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Home , Lipofuscin, Perls Prussian blue, Silver staining

766 Y Clin Pathol 1993;46:766-768 Combined Perls's stain for differential

of ringed sideroblasts and marrow J Clin Pathol: first published as 10.1136/jcp.46.8.766 on 1 August 1993. Downloaded from

K T Tham, J B Cousar

Abstract tain ringed sideroblasts were also included in During a study of nucleolar organiser the study. Three were from patients with regions, a modified silver stain was found , and two from to be a sensitive marker for the iron in patients with erythroleukaemia and acute ringed sideroblasts, more so than Perls's lymphocytic leukaemia, respectively. Paraffin stain when the marrow iron stores were wax sections were stained with a Mallory low. To enhance the usefulness of the sil- modification of Perls's stain and also with the ver stain, a combined silver Perls CSP silver and Perls's stain and examined method was developed. This stains the microscopically for ringed sideroblasts. ringed sideroblast iron black and hae- mosiderin blue, thus rendering the PROCEDURE FOR THE COMBINED SILVER AND detection of ringed sideroblasts easier PERLS'S STAIN even when marrow iron stores are exces- 1 Dewax paraffin sections and bring down sive. At the same time, it allows marrow to water with decreasing concentrations of iron content to be evaluated. The silver . reagent in this combined method proba- 2 If the marrow tissue is fixed in fixative bly shows phosphate rather than the iron containing , remove mercury with present in the abnormal mitochondria in Lugol's iodine for 5 minutes. Wash sections ringed sideroblasts. This facilitates the with tap water for 3 minutes and rinse with differential staining of ringed sideroblast distilled water. Do not use sodium thiosul- "iron" and haemosiderin. phate solution to remove iodine. 3 Cover each section with 3 drops (about (7 Clin Pathol 1993;46:766-768) 0-15 ml) of a freshly prepared solution (1 volume 50% , 2 vol- umes 1% formic acid in 2% aqueous gelatin

Evaluation of iron stores in and solution). http://jcp.bmj.com/ abnormal iron deposits in sideroblasts and 4 Drop a 40 x 24 mm coverslip on the plasma cells is an important part of bone staining solution to spread it evenly. marrow examination. Perls's 5 Allow to stain for 40 minutes in complete stain and its various modifications are tradi- darkness. tionally used for this purpose. All iron 6 Remove coverslips and rinse sections in deposits are stained blue. distilled water.

During a study of the nucleolar organiser 7 Stain section with a 1 in 1 mixture of 5% on September 28, 2021 by guest. Protected copyright. region (NORs) with a silver stain, iron parti- potassium ferrocyanide and 5% hydrochloric cles in ringed sideroblasts stained black (per- acid in a Coplin jar for 4 minutes. sonal observation). Application of the silver 8 Wash thoroughly in distilled water. stain to a series of marrow particle sections 9 with nuclear fast red showed that the stain was more sensitive than (Kemechtrot solution) for 3 minutes. Perls's stain for ringed sideroblasts, especially 10 Wash in tap water, dehydrate with when iron stores were low or absent.' To increasing concentrations of ethanol, clear enhance further the utility of the stain, a com- and mount sections. bined silver and Perns's (CSP) stain was developed to produce differential staining of ringed sideroblast iron and other iron Results deposits. The CSP stain was compared with The CSP stain showed the iron in Mallory's modification of Perls's stain for macrophages and plasma cells as coarse blue Departnent of sensitivity. Apart from its diagnostic useful- granules; that in ringed sideroblasts was seen , Veterans Administration ness, the differential staining capacity of CSP as uniform, small jet-black granules, scattered Medical Center and stain may have pathophysiological relevance around the cell nuclei (figure). Sometimes the Department of because it suggests that the chemical compo- black granules were clustered Pathology, Vanderbilt focally. University Medical sition of iron deposits in mitochondria are Occasionally aggregates of black granules Center, Nashville, different from those in macrophages. were present in red cells and intercellular Tennessee 37203 USA spaces adjacent to ringed sideroblasts (fig K T Tham IA). Cell nucleoli and were J B Cousar stained Methods dark brown. stain also worked Correspondence to: The on mar- Dr KT Tham Fifty consecutive marrow aspirates were row smears but not on decalcified marrow Accepted for publication selected without prior knowledge of the diag- aspirates. On marrow smears, the staining 17 March 1993 noses. Five marrow aspirates known to con- tends to be uneven due to variable thickness Combined silver Perls's stain for differential staining ofringed sideroblasts and marrow iron 767

