Immunohistochemical investigation of pulmonary surfactant in perinatal fatalities

Forensic

Forensic Science Intemidiid

Science International 83 (1996) 219-227

Immunohistochemical investigation of pulmonary surfactant in perinatal fatalities Bao-Li Zhu”‘“, Hitoshi Maeda”, Kazunori Fukita”, Masami Sakuraib, Yasutsugu Kobayashi’ of

“Department

Legal

Medicine,

Osaka

‘Department

of Pathology,

Osaka

‘Department

of Pathology,

Osaka

Received

29 May

City University Medical School, Asahi-machi l-4-54, Abeno. 545 Osaka. Japan City University Medical School, Asahi-mnchi l-4-54. Abeno, 54-f Osaka, Japan City General Hospital, Miyakojima-hondori 2-13-22, Miyakojima. 534 Osaka, Japan

1996: revised

16 August

1996; accepted

2X September

1996

Abstract In order to verify forensic pathological significance of immunohistochemical investigation of pulmonary surfactant, 11 forensic and 16 clinico-pathological cases of perinatal death were comparatively examined. Surfactant appeared in some infants of 31-32 weeks gestation and was usually positive thereafter, indicating maturity of fetal lungs, although it may not have fully developed until about the 36th week of gestation. It was negative in all cases of the hyaline membrane disease except for a full-term infant (secondary respiratory distress syndrome). In usual casts, surfactant coating the expanded alveolar epithelia with its diffuse deposit in the intra-alveolar spaces was considered to indicate duration of hypoxia under persistent respiration (agonal state). Such finding was most intensely observed in asphyxia and in severe respiratory failure from intrinsic causes in the infants over ca. 36 weeks of gestation. With reference to pulmonary micromorphology, the amount of intra-alveolar surfactant seemed to be most closely related to the alveolar septal (interstitial) edema. Keywords: Forensic death; Asphyxia

*Corresponding

pathology;

Pulmonary

surfactant;

Immunohistochemistry;

author.

0379.0738/96/$15.00 @ 1996 Elsevier Pff SO379-0738(96)02040-3

Science

Ireland

Ltd.

All rights

reserved

Perinatal

220

B. Zhu

et al. I Forensic

Science

International

83 (1996)

219-227

1. Introduction

Pulmonary surfactant appears in the late fetal period. Its congenital deficiency is considered to be a basic cause of the infantile respiratory distress syndrome (hyaline membrane disease) [l-4]. The amount of surfactant in the amniotic fluid is clinically used as an indicator for diagnosis of maturity of fetal lungs [5-71. In the forensic pathology, it has been reported that surfactant production and secretion were enhanced in asphyxia, and that the immunohistochemical examination was useful to clarify the cause of death in the newborn and infants [8-131. Forensic pathological diagnostic value for interpretation of the extrinsic causes of death should be established through investigation in comparison with the intrinsic disorders. However, the distribution of surfactant in deaths from natural causes, except for the hyaline membrane syndrome, has not been precisely reported. In forensic postmortem examination of perinatal fatalities, it is often difficult to obtain their precise case history, and the circumstantial evidence is usually very poor. On the other hand, more medical information is available in clinical cases. In this study, in order to more properly verify the medicolegal significance of immunohistochemical investigation of pulmonary surfactant, a comparative study between forensic and clinico-pathological autopsy materials was carried out.

2. Materials

and methods

2.1. Perinatal fatalities

Eleven forensic autopsy cases at the Department of Legal Medicine, Osaka City University Medical School, and 16 clinico-pathological autopsy cases at the Osaka City University Hospital and the Osaka City General Hospital during the recent 5 years were examined. Case profiles are shown in Table 1. The materials included fetuses and neonates of varying gestational ages (19-41 weeks) and fatalities from a variety of natural and violent causes; intra-uterine/prenatal death (case number, n=4), spontaneous/artificial abortion (n=4), ‘birth asphyxia’ (neonatal asphyxia or hypoxia at birth from intrinsic causes) without hyaline membranes (n=3), hyaline membrane disease (n=6), anomalies (n=2), sudden unexpected death after birth (n=3) and mechanical asphyxia (n=5). 2.2. Tissue specimens

Serial sections (4 km thick) were prepared from formalin-fixed paraffin-embedded tissue specimens of each lobe of the lungs. The tissue sections were used for hematoxylin-eosin and immunostaining.

