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1955 10: 160-166

The Application of the Prussian Blue Stain to Previously Stained Films of Blood and Bone Marrow
R. DOROTHY SUNDBERG and HARRIETTE BROMAN

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The

Application Stained
By R.

of the Prussian Films of Blood

DOROTHY SUNDBERG

Blue Stain to Previously and Bone Marrow

AND HAII1IIETTE BIIOMAN

SI1\IPLE
rocytes

METHOD

FOR

STAINING

non-hemoglobin

irOn and since films

within other 1951 of marrow

erythcells
.

aIld

their

precursors

and

within

macrophages

containing method
iron
ill

particulate has made the marrow as well

iron has been itl use in our laboratories possible rapid estimates of the amount and in the blood in both new and old
newT and old section material.

This
or

of non-hemoglobin

blood

as in both

LITERATURE

Gruneberg which were

fixation with

described positive for

absolute

iron cent

siderocytes as when subjected

alcohol, blood

erythrocytes to the prussian

films were

containillg granules blue reaction. After

treated with
cent

methyl

a freshly

mixed

solution

of 1 per

potassium then

ferrocyanide with Smith

and
(1945)2

1 per
used
cent

hydrochloric
methyl alcohol acid was the

acid for 3 to 5 minutes and Pappenheimer, Thompson,

fixation

counterstained Parker and mixed

illterest

biebrich

scarlet.
hydrochloric

(3

parts) basic that

followed by and 2 per fuehsin. they the blood

a freshly
ceilt potassium

solution
ferrocyanide

of 1 per (1 part).
study

They

counterstained in then

with fact applied of the

Of

particular
stain,

in their

of siderocytes

erythrocytes

photographed with the Giemsa blue with

gratlules They were not

and rod-like decolorized showed actually

(1945)2

structures with acid

demonstrable alcohol, and

prussian stained granules. prussian

reagent. Giemsa

that the stippled erythrocytes erythroeytes cotitaitiing some

also showed that the granules

type of iron which were

negative.

Pappenheimer positive did blood

large w-ere on is treated a brown i)lackened bodies

et al. take

the their
of

Gram cases
the

stain with

and high

were
bodies
lIl

Feulgen of
were

When
was Whell most the Since that

heparinized
sedimented, these took

from
numbers with by were

percetltages 0.9 per

et ouie

siderocytes recovered. salt found. felt

been

iron-cotltaining hydrogen

sulphuretted

cent were
al.2 might

solution,

color;

oiily
this
not

occasioulal
reaction, hemosideriti.

black
Pappenheimer Here

i)odies

hemosideriui iron-containing

comment that blackened.

by the study, iron felt

it is of some interest Further proof that

their erythrocytes et Giem.sa granules over the the and were

that occasional iron-containing siderocytes actually contained

toward to their many identical the slides with

bodies had iron was ohside

the Case hut

tamed while their the reason

demonstrating unaffected Pappenheimer stain that

migration settled al2 sent found not of

the
to

magnetized
During M. Case. present R.

of a tube
course applied for some of

bottom.

siderocytes siderotic

granules.

Rath
in

and cells
the

Finch

(1948)
total

have
amount films

made

extensive
stainable

use
iron of bone

of the
in

prussian or free

blue

reaction

estimating

fixed
marrow.

ret.iculoendo-

thelial
From

in unstained
University May 3,

of fragments
Hospital for publication 160

of Minnesota

Laboratories, July 6,

Minlseapolis,

Minn.

Submitted

1954;

accepted

1954.

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It.

DOROTHY

SUNDBERG

AND

HARRIETTE

BROMAN

161 in rubricytes similar

blue

Mills,
erythrocyt.es in

Huff,

Krupp
with

and the
and

Garcia
erythrocytes

(1950)

illustrated stain and also

to sui)jected

stipplitig showed
the prussian

and stippling
reaction

Wright-Giemsa

different

rubricytes

and

coutlter-stained Despite the early placed out the practice of counterst.ain row films which could
taitling
agaiil, Recetltly

with safranine. report of Pappenheimer of using a reaction Koszewski heeul stained Turnbull stained
with

et al. (1945)2 no real designed to demousstrate show-ed that May-Grunwald one could Giemsa

emphasis was iron as a type

uuitil had the which

(1952) with

decolorize maryears before and plasma Giemsa cells and conthen

t.hetl
after

apply

graulules
Kaplan,

blue reaction. first with the

Turuihull blue.

