Acta hi st ochom. Bd, 57, S. 1-13 (1976)

Institute of Morphology, Bulgarian Academy of Scien ces, Sofia. Bulgaria(Director: Prof. Dr. I VAN GORANOV)

A cytological method for the simultaneous stainingof nu cleoproteids and some cat hionic proteins

By ELISAVETA BORISOVA ZVETKOVA1) and IVAN BORISOV ZVETKOV2• 3)

With 25 figures and pl a te I

(R eceived Septem ber 1, 1975)

Summary

A cy tochemical method is sugges te d for the s im ultaneous a nd differential s ta in ing of cellu la r

nucleoproteid s [r ibonuc leop roteids (R NP) and desox yrib onucleoproteid s (DN P )], as well as for the

sim ultaneous contras t staini ng of some ba sic (arginine- a nd lysin -containing) protein s.The stain ing techn ique is ba sed on DN A-dena turati on proce dures and the application of mix­

tures of basic dye - m ethylen e blu e a nd acid dyes - eosin or fast g reen at low conce n trat ions.

The combina t ion of methyl en e blue with eos in is used for the stain ing of r ibonueleoproteids (RNP)whereas methylen e blu e-fast gree n fo r the simultane ous detection of ribonuclcoproteids and desoxy­

ri bonucleoproteids (R NP and DNP), as well as for the differential sta in ing of nuc lea r DNP (a ft er

cold hydroly sis with 5 N H CI). The acid dyes eosin an d fa st gree n stain in p ink resp. in green somecathionic protein s in the lysosomal (sp ecifi c) granules of t he neu trophi lic and eosinop hilic leucocytesi.n th e cytoplas m of erythrocytes. and a ft...r co ld hydrolysis in t.he cy to plasm of lynlphocy tes. Afluorescen t va riant of the method with sulfa flav in is also suggested for the fluorochromation of cyto­plasmi c ca thio n ic granu les in the leucocy tes .

Acid mucop oly sa cch a ride com ponents in the granu les of basophilic leucocy tes, t issue m ast ocytesa nd thrombocy tes are stain ed in ten sively pi nk-violet (y-metachromat ic) .

The p ossibil it ies for the a pplica t ion of the method in the quanti tative a nalysis of blood and ex­folia ted cells, as well as for purpose of h aematol ogy , immunology and exfoliative cytology a re di s­cussed .

Introduction

A cytologica l method is suggested with variants for the staining of nucleoproteids[ribonucleoproteids (RNP) and desoxyribonucleoproteids (DNP)), as well as for thesim ultaneous specific det ection of some basic (lysin. and arginine.cont aining) prot eins.

1) The method has been proposed by Dr. ELISAVETA ZVETKOVA at the IV European Congress

on Cy tology , Ljubljana , Jugoslavia, Sep tember 11th to 13th, 1974 .2) Technical Collaboration.

3) Institute of Techn ical Cybernetics, Bulgarian Aca dem y of Sciences (D irect or: P rof. Dr . N .

NAl'LATANOV).

1 Act a histochem. Ed . 57

2 E. B. ZVETKOVA and I. B. ZVETKOV

The cytological technique is based on DNA-denaturation procedures which in­clude the effect of temperature and the application of hot formalin va pours on cyto­logical smears. The method for DNA-denaturation has been described in collaborationwith HADJIOLOFF concerning the fluorescent cytochemistry of acid muoopolysao­charides and mucins and has been developed in connection with our methods fornuclear and chromosome fluorochromation with berberin sulfate (20, 21, 53).

The staining method includes the use of mixtures of basic and acid dyes, methyleneblue-eosin and methylene blue-fast green at low concentrations. The combination ofmethylene blue with eosin is used for staining of ribonucleoproteids (RNP) whereasmethylene blue-fast green for the simultaneous detection of ribonucleoproteida anddesoxyribonucleoproteids (RNP and DNP), as well as for the differential stainingof DNA following cold hydrolysis with 5 N HOI. The use of solutions, containingbasic and acid dyes at low concentrations aims at the reduction of the competitiveeffect of the oathionic proteins of nuoleoproteids with regard to optimal staining ofnucleic acids by the basic dye methylene blue (12,31,43). The simultaneously appliedacid dyes - eosin and fast green - stain in pink or in green some cathionic proteinsin the cytoplasm of erythrocytes and in the lysosomal granules of the neutrophilesand the eosinophiles (3, 7, 16).

