Allergy 1996:51: 614-620Printed in UK - all rights reserved

Copyright © Munksgaard 1996

ALLERGYISSN 0105-4538

Fixation with Carnoy's fluid reduces thenumber of chymase-positive mast cells: notall chymase-positive mast cells are alsopositive for tryptase

KleinJan A, Godthelp T, Blom HM, Fokkens WJ. Fixation with Carnoy'sfluid reduces the number of chymase-positive mast cells: not all chymase-positive mast cells are also positive for tryptase.Allergy 1996: 51: 614-620. © Munksgaard 1996.

Mast cells in the nasal mucosa can be studied by means of monoclonalantibodies (mAb) against tryptase (T^MC) and chymase (C^MC). Fixationwith acetone gives more positive cells than does fixation with Carnoy's fluid.In frozen biopsy specimens of allergic nasal mucosa fixed with acetone, thenumber of T^MC equals that of C^MC. When fixed with Carnoy's fluid,however, the number of T^MC is larger than the number of C^MC. Thedecrease in both T^MC and C^MC resulting from fixation with Carnoy'sfluid is time-related and depends on the type of mAb used. Carnoy fixationtime gives a decrease in the number of C^MC within 1 min, whereas thenumber of T'MC decreases only after 10 min. Within 1 min, the number ofC^MC decreases to a level where continued fixation no longer gives furtherdecreases in the number of cells. Two populations of mast cells can bedistinguished here: one sensitive and the other insensitive to Carnoy's fluid.When double-staining is used, fixation with acetone gives three populationsof mast cells: one positive for tryptase (T' C MC), another positive fortryptase and chymase (T^^C^MC), and a third one positive for chymase (T"C^MC). These three populations were found in lymph node, spleen, thymus,dermis, lung parenchyma, small intestinal submueosa, and nasal mucosa.

A. KleinJan^ , T.H. M. Blom\ W. J. FokkensDepartments of 'Otorhinolaryngology and^Immunology, University Hospital Dijkzigt andErasmus University, Rotterdam, The Netherlands

Key words: acetone; Carnoy's fluid;immunohistochemistry; mast-cell chymase;mast-cell tryptase; nasal mucosa.

A. KleinJanDepartment of OtorhinolaryngologyRoom Ee1655Erasmus University Rotterdam3015 GD RotterdamThe Netherlands

Accepted for publication 10 March 1996

Human mast cells may be classified on the basis oftheir neutral protease composition. Schwartz hasdeveloped highly effective monoclonal antibodies(mAb) against tryptase (G3) and chymase (B7) (1).On the basis of double-staining with these mAb,mast cells are subdivided into tryptase-positive andchymase-negative mast cells (T^C'MC) andtryptase-positive and chymase-positive mast cells(T^C^MC). These two types of mast cells are foundin the dermis, lung, small intestine, nasal mucosa,and conjunctiva (1-7). The ratio of T+C~MC mastcells to T' C^MC mast cells is tissue-related (1).Enerback et al. suggest that the ratio of T^MC toT^C^MC reflects a functional state of the mast cell(8).

Weidner & Austen, who recently discussed thetryptase-negative and chymase-positive mast cell(T"C^MC), could not demonstrate immuno-logically detectable tryptase. They did find these

mast cells in lung alveoli, bronchi, bowel mucosaand submueosa, axillary nodes, and breast skin (9).

Before the introduction of mAb against pro-teases, mast cells were stained by metachromatiestains, such as toluidine blue and Alcian blue. Inour laboratory, mast cells were stained by anti-IgEin combination with toluidine blue (10).

In metachromatie staining of mast eells, fixationis effected with Carnoy's fluid (11). In immuno-histochemieal staining of frozen biopsy specimens,mild fixatives such as acetone are generally used.Sehwartz and many others after him, however, usedCarnoy's fluid when staining with mAb againsttryptase and chymase instead of the more widelyused acetone. In our laboratory, we found thatCarnoy's fluid had a negative effect on the numberof mast cells.

To assess the effect of fixation with Carnoy'sfluid on immunohistochemical single mast cell

614

Chymase-positive mast cells

staining, we performed the following comparativestudies:

• The number of T" MC was compared to thenumber of C' MC after fixation with Carnoy'sfluid for 15 min as compared to fixation withacetone for 10 min.

