|Year : 2022 | Volume
| Issue : 1 | Page : 39-45
Chronic lymphocytic leukemia: Current approach to lab diagnosis
Devasis Panda, Narender Tejwani, Anurag Mehta
Department of Laboratory Medicine, Rajiv Gandhi Cancer Institute & Research Centre, New Delhi, India
|Date of Submission||19-Jul-2022|
|Date of Acceptance||26-Jul-2022|
|Date of Web Publication||02-Sep-2022|
Dr. Narender Tejwani
Department of Laboratory Medicine, Rajiv Gandhi Cancer Institute & Research Centre, Sir Chotu Ram Marg, Rohini Institutional Area, Sector 5, Rohini, New Delhi 110085
Source of Support: None, Conflict of Interest: None
Chronic lymphocytic leukemia (CLL) is the most common type of clinically encountered chronic lymphoproliferative disorder (CLPD). The disease was first found in 19th century and was described as asymptomatic leukemia which peacefully coexists with the host. The disease has an indolent clinical course and often picked up when complete blood count is done for an unrelated medical indication. CLL is a disease of elderly and the incidence increases as the age increases. However, Indian patient present early as compared to USA and Europe. There is a slight male preponderance reported worldwide. CLL is often preceded by a non-clinical phase called monoclonal B cell lymphocytosis (MBL) to which is closely related both immunophenotypically and spectrum of molecular aberrations. Recently describe low count MBL however appears to be a distinctive disease of unknown significance as there is not sufficient data to prove that they pose a higher risk of progression to MBL/CLL. Their genetic profile also is very different from MBL/CLL. The symptomatic cases may present with symptoms related to disease progression like cytopenias due to hematopoietic cells replacement or lymph node enlargement or liver/spleen enlargement. The symptoms may also be related to secondary autoimmune phenomena like autoimmune hemolytic anemia and autoimmune thrombocytopenia. The disease diagnoses mostly rely on the use of multi-parametric flowcytometry and peripheral blood morphology. The peripheral smear shows the presence of small cell lymphocytosis with numerous smudge cells. The flowcytometric immunophenotyping show loss of Pan-B cell markers like surface immunoglobulin, CD79b, CD22, and FMC-7. The expression of these markers is either negative or dimmer when compared to normal mature B cells. CD20 expression is also dim as compared to normal mature B cells. Along with the loss of Pan-B cell markers, there is a gain of CD5 (A T cell markers) and CD23. CD200 has emerged as a very useful marker to differentiate this neoplasm from Mantle cell lymphoma which characteristically is CD5 positive, does not show loss of Pan-B cell markers and is negative or Dim positive for CD200. The other differential diagnosis remains other causes of small cell CLPDs like hairy cells, marginal zone lymphomas, lympho-plasmacytic lymphoma, and follicular lymphomas. These cases can be easily differentiated when morphology is combined with flow cytometric findings. In difficult cases, one can use Matutes score where a score of 4 or 5 will indicate a diagnosis of CLL.
Keywords: CLL, CLPD, flow cytometry
|How to cite this article:|
Panda D, Tejwani N, Mehta A. Chronic lymphocytic leukemia: Current approach to lab diagnosis. J Curr Oncol 2022;5:39-45
| Introduction|| |
Chronic lymphoproliferative disorder (CLPD) constitutes a group of mature lymphoid neoplasms that primarily present with the involvement of peripheral blood, bone marrow, spleen, and liver. Involvement of lymph nodes or tissues is not seen in the initial stages of these neoplasms and may present later as a part of disease progression. These neoplasms can arise from both B cells and T cells. However, B-cell CLPDs are most common. Approximately 90% of all CLPDs are B cell in origin and chronic lymphocytic leukemia (CLL) is the most common among them. CLL is a disorder of old ages, twice as common in males compared with females, and often indolent in its clinical behavior. In our country where routine preventive health checkup is not a norm, these disorders are often picked up when complete blood count (CBC) is done for an unrelated medical condition. The disorder was first identified in the middle of nineteenth century and even than it was well recognized that this disorder mostly remains asymptomatic. Bethel et al. in 1955 described this as “leukemia may be said to coexist peacefully with its host.”
