Effect of amniotic membrane/collagen scaffolds on laryngeal cartilage repair.

Objectives: Laryngeal cartilage defects are a major problem that greatly impacts structural integrity and function. Cartilage repair is also a challenging issue. This study evaluated the efficacy of a collagen scaffold enveloped by amniotic membrane (AM/C) on laryngeal cartilage repair. Study Design: Experimental animal study. Methods: Fourteen Dutch rabbits were enrolled in the study. A 5 mm cartilage defect was created in the right and left thyroid lamina. The animals were divided into two groups randomly. Group 1 collagen scaffolds and group 2 AM/C were applied to the right side defects. Left side defects were not repaired, serving as control. Histologic evaluation was done 45 and 90 days following collagen and AM/C application with criteria of tissue and cell morphology, lacuna formation, vascularization, and inflammation. Results: Significant improvement in cartilage repair was observed in the AM/C side compared to the control side in all histologic criteria after 45 days (p<.05). After 90 days, cartilage repair improved in cell morphology, lacuna formation, and inflammation significantly (p<.05). Conclusion: The combination of amniotic membrane and collagen scaffolds provides a promising treatment modality for improving the repair of laryngeal cartilage defects. Level of Evidence: NA.

Transmission of high arterial pressure into renal microvessels during venous-clamping augments ischaemia/reperfusion-induced acute kidney injury in anaesthetized rats.

AIM: In two recent studies, we observed that a 30-min renal vein clamping caused formation of interstitial haemorrhagic congestion in ischaemic and ischaemic/reperfused kidney along with the development of severer acute kidney injury (AKI) than renal artery or pedicle clamping. It was suggested that the transmission of high arterial pressure into renal microvessels during vein occlusion probably causes the occurrence of interstitial haemorrhagic congestion that augments AKI. The present investigation aimed to evaluate this suggestion by reducing renal perfusion pressure (RPP) during renal venous occlusion. METHODS: Anaesthetized male Sprague-Dawley rats were divided into three groups (n = 8), which underwent a 2-h reperfusion period following 30-min bilateral renal venous clamping along with reduced RPP (VIR-rRPP group) or without reduced RPP (VIR group) and an equivalent period after sham-operation (Sham group). RESULTS: The VIR-rRPP group compared with VIR group had lower levels of kidney malondialdehyde and tissue damages as epithelial injuries of proximal tubule and thick ascending limb, vascular congestion, intratubular cast and oedema, along with the less reductions in renal blood flow, creatinine clearance, Na+ -reabsorption, K+ and urea excretion, urine osmolality and free-water reabsorption. Importantly, the formation of intensive interstitial haemorrhagic congestion in the VIR group was not observed in the VIR-rRPP group. CONCLUSION: These results indicate that the transmission of high arterial pressure into renal microvessels during venous occlusion leads to rupturing of their walls and the formation of interstitial haemorrhagic congestion, which has an augmenting impact on ischaemia/reperfusion-induced renal structural damages and haemodynamic, excretory and urine-concentrating dysfunctions.

Diagnosis of fast-growing thoracic aneurysm with microscopic evidence of dissection over 6 months follow-up in an asymptomatic middle aged gentleman: a case report.

BACKGROUND: Thoracic aortic aneurysm (TAA), is a pathological dilatation of the aortic segment with the tendency to expand, dissect or rupture, and risk of mortality. The progression rate is mainly slow. As the risk of rupture increases with the size of the aortic diameter, it is important to diagnose TAA appropriately to prevent mortality. CASE PRESENTATION: Here, we present a case with a fast-growing TAA, complicated by subclinical dissection in a middle-aged gentleman, associated with non-compaction left ventricle, diagnosed 6 months after the first diagnosis of this co-occurrence, successfully managed by an uneventful surgical procedure. The pathological examination was the key to the diagnosis of this concealed phenomenon, i.e. a fast-growing aortic aneurysm complicated by subclinical dissection. CONCLUSION: This case report emphasizes the importance of close follow-up of patients with fast-growing TAA for considering remote possibility of this silent life-threatening disease; subclinical dissecting aneurysm, especially in patients with other cardiac comorbidities. Although imaging modalities can help accurate diagnosis, in cases with fast-growing TAA, we should not wait for imaging signs of dissection and/or rupture.

