Background Biliary atresia (BA) is a neonatal cholestatic disease of unknown etiology. It is the leading cause of liver transplantation in children. Many similarities exist between BA and graft versus host disease suggesting engraftment of maternal cells during gestation could result in immune responses that lead to BA. The aim of this study was to determine the presence and extent of maternal microchimerism (MM) in the livers of infants with BA.
Background
Biliary atresia (BA) is a neonatal cholestatic disease of unknown etiology. It is the leading cause of liver transplantation in children. Many similarities exist between BA and graft versus host disease suggesting engraftment of maternal cells during gestation could result in immune responses that lead to BA. The aim of this study was to determine the presence and extent of maternal microchimerism (MM) in the livers of infants with BA.Methods Using fluorescent in situ hybridization (FISH), 11 male BA & 4 male neonatal hepatitis (NH) livers, which served as controls, were analyzed for X and Y-chromosomes. To further investigate MM in BA, 3 patients with BA, and their mothers, were HLA typed. Using immunohistochemical stains, the BA livers were examined for MM. Four additional BA livers underwent analysis by polymerase chain reaction (PCR) for evidence of MM.
Methods
Using fluorescent in situ hybridization (FISH), 11 male BA & 4 male neonatal hepatitis (NH) livers, which served as controls, were analyzed for X and Y-chromosomes. To further investigate MM in BA, 3 patients with BA, and their mothers, were HLA typed. Using immunohistochemical stains, the BA livers were examined for MM. Four additional BA livers underwent analysis by polymerase chain reaction (PCR) for evidence of MM.Results By FISH, 8 BA and 2 NH livers were interpretable. Seven of eight BA specimens showed evidence of MM. The number of maternal cells ranged from 2–4 maternal cells per biopsy slide. Neither NH specimen showed evidence of MM. In addition, immunohistochemical stains confirmed evidence of MM. Using PCR, a range of 1–142 copies of maternal DNA per 25,000 copies of patients DNA was found.
Results
By FISH, 8 BA and 2 NH livers were interpretable. Seven of eight BA specimens showed evidence of MM. The number of maternal cells ranged from 2–4 maternal cells per biopsy slide. Neither NH specimen showed evidence of MM. In addition, immunohistochemical stains confirmed evidence of MM. Using PCR, a range of 1–142 copies of maternal DNA per 25,000 copies of patients DNA was found.Conclusions Maternal microchimerism is present in the livers of patients with BA and may contribute to the pathogenesis of BA.
Conclusions
Maternal microchimerism is present in the livers of patients with BA and may contribute to the pathogenesis of BA.

Background Biliary atresia (BA) is a cholestatic disease of infancy characterized by the destruction of the biliary tree. [ 1 - 3 ] Both the intra-and extra-hepatic biliary ducts demonstrate evidence of a progressive destruction. This results in cholestasis, hepatic fibrosis and eventually cirrhosis. BA is associated with significant morbidity and mortality. Although the incidence of BA is 1 in 10,000 to 14,000 live births, [ 4 , 5 ] it accounts for over 40% of the neonatal cholestatic liver disease in Europe and the United States. [ 6 ] Prior to the hepatic portoenterostomy and liver transplantation, infants with BA had less than a 10% survival at 3 years of life and almost 100% mortality at 7 years of life. [ 7 ] In the United States, it is the leading cause of pediatric liver transplantation. [ 8 ] The etiology of biliary atresia remains unknown. Many hypotheses on the etiology of BA exist. Two leading hypothesis are that BA occurs as a result of ductal plate malformations occurring during development of the liver, or as a result of an immune mediated process triggered by yet to be determined stimulus. [ 9 ] As more knowledge accrues about the histology and immunologic characteristics of BA, the more it appears to be a progressive immune mediated process. Graft versus host disease (GVHD), seen after allogeneic hematopoietic stem cell transplantation, is an immune mediated process triggered by the transfer of alloimmune cells from a donor to a host. GVHD shares many similarities with BA. Both BA and GVHD are characterized by a lymphocytic infiltration around the portal triad and damage to the biliary ducts. [ 10 ] The predominant lymphocytes in both disease are CD4 +  T – helper (Th) 1 cells. [ 11 - 14 ] As with BA, in GVHD there is also an increase in cell adhesion molecules and human leukocyte antigen (HLA) class II markers. [ 11 ] Given the similarities between BA and GVHD, we hypothesized that a contributing etiology of BA could be an alloimmune reaction such as in GVHD triggered by maternal microchimerism. Maternal microchimerism occurs when a small number of maternal cells are transferred to the offspring during pregnancy. This is known to occur in up to 40% of normal pregnancies. [ 15 , 16 ] In addition, a clinical precedent of maternal microchimerism causing hepatic GVHD in children with severe combined immune deficiency (SCID) exists. [ 17 ] The aim of this study was to determine the presence and extent of maternal microchimerism in the livers of infants with BA.
Background
Biliary atresia (BA) is a cholestatic disease of infancy characterized by the destruction of the biliary tree. [ 1 - 3 ] Both the intra-and extra-hepatic biliary ducts demonstrate evidence of a progressive destruction. This results in cholestasis, hepatic fibrosis and eventually cirrhosis. BA is associated with significant morbidity and mortality. Although the incidence of BA is 1 in 10,000 to 14,000 live births, [ 4 , 5 ] it accounts for over 40% of the neonatal cholestatic liver disease in Europe and the United States. [ 6 ] Prior to the hepatic portoenterostomy and liver transplantation, infants with BA had less than a 10% survival at 3 years of life and almost 100% mortality at 7 years of life. [ 7 ] In the United States, it is the leading cause of pediatric liver transplantation. [ 8 ] The etiology of biliary atresia remains unknown.Many hypotheses on the etiology of BA exist. Two leading hypothesis are that BA occurs as a result of ductal plate malformations occurring during development of the liver, or as a result of an immune mediated process triggered by yet to be determined stimulus. [ 9 ] As more knowledge accrues about the histology and immunologic characteristics of BA, the more it appears to be a progressive immune mediated process.Graft versus host disease (GVHD), seen after allogeneic hematopoietic stem cell transplantation, is an immune mediated process triggered by the transfer of alloimmune cells from a donor to a host. GVHD shares many similarities with BA. Both BA and GVHD are characterized by a lymphocytic infiltration around the portal triad and damage to the biliary ducts. [ 10 ] The predominant lymphocytes in both disease are CD4 +  T – helper (Th) 1 cells. [ 11 - 14 ] As with BA, in GVHD there is also an increase in cell adhesion molecules and human leukocyte antigen (HLA) class II markers. [ 11 ]Given the similarities between BA and GVHD, we hypothesized that a contributing etiology of BA could be an alloimmune reaction such as in GVHD triggered by maternal microchimerism. Maternal microchimerism occurs when a small number of maternal cells are transferred to the offspring during pregnancy. This is known to occur in up to 40% of normal pregnancies. [ 15 , 16 ] In addition, a clinical precedent of maternal microchimerism causing hepatic GVHD in children with severe combined immune deficiency (SCID) exists. [ 17 ] The aim of this study was to determine the presence and extent of maternal microchimerism in the livers of infants with BA.Methods This study was performed with the approval of the Committee of Human Research at USCF (H9048-20247-01). Cases of BA were identified through a data search in the Anatomic Pathology CoPath system by diagnosis or during patient medical visits to the UCSF Division of Pediatric Gastroenterology, Hepatology, and Nutrition. An explanation of the study was given to each study candidate. Written consent was obtained from each study participant. Fluorescent in situ hybridization (FISH) of male BA livers We modified a previously reported method of FISH, [ 18 ] using X and Y-chromosomes probes. Y-chromosomes were stained with a green fluorescent dye, fluorescein isothiocyanate (FITC). X chromosomes were stained with a red fluorescent dye, cyanine 3 (Cy-3). Nuclear material was stained with a blue fluorescent dye 4", 6"-diamidino-2-phenylindole (DAPI). After initial digestion and staining of the liver specimens, we examined the slides for evidence of female cells, depicted by two red signals, i.e. X chromosomes, with no green signal within the blue nuclear material. Slides were analyzed in a blinded fashion. HLA typing Patients with BA and their mothers were HLA typed. The HLA typing HLA-A, -B, and -DRB1 alleles of the child and mother were determined by sequence specific PCR (SSP) (Pel-Freez Clinical Systems, LLC ® , Brown Deer, WI, USA). Immunohistochemistry Frozen tissues sections of BA patient's livers (5 μm) were fixed in acetone for 10 minutes at 4°C then washed in PBS (5 minutes centrifugation × 3). Sections were blocked with Protein Block (Dako, Carpinteria, CA, USA) for thirty minutes at room temperature. Sections were then incubated with anti-HLA-B14 mouse monoclonal antibody (US Biological, Swampscott, Mass. USA) then washed in PBS. Sections were incubated in goat anti-mouse conjugated with FITC for 30 minutes at room temperature. Sections were rewashed and then counterstained with DAPI. Slides were then examined using a fluorescent microscope. Kinetic Polymerase Chain Reaction (kPCR) Using the HLA types of the patients with BA and their mothers, and a previously reported method of kPCR, evidence of maternal DNA was explored within the liver biopsy specimens of explanted BA livers. [ 18 ] The minor modifications to the kinetic PCR protocol included use of iCyler from BioRad (Hercules, Ca, USA) for amplification.
