New autopsy manual section on liver

17 Jan

The latest post has my same complaints: I need a library to read or reread articles, and I lack many images that I had. The posted manuals are more like an outline that needs to be filled in.

The manual will be the basis for the stillbirth study, which I hope will be able to become multi-institutional. I have done over 2,000 perinatal autopsies in 40 years, 40 people doing 50 autopsies a year could achieve the same experience in a year if we pool information and images. With more careful protocols and insights gained in previous autopsies we have performed, and perhaps with more resources as part of a funded study, we will be able to provide even more meaningful information. Hope some of this will become reality. I am now Pittsburgh, to start work at Magee on Feb 1.

New autopsy manual page Thyroid

5 Jan

This page like others suffers from my inability to find some never digitized images, particularly in this case the gross photo of an enlarged thyroid in a stillborn infant of a mother with a history of Graves syndrome. I am moving to Pittsburgh this week if all goes well, so there will be a delay before the next blog.

New Page: Autopsy Manual: Spleen

3 Jan

            I have posted a new autopsy manual page on the spleen. It will need continued work, but I think it provides an atlas that will be useful in the stillbirth project. In relation to the physiologic mechanisms of death, I am interested in the weight and how it is affected by the balance of sinus congestion with possible decreased T lymphocyte population with stress induced cortisol. This would require an area measurement of the percentage of T cells times the spleen weight that could then be correlated with thymic weight, assuming a negligible difference in blood versus tissue density.

            Over the years, I have found rare lesions like hamartoma and HLH that I wish could have been evaluated genetically. Perhaps as such studies become less expensive, or even covered by maternal insurance, it will be possible to pursue such cases.

New Post, Autopsy Manual: Lung

27 Dec

I have added one more organ to the manual. I am still frustrated by having discarded my copies of papers with notes, and images on film, but I think the manual provides a start. I have continued to work on the proposal for the study of stillbirth (see research menu), and this manual will provide a basis for the autopsy protocol for that study. As always, I hope the manual will be useful to residents and fellows.

New Page of Autopsy Manual: Heart

20 Dec

I have added a page on the heart to the autopsy manual. I hope to expand this chapter with more references and examples as I resume work now at Magee Hospital. I spent more time on this page describing how I do the dissection than I have in other pages since it is more difficult with the small size and often soft texture of fetal hearts. As I hope this manual will be of value to residents of pathology, I would like someday to be able to create videos of the dissection technique, since I find learning this aspect of the autopsy to be difficult for novices.

Stillbirth: mechanisms and causes

9 Dec

This blog is a posting of the introduction section to Stillbirth, that I will publish as a page, but I wanted to have it on-line now, also as a useful introduction to the proposal for a study of stillbirth:

         A pathologist performing an autopsy is not looking for a single cause of death, but rather a chain of causation in which one event logically based on experimental and observational data links to another until a final cause of death is identified. Any terminology for the parts of that chain or overall classification system usually become tangled in these chains of causation. 

         Perhaps the most clearly defined term is the immediate or final cause of death. In utero this is relatively simple. Death is considered the irreversible cessation of the fetal heart pumping. The fetal-placenta-maternal system can keep the fetus alive despite absent function of many organ systems such as lungs, kidney, brain, etc. as is often demonstrated by infants born alive without those organs. The components needed for life are those needed for receiving oxygen, and for removing respiratory and metabolic acids. These systems include the blood, the heart pump, adequate patent vessels from fetus to umbilical cord to placenta, and a viable maternal circulation of the placenta. Anything that interferes with this system can cause death.