• The number of T^MC was compared to thenumber of C^MC after fixation with Carnoy'sfluid for 0, 0.01 (dip), 1, 5, 10, 15, 30, 60 min,and 24 h.

Furthermore, we performed double-staining,comparing fixation with Carnoy's fluid to acetonefixation.

Material and methodsPatients

In this study, biopsy specimens of the nasal mucosaof 10 grass-pollen-allergic patients were studied.The patients had a history of isolated grass-pollenallergy for a period of at least 1 year, confirmed bypositive skin prick test reaction with Alutard Solu-prick extract of 1 HEP/ml and no other positiveskin prick test reaction with 13 common allergens,a median (range) radioallergosorbent test (RAST)score of 4+ (from 3+ to 5+), and a median (range)total IgE value of 290 IE/ml (22-1900). Biopsies ofnasal mucosa were taken when patients had symp-toms of allergic disease.

Tissue

Biopsies of nasal mucosa were taken from thelower edge of the inferior turbinate, about 2 cmposterior to the edge, with a Gerritsma forceps (cupdiameter of 2.5 mm). Local anesthesia wasobtained by placing a cotton-wool carrier with 50-100 mg cocaine and three drops of epinephrine(1 : 1000) under the inferior turbinate withouttouching the biopsy site (12). The biopsy specimenswere embedded in Tissue-Tek II O.C.T. compound(Bayer, Mijdrecht, The Netherlands) in a gelatincapsule and immediately frozen.

Furthermore, normal tissue collected at autopsysuch as submucosa of the small intestine, skin(dermis), lung parenchyma, lymph node, thymus (3months), and spleen was used. Nasal polyp tissuecollected during endoscopic surgery was used as well.

Monoclonal antibodies (mAb)

Use was made of antibody B7 (Chemicon,Brunschwig, Amsterdam, The Netherlands) againstchymase and G3 (Chemicon, Brunschwig,Amsterdam, The Netherlands) against tryptase.

The antibody AAl (Dako, ITK. Uithoorn, TheNetherlands), also against tryptase, was tested aswell (fixed with 10-min acetone at room tempera-ture and optimal diluted concentration of the anti-bodies). We found that AAl gave a weaker signalthan G3.

Fixation

The nasal biopsy specimens were fixed withCarnoy's fluid (60% ethanol, 30% chloroform,10% glacial acetic acid (Merck bv, Amsterdam, TheNetherlands). A fixation time series was made,ranging from 0, 0.01 (dip), 1, 5, 10, 15, 30, and 60min to 24 h in Carnoy's fluid. Biopsy specimens ofother tissues were fixed in Carnoy's fluid for 15min. Control fixation was achieved by 10-minfixation in acetone (Fluka, Bornem, Belgium).

Staining procedure

Staining was performed by the long-chain biotinstreptavidine-alkaline phosphate method (super-sensitive Biogenix, Klinipath, Duiven, TheNetherlands). Each tissue specimen was cut intoserial 6-|xm-thick sections on a Reichert-Jung 2800eFrigocut cryostat, transferred to poly-L-lysine-coated (Sigma, Bornem, Belgium) microscopeshdes, dried, and stored at -80°C for a maximumof 3 months. The specimens were raised to roomtemperature, dried, and fixed in acetone for 10 minat room temperature, or fixed in Carnoy's fluid andrinsed in phosphate-buffered saline (PBS), pH 7.8.

The staining procedure was continued by placingthe slides in a semiautomatic stainer (Sequenza,Shandon Scientific, Zeist, The Netherlands). Then,the sections were incubated for 10 min with0.5-1% bovine serum albumin (BSA, Sigma,Bornem, Belgium) in PBS. Next, the sections wereincubated with normal rabbit serum (CLB,Amsterdam, The Netherlands) for 10 min andsubsequently for 60 min with the mAb antichymase(B7 1 |xg/ml) and antitryptase (G3 0.4 ixg/ml). Afterthis, they were rinsed with PBS for 5 min andincubated with Link (rabbit antimouse long-chain biotinylated supersensitive AP, BioGenexAZOOOUM, Khnipath, Duiven, The Netherlands)for 30 min, rinsed with PBS for 5 min, and incu-bated with Label (streptavidine-alkaline phos-phatase, BioGenex AZOOOUM, Klinipath, Duiven,The Netherlands) for 30 min. They were thenrinsed once more in PBS for 5 min and TRIS buffer(0.1 M, pH 8.5) for 5 min and incubated for 30 minwith a New Fuchsin substrate (Chroma, Kongen,Germany). Finally, the sections were rinsed indistilled water, counterstained with Mayer's hema-toxylin, and mounted in glycerin-gelatin.