Berberis integerrima hydro-alcoholic root extract and its constituent berberine protect against cisplatin-induced nephro- and hepato-toxicity.

BACKGROUND: This study determined the potential hepato- and renal protective role of berberine and hydroalcohol extract of Berberis integerrima (barberry) against cisplatin-induced acute liver and kidney injury. METHODS: Animals were dedicated into six groups (n = 7 per group): control, control+Ber (berberine, 0.4 mg/kg/day during 10 days, i.p.), control+B.E (barberry extract, 160 mg/kg/day during 10 days, i.p.), Cis (cisplatin, 8 mg/kg on 7th day, i.p.), Cis+Ber (berberine, 100 mg/kg/day during 10 days; cisplatin, 8 mg/kg on 7th day), Cis+B.E (barberry extract, 160 mg/kg/day during 10 days; cisplatin, 8 mg/kg on 7th day). After placing the rats in metabolic cages for 24 h, blood, urine, liver and kidney tissue samples were collected. RESULTS: Compared to control, control+Ber and control+B.E groups, cisplatin administration led to kidney and liver dysfunction. These happened with diminished activities of antioxidant enzymes, increased levels of malondialdehyde, Toll-like receptor 4 gene expression and histological damages in hepatic and renal tissues. Berberine and barberry extract administration decreased all the changes. CONCLUSIONS: An intensification in enzymatic oxidant status, decrease in lipid peroxidation with decrease in TLR4 gene expression level indicate that barberry extract may be a potential candidate in combating cisplatin-induced oxidative stress and inflammation in liver and kidney tissues through its constituent berberine.

Electron microscopy study of 496 cases of lupus nephritis: A single-center experience.

INTRODUCTION: Renal involvement is seen in about 40-82% of systemic lupus erythematosus (SLE) Asian patients. The exact diagnosis and classification of lupus nephritis are important for treatment and prognosis. This study aimed to investigate the value of electron microscopy (EM) in the diagnosis and classification of lupus nephritis compared with light microscopy. METHOD: In this cross-sectional referral-center 16-year study of lupus nephritis, the final diagnosis was based on the EM study. Primary light microscopy findings were compared with EM diagnosis. Moreover, Immunofluorescence patterns distribution was assessed. RESULTS: From 496 patients diagnosed with lupus nephritis based on EM, 225(45.4%) of patients were categorized in class IV, followed by 98(19.7%), 93(18.8%), 46(9.3%), and 14(2.8%) who were categorized into classes of II, III, V, and VI respectively. Only 1(0.2%) patient belonged to class I, and 19(3.8%) cases were diagnosed with mixed two classes. Using EM was essential for diagnosing 25.6% of cases taking the correct classification by light microscopy into account; however, disregarding correct classification, this could change to a 7.4% contribution rate of EM. The most common cause of misdiagnosis, disregarding incorrect classification, was inadequate or wrong tissue. Positive associations were detected between tubular atrophy and interstitial fibrosis of both electron and light microscopy with different classes (P < 0.001). CONCLUSION: While light microscopy is highly accurate for diagnosing lupus nephritis regardless of correct classification, EM contributes substantially to the correct classification of lupus nephritis types.

A 16-year Survey of Clinicopathological Findings, Electron Microscopy, and Classification of Renal Amyloidosis.

Background: Electron microscopy (EM) is a valuable tool in the diagnosis of renal amyloidosis, particularly in the early stages of the disease. In Iran, studies on EM and the clinical features of renal amyloidosis are scarce. The objective of the present study was to survey EM investigations, pathological classifications, and clinical features of renal amyloidosis. Methods: This cross-sectional study was performed in Shiraz, Iran, during 2001-2016. Out of 2,770 kidney biopsies, 27 cases with a diagnosis of renal amyloidosis were investigated. EM investigation and six staining procedures for light microscopy (LM) were performed. Two pathological classifications based on glomerular, peritubular, perivascular, and interstitial involvement were made. Finally, the association between these classifications and the clinical features was assessed. Chi-square, Fisher's exact, Independent t test, and Multiple logistic regression analysis were used. P values<0.05 were considered statistically significant. Results: In 51.9% of the cases, the clinical diagnosis was nephrotic syndrome. Proteinuria and edema were the most prevalent clinical manifestations. The role of EM investigation for diagnosis was graded "necessary" or "supportive" in 48.2% of the patients. In the classification based on glomerular classes, variables such as the mean blood pressure (P=0.003), history of hypertension (P=0.02), creatinine >1.5 (P=0.03), and severe tubular atrophy (P=0.03) were significantly higher in class B (advanced amyloid depositions). Conclusion: EM plays an important role in the diagnosis of renal amyloidosis. EM in conjunction with LM investigation with Congo red staining is recommended, to prevent misdiagnosis of patients with a clinical suspicion of renal amyloidosis. Among different pathological features of renal amyloidosis, the severity of glomerular amyloid depositions had a clear relationship with clinical presentations.

