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Bilirubin is formed as a result of hemoglobin degradation. Normally, senescent red blood cells are removed from circulation and the bilirubin that is formed is processed by the liver. The normal level of bilirubin in the serum of adults is 0.2-1mg/dl. Bilirubin levels increase with liver disorders and also in anemia that is a result of a hemolytic process. Patients may display jaundice when serum bilirubin levels exceed 2mg/dl.Persons with alpha thalassemia intermedia usually have an increased bilirubin level, because of ongoing hemolysis. This bilirubin is typically the unconjugated fraction of bilirubin.

Bilirubin is formed as a result of hemoglobin degradation. Normally, senescent red blood cells are removed from circulation and the bilirubin that is formed is processed by the liver. The normal level of serum bilirubin for adults is 0.2-1mg/dL.Bilirubin levels increase with some liver disorders and also in anemia that is a result of a hemolytic process. Patients may display jaundice when serum bilirubin levels exceed 2mg/dL.Persons with beta thalassemia major usually have an increased bilirubin level. This bilirubin is typically the unconjugated fraction of bilirubin.

Bilirubin, a product of hemoglobin breakdown, is characterized by its yellow pigment. The presence of bilirubin in urine is always abnormal. It is important to note that unconjugated bilirubin cannot be excreted by the kidneys because it is bound to albumin and is not soluble in water. In the liver, bilirubin combines with glucuronic acid through the action of a glucuronyl transferase to form water soluble bilirubin diglucuronide. Under normal circumstances, conjugated bilirubin passes from the bile duct and then to the intestinal tract. Intestinal bacteria reduce conjugated bilirubin to urobilinogen. Approximately half of the urobilinogen is excreted in the feces; most of the other half is recirculated through the liver. A small amount of urobilinogen bypasses the liver and is excreted in the urine.

Urobilinogen is a byproduct of hemoglobin breakdown. It is produced in the intestinal tract as a result of the action of bacteria on bilirubin. Almost half of the urobilinogen produced recirculates through the liver and then returns to the intestines through the bile duct. Urobilinogen is then excreted in the feces where it is converted to urobilin. As the urobilinogen circulates in the blood to the liver, a portion of it is diverted to the kidneys and appears as urinary urobilinogen. Up to 1 mg/dL or Ehrlich unit of urobilinogen is present in normal urine. A result of 2.0 mg/dL represents the transition from normal to abnormal and the patient should be evaluated further. It is important to note that the reagent strip cannot determine the absence of urobilinogen.

Bilirubin is formed as a result of the breakdown of hemoglobin from erythrocytes in the reticuloendothelial system. It becomes bound to albumin and transported through the blood to the liver. This free or unconjugated bilirubin is insoluble in water and cannot be filtered through the glomerulus of the kidney. In the liver, bilirubin becomes conjugated with glucuronic acid to form bilirubin diglucuronide. This conjugated bilirubin, which is also called direct bilirubin, is water soluble and is excreted by the liver through the bile duct and into the duodenum.

Ketone bodies are formed in the liver as intermediates in the catabolism of fatty acids. In normal, healthy individuals, ketone bodies are almost completely metabolized so that only negligible amounts appear in the urine.

By measuring ApoB we can quantify the amount of all atherogenic or potentially atherogenic lipoproteins that carry this apolipoprotein. Although lipoprotein particles other than LDL can carry ApoB, LDL accounts for the vast majority of ApoB; therefore, it is a good index of LDL particle number. Furthermore, the other particles that can have ApoB (such as IDL and Lp(a)) are also atherogenic and so it is not problematic if they are counted along with LDL, since they also contribute to cardiovascular risk. What about ApoA1? HDL-C is known as 'good cholesterol'. The role for HDL in the body is to sequester excess cholesterol and bring it back to the liver. Since HDL can remove cholesterol and transport it back to the liver for excretion or re-utilization it is indeed good. HDL is a negative cardiovascular risk factor; as its concentration goes up, a person's cardiovascular risk decreases. A person with low cardiovascular risk would have low ApoB levels and high ApoA1 levels. If we measure both ApoB and ApoA1 and express them as a ratio of ApoB/ApoA1 we get a powerful cardiovascular risk marker. The ratio should be approximately 0.3-0.9. Patients with a higher ratio have elevated ApoB (LDL) and/or low ApoA1 (HDL) and are thus at increased risk. By combining these two markers in a ratio, we get synergy and enhanced predictive power.

Disseminated Intravascular Coagulation (DIC) is best described as a disorder of consumption, because clotting factors are depleted from the blood. Basically, clotting occurs randomly throughout the body, as opposed to just in the localized areas where vascular damage has occurred, consuming clotting factors and other components such as platelets in the process. Symptoms may range from a mild bleed, to severe, profuse bleeding, primarily dependant upon the availability of clotting factors. As more and more coagulation factors and components are consumed, the disorder progresses and symptoms worsen. Most heavily impacted are the levels of factors I, V, and VIII as well as the number of available platelets. Clinically, DIC is detected via an elevated (positive) FDP, positive D-dimer test, a prolonged PT and APTT, plus the manifestation of hemorrhagic episodes. DIC is diagnosed as two primary types, acute and chronic. Acute DIC manifests in a few hours or a few days, has a high mortality rate, and is seen in infections, obstetric complications, liver disease, and tissue injury. Chronic DIC is a secondary condition to some other disease state. Once you treat the primary disease, this type of DIC will go away. Treatment is often factor replacement therapy through the use of fresh frozen plasma and/or cryoprecipitate.

Regulation of iron equilibrium occurs mainly through the process of absorption. Iron is absorbed through the mucosal cells lining the duodenum. A variety of proteins are involved in this process. Hepcidin, an antimicrobial protein primarily produced in the liver, has been recently found to be a major (negative) regulator of dietary iron absorption by disrupting cellular iron transport in the intestine. Decreased levels of hepcidin are related to increased iron absorption into the bloodstream. Hepcidin is increased in response to iron overload and inflammation. (4)Additional proteins involved in iron metabolism include transferrin (Tf), transferrin receptor (TfR), ferroportin, HFE protein, hemojuvelin, and others. Their roles in iron absorption are complex and in some instances incompletely understood.Factors affecting iron absorption include: Tissue stores, e.g., decreased stored iron is associated with a decrease in hepcidin and increase in iron absorption. Rate of hematopoietic activity, e.g., an increased rate of erythropoiesis is associated with a decrease in hepcidin and an increase in iron absorption. Oxygen concentration in tissues, e.g., hypoxia decreases hepcidin and increases iron absorption, thereby promoting increased erythopoiesis. Dietary intake, including form of iron ingested, e.g., heme iron is more readily absorbed than non-heme forms of iron. Condition of GI tract mucosal cells Intraluminal factors, e.g. intestinal motility

Several mutations of the HFE gene have been described. In the C282Y mutation, a base substitution leads to a change in the amino acid in position 282 from cysteine (C) to tyrosine (Y). The loss of the sulfhydryl-containing amino acid disrupts the tertiary structure of HFE so that it no longer binds to beta-2 microglobulin. Beta-2 microglobulin appears to act along with other proteins to chaperone the newly synthesized HFE out of the Golgi apparatus and to the cell surface where it can then bind to TfR. In the C282Y mutation, HFE remains in the Golgi, never making it to the cell surface. The result is that transferrin binding to TfR is enhanced and excessive amounts of iron enter the cells of the small intestine, liver, and other tissues. A second mutation, H63D, causes a histidine (H) residue in position 63 to be replaced by aspartic acid (D). The mechanism by which this mutation leads to increased iron uptake is less well understood when compared to the C282Y mutation. Unlike the C282Y mutation, the H63D mutation does not seem to affect the binding of beta-2 microglobulin and intracellular movement, since detectable concentrations of the mutated protein are found on cell membranes. Some researchers speculate that the H63D mutation affects the binding of proteins involved in iron regulation and uptake at the cell surface.A third mutation, S65C, leads to a serine-to-cysteine substitution in its associated protein. This mutation has been been found in some compound heterozygotes for C282Y or H63D, but is rarely associated with iron overload in HH.Additional mutations of HFE have been identified, but their clinical significance is unclear. Most laboratories performing molecular assays test for only the C282Y, H63D, and S65C mutations.

