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ß+ Large Granular Lymphocytes
From the Serviço de Hematologia Clínica,*
Unidade de Citometria, Hospital Geral de Santo António, Porto,
Portugal; and the Servicios de
Citometría
y
Hematología,
Hospital Universitario
de Salamanca and Centro de Investigación del Cáncer,
Universidad de Salamanca, Salamanca, Spain.
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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ß+ LGLs
and compared the results with those obtained in molecular studies of
TCR-ß gene rearrangements. Fifty-eight cases were considered to be
monoclonal in molecular studies whereas in the remaining 40 cases there
was no evidence for monoclonality (11 cases were considered oligoclonal
and 29 polyclonal). The TCR-Vß repertoire was biased to the
preferential use of one or more TCR-Vß families in 96% of
cases, a total of 124 TCR-Vß expansions being diagnosed: one
TCR-Vß expansion in 71 cases and two or more TCR-Vß expansions in
23 cases. The highest TCR-Vß expansion observed in each case was
higher among monoclonal (74 ± 19%) as compared to nonmonoclonal
cases (24 ± 14%) (P = 0.001), as did
the fraction of LGLs that exhibited a TCR-Vß-restricted pattern
(86 ± 16% and 42 ± 23%, respectively;
P = 0.0001); by contrast, the proportion of
cases displaying more than one TCR-Vß expansion was higher in the
latter group: 7% versus 48%, respectively
(P = 0.001). Results obtained in oligoclonal cases
were intermediate between those obtained in polyclonal and monoclonal
cases and similar results were observed for CD4+ as
for CD8+bright T-cell expansions. TCR-Vß
familiesexpressed in CD8+bright T-cell-LGL
proliferations showed a pattern of distribution that mimics the
frequency at which the individual TCR-Vß families are represented in
normal peripheral blood T cells. Assuming that a given proliferation of
LGLs is monoclonal whenever there is an expansion of a given TCR-Vß
family of at least 40% of the total CD4+ or
CD8+bright T-cell compartment, we were able to
predict clonality with a sensitivity of 93% and a specificity of 80%.
By increasing the cut-off value to 60%, sensitivity and
specificity were of 81% and 100%. In summary, our results
suggest that flow cytometry immunophenotypic analysis of the TCR-Vß
repertoire is a powerful screening tool for the assessment of T-cell
clonality in persistent expansions of TCR-ß+
LGLs.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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ß represent the majority of cases,
whereas TCR-
+ T-LGL leukemias are
relatively infrequent. From the clinical point of view whereas most
clonal T-LGL leukemias show a benign clinical outcome, in some cases
they behave as an aggressive condition. Either transient or persistent
expansions of polyclonal, oligoclonal, or even monoclonal LGLs are
relatively common in various disease conditions and such expansions of
LGLs have also been reported in otherwise normal healthy
individuals,3,4
although their neoplastic nature remains
unclear.5,6
In any case, at present, a major challenge in
the initial diagnosis of LGL leukemias is to establish the clonal
nature of the expanded population of LGLs, to distinguish between
polyclonal, oligoclonal, and monoclonal LGL proliferations.
Molecular investigation into the existence of monoclonal rearrangements
of the TCR-ß and TCR- genes, using Southern blot analysis is the
preferred method for investigation of T-cell clonality.7
Despite the high reliability of the Southern blot, this method has some
drawbacks that limit its routine use in diagnostic laboratories: it is
a labor-intensive and time-consuming method and large quantities of
high-quality DNA are needed to obtain reliable results. In recent years
alternative approaches have been developed for the assessment of T-cell
clonality. Of them, the immunophenotypic analysis of the repertoire of
the variable (V) regions of the TCR-, -ß, -, and - chains
represent one of the most attractive options. In humans, the V and
Vß gene segments are estimated to contain 
46 and 52 different
functional members that, based on nucleotide homology, can be grouped
into 32 and 25 different families, respectively.8,9
Polymerase chain reaction using TCR-Vß- and TCR-V-specific primers
and, more recently, flow cytometry using TCR-Vß- or TCR-V-specific
monoclonal antibodies (mAbs) can be used to investigate the TCR-Vß
and TCR-V gene usage. Flow cytometry is not only routinely available
in many laboratories but also offers several advantages: 1) it allows
both a quantitative and qualitative characterization of the T-cell
repertoire; 2) the T-cell repertoire can be specifically evaluated
within the population of interest by combining TCR-Vß and TCR-V
specific with other mAbs; and 3) a large panel of mAbs against
Vß-region determinants is now commercially available, making it
possible to access a large fraction of the T-cell repertoire. Although
the analysis of the TCR-V repertoire is now being used to indirectly
assess T-cell clonality, the finding of an expansion of T cells
restricted to a particular V-region family, does not necessarily mean
the presence of underlying monoclonal TCR gene rearrangements. Thus,
studies in which immunophenotyping is compared with molecular methods
are necessary to establish the value of the TCR-V repertoire analysis
in investigating T-cell clonality.