++ ++ ++ & ++ ++ ++ 2ru ,++ +++ ++ +fi + 3 + 2 + c ++ ++ + B 3 E % 9 s 4 8 ++ + 5 + +++ ++ *‘t + ++ ++ 2 + +++ ++ 2 + +++ ++ 2 g : ++: ++ 3 G 3

-

+M + + + + +

-

+

+

+ + +

A+ ++ +

+ -

-

+

-

+ +

+ + + + + +

-

+

+ +

++

+ + + +

+ + + + + + +

++

++ + ++ + ++

+ ++ + + + +

+ + ++ ++ ++ ++ it ++ ++ + ++ ++ ++ ++ ++ ++ ++

++ + + ++

++ ++ ++ +

+ + + ++ ++ + ++ ++ + ++ + + ++

++ ++ + + + + + + ++ ++ + ++

(3):: -

(311 i

++ T 5

&I)+1 (a)++ + + + + + I

+ + +

Y ZI ‘4 91

9E q oz q 01 4 01

q 11 q 81 9 PI q II q 9E qb q PI -

q1

q Zb 91 q 02 98 q IE AeP 1 -

OLPE nuns OZIE nm PLZE $011 9EIE OS91 OSEl SIX SW1 OL9 09P OE9 OIL 5-K E89Z OOZE EZhZ E9nE 6LPE OOZZ OL61 RPZZ 299 ES5 59s

avJUH “o!,mqc! ,e!xJ!11V qteap a”!la,“-elm, e!riqdss qwa uo!w!dse,e!xdqdsV e!xdqdse qwq/s!semlalsLa “o!wm33ns,e!xAqdsV Zu!~aq~~~s,e!xdqdsV auuaqt0ms,e!udqdsV uo!gel!dse,e!xdqdsV w.yeqdw,epdqdsV uo!,mqe snoaueluodg wx,mqe snoaueluods uo!wqe snoauewdg

avGVH am?IH aWH qmap a”!mln-e,,“I

w=P UaPPnS ql@aP UaPPTi (IWH (IWH W=JP uappns sa!,emo”v sayxlm”v qwap a”!lal”-ell”I eylqdse q,,!s

Ib UP UP UP 8E SE PC IE IE OE 62 9z sz PZ IS 61 09 UP UP 6E XE 9E ZE ZE PZ IZ IS

91-J SI-3 PL-3 EI-3 21-3 II-5 01-J 6-3 n-3 L-3 9-3 S-5 b-3 E-3 Z-3 l-2 11-d 01-d 6-d 8-J L-d 9-d S-d P-J E-d Z-J 1-d

222

B. Zhu

2.3. Immunostaining

et al. I Forensic

of pulmonary

Science

International

83 (1996)

219-227

surfactant

Anti-human pulmonary surfactant apoprotein A mouse monoclonal antibody PE-10 (Dako Japan, Kyoto) was used at a loo-fold dilution, with a 30-min incubation at room temperature, on a universal streptavidin / biotin immunoperoxidase detection system OmmiTags Kit (DAB) (Shandon/ Lipshaw / Immunon, Pittsburgh, PA, USA), according to the manufacture’s instruction (counterstaining with hematoxylin).

