THE AMERICAN JOURNAL

OF PATHOLOGY

VOLUME XXXV NOVBEDECE, 1959 NuMBER 6

THE GENESIS OF THE CHARCOT-LEYDEN CRYSTAL INTHE EOSINOPHILIC LEUKOCYTE OF MAN *

RONAw A. WEmS, M.D.From tke Eectron Microscope Laboratory, Department of Pathology, Louisiana State

University School of Medicine, New Orkans, La., axd the Electron MicroscopeLaboratory, Department of Anatomy, University of Texas Medic Braxck,

Galveston, TexsThe formation of protein crystals within the cytoplasm of human

cells of any type is actually an infrequent occurrence, the most note-worthy exceptions being the Charcot-Leyden crystals in the eosinophilicleukocytes, and the crystalloids of Reinke in the interstitial cells ofthe testis or hilar cells of the ovary.The development of the first named crystals represents an uncom-

mon fundamental intracellular process with rapid protein crystal for-mation that has no parallel in any other cell of the hutman body.Charcot-Leyden crystals have been descnbed in the tissues in manycases of eosinophilic granuloma of bone,' in the sputum of patientswith bronchial asthma,2 and in stools associated with amebic dysen-tery.2 The formation of these crystals is rapidly accomplished andcan be observed in vitro in eosinophilic leukocytes within a few min-utes after the application of surface-active wetting agents. Ayres andStarkey3 studied the formation of these crystals by phase microscopyand concluded that they arose in the region of the eosinophilic granules,but saw no evidence that the granules were the source of the crystals.They also postulated, on the basis of other evidence, that the crystalswere derived from the nucleus.The present investigation is the first report of electron microscopic

examination of developing crystals in eosinophils in ultrathin sections.New evidence indicates that a portion of the eosinophilic granule actsas a nidus in the genesis of the crystal and probably supplies the pre-dominant molecular building material for crystal growth.

* Supported by grants from the American Heart Association and the National Insti-tutes of Health (H-2549, B-69o; C4 and C4SI).

Received for publication, February is, i959.

I09I

MATEIAL AND METHODS

Blood was obtained from patients suffering from a variet of alergicdisorders. The best preparations were made from a patient with severebronchial asthma whose peripheral blood leukocyte conmt was 34,400per cubic mL with 75 per cent ein ls. Twenty mL of venous bloodwas mixed with 2.0 cC. of 3.8 per cent sodium citrate and 4.0 cc. of 6per cent dextran. The plasma with leukocytes was then separated aftersedimentation for 45 minutes at room temperature, according to themethod of Klein, Endani, Djerassi and Resnick.4 Aliquots of 5 mL ofthe pLasmaleukocyte susion were centrifuged for 5 minutes at2,00o r.pm. he su rtant was removed by aspiration, leaving onlya dense suspension of leukocytes in a smal amount of plasma Approx-imately 5 to io mg. of Aerosol OT, dioctyl sodium sulfosucinate(product of the American Cyanamid Company; obtainable from FisherScientific Company, Pittsburgh, Pennsylvania), on a glass sfirring rodwas added to one such concentrated aliquot, while a second similaraliquot was treated with a cle glass stirring rod as a control. Bothmixtures were stirred gently at about I minute interals Within 2minutees the aerosol-treated sample became viscid and ny in cOn-sistency. At the end of 5 minutes both the control mixture and theaerosol-treated mixture wer fixed in buered osmic addswith added sucrose" for 30 minutes. Each was thn dehydrated pro-gressively in alohols and embedded in thlate Ultathin sec-tions were red on a Porter-Blum microtome and examined in anRCA EMU 3 or an RCA EML 2 elect mioscope

Crystals were also produced by taking a similarly c tedsterile specimenof leukocytes and incutingat370 C. for3dayswithout the addition of wetfing a During this tme interal,Charcot-Leyden crystals formed sponaeously. The wasthen fixed ad prepard for electro micrscopy by thle procdureoutlined above.