Several studies have already been performed aimed at the
characterization of both TCR-Vß and TCR-V T-cell repertoires from
normal healthy individuals10-12
and the T-cell responses
occurring in various pathological conditions, including
autoimmune13-15
and infectious diseases,16
tumors,17-19
allogeneic transplants,20
and
other disease states.21
However, only a few reports on the
TCR-Vß or TCR-V repertoire in LGL leukemias are available and,
furthermore, in the majority of these studies the numbers of TCR-Vß
families analyzed were limited.22-27
Some of these
studies showed that several specific TCR-Vß and TCR-Jß genes are
randomly used,23,24,27
whereas others suggested a
preferential usage of a few TCR-Vß regions in LGL
leukemias.22,24
To the best of our knowledge, until now no
study has been reported in which the utility of using a relatively
broad panel of anti-Vß mAbs for the assessment of T-cell clonality in
a large series of consecutive individuals showing a persistent
expansion of peripheral blood (PB) TCR-ß+
LGLs has been evaluated in comparison to molecular techniques.
The aim of our study was to characterize the TCR-Vß repertoire from a
group of 98 consecutive patients displaying a persistent expansion of
either CD4+ or CD8+
TCR-ß+ T-LGLs in the PB and to establish its
utility in the diagnosis of clonality. For that purpose, a large panel
of 23 mAbs directed against 24 members of 19 different TCR-Vß
families was used and these results were compared with those obtained
with conventional molecular techniques.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Ninety-eight consecutive patients with persistent
TCR-ß+ T-cell proliferations of LGLs were
studied (51 males and 47 females; ages 12 to 90 years; median, 61
years). The diagnosis was established on the basis of a LGL morphology,
together with a typical
CD3+/TCR-ß+ LGL
phenotype, as defined by high FSC/SSC values, absence of
expression of CD28, and reactivity for NK-associated
antigens.2,3
From the phenotypic point of view, LGL
proliferations were classified into two groups:
TCR-ß+/CD8+bright/CD4-
(n = 72 cases) and
TCR-ß+/CD4+/CD8-/+dim
(n = 26 cases). The mean absolute number of LGLs
in the PB was 2856 ± 3086 x 106/L
(median, 1725 x 106/L) and the mean
percentage of LGLs within the CD4+ or
CD8+bright T-cell population was of 78 ±
19% (median, 83%). Increased numbers of LGLs were associated with
absolute lymphocytosis (>3.5 x 109/L) in
60 cases (61%). Neutropenia (<1.5 x
109/L) was observed in 34 cases (35%), anemia
(Hb < 10 g/dl) in 13 cases (13%), and thrombocytopenia
(<100 x 109/L) in 11 cases (11%).
Organomegalies were rarely detected (<10% of cases). Median follow-up
for the patients analyzed was of 29 months. During this period, 37
patients (38%) showed associated conditions, including autoimmune
disorders (14 cases), neoplastic diseases (15 cases), or other disease
states (8 cases). Ten age- and sex-matched healthy individuals (six
males and four females; median age, 47 years) were used as controls.
Immunophenotypic Studies
Ethylenediaminetetraacetic acid-anti-coagulated PB samples were stained using a direct immunofluorescence technique. Briefly, 100 µl of whole PB containing between 0.5 to 2.0 x 106 nucleated cells were incubated with saturating concentrations of mAbs for 15 minutes at room temperature in the dark. Then 2 ml of fluorescence-activated cell sorting lysing solution (Becton Dickinson, San Jose, CA) were added to lyse nonnucleated red cells. After another 10 minutes incubation at room temperature in the dark, cells were washed once and resuspended in 0.5 ml of phosphate-buffered saline (PBS).
