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The value of polyclonal carcinoembryonic antigen immunostaining in the diagnosis of microvillous inclusion disease
The Value of Polyclonal Carcinoembryonic Antigen lmmunostaining in the Diagnosis of Microvillous Inclusion Disease GABRIEL M. GROISMAN, MD, OFER BEN-IZHAK, MD, AUDREY SCHWERSENZ, MD, MOSHE BERANT, MD, AND BILLIE FYFE, MD Microvillous inclusion disease is a specific disorder recognized as a cause of intractable diarrhea of infancy. We studied three cases by light microscopy, electron microscopy, and immunostaining for polyclonal carcinoembryonic antigen (CEA). Histologically, all cases had villous atrophy and abnormal accumulation of periodic acidSchiff-positive material in surface enterocytes. Ultrastructurally, poorly developed brush-border and intracytoplasmic inclusions lined by intact microvilli were present in surface enterocytes. Crypt cells showed well-preserved surface microvilli. Carcinoembryonic antigen immunostaining showed prominent intracytoplasmic reactivity in surface enterocytes and linear brush-border reactivity in crypt cells. Normal and diseased small bowel biopsy specimens used as controls revealed linear brush-border reactivity without intracytoplasmic staining. Intracytoplasmic positivity for carcinoembryonic antigen in microvillous inclusion disease is explained by its presence in the glycocalyx within the microvillous inclusions. The demonstration of a distinct staining pattern for polyclonal carcinoembryonic antigen in routinely processed small bowel biopsy specimens provides a new useful criterion that complements other established techniques for accurate diagnosis of microvillous inclusion disease. HUM PATHOL 24:1232-1237. Copyright @ 1993 by W.B. Saunders Company
Microvillous inclusion disease (MID), also known as congenital/familial microvillous atrophy, is a specific disorder presenting shortly after birth as severe intractable secretory diarrhea.‘.’ The prognosis is extremely poor, as infants are completely dependent on total parenteral nutrition and usually die before the age of 18 months as a result of liver failure, sepsis, and dehydration.‘,’ The only effective therapy is multivisceral transplantation. Pathologists should be able to recognize MID and to distinguish it from other causes of intractable diarrhea of infancy. Presumptive diagnosis is based on the demonstration of villous atrophy associated with abnormal periodic acid-Schiff (PAS) and alkaline phosphatase staining. l-4 Definitive diagnosis, however, depends on the ultrastructural demonstration of intracyFrom the Lillian and Henry M. Stratton-Hans Popper Department of Pathology and the Division of Pediatric Gastroenterology, Mount Sinai School of Medicine, New York. NY; and the Departments of Pathology and Pediatrics B, Rambam Medical Center, Haifa, Israel. Accepted for publication June 28, 1993. Presented in part at the annual meeting of the United States and Canadian Academy of Pathology/Society for Pediatric Pathology, New Orleans, LA, March 1993. Kry wordx congenital microvillous atrophy, microvillous inclusion disease, carcinoembryonic antigen. Address correspondence and reprint requests to Gabriel M. Groisman, Department of Pathology, Mount Sinai Hospital, One Gustave L. I,evy Place. New York, NY 10029. Copyright 0 1993 by W.B. Saundel-s Company
004s-8177/93/2411-0013$5.00/0
1232
toplasmic microvillous inclusions and poorly developed brush-border microvilli of small and large intestinal surface epithelium.‘,‘.4-” We report three cases of MID and evaluate the potential usefulness of polyclonal carcinoembryonic antigen (CEA) immunostaining in the diagnosis of this disorder. MATERIALS
AND METHODS
Clinical Data The clinical characteristics are summarized in Table 1.
of the three patients with MID
Patient no. 1 was a boy with no Patients no. 2 and 3 were girls of
significant family history. Arab origin. Each had consanguineous parents and one sibling who died from intractable diarrhea in the first months of life. All patients presented early after birth with severe watery diarrhea that persisted after oral feeding was discontinued. The patients were supported on continuous total parenteral nutrition. Somatostatin, given to patient no. 1, had only minimal effect in reducing stool output and was discontinued after 2 months. Small bowel biopsies were obtained at 2 months in patient no. 1 and at 3 months in patients no. 2 and 3. Patients no. 2 and 3 died from sepsis attributable to long-term total parenteral nutrition. Patient no. 1 underwent multivisceral transplantation at 7 months of age. He is alive and well with normal small intestinal function 9 months after the procedure.