The disorder in current ages is recognized as a continuum of a spectrum that ranges from monoclonal B-cell lymphocytosis-low counts (MBL-LC) to MBL to full-blown CLL. Most of the patients with CLL progress through a phase of MBL. Although in our country most of the patients are diagnosed only when they have progressed to CLL, immunophenotypic studies are mostly not done for mild asymptomatic lymphocytosis. The cases of MBL have a 1%–2% risk of progression to CLL every year. These are the patients who require regular follow-up.
MBL-LC is still a controversial entity and no definitive link of progression to MBL or CLL has been confirmed till now. MBL-LC has the same immunophenotype as of MBL/CLL but in many instances they are found to be polyclonal/oligoclonal. They are also shown to use a different set of immunoglobulin heavy chain gene (IGHV) genes when compared with CLL. CLL is characterized by preferential usage of a restricted group of IGHV such as IGHV1-69, IGHV4-34, IGHV3-7, and IGHV3-23. CLL-LC cells rarely use these group of IGHV genes. Moreover, stereotyped receptors are rare in CLL-LC, whereas they are found in approximately 30% of the CLL cases.
| Incidence and Risk Factors|| |
CLL is a disease of adults and is often seen in patients over 60 years of age. The median age of diagnosis is ~70 years in West and Europe. In Indian population, the disease presents almost a decade earlier than it does in the USA with an incidence of 0.41 cases per lakh. This incidence is almost 10 times less as compared with the USA where CLL is the most common type of leukemia. However, due to the large population absolute number of new cases and prevalence is approximately three times.
Risk factors associated with development are few and almost none of them are preventable. The most important factors that predispose to CLL are increasing age, gender (more common in males), family history, and ethnicity. The incidence of disease is lower in Asian countries as compared with West and Europe. Migration studies have indicated that this difference in incidence is more attributable to environmental factors than genetics. There are also some links to the development of CLL in patients who are exposed to pesticides.,
| Clinical Presentation|| |
Most of the patients with CLL are asymptomatic as discussed earlier and diagnosis is often made after lymphocytosis found on CBC done for an unrelated condition. Some of the patients also present after they feel painless swelling of nodes (often in the cervical area). Approximately 10% of these patients have B symptoms at the time of diagnosis.
There can sometimes be uncommon presentations such as severe anemia or bleeding due to thrombocytopenia. This happens due to the presence of underlying autoimmune hemolytic anemia or immune thrombocytopenia.
On physical examination, nontender lymph-adenopathy is found in the majority of these patients. Most common group affected is the cervical and axillary region. Mostly the lymph nodes are firm and discreet., Spleen is the second most commonly affected lymphoid organ after lymph node and mild splenomegaly is seen in nearly a quarter of the patients. Hepatic enlargement is also common; however, other organ involvement is rare at initial diagnosis.,
| Laboratory Findings|| |
The most striking finding on CBC and peripheral smear is the presence of lymphocytic leukocytosis. The lymphocytes are usually small and resemble small mature lymphocytes morphologically. On Giemsa smears, the cells are small with scant cytoplasm and their nucleus roughly equal to the size of normocytic red cells. Nucleus is mostly rounded and the chromatin shows peculiar alternating light and dark areas (heterochromatin and euchromatin) giving it a checkerboard or football-type appearance. The cells are fragile and often get ruptured when they are spread on slide. These cells are seen as a bare disturbed nucleus on Giemsa stained smears and are called basket cells or smudge cells [Figure 1].
|Figure 1: Peripheral smear from a case of CLL showing small mature type lymphoid cell proliferation. Smudge cells (white arrows) are seen as a result of damage during smear spreading|
Click here to view
There can be a few pro-lymphocytes seen which will constitute <55% of the total lymphocytes. These cells show mild cellular and nuclear enlargement with prominent central nucleoli [Figure 1], inset].
Hemoglobin and platelets are usually not affected at the time of initial diagnosis. There are two important causes of anemia and thrombocytopenia in patients with CLL. One of the causes is the presence of underlying autoantibody causing autoimmune hemolytic anemia (AIHA) or autoimmune thrombocytopenia. A reticulocyte count can help to identify underlying AIHA in event of anemia. Peripheral smear done will show the presence of polychromasia and spherocyte. The antibody is most commonly IgG type; however, IgM type antibody is also not uncommon. Coombs test with anti-IgG and anti-C3d can be used to confirm the diagnosis. The diagnosis of autoimmune thrombocytopenia requires a bone marrow examination to show the presence of adequate or increased megakaryocytes. Both of these conditions are usually managed initially with steroids alone. The second cause of anemia and thrombocytopenia is disease progression leading to the replacement of hematopoiesis. Bone marrow done in these cases will show reduced hematopoietic elements due to replacement by small lymphoid cells. This group of patients is usually managed by combination chemotherapy. Pure red cells aplasia and agranulocytosis are rare complications and are seen in <1% of the cases.[6
| Bone Marrow Aspirate and Biopsy|| |
Bone marrow aspirate and biopsy are not compulsory to make a diagnosis of CLL as most of the investigations including FISH and IGHV mutations can be done on the peripheral blood itself. However],[ sometimes marrow may be done to identify the cause of thrombocytopenia. The cases with the immunological cause of thrombocytopenia will show normal to increased megakaryocytes in the marrow. Bone marrow aspirate invariably shows >30% lymphocytes and the pattern of infiltration on biopsy may be interstitial or nodular or diffuse. The diffuse pattern resembles the progression of the disease.,
| Flow-cytometry Findings|| |
Diagnosis of CLL requires persistent peripheral blood lymphocytosis of >5000 per cumm with a characteristic immunophenotype.,,
| Flow-cytometric Analysis|| |
The immunophenotype of CLL also has some very interesting findings such as it tries to lose most of the mature B-cell markers and instead gains a T-cell associated marker that is CD5. It may be noted that the percentage of the abnormal cells in flow cytometry may not be the same as in morphological analysis. This is both due to loss of the smudge cells and loss of fragile cells at the time of flow-cytometric processing. The following are the salient immunophenotypic findings:,
- Loss of Pan-B-cell markers including CD79b, CD22, FMC 7, and surface immunoglobulins. All these markers are negative or show dim fluorescence as compared with their normal counterparts. CD20 is often ignored when CLL is discussed; however, there is a characteristic downregulation of CD20 also in CLL cells [Figure 2].
- Gain of T-cell markers and CD23: CLL is a B-cell neoplasm that characteristically expresses CD5 (A T-cell antigen). The CD5 expression however is almost always weak as compared with normal T cells. CLL cells also show expression of CD23, which is a type of IgE receptor expressed on few normal mature B cells and also on a variety of other cells such as macrophages, eosinophils, megakaryocytes, and platelets.
- Role of CD200: There are two very important differential diagnoses when CD5 positive B-cell CLPDs are discussed. The diagnoses are CLL and mantle cell lymphoma/leukemia (MCL)., Historically Matutes score was used to differentiate between these two diagnoses. A score of 4 or 5 was required to call something as CLL [Table 1].
|Figure 2: Classical flow-cytometry findings in chronic lymphocytic leukemia. There is dim or absent expression of almost all B-cell markers including CD20 and CD19 on abnormal cells (red) when compared with normal B cells (green). CD5 expression also is dimmer as compared with normal T cells (blue) in sample|
Click here to view
|Table 1: Matutes score for CLL: A score of 4 or 5 is required for making a diagnosis of CLL|
Click here to view
The scoring system is very effective in differentiating MCL from CLL. However, there were a few drawbacks related to this methodology. First is a variable result for surface immunoglobulin depending on whether monoclonal or polyclonal antibody is used. Second is an inconsistent result with CD23 antibody across various manufacturers and clones of the same manufacturers. Irrespective of the score, most of the pathologists would do a Cyclin D1 antibody immunohistochemistry (IHC) to rule out mantle cells lymphoma. This process is both time-consuming and often required to do a bone marrow biopsy (If cell block preparation was not available).
In the current era, many centers are getting more and more dependent on CD200 to differentiate between the two. Moderate-to-bright expression of CD200 associated with CD5 expression is a characteristic signature for CLL [Figure 2] and [Figure 3].
|Figure 3: Classical immunophenotype in a case of mantle cell lymphoma. Note the absence of loss of Pan-B-cell markers as compared with CLL|
Click here to view
- 4. ZAP-70, CD38, and CD49d: These three markers are considered to be the surrogate markers of IGHV-unmutated phenotype. These markers do not directly test for the IGHV mutation but their expression is associated with unmutated phenotype of the CLL clones present. CD49d also is an independent strongest flow-cytometry-based predictor of treatment-free survival and overall survival.
| Differential Diagnosis|| |
The differential diagnosis of CLL comprises most of the small cell CLPDs and other non-Hodgkin lymphomas. The most important differential diagnosis on the flow cytometry is MCL and often marrow biopsy/cell block will be required for Cyclin D1/SOX11 IHC to differentiate.,
| Mantle Cell Lymphoma/Leukemia|| |
MCL can arise as a lymphoma outside the bone marrow, spleen, and liver or may present as CLPD with minimal nodal/tissue involvement. The cells are usually small in size and often show a small notch on the nucleus. These cases will have a characteristic immunophenotype and will not show downregulation of Pan-B-cell markers such as CD20, CD79b, CD22, FMC-7, and light chains. CD200 is another very useful marker that is consistently negative in most of these cases while remaining positive in CLL.
| Pro-Lymphocytic Lymphoma|| |
B-cell pro-lymphocytic lymphoma (PLL) is mostly a diagnosis that is based on morphology. These cases may arise de novo or may present as a part of progression in a case of CLL. The characteristic morphology with prominent central nucleoli [Figure 1], inset] is the primary clue to the diagnosis. The immunophenotype of these cases shows the presence of Pan B-cell markers like MCL. CD200 is also expressed weakly or remains negative like MCL. Thus, most of these cases need to get an IHC for Cyclin D1 and SOX11 if the patient is not a follow-up known case of CLL. The term however has now been removed from the recent fifth update of the World Health Organization (WHO) classification.[20
| Lympho-Plasmacytic Lymphoma|| |
Lympho-plasmacytic lymphoma (LPL) is another type of small-cell CPD that presents with an indolent course like CLL. LPL is called Waldenström macroglobulinemia when associated with an IgM-type monoclonal protein on electrophoresis/immunofixation. Four overlapping features are often present between CLL and LPL. First as explained earlier is the presence of indolent course],[ second is the expression of CD5 in a minority of cases],[ and third is the presence of mild plasmacytic differentiation in a small group of patients with CLL and presence of M protein in patients with CLL. The differentiation can be done based on the flow-cytometric findings where CD20],[ surface light chains],[ and IgM will be strongly expressed in LPL with the absence of CD23. In difficult cases],[ an MYD88 mutation analysis could be done. MYD88 mutations are present in >95% of these cases.,
| Follicular Lymphoma|| |
Peripheral blood lymphocytosis is uncommon in patients with follicular lymphoma (FL). The rare cases where peripheral blood involvement is seen will show the prominence of small lymphoid cells with a deeply cleaved nucleus which will give a coffee bean appearance. The flow cytometry is also very characteristic as these cases are CD10 positive and also show expression of Pan-B-cell markers. These cases are positive for CD10 and negative for CD5 and do not enter a differential diagnosis of CLL on flow cytometry. The marrow biopsy of these cases will show a very characteristic peri-trabecular band-shaped infiltration (not to be confused with peri-trabecular nodules) [Figure 4].
|Figure 4: Characteristic band-shaped peri-trabecular small cell infiltration in a known case of FL|
Click here to view
| Hairy Cell Leukemia and Splenic Marzinal Zone Lymphoma|| |
Hairy cell leukemia (HCL) and splenic marzinal zone lymphoma (SMZL) are another group of CLPDs that present with primarily small cell phenotype. Most of these cases will have a clinical and immunophenotypic picture that is very much different from CLL. The most important clinical differentiating feature is the presence of marked splenomegaly in most of these cases along with negativity for both CD5 and CD10. Both of these cases will show the presence of Pan-B-cell markers. In addition, HCL will show the presence of characteristic positivity for CD123, CD11c, CD25, and CD103. Most of the patients with HCL in addition will show characteristic abundant cytoplasm with projections. These projections may be very subtle in cases of SMZL or may be seen confined to two opposite poles of the cell.,,
| Summary|| |
CLPDs are a heterogeneous group of hematolymphoid malignancies, characterized by the involvement of peripheral blood or marrow, or spleen very early in its clinical course. CLL is the most common type of CLPD. CLL is often preceded by a nonclinical phase called MBL where the patient is asymptomatic and the abnormal cell counts in peripheral blood remain in the range of 500–5000 per cumm. Recently describe low count MBL however appears to be a distinctive disease of unknown significance. There is no definitive evidence till date that low count MBL pose as a risk for progression to MBL/CLL. Their genetic profile also is very different from MBL/CLL as they use different sets of IGHV genes and do not show stereotypy.
Patients with CLL often are asymptomatic and the disease is often pickup when CBC is done for an unrelated condition. The symptomatic cases may present with symptoms related to disease progression like cytopenias due to hematopoietic cell replacement or lymph node enlargement or liver/spleen enlargement. The symptoms may also be related to secondary autoimmune phenomena such as AIHA and autoimmune thrombocytopenia.
Laboratory diagnosis is often quite straight when a combined approach with peripheral smear morphology and flow-cytometric immunophenotyping is used. The peripheral smear shows the presence of small cell lymphocytosis with numerous smudge cells. Marrow is usually not required for diagnosis but may be done to evaluate the cause of cytopenia present in peripheral blood. The flow-cytometric immunophenotyping is very characteristic as the disease show loss of Pan-B-cell markers such as surface immunoglobulin, CD79b, CD22, and FMC-7. The expression of these markers is either negative or lesser when compared with normal mature B cells. CD20 expression is also dim as compared with normal mature B cells. Along with the loss of Pan-B-cell markers, there is a gain of CD5 (A T-cell markers) and CD23. CD200 has emerged as a very useful marker to differentiate this neoplasm from MCL which characteristically is CD5 positive, does not show loss of Pan-B-cell markers, and is negative or dim positive for CD200.
The other differential diagnosis remains other causes of small-cell CLPDs such as hairy cells, marginal zone lymphomas, LPL, and FLs. These cases can be easily differentiated when morphology is combined with flow cytometric findings. In difficult cases, one can use Matutes score where a score of 4 or 5 will indicate a diagnosis of CLL.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
| References|| |
Swerdlow SH, Campo E, Pileri SA, Harris NL, Stein H, Siebert R, et al
. The 2016 revision of the World Health Organization classification of lymphoid neoplasms. Blood 2016;127:2375-90.
Litz CE, Brunning RD Chronic lymphoproliferative disorders: classification and diagnosis. Baillieres Clin Haematol 1993;6:767-83.
Al-Katib AM, Mohamed AN Non-Hodgkin’s lymphoma and chronic lymphocytic leukemia. In: Runge MS, Patterson C, editors. Principles of molecular medicine. [Internet]. Totowa, NJ: Humana Press; 2006. p. 794–803. Available from: https://doi.org/10.1007/978-1-59259-963-9_82
Gribben JG Chronic lymphocytic leukemia: an historical perspective. Hematol Am Soc Hematol Educ Program 2008;2008:443.
Swerdlow SH, Campo E, Harris NL, Jaffe ES, 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. 585. (World Health Organization classification of tumours).
Vardi A, Dagklis A, Scarfò L, Jelinek D, Newton D, Bennett F, et al
. Immunogenetics shows that not all MBL are equal: the larger the clone, the more similar to CLL. Blood 2013;121:4521-8.
Dali N, Ait Ali H, Tibiche A, Belhadri F, Harieche F, Ahmed Nacer R, et al
. Epidemiology and clinical features of chronic lymphoid leukemia: review of the Algerian Chronic Lymphoid Leukemia Study Group. Blood 2015;126:5274.
Smith A, Howell D, Patmore R, Jack A, Roman E Incidence of haematological malignancy by sub-type: a report from the haematological malignancy research network. Br J Cancer 2011;105:1684-92.
Lad DP, Tejaswi V, Malhotra P, Varma N, Sachdeva MS, Naseem S, et al
. Establishment of a comprehensive chronic lymphocytic leukemia clinic at a tertiary referral center in India. Blood Adv 2018;2:33-4.
Haenszel W, Kurihara M Studies of Japanese migrants. I. Mortality from cancer and other diseases among Japanese in the United States. J Natl Cancer Inst 1968;40:43-68.
Yanagihara ET, Blaisdell RK, Hayashi T, Lukes RJ Malignant lymphoma in Hawaii–Japanese: a retrospective morphologic survey. Hematol Oncol 1989;7:219-32.
Hallek M, Cheson BD, Catovsky D, Caligaris-Cappio F, Dighiero G, Döhner H, et al
. iwCLL guidelines for diagnosis, indications for treatment, response assessment, and supportive management of CLL. Blood 2018;131:2745-60.
Rai KR, Sawitsky A, Cronkite EP, Chanana AD, Levy RN, Pasternack BS Clinical staging of chronic lymphocytic leukemia. Blood 1975;46:219-34.
Binet JL, Auquier A, Dighiero G, Chastang C, Piguet H, Goasguen J, et al
. A new prognostic classification of chronic lymphocytic leukemia derived from a multivariate survival analysis. Cancer 1981;48:198-206.
Diehl LF, Ketchum LH Autoimmune disease and chronic lymphocytic leukemia: autoimmune hemolytic anemia, pure red cell aplasia, and autoimmune thrombocytopenia. Semin Oncol 1998;25:80-97.
El Din Fouad NB, Ibrahim NY, Abdel Aziz RS, Ibrahim SK Cd200 expression in diagnostic and prognostic assessment of mature B-cell lymphophoproliferative neoplasms. Asian Pac J Cancer Prev 2018;19:3383-92.
Köhnke T, Wittmann VK, Bücklein VL, Lichtenegger F, Pasalic Z, Hiddemann W, et al
. Diagnosis of CLL revisited: increased specificity by a modified five-marker scoring system including Cd200. Br J Haematol 2017;179:480-7.
Haq H, Uddin N, Khan SA, Ghaffar S Prognostic markers in chronic lymphocytic leukaemia: a flow cytometric analysis. Pak J Med Sci 2020;36:338-43.
Bulian P, Shanafelt TD, Fegan C, Zucchetto A, Cro L, Nückel H, et al
. Cd49d is the strongest flow cytometry-based predictor of overall survival in chronic lymphocytic leukemia. J Clin Oncol 2014;32:897-904.
Alaggio R, Amador C, Anagnostopoulos I, Attygalle AD, Araujo IBO, Berti E, et al
. The 5th edition of the World Health Organization classification of haematolymphoid tumours: lymphoid neoplasms. Leukemia 2022;36:1720-48.
Alobeid B, Mears JG, Bhagat G Follicular lymphoma (in situ
) pattern in the bone marrow: Does it indicate an early stage in disease evolution? Clin Case Rep 2015;3:442-3.
[Figure 1], [Figure 2], [Figure 3], [Figure 4]