Methods
This study was performed with the approval of the Committee of Human Research at USCF (H9048-20247-01). Cases of BA were identified through a data search in the Anatomic Pathology CoPath system by diagnosis or during patient medical visits to the UCSF Division of Pediatric Gastroenterology, Hepatology, and Nutrition. An explanation of the study was given to each study candidate. Written consent was obtained from each study participant.Fluorescent in situ hybridization (FISH) of male BA livers We modified a previously reported method of FISH, [ 18 ] using X and Y-chromosomes probes. Y-chromosomes were stained with a green fluorescent dye, fluorescein isothiocyanate (FITC). X chromosomes were stained with a red fluorescent dye, cyanine 3 (Cy-3). Nuclear material was stained with a blue fluorescent dye 4", 6"-diamidino-2-phenylindole (DAPI). After initial digestion and staining of the liver specimens, we examined the slides for evidence of female cells, depicted by two red signals, i.e. X chromosomes, with no green signal within the blue nuclear material. Slides were analyzed in a blinded fashion.
Fluorescent in situ hybridization (FISH) of male BA livers
We modified a previously reported method of FISH, [ 18 ] using X and Y-chromosomes probes. Y-chromosomes were stained with a green fluorescent dye, fluorescein isothiocyanate (FITC). X chromosomes were stained with a red fluorescent dye, cyanine 3 (Cy-3). Nuclear material was stained with a blue fluorescent dye 4", 6"-diamidino-2-phenylindole (DAPI). After initial digestion and staining of the liver specimens, we examined the slides for evidence of female cells, depicted by two red signals, i.e. X chromosomes, with no green signal within the blue nuclear material. Slides were analyzed in a blinded fashion.HLA typing Patients with BA and their mothers were HLA typed. The HLA typing HLA-A, -B, and -DRB1 alleles of the child and mother were determined by sequence specific PCR (SSP) (Pel-Freez Clinical Systems, LLC ® , Brown Deer, WI, USA).
HLA typing
Patients with BA and their mothers were HLA typed. The HLA typing HLA-A, -B, and -DRB1 alleles of the child and mother were determined by sequence specific PCR (SSP) (Pel-Freez Clinical Systems, LLC ® , Brown Deer, WI, USA).Immunohistochemistry Frozen tissues sections of BA patient's livers (5 μm) were fixed in acetone for 10 minutes at 4°C then washed in PBS (5 minutes centrifugation × 3). Sections were blocked with Protein Block (Dako, Carpinteria, CA, USA) for thirty minutes at room temperature. Sections were then incubated with anti-HLA-B14 mouse monoclonal antibody (US Biological, Swampscott, Mass. USA) then washed in PBS. Sections were incubated in goat anti-mouse conjugated with FITC for 30 minutes at room temperature. Sections were rewashed and then counterstained with DAPI. Slides were then examined using a fluorescent microscope.
Immunohistochemistry
Frozen tissues sections of BA patient's livers (5 μm) were fixed in acetone for 10 minutes at 4°C then washed in PBS (5 minutes centrifugation × 3). Sections were blocked with Protein Block (Dako, Carpinteria, CA, USA) for thirty minutes at room temperature. Sections were then incubated with anti-HLA-B14 mouse monoclonal antibody (US Biological, Swampscott, Mass. USA) then washed in PBS. Sections were incubated in goat anti-mouse conjugated with FITC for 30 minutes at room temperature. Sections were rewashed and then counterstained with DAPI. Slides were then examined using a fluorescent microscope.Kinetic Polymerase Chain Reaction (kPCR) Using the HLA types of the patients with BA and their mothers, and a previously reported method of kPCR, evidence of maternal DNA was explored within the liver biopsy specimens of explanted BA livers. [ 18 ] The minor modifications to the kinetic PCR protocol included use of iCyler from BioRad (Hercules, Ca, USA) for amplification.
Kinetic Polymerase Chain Reaction (kPCR)
Using the HLA types of the patients with BA and their mothers, and a previously reported method of kPCR, evidence of maternal DNA was explored within the liver biopsy specimens of explanted BA livers. [ 18 ] The minor modifications to the kinetic PCR protocol included use of iCyler from BioRad (Hercules, Ca, USA) for amplification.Results Detection of female cells in male BA liver biopsies Using FISH, we examined 11 male BA liver biopsies and 4 male NH liver biopsies. After initial digestion and staining of the liver specimens, we were able to interpret 8 of the BA and 2 NH liver biopsy specimens (Table  1 ). Three BA and 2 NH biopsies underwent poor digestion and had no discernable signal. Figure  1  shows a liver from a male infant with BA. A male cell, depicted by a red signal, the X chromosome, and green signal, a Y chromosome within the blue nuclear material, clearly can be seen (A). In the same specimen, one can also see a female cell, depicted by two red signals, both X-chromosomes with no green signal within the blue nuclear material (B). 7/8 BA specimens had evidence of maternal microchimerism. The number of maternal cells, per biopsy slide, was 2 – 4 cells. Neither of the NH specimens had evidence of maternal microchimerism. Table 1 Overview of maternal microchimerism in patients with BA and NH Diagnosis Chimerism Method # of Maternal cells/DNA per slide (FISH) or per 25,000 copies of patient's DNA (kPCR) BA Present FISH 2 maternal cells per slide BA Present FISH 2 maternal cells per slide BA Present FISH 4 maternal cells per slide BA Present FISH 2 maternal cells per slide BA Present FISH+immunokistochemistry 3 maternal cells per slide BA Not interpretable FISH --- BA Present FISH 2 maternal cells per slide BA Present FISH 3 maternal cells per slide BA Not interpretable FISH --- BA Not interpretable FISH --- BA None detected FISH 0 maternal cells per slide BA Present kPCR 3 copies of maternal DNA per 25,000 copies of patient's DNA BA Present kPCR 1 copy of maternal DNA per 25,000 copies of patient's DNA BA Present kPCR 10 copies of maternal DNA per 25,000 copies of patient's DNA BA Present kPCR 142 copies of maternal DNA per 25,000 copies of patient's DNA NH None detected FISH 0 maternal cells per slide NH None detected FISH 0 maternal cells per slide NH Not interpretable FISH --- NH Not interpretable FISH --- Figure 1 A male cell, depicted by a red signal, the X chromosome, and green signal, a Y chromosome within the blue nuclear material, can be seen in both male BA liver specimens (A&B). In the same specimen, one can also see a female cell, depicted by two red signals, both X-chromosomes with no green signal within the blue nuclear material (arrow). Detection of maternal HLA antigen among BA liver samples To further investigate our findings, three patients with BA and their mothers were HLA typed. Using the HLA marker B14, we examined the BA livers for evidence of maternal microchimerism using immunohistochemistry techniques. An HLA-B14 positive patient served as our positive control (Figure  2 ). Specimen B, our negative control, was a patient who was, along with his mother, negative for HLA-B14. Specimen C, our test specimen, was HLA-B14 - , with an HLA-B14 +  mother. Specimen A (positive control) has a bright signal for HLA-B14 while there is only background staining for HLA-B14 in specimen B (negative control). Specimen C, the test specimen, has isolated scattered bright signals suggesting maternal microchimerism. Figure 2 Using the HLA marker B14, we examined the BA livers for evidence of maternal microchimerism using immunohistochemistry techniques. An HLA-B14 positive patient served as our positive control (A). Specimen B, our negative control, was a patient who was, along with his mother, negative for HLA-B14. Specimen C, our test specimen, was HLA-B14 negative, with an HLA-B14+ mother. Specimen A (positive control) has a bright signal for HLA-B14 while there is only background staining for HLA-B14 in specimen B (negative control). Specimen C, the test specimen, has isolated scattered bright signals suggesting maternal microchimerism. Detection of maternal DNA by kPCR Finally, using polymerase chain reaction, in conjunction with the HLA typing of patients and mothers, we were able to affirm that maternal microchimerism occurs in the BA livers. An additional four fresh frozen liver specimens of BA infants, obtained at the time of transplantation, were examined for maternal DNA. All the livers had evidence of maternal microchimerism, with the range of 1 to 142 copies of maternal DNA detected per 25000 copies of patients' DNA. Figure  3  is a representative kPCR for maternal HLA-B40 in the second liver sample. Figure 3 Representative kinetic PCR (kPCR) data for maternal HLA-B40 in BA liver sample. SYBR Green fluorescence is plotted on the y-axis as a function of amplification cycle number on the x-axis. The upper panels depicts results for standard dilutions of HLA-B40 [left to right curves: 25000, 10000, 1000, 100,10, and 1 genomic equivalence (gEq)]. A single copy of template produces fluorescent signal after approximately 36 cycles of amplification. The lower panel shows results from three liver replicates and two negative (no-template) controls. The three liver replicates have a positive signals at 2–5 gEq, while the negative controls remained negative through 50 cycles. In all eleven of twelve patients with BA had evidence of maternal microchimerism by FISH or kinetic PCR, while neither of the two neonatal hepatitis patients had evidence of maternal microchimerism (p = 0.03).