Comparison of ischemic and ischemic/reperfused kidney injury via clamping renal artery, vein, or pedicle in anesthetized rats.

PURPOSE: We recently observed that 30 min of bilateral renal arterial, venous, or pedicle occlusion with 2-h reperfusion differentially induced acute kidney injury (AKI), which was suggested to be probably resulted from their directly exerting dissimilar impacts on kidney during the ischemic period. The present study was further designed to evaluate and prove this suggestion. METHODS: Anesthetized male Sprague-Dawley rats were divided in two distinct supragroups with 30-min bilateral renal ischemia alone (BI) or followed by 30-min reperfusion (BIR), which each had four different groups (n = 8) of subjecting to renal artery, vein, or pedicle clamping and also sham operation. RESULTS: In the BI groups, artery clamping caused lower renal tissue injury than pedicle clamping but vein occlusion caused the highest levels of kidney histological damages along with the widespread hemorrhagic congestion. In the BIR groups, renal vascular congestion, intratubular cast, and edema decreased, but tubular epithelial injury did not significantly change in comparison to their equivalents BI groups. However, the orders of total renal tissue damages in the BIR groups were still as clamping renal veins > > pedicles > arteries and in association with their proportionally developed renal hemodynamic, excretory, and urine-concentrating dysfunctions. CONCLUSION: The transmission of high arterial pressure into renal microvessels and rupturing of their walls during venous-clamping augment, but the retrograde blood flow from veins into kidney during artery clamping attenuates induction of renal tissue injury with respect to pedicle clamping not only at the ischemic period but also at the early reperfusion period and along with the proportional development of renal dysfunctions.

Protective effect of genistein in a rat model of ischemic acute kidney injury.

BACKGROUND: This study determined the possible anti-inflammatory and antioxidant renal protective effect of genistein, a soy isoflavone, against kidney damage and functional disorders following renal ischemia/reperfusion (I/R) in male rats. MATERIALS AND METHODS: The animals were dedicated to five groups (n = 7 per group): Sham, Sham + Geni (genistein, 15 mg/kg in 1 ml 1% DMSO, i.p.), Sham + DMSO (1 ml 1% DMSO, i.p.), I/R (bilateral renal ischemia for 45 min followed by 24 h reperfusion), I/R + Geni (genistein, 15 mg/kg). 24-h urine samples, blood and tissue samples of the kidney were collected at the end of 24 h reperfusion period. RESULTS: Compared to sham, sham + Geni and sham + DMSO groups, IR injury (IRI) ended in kidney dysfunction (decreased creatinine clearance, and increased fractional excretion of sodium), increased levels of malondialdehyde, decreased activities of antioxidant enzymes (superoxide dismutase, gluthatione peroxidase, and catalase), increased gene expression levels of TLR4 (Toll-like receptor 4) and TNF-α (tumor necrosis factor-alpha), as well as histological damages in kidney tissue. Genistein administration decreased all the changes. Therefore, genistein apparently protects the kidney against IRI by mitigating both oxidative stress and inflammation. The antioxidant and anti-inflammatory properties of genistein probably exert important roles in improving functional disorders and offer renal protection against IRI.

Remote Ischemic Perconditioning Modulates Apelin Expression After Renal Ischemia-Reperfusion Injury.