In addition, smears of both the aerosol-treated suspension and con-trl suspension were made, allowed to air dry, and the stained withSudan black B according to the method of She T

RESULTS

In the control specimens the eosinophils were well preserved andshowed no evidence of crystal formation (Fig. I). The granules wereintact, and the characteristic inner, densely osmiophilic bar couldusually be distinguished from the outer, less dense substance. In afew cells a rare granule could be found showing a partial separation

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CHARCOT-LEYDEN CRYSTALS

of this outer substance from the inner bar. The eosinophils often con-tained one or more larger, very dense, oval bodies in the cytoplasm,representing the "basophilic bodies" descibed by Low and Freeman.8The nuclear membran of the eosnophil was intact, and the nudeo-plasm was finely dispersed.The e inophls from the emens tated with Aerosol OT were

Markedly altered. The cytoplasmic m rane was usually idistinctor had partally di The cytoplasmic granules showed definitealterations. One such change commonly seen was a swelling of thegranule leaving the central dense bar in a clear space surrounded by afragmented circular band (Fig. 2). In other regions, the granule wasswollen and o ntained numerous irregular spaces. The "basophilicbodies" were not altered. Charcot-Leyden cystals were foumd in allstages of development in the cytoplasm. The crystals showed a varying

al or elo ed dipyramidal shape, depending on the plane mwhich they were sectioned. The earliest forms, shown in Figures 3, 4,nd i,exhlbited alecence of several altered gramnules from which 3

or more sides of the crystal emerd. Large crystaLs, such as thoseshown in Figures 6 and 7, hibited incorporation of the granules intothe crystal proper. The altered granules and rystals had a similntenal arance. Many larger crystals wer fectin shape with-out evidence of included granules. High magnifications of the crysts,up to I9o,ooo, revealed an irregular separation of the crystal substanceinto dense, amorphous masses, between which there was a less densespace. Occasional dark ropy strands were intmingled (Fig. 8). Therewas no evidence of a discrete lattice or pattern of arrangement of thecrystal substance.The nuceus of the eosinophil was still distinct, but the nuclear

membrane was fragmented. The nucleoplasm was aggegated into saparticles or arranged coarse strands. The i varied in theseverity of the changes described above, as if the wettig agent didnot affect al the cll to an equal degree. A rare eosinophil had anintact ell membrane, and granules were tered No crystals wereseen in these better preserved cells. Crystals were found only in thosecells showing definite alterations in the granules. There was no evi-dence of crystal formation in the nucleus proper.The cells from the specmen not treated with wetting agents, but

incubated for 3 days, were poorly preserved; however, several featureswere noted. The eosinophils as individual cells could still be discernebut gran had disappeared almost completey. Crystals wereabundant, and could be seen in the cytoplasm. The nucleus was still

NovJ.-Dec, I959 I1093

present but was a dense shrunken structure. There was no evidence oforig of the crystals from the nucleus.Sudan black B stains of the air-dried smears produced a positive

black to brown staining of the eonophl granules in both the controland aerosol a. The granules were either a solid blak orappeared as black rings with clear cente Charcot-Leyden crystalsdid not stain with Sudan black. Ocsonaly, irrglar lacuas con-taining a granule could be faintlyd usually near the centerof a staL These intralr granes did not stain with Sudanhiack.

DIS;CUSSIONThe observations demonstre that there is a red structural

alteration of the eosiphl ganule as a prerequisite for crystal forma-tion. The altered granules become enlarged and vacuolated, showing atenldency to separation of an outer cortical rm from a central core.The Sudan black stains exhibit no apparent change in staining charac-teristics of this outer rtical layer, althoh in the crystals with visiblelcunas contaning granules, the granules did not stain with Sudanblack. It is possible that the lipid staining reaction of the granules inthe crystals was blocked in some manner, or it may be that only thecentral protein core of the granule was inorporated into the crystalAnalysis of pure crystal samples by Buddecke, Esseier and Marti'revealed that they were made of a zinc-containing polypeptide fromwhich I4 different amino ds were identified. The added fact that thecrystals were alwa negative ireaction to lipid stains makes it prob-able then that the outer lipid cortex was not incorporated when thegranule participated as a nidus for crystal formation.

