a comparative study of some staining properties of - blood

A Comparative Study of Some Staining Properties ofCrystals in a Lympho-plasmocytoid Cell, of Russell

Bodies in Plasmocytes, and of Amyloids-WithSpecial Emphasis on Their Isoelectric Points

Bg ARTHUR F. GOLDBERG AND HELEN WENDLER DEANE

T HIS STUDY was made to characterize intracellular crystals found in

unusual lympho-plasmocytoid cells, which have been described elsewhere’

and for convenience will be termed crystal cells. For comparison, an examina-

tion was made of the related inclusions in plasmocytes ( Russell bodies ) , and

also of primary, secondary, and senile cardiac amyloids. Their relative iso-

electric points were determined by a staining method that effectively titrates

the ionizable radicals of the compounds. Tile results suggest strongly that

the crystals and Russell bodies are gamma globulins, whereas the amyloids

studied resemble alpha glycoproteins. Two preliminary reports have been

made.2”

MATERIALS AND METHODS

The test objects were: (a) the intracytoplasmic crystals in lympho-plasmocytoid cells

found in a lymph node biopsy and in bone marrow smears;1 (b) Russell bodies in plasmo-

cytes present in the lamina propria of normal human stomach and small intestine (two

cases), as well as in the gastrointestinal tract of a patient with multiple myeloma (one

case) and in a biopsy of chronic inflammatory tissue from the external auditory canal;

(c) tissues containing secondary amyloids associated with chronic tuberculosis (three

cases), chronic osteomyeiitis (one case); and (d) specimens of amyloid associated with

multiple myeboma (two cases), as well as generalized primary amyloid and senile cardiacamyloid (one case each). The locations of the amyloids studied were as follows: liverand spleen in the tuberculosis cases; spleen in the osteomyclitis case; blood vessels in

stomach, intestine, and spleen of the cases of multiple myeloma; and the heart in general-

ized primary and senile cardiac amyloidosis.�

From the Departments of Pathology and Anatomy, Albert Einstein College of Medicine,

New York 61, N. Y.

This study was aided by a U.S.PJI.S. Training Grant (2G-97) to the Department ofPathology.

We wish to acknowledge the technical assistance of Irene Morvay and Ellen C. Driks.Submitted June 23, 1960; accepted for publication Aug. 13, 1960.

#{176}Additional data on the cases in which amyloid were found are as follows.(a) Cases associated with tuberculosis: (1) 73 year old male; total serum protein

4.9 Gm. per cent; albumin 2.8 Gm. per cent, globulin 2.1 Cm. per cent. Serum electro-

phoresis showed 29.3 per cent albumin, 7.1 per cent alpha-i, 17.7 per cent alpha-2, 11.8

per cent beta, and 34.1 per cent gamma globulin; BUN 60 mg. per cent; no proteinuria;(2) 23 year old female; total protein 7.2 Gm. per cent; albumin 4.5 Gm. per cent,

globulin 2.7 Cm. per cent; BUN 6 mg. per cent; no proteinuria; (3) 50 year old female;total protein 6.9 Cm. per cent; albumin 2.9 Cm. per cent, globulin 4 Cm. per cent. Serum

electrophoretic pattern showed 27 per cent albumin, 7 per cent alpha-i, 13.5 per cent

alpha-2, 11.5 per cent beta, and 41 per cent gamma globulin. Four months later totalprotein was 5.3 Gm. per cent; albumin 1 Cm. per cent, globulin 4.3 Gm. per cent. Serum

1708

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CRYSTALS, RUSSELL BODIES AND AMYLOIDS 1709

Since Russell bodies are believed to contain gamma globulin,1 human gamma globulin

was also studied.#{176} Mayer’s egg albumin, used for affixing sections to slides, was

examined in some situations.

All blocks of tissue and a portion of the gamma globulin were fixed in 10 per cent

formalin and embedded in paraffin. With the exceptions noted, the various staining pro-

cedures listed below were performed on deparaffinized, hydrated sections ( ca. 5 �s ) , andexcept for the preparations stained for lipides, all sections were mounted in Permount.