         We usually distinguish multiple mechanisms that are immediate causes of death, that is they stop cardiac function. These mechanisms are asphyxia, which is the interference with respiratory gases, shock which is the failure of adequate forward pumping of blood to support the needs of tissues, and heart failure in which the heart cannot fully empty, creating a back pressure which in utero is always elevates systemic venous pressure. These are useful concepts taught routinely to physicians, but clearly, they overlap, and even assigning such a mechanism as the immediate cause of death is not straight forward. For example, asphyxia causes hypoxia which increases systemic acidosis from anaerobic metabolism. The acidosis weakens cardiac contractions which then fails to pump blood forward, and develops failure with increasing umbilical venous pressure. The latter decreases umbilical venous flow and with failing cardiac forward flow increases hypoxia from decreased placental perfusion. Other starting points such a primary heart failure, for example from ventricular tachycardia, or hypovolemic shock, feed into the same final mechanisms as asphyxia. Assigning an immediate cause of death still requires a weighing of events. The pathologist, from the autopsy, is looking for anatomic evidence to clarify the order of events, the timing, and relative contribution of different factors that ended death, more than assigning an immediate cause of death*. 

         Another approach to classifying cause of death is to identify the initiating etiology. This can seem simple enough. Take the well-known example of Rh sensitization as a primary etiology. However, even in this straight-forward example, this etiology is not a complete description of all we would want to know about the fetal death. There is a complex chain of causation from sensitization to transfer of antibody to hemolysis of fetal Rh+ cells to timing of hypoxic heart failure and death. Understanding of each part of the chain has improved our therapies. The cause of death that matters depends on our objectives. If we want to look at failed prevention of Rh sensitization we may want to tabulate deaths from those who did not receive therapy because Rh was not screened for due to poverty or lack of medical resources, or those who were identified but did not receive therapeutic Rhogam on time, or we might want to look at those who became sensitized despite optimum prevention strategies, or we might want to look at infant who received in utero transfusion but died anyway or from complications of the procedure. The autopsy can provide some information on the extent of hemolysis, evidence of the severity of heart failure/hydrops, or identify therapeutic accidents. In some circumstances it may be important that the autopsy identify the cause of fetal hydrops as hemolysis from Rh sensitization.

         There are two points from this example. One is that the definition of cause may depend on the information needed to inform therapeutic practices or prevention practices, not just the biology. The second point is that the autopsy can identify some links in the chain of the autopsy from anatomy, but the investigation of death often requires a broader input.

In the case of Rh sensitization, the chain of causation is well understood. In many fetal deaths, understanding the chain is incomplete. 

         Another instructive example of a primary etiology of death is premature separation of the placenta, which may or may not be accompanied by a clinical placental abruption. The pathologist needs to evaluate the placental evidence in terms of how much placenta was acutely separated. If nearly complete, then the mechanism of death was primarily from asphyxia. If less of the placenta, but more than half, is separated of an otherwise normal placenta, then the mechanism of death is a progressive hypoxia/acidosis/ heart failure. The pathologist can estimate the timing of the separation by changes in the infarcted placental villi above the separation. If the separation is smaller than 50%, it could still be a contributing factor to immediate cause of death if the placenta already showed evidence of compromise of function, such as villous evidence of adaptation to utero-placental ischemia, of multiple placental infarctions, or severe loss of functioning perfused placentas. In such a situation, then the small separation may have caused sufficient hypoxia to be a final lethal asphyxia or to create a slower, progressive positive feedback of hypoxia/acidosis.

         Finding a placental separation and the chain of events leading to death is not the only goal of the autopsy. Discovering temporally proximal causes that initiated the placental separation are also important data. For example, evidence of fetal trauma and maternal history would point to mechanical forces separating the placenta, often an automobile accident. In other cases, there may be evidence clinically and pathologically in the placenta of preeclampsia/eclampsia in which case hemorrhage from spiral arteries is a suspected cause. There may be anatomic or clinical evidence of less well-established causes such as maternal coagulopathies, or vena caval compression. In some cases, placental separations have an increased recurrence risk unrelated to known causal factors, perhaps due to genetic differences in spiral artery remodeling or decidual trophoblast interactions. 

         Even looking for proximal causes does not completely define the chain of causation in premature placental separation. There are still many unanswered questions about the steps in pathogenesis of premature placental separation prior to and during the actual separation. If preeclampsia, coagulopathy, or recurrent/unexplained separation is present, are there anatomic predisposing reasons for the occurrence in the particular pregnancy with those risk factors. The pathologist looks for anatomic evidence of unusual changes in uninvolved basal or even parietal decidual spiral arteries that might explain a tendency to rupture and cause a hematoma that will separate the placenta. The fetal membranes are examined for venous engorgement that might indicate venous outflow obstruction. The autopsy also may yield other useful information such as the interval from the time of the separation, estimated from the timing of the overlying infarction, to the timing of intrauterine postmortem retention, to provide a estimate of the window of opportunity for rescuing the infant. The autopsy may show evidence for clues to the risk of separation, such as the presence of multiple placental infarctions. 

