ANNALS OF CLINICAL AND LABORATORY SCIENCE, Vol. 18, No. 6 Copyright © 1988, Institute for Clinical Science, Inc. Rhodamine B Fluorescence as a Stain for Amniotic Fluid Squames in Maternal Pulmonary Embolism and Fetal Lungs STANLEYH. SHAPIRO, M.S. and ZELMA WESSELY, M.D. Queens Hospital Center, Affiliation of Long Island Jewish Medical Center, Jamaica, NY 11432 ABSTRACT Rhodamine B fluorescence is presented as a simple, rapid, highly sensi­ tive, permanent method for the histologic detection of squames in mater­ nal amniotic fluid emboli and fetal lungs in cases of intrauterine asphyxia. The method may be used on alcohol fixed smears or formalin fixed paraffin sections. The application of this procedure allows for identification of sparsely distributed fetal squames which otherwise may be overlooked by less sensitive tinctorial methods which may also be more tedious in tech­ nical preparation and screening. Introduction tricular failure is the only hemodynamic abnormality consistently observed in Amniotic fluid containing squamous humans. This may be secondary to the cells shed from the surface of fetal skin entrance of amniotic fluid into the (vemix caseosa) can be found as emboli maternal circulation. in the maternal circulation or aspirated The characteristic histologic pattern of into the fetal lung. Although the patho­ lungs of a mature fetus may appear genesis of amniotic fluid embolism is not altered by intrauterine anoxia, external well established, it has been considered compression, or malfunction. If anoxia that a sudden and sometimes massive stimulates increased respiratory activity, obstruction of the pulmonary vasculature more amniotic fluid may be aspirated may occur in late pregnancy, during into potential air spaces. Concomitantly labor, or shortly after delivery, both in with increased fetal maturity, more des­ spontaneous delivery as well as during quamated epithelial cells and lanugo Caesarian section. This may lead to hairs are present in the amniotic fluid shock or in some cases even death sec­ and can be aspirated. Squames can be ondary to hypoxemia, circulatory col­ found in the lungs of stillborn fetuses, lapse, or coagulation defects. Left ven- particularly mature ones, and those expiring shortly after delivery. Intrauter­ ine asphyxia, a critical consequence of Address reprint requests to: Stanley H. Shapiro, obstruction of the placental circulation, Department of Pathology, Queens Hospital Center, initiates premature fetal respiratory 82-68 164th Street, Jamaica, NY 11432. movements and consequent aspiration of 451 0091-7370/88/1100-0451 $00.60 © Institute for Clinical Science, Inc. 45 2 RHODAMINE B STAIN FOR AMNIOTIC FLUID SQUAMES amniotic fluid with vernix caseosa and 1. Decerate three to six mu paraffin sec­ cornified epithelial cells contained there­ tions in three changes of xylene for in. The identification of cornified five minutes each. squames is essential as evidence of this 2. Hydrate through three changes of event.1-2-7-12’14-15’16 absolute alcohol; 95 percent alcohol; A technique utilizing a mixed stain of 70 percent alcohol; distilled water, at phloxin with alcian green has been three minutes each. Smears are reported to demonstrate fetal squames.1 washed in running tap water for three Routine hematoxylin and eosin, a modi­ minutes. fied Mallory trichrome,4 and a keratin 3. Stain in 0.1 percent for aqueous tolu- mucin procedure have been used in this idine blue for 10 minutes. laboratory to identify maternal pulmo­ 4. Rinse three times in distilled water. nary amniotic fluid embolism. Rhoda- 5. Stain 0.1 percent rhodamine B* in mine B fluorescence has been found to 0.1M Mcllvaine’s buffer at pH 3.6 for be a selective stain for keratin-like pro­ 10 minutes. teins in skin stratum corneum6 and in 6. Rinse three times in distilled water. Mallory bodies.18 The present report 7. Dehydrate rapidly in ascending eth- evaluates rhodamine B fluorescence as a anols starting with 95 percent alcohol. means of identifying amniotic sac 8. Clear in three changes of xylene. squames in maternal blood and fetal 9. Mount with permount. lungs. Fluorescence microscopy was carried out using a Leitz Orthoplan microscope with a 200 W Osram HBO lamp and Materials and Methods BG12 primary and K530 secondary filters. Two cases of women showing amniotic fluid embolism in venous blood smears were studied. Four cases of fetuses or Results stillborns with amniotic fluid aspiration in lungs were used. Ten cases of fetuses Venous blood samples from both cases or full-term stillborns uninvolved with presenting amniotic fluid embolism amniotic fluid aspirations, normal human showed squames as scattered irregular, skin, and six cases of squamous cell carci­ homogeneous eosinophilic, or red frag­ noma served as controls. Smears were ments between blood elements with made from maternal blood drawn from hematoxylin and eosin and Kreyberg the right pulmonary artery on acid methods, respectively. Rhodamine B cleaned slides by a technician