Human liver territories: Think beyond the 8‐segments scheme

Worldwide, compartmentalization of the human liver into portal venous territories today follows the eight‐segments scheme credited to Couinaud. However, there are increasing reports of anatomical, radiological and surgical observations that contradict this concept. This paper presents a viewpoint that enhances understanding of these inconsistencies and can serve as a basis for customized liver interventions. Clin. Anat. 30:974–977, 2017. © 2017 Wiley Periodicals, Inc.     

Liver segments: an anatomical rationale for explaining inconsistencies with Couinaud’s eight-segment concept

Background and purpose  

An increasing number of surgical and radiological observations call Couinaud’s concept of eight liver segments into question and such inconsistencies are commonly explained with anatomical variations. This paper was intended to demonstrate that, beyond variability, another anatomical principle may allow to understand supposedly differing concepts on liver segmentation.     

SINUSOIDAL STENOSIS – THE CAUSATIVE ANATOMICAL LESION OF PORTAL HYPERTENSION

In order to clarify the anatomical lesion being responsible for the development of portal hypertension without vascular occlusion, the correlation of the grade of portal pressure elevation and the histological findings of the surgical biopsy specimens of the liver (fibrosis—11 cases, cirrhosis—24 cases and intact livers—10 cases) were examined. The liver cell size of the portal hypertension case was significantly larger than the normotensive case and the sinusoidal space of the portal hypertension case was markedly diminished. There was a close reverse correlation between the degree of sinusoidal stenosis and of portal pressure elevation. The vascular lesions of both the portal venous tree and the hepatic venous tree were minimal. These results suggested that the sinusoidal stenosis was one of the important causative anatomical lesion of portal hypertension.     

Vascular Development and Differentiation During Human Liver Organogenesis

The vascular architecture of the human liver is established at the end of a complex embryological history. The hepatic primordium emerges at the 4th week and is in contact with two major venous systems of the fetal circulation: the vitelline veins and the umbilical veins. The fetal architecture of the afferent venous circulation of the liver is acquired between the 4th and the 6th week. At the end of this process, the portal vein is formed from several distinct segments of the vitelline veins; the portal sinus, deriving from the subhepatic intervitelline anastomosis, connects the umbilical vein, which is the predominant vessel of the fetal liver, to the portal system; the ductus venosus connects the portal sinus to the vena cava inferior. At birth, the umbilical vein and the ductus venosus collapse; the portal vein becomes the only afferent vein of the liver. The efferent venous vessels of the liver derive from the vitelline veins and are formed between the 4th and the 6th week. The hepatic artery forms at the 8th week; intrahepatic arterial branches progressively extend from the central to the peripheral areas of the liver between the 10th and the 15th week. Hepatic sinusoids appear very early, as soon as hepatic cords invade the septum transversum at the 4th week. They then progressively acquire their distinctive structural and functional characters, through a multistage process. Vascular development and differentiation during liver organogenesis is, therefore, a unique process; many of the cellular and molecular mechanisms involved remain poorly understood. Anat Rec, 291:614–627, 2008. © 2008 Wiley‐Liss, Inc.     

基于肝内肝门静脉解剖的肝脏右前叶分段新概念

目的根据肝内肝门静脉的走形分布,提出肝脏分段的新概念,为影像学和肝脏外科提供资料。方法采用60例正常的活体肝移植供肝影像资料,研究右前叶肝内肝门静脉的走形和分布以及肝静脉及其属支的回流范围,10例Mevis三维软件重建图像,探讨两者之间的关系。结果 Couinaud分段中的Ⅷ段门脉支可大致分为腹侧支和背侧支,最多可达4支;约90%的背侧支越过肝右静脉分布到Couinaud分段中的VII段。V段的门脉分支大多来自右前叶或Ⅷ段门脉的腹侧支。因此,可将右前叶分为腹侧段:Couinaud分段中的Ⅷ段的腹侧段(S8v)和V段(S5)背侧段:Couinaud分段中的Ⅷ段的背侧段(S8d)两个部分。结论新的划分方法不仅有利于肝内病变的精确定位,而且便于肝脏外科实施新的、更安全的术式。     

A comparison of hepatic segmental anatomy as revealed by cross‐sections and MPR CT imaging