In addition to hereditary hemochromatosis (HH), there are other conditions of iron overload that must be considered in a differential diagnosis. Disorders such as sickle cell disease, thalassemia, sideroblastic anemia, congenital dyserythropoietic anemia, and liver disease may also cause iron overload. Transfusion-dependant patients and persons who abuse iron-containing vitamin supplements are also at risk. These conditions are usually described as secondary iron overload, in contrast to the primary iron overload of HH.Patient history, clinical signs and symptoms, biochemical and hematologic laboratory analyses, and possibly results of a liver biopsy may be needed to establish a diagnosis of a condition causing secondary iron overload. DNA tests for common HFE mutations are very likely the most important diagnostic tool for identifying HH as the cause of iron overload. In some patients, both secondary causes and HH may be contributing to iron overload. Differentiating the secondary causes of iron overload from HH is heavily dependent on the results of laboratory assays, but a complete discussion is beyond the scope of this course.

DNA tests for HFE mutations associated with hereditary hemochromatosis (HH) are available in some clinical laboratories and reference laboratories. Testing for the presence of the C282Y is essential, although most labs also test for H63D and S65C mutations. Molecular testing is most appropriate for confirmatory testing of symptomatic individuals with altered iron studies (increased TS and SF), in pre-symptomatic individuals (increased TS, normal SF and liver function tests), and in family members of individuals diagnosed with HH. The use of genetic tests alone for routine screening of asymptomatic persons is not recommended for several reasons. A positive test indicating the presence of HFE mutations does not guarantee that an individual will develop clinically significant iron overload or predict severity of symptoms. A negative result (no HFE mutations present) does not rule out a diagnosis of iron overload because of genetic heterogeneity. Compared to biochemical analyses for iron, molecular assays are expensive. Finally, molecular testing may result in the diagnosis of a genetic disease, thus opening up the possibility for discrimination in health insurance coverage. Using molecular methods, DNA is extracted from leukocytes in whole blood samples or from buccal cells and analyzed for specific HFE mutations using polymerase chain reaction (PCR) with melt curve analysis. Currently there are no FDA-cleared products for HFE testing, and testing laboratories are using "home brew" reagents. This situation is expected to change as manufacturers submit products for FDA approval.

Deferoxamine (DFO), an iron chelating agent, may be used to reduce iron overload in patients for whom phlebotomy is contraindicated or not well tolerated. Examples include patients with sickle cell disease or thalassemia whose anemia would be exacerbated by phlebotomies. DFO is seldom used to treat hereditary hemochromatosis (HH) due to the low cost and efficacy of phlebotomy therapy. DFO is typically administered by intravenous or subcutaneous infusion.Patients with HH may be counseled to avoid alcohol use in order to avoid liver damage. With the exception of iron supplements, dietary restrictions on iron ingestion are rarely advised.

Before learning to examine bone marrow microscopically, it is important to understand the basic structure and function of the bone marrow. The bone marrow is one of the largest organs in the body. The normal adult marrow on a daily basis produces approximately 2.5 billion red cells, 2.5 billion platelets and 1.5 billion granulocytes per kilogram of body weight. The main function of this organ is the formation and development of blood cells. Hematopoiesis begins in the yolk sac in the first weeks of embryonic life; stem cells from the yolk sac travel first to the liver and then to the spleen. These organs are the only blood forming sites during the first three months of fetal life. At the beginning of the fourth month the bone marrow begins its life-long function of cell production.

Specialists in immunohematology perform all testing prior to blood transfusions and work to prevent transfusion infections. They also investigate any post-transfusion reactions. This specialty includes all lab procedures performed in the specialty of histocompatibility. Specialists in clinical chemistry analyze body fluids such as blood, urine, and spinal fluid to determine the chemical makeup, including the amount of carbohydrates, proteins, enzymes, and trace elements. The special covers urine microscopics and chemical evaluation of the liver, kidneys, lungs, heart, and other vital organ systems. This specialty also covers all testing performed in the specialties of radioassay and blood gas analysis. Specialists in blood banking can perform all immunohematology testing as well as testing from the specialties of clinical chemistry, hematology and serology/immunology that relates to donor blood. Clinical laboratory personnel who are licensed in the specialties of immunohematology, clinical chemistry, hematology, and serology / immunology may perform all tests in the blood banking specialty.

After the exposure, there is an incubation period that lasts between 45 and 180 days, with an average of 90 days.Many individuals with acute HBV will have no symptoms at all. Some will have a mild illness with loss of appetite, nausea and vomiting, and fatigue. About 30% of infected individuals will develop clinical hepatitis with jaundice (yellow discoloration of the skin and eyes due to liver dysfunction).

About 10% of adults who are infected with hepatitis B go on to chronic hepatitis, which lasts for years.Chronic hepatitis B eventually can cause scarring of the liver (known as cirrhosis), liver failure, and, more rarely, liver cancer.While these complications are uncommon, they serve to emphasize the need for proper techniques to prevent transmission of HBV.

There is no known cure for HCV disease. Some patients may require long-term therapy with a medication called Interferon.If patients develop liver failure due to HCV infection, they may require a liver transplant.

Laboratory specimens that are unlikely to cause disease and do not meet the criteria for category A or B substances are not subject to Division 6.2 regulations. Specimens for which the hazardous materials regulation (HMR) does not apply include human or animal samples (including, but not limited to, secreta, excreta, blood and its components, tissue and tissue fluids, and body parts) being transported for routine testing not related to the diagnosis of an infectious disease. This includes specimens that are being sent for: drug or alcohol testing cholesterol testing blood glucose level testing prostate specific antibody (PSA) testing testing to monitor kidney or liver function pregnancy testing tests for diagnosis of non-infectious diseases such as cancer biopsies

The liver plays a major role in converting lipophilic nonpolar molecules (drug molecules) to more polar, water-soluble forms through a series of enzymatic reactions. Drug molecules can be modified by either phase I or phase ll reactions. Phase I reactions alter chemical structure by oxidation, reduction, or hydrolysis. Phase ll reactions conjugate drugs to create products that are water-soluble.

Drug-binding proteins in serum can fluctuate in disease states. For example, if albumin levels fall, as can occur in liver failure or nephrotic syndrome, less albumin will be available for drug binding; a subsequent dose may produce a toxic concentration of free drug.The image on the right illustrates the loss of equilibrium between a protein-bound drug and a free drug when drug-binding proteins are diminished.Doses of drugs that are highly protein-bound may need to be adjusted in patients with lower drug-binding protein levels. Examples of some common drugs that are highly protein-bound include thyroxine, warfarin, diazepam, heparin, imipramine and phenytoin. �

To discuss PGx, we must first define two terms - polymorphism and cytochrome P450 (CYP450).A polymorphism is a variation in a gene (allele) that affects at least 1% of the population. CYP450 refers to a family of enzymes found predominantly in the liver. CYP450 enzymes work on a variety of substrates (drugs), altering their chemical structures to facilitate excretion in the urine and feces. There are many known polymorphisms in CYP450 enzymes.

The peripheral smear illustrated in the photograph was obtained from a patient with a recent renal transplant. The patient developed a rash, accompanied by nausea and diarrhea. Graft vs. host disease was clinically suspected. The peripheral smear findings are consistent with that diagnosis. The presence of spherocytes suggests a hemolytic process which is supported by the presence of nucleated RBCs. A few scattered schistocytes and the decrease of platelets suggests DIC. The presence of target cells presents the possibility of associated liver disease. Additional tests, particularly coagulation studies, should confirm the diagnosis of microangiopathic hemolytic anemia.

Another type of macrocyte can be seen in this image. Notice it appears larger than the lymphocyte but in contrast to the macrocytes (megalocytes) seen in megaloblastic anemias (vitamin B12 or folic acid deficiency), these macrocytes have an area of central pallor. These macrocytes are sometimes referred to as "pseudomacrocytes," since their size is the result of exaggerated flattening and thus the presence of central pallor. When this type of macrocyte is present on a blood slide, the MCV will most likely be within normal range. Pseudomacrocytes can be seen in peripheral blood smears from patients with cirrhosis of the liver, obstructive jaundice, post splenectomy and conditions that affect the red cell membrane.

Tyrosine crystals appear as fine silky needles arranged in sheaves or bundles in acid urine. They are rarely present and may appear together with leucine crystals in liver disease. Do not confuse tyrosine with crystals caused by x-ray dye. X-ray dyes will cause the urine specific gravity to be greatly increased (1.040), Tyrosine crystals are soluble in alkali or dilute mineral acid.