3. Results

Microscopic findings in hematoxylin-eosin and immunostaining of each case are summarized in Table 1. In the clinical cases, the alveoli were expanded in all cases from the 21st week of gestation to full-term infant, except for intra-uterine death. Surfactant was totally negative until the 25th week of gestation, when hyaline membrane disease was excluded, and appeared in a fetus of 31 weeks gestation (Table 1). It was observed also in the intra-uterine death (Case C-9; Fig. la). In

Fig. 1. Immunohistochemical distribution of pulmonary surfactant in the alveoli (arrows) and in the alveolar Type II cells (arrow heads) in clinical perinatal fatalities: (a) intra-uterine death of a premature fetus, 31 weeks gestation (Case C-9), diffusely positive: (b) hyaline membrane disease of a full-term infant (Case C-13), most intensely positive; (c) hyaline membrane disease of a full-term infant (Case C-14), negative; (d) sudden death of a full-term infant after birth (Case C-15) diffusely positive. Original magnification X200.

B. Zhu

ef al. I Forensic

Science

Internntional

83 (1996)

219-227

223

most instances, surfactant was densely, granularly demonstrated in the intraalveolar spaces, and also on the expanded alveolar epithelia membranously or linearly (Fig. la and Fig. Id). In the hyaline membrane syndrome of mature infant, surfactant was negative in a case (Case C-14; Fig. lc), whereas most intensely positive (partially amorphous) also on the hyaline membranes in the other one (Case C-13: Fig. lb). In forensic cases, the alveoli were expanded in five cases of the asphyxiated and a case of protracted ‘birth asphyxia’. Surfactant was totally negative until the 24th week of gestation. It appeared in the 32nd week of gestation. and thereafter positive in all cases (Table 1). Immunohistochemical distribution of surfactant was generally similar to those in clinical cases (Fig. 2a-d). All the asphyxiated over 36 weeks gestation showed a diffuse, very intense staining in the alveoli (Cases F-6, 7 and 8; Fig. 2a). A most intense staining was observed also in the still-preserved tissue structure of decomposing lungs (characterized by putrefactive gas bubbles, pyknosis, hyperchromatosis with partial chromatolysis, interstitial eosinophilic opacity and hemolysis) of a stillborn infant ca. 10 days after death (asphyxia due to aspiration of amniotic fluid; Case F-10; Fig. 2~). In those cases, some alveolar

Fig. 2. Immunohistochemical distribution of pulmonary surfactant in the alveoli (arrows) and in the alveolar Type II cells (arrow heads) in forensic perinatal fatalities: (a) the asphyxiated by smothering. 38 weeks gestation (Case F-7), most intensely positive; (b) a full-term infant dead from dystelectasis/ birth asphyxia (Case F-9), intensely positive; (c) intrauterine death (stillborn) of a full-term fetus from aspiration of amniotic fluid (Case F-10). most intensely positive; (d) protracted birth asphyxia of a full-term infant (Case F-11), positive mainly on the alveolar epithelia membranously or linearly. Original magnification X200.

224

B. Zhu

Table 2 Intensity of surfactant and forensic perinatal Intraalveolar surfactant

(+) (++I (+++I

Gestational (weeks)

31-3s 36 w 31-35 36 w 31-35 36 w 36 w -

et al. I Forensic

immunostaining fatalities age

w full-term w full-term w full-term full-term

Science

in relation

International

83 (1996)

to gestational

219-227

ages and causes

of death

Perinatal cause of death: clinical material

Forensic material

Intrauterine death (C-l) Artificial abortion (C-2) Hyaline membrane disease (C-3. 5, 6. 7) Intrauterine death (C-4) Birth asphyxia (C-8) Hyaline membrane disease (C-14) Anomalies (C-10. 11) Sudden death (C-12) Intrauterine death (C-9) Sudden death (C-15, 16) Hyaline membrane disease (C-13)

Spontaneous

Asphyxia

abortion

(aspiration)

in clinical

(F-l,

2, 3)

(F-4)

Asphyxia (Aspiration) (F-5) Protracted birth asphyxia (F-11) Dystelectasisibirth asphyxia (F-9) Asphyxia (smothering/ suffocation/ aspiration) (F-6, 7, 8, 10)

Intra-alveolar surfactant distribution: (-) negative, (+) weakly positive (sparse, small aggregates), ( + +) diffuse, granular staining, (+ + +) intense, diffuse positive with massive aggregates. Case numbers are shown in brackets (see Table 1 and Table 2 for details). Sudden death: pathologically unexplained death. Usually no detectable surfactant, under 30 weeks gestation; developmental stage of surfactant production, between the 31st and 35th week of gestation; mature surfactant production, over 36 weeks gestation.