To detect the cytoplasmic cathionic proteins in the Ieucocytes by fluorescentmicroscopy a fluorescent variant of the method with sulfaflavin is suggested, whichinduces a golden-yellow fluorescence in the granules of the granulocytes.

Material and methods

The staining method includes the following variants and procedures:1. DNA-denaturation, performed in situ on air-dried, nonfixed blood and cytological smears").

For that purpose the preparations are deep-frozen (- 20 DC to - 22 DC) for 2 to 3 h, thawed anddried at room temperature (5 min), followed by gradual heating of the slides on an electric drier for3 to 4 min up to 55 DC to 60 DC. The warm slides are then fixed by hot formalin vapours of 40 %formalin (pH = 4) for 12 min. The fixation is performed in a closed PETRI dish, in which the heat­

denatured preparation is introduced under formalin vapours (formalin heated to the point of bub­

bling in a small vessel with Ij3 of the diameter of the PETRI).

2. Following the action of formalin vapours the preparations are dried at room temperature

(5 min) without being washed. Immediately follows their staining for 50 to 55 min, by dropping

of one of the following staining mixtures:a) methylene blue (5 ml of 0,2 % aqueous solution) and eosin (5 ml of 0,2 % aqueous solution)

following dilution with 40 mljMj15 phosphate buffer with pH = 8 (see also 43).

b) methylene blue (5 ml of 0,2 % aqueous solution) and fast green (5 ml of 0,2 % aqueous solu­

tion) following dilution with 40 ml Mj15 phosphate buffer with 7 :::: pH :::: 8.c) the same mixture of methylene blue and fast green, applied after cold hydrolysis with 5 N HCI

(23). The cold hydrolysis is performed by dropping of cold 5 N HCl on denatured and fixed smears

for 15 to 25 min, followed by repeated washing in Mj15 phosphate buffer (7 :::: pH :::: 8), drying

of smears at room temperature and its staining.

1) The late treating of the cytological material does not damage the cell morphology. This allows

its safe transportation.

A cytologica l method 3

d) fluoroch romation with 0,1 % solu t ion of fluoroch rom e sulfa flavi n in bo racic buffer (0,1 Mb ori c acid, 0,1 N NaOH, 0,1 M K C1, diluted 1 : 10, p H = 8; see a lso 25).

3. Each st a in ing procedure is followed by severa l quick washin gs with the corresp on d in g bufferby drop let ting until t he staining solution is released no m ore. La ter the smears a re ai rdried , clearedin xylols (5 m in) and a re in cluded in im mersion or n on -fluorescin g oil.

Results and discussion

1. Concerning staining of the ce ll u l a r ribonucl eoprot eids (RNP) andd es ox y r i b onu cl eo pro t ei d s (DNP)

The use of the staining mixture, containing methylene blue a nd eosin is recommend­ed for the det ection of ribonuoleoproteids (RNP) which stain blue-vi olet . Th e desoxy­r ibonueleoproteids (DNP) do not stain with the except ion of cases of a lte red bas icityof the nu clear chromatin [(13,19); (fig. 1, 3, 4, 17-19,24,25; colour plate I: 1,2;9,10)].

Th e stain ing mixture of methylene blue and fast gree n is employ ed for t he simul­t aneous detection of cellu lar R NP a nd DNP. In t his case t he nuclear DNP are a lsostained by t he methylene blue in dark blue or dark blue-violet CO-metachromatic,fig. 5-7, 11, 14,20,21 ; colour plate I : 3, 11, 15, 16).

Both variants of the method result in ,B-metachromatic staining of the cytoplasmicRNP. They st ain in the form of granules in the cytoplasm of lymphocytes (fig . 1, 5-8,18,19 ; pl at e 1: 1- 3, 8) and diffusely in the cytoplasm of st imulated lymphocytes,lymphoblast s, myelobl ast s, neoplastic cells a nd other cellu lar types with an activecytoplasmic RNP-synthesis (fig . 10).

