412 J Clin Pathol 1992;45:412-415 Liver giant mitochondria revisited

N J Robertson, C H Kendall J Clin Pathol: first published as 10.1136/jcp.45.5.412 on 1 May 1992. Downloaded from

Abstract alcohol induced liver damage and the presence Aims: To examine the correlation be- of ICRBs. In a study by Chedid et al' in 1986, tween the severity of alcohol induced ICRBs were observed most frequently in liver liver damage and the presence of biopsies exhibiting mild degrees of alcohol intracytoplasmic red bodies (defined as induced damage. Junge et al2 in 1987 obtained periodic acid-Schiff diastase negative, similar results. However, in a study by globular, hyaline cytoplasmic inclusions Bruguera et al' in 1977 the frequency of larger in size than the hepatocyte ICRBs in liver biopsies from alcoholic nucleolus). To investigate the incidence patients was found to be unrelated to the of intracytoplasmic red bodies (ICRBs) nature of the histological changes present. in non-alcoholic liver . The significance of ICRBs with respect to Methods: Liver biopsy specimens from non- remains equally 53 patients with alcoholic liver disease unclear. The main purposes of this study are and 50 patients with a variety of non- therefore: (1) to examine the correlation be- alcohol related liver were tween the severity of alcohol induced liver examined by light microscopy for the damage and the presence of ICRBs; (2) to presence of ICRBs. For the 53 patients investigate the incidence of ICRBs in non- with alcoholic liver disease an assess- alcoholic liver disease. ment of recent alcohol consumption was Since the advent of transmission electron made indirectly from measurements of microscopy giant mitochondria have been red cell volume (MCV) and plasma identified in hepatocytes in liver biopsies y-glutamyl transferase (GGT). In addi- exhibiting both alcohol induced damage and tion, 10 liver biopsies with alcohol ICRBs.'" It has therefore followed that induced changes and ICRBs were exam- ICRBs seen in liver biopsies exhibiting alco- ined by electron microscopy for the hol induced damage have been interpreted as presence of mitochondrial aberrations being giant mitochondria, providing they are http://jcp.bmj.com/ including enlargement. periodic acid-Schiff diastase negative. There Results: ICRBs were detected in 18 of the has remained, however, some doubt as to the 53 liver biopsy specimens showing alco- exact nature of ICRBs, and a further aim of hol induced changes, and were more this study is to investigate liver biopsies con- abundant in those showing more advan- taining ICRBs ultrastructurally. ced changes. Those patients whose liver specimens contained ICRBs were found Methods on September 27, 2021 by guest. Protected copyright. to have a significantly higher mean The material consists of 103 liver biopsy plasma GGT activity and mean MCV specimens from 102 adults and one child; 53 than those individuals whose liver biopsy were from patients who either gave, or were specimens did not contain ICRBs. Two of strongly suspected of having, a recent history the 50 liver biopsy specimens showing of regular high alcohol consumption. Average non-alcohol induced changes contained daily intakes in excess of 60 g for men and ICRBs. Giant mitochondria were not 40 g for women are considered high and are detected by electron microscopy, but associated with increased morbidity and mor- this may reflect sampling. tality from a variety of diseases.5 And all 53 Conclusions: The results of this study biopsy specimens exhibited histological indicate that ICRBs are definitely as- changes consistent with an alcoholic aetiology: sociated with alcoholic liver disease and 50 liver biopsy specimens were from patients are more likely to be found in liver having a variety of non-alcohol related liver biopsy specimens showing more advan- diseases, and these included the following: ced alcohol induced damage, and when primary biliary cirrhosis (nine cases), chronic recent alcohol consumption has been active hepatitis (seven cases), chronic persis- Department of high. tent hepatitis (one case), sarcoidosis (two , Leicester Royal cases), miliary tuberculosis (one case), Reye's Infirmary, Leicester Globular intracytoplasmic red bodies (ICRBs) syndrome (one child case), ascending cholan- LE1 SWW have long been recognised by light micro- gitis (one case), chronic pericholangitis (one N Robertson scopy in hepatocytes in liver biopsies, and C H Kendall case), chronic large bile duct obstruction (one especially in those liver biopsies from patients case), a-l-antitrypsin deficiency (one case) and Correspondence to: with Dr N J Robertson alcoholic liver disease. There is, non-specific changes (25 cases). All biopsy Accepted for publication however, conflicting evidence in the literature specimens were obtained between 1983 and 8 October 1991 as to the correlation between the severity of 1989. Liver giant mitochondria revisited 413

Table I Histological category and the presence of ICRBs in liver biopsy specimensfrom patients with and without alcohol induced liver damage

Number of ICRBs Number Specimens

of positive J Clin Pathol: first published as 10.1136/jcp.45.5.412 on 1 May 1992. Downloaded from Histological category (-) (+ (+ +) (+ + +) biopsies for ICRBs Group I(n = 53) Fattychangealone 21 5 0 0 26 5 (19-2%) Patients with alcohol Fatty change with 12 5 0 1 18 6 (33 3%) induced liver damage alcoholic hepatitis or fibrosis or both Cirrhosis with or 2 3 1 3 9 7 (77-7%) without any other alcohol induced changes Group II (n = 50) Patients with non-alcohol A variety ofnon- 48 2 0 0 50 2 (4%) related liver damage alcohol related changes of various severity

All the liver biopsy specimens exhibiting ICRBs throughout the biopsy specimen were alcohol induced changes were categorised for denoted (+ + +), and (+ +) denoted an subsequent data analysis according to the intermediate number of ICRBs. An absence nature and severity of the histological changes of ICRBs was registered as (-). present. The categories were defined as For 10 cases with histological evidence of follows: fatty change alone, fatty change with both alcohol induced damage and ICRBs, tiny alcoholic hepatitis or fibrosis or both, and cubes of paraffin wax embedded tissue were cirrhosis with or without any other alcohol dewaxed in xylene, post fixed in 1% osmium induced changes. tetroxide and embedded in resin. Semi-thin The quantity of alcohol consumed around sections for orientation were stained with 1% the time of liver biopsy was assessed indirectly toluidine blue, and finally ultra-thin 90 nm from measurements of red cell volume (MCV) sections were stained with saturated uranyl and plasma y-glutamyl transferase (GGT). acetate/Reynold's citrate and examined in a Both these parameters are used clinically as Jeol 100 CX transmission electron micro- markers of alcohol consumption. Using upper scope. reference limits of 98 fl for MCV and 50 IU/l for GGT, Chick et al6 observed macrocytosis in 10 and increased GGT in 15 out of 30 Results company directors admitting to a daily alcohol In sections stained with haematoxylin and intake of more than 65 g. eosin ICRBs appeared as weakly eosinophilic All liver biopsy specimens were processed rounded bodies in otherwise normal for haematoxylin and eosin, chromotrope hepatocytes and in hepatocytes containing fat http://jcp.bmj.com/ aniline blue (CAB), Martius scarlet blue vacuoles. Their identification was much enhan- (MSB), and periodic acid-Schiff (PAS) before ced, however, on staining with CAB and MSB and after diastase digestion. Intracytoplasmic where they appeared bright red, and confusion red bodies (ICRBs) were defined as well cir- with red blood cells was eliminated as MSB cumscribed, globular, hyaline, intracytoplas- stained these cells yellow. milz bodies which stained bright red with CAB In all biopsy specimens where ICRBs were and MSB, and failed to stain with PAS after identified, they occurred in a minority of on September 27, 2021 by guest. Protected copyright. diastase digestion. Only red bodies larger than hepatocytes and their acinar distribution was the hepatocyte nucleolus were recorded and, entirely random. ICRBs ranged in size between in order to avoid confusion with red blood 2 and 10 gm and their number within cells, only red bodies seen to be clearly within hepatocytes varied markedly; some hepato- the hepatocyte cytoplasm and within the same cytes contained only one while others con- focal plane as the nucleus were recorded. tained two or more. Confusion with red blood cells was minimised Table 1 relates the presence of ICRBs to the with MSB staining as red blood cells appear histological categories defined earlier. Group I yellow with this staining method. comprises 53 liver biopsy specimens from The number of ICRBs per unit area of a patients with alcohol induced liver damage, section of liver biopsy specimen was variable, and group II comprises 50 patients with a both within the same biopsy and between variety of non-alcohol related liver diseases. different biopsies. ICRBs were subjectively ICRBs were observed in 18 liver biopsy semiquantified as (+ + +), (+ +), (+)), and specimens from group I and in two from group (-), according to their relative frequency in II, the latter being from two cases of primary the biopsy specimen. When only occasional biliary cirrhosis. ICRBs were seen in five of 26 ICRBs were detected in the entire biopsy liver biopsy specimens showing fatty change specimen this was denoted (+). Numerous alone and in seven of nine showing alcoholic

Table 2 Mean plasma GGT activity and mean MCV in relation to the presence and absence of ICRBs Mean plasma GGT activity Number of (IU/I) (SD) Mean MCV (fl) (SD) liver biopsy specimens ICRBs present (n = 18) 478-5 (98-2) 1059 (4 8) 18 ICRBs absent (n = 35) 225-5 (60 3) 94-0 (2 1) 35 414 Robertson, Kendall