Pathologic
Examination
Small bowel biopsy specimens from all patients were processed for light and electron microscopy. Tissue for light microscopy was fixed in 10% formalin, embedded in paraffin, and stained with hematoxylin-eosin and PAS reagent. Further sections were stained for CEA (polyclonal; dilution 1:500; Dako, Carpinteria, CA) using the avidin-biotin-peroxidase technique. For comparison, CEA staining was performed in small bowel biopsy specimens with histologically normal (six cases) and abnormal mucosa (seven cases): celiac disease (three cases), autoimmune enteropathy (one case), idiopathic intractable diarrhea of infancy (two cases), and cow’s milk proteininduced enteropathy (one case). Samples for electron microscopy were fixed in 3.5% cold gluteraldehyde, osmicated, dehydrated in graded alcohols, and embedded in Epon. Ultrathin sections were stained with uranyl acetate and lead citrate and were examined with a transmission electron microscope.
RESULTS Light Microscopy Small bowel mucosa in all MID cases revealed complete villous atrophy with normal or slightly increased
MICROVILLOUS
TABLE 1.
INCLUSION
DISEASE (Groisman
et al)
Clinical Features of Three Patients With Microvillous Inclusion Disease
Patient No.
Birth Weight
Gestational Age
Gender
Origin
(g)
(wk)
1 2 3
M F F
Hispanic Arabic Arabic
3,000 2,700 2,900
40 36 37
crypt depth (Fig 1). The surface epithelium showed focal cellular disarray and vacuolated cytoplasm (Fig 2). An abnormal accumulation of PAS-positive material was present within the apical cytoplasm of surface and upper crypt epithelium, whereas mid-crypt and basal enterocytes exhibited the usual brush-border staining (Fig 3). Electron Microscopy Ultrastructural examination revealed a similar appearance in all MID cases (Figs 4 and 5). Surface enterocytes had a severely abnormal apical membrane with short, disorganized, and infrequent microvilli. The most striking finding was the presence of numerous intracytoplasmic inclusions varying in size and shape with a complete brush-border lining their inner face. The inclusions were situated in the supranuclear and apical parts of the cytoplasm. Incompletely formed inclusions with microvilli restricted to the lower inner face and covered by apical cytoplasmic membrane material occasionally were seen. Numerous vesicular bodies containing amorphous material and forming single or multiple poorly developed microvillous projections surrounded larger, well-formed microvillous inclusions. Crypt epithelium had well-preserved brush-border microvilli with increased numbers of small membranebound vesicles containing electron-dense material (secretory granules) in the apical cytoplasm of mid and upper crypt cells. lmmunohistochemistry Polyclonal CEA staining of small bowel biopsies from patients with MID revealed a distinctive pattern. In surface and upper crypt cells the normal linear stain-
FIGURE 1. Small bowel mucosa with MID. There is total villous atrophy and mild chronic inflammation. (Hematoxylin-eosin stain; magnification X40.)
1233
Age at Onset of Diarrhea Birth 72 hr Birth
Outcome Alive at 16 mo Died at 5.5 mo Died at 6 mo
ing of the glycocalyx was absent and prominent intracytoplasmic reactivity was seen. Mid-crypt and basal enterocytes demonstrated normal brush-border positivity (Fig 6). All normal and disease control cases had linear brush-border reactivity without intracytoplasmic staining (except in goblet cells) (Fig 7). DISCUSSION The association of intractable diarrhea of infancy with enterocyte microvillous atrophy and intracytoplasmic microvillous inclusions has been termed “microvillous inclusion disease”‘s*5’6 and “congenital/familial microvillous atrophy.“2*4 Microvillous inclusion disease usually presents early after birth with severe, watery diarrhea. There is a slight female preponderance’,2 and the disease appears to be transmitted as an autosomal-recessive trait.‘,‘.” Microvillous inclusions have been described in colonic enterocytes,‘.‘,4 gastric antral epithelium,” gallbladder epithelium,7 and renal tubular cells.” The basic defect in MID is unknown. Carruthers et al” found decreased levels of cytoskeletal myosin in extracts of brush-border membrane from a small bowel biopsy in a patient with MID. These investigators suggested that this could be the underlying defect in the disease with failure of myosin to bind the base of the actin cables located in the microvilli and anchored to the plasma membrane, thus leading to perturbation of the membrane cytoskeleton at the terminal web. Cutz et al’ postulated an inborn error of intracellular transport leading to aberrant assembly of microvilli within intracytoplasmic vesicles, rather than at the apical cell
HUMAN PATHOLOGY
Volume 24, No. 11 (November
1993)
FIGURE 2. Close-up of surface enterocytes in MID showing cellular disarray and vacuolated cytoplasm. (Hematoxylin-eosin stain; magnification X400.)
