J Clin Pathol 1994;47:399-404

Clonality of CD3 negative large granularlymphocyte proliferations determined by PCRbased X-inactivation studies

A Kelly, S J Richards, M Sivakumaran, C Shiach, A D Stewart, B E Roberts, C S Scott

AbstractAims-To examine persistent CD3- largegranular lymphocytosis (LGL) cases forclonality, both by lineage specific (T cellreceptor) and lineage independent(X-inactivation) molecular methods; andto find out whether X-inactivation stud-ies are more appropriate than generearrangement studies for this subset ofLGL disorders.Methods-Patients were selected whohad LGL ofmore than six months' dura-tion and identified as CD3 - by immuno-phenotyping. T cell receptor studies and,where possible, X-inactivation studies ofthe phosphoglycerate kinase (PGK) genewere carried out. Analysis of subpopula-tions was carried out on cases heterozy-gous for PGK by the use of a polymerasechain reaction (PCR) method for X-inactivation.Results-Of 17 CD3- LGL cases studied,all were found to be germline for /1, y, andi T cell receptor studies, and immuno-globulin heavy chain genes. However, sixof these were analysed by X-inactivationof the PGK gene and two cases gaveclonal band patterns but only within theCD3- subpopulation.Conclusions-Clonal analysis by thelineage independent method of X-inactivation allows clonal expansionundetected by T and B cell specific mark-ers to be identified. It is therefore a more

appropriate method for the analysis ofCD3- LGL. This has implications fordiagnosis in CD3- LGL disorders.

(7 Clin Pathol 1994;47:399-404)

Molecular Pathologyand HaematologicalMalignancyDiagnostic Service,Algernon FirthBuilding, UniversityofLeeds, LeedsLS2 9JTA KellyS J RichardsM SivakumaranC ShiachB E RobertsC S Scott

Department ofGenetics, Universityof Leeds, Leeds LS29JTA D StewartCorrespondence to:Angela KellyAccepted for publication3 November 1993

Abnormally increased numbers of circulatinglarge granular lymphocytes (LGL) are associ-ated with a diverse range of disorders, includ-ing both reactive and malignant conditions.'Transient (acute reactive) LGL expansionsoccur primarily in response to viral infectionsand are often characterised by increases inboth CD3 + T cell and CD3 - natural killercell LGL.' In contrast, persistent lymphocy-toses with granular morphology are generallydue to an abnormal expansion of one LGLtype.' Many persistent LGL expansions withclinical and haematological features of"leukaemia" have been reported, and mostcases designated as such by the Morphology-Immunology-Chromosome (MIC) Coopera-tive Study Group are of the CD3 + subtype.4

However, a recent survey by the YorkshireLeukaemia Group (YLG) indicates that about25% of persistent LGL expansions are in factof CD3 - type, and a proportion of these arealso thought to be clonal in nature.5 6

Monoclonality is a fundamental character-istic of haemopoietic malignancies, and inves-tigations to establish the clonal nature ofabnormal proliferations are therefore impor-tant in differentiating reactive from malignantstates. The most widely used method forassessing clonality of lymphoid disordersinvolves molecular analysis of immunoglobu-lin and T cell receptor (TCR) gene rearrange-ments.27 TCR studies are informative formost cases of CD3 + malignancy,8 but TCRstudies of CD3 - proliferations have been oflittle value.' 9An alternative method of clonal analysis is

provided by the study of differential methyla-tion in certain X chromosome genes infemales.10 The inactivation of one X chromo-some with concomitant methylation of the 5'end of genes, such as PGK and hypoxanthinephosphoribosyl transferase (HPRT) duringearly embryogenesis, provides a stably inher-ited genetic marker. A normal population ofcells in an adult woman will comprise a ran-dom mixture of paternally and maternallyderived X-inactivated cells. A clonal popula-tion of cells, having originated from a singlecell, will feature inactivation of the same Xchromosome in each cell. The PGK gene canbe used for clonal analysis by a polymerasechain reaction (PCR) method where amplifi-cation ofDNA around the BstXI polymorphicrestriction site and a methylation site permitsanalysis of heterozygous females." The BstXIsite is polymorphic in about 33% of females.'0To examine the nature of persistent

increases in CD3 - LGIJnatural killer cells,we analysed X-inactivation at the PGK locusin six informative women from 17 femaleCD3 - cases identified in the YLG survey.5