Definite evidence is seen in Figure 6 that the granules are incorpo-rated into the crystal and may merge imperceptibly with it. Furthergrowth of the crystal probably does not occur through gross lumping ofthe granules on the original nidus but by an imperceptible molecularprocess. The molecular protein building terial could be suplied bythe other granules remain in the cell, although other molecules fromthe cytoplasm or nucleus may contribute. The dose similarity of thefine structure of the young crystal and the altered granules suggeststhat the eosinophil granule is probably the predominant source ofbuilding materiaL This investigation has not yielded evidence to indi-cate that the nucleus was an important contributor to the crystalsubstance. The best negative evidence was seen in the 3-day-old prepa-rations without added aerosoL In thlese, practically all the granules had

ared, crystals had formed, and yet the nucleus persisted as ashrunken dense mass. This would indi that little or no nuclear

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CHARCOT-LEYDEN CRYSTAIS

substance was utlized. Also, it should be noted that the nsophilicbodies" described by Low and Freeman' in the cytoplasm of theeosinophil are not involved in the nidus of crystal formation, and donot appear to alter significantly in the presence of aerosol.The formation of these crystals should be of interest to the investi-

gation of cystal growth of any type. Since the crystal can be sectioned,the stages of its development can be studied easily. It appeared thatthe genesis generally followed a pattern of nidus formation for theinitiation of crystalliation, with a subsequent uniform molecular build-up. This process appeared to be unique in that the nidus seemed toblend into the crystal imreptibly. Ayres3 showed that the crystalvaried in its solubility, depending on its age. When freshly formed, thecrystals were soluble in absolute methyl alcohol, but if allowed to stand6 to 24 hours, the crystals were insoluble in methyl akohol. This woulddite that the crystals were in a dynamic state of molecular arrange-

ment for optimum stability even in their initial stage. It is possiblethat high resolution studies of older crystals may show a more discretelattice or other arrangement within the crystal, although preliminaryinvestigations have not been satisfactory because of tehnical difficul-ties.

This study, of course, does not shed light on the function of thesecrystals in hmnnan disease processes; it is, however, a step further in theknowledge of specific intracellular functions of the eosinophiL Theeosinophil granule itself is an interesting and unique structure whenexamined by electron microscopy and by histochemical methods. Thefact that it undergoes such a strikng alteration before becoming thenidus for the formation of the crystalline structure represents a specificbasic intracellular process. This would justify further attention andinvestigation of possible applications to the general field of cellularbiophysical chemisry and understanding of the role of the eosinophilin allergic disorders.

SUARYThe formation of Charcot-Leyden crystals was examined pricilly

by electron microcopy. Ultrahin sections of human eosolicleukocytes treated 5 miutes with the wetting agent dioctyl sodiumsulfosuccinate were prepared. The eosinophlic granules showed swell-ing, vacuolation, and an apparent alteration of the outer cortical layer.The altered granules were observed in process of coalescence, actingas a nidus from which the crystal emerged. The internal structures ofthe developing crystal and of the altered granules were quite similar,and there was evidence that the granules merged with the crystalproper. The nucleus did not appear to be a source of crystal substance.

Nov.-DecE, I959 3I09S

iog6 WELSH Vol. 35, No. 6

It was proposed that the inner substance of the eosinophil granules wasthe nidus for and probably served as the basic protein building materialfor the crystal.

