Diagnosis of CLL by Dr Samir Agrawal Sep 2012

Diagnosis of CLL

Dr. Samir Agrawal MB ChB, BSc, FRCP, FRCPath, PhD Consultant Haematologist, St. Bartholomew’s Hospital, Barts Health NHS Trust, London; Blizard Institute, Queen Mary University of London.

Timothy Farren BSc (HONS) MIBMS, Clinical Scientist, St Bartholomew’s Hospital, Barts and The London School of Medicine, London.

The following article has been written in September 2012, exclusively for the CLL Support Association,  It is aimed at patients and their carers and assumes almost no prior medical knowledge.



A few words about chronic lymphocytic leukaemia (CLL) are necessary in order toexplain the diagnostic process. CLL is a malignancy of B lymphocytes, one of several types of cells of the immune system. Disorders of the other lymphocytes also occur – T cells and natural killer (NK) cells – but are much rarer. While CLL is the commonest adult leukaemia in the western world, there are many other types of malignancy of B lymphocytes, globally referred to as B-cell lymphoproliferative disorders (B-LPD).

As suggested by the term ‘leukaemia’, CLL is a disease characterised by an increase in the abnormal B-cells in the blood and bone marrow (BM). Rarely, CLL cells may not be detected in the blood (or BM), but only found in enlarged glands – lymph nodes (see below) – which is a form of CLL localised to the tissues and often referred to as small lymphocytic lymphoma (SLL). SLL is a subset of CLL and should be treated as any other case of CLL.

Despite CLL being termed a ‘leukaemia’, the most recent classification by the World Health Organisation (WHO) is as an indolent low grade lymphoma. The dominating feature of CLL is an increase in B lymphocytes in the blood – a ‘lymphocytosis’, however, infections may also lead to a lymphocytosis, usually of T or NK-cells, but also of B-cells. Most commonly, viral infections – which may or may not cause clinical symptoms – are the cause of a lymphocytosis, which resolves spontaneously with time.

Originally CLL was considered a disease of the elderly with limited chance of a successful remission following treatment. Whilst the median age at diagnosis is 71 years, more recently a new entity termed CLL-type Monoclonal B-cell lymphocytosis (MBL), in which cells biologically appear to be the same as in CLL, but with a far few number of cells in the blood (less than 5000 cells per microliter). For these patients active monitoring is recommended by means of a full blood count and physical examination by the consultant haematologist. In cases referred for subsequent investigation of a lymphocytosis, the risk of requiring treatment for progressive disease in approximately 1% per year.



Clinical features

The clinical presentation of CLL is entirely non-specific, with all the findings also seen in other B-LPD, as well as other malignant and non-malignant conditions. In fact, the commonest presentation of CLL today is as an ‘incidental’ finding on a routine blood test (full blood count) performed for some other reason, for example, prior to an operation or as part of a health check. In other words, the patients had no idea they were unwell! If patients do present because of the CLL itself, this may include: enlarged glands, which are called lymph nodes, in the neck, armpits, abdomen and groin (one or more areas may be affected); recurrent infections (related to the poorly functioning immune system in CLL); weight loss, fevers (not related to infection) and drenching night sweats – globally referred to as ‘B’ symptoms; excessive fatigue; a sensation of fullness in the abdomen (usually due to an enlarged spleen).


Full Blood Count

The full blood count (FBC) is one of the key tests in the diagnostic process and is the first step. The widespread availability of FBC analysers explains the increasing detection of CLL as an ‘incidental’ finding. The FBC reveals the increase in the lymphocytes in the blood – but cannot tell whether they are B-, T- or NK-cells. The other important information is the level of the haemoglobin (Hb) and platelets, as if they are low, this suggests bone marrow failure (usually due to the CLL) and is an indication for treatment. From the FBC sample, a drop of blood is spread on a glass slide, stained with dyes and examined under a microscope. This allows the appearance of the cells to be determined – CLL cells have a characteristic ‘clumped chromatin’ (which looks like cracked mud!) pattern to the nucleus (the central dark part of the cell) and from this a confident diagnosis of CLL can be made. BUT, some cases of CLL do not have this typical picture and equally other conditions can mimic CLL. The lymphocyte count derived from the FBC can provide a monitoring for the lymphocyte doubling time (LDT). This is defined as the time period during which the lymphocyte count is doubled and reflects the disease activity.


Down the microscope

CLL is diagnosed by looking at the cells in the blood down a microscope. The CLL cells have a classical picture being small dark purple blue cells, some of which are fragile and break when making the film termed ‘smear cells’. These smear cells are a hallmark feature of CLL. The red cells contain haemoglobin and function to provide oxygen to the surrounding tissues and organs. When there are not enough red cells – this is termed ‘anaemia’ which is used in determining clinical staging.


To confirm the diagnosis, additional techniques are required which may also be of prognostic significance. These additional tests are:



Having identified the lymphocytosis from the FBC, the next step is to determine the nature of the cells –B-, T- or NK-cells – in the blood. This is easily established by immunophenotyping. This test uses reagents (coloured antibodies) to label proteins on the cell surface. Specific antibodies only label specific proteins, so if an antibody sticks to a protein on a cell, it reveals which protein it is by emitting light. The machine that detects the cells is called a flow cytometer, which measures the light emitted when a laser beam hits the coloured antibody. In addition to proving the cells are B cells, immunophenotyping produces a profile of the different molecules expressed on the surface of the B-cells and establishes the typical pattern seen in CLL. This requires a panel of reagents, as no single marker can define a CLL cell, and the British Society of Haematology has published guidelines on this (see website for details 2002). It should be noted that different, but similar, panels may also be used to arrive at the same final conclusion. To assist in the diagnostic process with a panel of markers, Table 1 shows the five markers that constitute the so-called ‘CLL score’. A high CLL score of 4 or 5 indicates a true case CLL. However, this scoring system is increasingly being dropped as new antibodies have become available (van Dongen et al, {2012} Section 8 of “EuroFlow antibody panels for standardized n-dimensional flow cytometric immunophenotyping of normal, reactive and malignant leukocytes”. Leukemia; 26: 1908-1975). Recently, a tumour specific marker termed CD160 has been demonstrated to aid in the diagnosis of CLL and distinguishes it from other B-cell malignancies . A “mini CLL” score using CD160 offers a simplified diagnosis of CLL. (Farren TW et al {2011} Differential and tumor-specific expression of CD160 in B-cell malignancies. Blood; 118: 2174-83).


Table 1. Modified CLL scoring system
































A score of 4 or 5/5 supports a diagnosis of CLL.

Reference: Moreau EJ, Matutes E, A’Hern RP, et al (1997) Improvement of the chronic lymphocytic leukaemia scoring system with the monoclonal antibody SD8 (CD79b). Am J of Clin Pathol; 108: 378-382


Bone marrow

A bone marrow test does not have any role in the diagnosis of CLL. In general, a bone marrow biopsy is only performed if: treatment is to be started – as a baseline to then assess response; the platelet count is low and suggestive of idiopathic thrombocytopenia (ITP); anaemia is present and the blood film and Coomb’s test (or DAT) suggest autoimmune haemolytic anaemia (AIHA). Both ITP and AIHA are phenomena where the body destroys its own platelets and red cells, respectively, and such findings are relatively common in CLL. In such circumstances, a bone marrow biopsy is performed to help assess to what extent anaemia or a low platelet count are due to CLL infiltration of the bone marrow or to other causes. Characteristically, the bone marrow is comprised of more than 30% CLL cells.


Radiological imaging

Ultrasound and CT scanning allow more accurate documentation of the enlarged lymph nodes, liver and spleen – all possible features of CLL - than the physical examination. However, these tests do not provide diagnostic information and are usually onlynecessary as part of the work-up prior to instituting treatment.


Lymph node biopsy

A biopsy of an enlarged lymph node is not routinely performed in CLL, as the diagnosis is usually clear from analysis of the blood. In the majority of cases, a definitive diagnosis of CLL can be made from the FBC and immunophenotyping. However, if the cells in the blood are not typical under the microscope and the CLL score by immunophenotyping is not typical either, looking at the cells in a biopsy specimen under a microscope can be useful in definitively making a diagnosis – whether CLL or not.



The process of staging is calculated on a number of parameters derived from the above. In the UK, the Binet staging system is preferred. This system is based on the presence or absence of enlarged lymph nodes and other organs (such as spleen or liver), and whether there is a low haemoglobin and/or platelet count. The Binet stage provides the consultant with an idea on roughly how long a patient will be monitored before they require treatment.


Other tests at presentation

Other tests that can be performed at diagnosis are usually for prognostic purposes – see ‘Prognostic Factors’ also in this section on the website. Cytogenetic analysis (looking at the chromosomes in the cells) can also help in the diagnostic process in difficult cases. A number of cytogenetic changes are commonly detected in CLL and if present can support the diagnosis of CLL (as well as give prognostic information). Perhaps the most important role of cytogenetics in diagnosis is to specifically exclude mantle cell lymphoma (MCL) - which is generally a more aggressive disease than CLL. There can be significant diagnostic overlap between MCL and CLL, with either disease looking like the other, both under the microscope and by immunophenotyping!

In addition, another test performed by flow cytometry is ZAP-70, which correlates with patient prognosis and may affect patient management, although technical issues have dampened the initial enthusiasm for this marker.


Chromosomes analysed by cytogenetic analysis. Parts of one or more chromosomes may be deleted.



In general, the diagnosis of CLL is relatively straightforward and can be made confidently by a combination of the FBC, the appearances of blood cells under the microscope and definitively established by immunophenotyping. However, in difficult cases, a lymph node biopsy and cytogenetic analysis of blood or BM cells can help establish a definitive diagnosis. The commonest diagnostic confusion is with Mantle Cell Lymphoma and it is important to be aware of the overlap with CLL – with cytogenetics allowing separation of the two conditions. Diagnostic accuracy is essential, as all the prognostic markers available in CLL are only meaningful if the diagnosis really is CLL!

There are more and more markers being discovered to aid in the diagnosis of CLL where other techniques, such as cytogenetics, have failed. More recently, CD160 has been demonstrated to be a tumour specific marker for malignant B-cells and on going research is investigating into using this as a marker of low level disease following therapy (termed minimal residual disease). CD200 is another useful marker, which has proven beneficial in separating CLL from MCL.

All the above tests need to be performed not only for your diagnosis, but also for potential eligibility to enter into clinical trials.