         I could present similar arguments for many topics including other maternal immune disease, fetal hemorrhage, maternal diabetes, etc., but these are all topics that I anticipate covering in other pages of this site. 

         My suggestion is, rather than look for classifications of stillbirth, to develop studies focused on questions about particular links in the chain of causation in fetal death. This would require many different studies and would require large multi-institution collaboration. The autopsy and placental examination can make a valuable contribution to such studies, but that the investigation of stillbirth, and of any death, requires a broad approach beyond pathological findings. The pathologist needs to be part of a team of obstetricians, radiologists, scientists and other care givers, to optimize investigations that will improve our understanding and prevention of stillbirth.  

         However, for epidemiologic purposes, investigators have sought classification schemes for perinatal death and more specifically for stillbirth. An innovative scheme was that provided by the NIH funded SCRU study1. The published paper also has an excellent critical review of classification systems. The SCRU approach recorded detailed findings, both clinical and anatomic, in an attempt at standardization. What it does not do is attempt to quantify and link the various findings into a plausible chain of events leading to the fetal demise.

(For the purposes of gathering data for the project proposed on this web site, I think these data sheets would be useful, as well as point of comparison to the SCRU studies. I have not asked for permission to publish them on the web site or use them in the study, but that is the intent. I confirmed with the NIH that Dr. Reddy has not modified the forms. The stillbirth study I am proposing would still ask detailed questions about events around the time of fetal death relating to changes in fetal movements, how and where mother slept, her diet, her perception of any illness, activities, and any medications, prescribed or over the counter, that she was taking.)

*(Different rules may apply in classification of causes in forensic/legal cases)

1.      Dudley DJ, Goldenberg R, Conway D, et al. A new system for determining the causes of stillbirth. Obstet Gynecol 2010;116:254-60.

New Page on Pancreas

5 Dec

I have added the pancreas to the autopsy manual. My resources for creating these pages is currently limited. I am more than willing to have colleagues add their knowledge and suggestions to these pages by emailing me. I am posting the autopsy manual as a basis to develop a shared autopsy protocol for the stillborn research project (see research menu). Some conditions that cause perinatal death of liveborn infants, such as fetal hydrops, oligohydramnios and fetal growth restriction, also increase the risk of stillbirth. The manual considers findings and causes in these conditions, although briefly. The manual also does not develop an integrated picture of disease. I plan for the regular obstetrical pathology pages to accomplish that goal such as pages on maternal diabetes, macrosomia and the mechanisms of fetal growth restriction. 

            The stillbirth research protocol will include in situ MRI of the umbilical cord in fetal death, and a clinical history that looks in more detail at immediate factors that might in aggregate compromise the fetus and lead to a positive feedback loop that causes hypoxic or acidotic death. The primary goal of the study is to improve understanding and hopefully prevention of stillbirth of currently unknown cause. However, as in any perinatal autopsy, there is the hope that more general knowledge of intrauterine disease will also be found, such as insight into how intrauterine factors cause adult disease or how sublethal events might lead to or predispose to permanent neurologic injury. For an example of the former, I recall Dr. Mike Ross presenting at a NICHD Diabetes in Pregnancy meeting data on a rat model of maternal hyperglycemia that seemed to change the satiety response of the offspring. He presented slides showing associated brain abnormalities in the arcuate and related hypothalamic nuclei. I could not find a publication cite for this work, but if the results held, it would be possible to prospectively look for similar brain changes at autopsy of infants of diabetic mothers. 