He illustrated May-Grunwald

decolorization,

which
formaliui solution

they
vapor of

used
for 2 per

Zuelzer, and Mouriqualld on fresh films of marrow 30 minutes cent aqueous a brief wash with dilute

(1954)
.

have hour and

described
involved in equal

a method
fixation in

Their

method

prior t.o staining for one potassium ferrocyanide in tap

basic

2 per were

parts of a cent hydro-

chloric a(i(i. After for 30 to 60 seconds

water,
fuchsin.

their

slides

counterstaiuled

rFECHNIC Dry films of a large variety imprint iron \Vrights buffer stain in acid prussian appears way are the blue and comparable a vivid Individual been blue cells and and (3 parts) then of cellular fluids including blood, of various bone tissues marrow, or organs lymph, can

sputuni ttn(l ascitic fluid or be staitled for tsoishemoglobin

or touch preparations in the following manner.

1. The
2. state suitable 4. niixed The water Slides but amouist staining this The for

filni
slide stain to the

or iml)rint
is flooded is diluted strength are left in in this iron lsydrochloric the color

is dried
with with of the

rapidly.
stain and

the
to

stain

should

remaits

in

this

undiluted

4 minutes. allowed the and reagent to the or are remain of reagent cent to original blue still green, easily
nongranular

3. The

on the

the film. which

slide is

for made

a Period up in of

of time freshly (1 part). distilled stain, original of the of even than were

cellularity blue 2 per for allowed

The
1 l)C slides urstil particulate of

slides
cent are stained

immersed

prussian

potassium drain films and dry. stained there

ferrocyanidet washed briefly with has and

of

10 minutes4

a 1)iflk

Wrights been the many fragments blue or color we

is colored

a minimal

decolorization. Isas are as macrophages

It

recognizable
cytoplasm

reaction cytoplasm

altered color.
iron is

very
is often concerned, to attention various

little.
also

The
a vivid that

hemosiderirs-filled
Iiisoftr

blue which

or otsce

blue

green,

and deep

similar
s stain brought

particulate Wrigist

appeared blue or at The studied proving blue

purple * This att.em)tiulg

with

is changed to our

an

even by types many have

more Dr. of of a method Rudi

vivid

green when

paper
to

wts

Schmid

a time illustrations had t.he

distinguish many we it

between had was seen. desirable

stippling. the cases for

showed had previous of par-

cells resembling biopsies of the

ticulate iron in

Because
to

marrow,

presence

01(1 specimens.

0.5 per cent and 4 cent potassium ferrocyanide have also been used with apparently equal success. The proportions and strengths used were t.hose used by Pappenheimer et a!.2 They were recommended by Dr. Roy Holly who had consulted Dr. Clement Finch regarding the best stain. This reaction is reasonably clear at 2 minutes, but the granules seem less discrete than at 5 or 10 minutes. At 30 minutes (the time recommended for tissues), decolorization is still not too great, hut nuclei show some fading, and Jolly bodies may be very difficult to see.

From bloodjournal.hematologylibrary.org by guest on June 26, 2013. For personal use only.