surface. Alternatively, the inclusions may result from involution of performed surface microvilli. This hypothesis is supported by the observations that most microvillous inclusions are situated in the apical portion of the cytoplasm and that some superficial inclusions appear to be in continuity with the surface membrane.“.’ Regardless of pathogenesis, the normal appearance of mid-crypt and basal enterocytes suggests that the formation of microvillous inclusions and the depletion of surface microvilli are related to the process of differentiation and migration of enterocytes toward the small bowel lumen. Our light and electron microscopic analyses revealed typical features of MID in the three patients.
Carcinoembryonic antigen immunostaining showed a distinctive pattern resembling that seen with PAS and alkaline phosphatase staining.“*” Carcinoembryonic antigen is a 180,000-d glycoprotein of heterogeneous composition. First considered to be a specific marker for colon carcinoma, CEA was later found to be present in other epithelial tumors” as well as in normal small intestinal mucosa. I ’ Carcinoembryonic antigen has 10 to 15 epitopes, some of which are shared by one or more CEA-related proteins, such as nonspecific cross-reacting antigen and biliary glycoprotein.” To increase the specificity of CEA as a tumor marker, a number of monoclonal antibodies with restrictive reactivity have been devel-
FIGURE 3. Abnormal accumulation of PAS-positive material in surface cells (arrowheads). Crypt cells (arrows) and goblet cells (G) stain normally. (Small intestine. Periodic acid-Schiff stain; magnification X200.)
1234
MICROVILLOUS
INCLUSION
DISEASE (Groisman
et al)
FIGURE 4. Electron micrograph of surface enterocyte showing wellformed intracytoplasmic microvillous inclusions surrounded by several vesicular bodies (arrows>. Surface brush-border microvilli are severely diminished in an adjacent cell (upper left corner). (Patient no. 1. Magnification ~8,300.)
oped. Using nine different monoclonal CEA antibodies, Jothy et al I3 found diverse staining patterns in various normal and neoplastic tissues, including positive and negative reactivity of normal intestinal mucosa. We used a polyclonal CEA antiserum that also can react with CEA-related molecules. It is possible that the use of monoclonal CEA antisera would have produced different results. However, in a previous study normal small intestinal mucosa stained with nonspecific crossreacting antigen-free CEA antiserum revealed a staining pattern identical to that of our normal control cases, namely, positive staining over the surfaces of the villi,
FIGURE 5. Electron micrograph of crypt cells showing increased numbers of secretory granules and intact microvilli. (Patient no. 2. Magnification x10.000.)
1235
within goblet cells, and along the luminal borders of the crypts. I1 B y immunoelectron microscopy CEA was found within mucin granules of goblet cells and in the glycocalyx over the microvilli.“~14 The intracytoplasmic positivity for polyclonal CEA in MID is explained by the presence of CEA and/or CEA-related substances in the glycocalyx sequestered within the microvillous inclusions. Crypt enterocytes have linear brush-border CEA reactivity since the brushborder is intact and no intracytoplasmic microvillous inclusions are present in these cells.‘.‘,“.” The availability of multivisceral transplantation as a curative treatment makes the prompt and precise di-
FIGURE 6. lmmunostaining with polyclonal antiserum against CEA in the small bowel with MID shows intracytoplasmic reactivity in surface enterocytes (arrowheads). Crypt cells show linear brush-border positivity (arrows>. (Immunoperoxidase stain; magnification X 100.) (Inset) Higher-power view of intracytoplasmic CEA reactivity. (Immunoperoxidase stain; magnification X400.)
FIGURE 7. lmmunostaining with polyclonal antiserum against CEA in control cases. Brush-border staining with no intracytoplasmic reactivity (except in goblet cells) is seen in normal small bowel mucosa (top) and autoimmune enteropathy (bottom). (Immunoperoxidase stain. Top, magnification x100. Bottom. original magnification x400.)