Results
Detection of female cells in male BA liver biopsies Using FISH, we examined 11 male BA liver biopsies and 4 male NH liver biopsies. After initial digestion and staining of the liver specimens, we were able to interpret 8 of the BA and 2 NH liver biopsy specimens (Table  1 ). Three BA and 2 NH biopsies underwent poor digestion and had no discernable signal. Figure  1  shows a liver from a male infant with BA. A male cell, depicted by a red signal, the X chromosome, and green signal, a Y chromosome within the blue nuclear material, clearly can be seen (A). In the same specimen, one can also see a female cell, depicted by two red signals, both X-chromosomes with no green signal within the blue nuclear material (B). 7/8 BA specimens had evidence of maternal microchimerism. The number of maternal cells, per biopsy slide, was 2 – 4 cells. Neither of the NH specimens had evidence of maternal microchimerism. Table 1 Overview of maternal microchimerism in patients with BA and NH Diagnosis Chimerism Method # of Maternal cells/DNA per slide (FISH) or per 25,000 copies of patient's DNA (kPCR) BA Present FISH 2 maternal cells per slide BA Present FISH 2 maternal cells per slide BA Present FISH 4 maternal cells per slide BA Present FISH 2 maternal cells per slide BA Present FISH+immunokistochemistry 3 maternal cells per slide BA Not interpretable FISH --- BA Present FISH 2 maternal cells per slide BA Present FISH 3 maternal cells per slide BA Not interpretable FISH --- BA Not interpretable FISH --- BA None detected FISH 0 maternal cells per slide BA Present kPCR 3 copies of maternal DNA per 25,000 copies of patient's DNA BA Present kPCR 1 copy of maternal DNA per 25,000 copies of patient's DNA BA Present kPCR 10 copies of maternal DNA per 25,000 copies of patient's DNA BA Present kPCR 142 copies of maternal DNA per 25,000 copies of patient's DNA NH None detected FISH 0 maternal cells per slide NH None detected FISH 0 maternal cells per slide NH Not interpretable FISH --- NH Not interpretable FISH --- Figure 1 A male cell, depicted by a red signal, the X chromosome, and green signal, a Y chromosome within the blue nuclear material, can be seen in both male BA liver specimens (A&B). In the same specimen, one can also see a female cell, depicted by two red signals, both X-chromosomes with no green signal within the blue nuclear material (arrow).
Detection of female cells in male BA liver biopsies
Using FISH, we examined 11 male BA liver biopsies and 4 male NH liver biopsies. After initial digestion and staining of the liver specimens, we were able to interpret 8 of the BA and 2 NH liver biopsy specimens (Table  1 ). Three BA and 2 NH biopsies underwent poor digestion and had no discernable signal. Figure  1  shows a liver from a male infant with BA. A male cell, depicted by a red signal, the X chromosome, and green signal, a Y chromosome within the blue nuclear material, clearly can be seen (A). In the same specimen, one can also see a female cell, depicted by two red signals, both X-chromosomes with no green signal within the blue nuclear material (B). 7/8 BA specimens had evidence of maternal microchimerism. The number of maternal cells, per biopsy slide, was 2 – 4 cells. Neither of the NH specimens had evidence of maternal microchimerism.Overview of maternal microchimerism in patients with BA and NHDetection of maternal HLA antigen among BA liver samples To further investigate our findings, three patients with BA and their mothers were HLA typed. Using the HLA marker B14, we examined the BA livers for evidence of maternal microchimerism using immunohistochemistry techniques. An HLA-B14 positive patient served as our positive control (Figure  2 ). Specimen B, our negative control, was a patient who was, along with his mother, negative for HLA-B14. Specimen C, our test specimen, was HLA-B14 - , with an HLA-B14 +  mother. Specimen A (positive control) has a bright signal for HLA-B14 while there is only background staining for HLA-B14 in specimen B (negative control). Specimen C, the test specimen, has isolated scattered bright signals suggesting maternal microchimerism. Figure 2 Using the HLA marker B14, we examined the BA livers for evidence of maternal microchimerism using immunohistochemistry techniques. An HLA-B14 positive patient served as our positive control (A). Specimen B, our negative control, was a patient who was, along with his mother, negative for HLA-B14. Specimen C, our test specimen, was HLA-B14 negative, with an HLA-B14+ mother. Specimen A (positive control) has a bright signal for HLA-B14 while there is only background staining for HLA-B14 in specimen B (negative control). Specimen C, the test specimen, has isolated scattered bright signals suggesting maternal microchimerism.
Detection of maternal HLA antigen among BA liver samples
To further investigate our findings, three patients with BA and their mothers were HLA typed. Using the HLA marker B14, we examined the BA livers for evidence of maternal microchimerism using immunohistochemistry techniques. An HLA-B14 positive patient served as our positive control (Figure  2 ). Specimen B, our negative control, was a patient who was, along with his mother, negative for HLA-B14. Specimen C, our test specimen, was HLA-B14 - , with an HLA-B14 +  mother. Specimen A (positive control) has a bright signal for HLA-B14 while there is only background staining for HLA-B14 in specimen B (negative control). Specimen C, the test specimen, has isolated scattered bright signals suggesting maternal microchimerism.Detection of maternal DNA by kPCR Finally, using polymerase chain reaction, in conjunction with the HLA typing of patients and mothers, we were able to affirm that maternal microchimerism occurs in the BA livers. An additional four fresh frozen liver specimens of BA infants, obtained at the time of transplantation, were examined for maternal DNA. All the livers had evidence of maternal microchimerism, with the range of 1 to 142 copies of maternal DNA detected per 25000 copies of patients' DNA. Figure  3  is a representative kPCR for maternal HLA-B40 in the second liver sample. Figure 3 Representative kinetic PCR (kPCR) data for maternal HLA-B40 in BA liver sample. SYBR Green fluorescence is plotted on the y-axis as a function of amplification cycle number on the x-axis. The upper panels depicts results for standard dilutions of HLA-B40 [left to right curves: 25000, 10000, 1000, 100,10, and 1 genomic equivalence (gEq)]. A single copy of template produces fluorescent signal after approximately 36 cycles of amplification. The lower panel shows results from three liver replicates and two negative (no-template) controls. The three liver replicates have a positive signals at 2–5 gEq, while the negative controls remained negative through 50 cycles. In all eleven of twelve patients with BA had evidence of maternal microchimerism by FISH or kinetic PCR, while neither of the two neonatal hepatitis patients had evidence of maternal microchimerism (p = 0.03).
Detection of maternal DNA by kPCR
Finally, using polymerase chain reaction, in conjunction with the HLA typing of patients and mothers, we were able to affirm that maternal microchimerism occurs in the BA livers. An additional four fresh frozen liver specimens of BA infants, obtained at the time of transplantation, were examined for maternal DNA. All the livers had evidence of maternal microchimerism, with the range of 1 to 142 copies of maternal DNA detected per 25000 copies of patients' DNA. Figure  3  is a representative kPCR for maternal HLA-B40 in the second liver sample.In all eleven of twelve patients with BA had evidence of maternal microchimerism by FISH or kinetic PCR, while neither of the two neonatal hepatitis patients had evidence of maternal microchimerism (p = 0.03).Discussion BA is a progressive cholestatic disease of infancy, which is characterized by a mild to moderate lymphocytic infiltration within the extra and intrahepatic bile ducts. It is plausible, that maternal microchimerism may play a role in the etio-pathogenesis of BA by causing an alloimmune reaction given the similarities between BA and GVHD. We have shown that maternal microchimerism is present within the livers of patients with BA. Maternal microchimerism occurs when maternal cells reside in the body of offspring. Maternal-fetal lymphocytic transfer is known to occur during pregnancy starting as early as the tenth week of gestation and continuing up to delivery. [ 19 ] The number of cells that traverse the placenta increases throughout this period. Schroder et al [ 15 ] demonstrated that approximately 1/10 infants has about 0.07% maternal lymphocytes at the time of birth and more recent findings by Lo et al. [ 16 ] have indicated the presence of maternal cells in over 40% of fetal blood samples. Disease has been shown to occur in mothers who have had fetal microchimerism, and children/adults who have had maternal microchimerism. Children who have immunodeficiencies are at greater risk for graft verses host disease owing to engraftment of maternal lymphocytes. In both SCID and DiGeorge syndrome, maternal microchimerism and GVHD have been well described. In a study by Susanne Müller et al., 121 patients with SCID were evaluated for maternal microchimerism using HLA typing. Maternal cells were found in 48 patients, with 19 patients showing signs of GVHD. GVHD manifested itself in the skin and in the liver. [ 20 ] There have been numerous case reports showing GVHD in infancy of patients with DiGeorge. Manifestations of this disease include skin, gastrointestinal tract and liver involvement. [ 21 ] The histologic and immunologic similarities of BA and GVHD are striking. The site of damage in BA and GVHD after bone marrow transplantation is the same. In each, lymphocytes congregate around the bile ducts. The damage occurs to both the intra-and extrahepatic biliary tracts. In a mouse model of acute GVHD, Nonomura et al. showed that transfer of allogeneic cells set along minor HLA mismatch can cause damage to both the intra and extrahepatic biliary ducts. Interestingly, the timeframe in this mouse model of acute GVHD, in terms of lymphocytic infiltration and fibrosis, appear similar to that of BA. [ 14 ] Initially, a peak of lymphocytes around the bile ducts occurs about 2 weeks after transfer of allogeneic cells from donor to host mouse; as the lymphocytic infiltration subsides, liver fibrosis increases. This correlates with disease progression in BA, where initial diagnostic biopsies of BA livers usually show a larger lymphocytic infiltration, and less fibrosis, as compared to biopsies done later, i.e. at the time of liver transplantation. [ 22 ] The lymphocytic infiltrates in both BA and GVHD are predominantly CD4 +  T lymphocytes. The genes in BA livers showed differential lymphocytic function, with activation of osteopontin, a regulator of cell-mediated immunity in Th 1 cells, and interferon gamma. [ 23 ] Similarly, in GVHD, the predominant leukocyte is the CD4 +  T lymphocyte. Although a variety of complex mechanisms are involved in causing the end organ damage in GVHD, evidence exists showing similarities in effector mechanisms. In a SCID mouse model of acute GVHD, an increase in interferon-gamma secretion with a synchronized increase in activated Th cells was seen during acute GVHD. [ 24 ] The inflammatory responses in both BA and GVHD are also associated with increased expression of adhesion molecules such as CD54, and increased class II HLA markers, such as HLA-DR. HLA-DR and CD54, in both BA and GVHD, are seen predominantly around the bile ducts. [ 11 , 25 , 26 ] Maternal microchimerism is not a rare occurrence in healthy individuals, but is usually not associated with disease. The high occurrence of maternal microchimerism in healthy individuals has been suggested to have a tolergenic effect that may contribute to long-term microchimerism. [ 27 ]Additionally, in utero transplantation of haploidentical cells does not always lead to immune tolerance [28] and may lead to immune sensitization. Thus, the immunological consequences of the migration of maternal cells to the fetus appear to be variable. In most cases the maternal cells are likely to be cleared by the host's immune system or they may escape destruction by the immune system leading to engraftment. Engraftment of maternal immune cells in the fetal biliary tract may result in an immune reaction against the host cholangiocytes. This is what we believe may occur in infants with BA. Alternatively, engrafted maternal cells in the fetus may subsequently be rejected by the immune system of the offspring, leading to destruction of the biliary tree. Maternal microchimerism occurs in the livers of patients with BA. Given the previously well-described relationship between microchimerism and GVHD, and the similarities between BA and GVHD, these findings indicate a potential etio-pathogenisis for BA. Further investigations into the role of maternal microchimerism in BA are warranted.