REFERENCESi. Ayres, W. W., and Silhiphant, W. M. Charcot-Leyden crystals in eosinophilic

granuloma of bone. Am. J. Clin. Path., I958, 30, 323-327.2. Todd, J. C.; Sanford, A. Hi, and Wells, B. B. Clinical Diagnosis by Laboratory

Methods. A Working Manual of Clinical Pathology. W. B. Saunders Co.,Philadelphia, I953, ed. I2, pp. 3I-32.

3. Ayres, W., and Starkey, N. M. Studies on Charcot-Leyden crystals. Blood,I950, 5, 254-266.

4. Klein, E.; Eridani, S.; Djerssi, L, and Resnick, R. A simple method for theseparation of leukocytes from whole blood. Am. J. Clin. Path., I958, 29,550-552.

S. Palade, G. E. A study of fixation for electron microscopy. J. Exper. Med.,I952, 95, 285-298.

6. Caulfield, J. B. Effects of varying the vehicle for OsO in tissue fixation.J. Biophys. & Biochem. Cytol., 1957, 3, 827-830.

7. Sheehan, H L. The staining of leukocyte granules by Sudan black B. J. Path.& Bact., I939, 49, 58o-58i.

8. Low, F. N., and Freeman, J. A. Electron Microscopic Atlas of Normal andLeukemic Human Blood. Blakiston Division, McGraw-Eill Book Co., NewYork, I958, p. ii.

9. Buddecke, E.; Esselier, A. F., and Marti, Hi R. tber die chenische Natur derCharcot-Leydenschen Kristalle. Ztschr. f. phys. Chemie, I956, 305, 203-206.

ITe author wishes to acknowldge the technical mistance of Mr Yvonne Blocker atLoisiana State Universiy School of Medicne, and of Mr. Rikardo Monles at the Uni-versity of Texas Medial Branch.

LEGENDS FOR FIGURES

FiG. i. Untreated control eosinophilic leukocyte. There is an inner dense bandvisible in some of the specific granules. X 12,750.

FI. 2. Eosinophil granules 5 minutes after addition of Aerosol OT. Some of thegranules are swolle, leaving a central dense bar in a clea space surrounded bya fragmented circular band. Other granules are vacuolated. One granule ap-pears ualtd X I2,IOO.

CHARCOT-LEYDEN CRYSTALS

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Nov.-Decv, I959 IO097

Vol. 35, No. 6

FIG. 3. Arrow points to an area of coalescence of 4 or more altered granules withemergence of angular margins of a crystal as foot-like formations. A portionof the nucleus lies in the upper mid portion of the picture; there is fragmenta-tion of the nuclear membrane. X I5.900.

FIG. 4. In the center of the photograph an incompletely formed crystal is evidenLAlthough most of the sides have formed, the crystal is incomplete in its upperm n and appears to be reduplicating itself on the left. The other cytophsmic

granules are altered in outline and vacuolated. X i6.9oo.

i0o98 WTELSH

CHARCOT-LEYDEN CRYSTALS

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Nott,-cC, I959 I099

Vol. 35, No. 6

FIG. 5. A crystal develoing with an irregular ma, icorporating one distinctgranule. Within the crystal, above the incuded granul, there is a fine lineforming a partial crcle. This lies around what is probably another granule thathas merged almost completely with the crystaL X I5,99O.

FIG. 6. ThiS cryStal incorporates 3 distinct granules. X 12,750.

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Vol. 35, No. 6

FIG. 7. ODlY 3 sides of this crystal have formed. The undeveloped sides contain 2discrete ues X 5,990.

FIG. 8. An incompletely formed crystal partially incorporating a granule. X 5,990.

FIG. 9. Eosinopbilic leukocyte inad for 3 days at 370 C.; no wetting agentswere added. The nucleus persists as a vacolated structure; all of the cyto-plasmic granules have d ed. A h al crystal is present in thecytoplasm. X 12,000.

FIG. IO. Inner structure of crystal, showing dense amorphous strands irrgularlyseparated by less dense spaces. Scattered, darker, ropy strands are mamfestX 187,200,

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