The repertoire of the Vß chain of
TCR-ß+/CD8+bright/CD4-
and
TCR-ß+/CD4+/CD8-/+dim
lymphocytes was analyzed by combining either anti-CD4 or anti-CD8 with
the following panel of 23 mAbs specific against 24 members of a total
of 19 families of variable regions of the TCR-ß chain:
BV1S1 (Vß1.1), BV5S1 (Vß5.1), BV5S2 (Vß5.2), BV5S3 (Vß5.3),
BV6S1 (Vß6.1), BV7S1 (Vß7.1), BV8S1 + BV8S2 (Vß8.1 + 8.2), BV9S1
(Vß9.1), BV11S1 (Vß11.1), BV12S2 (Vß12.2), BV13S1 (Vß13.1),
BV13S6 (Vß13.6), BV14S1 (Vß14.1), BV16S1 (Vß16.1), BV17S1
(Vß17.1), BV18S1 (Vß18.1), BV20S1 (Vß20.1), BV21S3 (Vß21.3),
BV22S1 (Vß22.1), BV23S1 (Vß23.1) (Beckman-Coulter Immunotech,
Marseille, France), BV2S1 (Vß2.1) (Beckman-Coulter Immunotech or
Biodesign International, Kennebunk, ME), BV3S1 (Vß3.1)
(Beckman-Coulter Immunotech or Endogen, Woburn, MA) and BV6S7 (Vß6.7)
(Endogen). All anti-TCR-Vß reagents were tested in all samples except
for the anti-Vß1.1, -Vß6.7, -Vß7.1, and -Vß9.1 that were tested
in only 62%, 42%, 57% and 60% of the cases, respectively. In all
cases, isotype-matched fluorochrome-conjugated nonspecific
immunoglobulins were used as negative control.
Data acquisition was performed in two FACSCalibur flow cytometers (BD) using the Cell Quest software program (BD). Information on a minimum of 2 x 105 events was acquired for each reagent combination. Data analysis was performed using the Paint-a-Gate PRO software program (BD). For each TCR-Vß family, the proportion of positive cells within the CD8+bright or CD4+ lymphocytes was calculated as the percentage of cells stained above the negative isotype control value.
For those TCR-Vß families that were assessed with the panel of mAbs used in this study (direct identification), we considered that there is a TCR-Vß expansion whenever its representation exceeded by at least two standard deviations the mean value observed in CD4+ or CD8+ circulating T cells in normal healthy individuals.16 For the remaining TCR-Vß families that were not explored with the panel of mAbs used in this study, criteria used to define a TCR-Vß expansion (indirect identification) was based on the observation of a relative decrease in the fraction of either CD4+ or CD8+bright circulating T cells that were recognized with the panel of mAbs to values <85% of those observed for the same T-cell subsets in PB samples from normal adult individuals. These criteria rely on previous studies demonstrating that a value of 15% should be considered a T-cell expansion.28,29
Molecular Biology Studies
Rearrangements of the TCR-ß chain genes were evaluated by
conventional Southern blotting30
in all cases. Briefly,
mononuclear cells were obtained after fractionation on a Lymphoprep
(Nycomed Pharma AS, Oslo, Norway) density gradient, washed twice in
PBS, and cryopreserved. DNA was extracted using the
phenol/chloroform method and digested with EcoRI and
HindIII restriction enzymes. DNA fragments were
separated by 0.8% agarose gel electrophoresis and transferred to
nitrocellulose membranes by vacuum blotting, UV fixed, and hybridized
with 32P-labeled probes for the TCR-ß gene
region (Cß, TCRBC, and TCRBJ2; DAKO A/S, Glostrup, Denmark). In those
cases (n = 5) in which the
CD4+ or CD8+bright LGL
population represented <10% of the total nucleated cells present in
the sample and because all TCR-ß+ T-cell
malignancies have rearranged TCR- genes,31
the
clonality studies were performed by polymerase chain reaction analysis
of TCR- gene rearrangements, using the strategies and primers
previously described.32
Statistical Methods