To compare the areas of human liver horizontal sections with computed tomography (CT) images and to evaluate whether the subsegments determined by CT are consistent with the actual anatomy. Six human cadaver livers were made into horizontal slices with multislice spiral CT three‐dimensional (3D) reconstruction was used during infusion process. Each liver segment was displayed using different color, and 3D images of the portal and hepatic vein were reconstructed. Each segmental area was measured on CT‐reconstructed images, which were compared with the actual area on the sections of the same liver. The measurements were performed at four key levels namely: (1) the three hepatic veins, (2) the left, and (3) the right branch of portal vein (PV), and (4) caudal to the bifurcation of the PV. By dividing the sum of these areas by the total area of the liver, the authors got the percentage of the incorrectly determined subsegmental areas. In addition to these percentage values, the maximum distances of the radiologically determined intersegmental boundaries from the true anatomic boundaries were measured. On the four key levels, an average of 28.64 ± 10.26% of the hepatic area of CT images was attributed to an incorrect segment. The mean‐maximum error between artificial segments on images and actual anatomical segments was 3.81 ± 1.37 cm. The correlation between radiological segmenting method and actual anatomy was poor. The hepatic segments being divided strictly according to the branching point of the PV could be more informative during liver segmental resection. Clin. Anat. 2013. © 2012 Wiley Periodicals, Inc.     

A study of the accessory hepatic vein to segments VI and VII with a morphological reconsideration of the human liver

Introduction The liver is supplied by the common hepatic artery from the celiac trunk and by the portal vein from the gastrointestine. This double blood supply to the liver by the hepatic artery and the portal vein produced a complicated structure in the liver. For the blood outflow, we can see right, intermediate and left hepatic veins, and irregular veins: the accessory hepatic veins. These veins drain the blood in the liver into the inferior vena cava. In this study, we studied the layout of the accessory hepatic vein draining segments 6 and 7 in the human livers and attempted to reconsider the structure of the liver by the layout of the accessory hepatic vein. Methods Sixty livers were subjected in this study. They were prepared by using forceps to trace the layout of the blood vessels inside the livers. We carefully examined the relation between the layouts of the accessory vein to the segments 6 and 7 and of the portal vein. The confluence patterns of the accessory hepatic vein into the inferior vena cava were also examined to find the character of the vein. The relation between the accessory hepatic vein and standard hepatic veins was also studied. Results We found 2.2 accessory hepatic veins in one liver on average in our study. The vein was always within the area of segments 6 and 7, and did not surpass the boundary. We found at most five accessory hepatic veins in a liver in two cases. The accessory hepatic vein to the segments 6 and 7 always had its stem on the dorsal side to the portal vein. Different from the stem, the periphery of the accessory hepatic vein freely distributed with the peripheral branches of the portal vein. The area distributed by the accessory vein was also always dorsal part within the segments 6 and 7. The vein was small usually, but was big in few cases. When the vein was big, the area became solely drained by the accessory vein, because the standard hepatic veins (right and intermediate hepatic veins) did not reach the area, and we did not find any communication between the accessory vein and the standard veins. As the remaining region in the segments 6 and 7 became smaller, the draining right standard hepatic vein became shorter and smaller. Discussion The region drained by the accessory hepatic vein excluded the standard hepatic veins. Therefore, there are two different draining venous networks in the area of segments 6 and 7 classified by Couinaud. Conclusion The accessory hepatic vein draining segments 6 and 7 distributed somewhere dorsal side in the segments 6 and 7. The area where the accessory vein distributed was the region where standard hepatic veins did not reach. This would suggest that the region drained by the accessory hepatic vein makes an isolated segment in the liver in the segments 6 and 7 by the Couinaud’s Classification. The area might have a unique blood circulation system.     

Arterial supply and biliary drainage of the dorsal liver: A dissection study using controlled specimens