MICROVILLOUS
INCLUSION
DISEASE (Groisman
agnosis of MID by the pathologist imperative. The demonstration of a distinct staining pattern for polyclonal CEA in routinely processed small bowel biopsy specimens provides a new, useful criterion that complements other established techniques for accurate diagnosis of MID. Acknowledgment. The authors thank Norman Katz and Sharon Smith for photographic assistance and Marva Barbee
for preparing
this manuscript.
REFERENCES 1. Cutz E, Rhoads JM, Drumm B, et al: Microvillous inclusion disease: An inherited defect of brush-border assembly and differentiation. N Engl J Med 320:646-651, 1989 2. Phillips AD, Schmitz J: Familial microvillous atrophy: A clinicopathological survey of 23 cases. J Pediatr Gastroenterol Nutr 14: 380-396, 1992 3. Lake BD: Microvillous inclusion disease: Specific diagnostic features shown by alkaline phosphatase histochemistry. J Clin Path01 41:880-882, 1988 4. Phillips AD, Jenkins P, Raafat F, et al: Congenital microvillous atrophy: Specific diagnostic features. Arch Dis Child 60: 135-l 40, 1985
1237
et al)
5. Bell SW, Kerner JA, Sibley RK: Microvillous inclusion disease. The importance of electron microscopy for diagnosis. Am J Surg Path01 15:1157-1164, 1991 6. Schofield DE, Agostini RM, Yunis EJ: Gastrointestinal microvillous disease. Am J Clin Path01 98:119-124, 1992 7. Rhoads JM, Vogler RC, Lacey SR, et al: Microvillous inclusion disease. In vitro jejunal electrolyte transport. Gastroenterology 100: 811-817, 1991 8. Cutz E, Thorner P, Sherman P: Microvillous inclusion disease (MID)-Generalized defect of brush-border membranes. Lab Invest 58:3P, 1988 (abstr) 9. Carruthers L, Phillips AD, Dourmashkin R, et al: Biochemical abnormality in brush border membrane protein of a patient with congenital microvillous atrophy. J Pediatr Gastroenterol Nutr 4:902-907, 1985 10. Taylor CR: Immunomicroscopy: A Diagnostic Tool for the Surgical Pathologist. Philadelphia, PA, Saunders, 1986 11. Isaacson P, Judds MA: lmmunohistochemistry of carcinoembryogenic antigen in the small intestine. Cancer 42:1554-1559, 1978 12. Rogers GT: Carcinoembryonic antigens and related glycoproteins: Molecular aspects and specificity. Biochim Biophys Acta 695: 285-290, 1984 13. Jothy S, Brazinsky SA, Chin-A-Loy M, et al: Characterization of monoclonal antibodies to carcinoembryonic antigen with increased tumor specificity. Lab Invest 54:108-l 17, 1986 14. Ahnen DJ. Nakane PK, Brown WR: Ultrastructural localization of carcinoembryonic antigen in normal intestine and colon cancer. Abnormal distribution of CEA on the surfaces of colon cancer cells, Cancer 49:2077-2090, 1982
Microvillous inclusion disease (MID), also known as congenital/familial microvillous atrophy, is a specific disorder presenting shortly after birth as severe intractable secretory diarrhea.‘.’ The prognosis is extremely poor, as infants are completely dependent on total parenteral nutrition and usually die before the age of 18 months as a result of liver failure, sepsis, and dehydration.‘,’ The only effective therapy is multivisceral transplantation. Pathologists should be able to recognize MID and to distinguish it from other causes of intractable diarrhea of infancy. Presumptive diagnosis is based on the demonstration of villous atrophy associated with abnormal periodic acid-Schiff (PAS) and alkaline phosphatase staining. l-4 Definitive diagnosis, however, depends on the ultrastructural demonstration of intracyFrom the Lillian and Henry M. Stratton-Hans Popper Department of Pathology and the Division of Pediatric Gastroenterology, Mount Sinai School of Medicine, New York. NY; and the Departments of Pathology and Pediatrics B, Rambam Medical Center, Haifa, Israel. Accepted for publication June 28, 1993. Presented in part at the annual meeting of the United States and Canadian Academy of Pathology/Society for Pediatric Pathology, New Orleans, LA, March 1993. Kry wordx congenital microvillous atrophy, microvillous inclusion disease, carcinoembryonic antigen. Address correspondence and reprint requests to Gabriel M. Groisman, Department of Pathology, Mount Sinai Hospital, One Gustave L. I,evy Place. New York, NY 10029. Copyright 0 1993 by W.B. Saundel-s Company
004s-8177/93/2411-0013$5.00/0
1232
toplasmic microvillous inclusions and poorly developed brush-border microvilli of small and large intestinal surface epithelium.‘,‘.4-” We report three cases of MID and evaluate the potential usefulness of polyclonal carcinoembryonic antigen (CEA) immunostaining in the diagnosis of this disorder. MATERIALS
AND METHODS
Clinical Data The clinical characteristics are summarized in Table 1.
of the three patients with MID
Patient no. 1 was a boy with no Patients no. 2 and 3 were girls of
significant family history. Arab origin. Each had consanguineous parents and one sibling who died from intractable diarrhea in the first months of life. All patients presented early after birth with severe watery diarrhea that persisted after oral feeding was discontinued. The patients were supported on continuous total parenteral nutrition. Somatostatin, given to patient no. 1, had only minimal effect in reducing stool output and was discontinued after 2 months. Small bowel biopsies were obtained at 2 months in patient no. 1 and at 3 months in patients no. 2 and 3. Patients no. 2 and 3 died from sepsis attributable to long-term total parenteral nutrition. Patient no. 1 underwent multivisceral transplantation at 7 months of age. He is alive and well with normal small intestinal function 9 months after the procedure.
Pathologic
Examination
Small bowel biopsy specimens from all patients were processed for light and electron microscopy. Tissue for light microscopy was fixed in 10% formalin, embedded in paraffin, and stained with hematoxylin-eosin and PAS reagent. Further sections were stained for CEA (polyclonal; dilution 1:500; Dako, Carpinteria, CA) using the avidin-biotin-peroxidase technique. For comparison, CEA staining was performed in small bowel biopsy specimens with histologically normal (six cases) and abnormal mucosa (seven cases): celiac disease (three cases), autoimmune enteropathy (one case), idiopathic intractable diarrhea of infancy (two cases), and cow’s milk proteininduced enteropathy (one case). Samples for electron microscopy were fixed in 3.5% cold gluteraldehyde, osmicated, dehydrated in graded alcohols, and embedded in Epon. Ultrathin sections were stained with uranyl acetate and lead citrate and were examined with a transmission electron microscope.
RESULTS Light Microscopy Small bowel mucosa in all MID cases revealed complete villous atrophy with normal or slightly increased
MICROVILLOUS
TABLE 1.
INCLUSION
DISEASE (Groisman
et al)
Clinical Features of Three Patients With Microvillous Inclusion Disease
Patient No.
Birth Weight
Gestational Age
Gender
Origin
(g)
(wk)
1 2 3
M F F
Hispanic Arabic Arabic
3,000 2,700 2,900
40 36 37
crypt depth (Fig 1). The surface epithelium showed focal cellular disarray and vacuolated cytoplasm (Fig 2). An abnormal accumulation of PAS-positive material was present within the apical cytoplasm of surface and upper crypt epithelium, whereas mid-crypt and basal enterocytes exhibited the usual brush-border staining (Fig 3). Electron Microscopy Ultrastructural examination revealed a similar appearance in all MID cases (Figs 4 and 5). Surface enterocytes had a severely abnormal apical membrane with short, disorganized, and infrequent microvilli. The most striking finding was the presence of numerous intracytoplasmic inclusions varying in size and shape with a complete brush-border lining their inner face. The inclusions were situated in the supranuclear and apical parts of the cytoplasm. Incompletely formed inclusions with microvilli restricted to the lower inner face and covered by apical cytoplasmic membrane material occasionally were seen. Numerous vesicular bodies containing amorphous material and forming single or multiple poorly developed microvillous projections surrounded larger, well-formed microvillous inclusions. Crypt epithelium had well-preserved brush-border microvilli with increased numbers of small membranebound vesicles containing electron-dense material (secretory granules) in the apical cytoplasm of mid and upper crypt cells. lmmunohistochemistry Polyclonal CEA staining of small bowel biopsies from patients with MID revealed a distinctive pattern. In surface and upper crypt cells the normal linear stain-
FIGURE 1. Small bowel mucosa with MID. There is total villous atrophy and mild chronic inflammation. (Hematoxylin-eosin stain; magnification X40.)
1233
Age at Onset of Diarrhea Birth 72 hr Birth
Outcome Alive at 16 mo Died at 5.5 mo Died at 6 mo
ing of the glycocalyx was absent and prominent intracytoplasmic reactivity was seen. Mid-crypt and basal enterocytes demonstrated normal brush-border positivity (Fig 6). All normal and disease control cases had linear brush-border reactivity without intracytoplasmic staining (except in goblet cells) (Fig 7). DISCUSSION The association of intractable diarrhea of infancy with enterocyte microvillous atrophy and intracytoplasmic microvillous inclusions has been termed “microvillous inclusion disease”‘s*5’6 and “congenital/familial microvillous atrophy.“2*4 Microvillous inclusion disease usually presents early after birth with severe, watery diarrhea. There is a slight female preponderance’,2 and the disease appears to be transmitted as an autosomal-recessive trait.‘,‘.” Microvillous inclusions have been described in colonic enterocytes,‘.‘,4 gastric antral epithelium,” gallbladder epithelium,7 and renal tubular cells.” The basic defect in MID is unknown. Carruthers et al” found decreased levels of cytoskeletal myosin in extracts of brush-border membrane from a small bowel biopsy in a patient with MID. These investigators suggested that this could be the underlying defect in the disease with failure of myosin to bind the base of the actin cables located in the microvilli and anchored to the plasma membrane, thus leading to perturbation of the membrane cytoskeleton at the terminal web. Cutz et al’ postulated an inborn error of intracellular transport leading to aberrant assembly of microvilli within intracytoplasmic vesicles, rather than at the apical cell
HUMAN PATHOLOGY
Volume 24, No. 11 (November
1993)
FIGURE 2. Close-up of surface enterocytes in MID showing cellular disarray and vacuolated cytoplasm. (Hematoxylin-eosin stain; magnification X400.)
surface. Alternatively, the inclusions may result from involution of performed surface microvilli. This hypothesis is supported by the observations that most microvillous inclusions are situated in the apical portion of the cytoplasm and that some superficial inclusions appear to be in continuity with the surface membrane.“.’ Regardless of pathogenesis, the normal appearance of mid-crypt and basal enterocytes suggests that the formation of microvillous inclusions and the depletion of surface microvilli are related to the process of differentiation and migration of enterocytes toward the small bowel lumen. Our light and electron microscopic analyses revealed typical features of MID in the three patients.
Carcinoembryonic antigen immunostaining showed a distinctive pattern resembling that seen with PAS and alkaline phosphatase staining.“*” Carcinoembryonic antigen is a 180,000-d glycoprotein of heterogeneous composition. First considered to be a specific marker for colon carcinoma, CEA was later found to be present in other epithelial tumors” as well as in normal small intestinal mucosa. I ’ Carcinoembryonic antigen has 10 to 15 epitopes, some of which are shared by one or more CEA-related proteins, such as nonspecific cross-reacting antigen and biliary glycoprotein.” To increase the specificity of CEA as a tumor marker, a number of monoclonal antibodies with restrictive reactivity have been devel-
FIGURE 3. Abnormal accumulation of PAS-positive material in surface cells (arrowheads). Crypt cells (arrows) and goblet cells (G) stain normally. (Small intestine. Periodic acid-Schiff stain; magnification X200.)
1234
MICROVILLOUS
INCLUSION
DISEASE (Groisman
et al)
FIGURE 4. Electron micrograph of surface enterocyte showing wellformed intracytoplasmic microvillous inclusions surrounded by several vesicular bodies (arrows>. Surface brush-border microvilli are severely diminished in an adjacent cell (upper left corner). (Patient no. 1. Magnification ~8,300.)
oped. Using nine different monoclonal CEA antibodies, Jothy et al I3 found diverse staining patterns in various normal and neoplastic tissues, including positive and negative reactivity of normal intestinal mucosa. We used a polyclonal CEA antiserum that also can react with CEA-related molecules. It is possible that the use of monoclonal CEA antisera would have produced different results. However, in a previous study normal small intestinal mucosa stained with nonspecific crossreacting antigen-free CEA antiserum revealed a staining pattern identical to that of our normal control cases, namely, positive staining over the surfaces of the villi,
FIGURE 5. Electron micrograph of crypt cells showing increased numbers of secretory granules and intact microvilli. (Patient no. 2. Magnification x10.000.)