BACKGROUND: Renal ischemia/reperfusion injury (IRI) can result in impaired ability of urine concentration and increased sodium fractional excretion. Apelin, a (neuro) vasoactive peptide, enhances diuresis by increasing the renal microcirculation and by counteracting the antidiuretic effect of arginine vasopressin on the tubules. However, changes in renal apelin expression in renal IRI rat model have not been elucidated. Remote ischemic perconditioning (RIPerC) improves renal sodium and water handling after IRI. Here, we investigated whether RIPerC prevents dysregulation of renal sodium and water handling in response to IRI by apelin signaling pathway in rats. MATERIALS AND METHODS: Renal IRI was induced by 45-min clamping of renal arteries followed by 24 h reperfusion. RIPerC was created by applying four cycles of 2-min ischemia of the left femoral artery followed by 3-min reperfusion at the start of renal ischemia. Rats were randomly divided into sham, ischemia/reperfusion, and RIPerC + ischemia/reperfusion groups. Urine and blood were sampled after reperfusion period. The kidney was harvested for mRNA isolation and histopathological study. RESULTS: IRI resulted in decreased clearance of creatinine, increased sodium fractional excretion, and reduced urine osmolality compared with sham animals. This occurred with an increase in mRNA expression levels of apelin and histological damages in both cortical and medullary regions of kidney tissues. RIPerC treatment ameliorated all these changes. CONCLUSIONS: This study showed that RIPerC has protective effects against dysregulation of renal sodium and water handling after renal IRI, which might be related with inhibition of apelin signaling pathway.

Comparison of ischaemia-reperfusion-induced acute kidney injury by clamping renal arteries, veins or pedicles in anaesthetized rats.

NEW FINDINGS: What is central question of this study What are the differences between a traditional renal pedicle-clamping model of acute kidney injury and models with occluded renal artery or vein alone in rats? What is main finding and its importance? During renal venous occlusion, transmission of high arterial pressure into renal capillaries is likely to have caused the rupture of their walls and the occurrence of haemorrhagic congestion that led to higher kidney tissue damage and dysfunction than with pedicle and artery clamping. ABSTRACT: Animal models of ischaemic acute kidney injury (AKI) are valuable tools, but their therapeutic outcomes are not usually translated to humans. Ischaemic AKI in murines is mostly induced via renal pedicle clamping, which is different from patients with AKI that is due to renal artery hypoperfusion or vein thrombosis. This study was designed to compare the traditional pedicle-clamping with artery or vein occlusion alone in rat models of bilateral renal ischaemia-reperfusion (BIR). Twenty-eight anaesthetized male Sprague-Dawley rats were divided into four groups, a sham-operation group and groups that underwent 2 h reperfusion following 30 min clamping of renal arteries (BIR-A group), veins (BIR-V group) or pedicles (BIR-P group). The levels of epithelial injury in proximal tubules and thick ascending limb, intratubular casts and vascular congestion as well as renal malondialdehyde were moderately lower in the BIR-A than BIR-P group, while the BIR-V group showed much higher degrees of these damages than both these groups along with massive haemorrhagic congestion. Accordingly, renal blood flow, glomerular filtration, Na+ reabsorption, K+ and urea excretion, free water reabsorption and urine osmolality were lower in the BIR-V group than in the BIR-A and BIR-P groups, while the BIR-P group had slightly worse renal functional disorders than the BIR-A group. It seems that transmission of high arterial pressure into renal microvessels during venous occlusion causes rupture of capillary walls and haemorrhagic congestion, which leads to intensive kidney injury. In conclusion, the differences in renal disturbances induced by artery, vein and pedicle clamping strongly suggest use of a proper experimental model for each type of human ischaemic AKI.

The role of nitric oxide in the protective action of remote ischemic per-conditioning against ischemia/reperfusion-induced acute renal failure in rat.

OBJECTIVES: We investigated the role of nitric oxide (NO) in the protective effects of remote ischemic per-conditioning (rIPerC) on renal ischemia/reperfusion (I/R) injury in male rats. MATERIALS AND METHODS: I/R treatment consisted of 45 min bilateral renal artery ischemia and 24 hr reperfusion interval. rIPerC was performed using four cycles of 2 min occlusions of the left femoral artery and 3 min reperfusion at the beginning of renal ischemia. The animals were given normal saline (vehicle), NG-nitro-L-arginine methyl ester (L-NAME) or L-arginine. Following the reperfusion period, renal functional- and oxidative stress- parameters, as well as histopathological changes were assessed. RESULTS: In comparison with the sham group, I/R resulted in renal dysfunction, as indicated by significantly lower creatinine clearance and higher fractional excretion of sodium. This went along with decreased glutathione peroxidase (GPX) and catalase (CAT) activity in the I/R group, increased malondialdehyde (MDA) contents and histological damages. In comparison with the I/R group, the rIPerC group displayed improved renal function, increased activity of GPX and CAT enzymes, and decreased MDA level. However, these effects were abrogated by L-NAME injection and augmented by L-arginine treatment. CONCLUSION: According to the results, the functional and structural consequences of rIPerC against I/R-induced kidney dysfunction, which is associated with reduction of lipid peroxidation and intensification of anti-oxidant systems, is partially dependent on NO production.

