Fine-needle Aspiration Biopsy Of Liver Research Articles

The Authors' Reply: Organoid Technology: Are Human Cholangiocyte Organoids Immune Protected?

We thank Ekser et al1 for reading our commentary2 on the groundbreaking Science publication by Sampaziotis et al3 on repair of bile ducts after transplantation of cholangiocyte organoids in human liver grafts. Ekser et al1 comment that if allogenic cholangiocyte organoids would be used for graft repair, this potentially can provoke an alloimmune response. The authors are “puzzled by the outcome of the original paper3 as nonautologous organoids were used and no protective immunosuppressive medication is used during the normothermic machine perfusion of the liver grafts.” Indeed, cholangiocytes or cholangiocyte organoids, in this matter, are not blinded from the immune system as they normally express major histocompatibility complexes (HLA) on their cell surface. However, in this study, like many studies with human liver normothermic machine perfusion,4 leukocyte-depleted packed red blood cells in gelofusine were used for oxygenation of the graft and therefore completely lacked alloreactive immune cells. Therefore, no acute rejection of the engrafted organoids was observed, even despite no immunosuppressive treatment was given. In case such a cell-therapy repaired graft would be actually transplanted, immunosuppressive medication is inevitably needed to prevent rejection of the donor allograft and the third party allogenic organoid cell alike. Exposing the immune system to extra allogenic cells might nonetheless increase the risk of an alloimmune response. Therefore, as mentioned in our Game Changer commentary: “For graft repair, ideally, autologous cells of the recipient should be used to diminish the risk of alloimmune responses.”2 Safe means to obtain autologous cells for organoid initiation are required. Indeed, cholangiocyte organoids can now be cultured from liver fine-needle aspiration biopsies or from bile, obtained via percutaneous drainage of the gallbladder or via endoscopic retrograde cholangiopancreatography,5 with very limited risks. The future recipient’s cholangiocyte organoids could thus be stored in a biobank at enlistment for liver transplantation, making patient-derived cells directly available for regenerative purposes at the time of a donor liver offer. The bottleneck in using autologous cholangiocyte organoids is not so much culturing the recipient’s own cells but the logistics of having sufficient numbers of cells at the moment of transplantation. Sampaziotis et al3 used approximately 10 million cells for injecting single liver segment 3. To treat the whole graft, at least 10 times this number of cells would be required to treat the entire biliary tree of a full-size liver. To reach high amounts of organoid cells in a short time, more efficient culturing methods are being developed.6 Cryopreservation protocols should be optimized to store large quantities of cholangiocyte organoids, which could be directly administered to the liver after thawing. Taken together, we consider that ideally, autologous cells should be used in graft repair to minimize the risk of an alloimmune response. Logistic problems need to be solved before this regenerative approach can be clinically implemented. We foresee that this way of repairing the biliary tree will evolve from bench to bedside in the upcoming years.

A RARE PNEUMOBILIA CASE CAUSED BY LIVER ABSCESS

Pneumobilia is the accumulation of gas in the biliary tract. A 72-year-old woman was brought tour emergency department with complaints of abdominal pain and general condition. The patient, who described pain in the right lumbar region, had mild distension and right hypochondriac tenderness during the abdominal examination. In contrast-free abdominal tomography, one hypodense lesion was observed at the right and left lobe junction level of the liver. There was an air view in the main bile duct and intra-hepatic bile ducts. The patient was admitted to the general surgery intensive care unit. Liver abscess was found as a result of abdominal imaging and liver fine needle aspiration biopsy performed. After a 2-week intensive care follow-up the patient was discharged with oral antibiotics due to the complete absence of regression and general condition of the patient. Pneumobilia is a rare condition with a high mortality and liver abscess should also be considered among its causes.

