J. clin. Path., 1977, 30, 541-546

Intracellular lysozyme and lactoferrin inmyeloproliferative disordersD. Y. MASON

From the Department ofHaematology, Gibson Laboratories, Radcliffe Infirmary, Oxford

SUMMARY Samples from 49 cases of myeloproliferative diseases were tested by an immunocyto-chemical technique for leucocyte lysozyme and lactoferrin. The presence of these constituents inmyeloid precursors from cases of acute and chronic myeloid leukaemia reflected the degree of cellu-lar maturation, lysozyme appearing (as it does in normal myeloid cells) at the stage of primarygranule production (in promyelocytes), while lactoferrin was detectable only in more mature,secondary granule-containing myeloid cells. Auer rods stained positively for lysozyme, in keepingwith their relationship to primary granules.Monocytes from five cases of leukaemia showing predominantly monocytic differentiation were

indistinguishable from normal monocytes in their staining reactions for lysozyme despite the pre-sence of raised serum and urinary lysozyme levels.

In four cases of acute myeloid leukaemia circulating polymorphs deficient in lactoferrin weredetected: in one of these cases a similar percentage of polymorphs was lysozyme negative.

In a recent report from this laboratory an immuno-cytochemical technique was described for the de-monstration of cytoplasmic lysozyme and lacto-ferrin in human white cells (Mason et al., 1975). Thefirst of these constituents is present in both myeloidand monocytic cells; the second is restricted tomyeloid cells.The present report is concerned with the distribu-

tion of these two substances in leucocytes from casesof myeloproliferative diseases. This study was de-signed, first, to investigate whether the demonstra-tion of lysozyme and lactoferrin might aid the identi-fication or classification of proliferating cells inthese disorders; secondly, to study whether cases ofmonocytic leukaemia associated with marked in-creases in blood and urinary lysozyme are character-ised by abnormally strong reactions for intracellularlysozyme: and, finally, to look for evidence ofacquired deficiencies of intracellular lysozyme orlactoferrin in myeloid leukaemia and related dis-eases.

Patients and methods

PATIENTSSamples were obtained from patients attending the

Received for publication 28 October 1976

Radcliffe Infirmary, Oxford. The diagnosis ofleukaemia and of other myeloproliferative disorderswas based upon standard clinical and laboratorycriteria. The number of patients in each diagnosticcategory is given in Table 1. It should be noted thatthree cases of primary sideroblastic anaemia andparoxysmal nocturnal haemoglobinuria (PNH) havebeen included (under the category of chronic myelo-proliferative disorders). Although not conventionally

Table 1 Patients tested by the immunocytochemicaltechnique for intracellular lysozyme and lactoferrin

Diagnosis No. ofpatients

Chronic myeloproliferative disorders1 Chronic myeloid leukaemia 142 Myelofibrosis 63 Polycythaemia rubra vera 74 Primary sideroblastic anaemia 25 Paroxysmal nocturnal haemoglobinuria 1

Acute/subacute myeloproliferative disorders6 Acute myeloblastic/myelomonocytic/promyelocytic

leukaemia 147 Acute monocytic/monoblastic leukaemia 28 Subacute myelomonocytic leukaemia 3

All cases were tested for both lysozyme and lactoferrin reactivity, withthe exception of two patients in group 6 and one in group 7 who weretested for lysozyme only.

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classified in this way their association with acuteleukaemia, and the neutrophil alkaline phosphatasedeficiency which characterises PNH justifies theirinclusion.

All cases of acute leukaemia were studied beforethe initiation of treatment, whereas the majority ofcases of chronic myeloproliferative diseases were in-vestigated while under treatment. However, allpatients in this category had evidence of activedisease at the time of testing.

SAMPLES

Smears of whole blood or buffy coat preparationswere made from EDTA anticoagulated venoussamples within four hours of specimen collection.These smears, and direct smears ofmarrow aspirates,were stored at room temperature for a maximum offive days before being stained. Previous studies hadestablished that no loss of lysozyme or lactoferrinreactivity occurs during this period.

IMMUNOPEROXIDASE STAINING OF LYSOZYME

AND LACTOFERRIN

The technique used for the demonstration of theseconstituents has been described previously and wasapplied without modification (Mason et al., 1975).In summary, the method involves the successiveapplication to fixed cell smears (after inhibition ofendogenous peroxidase) of: (1) specific rabbit anti-serum to lysozyme or lactoferrin; (2) swine anti-serum to rabbit immunoglobulin; and (3) solublecomplexes of horseradish peroxidase and rabbitantibody to this enzyme. The swine antiserum (2)acts as a 'bridge' between the primary antibody andthe peroxidase-containing complexes. The develop-ment of the peroxidase activity by a cytochemicaltechnique (using diaminobenzidine and hydrogenperoxide as substrates) allows identification of lyso-zyme or lactoferrin containing cells. Slides were

counterstained with haematoxylin and mounted inDPX.