162
that green obtained with
separate

PRUSSIAN

BLUE

STAIN

with Wrights

the
l)hase

prussian takes
of

blue on

reaction a more

alone, intense

and color iron

that than
.

which it

appeared showed

blue

or

blueNo in-

stain the method, success ago* reagetst. atsd

originally.

tentional colorizes
identifiable. been as the marrow iron long used prussian

decolorization
of the the films films on on particulate prussiali originally stained

is necessary
blue with reagent

The
as

prussian
to a type make of

blue
the

reagent
iron

deeasily has stains

and
with

changes Tisis equal blue

color using

sufficiently with stain Wrights

counterstain, Gienlsa prior to

stained Wrights

or the

as 30 years

immediately

using

Controls
having

of this
inaiss-

nsethod have macrophages

as well as many

included laden
siderotic

various studies. with phagocytosed

isormoblasts and the prussian by Wrights

Comparable erythrocytes
and blue or siderocytes reagent the

films of the same and particulate

have been sub-

(hemosiderin?) to: alcohol 1. Methyl 2.

jected

fixation prussian fuchsin.

followed

(4 minutes) blue
by

(10
Giemsa

minutes).

The

outlined
or basic
stain

reaction the Prussian

followed blue

stains

or

by

safranine

3. Wrights

reaction.

In are and two

is

these similar cytoplasm

types

three and
normoblasts

types

of preparations

the

numbers

of

iron-laden

macrophages

the

blue
and

to

blue-green is similar
siderocytes

color
are type

of the
similar in

prussian types

positive

granules The latter

to be

of macrophages
of specimens. In granules precursors, the

in each

of the

siderotic used,

of specimens. percentages in the

in w-hich of cells not clearly
HO count-erstain

first are but the

of specimetl in large cell types numbers are

the

siderotic and

visible

i)elieved recoguiizable.

erythrocytes

The
tology.

Wrights Efforts

stain

follow-ed which and

by

the

prussiatl

blue

reagent

affords or which
blue positive

the type

best

cy-

to predict
erythrocytes

pling

ill

of the granules iii macrophages normoblasts will be prussian

of stiphave not

been uniformly successful. Much of the phages or usormoblasts and eryt.hrocyt.es

green blue-green, normoblasts

deep l)lue granulation in either will become a brilliant blue Numerous

and numerous

macroor blueblue
and

with

the
and have

prussian
olive i)eett

blue
green circled,

reagent.
pigments

macrophages
stippled

containing
erythrocytes

sketched,
not

subjected
blue or

to the
blue-green

prussian
pigmetlt

blue
ill

reagent
macro-

and reidentified. Some, phages or normoblast.s

olive-green pigmetit-

hut auld
remains

all olive

of the greeu

erythrocytes

proves
or

prussian

positive.
Some of

Some
the

of the

disappears.

stipplitlg

disappears.
which

Although
disappears,

most
occasionally

often it is the fine blue-green basophilic the cell which contained that stippling 1)lue-green
found

stippling will show reaction.

and

a somewhat Particulate
plasma blue-green cells

coarser,
iFOtl

brilliant
has as 1)een

stippling
and are

after

the

prussian
and

in neutrophils, prussian

lymphocytes, in erythrocytes

monocytes,
precursors.

as well

iui macrophages of neutrophils

The (riboof eryth-

D#{246}hlebodies

negative. basophilic hasophilic spongioplasm stippling

Because it seemed nucleic acid bearing rocytes and reticulocyt.es brilliant stain also cresyl

that it was cytoplasm) which studied. when

pernicious

the precipitated or the ordinary disappeared with The reticulation

normoblasts were also blue disappeared

the prussian of ret.iculocytes blue plus reagent

Downey.

reagent, films of staiuled with counterfor 10 minutes.

or stained

with subjected
anemia

brilliant

cresyl

a Wrights

to t.he prussian
from files of 1)r.

* Blood

film

from

Hal

From bloodjournal.hematologylibrary.org by guest on June 26, 2013. For personal use only.

R.