For all variables under study, median, mean, SD, and range values were calculated. Comparison between groups was calculated using the Mann-Whitney U and chi-square tests for continuous and dichotomic variables, respectively (SPSS 9.0; SPSS, Chicago, IL). P values <0.05 were considered to be associated with statistically significant differences.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The panel of 23 mAbs used in the present work allowed us to
identify 60.3 ± 4.3% (range, 53.3 to 65.5%) and 46.1 ±
5.6% (range, 37.4 to 53.5%) of all CD4+ and
CD8+bright T cells in the PB of 10 normal healthy
controls. The specific distribution of each of the different TCR-Vß
families on both CD4+ and
CD8+bright T cells from normal individuals is
displayed in Table 1
. As shown in this
table, certain TCR-Vß families are represented more than others,
either within the CD4+ or the
CD8+bright normal T lymphocytes. Overall, the
relative distribution of the different TCR-Vß families within the
CD4+ and CD8+ subsets was
comparable; however, some TCR-Vß families were preferentially
expressed either within the CD4+ or the
CD8+bright subsets, with differences only
statistically significant for a preferential expression of TCR-Vß2.1,
-Vß5.1, -Vß5.3, -Vß6.7, -Vß12.2, -Vß13.1, -Vß18.1,
-Vß20.1, and -Vß22.1 on CD4+ T cells and
TCR-Vß7.1 on CD8+bright T cells. Molecular
studies showed a polyclonal pattern for the TCR-ß gene rearrangements
in all control individuals.
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From the molecular point of view, 58 of the 98 cases studied (59%) were found to be monoclonal by either Southern blot and/or polymerase chain reaction whereas in the remaining 40 cases there was no evidence of monoclonality. Among these latter 40 cases, 11 were classified as oligoclonal whereas 29 were classified polyclonal.
The immunophenotypic analysis of the TCR-Vß repertoire of both
CD4+ and CD8+bright PB T
cells from individuals showing LGL expansions revealed the existence of
an expansion of at least one Vß family in all but four cases
(n = 94; 96%), with a total of 124 expansions
detected. Ninety-one of these TCR-Vß expansions (73%) were
directly identified with the panel of mAbs used whereas in the
remaining 33 cases the identification was indirect. Seventy-one
patients (72%) showed an expansion of a single TCR-Vß family whereas
23 cases (23%) displayed expansions of two or more TCR-Vß families:
two TCR-Vß families were simultaneously expanded in 17 patients,
three in 5 patients, and four in 1 case. The highest TCR-Vß family
expansion found in each case was highly variable, ranging from 4 to
96% of the total PB CD4+ and
CD8+bright T cells (mean, 55 ± 30%;
median, 57%) (Figure 1)
. The other
TCR-Vß expanded families represented 4 to 28% (mean, 9 ± 5%)
of the total PB CD4+ and
CD8+bright T cells. Interestingly, no significant
expansions of TCR-ß+ T cells not included in
the suspected LGL population were detected in cases analyzed here.
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shows the relative incidence of
expansions for each of the TCR-Vß families within the
CD4+ (Figure 2A)
and the
CD8+bright (Figure 2B)
T cells. As may be seen,
overall, the expanded TCR-Vß families within the
CD8+bright LGLs showed a similar distribution to
that observed in normal PB because the must frequently expanded
TCR-Vß families were those usually highly represented in normal
circulating CD8+bright T cells (Table 1
and
Figure 2A
). In contrast, among cases showing an expansion of
CD4+ LGLs only a few TCR-Vß families were found
to be expanded: TCR-Vß2.1, -Vß3.1, -Vß6.7, -Vß12.2, -Vß13.1,
and -Vß17.1. When considering only monoclonal expansions of
CD4+ LGLs, two cases (22%) corresponded to
TCR-Vß2.1, two cases (22%) to TCR-Vß3.1, and four cases (44%) to
TCR-Vß13.1, a frequency that exceeds the frequency that was expected
on the basis of the representation of each of these TCR-Vß families
among normal PB CD4+ T cells (Table 1
and Figure 2B
).
