third pattern has been seen on consecutive biopsies in the same patient.

Cytologically, AITL cells express pan-T-cell markers including CD2, CD3 and CD5 and the vast majority are CD4 positive. CD3 may be quantitatively decreased or absent by flow cytometry. There are a variable number of CD8 positive T-cells. The tumor cells also show the immunophenotyping of normal T follicular helper cells including CD10, CXCL13, ICOS, BCL6 and PD1 in 60-100% of cases. CXCL13 and CD10 are the most specific, whereas PD1 and ICOS are the most sensitive.

References

  1. Horne, C., Fraser, R., & Petrie, J. (1974). Angio-Immunoblastic Lymphadenopathy With Dysproteinemia. The Lancet, 304(7875), 291. doi:10.1016/s0140-6736(74)91455-x
  2. Harris, N.l. “A Revised European-American Classification of Lymphoid Neoplasms: a Proposal from the International Lymphoma Study Group.” Current Diagnostic Pathology, vol. 2, no. 1, 1994, pp. 58–59., doi:10.1016/s0968-6053(00)80051-4.
  3. Swerdlow, Steven H. WHO Classification of Tumours of Haematopoietic and Lymphoid Tissues. International Agency for Research on Cancer, 2017.
  4. “Angioimmunoblastic T Cell Lymphoma.” Pathology Outlines – PathologyOutlines.com, http://www.pathologyoutlines.com/topic/lymphomanonBAITL.html.

-Zachary Fattal is a 4th year medical student at the Central Michigan University College of Medicine. He is pursuing a career in pathology and has a special interest in hematopathology, cytopathology and blood bank/transfusion medicine. You can follow him on Twitter @Paraparacelsus.

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 36 Year Old Woman with an Incidental Neck Mass

Case History

A 36 year old female underwent thyroidectomy for multinodular goitre that led to the fortuitous discovery of a neck mass. The neck mass specimen submitted comprised two lymph nodes measuring 2.2 cm and 1.3 cm in the greatest dimensions, with a fleshy tan cut surface.

Biopsy Findings

H&E stained sections revealed numerous non-necrotizing granulomas effacing and replacing normal lymph node architecture. These consisted of pale epithelioid histiocytes and Langhans type of giant cells. The granulomas lacked a peripheral rim of lymphocytes. AFB and GMS stains were negative for microorganisms

Diagnosis

A diagnosis of non-necrotizing granulomatous lymphadenitis was rendered noting that in the correct clinical context the findings could represent sarcoidosis.

Discussion

Granulomatous inflammation is a special type of chronic inflammatory response characterised by the formation of discrete collections of histiocytes called granulomas. Activated histiocytes appear as epithelioid cells with round to oval nuclei, often with irregular contours and abundant granular eosinophilic cytoplasm with indistinct cell borders. They may coalesce to form multinucleated giant cells. When found in the lymph node, the reaction pattern is called granulomatous lymphadenitis. It can be caused by a variety of different conditions, and therefore, requires thorough workup to come to a conclusive diagnosis.

On the basis of presence or absence of necrosis, granulomatous lymphadenitis can be classified as necrotizing or non-necrotizing. Additionally, the presence of an abscess, usually central, indicates a suppurative lymphadenitis.

Non-necrotizing granulomatous lymphadenitis:

Sarcoidosis lymphadenitis is the prototype of non-necrotizing granulomatous lymphadenitis. It shows the presence of discrete granulomas without a peripheral rim of lymphocytes, called “naked granulomas”. The early phase shows follicular hyperplasia and sinus histiocytosis, followed by appearance of epithelioid cell nodules toward the end of this phase. The peak phase shows well-demarcated granulomas composed of epithelioid cells with scattered multinucleated giant cells observed throughout the lymph node. Granulomas may occasionally coalesce. In the late phase, increased collagen fibers result in fibrosis and hyalinization. There are no neutrophils and it is uncommon to find small foci of central necrosis. Numerous inclusions such as asteroid, Schaumann, or Hamazaki-Wesenberg bodies can be seen. In this case, we observed well-demarcated granulomas throughout the lymph node, typical of the peak phase without any caseous necrosis or suppuration.

Other causes of granulomatous lymphadenitis can be ruled out as follows.