MethodsTo date the YLG survey has identified 97people with persistently increased LGLand/or lymphocytes expressing natural killercell associated membrane determinants(defined as lasting more than six months).5 Ofthese, 23 were found with a primary increasein CD3 - natural killer cell associated + cells.Seventeen were women and were screened forheterozygosity at the polymorphic BstXI sitein the PGK gene (fig 1). LGL lymphocyteswere identified by conventional morphologi-

399

on Septem

ber 23, 2020 by guest. Protected by copyright.

http://jcp.bmj.com

/J C

lin Pathol: first published as 10.1136/jcp.47.5.399 on 1 M

ay 1994. Dow

nloaded from

Kelly, Richards, Sivakumaran, Shiach, Stewart, Roberts, Scott

Figure 1 Diagram of theexon 1 region of the PGKgene showing positions ofthe HpaII and BstXIendonuclease sites and theprimers used in the PCRmethod ofX-inactivationanalysis.

ENZYME

SITES Hpa2Bst Xi (site 7)

Hpa2(site 8)

EXON 19

AlE0I_

PRIMERS

cal examination and benzylcarbonyl-L-lysinethiobenzyl (BLT) esterase cytochemistry.'2Lymphocyte counts and immunophenotypicdata are shown in table 1. Age and clinicaldetails of the six heterozygous cases identifiedby this screen are shown in table 2.

LYMPHOCYTE FRACTIONATION ANDIMMUNOMAGNETIC MODIFICATIONPROCEDURESMononuclear cells were initially fractionatedfrom 30 ml of EDTA anticoagulated venousblood by density sedimentation withLymphoprep (Nycomed, UK). Cells isolatedfrom the interface were washed twice in phos-phate buffered saline and azide (0-01%)before immunophenotyping and immuno-magnetic depletion procedures.Washed mononuclear cells were pelleted

and mixed with the following combination of

monoclonal antibodies at saturating concen-trations: 63D3 (CD14); HD37 (CD19); andT3 (CD3). After a 30 minute incubation at4°C, with occasional mixing, the cells werewashed free of excess monoclonal antibodiesand resuspended in 0 5 ml Hanks' balancedsalt solution (HBSS), supplemented with 1%bovine serum albumin (BSA). Washed sheepanti-mouse immunoglobulin coated M450magnetic particles (Dynabeads; Dynal, UK)were added (50 ,ul). The leucocyte/Dynabeadmixture was incubated for 30 minutes at roomtemperature with gentle agitation, then resus-pended to 1 ml in 1% BSA/HBSS and placedon a magnetic separator. The unbound cellswere removed and tested for residual antibodycoated cells by indirect immunorosetting.13The depletion procedure was repeated withfurther 50 ,ul aliquots of Dynabeads until thenumber of residual monoclonal antibody cells

Table I Summary ofhaematological and immunophenotypc features of six CD3- natural killer cel associated +persistent expansions examined in this study*

AbsoluteLymphocyte %LGL CD2+ CD3- Primary natural killer cell

Case No count x 10'fl IBLTt x 1091l associated abnormnaitt Natural killer cell associatedphenotype*

AB 1-7 32/61 0-73 CD2+CD3-CD4-CD8- CD1lb+CD16+CD56+CD57+ZB 6-3 06/39 095 CD2+CD3-CD4-CD8- CDllb+CD16v CD56v CD57vWD 4-7 20/52 1-41 CD2 + CD3-CD4-CD8dim+ CDllb+CD16+CD56+CD57vWF 4-4 36/69 0-97 CD2 + CD3 - CD4-CD8dim+ CDlb+CD16v CD56+CD57vLG 5 9 30/75 2-42 CD2+CD3-CD4-CD8- CD1lb+CD16+CD56v CD57vAW 6-0 42/nt 1-56 CD2 +CD3-CD4-CD8 - CD1 lb + CD16 + CD56 + CD57 +

*These cases represent the six (of 17 tested) women who were shown in preliminary studies to be heterozygous for the PGK gene.t Indicates the percentages of lymphocytes with LGL morphology (Romanowsky) and positive BLT esterase staining.t Indicates the composite (CD2/CD3/CD4/CD8) phenotypes of the natural killer cell associated populations that were the pri-mary abnormalities in these patients.**Composite natural killer cell associated phenotypes of the abnormally increased CD2 + CD3 - fractions in these patients (+,v and - indicate > 60%, 30-60%, and < 30% of the CD2 + CD3 - fraction expressing the designated natural killer cell associateddeterminant).

Table 2 Summary of clinical data and DNA analysisX-inactivaton

AgeCase No (Cy) Clinicalfeatures TCR studyt CD3 + CD3-

AB 72 No important medical history Germline Poly PolyZB 40 No important medical history Germline Poly PolyWD 70 Polymyalgia rheumatica, gall stones, diverticulitis,

hysterectomy Germline Poly(s) Poly(s)WF 55 Hypothyroidism, maturity onset diabetes mellitus,

iron deficiency, menorrhagia, hysterectomy Germline Poly MonoLG 72 Transient headaches-resolved without treatment,

diverticulitis Germline Poly MonoAW 28 Clinically documented viral infecdon-monospot

negative. Now well Germline Poly Poly* These cases (six of 17 tested) are heterozygous for the BstXI polymorphism in the PGK gene.t Germline configuration of TCR genes was also found in 11 of 17 CD3 - NKa + cases tested, which were not polymorphic forPGK.t CD3 + denotes a composite population of cells comprising (CD3 + /CD19 +/CD14 +) separated by immunodepletion.Poly, polyclonal; Mono, monoclonal; Poly(s), skewed X-inactivation in this patient.

(s1 )Bst X l

INT3_

3'

.4 B3

400

on Septem

ber 23, 2020 by guest. Protected by copyright.

http://jcp.bmj.com

/J C

lin Pathol: first published as 10.1136/jcp.47.5.399 on 1 M

ay 1994. Dow

nloaded from

Clonal CD3- LGL proliferations

5:6 78:.: ::::.CD3.+

XH, 9 10

309 bp;249:b-

A B

11- 12

ol309 bp249 b.p

Figure 2 X-inactivation analysis by the PCR method. Lanes 1, 3, 5, 7, 10, and 12 show control products amplified without predigestion with HpaII.Lanes 2, 4, 6, 8, 9, and 1 I show X-inactivation patterns when PCRfollows predigestion with HpaII. (A) Example ofpolyclonal band patterns in bothCD3+ and CD3- cell fractions (case AB). (B) Example ofmonoclonal band pattern in CD3- cellfraction only (case WF). (C) Example ofskewedX-inactivation in both CD3+ and CD3- cellfractions (case WD).

was less than 2%. In practice, this rarelyrequired more than two depletion steps. TheDynabead bound fraction comprised CD1 4 +(monocyte), CD19 + (B lymphocyte), andCD3 + (T lymphocyte) cells. For simplicitythis fraction is referred to as CD3 + in fig 2.

DNA EXTRACTIONFor TCR analysis extraction was performedby a standard phenol/chloroform method after0-86M ammonium chloride lysis of red cellsand overnight digestion with proteinase K.'4 Asimplified protocol was used for X-inactiva-tion analysis following lymphocyte fractiona-tion. Cells were resuspended in 1 ml ofsodium chloride/TRIS/EDTA (pH8) (STE)buffer, lysed with 20 jul sodium dodecyl sul-phate (SDS) (20% w/v) and treated with100 jug proteinase K for two hours, thenextracted once with equal volumes of phenoland chloroform, ethanol precipitated, and pel-leted. The dried pellet was resuspended in100-200,ul of TRIS/EDTA (pH7-5) (TE)buffer.

T CELL RECEPTOR AND IMMUNOGLOBULINHEAVY CHAIN GENE REARRANGEMENTSDNA (10 1ug) was digested overnight withrestriction enzymes HindIII and BamHI (andEcoRI when results were inconclusive). TheDNA fragments were separated using 0-8%agarose gel and transferred to nylon mem-branes (Hybond N, Amersham International,UK) using vacublotting method (Pharmacia,UK). The membranes were fixed by ultravioletlight and prehybridised at 65°C in buffer solu-tion containing 10% dextran sulphate, 3 x

sodium citrate and sodium chloride (SSC),1% sodium dodecyl sulphate (SDS), anddenatured salmon sperm DNA, 10 mg/ml, fortwo hours. Denatured 32P labelled cDNAprobes were added to the mixture andhybridised overnight. The probes used in thisstudy were: Jur-fl2," T-y (pH60),'6 T-(R2 1XH),17 and JH (pUC 19).18 The mem-branes were washed twice in 2 x SSC solutionfor five minutes at room temperature and in2 x SSC and 1% SDS for 30 minutes at

65°C. Further washes were given if the back-ground radioactivity was still high. The mem-branes were autoradiographed (Hyperfilm,Amersham International, UK) for three tofive days.

X-INACTIVATION CLONAL ANALYSIS BY THEPCR METHODPrimers A1/B3 for the 619 base pair productfrom the PGK gene were made on AppliedBiosystems oligonucleotide synthesiseraccording to the sequence published else-where." Primer INT3 was also synthesisedwhich produces a fragment of 309 base pairsenabling greater separation of BstXI digestedfragments. The sequence of the 20 nucleotideoligomer INT3 is TTGGTGGTTTCTAGC-CGCATT (fig 1).The PCR was carried out in an LEP

Scientific, model Prem, thermal cycler.Temperatures were 95°C for one minute,denaturation, 56°C for one minute, annealingand 72°C for one minute extension.Amplification with primers Al and B3 was

for 25 cycles using 1.5 U Taq polymerase(Promega) per tube in a total volume of 50 jul.Primers INT3 and B3 were used by taking1 jul of the 619 base pair A1/B3 product and a

further 10 cycles of amplification carried outunder the same PCR conditions. Digests of0-5 ,ug DNA with 20 U HpaII enzyme(Northumbria Biologicals) were carried out in20 ,ul Taq buffer (Promega) before amplifica-tion. This digest (10 ul) was used for PCR.Control DNA was not digested with HpaIIbefore amplification. Following amplification20 ,ul of products from both control andHpaII digested samples were digestedovernight with 12 U BstXI enzyme(Northumbria Biologicals). Fragments were

separated using 2% agarose gel and visualisedwith ethidium bromide (fig 2).

ResultsIMMUNOPHENOTYPINGThe immunological characteristics of the sixpatients who were informative for PGK are

C

401

on Septem

ber 23, 2020 by guest. Protected by copyright.

http://jcp.bmj.com

/J C

lin Pathol: first published as 10.1136/jcp.47.5.399 on 1 M

ay 1994. Dow

nloaded from

KeUly, Richards, Sivakumaran, Shiach, Stewart, Roberts, Scott

}234 5 6 Z 8 9 }:o } } t}~~~~~~~~~~~~~~~~~~~~~~10I 2 3 1.4 5 1.6

* ...~~~~~~~~~~~~~~~~~~~~~~..~~~w;...................

..

:. 4, -....''|l*~~~~~~~~~~~~~~~~~~~~~~~.*: ~~~~~~~~~~~~~~~~~~~~~~.. .. ... .. .. ................ |-v....:.: .: .: :.' ........

...:............:........ :...........:.:.::..... ..:::

::;..

| .U........... '::..l_.... ... ......... ... ...... .....~~~~~. ..

BAU. Ni

I

T 2 3 4 5 6.

VAM 04

Figure 3 Southern blot analysis of T cell receptor genes. (A) f/ chain A analysis. HindIII and BamHI digests ofCD3- LGL cases WF andAB showgermline (G) configurations. For HindIII Lanes 1, 5, 6 negative controls; lanes 2-4 positive control samples showing rearranged (R) clonal bandpatterns. For BamHI Lanes 13, 14 negative controls; lanes 9-12 positive control samples. (B) y chain B analysis. BamHI digests ofCD3- LGL casesWF andAB again show gernline configuration. Lanes 1-3 are negative controls, lanes 6-8 are positive controls.

shown in table 1. Absolute lymphocyte countswere only slightly increased (range 4-7-6-3 x109/1) in four of these, the remaining twobeing within normal limits. Absolute increasesin CD2 + CD3 - lymphocytes (normal range,0'15-0-49 x 109/1), which are primarily asso-ciated with natural killer associated cells, werefound in all six cases, and multiple colour flowcytometry showed that the primary naturalkiller cell abnormality was associated withCD2 + CD3-CD4-CD8- (n = 4) orCD2 + CD3-CD4-CD8dim + (n = 2)fractions. Expression of natural killer cellassociated determinants showed some indi-vidual variability but the abnormally increasedCD2 + CD3 - cell numbers in all casesexpressed all four natural killer cell associatedmarkers examined (CD1 1b, CD16, CD56,and CD57) on at least 30% of the cell frac-tion.

T CELL RECEPTOR AND IMMUNOGLOBULINHEAVY CHAIN GENE REARRANGEMENTSDNA from the mononuclear cell fractions of17 patients with persistent expansions ofCD3 - natural killer associated cells wereinvestigated for the presence of monoclonalTCR and immunoglobulin gene rearrange-ments. Figure 3 illustrates germline andrearranged results obtained by this method.All of our 17 cases were found to havegermline configuration with each probe used,which is illustrated by results from cases WFand AB (fig 3). T and B cell gene rearrange-ment studies are carried out using a mono-nuclear cell fraction. Previous experience inthe laboratory indicates that other cell typespresent in the mixture do not obscure thepresence of a minor (as low as 1 %) populationof cells containing a single clonal rearrange-ment. Germline bands represent unre-arranged genes from most of the cells,whereas clonally rearranged cells appear as adistinct band of a different size (fig 3A; lanes2-4).

X-INACTIVATION ANALYSIS OFUNFRACTIONATED MONONUCLEAR CELLSInitially, DNA from the mononuclear cellfractions of 17 patients with persistent CD3 -lymphocytosis were screened for the presenceof a BstXI polymorphism at the PGK locus.Six heterozygotes were identified and X-inac-tivation clonal analysis was carried out bythe PCR method. None of these showed un-equivocal evidence of monoclonality, althoughcases WF, LG, and WI) (data not shown)produced highly "skewed" band patterns,with one product being significantly moreintense than the other. These observationscould either reflect an unusually high ratio ofmaternal:paternal X-inactivated or suggestthe existence of a mixture of clonal and non-clonal cells. Because X-inactivation studiescannot distinguish cell type, cells not involvedin the clone will interfere with the analysis.Therefore, the population of interest must beas pure as possible for meaningful analysis.

X-INACTIVATION ANALYSIS OFIMMUNOLOGICALLY FRACTIONATED CELLSTo isolate the abnormally increased CD3 -cell numbers, fresh samples of peripheralblood mononuclear cells from each patientwere separated into CD3 + and CD3 - frac-tions. Clonal analysis by the PCR method wasthen carried out on these subfractions. Resultsfrom case AB are shown in fig 2A, and show apolyclonal pattern in both CD3 + and CD3 -fractions, where both bands are retained afterHpaII digestion. Similarly, cases ZB and AWgave polyclonal patterns (table 2). In contrast,results from case WF in fig 2B showed a poly-clonal pattern of products in the CD3 + frac-tion and a monoclonal pattern in the CD3 -fraction (by the loss of one product from theCD3 - fraction after Hpal digestion). Like-wise, patient LG showed a polyclonal patternfor the CD3 + fraction and a monoclonal pat-tern for the CD3- fraction (table 2). Resultsfor case WD are shown in fig 2C. This case

402

on Septem

ber 23, 2020 by guest. Protected by copyright.

http://jcp.bmj.com

/J C

lin Pathol: first published as 10.1136/jcp.47.5.399 on 1 M

ay 1994. Dow

nloaded from

Clonal CD3 - LGL proliferations

shows evidence of uneven (skewed) X-inacti-vation in both the CD3 + and CD3 - frac-tions, shown by a reduction of intensity of thesmaller PCR product in the HpaII digestedsample compared with the control. Theobservation was consistent over three experi-ments (data not shown) and as the patternwas the same for both the CD3 + and CD3 -fractions we concluded that each comprisednormal polyclonal subpopulations. The skewtowards one X-inactivation product isassumed to be consistent for constitutionalDNA in this patient because of the compari-son with CD3 + cells. In this case there was aslight difference in band intensity in controllanes (fig 2C; lanes 10, 12). This was seenoccasionally and might have resulted from aPCR artefact due to template preference, ordifferences in ethidium bromide incorpora-tion within the agarose gel. However, therewas a clear difference between control lanesand HpaII digested samples (fig 2C; lanes 9,11).

DiscussionEvidence for monoclonality in LGL disorderswith CD3 + phenotype has been accumulat-ing for several years by the extensive use ofTCR gene rearrangement studies.8919 Thesestudies, however, have served to confirm theconsistent observation of germline TCR genesin LGL with CD3 - phenotype. This agreeswith our own finding of germline TCR genesin the mononuclear cells of 17 CD3 - LGLcases, whereas in CD3 + cases rearrange-ments can be clearly seen (fig 3). Con-sequently, a T cell specific genetic markerseems to be inappropriate in the assessment ofclonality in CD3 - LGL cases.As there is no specific clonal marker avail-

able for cells of natural killer cell lineage weapplied the lineage independent method ofX-inactivation analysis. A preliminary studyof mononuclear DNA from CD3 - LGLcases showed that polyclonal cells were pres-ent in the peripheral blood of these patients;but skewing of the bands suggested that insome cases there could be a subpopulation ofclonal cells present within the mononuclearfraction. This was confirmed when fresh sam-ples were divided into CD3 + and CD3 -subgroups and analysed separately. Of sixinformative cases examined, two patientsclearly showed a monoclonal pattern in theCD3 - cell fraction. Polyclonal patterns werefound for the CD3 + fraction of both of thesepatients, therefore effectively serving as tissuespecific internal controls, showing thatX-inactivation is not extreme within the nor-mal cells of these individuals. Extreme skew ofX-inactivation was, however, illustrated byanother of our patients (WD) who showedevidence of a skewed X-inactivation patternwithin both cell fractions. This highlights theneed for caution when interpreting reducedband intensity as a measure of clonality by theX-inactivation method, particularly if consti-tutional DNA is not available.20 Fortunately,CD3 + cells provided suitable constitutional

DNA for this study.The study of minor subpopulations of cells

within the peripheral blood has become practi-cable by the development of a PCR basedtechnique. This has the advantage of rapidityand has a reduced requirement for startingmaterial compared with the standardSouthern blotting method.' 011 This limitationalso applies to other Southern blotting basedmarkers, such as HPRT and M27fl. M27fJmay not be ideal as an alternative because thetest region suffers from hypermethylation insome leukaemic blast cells.20

Analysis of subpopulations makes it poss-ible to isolate and identify the clonal origin ofproliferating LGL cells and define the limitsof lineage involvement. The abnormal mono-clonal population in CD3 - LGL seems to berestricted to the natural killer cell subpopula-tion. Thus the target cell is shown to be com-mitted to the natural killer cell lineage, theremaining peripheral blood cells having apolyclonal pattern of X-inactivation. Otherevidence for monoclonality in CD3 - LGLhas been found by Taniwaki et al,2' whosepatients had chromosomal abnormalities, andby Kawa-Ha et al,22 who examined their casesfor the presence of monoclonal Epstein-Barrvirus termini and suggested the virus mayhave an aetiological role in some cases ofLGL. In contrast, a recent report by Nash eta123 did not identify clonal CD3 - popula-tions within the seven patients studied.The clinical relevance of a clonal popula-

tion of natural killer cells is uncertain. Neitherof our two patients had any other evidence ofan haematological malignancy. One patient(WF) had a strong history of autoimmune dis-ease. The association of clonality withautoimmune disease is well recognised and isseen both in the manifestation of autoantibod-ies and clonal proliferation of large granularlymphocytes.2s26 The other patient (LG) hadno clinical problems known to be associatedwith clonal proliferation. The increasing useof new molecular techniques is likely to per-mit identification of previously unidentifiedminor clonal populations. However, the clini-cal importance of this can only be ascertainedby continued clinical observation.

We are grateful to the Yorkshire Leukaemia Group whoparticipated in the survey of LGL proliferations. Thanks go toDr T H Rabbitts for generously supplying T-y, to Dr TVilliamy for JH and T-5, and to Dr J H Pringle for Jpl2 probes.Thanks also go to Mr A Speight for technical assistance. AKwas supported by the Yorkshire Cancer Research Campaign.

1 Newland AC, Catovsky D, Linch D, Cawley JC, BeverleyP, San Miguel JF, et al. Chronic T-cell lymphocytosis: areview of 21 cases. BrJ Haematol 1984;58:433-66.

2 Hamblin TJ. Interleukin-2 and lymphokine activated killercells. Transfusion Sci 1991;12:35-42.

3 Ohno T, Kanoh T, Arita Y, Fujii H, Kuribayashi K,Masuda T, et al. Fulminant clonal expansion of largegranular lymphocytes: characterisation of their morphol-ogy, genotype and function. Cancer 1988;62:1918-27.

4 Bennett JM, Juliusson G, Mecucci C. Morphologic,immunologic and cytogenetic classification of thechronic (mature) B and T lymphoid leukaemias: fourthmeeting of the MIC Co-operative Study Group. CancerRes 1990;50:2212.

5 Scott CS, Richards SJ, Sivakumaran M, Short M, ChildJA, Hunt KM, et al. Transient and persistent expansionsof large granular lymphocytes (LGL) and NK-associatedcells: the Yorkshire Leukaemia Group Study. Br JHaematol 1993;83:504-15.

403

on Septem

ber 23, 2020 by guest. Protected by copyright.

http://jcp.bmj.com

/J C

lin Pathol: first published as 10.1136/jcp.47.5.399 on 1 M

ay 1994. Dow

nloaded from

Kelly, Richards, Sivakumaran, Shiach, Stewart, Roberts, Scott

6 Scott CS, Richards SJ. Classification of large granular lym-phocyte (LGL) and NK-associated (NKa) disorders.Blood Rev 1992;6:220-33.

7 Rabbitts TH, Stinson A, Forster A, Foroni L, Luzzatto L,Catovsky D, et al. Heterogeneity of T-cell receptor betachain gene rearrangements in human leukaemias andlymphomas. EMBOJ 1985;4:2217-24.

8 Sivakumaran M, Richards SJ, Hunt KM, Steed AJ, BynoeAG, Morgan MM, et al. Patterns of CD16 and CD56expression in persistent expansions of CD3+ NKa+lymphocytes are predictive for clonal T cell receptorgene rearrangements. Br J Haematol 1991;78:368-77.

9 Loughran TP, Starkebaum G, Kidd P, Neiman P. Clonalproliferation of large granular lymphocytes in rheuma-toid arthritis. Arthritis Rheum 1988;31:31-6.

10 Vogelstein B, Fearon ER, Hamilton SR, Freisinger AC,Willard HF, Michelson AM, et al. Clonal analysis usingrecombinant DNA probes from the X-chromosome.Cancer Res 1987;47:4806-13.

11 van Kamp H, Janssen R, Willemze R, Fibbe WE,Landegent JE. Studies on clonality by PCR analysis ofthe PGK-l gene. Nucleic Acids Res 1991;19:2794.

12 Wagner L, Base W, Weisholzer M, Sexl V, Bognar H,Worman CP. Detection of BLT substrate-specific pro-teases in individual human peripheral blood leukocytesand bone marrow cells: applications to the classification ofleukaemia. J Immunol Methods 1991;142:147-55.

13 MacKarill ID, Scott CS, Stark AN, Limbert HJ, MasterPS, Jones RA, et al. A rapid method for the preparation ofchromic chloride solutions for use in the direct rosettingtechnique. Med Lab Sci 1987;44:401-44.

14 Sambrook J, Fritsch EF, Maniatis T. Purification ofnucleic acids. In: Molecular Methods: a laboratorymanual-2nd edn. New York: Cold Spring HarbourLaboratory Press. 1989. Appendix (E) 3.

15 Yoshikai Y, Anatoniou D, Clark SP, Yanagi Y, Sangster R,Van Den Elsen P, et al. Sequence and expression of tran-scripts of the human T-cell receptor ,-chain genes.Nature 1984;312:521-4.

16 Lefranc M-P, Rabbitts TH. Two tandemly organised

human genes encoding T-cell y constant regionsequences show multiple rearrangements in different T-cell types. Nature 1985;316:464-6.

17 Boehm T, Buluwela L, Williams D, White L, RabbittsTH. A cluster of chromosome 11pi13 translocationsfound via distinct D-D and D-D-J rearrangements of thehuman T cell receptor delta chain gene. EMBO J1988;7:2011-17.

18 Early P, Huang H, Davis M, Calame K, Hood L. Animmunoglobulin heavy chain gene is generated from 3segments ofDNA, VH, D and JH. Cell 1980;19:981-92.

19 Sansoni P, Girasole G, Manara GC, Snelli G, Passeri G,Allavena P, et al. Lymphocytes of a patient with lympho-proliferative disease of large granular lymphocytesexpress high natural killer, ADCC and LAK activity.Clin Immunol Immunopathol 1990;56:9-21.

20 Gale RE, Wheadon H, Goldstone AH, Burnett AK, LinchDC. Frequency of clonal remission in acute myeloidleukaemia. Lancet 1993;341:138-42.

21 Taniwaki M, Tagawa S, Nishigaki H, Horiike S, MisawaS, Shimazaki C, et al. Chromosomal abnormalitiesdefine clonal proliferation in CD3 - large granular lym-phocyte leukaemia. AmJfHematol 1990;33:32-8.

22 Kawa-Ha K, Ishihara S, Ninomiya T, Yumura-Yagi K,Hara J, Murayama F, et al. CD3-negative lymphoprolif-erative disease of granular lymphocytes containingEpstein-Barr viral DNA. .7 Clin Invest 1989;84:51-5.

23 Nash R, McSweeney P, Zambello R, Semenzato G,Loughran TP. Clonal studies of CD3 - lymphoprolifer-ative disease of granular lymphocytes. Blood 1993;81:2363-8.

24 Viard JP, Bach JF. Clonality in autoimmune diseases.Semin Haematol 1991;28:57-65.

25 Loughran TP, Starkebaum G, Aprile JA. Rearrangementand expression of T-cell receptor genes in large granularlymphocyte leukaemia. Blood 1988;71:822-4.

26 Stamenkovic I, Stegagno M, Wright KA, Krane SM,Amento EP, Colvin RB, et al. Clonal dominance amongT-lymphocyte infiltrates in arthritis. Proc Nad Acad Sci,USA 1988;85: 1179-83.

404

on Septem

ber 23, 2020 by guest. Protected by copyright.

http://jcp.bmj.com

/J C

lin Pathol: first published as 10.1136/jcp.47.5.399 on 1 M

ay 1994. Dow

nloaded from