Th e R NP- granules in the lymphocytic cytoplasm delineat e a negative ly stainedzone of G OLQI wher eas in medium-sized lymphocytes they are clearly visualized alongthe nucleo- cytoplasmic margin (fig. 1, 7, 8; plate I: 1, 8). Changes are observed inthe number a nd st aining properties of cyt oplasmic RNP, induced by reactive, patho­logic conditi ons a nd t herapeutic procedures.

An increase of the number of R NP cytoplasmi c granules is observed for examplein "act ivated" lymphocyt es at an increased cytoplasmic R NP- synthesis, as in t hecase of bact erial infections (28 ; fig. 18). In such cases a more intens ive stainingof the RNP-granules is also seen , and particularly of the granules localized alongthe nucl eo-cytoplasmic margin , whose ro le in prot ein synthesis ha s been confirmed(5, 44). Th e reduced qua ntity of cytoplasmic R NP (rare or missing R NP- granules)is observed in lymphocytes in viral infecti ons, im munosuppressive condit ions, chroniclympholeucosis and ot her changes in the immune st at us (11). Expulsion of RNP.granules by means of microolasmatoses is a nother possibil ity (fig . 19) .

Therefore , differing from the classical m ethods for detection of H N P (41, 42), the m ethod sug­

gested may dep ict n ot on ly an in creased , but a lso a n or m al or re duced R N P -cont cnts, which accor d­ing to ultrastrll~tural da ta could quantitatively cha rac terize th e immunologica l activ ity of cer taintypes of lymphocyt es (51) in view of ribosom al an t ibody syn thesis and wit h regard to the ro le ofR NA in th is process (26 , 27). . .

I'

4 E . B. ZVETKOVA and 1. B. ZVETKOV

Figv I . Lymphocyte; m ethylen e blue-eosi n . N ot e the presen ce of blue-violet (,B.meta chromati c) RNp·

gr anules in the cytoplasm. The n ucleus is n ot stained. Immer sion.

F ig. 2. Neutrophilic granulocyt e fr om the peripheral blood of a pati ent with infectious-allergic

vasculitis; methylene blue-fast gr een, The cathionic granules in the cyto pla sm are stained inten­

siv ely gr een. The nuclear chromatin is h omogeneous and pale. Immer sion .

Fig. 3. Neutrophilic granulocyte fr om the peripheral blood of a patient with allergic disease; m ethy­

lene bl ue-eosin. The cathionic gran ules in the cy t oplasm are intensively pinkish. Note the stainingof the nuclear chromatin in this case. Immer sion.

Fig. 4. Eosinophilic granulocyte ; methylene blue-eosin. The cytoplasm ic granules, part of which

arc vi sualised above the nucleus, a re in ten siv ely pinkish , with clearly delineated peripheral zone.

Immersion.

Fig. 5, 6. Lymphocyte and neutrophilic granulocyte; m ethylene blue-fas t gr een . The cathionicgranules in the cyt opla sm of granulocyt e are gr een. Blue-violet (,B.metachromatic) RNP-granulesa rc visualised in the cytoplasm of lymphocy te (in fig. 6 see gr oss magnification). On e can see the di s­

tributi on of t he nuclear chro mat in ac cording t o it s degree of con densat ion or dispersion. Immersion.

A cy to logica l method 5

As was already mentioned , the cy to plasmic RNP cont ents is diffusely st a ined incells with high contents of pol yribosom es and fr eely dispersed cytoplasmic RNP (45):myeloblasts, lymphoblasts, para.l eueoblasts, neopl astic cells, imm unoblasts (trans­formed into blast-like cells lymphocytes and monocytes in cas es with humoral hyper­activity [(4 , 5, 15, 22, :~O); (fig. 10, 12)]. In these cellu lar types the cytoplasmic pro­trusions (uropodes) are clearly visualized and preserved as a result of preliminarytreatment. They are fill ed with diffusely stained RNP (fig. 10). An enla rged,' nega­tively stained zon e of G OLGI is often detected in these cellular t ypes.