Sarcoid-like lymphadenitis: It shows a similar pattern of non-necrotizing lymphadenitis like sarcoidosis. However, classically sarcoid like reaction shows scattered small epithelioid granulomas with sparsely arranged epithelioid cells. The border of the granulomas is usually obscure. The CD4:CD8 ratio ranges from 0.8 to 2.25 while in sarcoidosis, it is >3.5. These findings help distinguish sarcoid-like lymphadenitis from sarcoidosis.

Sarcoid-like adenitis may be seen in numerous conditions such as carcinoma, Toxoplasmosis, fungal infections, tuberculosis, immunocompromised states, pneumoconiosis etc. The fact that tuberculosis and fungal infections can present with a non-necrotizing granulomatous lymphadenitis highlights the importance of performing fungal (PAS & GMS) and AFB (Ziehl Neelson) stains in non-necrotizing lymphadenitis as well. In this case, the granulomas had distinct borders, numerous epithelioid cells, no organisms were identified on special stains, nor was there any history of immune compromise; ruling out a sarcoid-like reaction.

Berylliosis: The lymph node picture in Berylliosis is identical to that of sarcoidosis. We may even see asteroid bodies or Schaumann bodies. A diagnosis can be established by eliciting a history of chronic exposure to Beryllium. Beryllium lymphocyte proliferation test (BeLPT) is a test that measures Beryllium sensitization and is very specific for Beryllium exposure. There was no known history of exposure to Beryllium in this case.

Toxoplasmosis: A classic triad of follicular hyperplasia, small granulomas composed of epithelioid cells within and around hyperplastic follicles and, monocytoid B cell hyperplasia, is observed in toxoplasmosis lymphadenitis. This case did not show follicular hyperplasia, ruling out toxoplasmosis.

Necrotizing granulomatous lymphadenitis

Even though we did not find any necrosis in this case, yet, it is worthwhile to review briefly the various causes of necrotizing lymphadenitis.

  • Non-suppurative

Tuberculosis: Histology of a tuberculous lymph node is characterised by central caseous necrosis surrounded by an epithelioid cell layer. The outermost layer is comprised of lymphocytes and fibrosis. Plasma cells are not observed. Diagnosis can be established by performing an AFB stain that demonstrates acid fast rod shaped bacteria in the areas of necrosis. Organisms can also be detected by PCR.

BCG lymphadenitis: About 0.7 to 2.3% of BCG vaccinated children may develop BCG lymphadenitis that is smaller than tuberculous lymphadenitis. Early phase shows follicular hyperplasia and sinus histiocytosis. Later, there is development of micronodules of epithelioid granulomas without necrosis and epithelioid cell granulomas with central caseous necrosis. Langhans giant cells are rare.

Fungal infections: Fungal infections by Histoplasma, Cryptococcus, coccidiodomycosis, pneumocystis may also cause a necrotizing granulomatous inflammation. There are numerous neutrophils, and fungal structures can be seen. GMS and PAS can be used in cases where it is difficult to the find the fungal elements on H&E.

  • Suppurative

Tularemia: There are three forms of histological changes, Abscess form, showing abscess with central necrosis and mononuclear cells, Abscess-granulomatous form with granulomas with central necrosis, which form large lesions with central abscesses, and granulomatous form with caseating necrosis at the centre of the granulomas.

Cat Scratch disease: Similar to tularemia, there are three phases of histologic presentation, an early phase of follicular hyperplasia, intermediate phase of microabscess, and a late phase of granulomatous inflammation. Monocytoid B cell clusters are observed close to the abscess.

Conclusion

Sarcoidosis is usually diagnosed by excluding other causes of granulomatous inflammation, as we did in this case. Characteristic non-necrotizing, discrete granulomas were seen throughout the lymph node. The age of the patient and female gender epidemiologically support the diagnosis. This case reflects an example work up of a granulomatous lymphadenitis that is a morphologic presentation of myriad diseases.

-Swati Bhardwaj, MD has a special interest in surgical pathology and hematopathology. Follow her on Twitter at @Bhardwaj_swat.

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.