1235
within goblet cells, and along the luminal borders of the crypts. I1 B y immunoelectron microscopy CEA was found within mucin granules of goblet cells and in the glycocalyx over the microvilli.“~14 The intracytoplasmic positivity for polyclonal CEA in MID is explained by the presence of CEA and/or CEA-related substances in the glycocalyx sequestered within the microvillous inclusions. Crypt enterocytes have linear brush-border CEA reactivity since the brushborder is intact and no intracytoplasmic microvillous inclusions are present in these cells.‘.‘,“.” The availability of multivisceral transplantation as a curative treatment makes the prompt and precise di-
FIGURE 6. lmmunostaining with polyclonal antiserum against CEA in the small bowel with MID shows intracytoplasmic reactivity in surface enterocytes (arrowheads). Crypt cells show linear brush-border positivity (arrows>. (Immunoperoxidase stain; magnification X 100.) (Inset) Higher-power view of intracytoplasmic CEA reactivity. (Immunoperoxidase stain; magnification X400.)
FIGURE 7. lmmunostaining with polyclonal antiserum against CEA in control cases. Brush-border staining with no intracytoplasmic reactivity (except in goblet cells) is seen in normal small bowel mucosa (top) and autoimmune enteropathy (bottom). (Immunoperoxidase stain. Top, magnification x100. Bottom. original magnification x400.)
MICROVILLOUS
INCLUSION
DISEASE (Groisman
agnosis of MID by the pathologist imperative. The demonstration of a distinct staining pattern for polyclonal CEA in routinely processed small bowel biopsy specimens provides a new, useful criterion that complements other established techniques for accurate diagnosis of MID. Acknowledgment. The authors thank Norman Katz and Sharon Smith for photographic assistance and Marva Barbee
for preparing
this manuscript.
REFERENCES 1. Cutz E, Rhoads JM, Drumm B, et al: Microvillous inclusion disease: An inherited defect of brush-border assembly and differentiation. N Engl J Med 320:646-651, 1989 2. Phillips AD, Schmitz J: Familial microvillous atrophy: A clinicopathological survey of 23 cases. J Pediatr Gastroenterol Nutr 14: 380-396, 1992 3. Lake BD: Microvillous inclusion disease: Specific diagnostic features shown by alkaline phosphatase histochemistry. J Clin Path01 41:880-882, 1988 4. Phillips AD, Jenkins P, Raafat F, et al: Congenital microvillous atrophy: Specific diagnostic features. Arch Dis Child 60: 135-l 40, 1985
1237
et al)
5. Bell SW, Kerner JA, Sibley RK: Microvillous inclusion disease. The importance of electron microscopy for diagnosis. Am J Surg Path01 15:1157-1164, 1991 6. Schofield DE, Agostini RM, Yunis EJ: Gastrointestinal microvillous disease. Am J Clin Path01 98:119-124, 1992 7. Rhoads JM, Vogler RC, Lacey SR, et al: Microvillous inclusion disease. In vitro jejunal electrolyte transport. Gastroenterology 100: 811-817, 1991 8. Cutz E, Thorner P, Sherman P: Microvillous inclusion disease (MID)-Generalized defect of brush-border membranes. Lab Invest 58:3P, 1988 (abstr) 9. Carruthers L, Phillips AD, Dourmashkin R, et al: Biochemical abnormality in brush border membrane protein of a patient with congenital microvillous atrophy. J Pediatr Gastroenterol Nutr 4:902-907, 1985 10. Taylor CR: Immunomicroscopy: A Diagnostic Tool for the Surgical Pathologist. Philadelphia, PA, Saunders, 1986 11. Isaacson P, Judds MA: lmmunohistochemistry of carcinoembryogenic antigen in the small intestine. Cancer 42:1554-1559, 1978 12. Rogers GT: Carcinoembryonic antigens and related glycoproteins: Molecular aspects and specificity. Biochim Biophys Acta 695: 285-290, 1984 13. Jothy S, Brazinsky SA, Chin-A-Loy M, et al: Characterization of monoclonal antibodies to carcinoembryonic antigen with increased tumor specificity. Lab Invest 54:108-l 17, 1986 14. Ahnen DJ. Nakane PK, Brown WR: Ultrastructural localization of carcinoembryonic antigen in normal intestine and colon cancer. Abnormal distribution of CEA on the surfaces of colon cancer cells, Cancer 49:2077-2090, 1982