Figure 1 (A) Marrow with increased iron store. Iron in macrophages is stained blue and that in ringed sideroblasts black. J Clin Pathol: first published as 10.1136/jcp.46.8.766 on 1 August 1993. Downloaded from Black granules are also present in afew red ceUs and intercellular spaces (CSP stain). (B) Fine black granules in earlyforms oferythroblasts (CSP stain). (C)Black granules in three ringed sideroblasts and in the cytoplasm ofa | > i mf 4 W s*;= ,; mitotic cellprobably of erythroid lineage .: Y j - A .w - Y .- . ^. t \ _ (CSP stain). $ _ ww;: s... z ....d " " .si.'i4}w; ss i'&iS i' >A' W <';t .^^. ' - .' 3 _ jii s 5 jr NFx XF ^ ,; ; ,4 _ 2: * Fe. _ e ,. -9 ;_LFXsFu ^_0 -ir 7'.St:FsiMto... f wttwi4;^ v -,"I I_ikr Gj||;S, w *- ilL . k.,- B^S-.<;X.e;...... >^uES.._ t#f stgi3S .- '..' j .9 .''l_Fi-.>.E#*..v__t' t9I

of the smears. For the same reason, quantita- Of the 50 consecutive marrow aspirates tion of iron stores and ringed sideroblasts selected for the study, two marrows had rare may not be as reliable as when paraffin wax ringed sideroblasts demonstrable by both sections of more uniform thickness are used. Perls's and CSP stains. Seven marrows had On the other hand, if marrow smears are well ringed sideroblasts demonstrable only by the spread and thin, more cytological details and CSP stain. The positive results together with finer iron deposits can be discerned. iron store content and diagnoses are listed in All five marrows known to be positive for the table. It was remarkable that in cases 3

ringed sideroblasts had increased iron stores. and 9, several ringed sideroblasts were seen http://jcp.bmj.com/ The recognition of ringed sideroblasts was with the CSP stain but none with Perls's facilitated with the CSP stain because of the stain. In case 9, electron also differential staining of ringed sideroblast iron failed to identify ringed sideroblasts. and other iron in macrophages or plasma cells (fig 1A). Of the five marrows, four gave equivalent results by both staining methods. Discussion In one, more ringed sideroblasts were identi- The silver stain used is a modification of that on September 28, 2021 by guest. Protected copyright. fied with the CSP stain than with Perls's first introduced by Howell and Black' and stain, due to the staining of fine iron granules improved by Ploton and colleagues2 to in the early forms of erythroblasts by the CSP demonstrate NORs. It stains iron, calcium, stain in addition to the more mature forms , lipofuscin and fungal elements (per- (fig 1B). Cells in mitosis were identifiable as sonal observation), in addition to the argy- ringed sideroblasts because they contained rophilic associated with NORs. black granules in the cytoplasm (fig 1 C). Therefore, it is not a specific stain. Never- Perls's stain showed only the more mature theless, in marrow tissues the pattern of peri- ringed sideroblasts with coarser iron particles. nuclear black granules in erythroblasts is so characteristic that misidentification is unlikely. The use of coverslips in the staining proce- Marrows positivefor ringed sideroblasts out of50 consecutive unknown marrows dures minimises the volume of silver staining even and reduces Positivity by Positivity by Iron solution, ensures staining, Marrows Pers's stain CSP stain stores Diagnoses non-specific silver precipitate. The potassium acid solution used 1 + + Increased Malignant lymphoma ferrocyanide/hydrochloric 2 + + + Increased Small cell carcinoma, lung, S/P in step 7 of the staining procedure serves both Chemotherapy a for the silver stain 3 - + Increased Plasma cell dyscrasia as differentiating agent 4 - + + Normal Mastocytosis and as a staining agent for iron in 5 - + Normal Focal hypocellularity, cause uncertain and cells. If overdiffer- 6 - + Normal Small cell carcinoma, lung macrophages plasma 7 - + Decreased Acute lymphocytic leukaemia, entiated, some or all of the granules in ringed chemotherapy day 28 sideroblasts blue. As a further modifi- 8 - + Decreased Microcytic anaemia turned 9 - + + Decreased Acute myelomonocytic leukaemia cation of the previous method3 the sodium which reduces the sen- + = occasional ringed sideroblasts present; + + = many ringed sideroblasts present; + ++ = thiosulphate solution, numerous ringed sideroblasts present in many microscopic fields. sitivity of the silver stain, may be omitted 768 8Tham, Cousar