Results

NORMALSThe immunocytochemical reactions of normal peri-

D. Y. Mason

pheral blood and bone marrow samples for lysozymeand lactoferrin have been reported previously(Mason et al., 1975) and are summarised in Table 2.

CHRONIC MYELOPROLIFERATIVE DISORDERS

Chronic myeloid leukaemiaAll 14 patients in this category gave normal stainingreactions for the two antigens in mature neutrophilsand monocytes. Neutrophil precursors showed a re-

action consistent with their stage of maturation, iemyeloblasts were negative for both antigens, pro-

myelocytes stained for lysozyme only, while bothconstituents were detected in metamyelocytes andband forms.

In two cases of chronic myeloid leukaemia circu-lating polymorphs were observed lacking eachantigen. However, both these cases were charac-terised by increased basophil counts, and the per-

centage of lysozyme and lactoferrin deficient poly-morphs correlated with the basophil count.

Polycythaemia rubra vera, myelofibrosis, paroxysmalnocturnal haemoglobinuria, and sideroblastic anaemiaIn each of the 14 cases in this category normalneutrophil and monocyte staining reactions were

obtained for both antigens.

ACUTE MYELOPROLIFERATIVE DISORDERS

Acute myeloblastic/myelomonocytic leukaemiaMyeloid precursors in the 14 cases in this groupshowed staining reactions for lysozyme and lacto-ferrin which paralleled theirdegreeofcellularmatura-tion as assessed on morphological grounds and fromSudan Black staining. The percentage of lysozymepositive precursors was consistently greater thanthat of lactoferrin positive cells.The highest percentage of lysozyme positive

leukaemic marrow cells was observed in a case ofacute promyelocytic leukaemia, in keeping with thelarge number of cells containing azurophil granules.In one case of acute myeloblastic leukaemia Auerrods were plentiful. These structures stained posi-tively for lysozyme but not for lactoferrin. Thelysozyme reaction in most cells in this case was

Table 2 Summary of immunoperoxidase reactions for lysozyme and lactoferrin in different classes ofhumanhite cells

Neutrophil myeloid Monocytic Erythroid Lymphoid

M'blasts Prom'cytes Myelocytes Metam'cytes Band and Monocytes Normoblasts Lymphocytessegmentedformns and red cells

Lysozyme - + + + +Lactoferrin - - + + +

- absent; + present; ± present in some cells, absent in others.

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Intracellular Iysozyme and lactoferrin in myeloproliferative disorders

Table 3 Details of leukaemic patients in whomlysozyme and/or lactoferrin deficient neutrophils weredetected

Diagnosis Lysozvme negative Lactoferrin negativeneutrophils neutrophils

Acute myelomonocyticleukaemia <5 36

Acute myelomonocyticleukaemia <5 60

Subacutemyelomonocyticleukaemia 29 30

Subacutemyelomonocyticleukaemia <5 8

confined to the margin of the Auer rods, outliningnegatively staining central clefts.

Circulating polymorphs showed normal stainingfor both antigens (more than 95% positive) in 12 ofthe 14 cases of acute myeloblastic and myelomono-cytic leukaemia studied. In the remaining twopatients (Table 3) a population of negatively stainingperipheral blood neutrophils was identified. TheFigure illustrates one of these cases, 36% of whoseperipheral polymorphs were deficient in lactoferrin.Careful examination of the Romanovsky stainedperipheral film from this patient did not reveal any

agranular or otherwise atypical polymorphs.Circulating monocytes from all 14 patients stained

normally for lysozyme. In cases where there was a

mixed peripheral population of monocytoid andmyelocytic cells (ie, acute 'myelomonocytic' leu-kaemia) lysozyme staining clearly differentiatedbetween the weak to negatively staining monocytoidpopulation and the intensely staining immaturemyeloid cells.

ACUTE MONOBLASTIC/MONOCYTICLEUKAEMIATwo patients were studied whose leukaemic cellsshowed predominantly monocytic differentiation,associated with greatly raised serum and urinary lyso-

zyme levels. In both cases the staining pattern of theleukaemic monocytes was very similar to that ofnormal monocytes, ie, a weak or negative stain forlysozyme and a negative reaction for lactoferrin. Nomonocytoid cells stained for lysozyme with an in-tensity comparable to that consistently seen formyeloid cells. Faint lysozyme positive 'halos' were

not infrequently observed around leukaemic mono-

cytes, suggesting that some lysozyme had escapedfrom these cells as the smears were prepared.