The supravitally preserved cellula r form an d differen ces in the cy to p lasm ic RNP-precipitat,ion,

diffuse or gra nula r , may bear a diagnostic sign if ican ce in t he differentiation of the sep a ra te types of

blood and ex folia ted cells according to th e specif ic patte rn s of the ir cytop lasm ic RNP-synthesis(e. g. in d ifferentiation of Iymphoproliferative from m yeloproliferative disord ers , as well as in ex­

amining the correla t ion between the functional state and the cy tochem ica l aspects of the peripheral

lymphocy tes in lymphoproliferative, autoimmune disorders an d other changes in the immune sta tus

[sec 10]) .In connection with the mechanism of cy to plasm ic staining for RNP and their specific aggr ega­

tion - diffuse or granular - the significance of fixation cou ld be pointed ou t (2, 32, 49), as well as

of the a lkaline buffer appl ied (37).

On e could supp ose tha t the topoch emi cal preservati on of ribon ucleop rot eids is cond it ione d bythe preliminary t re a tm ent , DNA-den atura ti on procedures an d the a ction of formaldeh yde, whi ch

stab ilize RNA by modifying its n itrogen bases and increa sin g it s rcsistance to the effect of RNA­

ase (18, 35).

On the other hand , the mechanism s of elect rosta t ic bin d ing of methylene blue with RNA in th ese

cases are cond it ion ed n ot. only by the affin ity of basic (thiazine) dyes t owards nucleic acid s (42) but

Fig. 7, 8. Lymphocyte and n eutrophilic granulocyte; methylen e blue-fast gr een (in fi g. 8 see grossmagnifica ti on) . No te the d ifference in the degree of chromatin con de nsa t ion: one ca n sec 11 palishand hom ogeneously stai ned chroma t in (di sp ersed ch rom at in ) in t he lymphocy te nucleu s. Immersion.

F ig. 9. B asophilic granulocy te; m ethylen e blue-eosin. p.metachromati c (blue-v iolet) a nd y -meta­ch romatic (re dd ish -v iolet) cy to plasm ic gra nules are vi su ali sed . Immersion.

Fig. 10. Lymphoblas t [blast -like cell) fr om the peripheral blood ; m ethyl en e blue-eosin. The cy to­plasm is d if fusely s tai ned for R NP. The cytop lasm ic urop od es are clea r ly seen . I mmersion .

Fig. II. Neut ro ph ilic gra n ulocy te ; methylene blue-fast green (gr oss magn if ica t ion) . Note blue-violetstaining of the nuclear DNP from condensed chroma t in and it s distribution (chromocente rs , peri­nuclear zone). Immersion.

Fig. 12. Exfoliated n eoplastic cells in cancer of the collum uteri; methylen e blue-eosin . Nuclei , nu­

eoli and cy top las m ic RNP are inten sively s ta ined . Immers ion .

Fig. 13. Lymphocyte ; m eth ylen e blue-fast green after the cold hydrolysis with a 5 N R eI. The dif­

feren ti a t ion of the nuclear DN P fr om RNP is p ossible a nd one can see the n uclea r chromatin d is­

tribution. Some ba sic prote in s of RNP are d iffusely green s ta ine d in the lymphocyti c cy to plasm.Immersion .

Fig. 14. No rmoblast fr om the peripheral blood of a pa ti ent with chron ic mycloleucosis ; methylen e

blue-fast. green . DNP from the conde nsed nuclear zones are deepl y stai ned by m ethylen e blue . Theca t.h ion ie proteins in the cy t op lasm are green. Immer sion .

Fig. 15. Neutrophilic granulocyte; fluorochromation with sulfa f'Iav ine. The ca th ion ic granules in thecytoplasm fluoresce golden-yellow. Immer sion.

6 E. B. ZVETKOVA and 1. B. ZVETKOV

Fig. 16. Eosinophilic granulocyte; methylene blue-fast green. The cytoplasmic granules are green

with a clearly stained peripheral zone. Some granules are visualised above the nucleus, whose chro­

matin is pale and homogeneously stained (dispersed). Immersion.

Fig. 17. Degranulated mastocyte in the imprint of rat thymus; methylene blue-eosin. One can see

the sites of the extracted cytoplasmic granules as stainless spaces. Immersion.

Fig. 18. Lymphocyte from the peripheral blood of a patient with bacterial infection; methylene

blue-eosin. Note an increase of the number and a more intensive staining of the RNP-granules in

the cytoplasm. Immersion.

Fig. 19. Lymphocyte; methylene blue-eosin. One can see an expulsion of RNP-cytoplasmic granulesin the extracellular space by means of microclasmatoses. Immersion.