Discussion
BA is a progressive cholestatic disease of infancy, which is characterized by a mild to moderate lymphocytic infiltration within the extra and intrahepatic bile ducts. It is plausible, that maternal microchimerism may play a role in the etio-pathogenesis of BA by causing an alloimmune reaction given the similarities between BA and GVHD. We have shown that maternal microchimerism is present within the livers of patients with BA.Maternal microchimerism occurs when maternal cells reside in the body of offspring. Maternal-fetal lymphocytic transfer is known to occur during pregnancy starting as early as the tenth week of gestation and continuing up to delivery. [ 19 ] The number of cells that traverse the placenta increases throughout this period. Schroder et al [ 15 ] demonstrated that approximately 1/10 infants has about 0.07% maternal lymphocytes at the time of birth and more recent findings by Lo et al. [ 16 ] have indicated the presence of maternal cells in over 40% of fetal blood samples.Disease has been shown to occur in mothers who have had fetal microchimerism, and children/adults who have had maternal microchimerism. Children who have immunodeficiencies are at greater risk for graft verses host disease owing to engraftment of maternal lymphocytes. In both SCID and DiGeorge syndrome, maternal microchimerism and GVHD have been well described. In a study by Susanne Müller et al., 121 patients with SCID were evaluated for maternal microchimerism using HLA typing. Maternal cells were found in 48 patients, with 19 patients showing signs of GVHD. GVHD manifested itself in the skin and in the liver. [ 20 ] There have been numerous case reports showing GVHD in infancy of patients with DiGeorge. Manifestations of this disease include skin, gastrointestinal tract and liver involvement. [ 21 ]The histologic and immunologic similarities of BA and GVHD are striking. The site of damage in BA and GVHD after bone marrow transplantation is the same. In each, lymphocytes congregate around the bile ducts. The damage occurs to both the intra-and extrahepatic biliary tracts. In a mouse model of acute GVHD, Nonomura et al. showed that transfer of allogeneic cells set along minor HLA mismatch can cause damage to both the intra and extrahepatic biliary ducts. Interestingly, the timeframe in this mouse model of acute GVHD, in terms of lymphocytic infiltration and fibrosis, appear similar to that of BA. [ 14 ] Initially, a peak of lymphocytes around the bile ducts occurs about 2 weeks after transfer of allogeneic cells from donor to host mouse; as the lymphocytic infiltration subsides, liver fibrosis increases. This correlates with disease progression in BA, where initial diagnostic biopsies of BA livers usually show a larger lymphocytic infiltration, and less fibrosis, as compared to biopsies done later, i.e. at the time of liver transplantation. [ 22 ]The lymphocytic infiltrates in both BA and GVHD are predominantly CD4 +  T lymphocytes. The genes in BA livers showed differential lymphocytic function, with activation of osteopontin, a regulator of cell-mediated immunity in Th 1 cells, and interferon gamma. [ 23 ] Similarly, in GVHD, the predominant leukocyte is the CD4 +  T lymphocyte. Although a variety of complex mechanisms are involved in causing the end organ damage in GVHD, evidence exists showing similarities in effector mechanisms. In a SCID mouse model of acute GVHD, an increase in interferon-gamma secretion with a synchronized increase in activated Th cells was seen during acute GVHD. [ 24 ] The inflammatory responses in both BA and GVHD are also associated with increased expression of adhesion molecules such as CD54, and increased class II HLA markers, such as HLA-DR. HLA-DR and CD54, in both BA and GVHD, are seen predominantly around the bile ducts. [ 11 , 25 , 26 ]Maternal microchimerism is not a rare occurrence in healthy individuals, but is usually not associated with disease. The high occurrence of maternal microchimerism in healthy individuals has been suggested to have a tolergenic effect that may contribute to long-term microchimerism. [ 27 ]Additionally, in utero transplantation of haploidentical cells does not always lead to immune tolerance [28] and may lead to immune sensitization. Thus, the immunological consequences of the migration of maternal cells to the fetus appear to be variable. In most cases the maternal cells are likely to be cleared by the host's immune system or they may escape destruction by the immune system leading to engraftment. Engraftment of maternal immune cells in the fetal biliary tract may result in an immune reaction against the host cholangiocytes. This is what we believe may occur in infants with BA. Alternatively, engrafted maternal cells in the fetus may subsequently be rejected by the immune system of the offspring, leading to destruction of the biliary tree.Maternal microchimerism occurs in the livers of patients with BA. Given the previously well-described relationship between microchimerism and GVHD, and the similarities between BA and GVHD, these findings indicate a potential etio-pathogenisis for BA. Further investigations into the role of maternal microchimerism in BA are warranted.Competing interests None declared.
Competing interests
None declared.Author's contributions DLS conceived of the hypothesis and contributed to the design and coordination of the study; he also drafted the manuscript. PR and MBH participated in the design of the study and manuscript preparation. DK carried out the kPCR and HLA typing analyses. GM performed the FISH analyses. LBL carried out the kPCR and HLA typing analyses and participated in the study design. MOM participated on the study design and coordination, immunohistochemistry stains and the manuscript preparation.
Author's contributions
DLS conceived of the hypothesis and contributed to the design and coordination of the study; he also drafted the manuscript. PR and MBH participated in the design of the study and manuscript preparation. DK carried out the kPCR and HLA typing analyses. GM performed the FISH analyses. LBL carried out the kPCR and HLA typing analyses and participated in the study design. MOM participated on the study design and coordination, immunohistochemistry stains and the manuscript preparation.Pre-publication history The pre-publication history for this paper can be accessed here:
Pre-publication history
The pre-publication history for this paper can be accessed here:


Background The demonstration of tissue-bound immunoreactants by direct immunofluorescence microscopy (DIF) is a valuable parameter in the diagnosis of various autoimmune and immunecomplex-mediated skin diseases. For preservation of tissue-bound immunoreactants, biopsies are usually fresh-frozen in liquid nitrogen or transported in Michel's fixative. But even optimally preserved tissue specimens are no guarantee for the correct diagnosis by DIF, especially when weak to moderate IgG fluorescence of the epidermal basement membrane zone is involved. In such cases false negative results are easily obtained due to the relatively high dermal "background" fluorescence produced by polyclonal anti-human IgG fluorescein conjugates.
Background
The demonstration of tissue-bound immunoreactants by direct immunofluorescence microscopy (DIF) is a valuable parameter in the diagnosis of various autoimmune and immunecomplex-mediated skin diseases. For preservation of tissue-bound immunoreactants, biopsies are usually fresh-frozen in liquid nitrogen or transported in Michel's fixative. But even optimally preserved tissue specimens are no guarantee for the correct diagnosis by DIF, especially when weak to moderate IgG fluorescence of the epidermal basement membrane zone is involved. In such cases false negative results are easily obtained due to the relatively high dermal "background" fluorescence produced by polyclonal anti-human IgG fluorescein conjugates.Methods In the present study we have compared the use of normal saline (0.9% NaCl) with liquid nitrogen and Michel's fixative as transport medium for skin biopsies. From 25 patients with an autoimmune skin disease (pemphigus, pemphigoid, lupus erythematosus and vasculitis) four matched skin biopsies were obtained and transported in either saline for 24 and 48 hours, liquid nitrogen, or Michel's fixative for 48 hours.