Fine-Needle Aspiration Biopsy of Neuroendocrine Neoplasms in the Liver: A 9-Year Retrospective, Single Institutional Study

Abstract Objectives Neuroendocrine neoplasms (NENs) are divided into two major categories according to the 2018 WHO expert consensus proposal: well-differentiated neuroendocrine tumors (NETs) and poorly differentiated neuroendocrine carcinoma (NECs). Primary liver NENs are very rare and majority of NENs in the liver are metastatic. In this study, we retrospectively reviewed NENs in the liver diagnosed by fine-needle aspiration biopsy (FNAB). Methods We retrospectively reviewed malignant neoplasms diagnosed by imaging-guided liver FNAB performed between 2010 and 2018 in our institution. Patients’ electronic medical records and selective slides were reviewed. Results A total of 624 liver FNAB cases with a diagnosis of positive for malignancy were identified. Among those cases, 56 cases (9%) were NENs. Poorly differentiated NECs constituted 71% (40/56) of cases, and well-differentiated NETs constituted 29% (16/56) of cases. The most common primary sites of poorly differentiated NECs were lung (35%, 13/40), followed by pancreas (20%, 8/40), GI tract (17.5%, 7/40), GU (7.5%, 3/40), breast (5%, 2/40), head and neck (2.5%, 1/40), GYN tract (2.5%, 1/40), and CNS (dedifferentiated glioblastoma, 2.5%, 1/40). The remaining three cases (7.5%, 3/40) were diagnosed as primary liver NECs based on the clinical presentation. All three patients presented with a single large liver mass with no other mass lesions identified in other organ systems. For well-differentiated NETs, majority were from the GI tract (81.3%, 13/16), followed by pancreas (6.25%, 1/16) and lung (6.25%, 1/16). One patient was diagnosed with metastatic NET of unknown primary. Conclusion NENs in liver are predominantly metastatic, and majority of them are high-grade NECs. Most of well-differentiated NETs are metastases from the GI tract, while more than one-third of poorly differentiated NECs are metastases from the lung. Primary liver NENs also occur and all three cases in this study are poorly differentiated NECs.

Clinico-hemato-biochemical profile of dogs with liver cirrhosis.

Aim:The aim of this study was to determine the relevant tools in the diagnosis of liver cirrhosis in dogs.Material and Methods:A total of 140 dogs presented at Veterinary Teaching Hospital, Guru Angad Dev Veterinary and Animal Sciences University, Ludhiana, showing clinical signs of hepatic insufficiency were subjected to clinico-hemato biochemical, urological, ultrasonographic (USG), and USG guided fine-needle biopsy examinations by standard methods. On the basis of these results, 6 dogs out of 140 dogs were found to be suffering from liver cirrhosis. Six clinically healthy dogs constituted the control group.Results:The dogs suffering from liver cirrhosis manifested inappetence, halitosis, abdominal distension, weight loss, melena, icterus, anemia, and neutrophilic leukocytosis with the left shift. Levels of hemoglobin, lymphocytes, packed cell volume, mean corpuscular volume, mean corpuscular Hb (MCH), and platelet count were significantly lower in liver cirrhosis group than control group while total leukocyte count, neutrophils, and MCH concentration were significantly higher. Glucose, total protein, albumin, A/G ratio, and fibrinogen were significantly lower, and creatinine, alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, prothrombin time, and APTT were significantly higher than the control values. Ultrasound revealed diffuse increase in echogenicity with rounded and irregular liver margins. Cytological examination of the ascitic fluid and fine-needle aspiration biopsy of liver was not fruitful in the diagnosis of liver cirrhosis.Conclusions:Liver cirrhosis causes clinical and hemo-biochemical alterations, which require special consideration when treating diseased animals. USG, diffuse increase in echogenicity of liver, rounding and irregularity of liver margins and microhepatica were the consistent findings. It is suggested that USG along with hemo-biochemical alterations may be used as a diagnostic tool for liver cirrhosis in dogs.

On‐site adequacy evaluations performed by cytotechnologists

Practice patterns regarding on-site assessment of the adequacy of image-guided fine-needle aspiration biopsies (FNABs) vary among laboratories, but in many laboratories primary responsibility rests with the cytotechnologists. On-site evaluation provides feedback on the need for additional passes and facilitates triaging of the specimen for time-sensitive ancillary studies. Prior studies have suggested that cytotechnologists can assess the initially obtained specimens correctly, but they are few in number and limited by small size. The purpose of this study was to assess the frequency with which our cytotechnologists were able to correctly assess specimens as adequate using a large-scale database that included a wide range of image-guided FNABs. The frequency that on-site adequacy assessments of 5241 image-guided FNABs were correct was determined by correlating the cytotechnologists' assessments of adequacy with the final cytologic interpretation. An adequacy assessment was considered correct if the FNAB was ultimately reported as satisfactory and unequivocally benign or malignant. An adequate reading on a case that was ultimately reported as unsatisfactory, atypical, or suspicious was deemed "incorrect." The effect of imaging modality was also analyzed. Of 5241 FNABs, 2784 (53%) were interpreted as adequate on site. Of these, 2637 (95%) were correctly considered adequate. Of the common biopsy sites sampled, the adequacy assessments for liver FNABs demonstrated the highest frequency for being correctly considered adequate (97%) and those for kidney FNABs showed the lowest (90%). Imaging modality had no effect on accuracy. Cytotechnologists are almost always correct when assessing initial FNAB samples as adequate.