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Nonmonoclonal (polyclonal plus oligoclonal) and monoclonal T-LGL
expansions differed in a number of aspects including the number of
expanded TCR-Vß families and the magnitude of the highest TCR-Vß
expansion. Table 2
summarizes the
immunophenotypic features of monoclonal versus nonmonoclonal
expansions by considering separately the CD4+ and
CD8+bright LGL proliferations.
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Similarly, all but three LGL proliferations classified as not being
monoclonal by molecular techniques displayed expansion of at least one
TCR-Vß family (93%). However, the frequency of cases with expansion
of more than one TCR-Vß family among nonmonoclonal proliferations of
LGLs (19 of 40 patients; 48%)—18 of 34 patients (53%) and 1 of 6
patients (17%) for CD8+bright and
CD4+ T-LGL, respectively—was much higher than
that observed in monoclonal cases (P = 0.001).
In addition, the magnitude of the highest TCR-Vß expansion observed
was also much lower (P = 0.001) among these
cases as compared to the monoclonal cases, accounting for only 24
± 14% of the expanded T-cell population: 22 ± 12% of
CD8+bright and 34 ± 19% of the
CD4+ T cells. Interestingly, the proportion of
the TCR-Vß expanded T cells was higher among the oligoclonal as
compared to the polyclonal cases (38 ± 15% versus
18 ± 9%). Also in contrast (P = 0.0001)
to what was observed in monoclonal proliferations, the percentage of
CD4+ or CD8+bright T cells
expressing a single TCR-Vß family exceeded 40% of the total
CD4+ or CD8+bright T cells
in only eight cases (20%), all of them being classified as oligoclonal
by molecular techniques. Interestingly, the TCR-Vß expansion did not
exceed 60% in any of them. On comparing the proportion of T cells
expressing the expanded TCR-Vß family from either the total
CD4+ or CD8+bright T cells
that fulfilled the phenotypic criteria for LGLs in monoclonal and in
oligoclonal/polyclonal T-LGL expansions (Figure 2)
, significantly
higher numbers (P = 0.0001) were observed in
monoclonal cases—86 ± 16% versus 42 ±
23%—the lowest values being observed for polyclonal T-LGL expansions
(32 ± 17%) whereas oligoclonal LGL expansions showed
intermediate values (64 ± 21%).
Taking 40% of the total CD4+ or
CD8+bright T cells as the cut-off value for
diagnosis of a monoclonal T-cell expansion, immunophenotyping showed a
sensitivity of 93% and a negative predictive value of 89%, with a
specificity and positive predictive value of 80% and 87%,
respectively. By increasing the cut-off value to 60% both the
specificity and positive predictive value increased to 100%, although
a large proportion of cases displaying a monoclonal expansion of T-LGL
proliferations were improperly classified as nonmonoclonal by flow
cytometry: sensitivity of 81% and negative predictive value of 78%
(Table 3)
.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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ß+ T-LGLs form a continuum spectrum of
polyclonal, oligoclonal, and monoclonal proliferations of
TCR-Vß-restricted T cells that differ both in type and number of
expanded TCR-Vß families, as well as in the magnitude of the TCR-Vß
expansion.
Overall, once cases displaying a monoclonal expansion of
TCR-ß+ T-LGLs based on molecular techniques
were compared to the other patients, major differences in the
immunophenotypic result were observed: 1) polyclonal and oligoclonal
expansions of TCR-ß+ LGLs frequently showed
expansions of more than one TCR-Vß family; 2) the proportion of the
most represented TCR-Vß family was usually much lower in these cases;
and 3) even in the presence of a TCR-Vß dominance, nonmonoclonal LGL
proliferations usually displayed a large fraction of residual LGLs with
a highly diversified T-cell repertoire.