To determine the apparent isoelectric points of the test objects, the method of Singer

and Morrison4 was employed. Sections were stained overnight ( i.e. to equilibrium ) atroom temperature, in aqueous solutions of eosin Y and methylene blue at concentrationsof 10-4 and 2 X 10-� M., respectively, at intervals of about one pH unit over the range

3. 1 to 9.3 ( see table 1 ) . These dye concentrations were chosen because they permitted

comparable staining of nuclei, cytoplasm, erythrocytes, and collagen fibers at pH 5 with

that obtained after ethanol or Zenker-acetic fixation.5 The Walpole acetate buffer, 0.1 M.,was used for the solutions from pH 3.1 to 7.5. Propanediol buffer, 0.1 M., was employed

for pH 9.3. The sections were dehydrated in a standardized fashion through tertiary butyl

alcohol and xylene. Sections containing the various test objects were stained together,

so that they could be compared with confidence.

Some sections were subjected to deamination by tile Alfert nlodlficatlon of the Van

Slyke method.6 Blockade of carboxyl groups was produced by n�ethylation.7 After these

pretreatments, the sections and appropriate controls were stained with eosin and methylene

blue as above.Other slides were stained by the periodic acid-Schiff (PAS) procedure, by tile PAS

method after saliva treatment, with crystal violet,8 and with Congo red.9 The last prepara-

tions were examined with a polarizing microscope10 as well as the ordinary light

microscope.

The Baker acid-hematein stain for phosphatides” was performed on material fixed

in calcium-formalin: (a) frozen sections of normal human stomach containing plasmocytes,

(b) bone marrow smears from the case with crystal cells and (c) aqueous, air-dried

smears of human gamma globulin and egg albumin. Control preparations were extracted

overnight with pyridine at 60 C. before mordanting and staining.Frozen sections of the formalin-fixed human stomach having numerous plasmocytes in

the lamina propria and bone marrow smears containing crystal cells were stained with

Sudan black B and with oil red 012 for 10 minutes at room temperature, and for 10

and 60 minutes at 60 C.

electrophoretic pattern showed 19 per cent albumin, 8 per cent alpha-i, 29.2 per cent

alpha-2, 11.8 per cent beta and 32 per cent gamma globulin; BUN 20 mg. per cent;

4 plus proteinuria.

(b) Case associated with osteomyelitis: 59 year old male; total protein 5.2 Gm. percent; albumin 2.2 Cm. per cent, globulin 3 Cm. per cent; BUN 28 mg. per cent; trace

of proteinuria.

(c) Cases associated with multiple myeloma: (1) 90 year old female; BUN 28 mg.

per cent, 3 plus proteinuria; (2) 72 year old female; total protein 8.5 Cm. per cent;albumin 3.4 Cm. per cent, globulin, 5.1 Cm. per cent; BUN 10 mg. per cent; 2 plus

proteinuria, Bence-Jones protein present.

(d) Case with generalized primary amyloidosis: 55 year old female; total protein5.4 Gm. per cent; albumin 4 Gm. per cent, globulin 1.4 Cm. per cent; BUN 40 mg.

per cent; no proteinuria.

(e) Case with senile cardiac amyloidosis: 77 year old male; BUN 24 mg. per cent; traceof proteinuria.

#{176}Asample of purified Fraction II was obtained from the Mann Research Laboratories,

New York.




1710 GOLDBERG AND DEANE

OBSERVATIONS

The intensity of staining of the various test objects with eosin and methylene

blue was judged visually, as indicated in table 1. In addition to the Russell

bodies ( fig. 1 ) , gamma globulin, the crystals ( fig. 2 ) , and amyloids ( fig. 3),

the tabulation includes collagen fibers, erythrocytes, eosinophile granules,

and the basophilic cytoplasm ( ergastoplasm ) of plasmocytes. These addi-

tional objects have been studied by others,�m5 and therefore provided com-

parisons for our results. For each object, the zone of equal, weak staining by

tile acid and basic dyes was taken to represent its isoelectric point under the

conditions of preparation.4

For the Russell bodies and gamma globulin, the average isoelectric range

appeared to lie between p11 6.2 and 7.5 (table 1). The slopes of the curves of

dye uptake fell off for the acid dye between pH 6.2 and 7.5 and for the basic

dye between pH 9.3 and 7.5 (fig. 1). There was no residual staining at pH

9.3 with eosin or at pH 5.1 with methylene blue. The crystals stained quite

similarly, but they seemed to have a slightly more acid isoelectric point (table

1, fig. 2).