cirrhosis. Of the 18 biopsy specimens showing ted of abusing alcohol. Notwithstanding this, fatty change with alcoholic hepatitis and/or the evidence strongly suggests that ICRBs are fibrosis six contained ICRBs. ICRBs were more generated mainly by processes which induce damage in alcohol contrast to abundant in those biopsies exhibiting more exposed livers in J Clin Pathol: first published as 10.1136/jcp.45.5.412 on 1 May 1992. Downloaded from advanced alcohol induced damage. processes which give rise to other liver diseases. Table 2 gives the mean plasma GGT activity This study also shows that ICRBs are found and the mean MCV in relation to the presence more often in liver biopsy specimens exhibiting and absence of ICRBs in 18 and 35 liver biopsy more advanced alcoholic damage; seven ofnine specimens respectively. The mean plasma specimens exhibiting cirrhosis contained GGT activity for the 18 patients whose liver ICRBs in contrast to only five of 26 exhibiting biopsy specimens were positive for ICRBs was fatty change alone. This is at variance with the 478-5 (SD 98-2) IU/1. This contrasts with a findings of Chedid et al' and Junge et al2 who mean plasma GGT activity of 225.5 (SD 60-3) observed ICRBs most frequently in liver biop- IU/l for the 35 patients whose specimens were sies exhibiting mild degrees of alcohol induced negative for ICRBs. The difference between the damage. mean plasma GGT activities in the two groups Rosalki introduced and popularised the is statistically significant (0-05 > p > 0-02). measurement of plasma GGT activity as an The mean MCV was calculated to be 105-9 (SD important biochemical index of alcohol 4-8) fl for the 18 positive biopsy specimens and consumption.78 The MCV is also known to 94-0 (SD 2-1) fl for the 35 negative biopsy correlate positively with average daily alcohol specimens. Again the difference between the intake.901' Therefore, as an indirect measure mean values here is statistically significant of alcohol consumption the plasma GGT (0-05 > p > 0.02). activity and the MCV were examined in each of Ultra-thin sections from 10 liver biopsy the 53 cases with alcohol induced liver damage, specimens, all of which exhibited alcohol and these parameters were then related to the induced changes and ICRBs by light micro- presence or absence of ICRBs. Those scopy (including the four (+ + +) cases shown individuals whose liver biopsy specimens con- in table 1), were carefully examined by trans- tained ICRBs were found to have a significantly mission electron microscopy. The integrity of higher mean plasma GGT activity and mean cellular organelles was not optimal in any ofthe MCV than those individuals whose liver biopsy sections examined because the liver biopsy specimens did not contain ICRBs. It can be specimens had initially been embedded in inferred from this that ICRBs are more likely to paraffin wax. Nevertheless, mitochondria were be found when recent alcohol consumption has easily identified and in the majority of cases been high. It is also reasonable to suggest that their internal membranous structure was satis- individuals with a high alcohol consumption factorily preserved. In none of the sections are likely to have more advanced alcoholic liver examined were giant mitochondria detected, damage. More ICRBs would therefore be ex- although occasional mitochondria showing pected in cirrhotic livers than in livers exhibit- http://jcp.bmj.com/ slight enlargement or abnormal shape were ing fatty change alone, as was found. found. What is the ultrastructural nature of ICRBs? As stated earlier, giant mitochondria have been found by electron microscopy in alcoholic Discussion livers which contained ICRBs at the light Intracytoplasmic red bodies, as defined by the microscope level.'234 Mitochondrial changes, strict criteria detailed elsewhere in this paper, including enlargement, have been observed in on September 27, 2021 by guest. Protected copyright. can readily be differentiated from a number of livers exhibiting alcoholic hepatitis'21314 and similar eosinophilic intracytoplasmic bodies alcoholic fatty change without .15 including Mallory's hyalin, a-l-antitrypsin Apparently giant mitochondria are not ex- globules and phagocytosed red blood cells. clusive to alcoholic liver disease, having been Invariably Mallory's hyalin has a clumped reported in a number of dietary'6 and toxic appearance with an irregular indistinct margin conditions,'7 and after starvation.'8 Giant mito- and stains blue/grey with CAB. a-l-Antitrypsin chondria reaching the size of the hepatocyte globules are easily differentiated as they are nucleus are said to have been induced in PAS positive after diastase digestion. Phago- experimental animals by various conditions cytosed and misplaced red blood cells stain such as the administration of isonicotinic acid 19 yellow with MSB and can also be compared derivatives" and dietary riboflavin deficiency.~~~~~~~~~~20 morphologically with red blood cells within In this current study sections from 10 liver sinusoids. biopsies containing -ICRBs were examined ICRBs are clearly an important finding in carefully by transmission electron microscopy liver biopsy specimens exhibiting alcohol for giant mitochondria, although none were induced damage. They were observed in 18 of found. This may simply reflect sampling 53 liver biopsy specimens from patients with problems, although occasional mitochondria alcoholic liver disease, in contrast to only two of showing slight enlargement or abnormal shape 50 from patients with other types of liver were apparent. The concept that IGRBs seen in disease. The histological diagnosis in each of alcoholic liver disease are giant mitochondria the two positive biopsy specimens from has largely gained acceptance in the literature, patients with non-alcoholic liver disease was but couldnot be further confirmed in this study. primary biliary cirrhosis. Both patients were On the basis of this study the evidence female and their sera were positive for anti- indicates that ICRBs are definitely associated mitochondrial antibodies; neither was suspec- with alcoholic liver disease, and are more likely Liver giant mitochondria revisited 415

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