Liver surgeons favor using the entity called the 'dorsal liver' (i.e. the caudate lobe and other paracavally located liver parenchyme of segments 7 and 8). According to minute dissection of 48 livers, we describe the territories of the left/right portal veins, hepatic ducts and hepatic arteries in the dorsal liver. In the caudate lobe, the right hepatic artery, rather than the left hepatic artery (23/48 vs 19/48 for right vs left, respectively), tended to supply the 'left' portal vein territory. Similarly, paradoxical drainage patterns, such as the right hepatic duct draining the left portal vein territory, were found in seven of 48 livers. In the territory of the hilar bifurcation, right hepatic artery dominance was also evident and various bile drainage patterns were found. These included double drainage by the bilateral hepatic ducts (3/48) and drainage into the confluence of bilateral ducts (6/48). In contrast, the arterial supply and biliary drainage of the paracavally located parenchyme of segments 7 and 8 usually depended on the proper segmental arteries and ducts and their variations were within the range of those found in other parts of the right lobe. Therefore, the dorsal liver concept may not be anatomical but, rather, simply aimed at usefulness in surgery. Nevertheless, clear subdivision of the caudate lobe according to biliary drainage and/or arterial supply seemed difficult because of the paradoxical relatioships among the portal vein, hepatic artery and bile duct. Consequently, the present results support extended surgery based on the dorsal liver concept for carcinomas involving the caudate lobe.     

A comparative histological and morphometric study of vascular changes in idiopathic portal hypertension and alcoholic fibrosis/cirrhosis

AIM: To examine the pathological changes of hepatic arteries in idiopathic portal hypertension (IPH) which is characterized by the obliteration of the intrahepatic portal vein branches and presinusoidal portal hypertension. METHODS AND RESULTS: Liver specimens (biopsied or surgically resected) from 20 patients with IPH, 20 patients with alcoholic fibrosis/cirrhosis (AF/C) and 20 histologically normal livers were used. The vascular lumina of arterial and venous vessels in portal tracts were morphometrically evaluated by an image analysis system. The ratio of portal venous luminal area to portal tract area (portal venous index) of IPH and that of AF/C were significantly reduced compared with normal liver. The portal venous index for IPH was significantly lower than that for AF/C. The ratio of hepatic arterial luminal area to portal tract area for AF/C was significantly higher than that in normal liver; however, that for IPH was similar to normal. The peribiliary vascular plexus was increased in AF/C but not in IPH. In AF/C, the number of mast cells and macrophages known to be the source of angiogenic substances was significantly increased in the portal tract compared with normal liver, while in IPH it was not increased. CONCLUSIONS: In AF/C, a reduction in portal venous lumen was associated with an increase of hepatic arterial lumen and of angiogenesis-related cells in portal tracts. However, such compensatory arterial changes were not evident in IPH, and this compensatory failure may be a feature of IPH.     

Quantitative analysis of hepatic macro‐ and microvascular alterations during cirrhogenesis in the rat

Cirrhosis represents the end‐stage of any persistent chronically active liver disease. It is characterized by the complete replacement of normal liver tissue by fibrosis, regenerative nodules, and complete fibrotic vascularized septa. The resulting angioarchitectural distortion contributes to an increasing intrahepatic vascular resistance, impeding liver perfusion and leading to portal hypertension. To date, knowledge on the dynamically evolving pathological changes of the hepatic vasculature during cirrhogenesis remains limited. More specifically, detailed anatomical data on the vascular adaptations during disease development is lacking. To address this need, we studied the 3D architecture of the hepatic vasculature during induction of cirrhogenesis in a rat model. Cirrhosis was chemically induced with thioacetamide (TAA). At predefined time points, the hepatic vasculature was fixed and visualized using a combination of vascular corrosion casting and deep tissue microscopy. Three‐dimensional reconstruction and data‐fitting enabled cirrhogenic features to extracted at multiple scales, portraying the impact of cirrhosis on the hepatic vasculature. At the macrolevel, we noticed that regenerative nodules severely compressed pliant venous vessels from 12 weeks of TAA intoxication onwards. Especially hepatic veins were highly affected by this compression, with collapsed vessel segments severely reducing perfusion capabilities. At the microlevel, we discovered zone‐specific sinusoidal degeneration, with sinusoids located near the surface being more affected than those in the middle of a liver lobe. Our data shed light on and quantify the evolving angioarchitecture during cirrhogenesis. These findings may prove helpful for future targeted invasive interventions.     