Fig. 20. Neutrophilic granulocytes; methylene blue-fast green. The cathionic granules in the cyto­plasm are green stained. The nuclear DNP are blue-violet and one can see the distribution of con­

densed chromatin (chromocenters). Immersion.

Fig. 21. Nuclear erythrocytes (normocytes); methylene blue-fast green (see also fig. 14). Note an

intensive nuclear staining according to the degree of chromatin condensation. Immersion.

A cytological method 7

mostly by the participation of the acid dyes, eosin and fast green at low concentrations, which bind

with the nonhistonic basic proteins in RNP (31), reduce their competitive effect (12) and perhapsmediate and facilitate the binding between the RNA and the basic dye.

One is impressed by the fact that staining for nuclear and nucleolar ribonucleo­proteids is achieved by both variants of the method in cellular types with high con­tents of double-stranded RNA: some lymphocytes, tumour cells [(36,46); (fig. 12)].Nuclear and nucleolar staining for RNP is missing in blast cells whose quantity ofmRNA is reduced (8).

The p-metachromatic staining of the nuclear and nucleolar RNP could be differen­tiated from staining for DNP after the variant with methylene blue-fast green appliedafter the cold hydrolysis [(12,23); (fig. 13; plate I: 4, 6, 7)]. As a result from the hy­drolysis extraction of RNA occurs and then the differentiation of the nuclear chromo­centres from the nucleoli becomes possible, including also the so-called "ring-shapednucleoli" [(33,40); (fig. 13)].

Following the acid extraction of RNA by hydrolysis, in the lymphocytic cytoplasmthe above mentioned basic proteins of RNP (6,31) are visualized as diffusely greenstained by fast green (fig. 13; plate I: 6, 7). One is impressed by the fact that acid ex­traction of RNA is more difficult in cells whose cytoplasm stains diffusely for RNP,wish a simultaneously reduced staining for basic proteins, as compared with the eel­lular types, whose cytoplasmic RNP precipitate into a granular form. In connectionwith this fact we voice the assumption that basic proteins take part in the precipita­tion of cytoplasmic RNP, diffuse or granular. One could also assume that in all casesof RNA-staining (cytoplasmic, nuclear, nucleolar) in the mechanism of the stainingreaction the nonhistonic basic proteins of RNP take part, whereas when no stainingresults the same components are possibly reduced on account of the increased con­tent of acid nonhistonic proteins [(nucleoli of blast cells, dispersed chromatin (17)].

To stain the nuclear DNP the variant of methylene blue-fast green is recommended,as well as the same variant applied after cold hydrolysis (fig. 5-7,11,13,14; 20,21;plate 1: .3-8, 11, 12, 15, 16).

Fig. 22. Partially degranulated basophilic granulocyte; methylene blue-eosin. Note the sites of the

extracted cytoplasmic granules as stainless spaces. Immersion.

Fig. 23. Mastocyte in the imprint of rat thymus; methylene blue-eosin. ,B-metachromatic (blue­

violet) ann y-metachromatic (reddish-violet) granules fill up the cytoplasm ann are visualised alsoabove the nucleus. Immersion.

Fig. 24. Neutrophilic granulocyte from the peripheral blood of a patient with bacterial infection;

methylene blue-eosin. The cytoplasmic contour is not clearly detectable and peripheral cytoplasmic

protrusions are visualised. A number of extracted granules is seen as well. Immersion.

Fig. 25. Neutrophilic granulocyte from the peripheral blood of a patient with autoimmune disorder(glomerulonephritis); methylene blue-eosin. An increased quantity of voluminous pinkish cytoplasmic

granules (eosinophile-like granules) is observed. The nucleus is hypersegmented and unstained. Im­mersion.

8 E . B. ZVETKOVA and 1. B. Z VETKOV

The applica ti on of the sta ining variant wit h methylene blue-fast green wit ho ut h ydrolysis con­t ributes for the simultaneous staining of RNP and DNP, as well as for increasing the stain ing d if ­ferences of the nuclea r chromatin in condensed and dispersed sta tes. The condensed chromat insta ins non-hom ogen eou sly in dark blue-v iolet (fJ-me tachromatic), whereas the dis pe rsed ch romat inis homogen eous and is sta ined pale blue -pinkish, probably due to its degree of deconden sa tion, a swell as t o the compet it ive effec t of the a cid nonhistonic prot eins (12).

