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Year : 2020  |  Volume : 63  |  Issue : 4  |  Page : 642-644
Diagnosis of the leukemic phase of ALK-positive anaplastic large cell lymphoma by immunohistochemistry on cell block prepared from peripheral blood buffy coat

Department of Pathology and Lab Medicine, All India Institute of Medical Sciences, Bhubaneswar, Odisha, India

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Date of Submission02-Jun-2019
Date of Decision19-Oct-2019
Date of Acceptance22-Oct-2019
Date of Web Publication28-Oct-2020


A leukemic phase of anaplastic lymphoma kinase positive anaplastic large cell lymphoma (ALK+ ALCL) is rare. The leukemic cells morphologically appear as small to intermediate-sized cells with cerebriform and cloverleaf-like nuclei and are misdiagnosed as other T-Cell lymphomas/leukemia with similar morphology. We describe a case where the diagnosis of leukemic ALK+ ALCL was aided by immunohistochemistry performed on the cell blocks prepared from the peripheral blood buffy coat specimen. The diagnosis of ALK+ ALCL was further confirmed on the biopsy of a cutaneous nodule of this patient. We found the method of immunohistochemistry on peripheral blood buffy coat cell block very useful and suggest that it may be used as an alternative method to flowcytometry in low resource settings.

Keywords: ALK+ ALCL, buffy coat, cell block, immunohistochemistry, leukemia

How to cite this article:
Kundoo A, Sethy M, Sable MN, Mishra P, Panigrahi A, Adhya AK. Diagnosis of the leukemic phase of ALK-positive anaplastic large cell lymphoma by immunohistochemistry on cell block prepared from peripheral blood buffy coat. Indian J Pathol Microbiol 2020;63:642-4

How to cite this URL:
Kundoo A, Sethy M, Sable MN, Mishra P, Panigrahi A, Adhya AK. Diagnosis of the leukemic phase of ALK-positive anaplastic large cell lymphoma by immunohistochemistry on cell block prepared from peripheral blood buffy coat. Indian J Pathol Microbiol [serial online] 2020 [cited 2021 Jul 25];63:642-4. Available from: https://www.ijpmonline.org/text.asp?2020/63/4/642/299308

   Introduction Top

Anaplastic lymphoma kinase positive anaplastic large cell lymphomas (ALK+ ALCL) are categorized as mature T Cell lymphomas, characterized by large pleomorphic cells with horseshoe-shaped nuclei, ALK gene translocations, and expression of ALK and CD30.[1] ALK+ ALCL occurs more frequently in the first 3 decades of life and involves both nodal and extra-nodal sites such as skin, bone, soft tissue, lungs, and liver.[2] Leukemic presentation is extremely rare and it may be confused with other lymphoma/leukemia and pose a considerable diagnostic challenge at its initial presentation. Immunophenotyping of the peripheral blood or bone marrow cells by flow cytometric analysis is the mainstay of diagnosis. We report the utility of immunohistochemistry on cell blocks prepared from the peripheral blood buffy coat of a 25-year-old patient presenting with the leukemic conversion of ALK+ ALCL. This method may be employed as an alternative to flow cytometry in low resource settings.