New Page

29 Nov

I have added a new page to the autopsy manual on the thymus. My goal with the autopsy manual is to provide a basis for analysis of stillborn autopsies. As such, topics of malformation are covered only superficially if at all. In infants with malformations, it is important to discover the mechanism of death as the malformation per se is not a cause of death. Some malformations may predispose to intrauterine death such as those causing oligohydramnios. However, I am also seeking to understand the biological significance of the organ findings in order to integrate them into a more complete picture of the timing and mechanism of death. The manual will reflect those concerns.

New page on adrenals

22 Nov

            There are now new menu “widgets” to make accessing the pages easier. 

            I have posted an addition to the autopsy manual on the adrenals. I want to expand the autopsy section as a basis for a protocol for the proposed stillbirth study. Unfortunately, I had already discarded most of the organ and malformation specific images that I had, including hydrops, triploidy, etc. I will need help to expand the types and numbers of images. I think published images are critical to helping standardize pathological diagnoses. A single example of a lesion does not in itself present the variety seen in actual cases.

            Some of the text is probably dated, and I do not yet have access to a library. I still wanted to start the process of posting subheadings to the manual, hence the new page. I also added the Chronic Histiocytic Intervillositis page to the new research menu. The proposed research project is at the end of the page. 

Chronic Histiocytic Intervillositis: Study Proposal

15 Nov

            I have updated the e-book work in progress, but I still need to improve the formatting and navigation. I have renamed the “page” as “Chronic Histiocytic Intervillositis”. It is not yet the time to create an open source digital placental pathology book. In writing the draft, I did see how important it is to have many case  illustrations, even better if they could be whole slides. I think for pathologists to understand CHIV, they need to see a broad sample of the overlying pathologies and of the difficulties in assigning percentage borders to the diagnosis.

I still want to find participants for a multi-institutional study of CHIV. As with the stillbirth study, I hope to pilot a study at Magee after I start there in February. A draft of the proposal for the study is in the  “Massive Chorionic Intervillositis” page. I am also posting same draft proposal below on this blog. I still need to add references, and the proposal is incomplete. I am open to comments and suggestions. Thanks. 

Draft of a Proposal to Study CHIV

Background: 

            The lesion is defined by a consensus to have clusters of monocytes in more than 5% of the intervillous space. The monocyte clusters should contain at least 80% monocytes. There  may be heterogeneity in the distribution of intervillous monocytes. The lesion has an association with increased perivillous fibrinoid and villitis of unknown etiology. There is no clear association of obstetrical outcome with these histological features, although there is some evidence that monocytes filling more than 50% of the intervillous space are associated with more severe outcomes.

            Associated clinical outcomes include a high incidence of fetal loss at all gestations, but greater in the first and second trimester, fetal growth restriction, and recurrence in multiple pregnancies. The growth restriction is often early onset and may be associated with oligohydramnios. There may be an elevation of maternal serum alkaline phosphatase above normal pregnancy values.

            An immune origin of the lesion has been hypothesized. There may be an association with maternal autoimmune disease, especially anti-phospholipid syndrome, and with anti-HLA antibodies. The microvillous border of syncytiotrophoblast can be immunostained with c4d, a marker of antibody initiated fixation of complement. 

            Many treatment regimens have been used to prevent recurrent fetal loss, and anecdotally have resulted in a live born infant. These have included combinations of low dose aspirin, low molecular weight heparin, prednisone and hydroxychloroquine. No treatments have been demonstrated to be superior. 

            There is no standard pre-delivery method to diagnosis the lesion in an index pregnancy. Measures of early onset growth restriction are consistent with the lesion in pregnancies after a primary histologic diagnosis. The histologic lesions show variety and it is possible that the lesion is a common pathway of different etiologies. Only one, malaria, has been specifically identified. A better understanding of the pathogenesis of the lesion is needed.

            The goal remains to find a specific test for the lesion prior to delivery, and an effective treatment. Even though the lesion is infrequent (less than .5% prevalence), it can cause recurrent fetal losses in some mothers. 