In this manner the differentiation becomes possibl e of apparently equally maturein their morphology cellular types, di ffer ing in biochemical maturation of the nu­clear chro ma t in (su bclasses ma ture lymphocytes, gra nulocytes in leucoses, eosino­philes in allergic and infectious-all ergic disorders, etc . [(4) ; (fig. 2, 3; plate I : 4, 12),as weIl as of blast cells (45). For example in the peripheral blood of heal thy individualsthere exist 2 popul at ions of lymphocytes with typical RNP-granules, differing indegree of chro mat in condensation, with condensed nu clear chromatin and with apalish stained dispersed chromat in (fig. 5-8; plate I: 3, 7, 8). Th e latter may cor­respond in tinctorial charact erist ics t o the so-call ed B-lymphocytes, which accordingto the hypothesis of KWAN and NORMAN (24) differ in the nucl ear chromat in flueres­cence.

The di fferentiation of cellular types with functionally differing chromatin, accord ing to its degreeof dispersion or con densation, creates p ossibilit ies for the appl ica ti on of the method in the quantita ­tive analysis - for determinat ion of the differential distribut ion and t ranscriptiona l a cti vity of thechromatin in cellular types closely related in surface and contours (38).

After the application of cold hydrolysis and acid extraction of RNA and non­histonic proteins, their compet it ive effect is removed and a complet e differential st a in­ing of DNP of chromatin is achieved according t o it s degree of condensat ion or dis­persion (fig. 13 ; plate I: 4, 6, 7). In this case in t he nu clei with dispersed chromat inconde nsed zones (perinuclear, perinucleolar, chromocentres) may be also established ,wh ich render possible t he detect ion of small changes in the degree of chro matin con­densation a nd detail s in its distribut ion.

II. Concer n i ng p os s i biliti es f or l igh t - a n d flu or es cen t mi cros copi c d e­t e c ti on of some cat h io n ic p r oteins

The acid dyes eosin a nd fast green stain simultaneously in pink, resp . in gr een som ecathionic (argyrine- and lysin-containing) proteins in the cytoplasm of blood a nd ex­foliated cells [(3,7,34) ; (fig. 2-5, 7, 11 ; 16,20, 24, 25; plate I : 3, 4, 7, 8, 9-12;15, 16)]. Th e cat hionic prot ein s in t he lysosomal (secondary) cytoplasmic gra nulesof eosinophilic and neutrophilic gra nulocy tes (3) are intensively stained with t he ex­ception of the stage of myeloblast s (50) in whose cytoplasm diffuse staining is some­times also observed. Th e same cathionic gra nules of t he granulocytes fluoresce golde n­

ye llow with sulfaflavin (fig. 15; pla te I: 14).The cytoplas mic granules of neutrophiles and the non-mature granules of eosino­

philes are chara cte rized with sma ll size a nd homogeneous sta ining a nd when the fluo­rescent variant is applied they fluoresce less conspicuously as compared with themature grtanules of the eosinophiles . The latter are oharacterized with a larger size

A cyto logica l m ethod 9

elonga ted form, intensively sta ined , resp. fluorescing periphery a nd unstained foroat hionie proteins central part (fig. 4, 16 ; plate 1: 4, 9). These gra nu lar character­istics correlate with the data for high contents of argynine in the peripheral zone of

mature eosinophilic granules a nd for the presence of crysta lic inclusions at the siteof t he cent ra l brightening (8, 39).

The er yt h rocy tes stain pinkish fr om eosin and green fr om fa st green depending upon thei r con ­ten ts of ca t h ion ic prot eins (8, 39) but they do not fluoresce with sulfaf'lavin .

Cold hydrolysis with 5 N H Cl does not affect staining for ca thionic proteins III

the lysosomal granules of the granulocytes and in the cytoplasm of eryth rocy tes andas was already mentioned it contributes for the detection of the basic proteins linkedin RNP in the cytoplasm of lymphocytes (plate I: 4, 6, 7).

After the application of the variant with m ethylene blue-eo sin , a d iffuse and sometimes granula r

pinkish sta in ing of some cathion ic cy toplasmic components in the m on onuclear cells [monocytes (7)," activa te d " and blast transformed lymphocytes (48)] is achieved.