   Case Report Top

A 25-year-old male patient presented with weakness, fatigue, and pallor. On examination, there was no significant hepatosplenomegaly or lymphadenopathy. The chest radiograph was normal. The complete blood count revealed hemoglobin concentration: 7.6 g/dl, total leucocyte count: 71,170/μL, platelet count: 1,55,000/μL, and a differential count that included 50% atypical lymphoid cells. The lymphoid cells showed a range of morphological features. Most of the lymphoid cells were 1.5–2 times the size of mature lymphocytes. Many of them showed irregular nuclear borders with nuclear clefting and folding with cerebriform and cloverleaf-shaped nuclei [Figure 1]a. Some of the cells were smaller, had opened chromatin, inconspicuous nucleoli, and scanty cytoplasm resembling lymphoblasts. A provisional diagnosis of leukemia/leukemic conversion of lymphoma was made and bone marrow examination was done. The bone marrow aspirates and biopsy showed normal morphology with adequate and proportionate representation of all hematopoietic elements. There was no evidence of any infiltration by leukemia/lymphoma. For the preparation of the buffy coat, the peripheral blood sample was placed in a Wintrobe tube and centrifuged at 2000 rpm for 10 min. The supernatant plasma was removed, and the buffy coat was aspirated out and put in another plastic tube. Citrated plasma was added to it and a clot was generated by adding 250 μL of calcium chloride. The resultant clot was taken out and put in a 10% buffered formalin. The formalin-fixed clot sample was then processed as per routine protocol and cell blocks were prepared. Hematoxylin and eosin-stained sections from the cell block revealed atypical lymphoid cells with morphology similar to the peripheral blood cells [Figure 1]b. However, the cell appeared smaller than the peripheral blood cells due to fixation and processing artefacts. Immunohistochemistry was done on the sections cut from the peripheral blood cell block by routine methods. The atypical cells were positive for CD2, CD3, CD5, CD7, ALK1, and CD 30, whereas the cells were negative for CD20, CD79a, CD23, BCL-2, BCL-6, Tdt, CD10, CD56, Anti MPO, CD34, CD13, CD33, Granzyme, and Perforin [Figure 1]c and [Figure 1]d. In the meantime, the patient was found to have a cutaneous nodule of size 1.2 cm diameter in the chest wall. A biopsy was done from the nodule. Biopsy sections showed features of an ALK-positive anaplastic large cell lymphoma, confirmed by immunohistochemical markers [Figure 2]a, [Figure 2]b, [Figure 2]c, [Figure 2]d. Immunohistochemistry for CD30 was performed on the bone marrow biopsy sections and it failed to reveal any infiltration by the lymphoma cells. The patient succumbed to his illness before the initiation of any specific therapy.
Figure 1: (a) Peripheral blood smear showing leukemic cells with flower-like nuclei, cloverleaf-like nuclei, and few smaller cells with round nuclei (1000× Leishman stain); (b) Section from the cell block prepared from peripheral blood buffy coat showing cells having similar morphology, cell morphology is well preserved (400×, H and E); (c) Immunohistochemistry on cell block sections showing strong positivity for CD30 in the leukemic cells, (400×); (d) Immunohistochemistry on cell block sections showing strong positivity for ALK1 in the leukemic cells, (400x)

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Figure 2: (a) Biopsy section from the skin nodule showing infiltration of lymphoma cells in the upper and lower dermis (100×, H and E); (b) Lymphoma cells show typical morphology of ALCL with large anaplastic cells and irregular nuclear membrane and embryo-like cells (400 × original magnification, H and E); (c) Immunohistochemistry showing strong positivity for ALK1 in the lymphoma cells, (400×); (d) Immunohistochemistry showing strong positivity for CD30 in the lymphoma cells, (400×)

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   Discussion Top

Leukemic conversion of ALK+ ALCL is very rare. We found 21 cases of ALK+ ALCL in the leukemic phase reported in the literature. The clinicopathological features of these cases have been reviewed and tabulated by Nguyen et al.[3] previously. A vast majority of them were small cell variant. Most of the reported cases were male patients younger than 30 years of age. Patients usually present with B symptoms such as fever and night sweats and have widespread organ involvement at the time of presentation.[4] The WBC count at presentation is usually < 100 × 109/L although unusually high counts have also been reported.[3] Bone marrow involvement in ALCL is seen in about 30% of cases when immunohistochemical stains are used.[5] The overall prognosis of these patients is poor, and most patients die of the disease usually within months of diagnosis.[6] The present case supports these findings.