Individual crystals and Russell bodies always stained uniformly. At a

given p11 of staining, however, some would appear darker than others. Near

neutrality, some were stained while others were almost unstained (e.g.,

figs. lb and c, 2b).

The eight cases of amyloids studied reacted essentially alike and had ap-

l)�tre11t isoelectric points at about pH 5 (table 1, fig. 3). The slopes of theirstaining curves seemed more gradual than those of the crystals and Russell

bodies.

The remaining data listed in table 1 are compatible with the results of

others who have used the controlled-pH staining method, in that the isoelectric

point of collagen appeared to be near pH 613 (fig. 3); the acidophilia of ery-

throcytes and eosinophile granules extended to high pH’4 (fig. 2); and staining

of ergastoplasm with methylene blue dropped between pH 4 and 315 (fig. 1).

Deamination and methylation treatments were performed on sections con-

taining crystals (lymph node), Russell bodies (gut and external ear), three

examples of secondary amyloid (spleen, in cases with tuberculosis), and two

Table 1.-Staining of various objects with a cid and basic dyes at different pH’s#{176}t

pH 3.1 pH 4.1MB E MB

pH 5.1E MB

pH 6.2E MB

pHE

7.5MB

pE

H 9.3MBOBJECTS

CrystaLs

Russell bodies

Camma globulin

All amyloids

Collagen fibers

Erythrocytes

Eosinophile

granules

Ergastoplasrn of

plasmocytes ± 0

Sections stained with eosin (E) at 10-” M., and methylene blue (MB� at 2 >‘ 1O�1 M.

tScale of dye binding: 0 = no dye binding; ± = negligible dye binding: + to ��--�-�- = slight to veryintense dye binding.

0 ++4- 0 +±+ 0 + ± ± ± 0 4:++0 ++ 0 ++ 0 -f-f--’-�. 0-± + + 0 +++0 ++ 0 ++ 0 + ± + ± 0 ++0 ++ 0-± ± ± ()-± + +± 0 �-�--�-

0 ++ 0 + 0 ± ± 0 ± 0 ++0 ++++ 0 +++ 0 ++ 0 ++ 0 + ±

0 ++++ 0 +++ 0 m+ 0 ++ 0 ++ 0

+ 0 ++ 0 +++ 0 +++ 0 ++++




Fig. 1.-Degree of basophilia of Russell bodies in plasmocytes present in chronic

inflammatory tissue in external ear. Methylene blue, 2 X 10� M.; X 900. Staining is

(a) absent [0] at pH 5.1, (b) negligible I ±] at pH 6.2, (c) slight 1+] at pH7.5, and (d) intense [+++I at pH 9.3. Some plasmocytes contain one or two large

Russell bodies, others contain many smaller ones (arrows). The nucleus tends tobe displaced to the periphery of the cell. Strands of basophilic cytoplasm (ergasto-plasm) surround the Russell bodies; this material remains basophilic at pH 5.1.


cases of amvloid associated with multiple myeloma (spleen). For all objects.

deamination consistently increased uptake of metilviene blue at etdl p11.

producing detectable staining even at pH :3.5. In contrast, blockade of car-

boxy! groups increased the uptake of eosin, so that all these Oi)jectS stained

at pH 9:3. No changes appeared in the relative intensity of staining of tile

several test objects.

All of the amyloids stained red-orange with Congo red and exhibited the

typical birefringence 111 � light.’’1’ The crystals, Russell bodies, and

gamma globulin stained univ light orange with Congo red all(l (lid not i)e-

come birefringent.

Likewise, all the ainvloids stained ,netachroniaticallv with crystal violet.

There were no significant differences in the reactivity of these S cases. The




.../�.

�

�e �� -.

�

‘&� �

ky.- .% �

.�s,\

-p..

w

Fig. 2.-Degree of acidophilia of crystals in the lympho-plasmocytoid cellspresent in a lymph node biopsy. Eosin, 10’ M.; X 900. Dye uptake is (a) intenseat pH 5.1, (b) slight at pH 6.2, and (c) negligible at pH 7.5. Arrows point torepresentative crystal cells. Ervthrocytes (e) are still acidophilic at pH 7.5.


crystals, Russell bodies, and gamma globulin stained orthochromatically with

crystal violet.

Sections containing crystals and anlyloids and smears of gamma globulin

all stained with moderate intensity by the PAS method (fig. 4). Many Russell

bodies reacted more intensely than the other objects when stained in the

same run.

Russell bodies and crystals showed no dye uptake when exposed to either

Sudan black B or oil red 0. A negative reaction for Russell bodies has been

reported by many others.’62#{176} Grundner-Culemann and Diezel,20� however,

found that some Russell bodies do stain with Sudan black B.It has been reported that cell inclusions in plasmocytes stain by the Smith-

Dietrich procedure for phosphatides.21 Because we believe these inclusions

to have been Russell bodies, we also applied a method for phosphatides. The

Baker method11 has the advantage that it uses a parallel control preparation

in which lipides are pre-extracted, so that staining attributable to nonlipide

acidic substances may be recognized. Frozen sections of stomach including

plasmocytes, smears of bone marrow containing crystal cells, and smears of

gamma globulin and egg albumin were stained by this method. All of the

test objects except egg albumin reacted positively. Moreover, Russell bodies

(fig. 5) and crystals became intensely stained even when the preparations

were extracted with hot pyridine before being exposed to the mordant and




Is’.�’.

(:RYSTALS, RUSSELL BODIES AND AMYLOIDS 171:3

Fig. 3.-Degree of basophilia of amyloid occurring in the walls of splenicarteries from a patient who had multiple myeloma. Methylene blue, 2 X l0� M.;X 400. Staining of the amvloid is (a) virtually absent at pH 4.1, (b) negligible at

pH 5.1, (c) moderate at pH 6.2, and (d) fairly intense, although still much weakerthan that in nuclei, at pH 7.5. The collagen fibers in the adventitia of the arteriesare consistently less basophilic than the amyloid.

stain. Differentiation was conducted for the standard 18-hour period. Pre-

extracted smears of gamma globulin remained stained if differentiated for

only 5 hours but had lost all stain if differentiation was extended to 18 hours.

We did not utilize differentiation periods between 5 and 18 hours.

DIscusSIoN

Staining with dilute solutions of acid and basic dyes at a series of pH’s

allows the determination of the relative isoelectric points of various com-

pounds. It should be emphasized that to do this, staining must be continued

until equilibrium has been attained (18-24 hours ) � Also, the subsequent

washing and dehydration of the sections must be kept uniform so that dye is

not removed in differing amounts. If such preparations are analyzed with a

spectrophotometer, accurate pH “signatures” (titration curves) may be oh-




1714 (X)LI)BEII(. .“. NI) l)EA N i

Fig. 4.-PAS positive reaction in (a) crystals [lymph node biopsy I, (b) Russellbodies I chronic inflammatory tissue in external ear], (c) amyloid associated withmultiple myeloma [wall of splenic artery], and (d) secondary amyloid [spleen,chronic pulmonary tuberculosis]. All preparations lightly counterstained with diluteHarris’s hematoxylin; (a) and (b) X 900, (c) and (d) X 400.

tained,’’’’’-� and these provide even more information about the kinds of

ionizable groups presellt. In this study, however, we have hmite(l ourselves

to visual assessment.

Russell Bodies (Ifl (I (;r!/St(Il.5

For Russell bodies, the isoelectric points ranged between 6.2 and 7.5. This is

appro�a mately the range exhibi ted by undenatu red human gamma glob-

itliiis.�-2� Furthermore, \\‘hell we tested fixed gamma globulin by our pro-

cedure, it exhibited staining ProPerties similar to those of Russell bodies. That

its isoelectric range pro�’ec1 to be the sanie with the staining nlethO(15 aS

biophvsical measurement indicated ti iat the (he concentrations selected were

appropriate. Otherwise the rclatiue isoelectric points of the various objects

in the sections, although valid, might not have conformed to the range exhibited

1w undenatured proteins.




CRYSTALS, RUSSELL BOl)IES ANI) AMYLOIDS 1715

#{149}�P;� %‘�,

,e�

,j s�- ,, S

. � � ;� . S

Si � � �

:� �-‘-p �

‘�‘3. I ..

�4 -“ ..�,

� �S, ,

Fig. 5.-Russell bodies in plasmocytes in a human stomach. Pyridine-extractedfrozen section; Baker’s acid-hematein reaction; X 900. The Russell bodies remain

stainable. The base of a gastric gland occurs at (g).

There is al)undant evidence froni other ty�)es of methods that Russell bodies

contain gamma globulin,’ tile most direct being that they react with fluores-

cein-labeled antibodies to human gamma globulin.’�’ The variation of stain-

ability observed in different Russell bo(lies is consistent with the known fact

that there are a number of gamma globulins with a range of isoelectric points

between pH 6 and 8.23� These variations, which sometimes occur within a

single cell, have been seen by others using different staining methods.”

Pearse3’ was perhaps the first cytologist to show that Russell bodies consist

of glycoprotein. He found that this substance ceased to hind methylene blue

in dilute solution at pH 6.0 and that it was PAS positive. Human gamma

globulin contains about 3 Ier cent carbohydrate.24

The crystaLs in the lympho-plasmocytoid cell exhibited nearly the same

isoelectric range as the Russell bodies and gamma globulins. This, result sug-

gests that the crystalline material was a gamma globulin or a protein with a

similar isoeiectric range. The supposition of identity is reinforced h�’ the

other similarities in staining properties. Thus, the crystals, like Russell

hodies�’2”�’3#{176} and gamma globillin, also give tile PAS reaction. Furthermore,

the crystals, like Russell bodies, stained by tile Baker acid-hematem test, even

after pyridine extraction. The significance of this reactwity remains obscure,

but for our present purposes it indicates tile similarity of these two intracellu-

lar inclusion bodies. Gamma globuin seemed to react in the same way,

although the thin smears used dccolorized more readily than did the intra-

cellular inclusions in sectioned material. Ordinarily it is assumed that materials

stainable after pyridine extraction are nonlipides having strong acid radica1s.�





The high isoelectric point of the Russell bodies and crystals makes this explana-

tion improbable. And the complete absence of staining by oil-soluble dyes

makes it extremely unlikely that an unextractable acidic lipide accounts for

the stainability with acid hematein. Certainly the results do not support the

assumption that inclusions appearing to be Russell bodies contain phospho-

Iipide.2’

�‘Basophilia” and “acidophilid’

The conflicting reports in the literature concerning the staining properties

of inclusions in plasmocytes warrant comment. It seems to us that the van-

ability in tile staining properties of Russell bodies has been unduly exaggerated.

( a ) As we have shown here, in any one preparation, Russell bodies may

exhibit slightly differing dye uptakes ( fig. 1 ), probably because the constituent

gamma globulins vary slightly in isoelectnic point. (b ) More importantly, the

influence of pH on stainability with Romanovsky stains ( mixtures of methy-

lene blue, azures, and eosin ) is inadequately recognized. Russell bodies have

been described or illustrated as staining pale blue,’9’2029’3234 darker

blue,16’’2”’�3� violet or purple,’7’21”’’1#{176} 1)ink,.�i).:)(),.i7 or red,29’32’M Knowing

the isoelectric point of gamma globulin helps explain these discrepancies,

since Romanovsky mixtures (if buffered at all) are used anywhere between

pH 5.5 and 7. Frequently a buffer is not employed, and \Vnight’s stain may

he quite alkaline because of the bicarbonate present. Thus it is understandable

that sometimes the dye bath is on the alkaline side of the isoelectnic points of

Russell bodies, making them stain basophilically (blue); sometimes near the

isoelectric point, making them appear amphoteric (violet or purple); some-

times on the acid side, making them acidophilic (pink or red). Furthermore,

the Romanovsky mixtures are concentrated, so that staining is not carried to

equilibrium; consequently relative stainability may not be valid.0

\Ve believe, as Bessis concluded on other grounds,3t) that the tendency to

distinguish between the inclusions in plasinocytes associated with different

diseases is unwarranted on the basis of present information. This tendency

still continues.�#{176}’34 Unless preparations have been fixed and stained exactly

alike, tinctorial differences may not be significant. Certainly it is impossible to

compare preparations that have been made in different laboratories when no

standardized procedures exist.

Finally, the fact that Russell bodies often stain somewhat basophilically

with Romanovsky mixtures but are uniformly acidophilic in H & E preparations

is explicable on two grounds. (a) Hematoxylin is not a basic type and general-

ly stains chromatin only. Unless the solution is very old (S. H. Bensley, per-

sonal communication), it has little tendency to stain other basophilic sub-

stances. (b) H & E preparations are generally stained with a strong, alcoholic

solution of eosin, which is taken up by almost all materials in sections, re-

gardless of their isoelectric points.

Amyloids

All specimens of amyloid studied reacted similarly, having an apparent

isoelectnic point at about pH 5. Earlier, Games and Forker4#{176} showed that





uptake of another basic dye, toluidine blue, by both primary and secondary

amyloids was extinguished at pH 4.5. These results support the concept that

various amyloids are closely related and that their classification into different

types, although useful, is still quite arbitrary.

The plasma proteins having similar isoelectric points are the alpha-i and

alpha-2 glycoproteins and beta-i globulins.23 In extraction studies on tissues

containing amyloids,4143 it has been shown that amyloids consist of a glyco-

protein fraction and an acid mucopolysaccharide. The glycoprotein stains

by the PAS technic and the acid mucopolysaccharide stains metachro-

matically with toluidine blue or crystal violet. Both of these components

migrate electrophoretically like the alpha and beta globulin fractions. There

is at least one case in the literature in which amyloid was analyzed chem-

ically and found to have an amino acid composition similar to beta globulin.44

Current studies reveal that the acid mucopolysaccharide component of

amyloids does not contain chondroitin sulfate or hyaluronic acid,40’43’45 and

that the acidic moiety is probably carboxylated.45 A known acidic component

of certain mucoproteins and mucopolysaccharides is sialic acid.’” This sub-

stance is present as a significant constituent of the plasma alpha-2 globulins,

and as a lesser constituent of the alpha-i and beta globulin fractions.47 Sialic

acid has been found in extracts of organs containing amyloids as well as in the

appropriate globulin fractions.43’48 Since sialic acid may induce metachromasy

of basic dyes,46 it may be the major substance responsible for the characteristic

metachromatic staining of amyloid.

One of the inherent defects of the controlled-pH staining method, employed

here to determine the isoelectric point of amyloids, is that it measures only the

net available ionizable groups. When a test object contains a mixture of sub-

stances, it is impossible to determine how many ionizable radicals are con-

tributed by each component. Without differential extraction, it would be

impossible to determine the isoelectric points of the protein and carbohydrate

moieties separately.

A long-standing hypothesis has been that tissue amyloids may result from

the local deposition of certain circulating plasma globulin fractions. In the

cases just listed,4’44 the patients had elevated serum globulin fractions with

properties corresponding to those of the amyloids extracted from the tissues.

These cases represented both primary and secondary amyloids. In two of

our cases of secondary amyloidosis, on whom serum electrophoretic patterns

were performed, the alpha globulin fraction was elevated.

Other clinical and laboratory studies on patients or experimental animals

with amyloidosis have shown similar serum electrophoretic patterns.3”4954

Besides the slight-to-moderate elevation of the alpha or beta globulin frac-

tions, the serum albumin fraction is frequently reduced in such patients. The

gamma globulins are usually slightly elevated early in the disease. In long

standing cases, the total globulin is generally not elevated. This is particularly

true in cases of multiple myeloma with amyloidosis.”55

If the protein component of amyloid is an alpha or beta globulin, then

what is its origin? It has long been suggested, especially for cases of amyloid-

osis associated with multiple myeloma, that the amyloid is a product of





plasmocytes. While there is no definitive evidence that the plasmocyte pro-

duces alpha or beta globulins, there is indirect evidence that this is so. In

agammaglobulinemia, where plasmocytes are absent, some alpha and beta

globulins may not be produced.’ In multiple myeloma, the reverse situation

may occur, in that there is a marked elevation of serum alpha or beta globu-

lins. It is usually presumed that these abnormal proteins arise from the prolif-

erating myeloma plasmocytes. Even in those cases of multiple myeloma in

which the major protein elevation is gamma or beta globulin, the alpha

globulins may also be moderately elevated,3�’56’57 suggesting that these glob-

nuns too are being produced by the myeloma plasmocytes. In the same cate-

gory are the rare cases of multiple myeloma associated with amyloidosis in

which the amyloid is surrounded by myeloma cells,58’59 suggesting its local

production. Protein inclusions taking positive amyloid stains have also been

described in myeloma cells.58’60 Furthermore, since in multiple myeloma with

amyloidosis there is usually a fail in serum globulins,33’5� there may be a

block in the production of these plasma proteins by myeloma plasmocytes

and possibly a shift in ratio of production of the several globulins.

On the other hand, the liver has been implicated as the primary source of

most alpha and beta globulins.61”12 It is possible, therefore, that certain forms

of amyloidosis may result from a disturbance of protein metabolism in that

organ. The low serum albumin, so frequently found in amyloidosis, may like-

wise be a reflection of this derangement. Possibly hepatic cells also produce the

mucopolysaccharides associated with the globulin fractions and with amyloid.

Finally, it should be pointed out that there is some evidence in the literature

that amyloids may differ from one another. Notably, primary amyloid asso-

ciated with multiple myeloma sometimes exhibits little or no metachromatic

staining (e.g., with crystal violet) � Since patients with multiple myeloma

frequently exhibit reduced serum mucoprotein levels,63 it seems reasonable

to assume that in such cases as described above, little acid mucopolysaccharide

was available to be deposited in the amyloid complex. Our particular cases,

however, all exhibited significant metachromasy with crystal violet. The con-

ditions that may produce secondary amyloidosis all appear to be associated

with an elevation of such serum mucopolysaccharides, thus explaining the

consistent intense metachromasy of the deposits with crystal violet.

SUMMARY

A staining procedure for determining the approximate isoelectric points of

substances has been applied to tissue sections containing (a) intracytoplasmic

crystals in a lympho-plasmocytoid cell, (b) Russell bodies of plasmocytes, and

(c) secondary and primary amyloids. The same method was also applied to

sections of human gamma globulin.

The results indicate the similarity of Russell bodies, the crystals, and gamma

globulin, all of which showed an isoelectric range between pH 6 and 7.5.

Other staining methods employed tended to confirm this identity.

The discrepancies reported in the literature on the staining properties of

Russell bodies are discussed.




CRYSTALS, RUSSELL BODIES ANI) AMYLOIDS 1719

All of the amyloids tested had an apparent isoelectric point near pH 5.

Various suggestions in the literature about the nature and origin of amvloid

are summarized.

SusIsIAmo IN INrERLINGuA

Un methodo de tincturation P”� determinar le approxiniative punctos iso-

electric (Ic substantias esseva applicate a sectiones tissular que contineva (a)

crystallos intracytoplarmic in un cellula lympho-plasmocytoide, (b) corpores

de Russell in plasmocytos, e (c) amyloides secundari e primari. Le mesme

methodo esseva etiam applicate a sectiones de globuhina gamma human.

Le resultatos indica le similaritate de corpores de Russell, de crystallos, e de

globulina gamma que omnes monstrava un gamma isoelectric inter pH 6 e pH

7,5. Altere methodos tincturatori que esseva empleate tendeva a confirmar iste

identitate.

Es discutite le discrepantias que se trova reportate in le litterat� ira con

respecto al ProPrietates tincturatori de corpores de Russell.

Omne le amyloides testate habeva un apparente puncto isoelectric in le

vicinitate de pH 5. Es summarisate he suggestiones in Ic litteratura relative

al natura e al origine de amyloide.

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1960.

2. -- and 1)eane, H. \V.: Staining Pr�Pr-

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:3. - and -: Staining properties of Rus-

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stals ill a !yinpho-plasmocyte in com-

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‘iork Ac�l(lJ5led. (ill ptt’ss).

4. Singer, \I. and Morrison, P. R.: The in-

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fied aul(l unmodified fibrin. j.Bioi.

Chem. 175:133-145, 1948.

5. Fawcett, I). \V. and I)eane, H. XV.: The

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1960 16: 1708-1721

ARTHUR F. GOLDBERG and HELEN WENDLER DEANE Amyloids-With Special Emphasis on Their Isoelectric PointsLympho-plasmocytoid Cell, of Russell Bodies in Plasmocytes, and of A Comparative Study of Some Staining Properties of Crystals in a

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