Aims of the study

            Part 1: Retrospective study of the histology: 

  1. Develop a quantifiable method of measuring intervillous monocytes, perivillous fibrin/fibrinoid, and VUE
  2. Utilize CD 68, anti T cell, and c4d staining on cases and develop quantifiable measures.
  3. Correlate the quantified measures of part 1.2  with obstetrical outcomes: first trimester fetal death, second trimester fetal death, third trimester fetal death, fetal growth restriction in the second trimester, fetal growth restriction in the third trimester, history of recurrent pregnancy loss, and history of recurrent CHIV. 
  4. Correlate quantified histologic measures with treatment, and if possible from placental samples from the same patient before and after treatment.

            Part 2: Prospective study of mothers with a history of previous CHIV or with early onset severe fetal growth restriction.

  1. Obtain maternal serum at first visit, at time of delivery, and before and after start of treatment to test for markers of the disease including alkaline phosphatase, serum complement panel, and red cell c4d. 
  2. Perform an auto-antibody panel including anti-phospholipid and anti-HLA
  3. Save serum to explore other markers of monocyte activation, of syncytial trophoblast injury, or of auto/allo-antibodies to trophoblast antigens
  4. In addition to routine specimen processing, prepare tissue of further basic research on CHIV including frozen placental tissues, and monocyte extraction from placenta

Part 3: Prospective Clinical trial

  1. Trial comparison of at least two therapy regimes against historical controls 

Methods:

Part 1.1 

            The slides from cases of previously diagnosed CHIV (and gestation matched controls) are scanned and areas are selected that do not contain lesions or non-placental tissue. The size of the areas on each sample slide are calculated. The number of intervillous monocytes, foci of VUE, and area of pervillous fibrinoid are calculated for each area and summed area of each case. A shared software solution will be developed to use image analysis to perform this procedure. The range of values for the measured variables for each case in different slides of the same sample will also be recorded.

Part 1.2 

            From the same cases above, additional slides will be cut for immuno-staining with CD68, T-cell markers, and c4d along with controls. The same procedures will be used to select areas to evaluate as in part 1.1 . Then, software will be developed to count each of the CD68 cells in the intervillous space, the T-cells in the villi, and linear extent of c4d staining along the villous surface.

Part 1.3

            The various histological measures will be compared to the major clinical outcomes:  first trimester fetal death, second trimester fetal death, third trimester fetal death, fetal growth restriction in the second trimester, fetal growth restriction in the third trimester, history of recurrent pregnancy loss, and history of recurrent CHIV. Statistical analysis will look at independence of the variables, and the best cluster of variables to “predict” the clinical outcome and, if possible, using ROC analysis to create a diagnostic score.

Part 1.4 

            Using the best cluster of variables to “predict” outcome, the measure of these variables in the specimens before and after treatment in an individual patient are tested against the null hypothesis that treatment did not alter the diagnostic score. 

Part 2.1

            The serum values will be compared to controls without evidence of early growth restriction, or history of CHIV, and to cases with evidence of early growth restriction with and without the diagnosis of CHIV. The goal is to find a correlation of the serum markers with the diagnosis of CHIV.

Part 2.2

            These antibody screening will be from routine clinical laboratory procedures. The prevalence of such antibodies in CHIV will be calculated. In addition, cases with autoantibodies will be stratified in comparisons with obstetrical outcomes, measures of alloantigen response, and of treatment outcomes in part 3.

Part 2.3 and part 2.4 

            Future studies into the pathogenesis of the disease depend on researcher initiative. A sample proposed study that hypothesized an alloantibody to a paternal microvillous antigen, could utilize frozen section slides of placentas including those from the patients and controls. An Fc receptor blocker would be applied to the slides, and then CHIV and control serums applied to the slides. They would then be rinsed, and a fluorescent labeled anti-human IgG would be applied. Positive fluorescent staining would be evidence of specific anti-trophophoblast antibody if the control serums were negative. Further studies could be designed to identify the antigen.

Part 3: The high recurrence of fetal death in mothers with identified CHIV in a prior pregnancy precludes an untreated group given anecdotal reports of successful treatment. A panel of obstetricians would decide on the best way to approach comparing randomly assigned treatment groups. The results of the study would also be stratified by various anatomic and clinical pathological variables elucidated in parts 1 and 2. 

Statistical analysis:

Institutional resources:

Sample patient consent: