Combined method for simultaneous morphology , immunophenotype and karyotype ( MAC ) in leukemias

In the present study, a combined method (CM) for attaining simultaneous identification of leukemic cell morphology, karyotype and immunophenotype has been evaluated in 21 patients with acute leukemia and 1 with CML in blast crisis were studied for morphology, citochemistry, immunophenotype and karyotype. Karyotype was performed in abone marrow sample by using conventional techniques. In each case, direct method (DM) and/or three cultures were tried. The CM consisted in separating a small part of the material resulting from any of the cultures or DM, preparing slides through cytospin and immunophenotyping through APAAP method using the same monoclonal antibodies (MoAb) as for diagnosis. In 14 cases, the metaphases proved positive to the MoAb: in 4, the cells with abnormality had their origin defined; in other 4 the karyotype was normal preventing any identification; 6 cases had minimal abnormalities not visible through CM; and in two cases abnormal karyotypes were detected only in the cultures with GM-CSF: ' This study showed that CM is feasible in cases where evident numerical or structural chromosomal abnormalties are present.


S
everal of the cytogenetic abnormalities in acute leukemias are of clinicaI importance, once they .associatewith distinctive modes in which the disease presents itself as well as to prognostic factors.1.2 The acquisition of immunophenotyping and cytogenetic data has led to a morphologic, immunophenotypic and chromosomal (MIC) classification 3.4.5 which relates morphological subgroups within FAB subtypes with specific karyotype abnormalities.
Methods that allow the simultaneous analysis of morphology, immunophenotype and karyotype (MAC) have been described and deserve particular attention.6.7.X.9.1O The idea of concomitantly studying such aspects is of major interest especially in leukemias, as it could possibly indicate the cell lineagers which present chrolnosolnal abnormalities.It may as well indicate the involvelnent of different celllineages thus corroborating the heterogeneity of leukemias.lO And finally, the combined Inethod lnakes the analysis of a particular cell possible.
The main differences of the cOlnbined lnethod when compared to the conventional technique are: hypotonic treatment (using distilleq water instead of KCl), which leaves cytoplasln and cell membrane intact, through the utilization of cytospin in the place' of air drying Slnears and the abandon of acid fixatives.lO Few studies on th~application of this method to leukemic samples have been published up to the present moment, mostly analysing cases where nUlnerical chromosomal abnormalities occurred with results of lnuch consequence. 9• IO  By means of this technique the involvement of multiple lineages (i.e.erythrocytic, myelomonocytic ar megakaryocytic) has been elelTIOnstrateel in some cases of acute myeloiel Ieukemia (AML) with lTIOnOsomy 7.However, not alI the cases of AML with trisomy 8 elemonstrateel such involvement.7 We believe that the regular application of this lTIethoel, though tilTIe consuming, woulel proviele invaluable information on leukelTIic cell origino Thus, we inteneleel to .elevelop anel evaluate the feasibility ofthis CM as a regular proceelure, using a alkaline phosphatase anti-alkaline phosphatase (APAAP) kit.

MATERIAL AND METHODS
Twenty-one patients with acute leukemia (18 AML, 2 acute IYlTIphoielleukelTIia -ALL-anel 1 lTIixeelleukelTIia) anel 1 with chronic myeloiel leukelTIia (CML) in blast crisis who were aellTIitteel to Hospital S. Paulo have been investigateel, or those whose samples (cases 16 anel 20) had been sent to us for analysis, from June to December 1992.The average age of these subjects was 32 years (ranging frOlTI2 lTIonths to 65 years), being 9 male anel 13 felTIale.
The acute leukemia was diagnoseel in the presence ofblast cells in the patients' peripheral blood or more than 30% of thelTI in bone marrow. 1 1. 12 Bone lTIarrOW and peripheral blood Sl11earSwere stained for Leishman, SuelalTI Black B (SBB), Peroxielase (POX), periodic aciel Schiff (PAS), naftol-ASD esterase (ASD), alfa naftil acetate esterase (aES), NaF anel acid phosphatase (Fác)13 for lTIorphological and cytochemical diagnosis of the acute leukemia and classified according to FAB.I).Positivity was considered when more than 20% of cells were stained.Bone lnarrow cells separated by Ficoll-Hypaque gradient were washed 3 tilnes in phosphate buffered solution (PBS), resuspended in RPMI 1640 medium (final concentration= 2x 1Oócells/ lnl) and divided into different tubes with mouse antibody and incubated for 30 lTIinutes at 4°C.Then fIuorescent anti-1110useantibody was added for 30 min in a cold CalTIera.The ceIls were washed in PBS for 2 tilTIeS, the slides prepared and 100 ceIls were counted in fluorescent microscope.17 For cytogenetic and CM studies, bone lnarrow cells were cultivated in a 25 ClTI 3 tissue culture flask with Sml 1337 RPMI 1640 (Sigma) medium, pH 7.0,200/0 fetal calf serUln (Cultilab), L-glutamine 2mM (Sigma) and penicillinstreptomicin (1OOU; 1OOug/mI).Depeneling on the amount of lTIaterial obtained (visual observation), 2 or 3 cultures were set up: a short term culture (S), a synchronised culture (MTX), a culture with GM-CSF (GM) and/or a elirect methoel (DM).The cultures were prepareel as follows: S culture: was cultivated for 24 hours at 37°C; MTX culture: ametopterine (MethotrexateR-Leelerle) was aelded 5 hours after set up (final concentration=10-7 M) for 17 hours at 37°C anel thymidine (Sigma) was added (final concentration= 10-5 M) for the last 5 hours; GM-culture: GM-CSF (Amgen)(1 Oul/ml) was aeleleelfor 24 hours; DM: colchicine (Colcemiel-Gibco) was ilTIlTIediatly added.' x Colchicine (0,4ul/ml) was added for the last two hours to alI cultures anel then the material was spun at 1200r/nlin for 8 min anel the sobrenaelant elischarged.For the CM 5, elrops (Pasteur pipette) were taken out froln the lTIaterial left from each culture or DM and put in 0.51TIIhypotonic solution (elistilleel water).The cells were counteel in Cehn CC 510 eletronic counter and adjusted to a final concentration of 2 to 20xl0 3 cellshTIlTI.3 50 uI of this solution was put into cytospin CalTIeraSand spun at 400r/ lTIin for 4 mino Meanwhile, cells were being counted (around 5 to S minutes) hypotonization was taking place.The slides were fixeel in cold fonTIalin-acetone solution for 1 lTIin at the moment of use and then washed in Tris buffered solution (TBS) pH 7.6 with 2.5% hUlTIan AB serum (4:1) for 40 mino Then 20 uI OflTIOUSetnonoclonal antibody (diluteel 1: 10) was dropped over the cells and incubated for 60 lnin.After TBS wash rabbit anti-tnouse Ig antibody (Dako Z 259) diluted 1: 10 in TBS with hunlan AB SerUlTI (4: 1) for 40 min was added.Another TBS wash and the APAAP complex (Dako D 651) was put over the celIs for 40 lTIin diluteel I :50 in TBS.After wash alkalin phophatase substrate was added diluteel in TBS with levamisole I mM for 20 mino After final wash, the slides were dried and stained for Harris Helnatoxilin for 5 lnin.Blast celIs positivity was observed at the lnicroscope as welI as positive and negative lnetaphases.Metaphases that had chromosomal abnormalities were identifieel.Positive control was done with lnarrow slides frotn the satne patient.The specimens left over frotn the cultures or DM were handled for conventional cytogenetic analysis, that is: hypotonization with KCI 0.075M (I Otnl) for 20 tnin, fixation with methanol-acetic acid solution (I :3)( 1OtnI), 3 washes in fresh made fixative, anel in the next elay the slides were made, aged and bandeei for trypsin G banding.19   Among the 22 patients studied, 18 were AML, being Ml, M2 and M4 equally frequento Few cases of ALL in this series is explained by the preponderance of adult patients admitted to our infirmary.
There was 63% of karyotype abnonnalities.In The Fourth International Workshop in Leukelnias,24 50% of AML presented karyotype abnormalities.In later stuelies, approximately 70 to 80% of cases of AML and 50% of ALL have clonal acquired chromosolnal aberration of leukemic cellS.1. 24.25.26.27MAC method identifies the Inalignant cell anel determines its lineage simultaneously in the intact cells.This has been well demonstraded by other authors7.~.I() Using MAC method with an APAAP kit, we wanteel to check its feasibility as a rotinary procedure.We coulel define the lineage as myeloiel in karyotypically abnonnal cells only in few cases among 22 consecutevely stuelieel.It becarne evident that cases with normal karyotype are not suitable for this procedure since there remains as to doubt whether the cytogenetically normal cell belongs to the malignant clone or if it represents the nonnal residual clone.In cases 15 and 18, wich did not present karyotype abnormalities, we observed that all metaphases in the slieles with CD 13 and CD33 were positive to these Inyeloid markers.So we could imagine that in these cases nonnal karyotype was representing malignant clone.Although one can not be certain that cytogenetically nOrInal cells isolated 1339 structural abnormalities.Case 6 was a Ml leukemia with a normal karyotype.Case 7 was a M2 AML with 8% of marrow eosinophils and showed ano entirely normal karyotype.Case 8 was a Ml subtype with loss of part of the long arm of chromosolne 5. Case 9 was a variant M3 but t(15; 17) was not seen.Case 10 was an infant with leukemia presenting t( 4; 11) and classified as monocytic subtype with lymphoid component following HURWTZ& MIRRO 21 criteria.Case 11 was a B cell ALL subtype LI with loss of a chromosome of group C and translocation involving long arm of chrolnosome 1. Case 12 was a M3 with deletion of part of long arm of chrOlnosome 6 and additionaly, in some metaphases monosomy 5. Case 13 was a

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8 were Ml and M4 respectively, with normal karyotypes.Case 16 has been published and was an ALL diagnosed and treated with epipodophyllotoxin some years early and now presenting relapse or a secondary mixed leukemia with t(9;22) and additionaly, in some lnetaphases, lnonosolny 13 22 .Case 17 was M2 with hypodiploidia.Case 19, Ml with normal karyotype, though negative showed a imlnunophenotype presented overt positive peroxidase (94%).Case 20 was a typical M4 with eosinophilia and central nervous system infiltration and inversion of chromosome 16.Case 21 was a CML in myeloid blast crisis with only the persistence of Philadelphia chromosome.Case 22 was a M2 in a 53-year-old patient with a complex karyotype with monosomies 2 and 7, translocations involving 1q and the presence of a marker chrolnosome.
Some of these patients (cases: 1,3,4,6,12,14,15,17,18 and 19) have been studied correlating survival tÍlne and karyotype abnormalities.23 Table 2 shows the karyotype and the results of CM.In 14 cases there was some positivity to one or more MoAb applied on the Inetaphase.But only in 4 cases (13,17,21 and 22) (Fig. 1) could we precisely define to which lineage belonged the cell with cytogenetic abnormality.Among the resting 10 cases, in 4 the karyotype was normal (cases 7, 9,15 and 18) avoiding identifications and in 6, small abnonnalities were impossible to be seen through CM (cases 1, 8,12,14,16 and 20).In cases 8 and 12, karyotype abnormality was seen only in GM cultures.It became clear that unevident aberrations ar diploid karyotype are very difficult to be seen through MAC method, and in these cases probes for fluorescent in situ hybridization (FISH) would help to point out the katyotype abnormality (MACFISH technique).
SOlne cases, particularly the lymphoid ones, presented very few metaphases to analyse through conventional Inethod as weIl as through MAC.Pe~haps this was due to different reasons such as low mitotic index, slnaIl amount of ceIls aspirated from the patient, smaIl nlllnber of metaphases put into cytospin, among other technical problems.
Another interesting point to discuss was the observation that MoAb positivity in MAC method using APAAP was, lower when compared to indirect immunofluorescence (IF) dane at diagnostic probably due to a different pattern of reaction.2X IF was dane immediatly after aspiration ar coIlection of the samples and generaIly presented positivity, while APAAP took a longer time to 1341 be dane and could present disappearence of cytoplaslnic ar surphace antigen recognized by CD 33 (My 9) for instance.2X Besides that, the ceIls stained for APAAP hael been cultivated ar submitted to DM, ar in other worels, had gane through handling that coulel explain the altereel bindings to MoAb.Yet these cells had gane through hypotonia and sweIli ng anel thus had s uffereel modifications that could alter surphace antigens.
In 2 cases (8 and 12), karyotype abnonnalities were found only in GM culture.It has already been shown that GM-CSF may induce proliferation of abnormal clonogenic cells in human myeloid diseases such as AML anel myelodysplastic syndromes.2ó Leukemic cells froln bane marrow have an increased response to GM-CSF in a pattern caIled "leukemic specific", where there are stimulation of cytogeneticaIly abnormal ceIls, prolif~rative advantage to malignant ceIls in a mixed population (nonnal-abnonnal) and a possible influence in the karyotype.27.2lJ.30This cOlllel mean that GM induced the proliferation of Inalignant clonal ceIls, and this aspect should be better studieel in fllture works.
This study showed that the cOlnbined Inethoel is feasible in cases with numerical ar evident.structural chromosomal abnormalities', and not as a routine procedure.Associated with probes for FISH, it will bring much more interesting details.

Figure 1 -
Figure 1 -Example of case 21 showing a metaphase positive to CO 33.The arrow M4 leukemia with monosomy 7 in a indicates the Philadelphia chromosome.Positive and negative interphases are also patient with previous exposition to toxic seen.agents.Case 14 was a Ml with material added to the short arm ofchromosome 16.Cases 15 and .L.F.; COUTINHO, v.; YAMAMOTO, M. & KERBAUY, J. -Combined method for simultaneous morphology, immunophenotype and karyotype (MAC) in leukemias São Paulo Medicai Journal/RPM 115(1): 1336-1342, 1997