الفهرس 
Chronic Lymphocytic Leukemia (CLL)
Patients and Methods .Only 14 pages are availabe for public view
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Abstract
Chronic lymphocytic leukemia (CLL) is considered the most common form of leukemia in adults
over sixty, and rarely occurs below forty. The CLL is a malignant proliferation of monoclonal
lymphocytes, most commonly of B-cell origin (B-CLL). It is characterized by accumulation of small,
mature appearing -functionally incompetent- lymphocytes in bone marrow (BM), peripheral blood
(PB) and lymphoid tissue. The diagnosis of CLL requires a lymphocyte count of 5000 or more per
cubic millimeter, and a characteristic cell surface phenotype of clonal B-cells; the presence of
CD19, CD5, CD23 and weak
expression of CD20, CD79b, and either kappa (κ) or lambda
(λ) immunoglobulin light chain (LC).
The flow cytometry (FCM) offers important advantages over competing laboratory technologies in
the diagnostic workup of CLL. The Ig (LC) expression analysis has a critical role in the FCM
evaluation of CLL. The presence of a distinct B-cell population expressing only one type of Ig
(LC) (κ) or (λ) essentially establishes a clonal B- cell process, and supports a diagnosis of CLL.
Few published studies have suggested that the choice of anti-LC antibody is
one of the main factors determining whether expression of surface LC can be demonstrated.
Currently, monoclonal ”mAbs” and polyclonal Abs ”pAbs” are commercially
available for the clinical assessment of LC expression by FCM, but neither has been consistently
shown to be superior to the other.
This study aimed to: 1) evaluate LC expression in CLL patients by FCM using monoclonal (mAbs) and
polyclonal (pAbs), 2) to evaluate and compare diagnostic utility, sensitivity and specificity of
both mAbs and pAbs and 3) to consider the use of one set for screening, with the addition of the
other set if LC expression is not identified initially.
This study included 40 newly diagnosed CLL adult patients who were attending the
Haematology/Oncology Unit of Ain-Shams University Hospitals, during the period from June 2013 to
July 2014. Written consent was taken from all CLL patients. Their ages ranged between 50-75 years,
with mean age of 61.82 ± 6.39 years. They were 23 (57.5%) males and 17 (42.5%) females, with male
to female ratio 1.4:1.
All the patients were subjected to thorough history taking and clinical examination (for
organomegaly and/or lymphadenopathy), complete blood count (CBC), with examination of
Leishman-stained smears, BM aspiration with morphological examination of Leishman-stained PB
smears, examination of biopsy specimens, including BM trephine biopsies and/or lymph nodes
histopathology in patients with accessible lymph node enlargement, immunophenotyping of
PB or BM samples using a standard panel of monoclonal antibodies (Matutes et al., 2012), and light
chain restriction (clonality) was assessed using mAbs and pAbs against (κ) and (λ) light chains
[eBioscience, San Diego, CA, USA] by FCM.
Regarding CBC parameters, the mean hemoglobin concentration ranged from 3.9 to 15.4 g/dl, with a
mean of
11.12 ± 2.29 g/dl. While median TLC was 37.0 x 109/L
(range 17-81 x 109/L), and platelet count ranged from 31 to
298 x 109/L, with a mean of 150.95 ± 74.48 x 109/L.
The LC restriction was detected in (34/40) (85%) of the presently studied CLL patients, (κ) LC
restriction was detected in (31/34) (91%) of studied positive CLL patients, pAbs (κ) were able to
detect clonality in 27/34 (79.5%) of patients, while mAbs (κ) detected clonality in 28/34 (82.5%)
of patients, with no statistically significant difference on comparing LC restriction with pAbs and
mAbs (P>0.05). Meanwhile, a highly statistically significant difference was detected on comparing
median expression of both pAbs (κ) and mAbs (κ) (80.1-41.7), respectively, being higher in pAbs
(κ).
On the other hand, (λ) LC restriction was detected in (3/34) (9%) of studied positive CLL patients
by both pAbs and mAbs, with no statistically significant difference regarding the number of CLL
patients or the median
expression. In (6/40) (15%) LC restriction by both pAbs and mAbs failed to prove clonality.
On comparing the diagnostic utility of pAbs versus mAbs in FCM detection of LC restriction in all
studied CLL patients, mAbs (κ + λ) were able to prove clonality in (31/40) (77.5%), while
pAbs proved clonality in (30/40) (75%), the difference was not statistically significant (p=
0.005). 85% of studied CLL patients (34/40) showed LC restriction by concurrent use of both Abs
(mAbs & pAbs). The difference was statistically highly significant (p= 0.001), signifying the
importance of concurrent use of both mAbs and pAbs.
A discrepant pattern of LC expression by 4-color FCM was documented in a small but significant
percentage of CLL patients. Clonality was detected in (3/40) of the studied CLL patients by pAbs
(κ) alone, versus (4/40) of patients by mAbs (anti-κ) alone. The difference was statistically
non- significant (p= 0.005). Meanwhile, a highly statistically
significant positive correlation was detected between both polyclonal and monoclonal (κ), and
polyclonal and monoclonal (λ) (r= 0.581 and r= 0.740, respectively).
On correlating pAbs and mAbs with CBC parameters, a positive correlation was found between (κ) mAbs
and TLC (r=
0.512, p= 0.001), and a negative correlation was documented
between (κ) mAbs and Hb (r= -0.346, p= 0.029). This
suggested that the mAbs (κ) positivity may have a prognostic significance. On the other hand, a
high statistically significant negative correlation was detected between pAbs (λ) and platelets (r=
-0.366, p= 0.020). This suggested that the pAbs (λ) positivity may have a prognostic significance.
Finally, the present study documented clonality -as proved by LC restriction- in 85% of studied CLL
patients, of which (κ) LC restriction was detected in 91% of patients and (λ) LC restriction was
detected in 9% of patients, with no statistically significant difference between the used pAbs
(positive in 75%) and mAbs (positive in 77.5%). Moreover,
the simultaneous use of both pAbs and mAbs proved clonality in 85% of CLL patients, with
statistically highly significant difference, high-lightening the importance of using both
reagents to increase the sensitivity of LC detection by FCM in CLL. Furthermore, a
highly statistically significant difference was documented on comparing the
median expression of both pAbs κ (80.1) and mAbs κ (41.7),
being higher in pAbs κ. Thus, the present results states that the dim LC expression κ, in CLL, is
best detected with pAbs. To reduce the cost, the use of pAbs κ for screening is considered first,
with the addition of the either mAbs (λ) or pAbs (λ), if LC expression was not identified
initially, instead of concurrent use of both surface antibody sets.