SUBACUTE/CHRONIC MYELOMONOCYTICLEUKAEMIAThe three patients in this category conformed to the

clinicopathological pattern described by Sexauer etal. (1974) and Geary et al. (1975) in that they wereelderly patients with a peripheral monocytosis,myelomonocytic bone marrow infiltration, and in-creased serum lysozyme levels.The monocytoid cells from these patients showed

weak staining reactions for lysozyme resemblingnormal monocytoid cells; as in the case of acutemonocytic leukaemia (see section above), no mono-cytes stained as strongly for lysozyme as did mye-loid cells. Monocytes from these patients were lacto-ferrin negative.

In two of these patients lactoferrin negative maturepolymorphs were detected, associated in one casewith a similar percentage of negative polymorphs inthe preparation stained for lysozyme (Table 3).

Discussion

One of the aims of this study was to assess whetherstaining for lactoferrin and lysozyme may be ofvalue in the classification or identification of leukae-mic cells. The results obtained suggest that these re-actions yield no more information than can beobtained from conventional techniques such as theassessment of cell morphology and the use of cyto-chemical stains. Lysozyme positivity appeared toparallel sudanophilia (and hence, presumably,peroxidase activity) in leukaemic precursors.Furthermore, the reaction of Auer rods for lysozymeresembled that previously described for myelo-peroxidase.

This is in keeping with the fact that myeloperoxi-dase and lysozyme appear simultaneously in thecourse of normal myeloid maturation (at the pro-myelocyte stage), being both primary granule con-stituents (Spitznagel et al., 1974; Baggiolini et al.,1974; Bainton, 1975a).Leukaemic myeloblasts (which lack primary

granules) were lysozyme negative, a finding whichaccords with the report by Asamer et al. (1971) thatlysozyme could not be visualised by immuno-fluorescence in leukaemic myeloblasts, although itconflicts with the observation of Karle et al. (1974)that lysozyme can be extracted from leukaemicmyeloblasts.

Lactoferrin, as would be expected from its associa-tion with secondary granules in normal human mye-loid cells (Baggiolini et al., 1974; Spitznagel et al.,1974; Bainton, 1975a) was detected only in moremature leukaemic myeloid precursors. The lacto-ferrin reaction permitted a clear distinction betweenlate myeloid precursors (positive) and monocytoidcells (negative). However, the same differentiationcan equally well be achieved by methods such asesterase staining.

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V.:

Figure Peripheral bloodpolymorphs from a case of acute myelomonocytic leukaemia stainedfor lactoferrin. Noteone positive cell (arrowed), characterised by granular cytoplasmic labelling, and an adjacent lactoferrin negativepolymorph.

One possible advantage offered by these immuno-cytochemical techniques is that lactoferrin and lyso-zyme in blood or marrow smears are relativelystable on storage at room temperature (Mason et al.,1975), so that immunoperoxidase staining may be ofvalue when studying leukaemic samples retro-spectively.A further intention of this investigation was to see

whether high levels of serum and urinary lysozymeencountered in cases of leukaemia showing mono-cytic differentiation would be paralleled by ab-normally strong cytochemical reactions for intra-cellular lysozyme. In the five cases of monocyticleukaemia studied in the present survey, only weakreactions were observed, comparable in their in-tensity with the reactions of normal monocytes. Thereport by Perillie et al. (1968) that the intracellularlysozyme content of leukaemic monocytes is lessthan that of chronic myeloid leukaemic myelocytesand granulocytes accords with this finding. Theseresults pose the paradox that leukaemia cells whichare presumably liberating large amounts of lyso-zyme apparently contain relatively little of thismaterial. One possible explanation is that the im-

munocytochemical technique is for some reason in-efficient at detecting lysozyme in monocytes. Amore probable explanation is that leukaemic mono-cytes liberate lysozyme rapidly after synthesising itso that no intracellular lysozyme pool accumulates.In contrast, myeloid cells, which store their comple-ment of lysozyme in cytoplasmic granules and liber-ate it only at cell death or degranulation, reactstrongly for this protein.

Evidence of continuous liberation of lysozymefrom cultured normal human monocytes, and for agreater intracellular content of this material innormal polymorphs than in normal monocytes, hasbeen provided by Gordon et al. (1974). Furthermore,Farhangi and Osserman (1974), studying a case ofmonocytic leukaemia, were able to demonstrate, afterincubation of bone marrow with labelled leucine,that newly synthesised lysozyme accumulated in themedium rather than in the leukaemic cells. Addi-tional evidence for the readiness with which lyso-zyme is liberated from monocytes is found in theirability to lyse adjacent organisms of Micrococcuslysodeikticus in the cytobacterial technique de-scribed by Syren and Raeste (1971), and in the 'halo'

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Intracellular lysozyme and lactoferrin in myeloproliferative disorders

effect noted in the present study when staining mono-cytes for lysozyme by the immunoperoxidasemethod (see results).The observation by Asamer et al. (1971) of strong

immunofluorescent staining for lysozyme in mono-cytes from three cases of monocytic leukaemiaassociated with heavy lysozymuria is in conflict withthis concept. This discrepancy is conceivably ex-plained by the presence of strongly staining myeloidcells among the monocytic population in their cases:the greater morphological cell detail visible in im-munoperoxidase (as opposed to immunofluorescent)preparations is of value in correctly identifyingmonocytic and myelocytic cells.