The technique suggested creates possibilities for the quantitative characterizationof the cathionic components in the leucocyte cytoplasm, whose significance as bac­t eri cidal subst ances in infections and vasopermeable factors in immune responses is

well established (14, 52). F or exa mple an increased quantity of cat hionic granulesa nd accessible extract ibility of cathion ic cytoplasmi c comp onents is established int he granulocy tes in bacterial infect ions (fig. 24); the presen ce of eosinophile-like largegra nules in the neutrophiles [(1, 29) ; (fig. 25)] in allergic and some immune disorders ;a reduced quantity and small-sized cathionie granules in viral diseases, unfavourablyadvancing bac terial infections, myeloproliferative and neoplastic processes (52). Ina ll ab ove-mentioned instances a compar ison becom es possible betw een the character­

istics of the cathionic granules with the dynamics of the chang es in the phosphataseindex in view of the localizati on of the cathionic proteins and t he alkaline-phosphat­ase ac t ivity in som e and the sa me cellular st ructures, t he so-ca lled specific granules (3)a nd with regard to the differential diagnostic significance of the method suggested (8).

Analogou s to othe r ex ist ing techn iques for the detecti on of cellula r p olycathionic componen ts(16, 25, 39) one could a ssume that our sta in ing results are possibl y ba sed on an elect rosta t ic prin­cip le wh ich is practically conveni ent and allows for quantitative determination of cathionic pro­

teins (25).As advantages of the staining technique could be pointed out the possibiliti es for a simultaneous

detect ion of ca th ion io proteins in a ll blood cells and especially in mature granulocytes which contain

them (16), a s well as for f'luorochromation of the same cytopla smi c ca thi on ic granules in the granule ­

cy tes, whi ch represents the fi rst a t t em p t in this direction of resea rch .

After colorimetric and m icrof'luor imetrio data the applica t ion of the sta in ing m ethod becom es

p oss ible in the quantitative an d automati c blood cells ' analysis, in whi ch the sta in ing and fluorescen t ­

mi croscop ic p rop erties of the gra n ules m ay ser ve as simplified signs for cellula r detecti on. P articularlythe different iation of the 2 ma in classes of leucocytes - m on o- and p olynuclear leucocy tes - would

be p ossibl e (9). In this case a s an additi ona l sign appear t o be the blue sta ine d, forming a peripheral

margin, R NP -cytoplasmic granules of the lymphocytes.

10 E. B. ZVETKOVA and 1. B. ZVETKOV

III. Concerning staining for acid mucopolysaccharides in blood b a s o­philes, tissue m a s t o cy t es and throm bocytes

The acid mucopolysaccharide components of the granules of basophilic leucocytes

and tissue mastocytes (5, 8,16) are stained {J- and y-metachromatically (bluish-violet,resp. reddish-violet) after the light-microscopic variants of the method and fluoresce

reddish-gold with sulfaflavin (fig. 9, 23; plate I: 13). In degranulation (47) the sites

of the extracted cytoplasmic granules are visualized as stainless spaces similar tobee-hive cells (fig. 17, 22).

Reddish-violet (y-metachromatic) are stained also the granular components of the thrombocytes,

containing acid mucopolysaccharides (16). The phenomenon is particularly manifested in disordersaccompanied by thrombocytosis and a marked aggregation of thrombocytes.

Acknowledgements

I wish to thank my teacher, Academian A. 1. HADJIOLOFF, for the useful discussions and his

kind help in editing the text of this paper.I am also grateful to J. ZHECHEV, J. CHOLAKOVA, ST. BAIKOV and L. KOLEvA for technical

assistance.

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A cytological method 11

14. DIXON, F. J., and WILSON, C. B., Irrununological aspects of glomerulonephritis. In: Renal dis

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12 E . B . ZVETKOVA an d 1. B . ZVETKOV

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Address: Dr. ELISAVETA ZVETKOVA, Bulgarian Aca dem y of Sciences, Institute of Morp h ology,

kv. " Geo Milev", Aead. G. Bontch ev Stree t , Block I II, Sofia 13, Bulgaria.