DOROTHY

SUNDBERG

AND

HAIIIIIETTE

BROMAN

163
imprints of liver

In the course from a patient

types of preparations

of this investigation, films with malignant melanoma

golden yellow* to

of bone marrow and w-ere also studied.

brown stain, pigmeilt (riot

In

both

of these and melanot

With

distinguishable still was

from noma
stain,

unstained cells.
Oully Wheul

hemosiderin)
stained with

was

seen
Wrights

in many the a very prussian

of the
the

macrophages used
be

pigmetit

disthe

tinguishable simple phages

ous cells
application

from
very

hemosiderin.
occasional bright

With only

reagent
could

as
found.

a countermacronumer-

blue-greetl

granules

prussian were
of
itt

reaction found the the in the the prussian marrow

iron.

alone, films

few hemosideriul-containing or imprints of liver, although

of marrow

containing

golden brow-Il pigment reagent. These studies which has

tissues.

had heetl identified prior to afforded further evidence that hemoglobin

Oil sections

all pigment
does not

resembles also been

Here

some used

i)reakdow-n
of particles

product
of

contain

The
marrow

prussian
and,

blue
of course,

reagent
other

boule

1. Take tilled
2.

the

tissue

through with

xylol the

and prussian

the technic is as follows: decreasing strengths of alcohol reagent. 2 minutes and eosin for a few

to

dis-

water. Stain for

30 minutes water with

3. 4. 5.

Wash in tap Counterstain Do not

for 20 minutes. hematoxylin for the hematoxylin.

secoulds.

decolorize

6. Dehydrate. Coverslip With this technic bright macrophages and phagocytic rocytes. extremely Some of the masses large ; their color and section the prussiatl material passed reagent

from xylol. blue-green granules of varyiusg sizes are visible in cells of all types atld also ill nornoblasts and erythof prussian is, how-ever, is not applied. available, Restaining positive similar the material to that coverslips strengths w-ith and hematoxylin an additional method7
atid seem similar
itt

the
itl

seen can

macrophages the erythrocytes be removed to is getierally passage through uistained

obtaitled

are

siderocytes)

If new old and slides, the

from water, not

tissue

through

decreasing

of alcohol

but the original eosin usually fades, eosill is desirable. Because Kaplaul, Zuelzer, and Mouriquatlds
films
of

necessary,

used
to

ott
those

of marrow
various

allowed types, and

estimation because

of normoblasts their results

reasouiahle

identification

cell

with stained Zuelzer,

and

the

method films and

described of marrow
.

here, It can
original

we decided found
stain was is

to use the to previously

generally

their

method described

on previously by Kaplan, blood and pertwo gastrecas au of

by

was

that

method

Mouriquand
films. The 1000

be applied vivid

marrow

fixed and stained fairly well maintained, color.

on blood using that

the
tients

particulate based
with

iron
Oil

is the
cell

expected
counts,

blue-greeti
estimated atlemia).

The
films these

siderocyte
from atid counts percentage

centage,
tomized objective
* The

pa-

remarkable splenectomized means of seemed

to t Isat seen

siderocytoses megaloblastic comparisous, very similar it was

in

(exogenous determined
to that

hemochromatosis By a higher
as

pigment.
aIs(!

color anemias,

described

hemosiderits

Rath

and
alid

Finds3
refract

us hemolytic

hemocisromat

osis,

megaloblast

Ic ausemias

orv

aiiemuas.

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164

PRUSSIAN

BLUE

STAIN

TABLE

1 . -Iatient Fe, P10 B

with F

Exogenous P60 B

Hemochromatosis-Siderocytes KZM wP60

in

Blood \V Fso P60

RDS

9.6

13.5

10.1

7.0

6.4

9.6

LMG
Gastrectomized Splenectom F P10 B
r

ized

Patient

with
KZM

Megalobiastic
WP10

4 nemia-Siderocytes
W Fo P,o W F p10

in

Blood
K

Fio P10 B

\V Fo

C 39.7 35.5 35.1

%
37.3 34.9 38.5

%
19.6 22.7 18.8 21.6

%
38.6 39.9 39.0

%
37.1 36.7 36.8

%
40.2 34.9 34.6

RDS HB LMG

40.9 37.5 39.3 38.0

F30-Formol gent-concentration
same lined et
.

vapor-30 minutes. as described

minutes. B-Basic
.

in

F10-Formol vapor-lO minutes. P,o-Prussian reapreseist report-lO minutes. P60-Prussian reagentfuchsin. KZM-Method of Kaplan described et al.7 by as out-

as al.7-60

above-60 minutes.

