anelectron microscopic description ofbasophilic stippling inred cells

An Electron Microscopic Description of Basophilic

Stippling in Red Cells

By WALLACE N. JENSEN, GIULIANA D. MORENO AND MARCEL C. BEssis

I HE FIRST DESCRIPTION of basophihic stippling in erythrocytes was

made by Ehrhich1 and the association of basophilic stippling with lead

intoxication followed shortly thereafter.2 Numerous studies of basophilic

stippling and its occurrence in a variety of hematologic disorders followed

these original descriptions. Various early investigations indicated that the

substance which forms the punctate basophilia in the red cell is derived

from the nucleus or from various cytoplasmic components or from the red cell

membrane.3’4 More recent studies suggest that basophilic stippling occurs only

in young erythrocytes and that cells with hasophibic stippling have common

properties with reticubocytes.a� The comprehensive studies of Dustin7 resulted

in the conclusion that the stipple material is composed exclusively of ribo-

nucleoproteins. Others have suggested that punctate stipple material is corn-

posed of ribonucleoproteins plus mitochondria or ribonucleoproteins and

portions of the red cell membrane. Others have suggested that nonheme iron

may constitute a part of the basophihic stipple substance.8’#{176} The work of

Bruckner1#{176} and that of Dustin7 indicate that basophihic stippling does not

exist in cells suspended in plasma, but occurs when cells which contain the

necessary precursor components are subjected to certain manipulations during

the preparation of the blood for microscopic examination.

These studies describe basophilic stippling as observed with the combined

use of supravital stains, light microscopy and electron microscopy. A pre-

liminary report of the findings has appeared elsewhere.�’

MATERIALS AND METHODS

Lead intoxication was produced in guinea pigs, rabbits and rats by the subcutaneous

injection of 4 per cent lead acetate in water in amounts of 200 to 400 mg. per Kg., given

in single or multiple injections during a period of less than 7 days. Hematologic abnormal-

ities appeared within 2 to 4 weeks. Blood was also examined from patients with lead in-

toxication and other types of hemolytic anemia.

Blood was used, with or without heparin, for routine smears and electron microscopy.

When advantageous, young cells were concentrated by centrifugation (600 g. for 10

minutes). Reticulocyte preparations were made by mixing equal amounts of blood and

dye (0.5 per cent New Methylene Blue dye in a 0.7 per cent solution of sodium chloride)

for 5 minutes, after which smears were made, fixed with alcohol and stained with Giemsa

solution.12 For the preparation of stippled cells by the action of dye, equal amounts of

New Methylene Blue of various concentrations (0.025 to .008 per cent) and blood were

mixed for 45 seconds following which smears were made for light microscopy, or the

From the Centre National de Transfusion Sanguine, Paris, France and the Department

of Medicine, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania.

Supported in part by grant (HE-06270) from the U. S. P. H. S., Bethesda, Maryland.Submitted July 3, 1964; accepted for publication Oct. 26, 1964.

933

BLOOD, VOL. 25, No. 6 (JuNE), 1965

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934 JENSEN, MORENO AND BESSIS

dye and blood mixture was centrifuged and the cells resuspended in native plasma prior

to fixation for electron microscopy. Blood and bone marrow were fixed in Daltons’ solu-

tion, dehydrated in alcohols and embedded in Epon. The sections were mounted on

formvar, stained with lead acetate and examined by electron microscopy. Dried fixed bloodsmears were stained with LOfflers methylene blue dye for basophilic stippling’3 and by the

Prussian blue reaction for nonhenie iron (leposits. Ribonuclease studies were done by

treatment of cells on dried smears with an 0. 1 per cent solution of a commercially avail-

able enzyme#{176} at pH 6.4, for 15 to 30 mnintites at 45 C.

RESULTS

Blood or bone marrow cells from 20 rabbits, 20 guinea pigs and 60 rats were

examined by one or more technics, usually on multiple occasions. There was

marked variation in the time of appearance and in the number of stippled

cells present. Stippling was always found in the bone marrow when it was

present in the peripheral blood, b�t was found only in the orthochromatophilic

normoblasts which were relatively few in the animals with severe lead in-

toxication.