The fate of the vitelline and umbilical veins during the development of the human liver

Differentiation of endodermal cells into hepatoblasts is well studied, but the remodeling of the vitelline and umbilical veins during liver development is less well understood. We compared human embryos between 3 and 10 weeks of development with pig and mouse embryos at comparable stages, and used Amira 3D reconstruction and Cinema 4D remodeling software for visualization. The vitelline and umbilical veins enter the systemic venous sinus on each side via a common entrance, the hepatocardiac channel. During expansion into the transverse septum at Carnegie Stage (CS)12 the liver bud develops as two dorsolateral lobes or ‘wings’ and a single ventromedial lobe, with the liver hilum at the intersection of these lobes. The dorsolateral lobes each engulf a vitelline vein during CS13 and the ventromedial lobe both umbilical veins during CS14, but both venous systems remain temporarily identifiable inside the liver. The dominance of the left‐sided umbilical vein and the rightward repositioning of the sinuatrial junction cause de novo development of left‐to‐right shunts between the left umbilical vein in the liver hilum and the right hepatocardiac channel (venous duct) and the right vitelline vein (portal sinus), respectively. Once these shunts have formed, portal branches develop from the intrahepatic portions of the portal vein on the right side and the umbilical vein on the left side. The gall bladder is a reliable marker for this hepatic vascular midline. We found no evidence for large‐scale fragmentation of embryonic veins as claimed by the ‘vestigial’ theory. Instead and in agreement with the ‘lineage’ theory, the vitelline and umbilical veins remained temporally identifiable inside the liver after being engulfed by hepatoblasts. In agreement with the ‘hemodynamic’ theory, the left–right shunts develop de novo.     

Protective Effect of Glutathione Against Liver Warm Ischemia‐Reperfusion Injury in Rats is Associated with Regulation of P‐Selectin and Neutrophil Infiltration

We examined the effects of glutathione (GSH) preconditioning through the portal vein on rat warm liver ischemia reperfusion injury (I/R injury) and investigated the mechanisms involved. In rats with warm liver I/R injury, administration of GSH by means of the portal vein before ischemia increased the 7‐day survival rates of rats after liver I/R from 38% to 75%. This effect was correlated with significantly improved liver function, depressed MDA content in the liver and fewer histologic features of hepatocyte injury. Intrahepatic expression of P‐selectin and infiltration of neutrophils were increased significantly after liver I/R. GSH pretreatment decreased intrahepatic MPO content and the expression of P‐selectin. However, it did not significantly affect the mRNA levels for P‐selectin after liver I/R. Thus, preconditioning with GSH protects the liver against I/R injury by a mechanism dependent on free radical species scavenging, down‐regulation of adhesion molecule expression and inhibition of neutrophil accumulation. These findings document the potential clinical utility of GSH to improve the overall success of diverse procedures, such as liver surgery and liver transplantation. Anat Rec, 291:1016–1022, 2008. © 2008 Wiley‐Liss, Inc.     

An autopsy case of metastatic foci of hepatocellular carcinoma in adenomatous hyperplasias of the liver.

Adenomatous hyperplasia of the liver is known as a preneoplastic or early developmental stage of hepatocellular carcinoma, in which overt malignant foci occasionally develop. We have recently experienced an autopsy case (a 70-year-old male) of liver cirrhosis with hepatocellular carcinoma and two nodules of adenomatous hyperplasia. The latter two nodules contained several microscopic foci of moderately differentiated hepatocellular carcinoma. There were a number of tumor microemboli in portal vein branches within areas of adenomatous hyperplasia in addition to areas surrounding cirrhotic liver, some of which grew into the parenchyma of adenomatous hyperplasia and cirrhotic regenerative nodules. These findings and the fact that adenomatous hyperplasia contained portal tracts including portal venous branches, suggest that malignant foci in adenomatous hyperplasia contained portal tracts including represent metastases from hepatocellular carcinoma in other parts of the liver via the intrahepatic portal venous system.     

An Autopsy Case of Metastatic Foci of Hepatocellular Carcinoma in Adenomatous Hyperplasias of the Liver

Adenomatous hyperplasia of the liver is known as a preneoplastic or early developmental stage of hepatocellular carcinoma, in which overt malignant foci occasionally develop. We have recently experienced an autopsy case (a 70-year-old male) of liver cirrhosis with hepatocellular carcinoma and two nodules of adenomatous hyperplasia. The latter two nodules contained several microscopic foci of moderately differentiated hepatocellular carcinoma. There were a number of tumor microemboli in portal vein branches within areas of adenomatous hyperplasia in addition to areas surrounding cirrhotic liver, some of which grew into the parenchyma of adenomatous hyperplasia and cirrhotic regenerative nodules. These findings and the fact that adenomatous hyperplasia contained portal tracts including portal venous branches, suggest that malignant foci in adenomatous hyperplasia of the liver in this case might represent metastases from hepatocellular carcinoma in other parts of the liver via the intrahepatic portal venous system. Acta Pathol Jpn 41: 911 915, 1991.     

Anatomy of the Subcutaneous Lymph Vascular Network of the Human Leg in Relation to the Great Saphenous Vein

The anatomical relationship between lymphatic collectors and veins is of clinical importance for preventing lymphedema secondary to lymphatic collector injury during surgical procedures. To identify areas at risk during surgical interventions, we performed an anatomical study of human legs. The lymphatic collectors of 42 legs of human cadavers were injected with Berlin Blue solution or contrast medium. After fixation, the collectors were dissected and their distances from the great saphenous vein were determined. We found that the lymphatic collectors on the dorsum of the foot ran in close parallel with the corium, whereas in the groin a greater number of lymphatic collectors clustered around the great saphenous vein. The ventromedial bundle that drains into the superficial inguinal nodes included 5–20 lymphatic collectors. The average width of the ventromedial bundle varied between 116 mm at the middle of the lower leg and 32 mm at the groin. Our study cannot confirm the previous observation of a bottleneck of the ventromedial bundle occurring at the knee, but does support the finding of an elongated bottleneck at the thigh and groin draining into the superficial inguinal lymph nodes. In addition, the idea of one sentinel lymph node for a specific region of the leg is not supported by these data. These observations will help surgeons to plan incisions and dissections with respect to lymphatic collectors, thereby minimizing damage to them and reducing complications resulting from unnecessary lymphatic excisions. Anat Rec, 2009. © 2008 Wiley‐Liss, Inc.     

Lymph Circulation in the Liver

The liver produces a large amount of lymph, which is estimated to be 25 to 50 % of lymph flowing through the thoracic duct. The hepatic lymphatic system falls into three categories depending on their locations: portal, sublobular, and superficial lymphatic vessels. It is suggested that 80 % or more of hepatic lymph drains into portal lymphatic vessels, while the remainder drains through sublobular and capsular lymphatic vessels. The hepatic lymph primarily comes from the hepatic sinusoids. Our tracer studies, together with electron microscopy, show many channels with collagen fibers traversing through the limiting plate and connecting the space of Disse with the interstitial space either in the portal tracts, or around the sublobular veins. Fluid filtered out of the sinusoids into the space of Disse flows through the channels traversing the limiting plate either independently of blood vessels or along blood vessels and enters the interstitial space of either portal tract or sublobular veins. Fluid in the space of Disse also flows through similar channels traversing the hepatocytes intervening between the space of Disse and the hepatic capsule and drains into the interstitial space of the capsule. Fluid and migrating cells in the interstitial space pass through prelymphatic vessels to finally enter the lymphatic vessels. The area of the portal lymphatic vessels increases in liver fibrosis and cirrhosis and in idiopathic portal hypertension. Lymphatic vessels are abundant in the immediate vicinity of the hepatocellular carcinoma (HCC) and liver metastasis. HCCs expressing vascular endothelial growth factor‐C are more liable to metastasize, indicating that lymphangiogenesis is associated with their enhanced metastasis. Anat Rec, 291:643–652, 2008. © 2008 Wiley‐Liss, Inc.     

Regulation of Sinusoidal Perfusion in Portal Hypertension

Portal hypertension, a major complication of cirrhosis, is caused by both increased portal blood flow and augmented intrahepatic vascular resistance. Even though the latter is primarily caused by anatomical changes, it has become clear that dynamic factors contribute to the increased hepatic vascular resistance. The hepatic sinusoid is the narrowest vascular structure within the liver and is the principal site of blood flow regulation. The anatomical location of hepatic stellate cells, which embrace the sinusoids, provides a favorable arrangement for sinusoidal constriction, and for control of sinusoidal vascular tone and blood flow. Hepatic stellate cells possess the essential contractile apparatus for cell contraction and relaxation. Moreover, the mechanisms of stellate cell contraction are better understood, and many substances which influence contractility have been identified, providing a rationale and opportunity for targeting these cells in the treatment of portal hypertension in cirrhosis. Anat Rec, 291:693–698, 2008. © 2008 Wiley‐Liss, Inc.     

Analyzing the human liver vascular architecture by combining vascular corrosion casting and micro‐CT scanning: a feasibility study

Although a full understanding of the hepatic circulation is one of the keys to successfully perform liver surgery and to elucidate liver pathology, relatively little is known about the functional organization of the liver vasculature. Therefore, we materialized and visualized the human hepatic vasculature at different scales, and performed a morphological analysis by combining vascular corrosion casting with novel micro‐computer tomography (CT) and image analysis techniques. A human liver vascular corrosion cast was obtained by simultaneous resin injection in the hepatic artery (HA) and portal vein (PV). A high resolution (110 μm) micro‐CT scan of the total cast allowed gathering detailed macrovascular data. Subsequently, a mesocirculation sample (starting at generation 5; 88 × 68 × 80 mm³) and a microcirculation sample (terminal vessels including sinusoids; 2.0 × 1.5 × 1.7 mm³) were dissected and imaged at a 71‐μm and 2.6‐μm resolution, respectively. Segmentations and 3D reconstructions allowed quantifying the macro‐ and mesoscale branching topology, and geometrical features of HA, PV and hepatic venous trees up to 13 generations (radii ranging from 13.2 mm to 80 μm; lengths from 74.4 mm to 0.74 mm), as well as microvascular characteristics (mean sinusoidal radius of 6.63 μm). Combining corrosion casting and micro‐CT imaging allows quantifying the branching topology and geometrical features of hepatic trees using a multiscale approach from the macro‐ down to the microcirculation. This may lead to novel insights into liver circulation, such as internal blood flow distributions and anatomical consequences of pathologies (e.g. cirrhosis).     

Boundaries between subsegments IVa and IVb in the human liver

Until now there has been no definitive anatomical study of the boundary between hepatic subsegments IVa and IVb. In this study, we used a multicolor segmental corrosion cast technique and a multicolor segmental technique on plastinated slices, in combination with helical CT three-dimensional (3D) reconstruction on 15 donated fresh isolated human liver specimens. The corrosion cast technique was carried out on eight of these specimens, and the remaining seven livers were used to make horizontal plastinated slices. Examination of these specimens and observation of 20 additional liver corrosion cast specimens showed that all boundaries between hepatic segments were complex, undulating scissures rather than simple, flat planes. We also found that there was an obvious boundary between subsegments IVa and IVb, such that subsegment IVa laid posterosuperior to subsegment IVb. A tributary of a hepatic vein passed through the boundary between subsegments IVa and IVb, and could serve as an anatomical landmark of this boundary.     

Histologic classification of microscopic portal venous invasion to predict prognosis in hepatocellular carcinoma

Portal venous invasion is one of the most important prognostic factors after surgical resection of hepatocellular carcinoma. Microscopic portal venous invasion can be evaluated histologically. We examined 280 hepatocellular carcinomas with microscopic portal venous invasion (n = 125) or without it (n = 155) for 3 characteristics: the number of invaded portal vessels, the maximum number of invading carcinoma cells, and the farthest distance from the tumor. Univariate analysis of overall and disease-free survival revealed that the number of invaded portal vessels and the number of invading carcinoma cells were poor prognostic factors. Therefore, we classified patients with microscopic portal venous invasion into 2 groups: a high–microscopic portal venous invasion group, in which there were multiple invaded portal venous vessels (≥2) and more than 50 invading carcinoma cells (n = 57), and a low–microscopic portal venous invasion group, in which microscopic portal venous invasion was observed but with invasion of only a single portal venous vessel or fewer than 50 invading carcinoma cells (n = 68). The high–microscopic portal venous invasion group showed significantly higher α-fetoprotein levels, larger tumor size, and higher frequencies of poorly differentiated histology, capsule infiltration, and intrahepatic metastasis compared with the low–microscopic portal venous invasion group (P = .0496, P < .0001, P = .0431, P = .0180, and P = .0012, respectively). The high–microscopic portal venous invasion group showed poorer overall survival and disease-free survival rates than the low–microscopic portal venous invasion group (P = .0004 and P = .0003), and the high–microscopic portal venous invasion group was an independent prognostic factor for disease-free survival (P = .0259). We proposed a new definition for classifying microscopic portal venous invasion and documented the necessity of definite histologic evaluation of it.