615

KleinJan et al.

Double-staining

After fixation with acetone, the sections were rinsedin PBS and incubated with normal goat serum(1:10) (CLB, Amsterdam, The Netherlands) for 10min and subsequently for 60 min with the mAbantitryptase (G3 4 fxg/ml). They were then rinsedwith PBS for 5 min, incubated with goat antimouseP-galactosidase (1 : 10, Southern Biotechnologies,ITK, Uithoorn, The Netherlands) for 30 min, rinsedwith PBS for 5 min, and blocked with 10% normalmouse serum (CLB, Amsterdam, The Netherlands)for 10 min. Next, the sections were incubatedwith antichymase biotin (3.3 (xg/ml, Chemicon,Brunschwig, Amsterdam, The Netherlands) for 60min, rinsed with PBS for 5 min, and incubated for30 min with alkaline phosphatase-conjugated goatantibiotin (1 : 100, Sigma, Bornem, Belgium). Theywere subsequently rinsed with PBS for 5 min, incu-bated with conjugated avidine-biotin-complex AP(ABC-AP Vector, Brunschwig Chemie, Amsterdam,The Netherlands) for 30 min, rinsed again withPBS for 5 min, and incubated for 30 min withP-Gal substrate (5-bromo-4-chloro-3-indolyl p-D-galactopyranoside, Sigma, Bornem, Belgium) (13).Finally, they were rinsed with TRIS buffer 0.1 M,pH 8.5, incubated for 30 min with a New Fuchsinsubstrate, rinsed in distilled water, counterstainedwith Gill's hematoxyhn (Polysciences, BrunschwigChemie, Amsterdam, The Netherlands), andmounted in glycerin-gelatin.

Toluidine blue staining

Toluidine blue, an aniline dye, stains mast cellsmetachromatically. Tissue sections were immuno-histochemically stained with toluidine blue at pH0.5 for at least 5 min and observed immediately.

Microscop ic e va h i a tio n

After evaluation of the sections, the stained cellswere counted under light microscopy at x250(single-staining) or x400 (double-staining). Thetotal surface area of each section was estimated bythe Kontron Image Analysis System Videoplan.

Statistical analysis

The coefficients of the regression lines wereworked out by logarithmic transformation, thusacquiring a linear relation. The nonparametricMann-Whitney U-test was used to compare thedifferences in cell counts between the groups andto compare vector coefficients of the regressionlines. A P value under 0.05 was considered toindicate a significant difference.

Results

General description

The sections of nasal mucosa had an averagesurface area of 2 mm^ and usually showed a hningof ciliated columnar epithelium with or withoutgoblet cells and/or partially stratified cuboidal epi-thehum. The lamina propria usually consisted of alooser, subepithelial, cell-rich layer with most ofthe mucous glands and a deeper, collagenous, cell-poor layer on the bone. The number of mast cellsin the epithelium was small; thus, only the laminapropria was used.

Acetone in comparison with Carnoy's fluid

After fixation with acetone, staining procedureswith antitryptase and antichymase gave identicalnumbers of positive cells (Fig. 1 and Table 1). Boththe number of T' MC and the number of C' MChad decreased significantly after fixation withCarnoy's fluid in relation to fixation with acetone(T^MC, P = 0.01; C+MC P = 0.0001). Fixation withCarnoy's fluid, however, gave a significantly moremarked decrease in the number of C^MC than inthe number of T+MC {P = 0.05).

Effects of Carnoy 's fluid over time

Fixation with acetone for 10 min gave no changein the number of C" MC and T" MC compared tononfixation. On the other hand, fixation withCarnoy's fluid for 1 min showed a significantdecrease in the number of C^MC (Fig. 2): for 5 min,median (range) cell number/mm-^ 74 (50-104)T^MC and 60 (20-169) C+MC; for 10 min, 90 (62-142) T^MC and 54 (22-102) C^MC; and for 30 min,106 (74-139) T^MC and 57 (16-97) C^MC. Fixationfor more than 1 min caused only a slight furtherdecrease in the number of C^MC. Fixation withCarnoy's fluid gave a reduction in the number ofT^MC as well. After fixation for 10 min, a markeddecrease was observed compared to before fixa-tion. The coefficients of the C" MC regression linesshowed a significantly larger decrease than thoseof the T+MC regression lines (P = 0.01).

Other tissues

Table 2 indicates the number of C^MC and T^MCin a few other tissues, such as the submucosa of thesmall intestine, skin (dermis), lung parenchyma,lymph node, thymus (3 months), spleen, and nasalpolyp, after fixation with Carnoy's fluid for 15 minand fixation with acetone for 10 min. In all tissues,the number of C^MC was found to be smaller after

616

Chymase-positive mast cells

tryptase * chymase

200

##%V)a>

jQ 150E3C

— 100

a>o

50

n

P=0.011

P=0.0001

1

•

t

• + + •• •

+ •

1

P=0051 1

•

• +

acetone10 minutes

Carnoy15 minutes

Fig. L Score of tryptase- and chymase-positive mast cells/mm" in lamina propria: acetone fixation for 10 min and fixation with

Carnoy's fluid for 15 min.

Table 1. Number of tryptase-positive and chymase-positive mast

cells/mm^ in lamina propria of nasal mucosa of patients with isolated

grass-pollen allergy

No fixation(cells/mm^)

Acetone, 10 min(celis/mm^)

Carnoy's fluid, 15 min(cells/mm^)

mAb used

AntitryptaseAntichymase

Median

102130

Range

77-17957-216

Median

124123

Range

83-17273-156

Median

9060

Range

75-15430-107

Statisticsacetone-C^MC acetone: P= NS.acetone-rMC Carnoy: P = 0.01.

C MC acetone-C^MC Carnoy: P= 0.0001.C MC Carnoy-rMC Carnoy: P= 0.05.C+MC acetone-C^MC no fixation: P= NS.T MC acetone-rMC no fixation: P = NS.

fixation with Carnoy's fluid than after acetonefixation. There were no consistent differences inthe number of T^MC between fixation withCarnoy's fluid and acetone.

Double-staining

Double-staining showed three different types ofmast cells. Ten nasal biopsy specimens of sympto-matic allergic patients gave the following ratios:74% (50-88%) TX^MC, 15% (6^3%)and 7% (1-32%) T

Evaluation of the other tissues (Table 3) showedlarge ratio differences. In the dermis, for instance,T" C^MC predominated (87%), whereas only smallpercentages of T+C'MC (5%) and TC^MC (8%)were found. In the lung, however, T+C"MC (90%)was mainly found, with only a small percentage ofT+C+MC (9%) and hardly any 'TC+MC (1%).After fixation with Carnoy's fluid, no T^C^MCwere demonstrated in any of the previously men-tioned tissues.

Toluidine blue

Double-staining with toluidine blue and chymaseshowed most cells to be double positive for tolui-dine blue and chymase, and the existence of weaklychymase-positive, toluidine-negative stained cellscould be explained by the fact that the sensitivityof the toluidine blue method is much lower thanthat of the immunohistochemical method of stain-ing chymase. Double staining with toluidine blueand tryptase showed most cells to be double posi-tive for toluidine blue and tryptase.

DiscussionIn the literature, mast cells are divided into T^OMCand T+C^MC (4,5). Carnoy's fluid is used as fixative.In our laboratory, we found that Carnoy's fluid hada negative effect on the number of mast cells.

617

KleinJan et al.

• tryptase * chymase

250

2CX)

0)150

E3C

_ . 100a>o

50

0

Hh

•

t

+•

• +••

*

••

••

•

s

• »

t

•

•

•

j *ti

•

•

• * .' { : * • ;

not fixed dip 1 min 15 min 1 h

log fixation time24 h fixed

Fig. 2. Score of tryptase- and chymase-positive mast cells/mm- in laminia propria, given different (0, dip, 1 min, 15 min, 1 h,and 24 h) Carnoy's fluid fixation times plotted on log scale.

Table 2. Number of tryptase-positive (T*MC) and chymase-positive (C^MC)

mast cells/mm^ in serial biopsy samples of various tissues by single staining

with either antitryptase (G3) or antichymase (B7)

T+MC (cells/mm^ C+MC (cells/mm^)

Acetone Carnoy's fluid Acetone Carnoy's fluid

Spleen

Dermis

Lung parenchyma

Thymus (3 months)

Small-intestine

submucosa

Lymph node

Nasal polyp

0.8138.6

>500.9

32.2

15.7

4.9

1.2107.6

>501.1

28.8

9.76.2

0.7187.3

4.81.8

36.4

17.6

5.2

0.4123.5

0.90.9

10.4

11.4

1.0

The present study on nasal biopsies of sympto-matic allergic patients showed that fixation withacetone gives a significantly larger number ofT^MC and C+MC than fixation with Carnoy's fluid(Fig. 1). This difference may be explained by thefact that Carnoy's fluid fixation weakens the bind-ing of the mAb to the epitope against which it isdirected, as the epitope is either changed or dam-aged. Variations in the degree of sensitivity of theepitopes to Carnoy's fluid may explain the fact thatthe number of chymase-positive cells shows a moremarked decrease than the number of T

Table 3. Percentage of single tryptase-positive (VZ MC), tryptase-positive/

chymase-positive (rC*MC), and single chymase-positive (T C+MC) mast cells

in serial biopsy samples of various tissues by double-staining technique

Tissue T+C-MC (%) T+C+MC (%) r C + M C (%)

Spleen

Dermis

Lung parenchyma

Thymus (3 months)

Small-intestine submucosa

335

901968

49

87

9

67

31

18

8

1

15

1

This finding is true not only of the nasal mucosa,but also of the small-intestine submucosa, thymus,dermis, lung parenchyma, lymph node, and spleen(Table 2). Carnoy fixation gives a decrease in thenumber of C^MC' for all tissues. In relation to thenumber of T^MC, the picture is not as clear.

For further study of the effects of Carnoy's tluidon tryptase and chymase, different fixation timeseries were compared (Fig. 2). Fixation withCarnoy's fluid was shown to induce a time-relateddecrease in the number of both T' MC and C"* MC.Acetone fixation did not give changes in the num-bers of T^MC and C^MC after the standard 10-minfixation period in relation to nonfixation. For thisreason, it was decided not to use a time series foracetone fixation. Although Carnoy's fluid fixation

618

Chymase-positive mast cells

T+C*MC

3

3. Double staining of human nasal mucosa with bio-tinylated-antichymase B7 antibodies and antitryptase G3 anti-bodies. Chymase-positive mast cells stained red, tryptase-positive mast cells stained green, and red- and green-stainedcells are chymase- and tryptase-positive mast cells. Inset showstoluidine blue-positive T"C" MC. e: epithelium; bm: basalmembrane; lp: lamina propria.

Double-Staining with chymase and tryptase hasshown that, besides T+C^MC and T+C-MC, T"C" MC also occur (15), contrary to the findings ofSchwartz (4, 5) and others after him. T'C^MC cellswere demonstrated in all tissues examined. For thedifferent tissues, however, cor^siderable differenceswere found in the ratios among the T^C^MC, T^C~MC, and TX^^MC cells (Table 3). This findingagrees with a recent study by Weidner on lung, skin,and bowel mucosa. It must be added, however, thatWeidner used a polyclonal antibody against chy-mase (developed by Schechter [9]). Recently,Bradding et al. noted that in normal nasal mucosalbiopsies, cells occasionally expressed immunoreac-tivity for interleukin-4 and chymase, but not tryp-tase (16).

To be sure that the T~C" MC cells are mast cells,a few double stainings were done. A small numberof the T~C^MC cells were stained with toluidineblue, and only the strongly positive cells staineddouble. This effect could be the result of the limitedsensitivity of the toluidine blue staining. This stain-ing detects only the mast cells that are not degran-ulated (10). Double-staining of allergic nasalmucosa with IgE and tryptase (data not shown)show all tryptase-positive cells that are doublepositive for IgE. Double-staining IgE and chymaseshow all chymase-positive cells that are doublepositive for IgE.

In our opinion, Schwartz and others failed toobserve the T"C^MC population because they usedCarnoy's fluid for fixation. In this situation, we didnot find any T"C"^MC in our laboratory either.Studies are in progress to evaluate further single-and double-tryptase- and chymase-positive cellsduring allergen provocation.

AcknowledgmentsThis work was supported in part by the Dutch AsthmaFoundation (grant no. 32-90-54) and Glaxo.

gives a reduction in the number of both C"* MC andT^MC, the number of C^MC decreased much moregreatly than the number of T^MC. Within 1 min,the number of C^MC had fallen to under 50% ofthe number which had not been fixed. It seemsreasonable, then, to assume that there are twopopulations of C^MC, one sensitive and the otherinsensitive to Carnoy's fluid. A similar differencein response to fixatives - in this case, formalin -was reported by Befus et al. (11) for metachromaticstaining of mast cells. A similar difference in sen-sitivity to formalin was also reported for the T^MCpopulation (2, 14).

References1. Irani AA, Schechter NM, Craig SS, DeBlois G,

Schwartz LB. Two types of human mast cells that havedistinct neutral protease compositions. Proc Natl Acad SciU S A 1986;83:4464-8.

2. Craig SS, DeBlois G, Schwartz LB. Mast cells in humankeloid, small intestine, and lung by an immunoperoxidasetechnique using a murine monoclonal antibody againsttryptase. Am J Pathol 1986;124:427-35.

3. Craig SS, Schwartz LB. Tryptase and chymase, markers ofdistinct types of human mast cells. Immunol Res1989;8:130-48.

4. Igarashi Y, Kaliner MA, Hausfeld JN, Irani AA,Schwartz LB, White MV. Quantification of resident inflam-matory cells in the human nasal mucosa. J Allergy ClinImmunol 1993;91:1082-93.

619

KleinJan et al.

5. Irani AM, Bradford TR, Kepley CL, Schechter NM,Schwartz LB. Detection of MCT and MCTC types ofhuman mast cells by immunohistochemistry using newmonoclonal anti-tryptase and anti-chymase antibodies.J Histochem Cytochem 1989;37:150945.

6. Kawabori S, Denburg JA, Schwartz LB, et al. Histochem-ical and immunohistochemical characteristics of mast cellsin nasal polyps. Am J Respir Cell Mol Biol 1992;6:1992.

7. Schwartz LB, Irani AM, Roller K, Castells MC,Schechter NM. Quantitation of histamine, tryptase, andchymase in dispersed human T and TC mast cells. JImmunol 1987;138:2611-15.

8. Enerback L, Aldenborg F, Juliusson S. Lack of chymase: afunctional rather than a phenotypic marker of mucosalmast cells [Abstract]? Allergy 1993;48 Suppl 16:110.

9. Weidner N, Austen KF. Heterogeneity of mast cells atmultiple body sites. Fluorescent determination of avidinbinding and immunofluorescent determination of chymase,tryptase, and carboxypeptidase content. Pathol Res Pract1993;]89:I56-62.

10. Fokkens WJ, Godthelp T, Holm AF, et al. Dynamics ofmast cells in the nasal mucosa of patients with allergicrhinitis and non-allergic controls: a biopsy study. Clin ExpAllergy 1992;22:70M0.

11. Befus D, Lee T, Goto T, Goodacre T, Shanahan F,Bienenstock J. Histologic and functional properties of mastcells in rats and humans. In: Befus AD, Bienenstock J,Denburg JA, editors. Mast cell differentiation and hetero-geneity. New York: Raven Press, 1986:205.

12. Fokkens WJ, Vroom TM, Gerritsma V, Rijntjes E. A biopsymethod to obtain high quality specimens of nasal mucosa.Rhinology 1988;26:293-5.

13. Bondi A, Chieregatti G, Eusebi V, Fulcheri E, Bussolati G.The use of ^-galactosidase as a tracer in immuno-cytochemistry. Histochemistry 1982:76:153-8.

14. Walls AF, Bennett AR, McBride HM, Glennie MJ,Holgate ST, Church MK. Production and characterizationof monoclonal antibodies specific for human mast celltryptase. Clin Exp Allergy 1990;20:581-9.

15. Blom HM, Godthelp T, Fokkens WJ, et al. Mast cells,eosinophils and igE-positive cells in the nasal mucosa ofpatients with vasomotor rhinitis. An immunohistochem-ical study. Eur Arch Otorhinolaryngol 1995; Suppl 1:S33-9.

16. Bradding P, Okayama Y, Howarth PH, Church MK,Holgate ST. Heterogeneity of human mast cells based oncytokine content. J Immunol 1995;155:297-307.

620