Methods
In the present study we have compared the use of normal saline (0.9% NaCl) with liquid nitrogen and Michel's fixative as transport medium for skin biopsies. From 25 patients with an autoimmune skin disease (pemphigus, pemphigoid, lupus erythematosus and vasculitis) four matched skin biopsies were obtained and transported in either saline for 24 and 48 hours, liquid nitrogen, or Michel's fixative for 48 hours.Results Direct IF microscopy showed significant reduction of background fluorescence (p < 0.01) and relatively enhanced desired specific (IgG, IgA) staining in biopsies transported in saline. A conclusive or tentative IF diagnosis was reached in 92% after 24 h saline, 83% after 48 h saline, 68% after freezing in liquid nitrogen, and 62% after 48 h Michel's medium (n = 25).
Results
Direct IF microscopy showed significant reduction of background fluorescence (p < 0.01) and relatively enhanced desired specific (IgG, IgA) staining in biopsies transported in saline. A conclusive or tentative IF diagnosis was reached in 92% after 24 h saline, 83% after 48 h saline, 68% after freezing in liquid nitrogen, and 62% after 48 h Michel's medium (n = 25).Conclusions We conclude that transporting biopsies without freezing in normal saline for 24 hours is an adequate and attractive method for routine IF diagnosis in autoimmune and immune complex-mediated dermatoses. The superior results with saline incubation are explained by washing away of IgG background in dermis and epidermis.
Conclusions
We conclude that transporting biopsies without freezing in normal saline for 24 hours is an adequate and attractive method for routine IF diagnosis in autoimmune and immune complex-mediated dermatoses. The superior results with saline incubation are explained by washing away of IgG background in dermis and epidermis.

Background The demonstration of tissue-bound immunoreactants by direct immunofluorescence microscopy (DIF) is a valuable parameter in the diagnosis of various autoimmune skin diseases[ 1 ]. Reliable diagnosis by DIF not only requires an experienced observer, but first of all proper skin (or mucosal) biopsies with well-preserved immunoreactants. For the latter purpose biopsies are usually snap-frozen in liquid nitrogen or, alternatively, placed in Michel's fixative that facilitates transport of biopsies from outside hospitals [ 2 - 6 ]. But even representative and optimally preserved tissue specimens are no guarantee for the correct diagnosis by DIF, especially when weak to moderate desired specific staining (DSS) of the epidermal basement membrane zone (BMZ) is involved [ 7 ]. In such cases, specific IgG fluorescence is easily masked by the relatively high dermal "background" fluorescence produced by polyclonal anti-human IgG fluorescein conjugates. The background fluorescence, consisting of both undesired specific staining (USS) and non-specific staining (NSS), largely determines the signal-to noise ratio [ 7 ]. This ratio in turn determines the detection threshold and thereby the diagnostic sensitivity of the DIF technique. A low ratio for IgG resulting from weak DSS and high USS plus NSS will undoubtedly yield false negative cases. So far, the signal- to noise ratio in diagnostic IF has received little attention. The present study was initiated by the unexpected finding of significant increase of the signal-to noise ratio in a skin biopsy submitted for DIF and accidentally kept overnight in normal saline. The biopsy, obtained from a patient suspect of pemphigoid, showed substantial reduction of IgG background fluorescence and relatively bright specific IgG fluorescence along the BMZ. This finding encouraged us to compare diagnostic results of DIF in matched skin biopsies using standard snap-freezing, Michel's fixative and normal saline.
Background
The demonstration of tissue-bound immunoreactants by direct immunofluorescence microscopy (DIF) is a valuable parameter in the diagnosis of various autoimmune skin diseases[ 1 ]. Reliable diagnosis by DIF not only requires an experienced observer, but first of all proper skin (or mucosal) biopsies with well-preserved immunoreactants. For the latter purpose biopsies are usually snap-frozen in liquid nitrogen or, alternatively, placed in Michel's fixative that facilitates transport of biopsies from outside hospitals [ 2 - 6 ]. But even representative and optimally preserved tissue specimens are no guarantee for the correct diagnosis by DIF, especially when weak to moderate desired specific staining (DSS) of the epidermal basement membrane zone (BMZ) is involved [ 7 ]. In such cases, specific IgG fluorescence is easily masked by the relatively high dermal "background" fluorescence produced by polyclonal anti-human IgG fluorescein conjugates. The background fluorescence, consisting of both undesired specific staining (USS) and non-specific staining (NSS), largely determines the signal-to noise ratio [ 7 ]. This ratio in turn determines the detection threshold and thereby the diagnostic sensitivity of the DIF technique. A low ratio for IgG resulting from weak DSS and high USS plus NSS will undoubtedly yield false negative cases. So far, the signal- to noise ratio in diagnostic IF has received little attention.The present study was initiated by the unexpected finding of significant increase of the signal-to noise ratio in a skin biopsy submitted for DIF and accidentally kept overnight in normal saline. The biopsy, obtained from a patient suspect of pemphigoid, showed substantial reduction of IgG background fluorescence and relatively bright specific IgG fluorescence along the BMZ. This finding encouraged us to compare diagnostic results of DIF in matched skin biopsies using standard snap-freezing, Michel's fixative and normal saline.Methods Patients The 25 patients included in this study were selected on the basis of previously confirmed positive direct immunofluorescence (IF) in skin biopsies transported in liquid nitrogen. The final diagnosis was reached by clinical, routine laboratory, histological and direct IF findings. In case of bullous autoimmune diseases, serum samples were characterized by indirect IF on 1.0 M NaCl-split skin [ 8 , 9 ], immunoblotting [ 10 ], and ELISA (desmoglein 1 and 3) [ 11 ]. The patients had one of the following diagnoses: bullous pemphigoid (BP; n = 5); mucous membrane pemphigoid with skin involvement (MMP; 1); linear IgA dermatosis (LAD; 1), anti-epiligrin cicatricial pemphigoid (AECP; 1); epidermolysis bullosa acquisita (EBA; 1); dermatitis herpetiformis (DH; 1); pemphigus vulgaris (PV; 3); pemphigus foliaceus (PF; 3); subacute and systemic lupus erythematosus (LE; 5); and small vessel IgA vasculitis (4). Skin biopsies and processing From each patient four skin specimens were obtained by punch biopsy (4 mm) using lidocaine as the local anaesthetic. The biopsies were taken from perilesional skin within an area of 2 cm 2  to minimize the risk of local variation of immunoreactants. The matched skin specimens from each patient were immediately placed in one of the following transport media: (a) liquid nitrogen, (b) Michel's fixative 48 hours with appropiate pH, (c) saline 24 hours and (d) saline 48 hours. We used 5 ml screw-capped polypropylene vials for transporting biopsies in Michel's fixative and saline. Freezing Biopsies were placed in aluminum vials, snap-frozen in liquid nitrogen and stored at -80°C until further processing within two weeks for DIF (see below). Fixative Michel's fixative and buffer solution were prepared monthly according to the original description. 1  Biopsy specimens were kept in 5 ml Michel's fixative for 48 hours (Mi48) at room temperature, followed by washing for 30 minutes in Michel's buffer solution. The specimens were then blotted on filter paper to remove excess moisture, and stored at -80°C until further processing. Saline We used normal saline solution (0.9% NaCl in aqua dest.) without addition of calcium or magnesium. Skin specimens were kept in 5 ml saline solution for 24 hours (S24) and 48 hours (S48) at room temperature. Preliminary experiments with saline time of 6 hours did not result in improvement of the signal-to noise ratio. The specimens were then blotted on filter paper and stored at -80°C until further processing for DIF. Direct immunofluorescence microscopy For comparative purposes, matched skin specimens of each patient were processed for direct IF microscopy at the same occasion. Cryosections of 4 μm thickness were mounted on polysine™ glass slides, air-dried for 30 min before a fan, and encircled with a hydrophobic emulsion (PAP-pen; DAKO; Glostrup). The sections were then stained for 30 min in a moist chamber at room temperature, using fluorescein (FITC)-labeled, Fc-specific goat F(ab') 2  antibodies against human IgG, IgA and IgM (Protos Immunoresearch, Burlingame CA), and rabbit antibodies against human C3c and fibrinogen/fibrin (DAKO; Glostrup). Proper conjugate dilutions were made in phosphate-buffered saline (0.01 M PBS, pH 7.3) supplemented with bovine serum albumin 1%. After washing in 1000 ml PBS for 30 min, the sections were coverslipped under fresh PBS/glycerol (50% v/v). The slide preparations were kept at 4°C until microscopic examination within two days. The sections were examined with a Leica DMRA microscope (Leica, Wetzlar, Germany) for selective incident light fluorescence using a xenon arc (XBO 75W) as light source and PL Apo ×40/0.80 dry objective. The fluorescent staining was graded as follows: - (negative), ± (doubtful), + (weak), ++ (moderate), +++ (bright). The desired specific staining (DSS) of the following target structures was scored 12;13 .: (a) epidermal in vivo ANA, (b) epidermal intercellular spaces, (c) basement membrane zone (dermo-epidermal junction), and (d) blood vessel walls. In addition, the background staining (USS plus NSS) of the upper dermis was scored. All sections were read blindly by the same experienced observer (MCJMdJ). The diagnosis made by direct IF was regarded as conclusive if the DSS score of the relevant target structure was at least weak but definite (Table). The diagnosis was regarded as tentative in case of weak staining that was not consistently distributed at the relevant target structure. A case was regarded as non-diagnostic if target structures showed negative to doubtful DSS scores. Statistical analysis of IgG and IgA background fluorescence in matched biopsies was done by the McNemar test.