Optical Microscopy

Relations of Basophilic Stippling, Reticulocytes and Polychrornatophilic

Cells. Examinations were made on samples of blood which had greatly varied

numbers of reticulocytes and degrees of pol�chromatophilia. The number of

basophilic stippled cells was always less than the number of reticubocytes.

In a single sample of blood the number of stippled cells varied from one

slide to another and in various parts of the same smear. A greater number of

stippled cells was present in thicker than in thinner parts of the smear, and

blood films which were dried rapidly had fewer stippled cells than those dried

slowly. The difference in numbers of stippled cells in a sample of blood was

accentuated by drying blood films very rapidly and very slowly. Slow drying

was accomplished by use of a humid chamber as described by Dustin.7 Stippled

cells induced by the humid chamber had morphologic characteristics identical

to those found on randomly prepared or rapidly dried smears. Stippling was

produced by slow drying not only in lead intoxicated animals, but also in

the bloods of animals with phenylhydrazine and phlebotomy-induced reticulo-

cytosis.

The number of stippled cells was also increased by the incubation of blood

with various concentrations of New Methylene Blue dye (less than O.2�5 per

cent). Basophilic stippled cells were produced when an abundance of young

cells were present whether due to lead poisoning, phenylhydrazine intoxica-

tion or prior phlebotomy. The appearance of the stippling was identical to

that of blood specimens prepared by conventional methods. The converse or

production of reticulocytes to the exclusion of cells with basophihic stippling

was attempted. Samples of bbocd were incubated with concentrated New

Methylene Blue dye (0.25 per cent) for 5 minutes. In dried smears prepared

#{176}L.Light and company Ltd., Colnbrook, England.




BASOPHILIC STIPPLING IN RED CELLS 935

from such blood samples, only cells which had the typical appearance of

reticubocytes were seen. Thus the arrangement of basophilic material in

either round multiple regular masses, as seen in stippled cells ( fig. 1A ) or in

irregular serpentine networks as seen in reticulocytes ( fig. 1 B ) may be

produced by the interaction of appropriate proportions of New Methylene

Blue dye and the basophilic material within young red cells.

There was increased ultraviolet light absorption of the stippled points and

of a diffuse nature throughout the cytoplasm of the polychromatophilic cells

in dried blood smears. Punctate basophilia was not seen with ultraviolet

light in plasma suspended cells. Examination of dried smears with light in

the Soret band showed negative absorption scattered in the cell in punctate

arrangements.

Treatment of blood smears with ribonuclease, caused a loss of the basophilic

punctuation, diffuse basophilia and reticubocyte skeins.

The Relationship between Baso’philic Stippling and Siderocytes. The rela-

tion of nonheme iron and basophilic stippling was examined in two ways.

A comparison of the number of siderocytes and cells with basophilic stippling

was made, and single intracellular basophilic stipple points were localized

and restained for nonheme iron. There was no constancy between the number

of siderocytes and cells with hasophilic stippling. In single cells, it was not

always possible by light microscopy to determine whether a basophilic stipple

point, when restained for iron, was of the exact same size and location. Single

basophilic points were found which were positive, and others negative for

iron. There were iron positive deposits which were negative with the baso-

philic stipple stain. Thus, by supravital stains and by light microscopy there

was no constant association between nonheme iron deposits and basophilic

stippling.

Electron Microscopy

Abnormalities in normoblasts and reticubocytes of animals with lead poison-

ing were of multiple types and frequent occurrence. Those which do not

specifically constitute a part of basophilic stippling will only be listed here

and will be described in detail in another publication. There were abnormal

densities, irregular in outline and eccentrically located in the erythroblast

nuclei. There was widening of nuclear membrane pores with concentric

lamellated myelin bodies at the juncture of the nuclear membranes and mito-

chondria. The Golgi apparatus was greatly enlarged. Many mitochondria

were seen, which were greatly enlarged, dilated, devoid of normal cristae, and

often contained large amounts of ferritin or ferruginous micelles. Ferritin in

scattered and aggregate forms was present in cytoplasm and in discrete

vacuoles or in double membrane lined tracts and in invagmations of the cell

membrane.