wearing fluorescence demonstrated these moie­ surgical gloves to prevent introduction of ties and lanugo as vivid bright yellow or exogenous squames. Smears of venous yellow orange tissue fragments against a blood and formalin fixed tissue blocks of dark background (figure 1). Paraffin sec­ lungs were stained with hematoxylin and tions from four cases of full term infant or eosin, Kreyberg technique, and the rho­ fetal lungs, which contained material damine B procedure. from the amniotic sac, showed some or many squames within alveoli. These were also identified in all cases with F ixation hematoxylin and eosin and Kreyberg stains. One case required extensive Ten percent neutral buffered formalin is used for tissue, and absolute ethanol is * Allied Chemical Company, National Anilino used for smears. Division C.I., Sinking Spring, OH 54170. RHODAMINE B STAIN FOR AMNIOTIC FLUID SQUAMES 4 5 3 F igure 1. Fluorescence microscopy of a venous smear from a patient with amniotic fluid embolism showing brightly stained squames. (x 1200) F igure 2. Fluorescence microscopy of a paraffin processed portion of a lung from an infant with sparce amniotic sac contents in alveoli showing bright yellow orange squames in alveoli against pale green back­ ground of lung parenchyma. (X 600) screening to localize the squames after embolism show amniotic fluid contain­ conventional stains. In contrast in all ing particulate matter, such as fetal des­ cases screened with rhodamine B fluo­ quamated squamous cells, sebaceous rescence, the squames, even though material, and mucous, which can be they were sparse in distribution, were identified in venous blood. Although a rapidly identified as needle-like or irreg­ direct connection between amniotic ularly rounded bright yellow orange tis­ fluid embolism and circulatory collapse sue fragments against the pale orange or has not been elucidated, the histologic green background of lung parenchyma detection of squames in maternal circu­ (figure 2). Lungs from fetuses or full- lation and prompt therapeutic interven­ term stillborns used as controls showed tion is crucial. It is therefore essential to no squames. The stratum corneum of identify amniotic fluid embolism, which normal skin and all six cases of squamous may occur in late pregnancy during labor cell carcinoma stained positively with or shortly after delivery, both in sponta­ rhodamine B fluorescence. neous delivery as well as in Caesarian sections. Discussion Amniotic sac contents in fetal lungs stained with hematoxylin and eosin are Pregnant women at term or during not always readily evident in microscopic delivery with clinical signs of pulmonary sections and may be occasionally over­ 454 SHAPIRO AND WESSELY 2. Attwood, H. D.: Amniotic fluid embolism. looked with conventional stains for kera­ Pathol. Ann. 7:145-172, 1972. tin and acid mucopolysaccharides. 3. AYOUB, P. and Shklar, G.: A modification of the Rhodamine B has been introduced as a Mallory connective tissue stain as a stain for ker­ specific stain for comification at the light atin. Oral Surg. Oral Med. Oral Pathol. 26:580- microscopic level.11 The mechanism by 581, 1951. 4. B O ER N E R , D.: Fluoreszenz Mikroskopische which this stain binds keratin is not pres­ Untersuchungen an Lipoiden. Protoplasma ently known. It has been reported that 42:168-177, 1952. the intensity of this weakly basic xan- 5. C lark , S. L ., Pavlo va, Z ., G reenspo o n, J., H o r e n st ein , J., and Ph e l a n , J. P.: Squamous thene derivative could be greatly cells in maternal pulmonary circulation. Am. J. enhanced by the use of fluorescent Obstet. Gynecol. 254:104-106, 1986. microscopy.6 In the present techniques, 6. Clausen, F. P., and Dabelstein, E.: Increase in sensitivity of rhodamine B method for kerati- toluidene blue binds nucleic acid and nization by the use of fluorescent light. Acta mucopolysaccharide moieties, some of Pathol. Microbiol. 77:169-171, 1959. which also show affinity for the fluoro- 7. Färber, S. and Sweet, L. K.: Amniotic sac con­ tents in the lungs of infants. Amer. J. Dis. Child chrome. In the past, rhodamine B has 42:1372-1383, 1931. been reported to stain lipids,4 certain 8. Garland, M C. and Thompson, W. D.: Diag­ connective tissue elements,17 and myco­ nosis of amniotic fluid embolism using an anti­ bacteria. 10 In this laboratory, the dye has serum to human keratin. J. Clin. Pathol. 36:625-627, 1983. been found to demonstrate Mallory 9. Kreyberg, L.: Main histological types of pri­ bodies sensitively and selectively in mary epithelial lung tumors. Brit. J. Cancer murine and human liver.18 25:206-210, 1961. 10. KUPER, S. W. A. and May, J. R.: Detection of Special stain procedures, such as Att- acid fast organisms in tissue sections by fluores­ wood’s stain,1 have been utilized in an cence microscopy. J. Pathol. Bact. 79:59-681, attempt to facilitate identification of 1960. amniotic squames. An immunoperoxi- 11. LlISBERG, M. F.: Rhodamine B as an extremely specific stain for comification.