Methods
Patients The 25 patients included in this study were selected on the basis of previously confirmed positive direct immunofluorescence (IF) in skin biopsies transported in liquid nitrogen. The final diagnosis was reached by clinical, routine laboratory, histological and direct IF findings. In case of bullous autoimmune diseases, serum samples were characterized by indirect IF on 1.0 M NaCl-split skin [ 8 , 9 ], immunoblotting [ 10 ], and ELISA (desmoglein 1 and 3) [ 11 ]. The patients had one of the following diagnoses: bullous pemphigoid (BP; n = 5); mucous membrane pemphigoid with skin involvement (MMP; 1); linear IgA dermatosis (LAD; 1), anti-epiligrin cicatricial pemphigoid (AECP; 1); epidermolysis bullosa acquisita (EBA; 1); dermatitis herpetiformis (DH; 1); pemphigus vulgaris (PV; 3); pemphigus foliaceus (PF; 3); subacute and systemic lupus erythematosus (LE; 5); and small vessel IgA vasculitis (4).
Patients
The 25 patients included in this study were selected on the basis of previously confirmed positive direct immunofluorescence (IF) in skin biopsies transported in liquid nitrogen. The final diagnosis was reached by clinical, routine laboratory, histological and direct IF findings. In case of bullous autoimmune diseases, serum samples were characterized by indirect IF on 1.0 M NaCl-split skin [ 8 , 9 ], immunoblotting [ 10 ], and ELISA (desmoglein 1 and 3) [ 11 ]. The patients had one of the following diagnoses: bullous pemphigoid (BP; n = 5); mucous membrane pemphigoid with skin involvement (MMP; 1); linear IgA dermatosis (LAD; 1), anti-epiligrin cicatricial pemphigoid (AECP; 1); epidermolysis bullosa acquisita (EBA; 1); dermatitis herpetiformis (DH; 1); pemphigus vulgaris (PV; 3); pemphigus foliaceus (PF; 3); subacute and systemic lupus erythematosus (LE; 5); and small vessel IgA vasculitis (4).Skin biopsies and processing From each patient four skin specimens were obtained by punch biopsy (4 mm) using lidocaine as the local anaesthetic. The biopsies were taken from perilesional skin within an area of 2 cm 2  to minimize the risk of local variation of immunoreactants. The matched skin specimens from each patient were immediately placed in one of the following transport media: (a) liquid nitrogen, (b) Michel's fixative 48 hours with appropiate pH, (c) saline 24 hours and (d) saline 48 hours. We used 5 ml screw-capped polypropylene vials for transporting biopsies in Michel's fixative and saline.
Skin biopsies and processing
From each patient four skin specimens were obtained by punch biopsy (4 mm) using lidocaine as the local anaesthetic. The biopsies were taken from perilesional skin within an area of 2 cm 2  to minimize the risk of local variation of immunoreactants. The matched skin specimens from each patient were immediately placed in one of the following transport media: (a) liquid nitrogen, (b) Michel's fixative 48 hours with appropiate pH, (c) saline 24 hours and (d) saline 48 hours. We used 5 ml screw-capped polypropylene vials for transporting biopsies in Michel's fixative and saline.Freezing Biopsies were placed in aluminum vials, snap-frozen in liquid nitrogen and stored at -80°C until further processing within two weeks for DIF (see below).
Freezing
Biopsies were placed in aluminum vials, snap-frozen in liquid nitrogen and stored at -80°C until further processing within two weeks for DIF (see below).Fixative Michel's fixative and buffer solution were prepared monthly according to the original description. 1  Biopsy specimens were kept in 5 ml Michel's fixative for 48 hours (Mi48) at room temperature, followed by washing for 30 minutes in Michel's buffer solution. The specimens were then blotted on filter paper to remove excess moisture, and stored at -80°C until further processing.
Fixative
Michel's fixative and buffer solution were prepared monthly according to the original description. 1  Biopsy specimens were kept in 5 ml Michel's fixative for 48 hours (Mi48) at room temperature, followed by washing for 30 minutes in Michel's buffer solution. The specimens were then blotted on filter paper to remove excess moisture, and stored at -80°C until further processing.Saline We used normal saline solution (0.9% NaCl in aqua dest.) without addition of calcium or magnesium. Skin specimens were kept in 5 ml saline solution for 24 hours (S24) and 48 hours (S48) at room temperature. Preliminary experiments with saline time of 6 hours did not result in improvement of the signal-to noise ratio. The specimens were then blotted on filter paper and stored at -80°C until further processing for DIF.
Saline
We used normal saline solution (0.9% NaCl in aqua dest.) without addition of calcium or magnesium. Skin specimens were kept in 5 ml saline solution for 24 hours (S24) and 48 hours (S48) at room temperature. Preliminary experiments with saline time of 6 hours did not result in improvement of the signal-to noise ratio. The specimens were then blotted on filter paper and stored at -80°C until further processing for DIF.Direct immunofluorescence microscopy For comparative purposes, matched skin specimens of each patient were processed for direct IF microscopy at the same occasion. Cryosections of 4 μm thickness were mounted on polysine™ glass slides, air-dried for 30 min before a fan, and encircled with a hydrophobic emulsion (PAP-pen; DAKO; Glostrup). The sections were then stained for 30 min in a moist chamber at room temperature, using fluorescein (FITC)-labeled, Fc-specific goat F(ab') 2  antibodies against human IgG, IgA and IgM (Protos Immunoresearch, Burlingame CA), and rabbit antibodies against human C3c and fibrinogen/fibrin (DAKO; Glostrup). Proper conjugate dilutions were made in phosphate-buffered saline (0.01 M PBS, pH 7.3) supplemented with bovine serum albumin 1%. After washing in 1000 ml PBS for 30 min, the sections were coverslipped under fresh PBS/glycerol (50% v/v). The slide preparations were kept at 4°C until microscopic examination within two days. The sections were examined with a Leica DMRA microscope (Leica, Wetzlar, Germany) for selective incident light fluorescence using a xenon arc (XBO 75W) as light source and PL Apo ×40/0.80 dry objective. The fluorescent staining was graded as follows: - (negative), ± (doubtful), + (weak), ++ (moderate), +++ (bright). The desired specific staining (DSS) of the following target structures was scored 12;13 .: (a) epidermal in vivo ANA, (b) epidermal intercellular spaces, (c) basement membrane zone (dermo-epidermal junction), and (d) blood vessel walls. In addition, the background staining (USS plus NSS) of the upper dermis was scored. All sections were read blindly by the same experienced observer (MCJMdJ). The diagnosis made by direct IF was regarded as conclusive if the DSS score of the relevant target structure was at least weak but definite (Table). The diagnosis was regarded as tentative in case of weak staining that was not consistently distributed at the relevant target structure. A case was regarded as non-diagnostic if target structures showed negative to doubtful DSS scores. Statistical analysis of IgG and IgA background fluorescence in matched biopsies was done by the McNemar test.