Polyribosomes and monoribosomes were evenly distributed in the cytoplasm

of the reticubocytes. It was not possible in these studies to accurately quantitate

the proportions of each type of rihosome.




A B

JENSEN, MORENO AND BESSIS

09

Fig. 1.-(A) Basophilic stippling produced in young, non-nucleated red cells bysupravital stain (dilute New Methylene Blue). Smears of cells were stained withGiemsa stain and examined with a low phase contrast objective. (B) Reticulocyteswith supravital stain (concentrated New Methvlene Blue). The blood smears werestained with Giemsa stain.

Blood specimens from all animals were searched by electron microscopy

for intracellular structures which might be interpreted as basophilic stippling.

Although basophilic stippling was present in large numbers when smears were

prepared and examined with the optical microscope, they were never seen

when the blood was prepared in routine fashion for electron microscopy.

Blood with high percentages of reticulocytes, from animals with lead

poisoning, phenylhydrazine-induced anemia and postphlebotomy anemia were

incubated with various concentrations of new methylene blue dye, and when

basophilic stippling was found by optical microscopy, similar preparations

were made for electron microscopy.

Blood samples were examined with and without prior incubation with new

methylene blue dye. In the specimens without supravital staining, there were

reticulocytes with mitochondria and evenly dispersed ribosomes (fig. 2). In

the specimens with supravitally stained cells, there were aggregated ribo-

somes, which formed irregular networks (fig. 3). In the supravital stained

preparations still other cells were seen, which contained multiple, round or




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BASOPHILIC STIPPLING IN RED CELLS

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937

Fig. 2.-Electron microscopic photograph of a reticulocyte without supravital

stain. Ribosomes are evenly scattered in the cytoplasm, mitochondria are present

and the cell outline is irregular. Mag. x 32,600. (Reproduced from1 4)

irregularly round aggregates of rihosomes without other scattered cvtoplasmic

ribosornes (fig. 4A, B).

The majority of cells with (lot-like aggregates of ribosornes also had

mitochondria. The mitochondria had no constant positional relation to the

ribosomal aggregates. The hasophilic stipple material of the cell does not,

therefore, include mitochondria. Not infrequently, nonheme iron-laden mito-




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938 JENSEN, MORENO AND BE.SSTS

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Fig. 3.-Electron microscopic photograph of a reticulocyte after supravital stain(concentrated New Methylene Blue). The ribosomes are aggregated in linear and

skein-like networks. Mitochondria are present. (Reproduced from’4)

chondria were located adjacent to ribosomal aggregates, but were clearly

separate from the ribosomes (fig. 5). The two structures could be separately

identified only with magnifications afforded by electron microscopy. Blood

films were prepared on glass slides, removed by scraping with a razor and

prepared for electron microscopy. These cells were greatly distorted, not ideal

for examination and showed nonheme iron deposits, but were without identi-

fiable mitochondria or ribosomes.




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Fig. 4.-(A) Electron microscopic photograph of a young reticulocyte after supra-vital stain with dilute New Methylene Blue dye. The ribosomes are aggregated andin punetate forms but are independent of mitochondria which are also clearly seen.

Discussiox

The affinity of the stippled material of young erythrocytes of lead intoxicated

animals for basic dyes, the absorption of ultraviolet light and the disappear-

ance of the basophilic material following treatment of the cells with ribo-

nuclease have been describe(l previously.7 Identical findings have been

described in the reticulocyte,14 and indicate strongly that stippling is composed

either wholly or in part of ribonucleoproteins. The production of greater or

smaller numbers of basophilic stippled cells by slow or rapid dessication of

the blood film and by incubation with various concentrations of new

methylene blue strongly suggests that the three morphologic entities (retic-

ulocytes, diffuse polychromasia and punctate hasophilic stippling) are the

result of different manipulations of the cell in preparation for optical or

electron microscopy. These same factors may partially explain the variable

numbers of cells with punctate basophilia which may be found on different

blood films made from the same blood.5’7’10 The number of siderocytes and

their lack of constancy as well as the number of basophilic stippled cells in

a sample of blood and the different locations within a cell of basophilic




Fig. 4.-(B) The same section as figure 4(A) but with greater magnification,which allows a more discrete view of aggregate ribosomes and mitochondria.


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stippling and iron have been noted in previous studies and was confirmed.15

The arrangement of iron-laden mitochondria and ribosomal aggregates

within the cell was sometimes such that they could not be resolved as two

distinct structures by light microscopy. The occurrence of nonheme iron in

a stipple point is therefore fortuitous and results from the superimposition or

juxtaposition of the two separate structures. This is best appreciated when the

cells are examined by electron microscopy. Prior studies7’15 and the present

one indicate strongly that nonherne iron is not an essential component of

basophilic stippling.

It seems that the multiplicity of different conclusions which have been

reported concerning the nature of basophilic stippling3’4’6”548 may have

derived in part from the variable methods used for preparation and examina-

tion of cells. In the present study, the evidence indicates that only the

ribosomal aggregates seen by electron microscopy in the young red cells from

animals with lead poisoning, constitute the hasophilic stipple.

The peculiarity of the basophilic stippling which occurs classically in lead

poisoning, but also in a variety of other diseases is that it occurs “spontane-

ously.” It might, therefore, he properly suggested that the occurrence of baso-

philic stippling is evidence of ribosomal abnormality.

The abnormalities of function of ribosomes in thalassemia described by

Burka and Marks19 and other biochemical and clinical similarities between

lead poisoning and thalassemia make attractive a suggestion of the possibility




‘�1

14�.

‘1.1

- S.

#{149}m�’�’; �

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Fig. 5.-Electron microscopic photograph of a young non-nucleated erythrocyte

from a rat with lead intoxication. The cell was stained supravitallv with dilute New

Methvlene Blue dye prior to fixation for electron microscopy. Punctate aggregates

of ribosomes are adjacent to dilated mitochondria which contain iron deposits. Amass of cvtoplasmic ferritin is also present.


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#{189}

of functional abnormalities of ribosomes in erythrohlasts and reticulocytes of

animals with lead poisoning. However, the known multiple biologic actions

of lead also allow the possibilities of a primary alteration of red cell mem-

brane20’2’ or nonribosomal organelles22 or other substances in the interior of

the cell23 which then cause ribosomes to agglutinate. Qualitative structural

abnormalities of red cell ribosomes are difficult to evaluate by electron

microscopy since the ribosomes vary in type and numbers with stage of

maturation of the red cell.

CONCLUSION

Red cells which contain ribosomes presumably have the potential to form

stippled cells. The rihosomes in cells from animals with phenylhydrazine and

postphlebotomy anemia may form stippling when blood films are dried slowly

or when stained supravitally with dilute concentrations of New Methylene Blue

dye. The ribosomes of cells from animals with lead intoxication have a greater

propensity to form aggregates and therefore to result in stippling.

Since the formation of stippling in cells is dependent on the dessication of

cells, they are not seen in plasma suspended cells nor in cells prepared in

routine fashion for electron microscopy. The basophilic stippled material





produced in certain blood cells by the action of supravital dye consists exclu-

sively of ribosomes. The association of nonheme iron with basophilic stippling

is usually the consequence of the presence of two separate morphologic struc-

tures within the cells which may not be resolved, one from the other, in light

or phase microscopy, but may be distinguished by the use of electron micro-

scopy.

The “spontaneous” occurrence of the hasophilic stippling may be interpreted

as evidence of ribosomal abnormality, hut the multiplicity of cellular injuries

induced by lead, other chemicals, drugs or disease processes may indicate that

rihosomal aggregation results from alteration of nonribosomal organelles or

other cellular constituents.

SUMMARIO IN INTERLINGUA

Erythrocytos que contine ribosomas ha presumitemente le potential de

formar cellulas marmorate. Le ribosomas in cellulas ab animales con anemia

per phenylhydrazina o phiebotomia pote causar marmoration quando pel-

liculas de sanguine es siccate lentemente o quando illos es tincturate supra-

vitalmente con diluite concentrationes de nove blau methylenic. Le ribosomas

de cellulas ab animales con intoxication per plumbo ha un plus grande diathese

a formar aggregatos e, per consequente, a manifestar marmoration.

Viste que le formation de iste phenomeno in le cellula depende del desicca-

tion cellular, illo non occurre in cellulas suspendite in plasma, e non in cellulas

preparate routinarimente pro le microscopia electronic. Le marmorate material

basophilic producite in certe cellulas sangilinee per le action de tincturation

supravital consiste exclusivemente de ribosomas. Le association de ferro

nonhemic con le phenomeno de marmoration basophilic es usualmente le

consequentia del presentia de duo separate structuras morphologic intra le

cellulas. Iste duo structuras intracellular non pote esser resolvite, le un ab le

altere, per microscopia a lumine o de phase, sed illos pote esser distinguite

per Ic uso de un microscopio electronic.

Le occurrentia “spontanee” de marmoration basophilic pote esser inter-

pretate como evidentia de un anormalitate ribosomal, sed le multiplicitate de

lesiones cellular que es inducite per plumbo, per altere substantias chimic, per

pharmacos, o per processos pathologic, indica possibilemente que aggregation

ribosomal resulta ab le alteration de organellas nonribosomal o de altere

constituentes in le cellula.

REFERENCES

1. Ehrlich, P.: Uber einige beobachtungen 1. Milano, Societa Editrice Libraria,

am an#{228}mischen blut. Berl. Klin. 1933, p. 22.

\Vschr. 1881. 5. Brookfield, R. W.: Blood change oc-

2. Behrend: In Ucber basophile kornungen curring during the course of treat-

in rother blutkorpen by Litten, M. ment of malignant disease by lead,

Dtsch. Med. Wschr. 25:717, 1899, with special reference to punctate

and Vereins-Beilage 25:253, 1899. basophilia and the platelets. J. Path.

3. Naegeli, 0.: Blutkrankheiten und Blut- Bact. 31:277, 1928.

diagnostik. Berlin, Julius Springer, 6. Whitby, L. E. H., and Britton, C. J.1931, p. 122. C.: The relation of the stippled cell

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reticulocyte. Lancet 1:1173, 1933.7. Dustin, P., Jr.: Contribution a l’#{233}tude

histophysiologique et histo chimique

des globules rouges des vert#{233}br#{233}s.

Arch. Biol. 60:285, 1943.

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roten blutkorperchen II. Uber die

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der basophilen substanz in den jug-

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98:95, 1927.

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philes des #{233}rythrocytes dans l’intox-

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12. Brecher, G.: New methylene blue as a

reticulocyte stain. Amer. J. Clin.

Path. 19:895, 1949.

13. Haeger-Aronsen, Birgitta: Studies on

urinary excretion of 8-amino-laevul-

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Le r#{233}ticulocyte. Colorations vitales

et microscopic #{233}lectronique. Nouv.

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Etude au microscope #{233}lectronique dim

sang et des organes h#{233}mopoietiques

dans le saturnisme experimental.

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19. Burka, E. R., and Marks, P. A.: Ribo-

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human reticulocytes: a defect in thai-

assemia major. Nature 199:706, 1963.

20. Fratianne, R. B., Griggs, R. C., and

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Effect of lead, in vivo and in vitro

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Medicine, University of Paris, Head of Research Laboratories,

Centre National de Transfusion Sanguine, Paris, France.




1965 25: 933-943

WALLACE N. JENSEN, GIULIANA D. MORENO and MARCEL C. BESSIS An Electron Microscopic Description of Basophilic Stippling in Red Cells

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anelectron microscopic description ofbasophilic stippling inred cells

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