Utility of glypican‐3 and survivin in differentiating hepatocellular carcinoma from benign and preneoplastic hepatic lesions and metastatic carcinomas in liver fine‐needle aspiration biopsies

Glypican-3 (GPC-3), a membrane-anchored heparin sulfate proteoglycan, has been shown to be expressed in approximately 80% of hepatocellular carcinoma (HCC) but not in benign hepatic lesions. Survivin, a novel inhibitor of apoptosis, and a prognostic marker, has also been expressed in HCC. We evaluated these two immunomarkers (GPC-3 and survivin) in differentiating HCC from benign and preneoplastic hepatic lesions and metastatic carcinomas, comparing them to HepPar-1 (hepatocyte paraffin-1) in liver fine-needle aspiration biopsies (FNAB).Immunohistochemistry for GPC-3, survivin and HepPar-1 was performed on 92 FNAB including HCC, hepatic cirrhosis, focal nodular hyperplasia (FNH), hepatic adenoma, dysplastic hepatic nodules and metastatic carcinomas. Immunostaining was scored as positive, if > or =10% of tumor cells stained.GPC-3 is immunoexpressed in 56.8% of HCC, but not in benign and preneoplastic hepatic lesions, or metastatic carcinomas; whereas survivin is expressed in HCC (86.4%), benign hepatic lesions (85.7%), dysplastic hepatic nodules (100%) and metastatic carcinomas (94.3%). HepPar-1 is immunoexpressed in HCC (72.7%), benign hepatic lesions (100%), dysplastic nodules (100%) and metastatic carcinomas (2.9%). The sensitivity and specificity of GPC-3, survivin and HepPar-1 for detection of HCC are 56.8 and 100%, 86.4 and 6.3%, 72.7 and 70.8%, respectively.GPC-3 is a reliable and more specific immunohistochemical marker than survivin for the diagnosis of HCC in FNAB. HepPar-1, although a more sensitive marker than GPC-3, has a lower specificity for detection of HCC. Our data supports the potentially significant diagnostic utility of GPC-3 in FNABs in differentiating primary malignant from benign and preneoplastic liver lesions, and metastatic carcinomas.

HepPar1, MOC-31, pCEA, mCEA and CD10 for Distinguishing Hepatocellular Carcinoma vs. Metastatic Adenocarcinoma in Liver Fine Needle Aspirates

To investigate immunohistochemical staining of hepatocyte paraffin-1 (HepPar1), alpha-fetoprotein (AFP), polyclonal carcinoembryonic antigen (pCEA), monoclonal CEA (mCEA), MOC-31 and CD10 for differential diagnosis of hepatocellular carcinoma (HCC) from metastatic adenocarcinoma (MA) on fine needle aspiration biopsy (FNAB).Fifty-one archival, paraffin-embedded FNAB cell blocks, representing 18 HCCs and 33 MAs, were immunostained with antibodies for AFP, CD10, pCEA, mCEA, HepPar1 and MOC-31.HepPar1, AFP, canalicular pCEA and CD10 were positive in 78% (14 of 18), 28% (5 of 18), 72% (13 of 18) and 35% (6 of 17) of cases of HCC, respectively. The 33 MAs were negative for immunostaining of the above antibodies except for one AFP-positive MA. Ninety-seven percent (31 of 32) of the MAs and 6% (1 of 17) of the HCCs were positive for MOC-31. Monoclonal CEA was immunoreactive on 82% (27 of 33) of the MAs and negative on all the HCCs.HepPar1 was the most sensitive marker for HCC, followed by canalicular staining for pCEA. For MA, MOC-31 was the most sensitive marker; mCEA was slightly less sensitive but more specific. We suggest using HepPar1, pCEA, CD10, MOC-31 and mCEA as a panel for distinguishing HCC from MA in liver FNAB.