Direct immunofluorescence microscopy
For comparative purposes, matched skin specimens of each patient were processed for direct IF microscopy at the same occasion. Cryosections of 4 μm thickness were mounted on polysine™ glass slides, air-dried for 30 min before a fan, and encircled with a hydrophobic emulsion (PAP-pen; DAKO; Glostrup). The sections were then stained for 30 min in a moist chamber at room temperature, using fluorescein (FITC)-labeled, Fc-specific goat F(ab') 2  antibodies against human IgG, IgA and IgM (Protos Immunoresearch, Burlingame CA), and rabbit antibodies against human C3c and fibrinogen/fibrin (DAKO; Glostrup). Proper conjugate dilutions were made in phosphate-buffered saline (0.01 M PBS, pH 7.3) supplemented with bovine serum albumin 1%. After washing in 1000 ml PBS for 30 min, the sections were coverslipped under fresh PBS/glycerol (50% v/v). The slide preparations were kept at 4°C until microscopic examination within two days.The sections were examined with a Leica DMRA microscope (Leica, Wetzlar, Germany) for selective incident light fluorescence using a xenon arc (XBO 75W) as light source and PL Apo ×40/0.80 dry objective. The fluorescent staining was graded as follows: - (negative), ± (doubtful), + (weak), ++ (moderate), +++ (bright). The desired specific staining (DSS) of the following target structures was scored 12;13 .: (a) epidermal in vivo ANA, (b) epidermal intercellular spaces, (c) basement membrane zone (dermo-epidermal junction), and (d) blood vessel walls. In addition, the background staining (USS plus NSS) of the upper dermis was scored. All sections were read blindly by the same experienced observer (MCJMdJ). The diagnosis made by direct IF was regarded as conclusive if the DSS score of the relevant target structure was at least weak but definite (Table). The diagnosis was regarded as tentative in case of weak staining that was not consistently distributed at the relevant target structure. A case was regarded as non-diagnostic if target structures showed negative to doubtful DSS scores.Statistical analysis of IgG and IgA background fluorescence in matched biopsies was done by the McNemar test.Results A total of 95 biopsies from 25 patients were examined; three biopsies were missing, and two biopsies proved unsuitable at cryosectioning. Cutting of 4 μm cryosections was easiest with biopsies transported in saline and hardest with biopsies transported in Michel's fixative. Biopsies kept in saline for 24 and 48 hours showed statistically significant reduction of background fluorescence in the dermis, especially with IgG, as compared with fresh-frozen and fixed biopsies (p < 0.01). After 24 hours, this resulted in enhanced signal-to noise ratios and accordingly more easy detection of immunoreactants at target structures, in particular the epidermal basement membrane zone (BMZ) and subepidermal blood vessel walls (Fig.  1 ). In comparison, fresh-frozen and fixed biopsies showed a relatively poor signal- to noise ratio for IgG (and IgA). In these biopsies, weak (+) specific fluorescence of IgG at the BMZ or IgA in vessel walls was found to be masked easily by relatively high background staining. Biopsies kept for 48 hours in saline showed a variable degree of diminution of specific staining, and tended to become negative in case of weak (+) specific fluorescence. The IgG fluorescence of epidermal in vivo ANA, present in fresh-frozen and fixed biopsies of two cases with (S)LE, became negative in saline biopsies (both 24 h and 48 h). In general, biopsies with moderate to bright (++/+++) specific fluorescence remained positive in all transport media. The reduced background fluorescence in saline biopsies occasionally revealed doubtful to weak focal IgG fluorescence of the BMZ that was regarded as non-relevant. Furthermore, we observed dermo-epidermal split formation in saline biopsies, not present in matched fresh-frozen and fixed biopsies. The extent of split formation varied among saline biopsies and increased with time (48 h > 24 h). In cases with pemphigoid, IgG was found predominantly at the epidermal side of splits, in contrast with e.g. SLE where IgG was found at the dermal side (Fig.  2 ). In case an artificial split is induced because of saline incubation a BMZ signal stand more out because of the dark background of the blister cavity (Fig.  2 ). The overall morphology in saline biopsies was quite fair and largely sufficient for the purpose of diagnostic IF microscopy. Figure 1 Comparison of direct immunofluorescence in cryosections of matched skin biopsies transported in liquid nitrogen, Michel's fixative or saline. Note the substantially reduced background fluorescence in saline-transported biopsies. Pemphigus foliaceus showing characteristic IgG fluorescence at the epidermal intercellular space. Additional granular IgG staining at the basement membrane zone (arrow) stands out most clearly in saline transported biopsy. (obj. ×20) Mucous membrane pemphigoid with skin involvement showing weak linear IgG fluorescence at the basement membrane zone that is only visible in the saline transported biopsy (arrow). (obj. ×20) Lupus erythematosus showing granular IgG fluorescence at the dermo-epidermal junction. Additional IgG staining of subepidermal vessel walls is best visible in the saline-transported biopsy. (obj. ×20) Vasculitis showing fine-granular IgA fluorescence in subepidermal capillary walls (arrows) which is most distinct in the saline-transported biopsy. (obj. ×40) Figure 2 Direct immunofluorescence (IgG, combined with transmitted light) in saline transported skin specimen of lupus erythematosus. After 48 hours in saline there is subepidermal split formation, not present in fresh-frozen (N 2 ) and fixed (Mi48) skin. Note the still obvious granular IgG fluorescence at the dermal side of the split. (obj. ×40) The diagnostic results of direct IF in matched biopsies are summarized in Table  I . By interpreting these data it should be realized that the minority of cases (20%) showed bright (+++) specific fluorescence in standard frozen biopsies, whereas the majority (52%) showed only weak to moderate (+/++) specific fluorescence of the relevant target structure(s). Two originally positive cases, one pemphigus and one IgA vasculitis became doubtful or negative (non-diagnostic) in all transport media. The highest rate of conclusive cases by direct IF was obtained in biopsies kept in saline for 24 hours (S24, 84%), and the lowest in fixed biopsies (Mi48, 50%). The case of mucous membrane pemphigoid with weak IgG (+) and IgA (+) fluorescence at the BMZ was obvious in saline biopsies (S24, S48) but non-diagnostic in matched fresh-frozen and fixed biopsies. The highest non-diagnostic percentage was obtained in fixed biopsies (32%) and the lowest in S24 saline biopsies (8%). In one case, classified as misdiagnosed, the fresh-frozen biopsy led to the diagnosis of LAD as IgA (+) and C3c (+) were the only immunoreactants observed, whereas the matched fixed and saline biopsies showed additional linear IgG (+/++) fluorescence at the BMZ (suggestive of mixed IgG/IgA pemphigoid). After correction for the missing biopsies, the results were only statistically significant comparing Mi and S24 (p < 0.05). Other comparisons (N2 versus Mi, S24 and S48 or Mi versus S48 or S24 versus S48) were not significant. Table 1 Results of direct immunofluorescence (DIF) of matched skin biopsies transported in different media. N2 Mi48 S24 S48 Diagnosis by DIF DSS n = 25 n = 22 § n = 25 n = 23 § a. conclusive +/+++ 14 (56%) 11 (50%) 21 (84%) 16 (70%) b. tentative ±/+ 5 (20%) 4 (18%) 2 (8%) 3 (13%) c. non-diagnostic -/± 5 (20%) 7 (32%) 2 (8%) 4 (17%) d. mis-diagnosed 1 (4%) - - - N 2 , snap-frozen specimens in liquid nitrogen; Mi48, specimens in Michel's fixative for 48 hours; S24 and S48, specimens in saline for 24 and 48 hours respectively; DSS, desired specific staining; n, number of cases §  Two S48 and three Mi48 matched biopsies were either lacking or considered unsuitable.
Results
A total of 95 biopsies from 25 patients were examined; three biopsies were missing, and two biopsies proved unsuitable at cryosectioning. Cutting of 4 μm cryosections was easiest with biopsies transported in saline and hardest with biopsies transported in Michel's fixative.Biopsies kept in saline for 24 and 48 hours showed statistically significant reduction of background fluorescence in the dermis, especially with IgG, as compared with fresh-frozen and fixed biopsies (p < 0.01). After 24 hours, this resulted in enhanced signal-to noise ratios and accordingly more easy detection of immunoreactants at target structures, in particular the epidermal basement membrane zone (BMZ) and subepidermal blood vessel walls (Fig.  1 ). In comparison, fresh-frozen and fixed biopsies showed a relatively poor signal- to noise ratio for IgG (and IgA). In these biopsies, weak (+) specific fluorescence of IgG at the BMZ or IgA in vessel walls was found to be masked easily by relatively high background staining. Biopsies kept for 48 hours in saline showed a variable degree of diminution of specific staining, and tended to become negative in case of weak (+) specific fluorescence. The IgG fluorescence of epidermal in vivo ANA, present in fresh-frozen and fixed biopsies of two cases with (S)LE, became negative in saline biopsies (both 24 h and 48 h). In general, biopsies with moderate to bright (++/+++) specific fluorescence remained positive in all transport media. The reduced background fluorescence in saline biopsies occasionally revealed doubtful to weak focal IgG fluorescence of the BMZ that was regarded as non-relevant. Furthermore, we observed dermo-epidermal split formation in saline biopsies, not present in matched fresh-frozen and fixed biopsies. The extent of split formation varied among saline biopsies and increased with time (48 h > 24 h). In cases with pemphigoid, IgG was found predominantly at the epidermal side of splits, in contrast with e.g. SLE where IgG was found at the dermal side (Fig.  2 ). In case an artificial split is induced because of saline incubation a BMZ signal stand more out because of the dark background of the blister cavity (Fig.  2 ). The overall morphology in saline biopsies was quite fair and largely sufficient for the purpose of diagnostic IF microscopy.The diagnostic results of direct IF in matched biopsies are summarized in Table  I . By interpreting these data it should be realized that the minority of cases (20%) showed bright (+++) specific fluorescence in standard frozen biopsies, whereas the majority (52%) showed only weak to moderate (+/++) specific fluorescence of the relevant target structure(s). Two originally positive cases, one pemphigus and one IgA vasculitis became doubtful or negative (non-diagnostic) in all transport media. The highest rate of conclusive cases by direct IF was obtained in biopsies kept in saline for 24 hours (S24, 84%), and the lowest in fixed biopsies (Mi48, 50%). The case of mucous membrane pemphigoid with weak IgG (+) and IgA (+) fluorescence at the BMZ was obvious in saline biopsies (S24, S48) but non-diagnostic in matched fresh-frozen and fixed biopsies. The highest non-diagnostic percentage was obtained in fixed biopsies (32%) and the lowest in S24 saline biopsies (8%). In one case, classified as misdiagnosed, the fresh-frozen biopsy led to the diagnosis of LAD as IgA (+) and C3c (+) were the only immunoreactants observed, whereas the matched fixed and saline biopsies showed additional linear IgG (+/++) fluorescence at the BMZ (suggestive of mixed IgG/IgA pemphigoid). After correction for the missing biopsies, the results were only statistically significant comparing Mi and S24 (p < 0.05). Other comparisons (N2 versus Mi, S24 and S48 or