W-Wright

s st am

K-Prussian

reagent-concent.rat

ion

as

Kaplan,

siderocytes

was

fouuid

when

formalin

fLxat.iOIl

yeas

used

either

originally

or

after

the methyl alcohol fixatioul employed fixative was methyl alcohol. Formalin for a higher per(entage of siderocytes
followed by immersion in

with Wrights stain thaul when the only seemed to be the only variable responsible and fixation with formalin for 10 minutes reagent
(concentration as described in

the

prussian

presetlt
staiil fixation

report or as recommended by Kaplan et al.7) for 10 minutes provided with as great. a degree of positivity as that obtaitled with 30 minutes and reaction 60 minutes
by

a of

of
formaliti

exposure
fixation

to and,

the
seems

prussian
to be based

reagent.
upon

The
the

increased
blue-green

positivity

provided

(olor

of mitiute couuits as the done

particles
iti table

sometimes
an

delicate of the induced and

fuzzy

aggregates of even fixation. G.*)

of
1000

staiuial)le iron. The siderocyte cell counts Counts as well were

1 give

idea

variability by formalin L. M.

variability

apparetitly D. S.,

by three

of us (R.

H. B.,

DIsCusSION

the

The technic siderotic which blue

described has proved granules of siderocytes acid) aIld .Jolly bodies. resemi)les reagent.

valuable in the and normoblasts It has also shown

problem of differentiating from basophilic stippling that all pigment in macrothe the which stains with staining period,

(rihonucleic phages prussian

hemosiderin does Because, with an

not contain iron added 10 minute

prussian blue reagent can he applied after utes) have already been stained and still
* Lorraine Hospitals.

films, imprints, or sections provide a clear-cut method

Technology, University

(30 mmof dis-

M. Gonyea,

MS.,

Instructor

in

Medical

of Minnesota

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R.

DOROTHY

SUNDBERG

AND

HARRIETTE

BROMAN

165 comparison iron. It

the staining

tinguishitlg atld new useful

smears

particulate specimens study from

iron, the which may of iron marrow

method contain

is invaluable in the excesses of particulate

of old is also
of

in the made

deficiency particles

anemia, but in the latter, as recommended by Rath iron can coat layer

and

Finch3 films and masses to blue,

is

desirable since often virtually made from the myeloid-erythroid the

no particulate or huffy

be foutld in ordinary of cetitrifuged marrow. studied, and

Bone marrowand blood from a variety of conditiouls has been results are the subject of a separate report.8 The term particulate iron is used here to refer to the granules positive material which show which, similar
in in

of prussian to purple reagent,

studies siderin,

colors

varying

from

blue-green of the color.

siderocytes

with Wrights stain, but all assume a reasonably

suggest probably that the

with the additioul vivid blue-green

normoblasts and

prussian blue The present as hemoare also

if the
siderotic

material
granules

macrophages color in the

is to he regarded cytoplasm recently of

hemosiderin.

The

diffuse

blue-green

macrophages presented by

could be due to the more The method described Kaplan, Zuelzer gree of positivity to allow blue
counterstam,

soluble form of iron, has been compared

ferritin. with that

and Mouriquand.7 which appears of minute The

can

Their method to be based upon or fuzzy by Kaplan particles dry

shows a variably higher dethe ability of formalin vapor of iron films with the the basic marrow-, prussian fuchsin etc., et al.7 minus

staining

granules described

reagent. tiot

method

a feature

be used pointed out

on previously stained by these investigators.

SUMMARY

of blood,

A simple
blasts,

method fluids

for
and

staining
other

non-hemoglobin
cells containing

iron
particulate

itl

erythrocytes, iron in new

normoor old

macrophages,

films
consists

of cellular

or imprints

of tissues

and

organs

is presented.

This

method

ill using the prussian blue reagent as a type of counterstain; no separate decolorization is necessary. The preparations obtained resemble the original preparations except that iron stands out as a vivid blue-green material. The method is particularly useful in studying conditions accompanied by varyitlg degrees of iron excess or hemosiderosis of the marrow.