Evaluation of the CD34 and CD10 immunostains using a two‐color staining protocol in liver fine‐needle aspiration biopsies

Fine-needle aspiration (FNA) biopsies of liver masses may need immunohistochemistry to resolve difficult cases. We examined liver FNAs with CD10 and CD34 using a two-color staining protocol. Fifty-one cases of hepatocellular carcinoma (HCC) and 23 cases of liver metastases were stained first with CD10 using diaminobenzidine as a chromogen and then with CD34 using Fast Red. All cases were reviewed in a blinded fashion by two of the authors (R.I.Z. and Z.B.). Diagnoses were unblinded and staining patterns evaluated. Forty-eight of 51 cases of HCC stained for CD34 in a peripheral pattern and no cases of metastases stained for CD34 Seven of 23 cases of metastatic disease stained for CD10 in a diffuse cytoplasmic distribution and 29 of 51 HCCs stained in a canalicular pattern. Overall, the sensitivity of the CD10/CD34 combination was 98% and the specificity was 100% for HCC when appropriate staining patterns were observed. Two-color immunostaining with CD10 and CD34 may prove useful in distinguishing HCC from metastases.

Diagnostic value of hepatocyte paraffin 1 antibody to discriminate hepatocellular carcinoma from metastatic carcinoma in fine-needle aspiration biopsies of the liver.

Diagnosing liver tumors by fine-needle aspiration biopsy is safe and accurate. However, there are cases that prove diagnostically difficult. Traditionally, immunostains for alpha-fetoprotein and polyclonal carcinoembryonic antigen have been used to distinguish adenocarcinomas from hepatocellular carcinomas (HCCs). In poorly differentiated tumors, these immunostains have limitations in both sensitivity and specificity. An hepatocyte-specific immunostain has been described in the surgical pathology literature. To the authors' knowledge, this hepatocyte antibody has not been studied in liver fine-needle aspiration biopsies. The authors examined the Hepatocyte Paraffin 1 (HP1) antibody for its diagnostic utility in this cytologic setting. Cell-block material from 40 cases of HCC and 53 cases of metastatic adenocarcinoma were studied. Slides were stained for HP1 by the avidin-biotin complex method following antigen retrieval. The percentage of malignant cells that exhibited coarse granular staining in the cytoplasm was estimated for all cases of HCC, poorly differentiated HCC, and metastatic adenocarcinoma. HP1 was expressed in 83% of all HCCs but in only 56% of poorly differentiated HCCs. Only 2 of 53 (4%) of metastatic tumors expressed HP1. The overall sensitivity of HP1 was 79% and its specificity was 96%. HP1 was found to be a specific immunostain that may prove helpful in diagnosing all but the most undifferentiated liver tumors biopsied by fine-needle aspiration.

Spindle-cell lesions of the liver: diagnosis by fine-needle aspiration biopsy.

Rarely, spindle-cell lesions in liver fine-needle aspiration biopsies (FNABs) are encountered. A retrospective review of our experience with lesions that are mesenchymal in origin or appearance was undertaken to elucidate the frequency and spectrum of these lesions. Image-guided liver FNABs performed over a 3-year period (n = 585) at our institution (1996-1998) were retrospectively evaluated. Cytologic smears, cell block preparations, and clinical follow-up of lesions with spindle-cell morphology were reviewed. Twenty-nine of 585 cases were of spindle-cell morphology (5%). Hemangiomas (n = 12, 41%) and metastatic sarcomas (n = 6, 21%) comprised the largest categories, followed by granulomatous inflammation (n = 3, 10%). Other cases included primary angiosarcoma and fibrolamellar hepatocellular carcinoma. The most frequent spindle-cell liver lesion encountered is hemangioma, followed by metastatic leiomyosarcoma and granulomatous hepatitis. Awareness of diagnostic possibilities, special attention to specimen adequacy, and use of ancillary procedures can maximize diagnostic yield.

Immunocytochemical Staining of Kupffer and Endothelial Cells in Fine Needle Aspiration Cytology of Hepatocellular Carcinoma

To evaluate the significance of the presence of Kupffer and endothelial cells in distinguishing hepatocellular carcinoma (HCC) and adenocarcinoma (AC) on cytologic smears. Fine needle aspiration biopsies (FNABs) from 43 cases, 21 HCC and 22 AC (8 primary and 14 metastatic), were immunostained for Factor VIII and vimentin as markers for endothelial cells and Kupffer cells, respectively. Cytologic diagnosis was verified by histologic and/or clinical follow-up. Eighteen of the 21 cases (86%) of HCC and 11 (5 primary and 6 metastatic) of 22 cases (50%) of AC showed positive immunostaining for Factor VIII (P = .02). Vimentin immunostaining was positive in 55% of HCCs and 41% of ACs (P = .74). Forty-five percent of cases of HCC showed immunostaining for both Factor VIII and vimentin, while 22% of cases of AC showed immunostaining for both Factor VIII and vimentin (P = .18). Immunocytochemical identification of endothelial cells using Factor VIII may have important diagnostic value in separating HCC from adenocarcinomas in liver FNABs. The presence of Kupffer cells labeled with vimentin has no diagnostic significance in FNAB of these tumors.

MOC-31 aids in the differentiation of metastatic adenocarcinoma from hepatocellular carcinoma

Fine-needle aspiration biopsy (FNAB) is frequently used to diagnose mass lesions in the liver. Differentiating metastatic adenocarcinoma from primary hepatocellular carcinoma can be difficult. Despite a number of morphologic criteria, there remain occasional cases in which the cytologic features fail to resolve this differential reliably; in these cases ancillary studies may be useful. Recently, it has been reported that the antibody MOC-31 reliably separates metastatic adenocarcinoma from hepatocellular carcinoma. In this study we examine the utility of MOC-31 in liver FNAB material. Thirty-three archival, alcohol-fixed, paraffin-embedded cell blocks representing 17 cases of hepatocellular carcinomas and 16 cases of metastatic adenocarcinoma were retreived. After protease digestion, the sections were immunostained with the antibody MOC-31 (Dako, Carpinteria, CA) utilizing a modified avidin-biotin complex technique. Only membrane-based reactivity was considered positive. In five cases there was insufficient diagnostic material remaining in the cell block for immunohistochemical staining. Among the remaining cases, MOC-31 reactivity was observed in 10 of 12 metastatic adenocarcinomas and 2 of 16 hepatocellular carcinomas. For metastatic adenocarcinoma the presence of MOC-31 reactivity yields a sensitivity of 83%, a specificity of 87%, a positive predictive value of 83%, and a negative predicitive value of 87%. MOC-31 is useful in separating metastatic adenocarcinoma from hepatocellular carcinoma in FNAB cell block material. Cancer (Cancer Cytopathol)

Fine Needle Aspiration Biopsy of the Liver: Principal Diagnostic Challenges

Fine needle aspiration biopsy (FNAB) is the diagnostic procedure of choice for evaluation of liver lesions. Although primarily applied to malignant disease, it is also used in the evaluation of benign conditions. Improvements in imaging techniques and advances in cytologic interpretations, as well as production of new biopsy needles, have all contributed to the rapid increase in radiologically guided liver FNAB.

The vascular architecture of renal cell carcinoma in fine-needle aspiration biopsies

The morphologic similarities between renal cell carcinoma (RCC) and hepatocellular carcinoma (HCC) can cause diagnostic difficulty in fine-needle aspiration biopsy (FNAB) specimens. In the authors' prior study of liver FNAB, peripherally wrapping endothelium (PE) and arborizing transgressing endothelium (TE) were 100% specific for HCC relative to metastatic tumors, which included only three RCCs. In this study, the vascular patterns of RCC in FNAB were reviewed for comparison with HCC, to determine their usefulness in the differential diagnosis of HCC and RCC. FNAB of 49 RCCs (26 primary and 23 metastatic) from 46 patients were reviewed. Four vascular patterns were assessed: PE, TE, papillary endothelium (PAP) in fibrovascular cores of papillary fragments, and short nonbranching endothelium (SE) in small cell clusters. Each pattern was given a semiquantitative score: absent (0), focal (1), or extensive (2). Cellularity was categorized as low (< 20 groups), moderate (20-50 groups), or high (> 50 groups). Vessels were present in 19 of 26 (73%) primary and 9 of 23 (39%) secondary RCC. PE was not identified. TE was observed in 11 primary (42%) and 7 metastatic (30%) RCC. SE was present in 5 primary (19%) and 1 metastatic (4%) RCC. The TE and SE patterns were distributed among the clear cell, granular cell, and chromophobe RCC. PAP was observed in all four papillary RCC. The majority of the TE and all of the PAP were present in moderately to highly cellular FNABs, whereas SE was usually observed in FNABs with low cellularity. FNAB specimens of RCC commonly contain TE, as in HCC, but lack PE. TE was less frequent in metastatic than primary RCC. Other vascular patterns (SE, PAP), absent in HCC, were observed infrequently. Vascular patterns, especially PE, are useful in distinguishing HCC from RCC.