Type II cells contained strongly positive granular substances. A case of protracted ‘birth asphyxia’ (Case F-11) showed somewhat different surfactant distribution; more intensely positive on the alveolar epithelia than in the intra-alveolar spaces (Fig. 2d). Among the micromorphological findings (pulmonary congestion, edema and hemorrhages), the alveolar septal (interstitial) edema seemed to have the most significant relationship to the amount of intra-alveolar surfactant (Table 1). The intensity of surfactant immunostaining described above is summarized in relation to the gestational ages and causes of death in Table 2.

4. Discussion

Pulmonary surfactant is a lipoprotein which has the function for expansion of the alveoli, produced by alveolar Type II epithelial cells [14,15]. It is reported to appear in the 18-20th week, immunohistochemically demonstrated after the 31st week and remarkably increase in the 3.536th week of gestation [16-191. In this immunohistochemical study, surfactant was totally negative in the neonates until the 25th week of gestation, both in forensic and clinical cases. It appeared in some infants of 31-32 weeks gestation, and was usually positive thereafter both in the live-born and stillborn (Table 1). This is consistent with the observations

B. Zhu

et al. I Forensic

Science

International

83 (1996)

219-227

225

described in clinico-pathological reports [l&19]. Those findings would indicate that surfactant is a useful indicator for postmortem interpretation of lung maturity of neonates and viability, as described by Fujiwara [13]. In clinical cases of the hyaline membrane disease (respiratory distress syndrome), surfactant was usually negative. However, a case (Case C-13) showed intense positive staining, which was observed also on the hyaline membranes. It was not likely that artificial surfactant therapy caused the positive staining, since therapeutic surfactant product from bovine does not contain apoprotein; as expected, the immunostaining was negative in the other cases of death from the same syndrome after surfactant therapy (Cases C-3 and 5). Besides, our experimental study showed that the therapeutic substance was not detected by immunostaining with anti-human pulmonary surfactant apoprotein A (unpublished data). The above exceptional case of the hyaline membrane disease of mature infant with positive surfactant staining may be explained by the recovery course from respiratory distress [18] or by secondary alveolar destruction (inactivation of surfactant) similar to the adult respiratory distress syndrome [20,21]. Membranous or linear surfactant staining on the intra-alveolar interior surface was generally observed in the live-born except for a premature case with fatal anomalies (Case C-11) and considered to indicate normal production of surfactant. Intra-alveolar deposit of aggregated, granular reaction products observed in varying amounts from case to case may be regarded as a ‘spillover’ of surfactant apoprotein. This would not necessarily mean the presence or over-production of active surfactant. In forensic pathology, Morita and his co-workers [S-11] have reported that surfactant production and secretion were enhanced in asphyxia (intra-alveolar hypoxia). It is an interesting hypothesis for diagnosis of cause of death in perinatal fatalities, since positive evidence of violence is often difficult to find in routine postmortem investigation. From the observations obtained in clinical cases, surfactant coating the expanded alveolar epithelia with its deposit in the intraalveolar spaces (possible ‘spillover’ of surfactant apoprotein) may be considered to indicate duration of hypoxia under persistent respiration (agonal state). Such finding was most intensely observed in prenatal asphyxia due to aspiration of amniotic fluid (Case F-10) among forensic cases and in severe respiratory failure with hyaline membranes (Case C-14) among clinical cases over 36 weeks gestation (Table 2). Although intra-alveolar deposit of surfactant was also intensely observed in the fatalities due to mechanical asphyxia (smothering and suffocation: Cases F-6, 7 and 8), the diagnostic value should be investigated further and considered more carefully especially in relation to survival time, because the agonal period would run usually only a few minutes. Similar findings, although less intensely, were observed also in clinically unexpected sudden death after birth (Cases C-15 and 16; probably due to prolonged agony under critical medical care), premature intra-uterine death (Case C-9), and ‘birth asphyxia’ (Case F-9). In the infants under 36 weeks of gestation, surfactant was only weakly positive or not detectable in the alveoli even although in the asphyxiated (Case F-4 and S), except for a case of intra-uterine death (Case C-9). With reference to pulmonary