A cytological method

Explanation of Plate

Plate I

13

Fig. 1, 2. Lymphocyte; methylene blue-eosin. One can see the presence of blue-violet U~-metachro­

matic) RNP-granules in the cytoplasm. The erythrocytes are pink stained. Immersion.

Fig. 3. Lymphocyte and neutrophilic granulocyte; methylene blue-fast green. Note the presence

of blue-violet RNP-granules in the cytoplasm of lymphocyte. The cathionic granules in the cyto­plasm of granulocyte and the erythrocytes are stained green. Note the staining of the nuclear chro­

matin according to its degree of condensation. Immersion.

Fig. 4. Eosinophile, neutrophilic granulocyte and blast-like cell; methylene blue-fast green aftercold hydrolysis with 5 N HCI. The cytoplasmic granules of granulocytes are green and one can see

differences in the size and number of the 2 types of granules. Note also differences in the state of

chromatin condensation (condensed chromatin in the nucleus of neutrophilic granulocyte, and dis­persed in the nuclei of blast-like cell and eosinophile). Immersion.

Fig. 5. Lymphocytes; methylene blue-fast green. One can see differences in the chromatin distribu­tion according to its degree of condensation and dispersion (intensively stained is the condensed

chromatin of the small lymphocyte and pale and homogeneous is the nuclear chromatin in the

medium-sized lymphocyte with an abundant cytoplasm). Note the presence of RNP granules inlymphocytic cytoplasms. Immersion.

Fig. 6. Lymphocyte; methylene blue-fast green after cold hydrolysis with 5 N HCI. The cathionicproteins of RNP which stain diffusely green are visualized in the lymphocytic cytoplasm. Nuclear

DNP are blue-violet (p-metachromatic) stained and one can see the chromatin distribution accordingto its degree of condensation. Immersion.

Fig. 7. Lymphocyte and granulocyte; methylene blue-fast green after cold hydrolysis with 5 N HCI.After acid extraction of RNA some basic proteins of RNP are stained green in the lymphocyticcytoplasm. The cathionic proteins in erythrocytes and these in the cytoplasmic granules of granulo­

cytes are also green. One can see differences in the nuclear chromatin distribution. Immersion.

Fig. 8. (See also fig. 3 and 5) Lymphocyte and granulocyte; methylene blue-fast green. One can see

the differences in the state of condensation of the nuclear chromatin. The dispersed nuclear chromatinof lymphocyte is pale and homogeneously stained. Immersion.

Fig. 9. Eosinophilic granulocyte; methylene blue-eosin. The granules in the cytoplasm are intensivelypinkish with clearly delineated peripheral zone. Immersion.

Fig. 10. Neutrophilic granulocyte from the peripheral blood of a pateint with allergic disease (bron­chial asthma); methylene blue-eosin. The cytoplasmic cathionic granules are stained pink. In thiscase the nuclear chromatin is stained blue-violet by methylene-blue. Immersion.

Fig. 11, 15. Neutrophilic granulocytes (in different magnifications); methylene blue-fast green. Thecathionic cytoplasmic granules are green; the nuclear chromatin is blue-violet. One can see the dis­

tribution of the condensed and dispersed chromatin. Immersion.

Fig. 12. Neutrophilic granulocyte from the peripheral blood of a patinet with infectious-allergic

vasculitis; methylene blue-fast green. The cathionic granules in the cytoplasm are green. The nu­

clear chromatin is homogeneously stained and pale (pink-violet) as in the nucleus of eosinophile from

fig. 4. One can see the cytoplasmic protrusions (uropodes). Immersion.

Fig. 13. Basophilic granulocyte; methylene blue-eosin. Note y-metachromatic (reddish-violet) gra­

nules in the cytoplasm. Immersion.

Fig. 14. Neutrophilic granulocyte; fluorochromatin with sulfaflavine. The cathionic granules in the

cytoplasm fluoresce golden-yellow. Immersion.

Fig. 16. Normoblast; methylene blue-fast green. The cathionic proteins in the cytoplasm are stained

green. The condensed zones of the nuclear chromatin are blue-violet (p-metachromatic). One can seethe chromatin distribution. Immersion.

Acta histochem. Rd. 57

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Z w e t k o v a a n d Z w e t k o v

VES GUSTAV FISCHER VERLAG lENA