766 Y Clin Pathol 1993;46:766-768 Combined silver Perls's stain for differential staining of ringed sideroblasts and marrow iron 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 myelodysplastic syndrome, 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 ethanol. reagent in this combined method proba- 2 If the marrow tissue is fixed in fixative bly shows phosphate rather than the iron containing mercury, 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 silver staining solution (1 volume 50% silver nitrate, 2 vol- umes 1% formic acid in 2% aqueous gelatin Evaluation of iron stores in macrophages 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 Prussian blue 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 Counterstain 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 Pathology, 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 lipofuscin 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 FX;_L sFu ^_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 microscopy 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 melanin, 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 protein 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