Such observations would support the notion that monoclonal LGL
proliferations may arise as a consequence of an antigen-driven immune
response that would start as a polyclonal reactive T-cell response and
could thereafter subsequently evolve into oligoclonal and monoclonal
processes. This hypothesis would also be supported by the fact that
TCR-Vß families expressed in CD8+bright LGL
proliferations showed a pattern of distribution that mimics the
frequency at which individual TCR-Vß families are represented in
normal CD8+bright T cells. This would suggest
that CD8+bright T-LGLs are clonally transformed
in a random manner; the possibility of a bias to the preferential use
of some TCR-Vß families in cases showing a monoclonal expansion of
CD4+ LGLs needs further evaluation using a larger
number of cases. The notion that monoclonal expansions of T-LGLs may
represent an evolutionary end stage of an antigen-mediated
proliferation of LGLs is not new.5,6
In fact, there is a
great deal of evidence supporting this view: 1) clonal dominance with
preservation of a polyclonal reservoir is a typical feature in the
normal TCR-ß+ T-cell
repertoire;33
2) the immune TCR-Vß repertoire is
dynamic and can be continuously modulated;34
3) in normal
individuals, TCR-Vß-restricted T-cell expansions accumulate
with age;10-12
4) TCR-Vß-restricted T-cell expansions
are more frequently found in association with pathological conditions
characterized by chronic antigen stimulation;35,36
5)
preliminary reports suggest that a higher incidence of clonality seems
to be a frequent finding once TCR-Vß-restricted T-cell repertoires
are observed;10,37,38
this phenomenon seems to be
particularly frequent in the
TCR-ß+/CD8+bright
T-cell compartment, although expansions of
TCR-ß+/CD4+ cells,
TCR-ß+/CD4-/CD8-39
and TCR-+ T cells40
have also
been sporadically observed; 6) similarly to LGL-leukemia cells,
polyclonal T-LGLs displaying a restricted usage of TCR-Vß families
are usually CD28-,41
express
NK-associated antigens and NK receptors,42
suggesting that
they represent antigen-driven cytotoxic T cells; and 7) molecular
studies provided evidence that TCR-Vß-restricted T-cell expansions
may depend on an antigen-mediated selection
process.25,43,44
If this hypothesis is true, the higher the TCR-Vß expansion is, the greater the probability for a monoclonal T-cell proliferation, as found in the present study. In turn, this indicates that a large expansion of a TCR-Vß family would be highly predictive of clonal transformation of a chronic reactive process. Nevertheless, these phenomena do not always necessarily correlate. In fact, some cases displaying LGL expansions in which a single TCR-Vß family account for more than a half of the CD4+ or CD8+bright T-cell compartment proved to be oligoclonal by molecular techniques. Moreover, we have found a case of monoclonal T-cell proliferation in which a normal distribution of the different TCR-Vß families was observed in accordance to previous reports.45,46 It could be argued that such discrepancies could be because of problems related to technical uses such as the different sensitivity of the molecular and immunophenotypic techniques in cases in which minor populations of clonal LGLs are present.7 Alternatively, these observations could also suggest that monoclonal T-cell rearrangements could theoretically occur at any of the stages of the process of continuous T-cell stimulation. Based on the results presented here and in previous reports indicating that a TCR-Vß expansion representing >40% of the overall population of CD4+ and CD8+bright T cells is hardly ever found in normal individuals,10-12 we propose that, for routine purposes, expansions of a single TCR-Vß family representing >60% of the overall population of CD4+ and CD8+bright T cells could be considered as highly suggestive of monoclonal whereas those of <40% would be nonmonoclonal, pending confirmation by molecular techniques. In those cases in which the TCR-Vß expansion represents between 40% and 60% of the total CD4+ and CD8+bright T cells, molecular studies are essential to establish clonality. Further studies are necessary to clarify the utility of the immunophenotypic assessment of T-cell clonality based on the analysis of the TCR-Vß repertoire together with a characterization of the phenotypic profile of the expanded TCR-Vß family aimed at the identification of specific aberrant phenotypes, not only at the time of diagnosis, but also during the follow-up of patients, in particular if therapy is required.
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Acknowledgements |
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Footnotes |
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Supported in part by the Comissão de Fomento da Investigação em Cuidados de Saúde, Ministério da Saúde, Portugal (PI 51/99); Acções Integradas Luso-Espanholas do Conselho de Reitores das Universidades Portuguesas (E-31/99); and Acción Integrada Hispano-Portuguesa (HP 1998-0091), Dirección General de Enseñanza Superior e Investigación Científica, Ministerio de Educación y Cultura, Spain.
This work was done in the context of the BIOMED2 program (action BMH4-CT97-2611).
Accepted for publication August 3, 2001.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and CD8ß chains. Blood 1992, 80:1765-1773 but diverse J usage in one of five patients. Blood 1994, 83:767-775This article has been cited by other articles:
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