Hematology Case Study: A 69 Year Old Female with Breast Implants

Case History

A sixty nine year old female who underwent right breast reconstruction about 13 years ago due to breast cancer presents to the doctor office with right breast pain and right breast enlargement over the last two months. She has lost some weight and does not recall any trauma to this area. She had a textured saline implant. Examination reveals no definite palpable masses. MRI of right breast showed intact saline implant with moderate amount of fluid surrounding the implant within the intact external capsule. No adenopathy was noted. Right breast implant was removed and complete capsulectomy was performed.

Image 1. A. Section of breast capsule with rare atypical hyperchromatic cells (arrow). B. Cytospin preparation of the fluid surrounding the implant with numerous atypical lymphocytes. C. Cell block of the fluid with large atypical lymphocytes. D, E. Lymphocytes are positive for CD30 (image D) and negative for ALK-1 (image E). F. CD30 positive cells in the section of the implant.

Diagnosis

Breast implant-associated anaplastic large cell lymphoma.

Discussion

Breast implant associated anaplastic large cell lymphoma is a provisional entity that is morphologically and immunophenotypically similar to ALK-negative anaplastic large cell lymphoma. It arises primarily in association with a breast implant. It is a very rare entity with an incidence of 1 in 500,000 to 3 million women with implants. Tumor cells may be localized to the seroma cavity or may involve pericapsular fibrous tissue. Sometimes it can form a mass lesion. Locoregional lymph node may be involved. The mean patient age is 50 years. Most patient presents with stage 1 disease, usually with peri-implant effusion. The mean interval from implant placement to lymphoma diagnosis is 10.9 years. There is no association with the type of implant. Histologic examination shows two different types of proliferations. In patients with seroma, the proliferation is confined to the fibrous capsule (“in situ” iALCL). However, the distribution of neoplastic lymphocytes could be heterogeneous with some cellular areas with numerous large pleomorphic cells of varying size and some fibrotic areas with rare atypical lymphocytes. It is beneficial to look at the seroma fluid in addition to capsule sections, because sometimes the neoplastic lymphocytes are predominantly present in fluid (as in our case). Patients presenting with tumor mass show more heterogeneous proliferations infiltrating surrounding tissues (“infiltrative” iALCL). They consists of either sheets are clusters of large neoplastic cells accompanied by a large number of eosinophils. By immunohistochemistry, the tumor cells are strongly positive for CD30. CD2 and CD3 are more often positive than CD5. CD43 is almost always expressed. Most cases are CD4 positive. The prognosis is very good in patients with disease confined to the capsule. The median overall survival is 12 years. However, patients with a tumor mass could have a more aggressive clinical outcome.

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. Jaffe, E , Arber, D, et al. Hematopathology (second edition) 2017.

-Junaid Baqai, MD, was born in Chicago, IL but spent most of his life in Karachi, Pakistan. He graduated from DOW Medical College in Pakistan and did his residency in anatomic and clinical pathology at Danbury Hospital, CT followed by hematopathology fellowship from William Beaumont Hospital, Michigan and oncologic-surgical pathology fellowship from Roswell Park Cancer Institute, New York. He currently serves as Medical Director of hematology, coagulation and flow cytometry at Memorial Medical Center and Medical Director of Laboratory at Taylorville Memorial Hospital.

Hematopathology Case Study: A 76 Year Old Man with Lymphadenopathy

Case History

76 year old man with a history of chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL) with new anterior mediastinal mass and increasing lymphadenopathy.

Lymph Node Biopsy

H&E

Diagnosis

Tissue sections show a diffuse atypical lymphoid infiltrate that completely effaces the normal nodal architecture. The infiltrate is composed of numerous small lymphocytes with round to mildly irregular nuclei, clumped chromatin, inconspicuous nucleoli and scant cytoplasm. There are also expanded pale areas that contain intermediate sized cells with more open chromatin and distinct single to multiple nucleoli. These cells are most consistent with prolymphocytes/paraimmunoblasts and form the proliferation centers characteristic of CLL/SLL. Occasional centroblastic-type B-cells are noted within these proliferation centers. In addition, there are scattered single to multinucleated cells that have irregular nuclear membranes with pale, vesicular chromatin and prominent inclusion-like, eosinophilic nucleoli. These cells morphologically resemble Hodgkin cells, Reed-Sternberg cells, mummified forms and other variants. These large cells are more evident in areas with a histiocyte rich background and around foci of necrosis. Occasionally, apoptotic bodies and mitotic figures are seen.