Glucose and insulin responses to glucagon injection in dairy cows with ketosis and fatty liver.

The purpose of this investigation was to study the metabolic situation in clinical cases of bovine ketosis and to diagnose additional diseases. Extensive clinical examination, clinical biochemistry, haematology and fine-needle aspiration biopsy of liver was performed on 17 ketotic and eight control dairy cows in the field, and on seven hospitalized hyperketonaemic fatty liver patients. Additional findings in the ketotic group were heat (n = 7), indigestion (n = 5), endometritis (n = 2), cystic ovaries (n = 1), and mastitis (n = 1), and in the fatty liver group displaced abomasum (n = 4), abomasal ulcers (n = 3), mastitis (n = 2), laminitis (n = 1), bronchopneumonia (n = 1), and hypomagnesaemia (n = 2). There were no additional findings in the control group. Aspartate aminotransferase (AST) and creatine kinase (CK) were elevated in the ketosis and fatty liver groups. Total bilirubin, gamma-glutamyl transferase (GGT) and glutamate dehydrogenase (GD) were elevated in the fatty liver group and in some animals in the ketosis group. Total bile acid was not different between the groups. The free fatty acid/cholesterol ratio was higher in the fatty liver group compared with the control and ketosis groups. There was no or only slight fatty degeneration of the liver cells in the control and ketosis groups. Glucose and insulin preinjection concentrations and changes from basal values after glucagon injection were significantly lower in the ketosis group if compared with the control group. The responses in the fatty liver animals after glucagon injection were more heterogeneous than in the control and ketosis animals, a sign of disturbance in the metabolic adaptation, which together with high free fatty acid (FFA) levels can lead to fatty liver in cows with concurrent diseases.

Fine needle aspiration and liver cytology

Fatty liver in cattle is quite common around parturition. Diagnosis is difficult and often time and resource consuming. Hepatic FNA is a useful, fast and inexpensive diagnostic tool. This article describes in detail a method of fine-needle aspiration biopsy of liver. The fatty change/degeneration of liver is classified on a scale of 0 (normal) to 5 (fig. 2-7). Fatty change grade 3 or below gives a good prognosis. Fatty change grade 4 gives a guarded to poor prognosis, especially if primary illness is the cause of poor appetite.

Fine‐needle aspiration biopsies of metastatic neuroendocrine tumors in liver: Use of transmission electron microscopy and protein A gold technique in diagnosis

Five fine-needle aspiration biopsies of liver were studied to evaluate the use of electron microscopy and protein A gold technique in the diagnosis of metastatic neuroendocrine tumors. All five cases had cytologic features suggestive of neuroendocrine tumors and neurosecretory granules on ultrastructural examination. The aspirates were stained by the immunoperoxidase technique and the postembedding protein A gold technique using the antibody to chromogranin A. Three cases were positive for chromogranin A by both immunoperoxidase and protein A gold techniques. Two cases were positive by protein A gold technique and negative by immunoperoxidase technique. Confirmation of neurosecretory granules by immunochemistry is ideal, since ultrastructurally they can be mimicked by other electron-dense inclusions. Advantage of the protein A gold technique is the use of the same material for both ultrastructural evaluation and the protein A gold technique.

Letter: Fatality after fine-needle aspiration biopsy of liver.

Letter: Fatality after fine-needle aspiration biopsy of liver.

Methods for the determination in the nanomole range of lipids in liver fine-needle aspiration biopsies.

Triglycerides and phospholipids 0.5-2mg (wet weight) of human liver material obtained by the fine-needle aspiration biopsy technique can be estimated after chloroform:methanol extraction by the determination of lipid phosphorus and glyceride glycerol. The methods are based on the colorimetric determination of inorganic phosphate after oxidation of the phospholipids and the enzymatic fluorometric determination of glycerol after complete hydrolysis of the triglycerides, catalyzed by purified pancreatic lipase. The accumulation of lipids is then expressed as the increase in the molar ratio of triglycerides over phospholipids.