Finally, this study sought evidence of acquireddeficiencies of cellular lysozyme and/or lactoferrin inmyeloproliferative disorders. The deficiency ofneutrophil alkaline phosphatase, which is a featureof chronic myeloid leukaemia, suggested that addi-tional deficiencies of neutrophil constituents mightbe detected in this disorder by immunoperoxidasestaining. In particular, a depression or deficiency oflactoferrin reactivity might be expected on thegrounds that alkaline phosphatase has been localised(in common with lactoferrin) to neutrophil second-ary granules (Bainton, 1975a).

In the present study no abnormalities of eitherlactoferrin or lysozyme reactivity were observed inchronic myeloid leukaemic neutrophils. It may benoted that recent work (Bretz and Baggiolini, 1974;Spitznagel et al., 1974) has shown that alkalinephosphatase reactivity is in fact not associated witheither primary or secondary human neutrophilgranules but with a third subcellular fraction.Furthermore, electron microscopic examination ofchronic myeloid leukaemic neutrophils reveals anormal complement of primary and secondary gran-ules (Ullyot and Bainton, 1974) so that normal lacto-ferrin and lysozyme neutrophil reactivity in thisdisease is not an inconsistent finding. There has,however, been a recent report from Olofsson et al.(1975) suggesting that in a proportion of chronicmyeloid leukaemia patients intracellular levels ofneutrophil lysozyme and lactoferrin are reduced.

It is possible that the discrepancy between thefindings of Olofsson et al. and those of the presentstudy arises from the fact that immunocytochemicalmethods are only semiquantitative and, while beingable to reveal even a small subpopulation of grosslydeficient neutrophils, cannot detect a generalised re-duction in lysozyme or lactoferrin content affectingthe majority of cells.

In contrast to the findings in chronic myeloidleukaemia and other chronic myeloproliferative dis-orders, it was possible to demonstrate lysozyme andlactoferrin deficient neutrophils in a proportion of

cases of acute myeloid leukaemia. This is the firstoccasion on which deficiencies of these two whitecell constituents have been directly demonstrated inindividual cells, although Olsson's group (Odeberget al., 1976) has recently reported a marked reduc-tion in the quantity of lactoferrin extractable fromneutrophils in cases of acute myeloid leukaemia, anda similar observation was made by Karle et al. (1974)for neutrophil lysozyme in this disease. Furthermore,Bainton (1975b) has reported deficiencies of bothprimary and secondary granules on ultrastructuralexamination of granulocytes from cases of acutemyeloid leukaemia.A close precedent to this leukaemia-associated

deficiency of lysozyme and lactoferrin is provided bythe deficiency of neutrophil myeloperoxidase whichhas been reported by a number of authors in cases ofacute leukaemia (Hayhoe et al., 1967; Schmalzl etal., 1970; Davis et al., 1971; Catovsky et al., 1972),presumably representing the production of abnormalpolymorphs by the leukaemic myeloid clone. Onepoint of difference from leukaemia-associatedmyeloperoxidase deficiency is found in the factthat lysozyme and lactoferrin deficiency was found inthe present study in association with both myelo-blastic leukaemia, whereas Catovsky et al. (1972)emphasised that neutrophil myeloperoxidase de-ficiency was rare in cases showing predominantlymonocytic differentiation.

It remains to be determined whether deficiency ofneutrophil lysozyme or lactoferrin can be incrimi-nated as a cause of infectious complications in acuteleukaemia. One obvious practical implication ofthese neutrophil deficiencies is that their demonstra-tion may provide additional diagnostic informa-tion in the study of atypical myeloproliferative statessuch as 'preleukaemia' or refractory anaemia since ithas already been demonstrated that myeloperoxi-dase deficiency can occur in these disorders (Catovskyet al., 1971; Bessis et al., 1969). Future studies couldprofitably explore this avenue and also attempt tocorrelate, in individual cases of leukaemia, quantita-tive assays of granular proteins extracted from leu-kaemic cells with the pattern of immunocytochemicalreactions for the same constituents.

I am grateful to Caroline Farrell for skilful technicalassistance; to the Haematology Department andNuffield Department of Medicine, Radcliffe In-firmary for samples from patients under their care;and to Dr T. Parry for photographic assistance.

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