226

B. Zhu

et al. I Forensic

Science

International

83 (1996)

219-227

micromorphology, the amount of intra-alveolar surfactant seemed to be most closely related to the alveolar septal (interstitial) edema (microvascular injury). It was interesting in connection with characteristic histopathological alterations of lungs in fatal asphyxiation [22,23]. Specific, membranous (or linear) and granular immunostaining of surfactant was identifiable also in the decomposing lungs with apparent signs of putrefaction and autolysis, at least on condition that tissue structures (partially expanded alveoli filled with amniotic fluid elements) were still preserved (Case F-10; Fig. 2c) [13]. As to the observations described above, we would summarize the immunohistochemical localization and appearance of pulmonary surfactant in perinatal fatalities in relation to its diagnostic significance at the moment as follows: (a) surfactant were not detectable under 30 weeks gestation; (b) diffuse membranous or linear staining on the intra-alveolar interior surface or on the surface of intra-alveolar effusion would indicate normal production of surfactant; (c) diffuse deposit of granular reaction products in the expanded intra-alveolar spaces may mean a ‘spillover’ of surfactant apoprotein, possibly indicating asphyxia or intrinsic severe respiratory failure (duration of hypoxia under persistent respiration; agonal state): (d) between the 31st and 35th week of gestation, none or a small amount of surfactant would not necessarily exclude the death from asphyxiation (surfactant production may not have fully developed until about the 36th week of gestation), although further investigation is required for more precise quantitative evaluation. Additionally, postmortem stability of surfactant was confirmed. In conclusion, this study has shown that immunohistochemical investigation of surfactant would be useful for postmortem interpretation of maturity of lungs, viability and causes of death (asphyxia or respiratory failure) in perinatal fatalities over ca. 36 weeks gestation, although the distribution pattern is not specific to mechanical asphyxia.

Acknowledgments

The authors are deeply indebted to Prof. M. Morita, MD, Ph.D. (Director, Department of Legal Medicine, Sapporo Medical College, Sapporo, Japan) for his kind advice and to Mr. M. Imura, Ph.D., and Mr. K. Nagai, DDS, for their partial contribution.

References [l]

M.E. Avery and J. Mead, Surface properties in relation to atelectasis and hyaline membrane disease. Am. J. Dis. ChiM., 97 (1959) 517-523. [2] D. Schofield and R.S. Cotran, Respiratory distress syndrome in newborn. In R.S. Cotran, V. Kumar and S.L. Robbins (eds.), Robhins Pathologic Basis of Disease, 5th ed., Saunders, Philadelphia, 1994, pp. 444-446.

B. Zhu

et al. I Forensic

Science

International

83 (1996)