 Immunohistochemical studies show that the vast majority of the small-intermediate lymphocytes express B-cell markers CD20 (dim) and PAX5 and co-express CD5 and CD23 (subset). This is consistent with a background of CLL/SLL. The large atypical cells are positive for CD30, PAX5 and CD20 (variable). CD3 highlights numerous scattered background small T-cells, which are increased in the areas with the large cells. In situ hybridization for Epstein Barr viral RNA (EBER ISH) is mainly staining the large atypical cells. By Ki-67, the proliferation fraction is overall increased (40%) with increased uptake by the large atypical cells.

The morphologic and immunophenotypic findings are consistent with involvement by the patient’s known small lymphocytic lymphoma/chronic lymphocytic leukemia (SLL/CLL) with aggressive morphological features. The aggressive features include expanded proliferation centers and an elevated Ki-67 proliferative index (40%). Additionally there are histiocyte/T-cell rich areas composed of multiple EBV positive large atypical cells with morphologic and immunophenotypic features compatible with Hodgkin/ Reed-Sternberg cells. These areas are most in keeping with evolving classic Hodgkin lymphoma. Sheets of large cells indicative of large cell transformation are not seen, although increased scattered large centroblastic-type B cells are present.

Discussion

Lymph node involvement by CLL/SLL will typically show a diffuse proliferation of small lymphocytes with effacement of the normal nodal architecture.  The small lymphocytes have round nuclei, clumped chromatin and scant cytoplasm. Scattered paler areas known as proliferation centers are characteristic of this entity. The proliferation centers are composed of a mixture of cell types including small lymphocytes, prolymphocytes and paraimmunoblasts. Prolymphocytes are small to medium in size with relatively clumped chromatin, whereas paraimmunoblasts are larger cells with round to oval nuclei, dispersed chromatin, eosinophilic nucleoli and slightly basophilic cytoplasm. Some cases show increased and enlarged proliferation centers with a higher proliferation rate. This must be distinguished from large cell transformation.1

Aggressive features of CLL/SLL include proliferation centers that are broader than a 20x field or becoming confluent. An increased Ki-67 proliferation >40% or >2.4 mitoses in the proliferation centers can also portend a more aggressive course. These cases tend to have worse outcomes than typical CLL/SLL and better outcomes than cases that have undergone Richter transformation to diffuse large B-cell lymphoma (DLBCL). Transformation to DLBCL occurs in 2-8% of patients with CLL/SLL. Less than 1% of patients with CLL/SLL develop classic Hodgkin lymphoma (CHL). In order to diagnose CHL in the setting of CLL/SLL, classic Reed-Sternberg cells need to be found in a background appropriate for CHL, which includes a mixed inflammatory background. The majority of these CHL cases will be positive for EBV.1

Richter’s transformation is defined as an aggressive evolution of CLL. While the most common type of transformation is to a high-grade B-cell Non-Hodgkin lymphoma, other histological transformations have been described. This includes CHL, lymphoblastic lymphoma, hairy cell leukemia and high-grade T-cell lymphomas. The prognosis for patients who present with transformation to CHL is poor compared to de novo CHL.2 A large study from the M.D. Anderson Cancer Center described 4121 patients with CLL/SLL and found that only 18 patients or 0.4% developed CHL. The median time from CLL to CHL diagnosis was 4.6 years. Fourteen of the patients received chemotherapy. The overall response rate was 44% with a complete response rate of 19%. The median overall survival was 0.8 years and all patients eventually died from disease recurrence or progressive disease.3 This dismal prognosis is similar to patients with Richter transformation to DLBCL and much worse than patients with de novo CHL, which is curable in >85% of cases.1

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. Janjetovic S, Bernd HW, Bokemeyer C, Fiedler W. Hodgkin’s lymphoma as a rare variant of Richter’s transformation in chronic lymphocytic leukemia: A case report and review of the literature. Mol Clin Oncol. 2016;4(3):390–392.doi:10.3892/mco.2016.727.
  3. Tsimberidou, AM, O’Brien, S and Kantarjian, HM, et. al. Hodgkin transformation of chronic lymphocytic leukemia. Cancer. 2006;107(6).doi.org/10.1002/cncr.22121.

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.