from the mercury-removing sequence without contain phosphates of other elements in addi- adverse effect. tion to or instead of iron. Therefore, Perls's Our results indicate that the CSP stain is stain will be weakly positive or negative but more sensitive than Perls's stain alone for silver stain will still be distinctively positive. J Clin Pathol: first published as 10.1136/jcp.46.8.766 on 1 August 1993. Downloaded from ringed sideroblasts, particularly when the This explains the greater sensitivity of the sil- marrow iron stores are low or absent. A previ- ver stain for ringed sideroblasts in iron defi- ous study reported that a simple silver stain is ciency and also explains the loss of ringed also sufficient for the identification of ringed sideroblasts in severe iron deficiency reported sideroblasts in marrow aspirates with reduced by Stavem and colleagues.8 If the silver stain iron stores.3 Nikicicz and Norback reported is specific for abnormal mitochondrial "iron" that the silver stain alone could be used to deposits, then even a few black granules in an assess not only sideroblast but also bone mar- erythroblast may be enough to identify it as a row iron stores4 and to study other marrow ringed sideroblast, as opposed to the require- cells.' Our experiences indicate, however, that ment of finding at least five blue granules the silver stain is not specific for iron and is encircling more than one third of the circum- not recommended for the identification of ference of the nucleus to identify a ringed iron in macrophages. The CSP stain permits sideroblast by Perls's stain.9 The presence of more specific identification and evaluation of black cytoplasmic granules may help to iden- iron stQres. Furthermore, when iron stores tify a "blast" or mitotic figure as an erythroid are excessive, the CSP stain facilitates identi- cell (figs 1B and C). Furthermore, the pres- fication of ringed sideroblasts because of its ence of multiple black granules in red cells differential staining ability. and intercullular spaces (fig 1A) may be an In our study of 55 marrows 14 were posi- indication to search carefully for ringed tive for ringed sideroblasts with the CSP sideroblasts as they may be derived from stain. Twelve of the 14 were associated with ringed sideroblasts. The ability of the silver neoplastic or pre-neoplastic conditions. One stain to demonstrate fungal elements adds to of the two exceptions was from a 68 year old its usefulness. patient with a focally hypocellular marrow of uncertain importance. The other was from a 82 year old patient with iron deficiency Conclusion anaemia receiving multiple medications for The CSP stain is more sensitive than Perls's hypertension and chronic obstructive pul- stain for ringed sideroblasts. It also permits monary . evaluation of iron stores. The silver reagent of Iron in ringed sideroblasts is located in the the CSP stain probably demonstrates phos- mitochondria. Using energy dispersive x ray phate and not iron in mitochondria of ringed analysis of the mitochondria, Grasso and col- sideroblasts. If it is a specific marker for leagues detected a consistent presence of iron ringed sideroblasts, then the presence of a few and phosphorus.6 They suggested that iron black granules may be enough to identify an http://jcp.bmj.com/ was present in the form of ferric phosphate. erythroblast as a ringed sideroblast, and the Other elements variably detected included presence of black granules in red cells and the calcium, lead, potassium and . No phos- intercellular spaces may be an indication to phorus was detected in normal or unaffected search carefully for ringed sideroblasts. mitochondria in the same section. and haemosiderin contained relatively much on September 28, 2021 by guest. Protected copyright. less phosphorus. Their findings suggest a pos- 1 Howell WM, Black DA: Controlled silver staining of sible staining mechanism for the silver stain; nucleolus organiser regions with a protective colloidal developer: a one-step method. Experientia 1980;36: the stain shows the phosphate and not the 1014-5. iron moiety of ferric phosphate. A similar 2 Ploton D, Menager M, Jeannesson P, Himber G, Pigeon F, Adnet JJ. Improvement in the staining and in the mechanism is seen with von Kossa's stain visualization of the argyrophilic of the nucleolar which demonstrates the phosphate and car- organizer region at the optical level. Histochem J 1986;18:5-14. bonate but not the calcium in bone.7 Both 3 Tham KT, Cousar JB, Macon WR. Silver stain for ringed stains do not work on decalcified material. sideroblasts: Am J Clin Pathol 1990;94:73-6. 4 Nikicicz EP, Norback DH. The use of a collodial silver Von Kossa's stain, however, does not demon- (AgNOR) method in assessing iron stores strate ringed sideroblasts (personal observa- and sideroblasts. Mod Pathol 1991;4:363-7. 5 Nikicicz EP, Norback DH. Argyrophilic nucleolar organ- tion). The differential staining capability of iser region (AgNOR) staining in bone marrow cells: the CSP stain supports the above hypothesis. J Clin Pathol 1990;43:723-7. 6 Grasso JA, Myers TJ, Hines JD, Sullivan AL. Energy-dis- Haemosiderin in macrophages is stained blue persive X-ray analysis of the mitochondria of sidero- because of the excess of iron over phosphate. blastic anaemia. BrJ Haemawol 1980;46:57-72. 7 Bancroft JD. Histochemical techniques. 2nd edn. London: In ringed sideroblasts the black staining of the Butterworths, 1975:211. phosphate moiety masks the blue staining of 8 Stavem P, Rorvik TO, Rootwelt K, Josefsen JO. Severe iron deficiency causing loss of ring sideroblasts. Scand J the iron moiety. If overdifferentiated, the Haemawol 1983;31:389-91. black stain is removed and the blue stain 9 Juneja SK, Imbert M, Sigaux F, Jouault H, Sultan C: Prevalence and distribution of ringed sideroblasts in pri- becomes visible. In iron deficiency the abnor- mary myelodysplastic syndromes. J Clin Pathol 1983;36: mal mitochondria in ringed sideroblasts may 56(;-9. '