219-227

227

[3] R.H. Perelman, M.J. Engle and P.M. Farrell, Perspectives on fetal lung development. Lmzg, 159 (1981) 53-80. [4] D.E. deMello, S. Heyman, D.S. Phelps and J. Floros, Immunogold localization of SP-A in lungs of infants dying from respiratory distress syndrome. Am. J. Puthol., 142 (1993) 1631-1640. [S] M. Hallman, M. Kulovich, E. Kirkpatrick, R.G. Sugarman and L. Gluck, Phosphatidylinositol and phosphatidylglycerol in amniotic fluid: indices of lung maturity. Am. J. Obstet. Gynecol., 125 (1976) 613-617. [6] R.J. King, J. Ruth, E.G. Gikas, A.C.G. Platzker and R.K. Creasy, Appearance of apoproteins of pulmonary surfactant in human amniotic fluid. J. Appl. Physiol., 39 (1975) 735-741. [7] M. Hallman, P. Arjomaa, M. Mizumoto and T. Akino, Surfactant proteins in the diagnosis of fetal lung maturity. 1. Predictive accuracy of the 35 kD protein, the lecithin/sphingomyelin ratio, and phosphatidylgycerol. Am. J. Obstet. Gynecol., 158 (1988) 531-535. [8] M. Morita, Lung surfactant: its application to the lung in asphyxia. Jpn. J. Legal Med., 48 (1994) 395-402 (in Japanese with English abstract). [Y] M. Morita, N. Tabata and A. Maya. Studies on asphyxia: on the changes of the alveolar walls of rats in the hypoxic state. Forensic Sci. Int., 27 (1985) 81-92. [lo] M. Morita and N. Tabata, Studies on asphyxia: on the changes of the alveolar walls of rats in the hypoxic state. II. The hypoxic state produced by carbon dioxide and methane gases. Forensic Sci. Int., 39 (1988) 257-262. [ll] M. Morita and N. Tabata, Studies on asphyxia: on the changes of the alveolar walls of rats in the hypoxic state. III. The hypoxic state by carbon monoxide gas. Jpn. J. Legal Med., 44 (1990) 218-222. [12] M. Funayama, N. Kageyama, S. Ohtani, S. Tokudome, N. Tabata and M. Morita, An immunohistochemical study on pulmonary surfactant of infants diagnosed as sudden infant death syndrome. Jpn. J. Legal Med., 48 (1994) 225-230. [13] M. Fujiwara, An immunocytochemical study on human pulmonary surfactant and its application to legal medicine. Sapporo Med. J., 57 (1988) 379-393. [14] J. Goerke, Lung surfactant. Biochem. Biophys. Acta, 344 (1974) 241-261. [1.5] A.B. Fisher and A. Chander, Lung surfactant: phospholipids and apoproteins. Exp. Lung Res., 6 (1984) 171-174. [16] A. Khoor, M.E. Gray, W.M. Hull, J.A. Whitsett and M.T. Stahlman, Developmental expression of SP-A and SP-A mRNA in the proximal and distal respiratory epithehum in the human fetus and newborn. J. Hisiochem. Cytochem., 41 (1993) 1311-1319. [17] P. Gruenwald, Normal and abnormal expansion of the lungs of newborn infants obtained at autopsy. Lab. Invest.. 12 (1963) 563-576. [18] Y. Kuroki, K. Dempo and T. Akino, Immunohistochemical study of human pulmonary surfactant apoproteins with monoclonal antibodies? Pathologic application for hyaline membrane disease. Am. J. Pathol.. 124 (1986) 25-33. [ 191 K. Kataoka. Y. Murata and M. Satoh, Structure and localization of surfactant protein B (SP-B) in human lung. Sapporo Med. J., 60 (1991) 501-513 (in Japanese with English abstract). [20] U. Bleyl, Vergleichende Untersuchungen an Erwachsenen und Neugeborenen zur Entstehung pulmonaler hyaliner Membranen. Verh. Dtsch. Ges. Pathol., 54 (1970) 340-347. [21] W. Janssen. Specific organ findings in shock. In Forensic Histopathology Springer, Berlin, 1984, pp. 161-174. [22] B. Brinkmann and K. Piischel, Die Lunge als Erfolgsorgan der Strangulationsagonie. Eine vergleichende experimentalle Studie. Z. Rechtsmed., 86 (1981) 175-194. [23] W. Grellner and B. Madea, Pulmonary micromorphology in fatal strangulations. Forensic Sci. Int., 67 (1994) 109-125.