Mi versus S48 or S24 versus S48) were not significant.Results of direct immunofluorescence (DIF) of matched skin biopsies transported in different media.N2Mi48S24S48Diagnosis by DIF±/+-/±N 2 , snap-frozen specimens in liquid nitrogen; Mi48, specimens in Michel's fixative for 48 hours; S24 and S48, specimens in saline for 24 and 48 hours respectively; DSS, desired specific staining; n, number of cases§  Two S48 and three Mi48 matched biopsies were either lacking or considered unsuitable.Discussion Perilesional skin biopsies kept in saline for 24 hours yielded a higher diagnostic rate in direct IF than fresh-frozen biopsies in liquid nitrogen or biopsies kept in Michel's fixative. Several authors have described the positive effect of saline in increasing the sensitivity of IF analysis. Judd and Lever showed that skin biopsies stored for 24 hours in 0.15 M phosphate buffered saline prior to freezing gave a very high incidence of positive readings in direct IF [ 14 ]. Similarly, the use of 1.0 M NaCl split skin as a diagnostic tool for direct and indirect IF has been reported to increase the sensitivity of these methods [ 9 , 15 - 17 ]. It has been suggested that the increase of IF sensitivity by saline incubation is due to improved exposure of epitopes and/or by a decrease of background staining [ 16 ]. Our data suggest that the improved DIF sensitivity in saline biopsies is primarily due to decreased background staining resulting in better image contrast (signal-to noise ratio). We know that our visual perception is sensitive for light contrast and not for intensity [ 18 ]. The staining intensity perceived of a given specific fluorescent signal is strongly correlated with the intensity of the background (compare looking at the stars in daylight and at night; the intensity is the same, but seems multiplied at night). In this respect, IgG (and IgA) fluorescence in skin tissue is hampered by relatively strong background fluorescence in the dermis that may mask weak specific fluorescence at the BMZ and in vascular walls. Saline appears to reduce this background staining, resulting in relative increase of desired specific staining and thereby enhanced diagnostic sensitivity. A disadvantage of saline is the limited time of transport (24 h) for consistently reliable results. If biopsies are kept longer than 24 hours in saline, decreased fluorescence of tissue-bound immunoreactants may be encountered, although we have observed bright specific fluorescence of the BMZ in biopsies kept in saline for at least 5 days. Michel's fixative seems to have a similar limitation: Skeete and Black found that biopsies stored in this fixative should be received within 1 day of biopsy for consistently reliable results [ 4 ]. The second disadvantage of saline is the loss of at least some epidermal  in vivo  ANAs, possibly due to extraction or degradation of nuclear antigens. Another disadvantage of saline might be, from a histopathological point of view, morphological disturbances such as hydropic degeneration [ 19 ], and splitting at the dermo-epidermal junction, not found with Michel's fixative [ 6 ]. That is why saline is not suitable for antigen mapping in the diagnosis of genetic diseases [ 20 ]. Neither are biopsies kept in saline suited for immuno-electron microscopy [ 5 , 6 , 20 ]. However, for the diagnosis of autoimmune and immune complex-mediated diseases by DIF, it is not optimal morphology that counts, but the low detection threshold of immunoreactants. In this regard, (artificial) dermo-epidermal split-formation may add value to the method, rather than being a problem, by darkening of the juxtaposed background and mapping of the linear epidermal BMZ deposition [ 21 ]. Besides the diagnostic benefit, the preference would also go to the use of saline because it is a ready available, inexpensive, and convenient transport medium, and it certainly improves cutting properties of skin biopsies compared to biopsies fixed in Michels' medium. Saline can be used as standard medium at room temperature for express postal delivery of IF biopsies to the laboratory, if delivery is guaranteed within 24 hours. Transport of biopsies by express postal delivery or in-house airtube post is much more cost-effective than courier delivery necessary for biopsies transported in liquid nitrogen. If it is expected to tide over a longer period we advise to store the biopsy in saline for 24 hours and then to place it in Michel's fixative for further transport. Practically, place the biopsy in a screw-capped 5 ml polypropylene tube filled to the top with saline. The saline does not have to be sterile. If the specimen arrives in the laboratory the same day after biopsy, we advise to keep it overnight in saline at room temperature, followed by snap-freezing and (optional) storage at -80°C the next day.
Discussion
Perilesional skin biopsies kept in saline for 24 hours yielded a higher diagnostic rate in direct IF than fresh-frozen biopsies in liquid nitrogen or biopsies kept in Michel's fixative.Several authors have described the positive effect of saline in increasing the sensitivity of IF analysis. Judd and Lever showed that skin biopsies stored for 24 hours in 0.15 M phosphate buffered saline prior to freezing gave a very high incidence of positive readings in direct IF [ 14 ]. Similarly, the use of 1.0 M NaCl split skin as a diagnostic tool for direct and indirect IF has been reported to increase the sensitivity of these methods [ 9 , 15 - 17 ]. It has been suggested that the increase of IF sensitivity by saline incubation is due to improved exposure of epitopes and/or by a decrease of background staining [ 16 ].Our data suggest that the improved DIF sensitivity in saline biopsies is primarily due to decreased background staining resulting in better image contrast (signal-to noise ratio). We know that our visual perception is sensitive for light contrast and not for intensity [ 18 ]. The staining intensity perceived of a given specific fluorescent signal is strongly correlated with the intensity of the background (compare looking at the stars in daylight and at night; the intensity is the same, but seems multiplied at night). In this respect, IgG (and IgA) fluorescence in skin tissue is hampered by relatively strong background fluorescence in the dermis that may mask weak specific fluorescence at the BMZ and in vascular walls. Saline appears to reduce this background staining, resulting in relative increase of desired specific staining and thereby enhanced diagnostic sensitivity.A disadvantage of saline is the limited time of transport (24 h) for consistently reliable results. If biopsies are kept longer than 24 hours in saline, decreased fluorescence of tissue-bound immunoreactants may be encountered, although we have observed bright specific fluorescence of the BMZ in biopsies kept in saline for at least 5 days. Michel's fixative seems to have a similar limitation: Skeete and Black found that biopsies stored in this fixative should be received within 1 day of biopsy for consistently reliable results [ 4 ].The second disadvantage of saline is the loss of at least some epidermal  in vivo  ANAs, possibly due to extraction or degradation of nuclear antigens. Another disadvantage of saline might be, from a histopathological point of view, morphological disturbances such as hydropic degeneration [ 19 ], and splitting at the dermo-epidermal junction, not found with Michel's fixative [ 6 ]. That is why saline is not suitable for antigen mapping in the diagnosis of genetic diseases [ 20 ]. Neither are biopsies kept in saline suited for immuno-electron microscopy [ 5 , 6 , 20 ]. However, for the diagnosis of autoimmune and immune complex-mediated diseases by DIF, it is not optimal morphology that counts, but the low detection threshold of immunoreactants. In this regard, (artificial) dermo-epidermal split-formation may add value to the method, rather than being a problem, by darkening of the juxtaposed background and mapping of the linear epidermal BMZ deposition [ 21 ].in vivoBesides the diagnostic benefit, the preference would also go to the use of saline because it is a ready available, inexpensive, and convenient transport medium, and it certainly improves cutting properties of skin biopsies compared to biopsies fixed in Michels' medium. Saline can be used as standard medium at room temperature for express postal delivery of IF biopsies to the laboratory, if delivery is guaranteed within 24 hours. Transport of biopsies by express postal delivery or in-house airtube post is much more cost-effective than courier delivery necessary for biopsies transported in liquid nitrogen. If it is expected to tide over a longer period we advise to store the biopsy in saline for 24 hours and then to place it in Michel's fixative for further transport.Practically, place the biopsy in a screw-capped 5 ml polypropylene tube filled to the top with saline. The saline does not have to be sterile. If the specimen arrives in the laboratory the same day after biopsy, we advise to keep it overnight in saline at room temperature, followed by snap-freezing and (optional) storage at -80°C the next day.Conclusions The use of normal saline offers an attractive alternative to liquid nitrogen and Michel's fixative for diagnostic IF in autoimmune and immune complex-mediated dermatoses, but is only consistently reliable if the specimens are received within 24 hours after biopsy.
Conclusions
The use of normal saline offers an attractive alternative to liquid nitrogen and Michel's fixative for diagnostic IF in autoimmune and immune complex-mediated dermatoses, but is only consistently reliable if the specimens are received within 24 hours after biopsy.Competing interests None declared.
Competing interests
None declared.Abbreviations BMZ: basement membrane zone BP : bullous pemphigoid DH: dermatitis herpetiformis DIF: direct immunofluorescence microscopy DSS: desired specific staining EBA: epidermolysis bullosa acquisita IF: immunofluorescence microscopy LAD: linear IgA dermatosis LE : lupus erythematosus MMP: mucous membrane pemphigoid NSS: non-specific staining PF : pemphigus foliaceus PV : pemphigus vulgaris USS: undesired specific staining
Abbreviations
BMZ: basement membrane zoneBP : bullous pemphigoidDH: dermatitis herpetiformisDIF: direct immunofluorescence microscopyDSS: desired specific stainingEBA: epidermolysis bullosa acquisitaIF: immunofluorescence microscopyLAD: linear IgA dermatosisLE : lupus erythematosusMMP: mucous membrane pemphigoidNSS: non-specific stainingPF : pemphigus foliaceusPV : pemphigus vulgarisUSS: undesired specific stainingAuthors' contributions RV carried out the studies, participated in the design and data analysis and drafted the manuscript. MdJ participated in patient selection and carried out the immunofluorescence microscopy and imaging. HM performed the immunofluorescence technical work up. MW participated in material harvesting. HP participated in patient diagnosis. MJ conceived the study, and participated in its design and coordination. All authors read and approved the final manuscript.
Authors' contributions
RV carried out the studies, participated in the design and data analysis and drafted the manuscript. MdJ participated in patient selection and carried out the immunofluorescence microscopy and imaging. HM performed the immunofluorescence technical work up. MW participated in material harvesting. HP participated in patient diagnosis. MJ conceived the study, and participated in its design and coordination. All authors read and approved the final manuscript.Pre-publication history The pre-publication history for this paper can be accessed here:
Pre-publication history
The pre-publication history for this paper can be accessed here: