alpha-1-antitrypsin ( aat ) deficiency , an autosomal recessive disorder , is caused by inheritance of two severe deficient alleles of serpin 1 ( located on chromosome 14q31 - 32 ) encoding aat .
the reactive loop site in the -sheet contains the neutrophil elastase binding site with a methionine residue . the protease inhibitor ( pi)*mm wild - type allele is the most prevalent normal allele and has many variants .
the most important pathogenic pi*s and pi*z variants show mutations glu264val and glu342lys , respectively , whereas pi*null variant has no protein production . the patient can be homozygous or compound heterozygous for pathogenic variants .
the protein product contains variable amounts of abnormal polymers of aat molecules which retain in the endoplasmic reticulum ( er ) in the liver .
these retained abnormal polymers can lead to hepatic cell injury and result in haemorrhagic disease and or cholestasis in infancy and chronic liver disease in childhood or adulthood .
in addition , the uninhibited action of the neutrophil elastase in the lungs can cause alveolar damage resulting in emphysema in adulthood .
the highest pi*z variant prevalence was recorded in northern and western europe ( mean gene frequency , 0.0140 ) .
about 12% of zz infants develop neonatal hepatitis and cholestasis within the first month of life .
the presenting symptom in neonates can vary from harmless - looking ( warning ) bleeds or earlier recognised jaundice to cholestasis and full - blown bleeding diathesis with dramatic consequences [ 4 , 6 , 11 ] . to diagnose aat deficiency , three different methods can be used 
level testing is not specific , phenotyping shows the mutated protein and genotyping detects the common selected mutations .
a 4-week - old baby girl was admitted to the neonatology department of our hospital because of respiratory insufficiency , convulsion and a large intracranial bleeding .
she was the second child of healthy non - consanguineous parents born after an uneventful pregnancy and delivery at 40-weeks gestational age .
vitamin k prophylaxis was given at birth and until hospital administration according to the standard dutch protocols . from birth , she was only breastfed .
the mother was instructed to follow a cow milk protein - free diet for herself . in the following weeks , oozing of the umbilical cord and bruising of the palate were noted .
both symptoms were reported separately and considered not to be alarming by the different general physicians .
nearly 4  weeks after birth she became lethargic , started vomiting and refused breastfeeding .
she was referred to the emergency department of the referral hospital . at first , an infection was suspected for which intravenous antibiotics were started . a few hours after admission ,
she became respiratory insufficient for which she was intubated , ventilated and transported to our university hospital .
laboratory results revealed total bilirubin , 85  mol / l ; conjugated bilirubin , 58  mol / l ; asat , 94  u / l ; alat , 40  u / l ; haemoglobin , 4.6  mmol / l ; thrombocytes , 388    10/l ; prothrombin time ( pt ) , > 100  s and partial thromboplastin time ( ptt ) , > 300  s. vitamin k - dependent clotting factors were all below 40% . because factor v and factor viii levels at the same time were 160% and 200% , respectively , clinical vitamin k - dependent bleeding disorder ( vkdbd ) was suspected .
vitamin k ( 1  mg ) was given intravenously and within 2-h pt and ptt times , factor v and factor viii levels normalized .
ct scan showed large intraventricular bleedings on both sides and a large intraparenchymal bleeding in the left temporal lobe with severe oedema .
differential diagnosis for the background pathology leading to cholestasis , liver dysfunction and further vkdbd was biliary atresia , alpha-1-antitrypsin deficiency , cystic fibrosis , inborn error in bile acid synthesis , haematochromatosis , liver pathology such as viral neonatal hepatitis or one of rare autosomal recessive forms of vitamin k - dependent clotting factor deficiency 1 and 2 .
both parents gave informed consent for complete autopsy . at brain autopsy , left temporal lobe showed haemorrhage with intraventricular extension .
this resulted in increased intracranial pressure and herniation of the cerebellar tonsils . at body autopsy ,
the gallbladder , the extrahepatic bile ducts and the papilla of vater could be identified and opened .
the mucosal lining showed bile staining . with these findings we excluded biliary atresia macroscopically .
the new pathologic differential diagnosis for the primary source of the problems leading to vkdbd in this patient was neonatal hepatitis / viral neonatal hepatitis , haematochromatosis or aat deficiency .
microscopic examination of the liver displayed acute hepatitis with portal tracts showing a mixed inflammatory reaction with variable destruction of bile ducts and infiltration of the parenchyma .
the parenchyma showed individual hepatocyte necrosis with accumulation of waste products , bile and iron in hepatocytes and kupfer cells . among the waste products were fine cytoplasmic granules located especially in the periportal hepatocytes which stained with periodic acid - schiff ( pas ) before and after diastase pretreatment ( fig .  1 ) .
these pas ( + ) granules were not abundant enough to diagnose the primary pathology with certainty ; however , suspicion rose on aat deficiency . 

h & e , 400 the additional immunohistochemical anti - cytokeratin 7 and anti - cytokeratin 19 stainings ( biogenex ) showed focal paucity of the premature and mature bile ducts , respectively . this paucity could be due to periductal inflammation and cholestasis .
finally , anti - aat protein staining ( dako , concentration 1/40,000 without antigen retrieval in dako immunostainer ) showed clearly the accumulation of aat protein polymers in the hepatocytes ( fig .  2 ) . 

2immunohistochemical staining showing aat protein deposition in the hepatocyte ( 200 ) immunohistochemical staining showing aat protein deposition in the hepatocyte ( 200 ) pcr studies to identify viral agents showed no cytomegalovirus in liver .
mutation analysis of serpin 1 was performed by direct sequence analysis of the coding exons ( 25 ; exon 2 split into two ) .
primers , containing an m13 tail , used were : 
 exon 2 - 1forward : 5 cacttccacgtggtgtcaatreverse : 5 ggttgagggtacggaggagtexon 2 - 2forward :
5 ttcttctccccagtgagcatreverse : 5 gaatccacgctgaaaagcatexon 3forward : 5 ggaggggactcatggtttctreverse : 5 tagcagtgacccagggatgtexon 4forward : 5 tagtgtgggtggaggacacareverse : 5 cagcctgggtcttcatttgtexon 5forward : 5 gtccacgtgagccttgctreverse : 5 ggaccagctcaacccttctt pcr reactions were performed using standard conditions ( 25-l reaction , containing 100-ng genomic dna ; 2 at 94c , followed by 29 cycles of 30 at 94c , 30 at 60c and 30 at 72c and a final extension of 10 at 72c ) .
sequence analysis of exon 5 of serpin 1 showed a homozygous pattern for p.glu342lys in our patient . in agreement with the homozygous pattern , the parents of the patient were heterozygous for this pathogenic mutation .
both parents gave informed consent for complete autopsy . at brain autopsy , left temporal lobe showed haemorrhage with intraventricular extension .
this resulted in increased intracranial pressure and herniation of the cerebellar tonsils . at body autopsy ,
the gallbladder , the extrahepatic bile ducts and the papilla of vater could be identified and opened .
the mucosal lining showed bile staining . with these findings we excluded biliary atresia macroscopically .
the new pathologic differential diagnosis for the primary source of the problems leading to vkdbd in this patient was neonatal hepatitis / viral neonatal hepatitis , haematochromatosis or aat deficiency .
microscopic examination of the liver displayed acute hepatitis with portal tracts showing a mixed inflammatory reaction with variable destruction of bile ducts and infiltration of the parenchyma .
the parenchyma showed individual hepatocyte necrosis with accumulation of waste products , bile and iron in hepatocytes and kupfer cells . among the waste products were fine cytoplasmic granules located especially in the periportal hepatocytes which stained with periodic acid - schiff ( pas ) before and after diastase pretreatment ( fig .  1 ) .
these pas ( + ) granules were not abundant enough to diagnose the primary pathology with certainty ; however , suspicion rose on aat deficiency . 

h & e , 400 the additional immunohistochemical anti - cytokeratin 7 and anti - cytokeratin 19 stainings ( biogenex ) showed focal paucity of the premature and mature bile ducts , respectively . this paucity could be due to periductal inflammation and cholestasis .
finally , anti - aat protein staining ( dako , concentration 1/40,000 without antigen retrieval in dako immunostainer ) showed clearly the accumulation of aat protein polymers in the hepatocytes ( fig .  2 ) . 

2immunohistochemical staining showing aat protein deposition in the hepatocyte ( 200 ) immunohistochemical staining showing aat protein deposition in the hepatocyte ( 200 )
both parents gave informed consent for complete autopsy . at brain autopsy , left temporal lobe showed haemorrhage with intraventricular extension .
this resulted in increased intracranial pressure and herniation of the cerebellar tonsils . at body autopsy ,
the gallbladder , the extrahepatic bile ducts and the papilla of vater could be identified and opened .
the mucosal lining showed bile staining . with these findings we excluded biliary atresia macroscopically .
the new pathologic differential diagnosis for the primary source of the problems leading to vkdbd in this patient was neonatal hepatitis / viral neonatal hepatitis , haematochromatosis or aat deficiency .
microscopic examination of the liver displayed acute hepatitis with portal tracts showing a mixed inflammatory reaction with variable destruction of bile ducts and infiltration of the parenchyma . slight fibrosis with septum formation was present .
the parenchyma showed individual hepatocyte necrosis with accumulation of waste products , bile and iron in hepatocytes and kupfer cells . among the waste products were fine cytoplasmic granules located especially in the periportal hepatocytes which stained with periodic acid - schiff ( pas ) before and after diastase pretreatment ( fig .  1 ) .
these pas ( + ) granules were not abundant enough to diagnose the primary pathology with certainty ; however , suspicion rose on aat deficiency . 

h & e , 400 the additional immunohistochemical anti - cytokeratin 7 and anti - cytokeratin 19 stainings ( biogenex ) showed focal paucity of the premature and mature bile ducts , respectively
finally , anti - aat protein staining ( dako , concentration 1/40,000 without antigen retrieval in dako immunostainer ) showed clearly the accumulation of aat protein polymers in the hepatocytes ( fig .  2 ) . 

2immunohistochemical staining showing aat protein deposition in the hepatocyte ( 200 ) immunohistochemical staining showing aat protein deposition in the hepatocyte ( 200 )
pcr studies to identify viral agents showed no cytomegalovirus in liver . additional studies in microbiology showed no growth of bacterial of viral agents .
mutation analysis of serpin 1 was performed by direct sequence analysis of the coding exons ( 25 ; exon 2 split into two ) .
primers , containing an m13 tail , used were : 
 exon 2 - 1forward : 5 cacttccacgtggtgtcaatreverse : 5 ggttgagggtacggaggagtexon 2 - 2forward : 5 ttcttctccccagtgagcatreverse : 5 gaatccacgctgaaaagcatexon 3forward : 5 ggaggggactcatggtttctreverse : 5 tagcagtgacccagggatgtexon 4forward : 5 tagtgtgggtggaggacacareverse : 5 cagcctgggtcttcatttgtexon 5forward : 5 gtccacgtgagccttgctreverse : 5 ggaccagctcaacccttctt pcr reactions were performed using standard conditions ( 25-l reaction , containing 100-ng genomic dna ; 2 at 94c , followed by 29 cycles of 30 at 94c , 30 at 60c and 30 at 72c and a final extension of 10 at 72c ) .
sequence analysis of exon 5 of serpin 1 showed a homozygous pattern for p.glu342lys in our patient . in agreement with the homozygous pattern , the parents of the patient were heterozygous for this pathogenic mutation .
alpha-1-antitrypsin ( aat ) deficiency has an incidence of 0.05% and can be caused by homozygosity for pi*z .
the mutant z protein can form abnormal polymers of aat within the rough er of hepatocytes .
chains of these polymers become interwoven to form insoluble inclusions resulting in reduced levels of this protein in the bloodstream .
clinical progression to liver disease is complex , with z allele homozygous neonates developing hepatitis and cholestasis in about 10% of the cases . here , we reported our findings in a 4-week - old female neonate who showed harmless - looking warning bleeds at the age of 2  weeks and cascade of complications leading to her death as a result of intracranial vkdbd under standard vitamin k prophylaxis .
although vkdb is described earlier as a complication of aat deficiency in neonates , there are still two important lessons we can learn from this patient .
the clinical lesson is the importance of early recognition of the warning bleeds or the cholestasis .
it is also reported that adjustment of the standard vitamin k prophylaxis protocols could prevent the complications due to vkdb which also could have been the case in this patient .
the diagnostic lesson is to use pathology as an instrument in combination with verifying genotyping to diagnose aat deficiency .
this statement contrasts with the earlier studies where the cytoplasmic inclusions were not seen in children younger than 3  years [ 5 , 7 ] or the false - positive results which can be observed with immunohistochemistry in this age group . in conclusion , however , early recognition of the warning bleeds or cholestasis , correct diagnosis using pathology as a diagnostic instrument in addition to the existing methods and adjustment of the standard vitamin k prophylaxis protocols to prevent vkdb complications can be life saving in aat deficiency patients especially in the neonatal period .
 alpha-1-antitrypsin ( aat ) deficiency is a rare genetic disorder characterized by hepatitis in neonates , childhood and adulthood ( protease inhibitor ( pi)*zz ) and emphysema with or without hepatitis ( pi*zz)/(pi*ss , sz or null ) in adulthood . 
 we report the case of a female neonate born at 40  weeks of gestation who presented with vitamin k deficiency - related intracranial bleeding and cholestasis of which she died at 28  days of age . at autopsy , 
 the infant was found to have intracranial bleeding , hepatomegaly , and cholestasis with paucity of bile ducts in the liver . 
 small periodic acid - schiff diastase positive intrahepatic granules and positive staining with antibodies against aat protein suggested an aat deficiency . 
 aat is a glycoprotein that has a protease inhibitor function . 
 its deficiency can be the result of various point mutations in serpin 1 located on chromosome 14 . 
 the diagnosis aat deficiency was confirmed by mutation analysis showing the pi*zz genotype in the neonate . in conclusion , 
 aat deficiency is a rare genetic disorder that can lead to a serious bleeding disorder in the neonatal period if not recognised on time . 
 pathological diagnosis together with verifying molecular analysis can be used to identify index patients . 
Introduction
Case report and results
Pathology
Autopsy
Microscopy
Microbiology
Molecular analysis
Discussion