Microscopically, the leukemic cells of ALK+ ALCL show a varied morphology in the peripheral blood smears, ranging from small cells with inconspicuous nucleoli to cerebriform cloverleaf-like cells or flower-like cells to large anaplastic cells. This does not resemble the usual morphology of ALCL in the tissue sections. This raises many differential diagnoses at the time of the initial presentation. The cerebriform nuclear morphology overlaps with the cells of Mycosis fungoides/Sezary cell leukemia and the cloverleaf-like nuclei resemble the cells of adult T-cell lymphoma/leukemia. The smaller cells with opened chromatin may be mistaken for the cells of a low-grade B-cell or T-cell lymphoma/leukemia such as chronic lymphocytic leukemia, prolymphocytic leukemia, leukemic conversion of mantle cell lymphoma, and acute lymphoblastic leukemia. These suggestions have been made previously by others[3],[7],[8],[9] and our case supports this interpretation.

The immunophenotype of the cells and distinct clinical manifestations of these disorders are of utmost importance in their differential diagnosis. The usual method of immunophenotyping of the leukemic cells of peripheral blood is flowcytometry.[10],[11] The diagnosis of leukemic conversion of ALCL can be missed on flow cytometry, as the usual leukemia panel does not include CD 30 and ALK1. These cases may be misdiagnosed as another T-cell leukemia/lymphomas such as Sezary syndrome or adult T-cell leukemia/lymphoma. We did not have the facility for flow cytometry, and the bone marrow biopsy did not show any lymphoma infiltration. Thus, we performed immunohistochemistry on the cell blocks prepared from the buffy coat of the peripheral blood. Sections cut from the cellblock were stained with hematoxylin and eosin. Although the cells appeared a little smaller as compared to the cells in the peripheral smears, the morphology of the cells appeared to be well preserved in the sections. The cells showed cerebriform and cloverleaf-like nuclei similar to the morphology on peripheral blood smears. The morphology of immunohistochemistry on the sections cut from the cell blocks was satisfactory and easily interpretable. In the present case, we had a concurrent biopsy of a skin nodule which showed the typical morphological (large anaplastic cells with twisted embryo-like nuclei) and immunohistochemical features (CD30+ and ALK+) of an ALK+ ALCL. This enabled us to incorporate ALK1 and CD30 in our panel for evaluation of the cell block sections prepared from the peripheral blood buffy coat and make a correct diagnosis. In the absence of prior knowledge of concurrent ALCL, it is difficult to diagnose the leukemic phase of ALCL in the peripheral blood or bone marrow sections.

Buffy coat preparations of peripheral blood are usually done for the identification of parasites such as microfilaria for demonstration of LE cell phenomenon or to concentrate neoplastic cells such as cells of solid tumors.[12],[13] Cellblock immunohistochemistry of buffy coat preparation of the bone marrow aspirate has been demonstrated to be useful in the detection of metastatic malignancy.[13] The use of cell blocks prepared from the peripheral blood buffy coat for immunophenotyping of leukemia cells by immunohistochemistry method has not been documented previously. We found this method very useful in our case. A further large-scale study is needed to properly evaluate its efficacy. Buffy coat and cell blocks can be easily prepared in clinical laboratories and it does not require sophisticated instruments. This method may be employed for immunophenotyping of leukemic cells in low resource settings where the facility for flowcytometry is not available.

In conclusion, we describe a rare case of ALK+ ALCL presenting in a leukemic phase. The diagnostic problems and pitfalls are highlighted and the possibility of using cell blocks prepared from peripheral blood buffy coat for immunophenotyping of leukemic cells is demonstrated. Considering the possibility of leukemic conversion of ALCL in the proper clinical and hematological context and inclusion of ALK1 and CD30 in the immunohistochemical profile leads to a correct diagnosis.

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Conflicts of interest

There are no conflicts of interest.

   References Top

Falini B, Lamant-Rochaix L, Campo E, Jaffe ES, Gascoyne RD, Stein H, et al. Anaplastic large cell lymphoma, ALK-positive.  Back to cited text no. 1
In: Swerdlow SH, Campo E, Harris NL, Jaffe SE, Pileri SA, Stein H, et al., editors. WHO Classification of Tumours of Haematopoietic and Lymphoid Tissues. Revised 4th ed. Lyon: International Agency for Research on Cancer; 2017. p. 413-8.  Back to cited text no. 2
Stein H, Foss HD, Durkop H, Marafioti T, Delsol G, Pulford K, et al. CD30(+) anaplastic large cell lymphoma: A review of its histopathologic, genetic, and clinical features. Blood 2000;96:3681-95.  Back to cited text no. 3
Nguyen JT, Condron MR, Nguyen ND, De J, Medeiros LJ, Padula A. Anaplastic large cell lymphoma in leukemic phase: Extraordinarily high white blood cell count. Pathol Int 2009;59:345-53.  Back to cited text no. 4
Medeiros LJ, Elenitoba-Johnson KS. Anaplastic large cell lymphoma. Am J Clin Pathol 2007;27:707-22.  Back to cited text no. 5
Fraga M, Brousset P, Schlaifer D, Payen C, Robert A, Rubie H, et al. Bone marrow involvement in anaplastic large cell lymphoma. Immunohistochemical detection of minimal disease and its prognostic significance. Am J Clin Pathol 1995;103:82-9.  Back to cited text no. 6
Onciu M, Behm FG, Raimondi SC, Moore S, Harwood EL, Pui CH, et al. ALK-positive anaplastic large cell lymphoma with leukemic peripheral blood involvement is a clinicopathologic entity with an unfavourable prognosis. Report of three cases and review of the literature. Am J Clin Pathol 2003;120:617-25.  Back to cited text no. 7
Grewal JS, Smith LB, Winegarden JD, Krauss JC, Tworek JA, Schnitzer B. Highly aggressive ALK-positive anaplastic large cell lymphoma with a leukemic phase and multi-organ involvement: A report of three cases and a review of the literature. Ann Hematol 2007;86:499-508.  Back to cited text no. 8
Dalal BI, Chhanabhai M, Horsman DE, LeHuquet J, Coupland R. Anaplastic large-cell lymphoma presenting as acute leukemia. Am J Hematol 2005;79:164-5.  Back to cited text no. 9
Takahashi D, Nagatoshi Y, Nagayama J, Inagaki J, Itonaga N, Takeshita M, et al. Anaplastic large cell lymphoma in leukemic presentation: A case report and a review of the literature. J Pediatr Hematol Oncol 2008;30:696-700.  Back to cited text no. 10
Kesler MV, Paranjape GS, Asplund SL, McKenna RW, Jamal S, Kroft SH. Anaplastic large cell lymphoma: A flow cytometric analysis of 29 cases. Am J Clin Pathol 2007;128:314-22.  Back to cited text no. 11
Muzzafar T, Wei EX, Lin P, Medeiros LJ, Jorgensen JL. Flow cytometric immunophenotyping of anaplastic large cell lymphoma. Arch Pathol Lab Med 2009;133:49-56.  Back to cited text no. 12
Bhandari PL, Raghuveer CV, Rajeev A, Bhandari PD. Comparative study of peripheral blood smear, quantitative buffy coat and modified centrifuged blood smear in malaria diagnosis. Indian J Pathol Microbiol 2008;51:108-12.  Back to cited text no. 13
[PUBMED]  [Full text]  
Dass J, Mittal S, Gupta A, Radhakrisnan N, Bhargava M. Bone marrow buffy coat cell block for confirmation of isolated bone marrow relapse in medulloblastoma. Indian J Hematol Blood Transfus 2017;33:438-40.  Back to cited text no. 14

Correspondence Address:
Amit K Adhya
All India Institute of Medical Sciences, Bhubaneswar - 751 019, Odisha
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/IJPM.IJPM_433_19

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