The of the of iron,

stainable cytoplasm ferritin.

iron

iron is all virtually of macrophages

is visible

the might

same color. be attributed and

The

diffuse to the

more

blue-green soluble

color form

Stainable

in normoblasts to the prussian using formalin

erythrocytes react-ion. on fresh

as well films

as in macrohas preto

phages in sections A prussian blue

subjected method

blue fixation

of marrow-7 iron iii appears

also i)een shown to viously stained films bring about a higher

be useful in the of marrow and percentage

demonstration blood. The

of particulate formalin fixation

of siderocytes.
IN IXTERLLNGUA pro frotis colorar e altere ferro cellulas cellular non-hemoglohinic a particulas como etiam iui erythcoust.ineuste in impres-

SUMMARIO Es rocytos, ferro-si presetitate hen un simple methodo macrophagos, e preservate

normoblastos, ill flO\C

de fluido

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166 siones Prussia

colorante

PRUSSIAN

BLUE

STAIN

de

texitos
Un requirite. es

e organos.
specie

Iste

methodo

consiste

ill

usar

le

reagente

blu

de

como

except.e Le
vane

Clue ferro

de cont.ra-coloratlte. Nulle processo specificamente deLe preparatos obteuiite es simile al preparatos original se distingue como materia de color vivement-e blu-verde. utile in studiar o de hemosiderosis
exhibi forma in dine in conditiones accompaniate le mesme de ferro. nuance.

met.hodo es specialmente grados de excesso de ferro

le ferro colorate per

de

del

medulla. es possibilee etiam

in

Omne

le methodo es un
normoblastos

pract.icameiit.e

Le
mente

diffuse Ferro

i)lu-verde

que

appare

le cytoplasma
plus

de macrophagos
solubile
e erythrocyt.os

at.tribuibile colorabile
in sectiones

a ferritina es visibile
subjicite

macroe

phagos
Mouriquand
tiOtl

al reactioul

a blu

de Prussia.

Le met.hodo de ferro

a blu de Prussia ha usate pro nove itltraparticular

a formalina pare

con fixation a formalina que Kaplan, Zuelzer, frotis de medulla es etiam utile in le demonstracolorate
uti frotis

in previemente distitictiuicar

de medulla

e sanguine. de siderocytos.

Le fixation

plus

alte

procentage

REFERENCES
1
GRIJNEBERG,

H.
with 1945
AND

: Siderocytes:

A new
THOMPSON,

kind

of erythrocytes. PARKER, inclusions D. P., aft.er

Nature,
AND

London SMITH,

148:

114,

1941.

2 PAPPENHEIMER,

A. M.,

\V. P.,
erythrocytic Sternal marrow

K.

E. : Anaemia

associated 14: RATH, tion 75,

unidentified FINCH, iron

splenectomy. A method for

Quart. the

J. Med.
determina-

C. E.

C. A.:
stores

hemosiderin.

in man. J. Lab. & Clin. Med. 33: 81, 1948. MILLS, H., HUFF, R. L., KRUPP, M. A., AND GARCIA, J. F.: Hemolytic anemia secondary to a familial (herditary) defect in hemoglobin synthesis. Arch. Int. Med. 86: 711, 1950. ItoSZEWSKI, B. J.: Zur Methodik des H#{228}mosiderinnachweises in den Blut- und Knochenmarksausstrichen. Klin. Wchnschr. 30: 926, 1952. 6 -: The occurrence of megaloblastic erythropoiesis in patients with hemochromatosis.
of available Blood KAPLAN, GONYEA, 7: 1182, 1952.

E.,

ZUELZER, iron
AND

W. W.,
in marrow
SUNDBERG,

AND

MOURIQUAND,

C.:
Blood 9:

Sideroblasts. 203,

A study iron

of

stainable and

nonhemoglobin
8

isormoblasts.

1954.
in erythrocytes

L. M.,

It. bone

D.: marrow.

A study (To

of stainable

lrecursors

in the

blood

and

be published.)