The protective effect of hydroalcoholic extract of Ginger (Zingiber officinale Rosc.) against iron-induced functional and histological damages in rat liver and kidney.

OBJECTIVE: Iron overload in the body is related with toxic effects and threatens the health. The aim of this study was to evaluate the protective role of hydroalcoholic extract of ginger (Zingiber officinale) against ferrous sulfate-induced hepatic and renal functional disorders and histological damages in rats. MATERIALS AND METHODS: The rats were divided into four groups (n=7): Sham, Sham + G.E (ginger extract, 400 mg/kg/day for 14 days), FS (ferrous sulfate, 30 mg/kg/day for 14 days), FS+G.E (ferrous sulfate, 30 mg/kg/day for 14 days; ginger extract, 400 mg/kg/day for 11 days from the fourth day of ferrous sulfate injection). After 24 hr, blood, urine and tissue samples were collected. RESULTS: Compared with Sham and Sham + G.E groups, administration of ferrous sulfate resulted in liver and kidney dysfunction as evidenced by significantly higher levels of serum hepatic markers and bilirubin, and lower levels of serum albumin, total protein, triglyceride, cholesterol and glucose, as well as lower creatinine clearance and higher fractional excretion of sodium (p<0.001). This was accompanied by increased malondialdehyde levels and histological damages (p<0.001). In the FS + G.E, ginger extract significantly (p<0.01) reversed the levels of serum hepatic markers, renal functional markers and lipid peroxidation marker. Furthermore, it restored the levels of serum total protein, albumin, glucose, triglycerides and cholesterol and decreased bilirubin concentration in the blood. All these changes were corroborated by histological observations of liver and kidney. CONCLUSION: In conclusion, ginger extract appears to exert protective effects against ferrous sulfate-induced hepatic and renal toxicity by reducing lipid peroxidation and chelating iron.

Renal ischemia/reperfusion against nephrectomy for induction of acute lung injury in rats.

PURPOSE: Acute kidney injury (AKI) induces acute lung injury (ALI) through releasing injurious mediators or impairing clearance of systemic factors. To determine the links between AKI and ALI, pulmonary and blood variables were evaluated following induction of AKI via different experimental models of bilateral renal ischemia/reperfusion (BIR: renal ischemia with uremia), unilateral renal ischemia/reperfusion (UIR: renal ischemia without uremia), bilateral nephrectomy (BNX: uremia without renal ischemia), and unilateral nephrectomy (UNX: without uremia and renal ischemia). METHODS: Ninety male Sprague-Dawley rats were divided into six groups. Animals had 1-h bilateral or 2-h unilateral renal ischemia followed by 24-h reperfusion in the BIR and UIR groups, respectively, and 24-h period following bilateral or unilateral nephrectomy in the BNX and UNX groups, respectively. There were also sham and control groups with and without sham-operation, respectively. RESULTS: Plasma malondialdehyde and nitric oxide were elevated by BIR more than UIR, but not changed by UNX and BNX. UIR slightly increased plasma creatinine, whereas BIR and BNX largely increased plasma creatinine, urea, K+ and osmolality and decreased arterial HCO3-, pH, and CO2. UNX and UIR did not affect lung, but BIR and BNX induced ALI with equal capillary leak and macrophages infiltration. However, there were more prominent lung edema and vascular congestion following BNX and more severe neutrophils infiltration and PaO2/FiO2 reduction following BIR. CONCLUSION: Acutely accumulated systemic mediators following renal failure in the absence of kidneys vary from those due to combined renal failure with ischemic-reperfused kidneys and consequently they induce ALI with distinct characteristics.

A1 -Adenosine receptor activation has biphasic roles in development of acute kidney injury at 4 and 24 h of reperfusion following ischaemia in rats.

NEW FINDINGS: What is the central question of this study? A1 -Adenosine receptor (A1 AR) blockade before renal ischaemia aggravated kidney injury after 24 h reperfusion in several studies, whereas we previously observed a renoprotective effect of A1 AR blockade during a 4 h reperfusion period. What are the underlying mechanisms for this biphasic effect of pretreatment with an A1 AR antagonist at 4 and 24 h reperfusion? What is main finding and its importance? A1 -Adenosine receptor blockade protects the kidney against ischaemia-induced injury during the early hours of reperfusion by attenuating the reduction in renal blood flow and lowering energy expenditure, whereas its inflammatory effects gradually dominate over 24 h reperfusion to intensify kidney injury. We previously reported that selective blockade of the A1 -adenosine receptor (A1 AR) with an antagonist, 8-cyclopentyl-1,3-dipropylxanthine (DPCPX), had protective effects on renal ischaemia-induced structural and functional disruption during a 4 h reperfusion period. In contrast, several studies demonstrated that endogenous and exogenous A1 AR activation before renal ischaemia had a renoprotective role 24 h after reperfusion, through mechanisms that reduced inflammation, necrosis and apoptosis. In this study, we investigated potential mechanisms underlying this biphasic action of A1 AR in renal ischaemia-reperfusion injury. Anaesthetized male Sprague-Dawley rats underwent 30 min of bilateral renal ischaemia, and biphasic effects of pretreatment with DPCPX at 4 and 24 h reperfusion were studied on the kidney injury. Pretreatment with DPCPX attenuated at 4 h but augmented at 24 h reperfusion the renal ischaemia-induced histological damage, reductions in creatinine clearance, urea excretion and free-water reabsorption, and increases in bicarbonate excretion and tissue malondialdehyde. The DPCPX increased tumour necrosis factor-α expression and migration of lymphocytes in the postischaemic kidney at both time points, but with a different pattern; lymphocytes mostly aggregated in cortical periarterial spaces at 4 h reperfusion but had infiltrated into the interstitium at 24 h reperfusion. In conclusion, A1 AR activation contributes to ischaemia-induced acute kidney injury during the early hours of reperfusion by causing a greater reduction in renal haemodynamics and by elevating tubular energy expenditure, which overcome its anti-inflammatory effect. However, its anti-inflammatory actions are exerted by reducing lymphocyte infiltration and cytokine production that begins to dominate from 4 to 24 h of reperfusion, which is reflected in attenuation of renal structural and functional disruption.

Morphologic changes of corneal epithelial flap: ethanol-mediated versus mechanical removal.

PURPOSE: To evaluate morphologic changes in human corneal epithelial flap removed mechanically or after ethanol application. METHOD: Epithelial corneal flap was removed after ethanol application (20 eyes) or mechanically (19 eyes). Any changes were studied by transmission electron microscopy. RESULTS: Thirty-nine eyes were enrolled in the study. The following changes were found in the alcohol-applied group: apoptotic cells, membrane-bound blebs with marked dilatation of endoplasmic reticulum, and short intercellular cleavage with approximately one-third of cell length depth. In mechanical debridement, cleavages extended more than half of the cell length by tearing hemidesmosomes. CONCLUSION: Alcohol application leads to cell damage in basal epithelial cells but cleavage plane remains smooth. Generally, none of the methods caused trauma to the basement membrane.

Accuracy of intra-operative frozen section consultation in south of Iran during four years.

BACKGROUND: Accuracy of intraoperative frozen section diagnosis is an important part of quality control in surgical pathology. In this study we try to evaluate the frozen section diagnosis in our center, a referral center in southern Iran. MATERIALS AND METHODS: During the four-year-period of study, all the frozen sections in the affiliated hospitals of Shiraz University of Medical Sciences were evaluated. Discrepant cases were studied to find out reasons for their inaccuracies. RESULTS: In the four years, 759 frozen sections have been done, 25 of which showed discordant results. The most common site of frozen section and discrepancy was in central nervous system tumors. The reason for inaccuracy in frozen section diagnosis in 52% of cases was proved to be interpretative, 44% sampling error and the remainder due to lack of clinical information of the pathologist. CONCLUSION: Accuracy of our intraoperative consultation is comparable with other centers in Western countries. Most of the discrepancies can be prevented by providing more clinical information for the pathologist and more accurate sampling.