Peak light absorption for NAD is ___ nm, while for NADH it is ___ nm.
Peak light absorption for NAD is 260 nm, while for NADH it is 340 nm.
While the terms “enzyme concentration” and “enzyme activity” are often used interchangeably, there is often a discordance due to…
While the terms “enzyme concentration” and “enzyme activity” are often used interchangeably, there is often a discordance due to inhibitors, macroenzymes, lack of cofactors, proteolytically-inactive enzymes, etc. Hence, enzyme activity usually underestimates the immunoassay.
What is a macroenzyme?
Macroenzymes are enzymes bound to antibodies, which inhibit enzyme function and block enzyme clearance.
What is plotted on a Lineweaver-Burke plot?
1/[S] (x-axis) vs. 1/v (y-axis). A Lineweaver-Burke plot is a means to express a non-linear asymptotic concept (how the rate of the reaction varies with the substrate concentration) in a linear fashion by using the reciprocals of substrate concentration and reaction rate.
Lineweaver-Burke plots, competitive inhibition, noncompetitive inhibition, uncompetitive inhibition.
Lineweaver-Burke plots are a means to express a non-linear asymptotic concept (how the rate of the reaction varies with the substrate concentration) in a linear fashion by using the reciprocals of substrate concentration (1/[S], x-axis) and reaction rate (1/v, y-axis). The x intercept is -1/Km, the y intercept is 1/Vmax, and the slope is Km. In competitive inhibition, the enzyme’s substrate and the inhibitor compete for the same binding site. This type of inhibition can be overcome by increasing substrate concentration. On the Lineweaver-Burke plot, the y intercept is unchanged and the slope is increased. In noncompetitive inhibition, the inhibitor binds the enzyme at a different site from the substrate binding site, effectively decreasing the amount of useful enzyme without affecting the binding of substrate to enzyme. On the Lineweaver-Burke plot, the Km (a function of binding) is unaffected, but the reaction rate decreases. In uncompetitive inhibition, the inhibitor binds and stabilizes the enzyme-substrate complex. On the Lineweaver-Burke plot, the Km is decreased (can’t bind enzyme if it’s in a stable complex), and the Vmax is decreased (product can’t be formed if the substrate and enzyme are stuck together).
What are the x-intercept and y-intercept on a Lineweaver-Burke plot, respectively?
-1/Km and 1/Vmax.
How is the International Unit (IU) defined?
An IU is the amount of enzyme that catalyzes the conversion of 1 micromole of substrate per minute. A katal is the amount of enzyme that catalyzes the conversion of 1 mole of substrate per second. 1 IU = 16.7 nanokatals.
AST and ALT. Which is more liver-specific?
ALT. ALT is mainly confined to the mitochondria (80%) of liver and kidney. AST is found in highest concentration in the heart.
Which LDH isoenyme is in highest concentration in serum?
LD2.
What is a flipped LD ratio?
Normally, the LDH isoenzyme LD2 is in a higher concentration in serum than LD1. The flipped ratio happens when LD1>LD2; this can be seen with acute MI, hemolysis, or renal infarction.
What conditons can be associated with a mildly increased alkaline phosphatase?
Unrecognized pregnancy. Some drugs, such as ibuprofen and acetaminophen. CHF. Hyperthyroidism. Hepatic metastases. Non-fasting Lewis (+) type B or O secretors after a meal.
In what 2 areas is GGT concentrated?
The biliary epithelial cells that line the small interlobular bile ducts and ductules. Smooth endoplasmic reticulum of hepatocytes.
What are the two main sources of ammonia in the body?
The gut and skeletal muscle. And the liver clears it.
Unconjugated bilirubin is a heme breakdown product; it is tightly bound to (what protein) in the bloodstream when it comes to the liver for (what enzyme process). The now water-soluble conjugated bilirubin can be excreted in bile, which intestinal bacteria convert to (what substance) for excretion in feces and urine.
Unconjugated bilirubin is a heme breakdown product; it is tightly bound to albumin in the bloodstream when it comes to the liver for glucuronidation. The now water-soluble conjugated bilirubin can be excreted in bile, which intestinal bacteria convert to urobilinogen for excretion in feces and urine.
What is dysproteinemia?
Dysproteinemia is the clinical state characterized by excessive synthesis of immunoglobulin molecules or subunits, resulting from clonal plasma cell proliferations or B-cell lymphoproliferative disorders.
What are the casts in light chain cast nephropathy AKA myeloma cast nephropathy composed of?
The are composed predominantly of a single monoclonal light chain, which is typically admixed with Tamm-Horsfall protein secreted by the thick ascending limb of Henle.
How can calcium phosphate and calcium oxalate crystals be distinguished by: H&E, von Kossa stain, Alizarin red S stain, and polarized light?
On H&E, calcium phosphate crystals often appear blue or purple, while calcium oxalate crystals appear translucent. With von Kossa stain, the stain reacts with the phosphate moiety of calcium phosphate, and does not stain calcium oxalate. Alizarin red S stains calcium specifically, and reacts with calcium phosphate at pH 7 and pH 4.2 while reacting with calcium oxalate at pH 7 (but not pH 4.2). Calcium phosphate is not birefringent under polarized light, while calcium oxalate is.
What conditions can lead to oxalate nephropathy?
Enteric hyperoxaluria, toxic exposures (such as ethylene glycol ingestion or excessive vitamin C intake (vitamin C is metabolized to oxalate)), excessive dietary intake of oxalate, and inborn errors of metabolism.
Pathogenesis/immunology of celiac disease.
Among autoimmune diseases, CD is one of the few where the offending antigen is known (gluten). Gluten is mostly made up of 2 groups of proteins: ethanol-soluble gliadins and ethanol-insoluble glutenins. Gliadin contains large amounts of the amino acids proline and glutamine. It is known that alpha-gliadin among other peptides is toxic to celiac patients. The pathogenesis of CD involves a CD4+ T-cell mediated immune response to gliadin peptides, activation of a CD8+ T-cell intraepithelial innate immune response, and production of antibodies against tissue transglutaminase, as well as anti-gliadin, anti-reticulin, and anti-endomysial antibodies. In 1997, tissue transglutaminase type II was identified as the major autoantigen of CD (and also the epitope recognized by anti-endomysial antibodies). Tissue transglutaminase is an 85-kDa enzyme that is expressed in multiple tissues. Gliadin can serve as a substrate of transglutaminase, which is activated upon injury of inflammation of the small bowel, and in the process becomes cross-linked to transglutaminase, thereby creating a neoantigen, which induces an immune response to the self-protein (transglutaminase). Moreover, tissue transglutaminase can selectively deamidate gliadin peptides, leading to enhanced T-cell stimulatory activity. The ensuing inflammatory cascade produces inflammatory cytokines, proteinases, and other tissue-damaging mediators that induce mucosal tissue damage, leading to the characteristic histopathologic alterations.
Serologic testing in celiac disease.
The most sensitive antibody tests detect IgA-class antibodies against tissue transglutaminase (tTGA) and endomysium (EMA). The anti-gliadin antibodies are no longer considered sensitive or specific enough to be used for routine clinical detection of CD, except in children younger than 18 months of age, because anti-gliadin IgA antibodies are considered to be the first autoantibodies to appear after intestinal exposure to a gluten-containing diet. Serologic IgA tTGA antibody testing is considered to be the most sensitive method for detecting CD, with sensitivity approaching 97% in clinical practice, while IgA EMA antibodes are highly specific markers for CD, approaching 100%. The presence of titers of both tTGA and EMA antibodies have been shown to correlate with the degree of mucosal damage. Seronegative CD does occur, accounting for up to 15% of all CD patients. Recently, a new test for detecting antibodies against deamidated gliadin peptides (DGP) was introduced, which displays promising results and a high specificity (99%). DGP IgA testing may have greater sensitivity for the detection of adequate adherence to a gluten-free diet.
What laboratory monitoring tests are used for the following anticoagulants: Coumadin, Heparin, LMW heparin, Fondaparinux, Rivaroxaban, Argatroban, Lepirudin, Bivalirudin, Dabigatran.
Coumadin: INR. Heparin: PTT, anti-factor Xa. LMW heparin: Anti-factor Xa if needed. Fondaparinux: Anti-factor Xa if needed. Rivaroxaban: Anti-factor Xa if needed. Argatroban: PTT. Lepirudin: PTT. Bivalirudin: ACT. Dabigatran: Thrombin time to test for residual drug. Dilute thrombin time to assess extent of anticoagulation or test for residual drug.
Coumadin reduces the synthesis of what coagulation factors?
Factors II, VII, IX, and X.
How are heparin and the LMW heparins different in mechanism of action?
Heparin inhibits the coagulation cascade at multiple sites; all of the activated coagulation factors, except factor III, are targets. The LMW heparins inhibit the action of factors II and X, and the different LMW heparins may inhibit them to different extents.
How is INR calculated?
INR = [Patient PT / Mean of normal PT range] ^ ISI. ISI = International Sensitivity Index for thromboplastin, used for PT determination. A patient with high ISI (ISI = ~2) will have low sensitivity to factor deficiencies. A patient with low ISI (ISI = ~1) will have high sensitivity to factor deficiencies.
What is the algorithm for treating a high INR in a patient on warfarin (above 2.0-3.0 or 2.5-3.5, depending on underlying condition)?
If there is significant bleeding, discontinue warfarin, give vitamin K (most likely IV or sub-Q), and give plasma to replace the missing coagulation factors. If there is no significant bleeding, and there is a desire to regain the anticoagulated state as soon as possible OR the INR is less than 10, discontinue warfarin and allow INR to decline. If there is no significant bleeding, and there is no desire to regain the anticoagulated state as soon as possible OR the INR is greater than 10, discontinue warfarin and administer vitamin K (most likely oral).
How does protamine sulfate work to neutralize heparin?
Heparin is a very negatively charged compound, and protamine sulfate is a positively charged compound. They bind together and heparin is neutralized.
In what situations should heparin (via PTT or anti-factor Xa) be monitored in patients?
If it is LMWH used for prophylaxis or LMWH therapy in normal weight, normal renal function, non-pregnant patients, no monitoring is needed. If unfractionated heparin is used, target PTT to >2.0x mean of normal range (varies widely depending on reagents and instruments).
What are nonmalignant medical conditions associated with reactive eosinophilia?
Allergic reactions (drug reactions, asthma), parasitic infections (strongyloidiasis, schistosomiasis, filariasis, toxocariasis), metabolic abnormalities (adrenal insufficiency), humoral immunodeficiency (hyperimmunoglobulin E syndrome (Job syndrome), Wiskott-Aldrich syndrome, hyperimmunoglobulin M syndrome, immunoglobulin A deficiency), pulmonary eosinophilias (eosinophilic granulomatosis with polyangiitis (formerly Churg-Strauss syndrome), allergic bronchopulmonary aspergillosis, chronic and acute idiopathic eosinophilic pneumonias), autoimmune blistering skin diseases (dermatitis herpetiformis, bullous pemphigoid).
Most eosinophilias are reactive, polyclonal processes. In all these reactive processes, the increase in AEC appears to be mediated by cytokines, principally ___, which promotes proliferation of eosinophils and their precursors. Reactive eosinophilias may also involve overproduction of additional cytokines, such as ___ and ___, which can lead to concomitant elevation of IgE in reactive eosinophilias.
Most eosinophilias are reactive, polyclonal processes. In all these reactive processes, the increase in AEC appears to be mediated by cytokines, principally IL-5, which promotes proliferation of eosinophils and their precursors. Reactive eosinophilias may also involve overproduction of additional cytokines, such as IL-3 and IL-4, which can lead to concomitant elevation of IgE in reactive eosinophilias.
Why do many patients with Addison disease/adrenal insufficiency have blood eosinophilia?
This association exists because glucocorticoids inhibit eosinophil proliferation and survival, an effect that is exploited clinically when steroids are used as treatment of reactive eosinophilias. In adrenal insufficiency, the converse occurs; low glucocorticoid levels allow increased eosinophil proliferation and survival. This scenario is especially important in critically ill patients, in whom eosinophilia due to adrenal insufficiency is common. Given the inverse relationship between glucocorticoid levels and eosinophil count, the standard evaluation of eosinophilia should include attention to clinical signs of adrenal insufficiency (orthostatic hypotension, skin discoloration); routine chemistries, which may be abnormal in Addison disease; and, in some cases, morning cortisol levels.
Among the reactive eosinophilias in the developed world, allergic reactions are most common, comprising ___% of cases, and parasitic infections represent the second most-common cause, comprising ___% of cases.
Among the reactive eosinophilias in the developed world, allergic reactions are most common, comprising ~80% of cases, and parasitic infections represent the second most-common cause, comprising ~8% of cases.
Paraneoplastic eosinophilia is frequently observed in association with solid organ malignancies, with reported incidences ranging from ___ to ___%. The phenomenon is not specific to any particular tumor type, and a broad spectrum of primary tumors have been implicated. Instead, paraneoplastic eosinophilia appears to be most closely related to ___. Various mechanisms have been proposed to explain this association.
Paraneoplastic eosinophilia is frequently observed in association with solid organ malignancies, with reported incidences ranging from 0.5% to 7%. The phenomenon is not specific to any particular tumor type, and a broad spectrum of primary tumors have been implicated. Instead, paraneoplastic eosinophilia appears to be most closely related to tumor stage. It has been noted that blood eosinophilia is a poor prognostic sign and, in almost all cases, is associated with advanced metastatic disease. Various mechanisms have been proposed to explain this association. The eosinophilia may disappear after surgical excision and reappear with recurrence.
(Direct or Indirect?) bilirubin is measured in a diazo-colorimetric assay.
Direct bilirubin is measured in a diazo-colorimetric assay. It is measured in the reaction with the diazo compound in the absence of an accelerator. Addition of an accelerator, such as alcohol, results in a measurement of total bilirubin. Indirect is calculated as total minus direct.
Cirrhosis, hemolysis, Gilbert syndrome, and Crigler-Najjar syndrome all cause (conjugated or unconjugated?) hyperbilirubinemia.
Cirrhosis, hemolysis, Gilbert syndrome, and Crigler-Najjar syndrome all cause unconjugated hyperbilirubinemia.
Which coagulation factor has the shortest half-life?
Factor VII, at 4-7 hours. Factor I is 72-120 hours, factor II is 48-120 hours, factor V is 12-36 hours, factor VIII is 8-12 hours, factor IX is 18-24 hours, factor X is 24-48 hours, factor XI is 40-84 hours, factor XII is 48-52 hours, and factor XIII is 9-12 days.
Which coagulation factor has the longest half-life?
Factor XIII, at 9-12 days. Factor I is 72-120 hours, factor II is 48-120 hours, factor V is 12-36 hours, factor VII is 4-7 hours, factor VIII is 8-12 hours, factor IX is 18-24 hours, factor X is 24-48 hours, factor XI is 40-84 hours, factor XII is 48-52 hours.
What is the leading cause of neonatal jaundice?
Physiological jaundice due to hepatic enzymes not being up to full capacity; the infant’s liver is not able fully to conjugate bilirubin for excretion. This can be exacerbated by peripartum hemolysis, which leads to an increased bilirubin load, or breast milk jaundice, since breast milk contains inhibitors of bilirubin conjugation.
What signs indicate that an infant may not have just physiological jaundice?
Jaundice within 24 hours of birth, rising bilirubin after 1 week, persistence past 10 days, a total bilirubin >12 mg/dL, a single day increase in bilirubin >5 mg/dL, or a direct bilirubin >2 mg/dL.
Out of HAV, HBV, HCV, autoimmune, toxic, and ischemic causes of liver injury, which 2 cause the largest increase in transaminases?
Ischemia and toxic injuries lead to the most profound elevations in transaminases, sometimes >100x the upper limit of normal.
How does the AST:ALT ratio help in distinguishing toxic/ischemic/alcoholic hepatitis from viral hepatitis?
An AST:ALT ratio greater than 2 implies toxic/ischemic/alcoholic hepatitis, while with viral hepatitis it is often less than 1.
What lab test is the most consistent indicator of prognosis in acute hepatic injury?
PT. A PT prolongation >4.0 seconds indicates severe injury and an unfavorable prognosis. The PT test is a test of loss of function, while viral titers, bilirubin, AST:ALT, and GGT are tests of injury.
There are __ isoenzymes of serum amylase as analyzed by serum electrophoresis. The fastest migrating are ___; the slowest are ___.
There are 6 isoenzymes of serum amylase as analyzed by serum electrophoresis. The fastest migrating are pancreatic; the slowest are salivary.
The salivary isoenzyme of serum amylase is (inhibited/uninhibited) and the pancreatic isoenzyme is (inhibited/uninhibited) by the effect of wheat germ lectin Triticum vulgaris.
The salivary isoenzyme of serum amylase is inhibited and the pancreatic isoenzyme is uninhibited by the effect of wheat germ lectin Triticum vulgaris.
What is the most common cause of pancreatitis associated with normal serum amylase levels?
Hypertriglyceridemia.
In macroamylasemia, why is the serum amylase increased and the urine amylase decreased?
Ig-amylase is referred to as macroamylase. Macroamylase cannot be cleared by the kidney due to its large size, which leads to increased serum amylase and decreased urine amylase.
Why is lipase better than amylase for the diagnosis of acute pancreatitis?
Lipase is more pancreas-specific, is less reliant on renal clearance, and while it rises in parallel to amylase it remains elevated for longer.
What are some advantages of using an interferon-gamma-release assay over a tuberculin skin test?
It is a blood test and patients do not need to return to have their skin test read. The results are more objective than those of a skin test, since with the TST there can be reader variability. Also, the test is not affected by previous vaccination with BCG (the TST is).
What are the two commercial choices for interferon-gamma-release assay testing?
QuantiFeron-TB Gold and its newer In-Tube version (Cellestis, a Qiagen company, Valencia, California) and T-Spot.TB (Oxford Immunotec Limited, United Kingdom). T-Spot requires isolated WBCs rather than whole blood, which can be more technically demanding; however, the T-Spot may be more sensitive in those who are immunocompromised.
Analysis of postmortem glucose, enzymes, and drugs shows significant differences between specimens taken from the right side of the heart, the left side of the heart, and the peripheral blood vessels. ___ or ___ specimens best approximate the antemortem values.
Analysis of postmortem glucose, enzymes, and drugs shows significant differences between specimens taken from the right side of the heart, the left side of the heart, and the peripheral blood vessels. Peripheral venous or peripheral arterial specimens best approximate the antemortem values.
What are filter blots obtained from autopsy cases used for?
300 uL of blood may be spotted on specialized filter papers. Several samples can be collected, dried overnight, wrapped in plastic wrap, and stored at -20 C. Such samples are useful for genotype and protein analyses available through commercial and academic laboratories. An example would be using tandem mass spectrometry on postmortem blood spotted on filter paper to detect specific enzyme defects. Filter blots of liver tissue, bile, and vitreous humor may also be used.
What is the best fluid for postmortem chemical analysis?
Vitreous humor provides one of the best samples for postmortem chemical analysis because it comes from a closed space and postmortem values often approximate the antemortem levels. Vitrous humor may not become contaminated after embalming, so it may still provide material for analysis in these cases. However, a sample of the embalming fluid should also be submitted to the laboratory as a control.
Postmortem determination of premortem glucose levels. Discuss.
Postmortem serum glucose decreases rapidly because of glycolysis, preventing detection of antemortem hypoglycemia. Even elevated levels of postmortem blood glucose require careful interpretation. Death from asphyxia, cerebral hemorrhage, CHF, electrocution, or terminal CPR may increase postmortem peripheral vascular glucose and falsely indicate hyperglycemia. Glycosuria, ketonuria, or elevated serum acetone can help confirm diabetic ketoacidosis. Blood samples taken from the right atrium or IVC may have a high glucose content because of glycogenolysis in the liver and subsequent diffusion of glucose into adjacent vessels. Thus, a low glucose level in blood from the right atrium and a positive test for ketones may support starvation. Vitreous humor provides more reliable data for determination of antemortem hyperglycemia. Glycolysis reduces the postmortem concentration of vitreous humor glucose; however, values greater than 200 mg/dL usually indicate that the decedent had uncontrolled diabetes.
List the four general patterns of postmortem vitreous humor concentrations of sodium, chloride, potassium, and BUN/Cr, which allow some assessment of the terminal metabolic condition of the decedent.
Dehydration Pattern: Increased sodium and chloride concentrations. No significant increase in potassium. Moderate elevation of BUN/Cr levels. Uremic Pattern: No substantial increase in sodium, chloride, or potassium. BUN/Cr levels increased. Low-salt Pattern: Low sodium, chloride, and potassium concentrations. BUN/Cr stable. Decomposition Pattern: Low sodium and chloride concentrations. Increased potassium. BUN/Cr stable.
What is the most stable postmortem chemistry blood constituent?
Urea nitrogen is perhaps the most stable blood constituent after death as it approximates premortem levels even after moderate decomposition. Urea nitrogen also remains stable in CSF, vitreous humor (even after embalming), and synovial fluid. In addition to their use in assessing renal function, urea nitrogen concentrations aid in the interpretation of hypernatremia. Similarly, creatinine levels in the blood remain stable after death, as they do in CSF and vitreous humor, making creatinine a valid postmortem marker of nitrogen retention and renal function.
Of the cytokines, IL-__ is the most specific for the eosinophil lineage.
Of the cytokines, IL-5 is the most specific for the eosinophil lineage. It is responsible for the selective differentiation of eosinophils and release of eosinophils from bone marrow.
What hematologic abnormality is a serious potential complication of antithyroid drugs (carbimazole, methimazole, and propylthiouracil)?
Agranulocytosis. The effect is not dose-dependent. The incidence is 0.05-0.5% of all users. In most patients, the agranulocytosis occurs within the first 3 months of treatment. This often presents with pharyngitis, and patients are instructed to seek attention if they develop sore throat, fever, an oral ulcer, or other symptoms of infection (UTI, PNA). Minor depressions in the neutrophil count are much more common than true neutropenia.
What are etiologies of HCC?
Multiple risk factors can contribute to the development of this neoplastic process, such as viral hepatitis infection with HBV or HCV, cirrhosis (independently of cause), exposure to aflatoxin, use of anabolic steroids and tyrosemia.
What are some nonmolecular methods and molecular methods for detecting M. tuberculosis complex (with or without the simultaneous detection of genes conferring drug resistance)?
Nonmolecular methods: Microscopic observation direct susceptibility assay (MODS assay). Light-emitting diode microscopy. MDR-XDRTB Colour Test. Colorimetric assays. Phage amplification assays. Molecular methods: Line probe assays. Automated nucleic acid amplification tests. Loop-mediated isothermal amplification. Oligonucleotide microarray.
Where in the body is AST present?
In decreasing order of concentration: cardiac muscle, liver, skeletal muscle, kidney, brain, lung, pancreas. ALT is more specific for the liver than AST.
Where in the body is ALT present?
ALT is more specific for the liver than AST, and is found mainly in the liver and kidney.
AST is found both within cytoplasm (~__%) and mitochondria (~__%). ALT is found in ___.
AST is found both within cytoplasm (~20%) and mitochondria (~80%). ALT is located entirely within the cytoplasm.
How do the activity levels of AST and ALT change with age? How are activity levels different by gender and race?
In children, AST activity is slightly higher than the ALT, with this pattern reversing by age 20. In adults, AST activity tends to be a little lower than ALT. This ratio may again reverse in old age. Both AST and ALT activities are higher in adult males than adult females, and both are higher in African Americans.
Intra-individual variation is more significant for (AST/ALT).
Intra-individual variation is more significant for ALT than AST, with marked diurnal variation (highest in the afternoon) and day-to-day variation of up to 30%.
Both AST and ALT may be elevated in heparin therapy to around __x of baseline.
Both AST and ALT may be elevated in heparin therapy to around 3x of baseline.
In renal failure, AST and ALT are (lower/higher) than in healthy individuals.
In renal failure, AST and ALT are significantly lower than in healthy individuals.
LDH is found in numerous tissues and is traditionally separable into 5 isoenzymes by electrophoresis. In what tissues are each of the isoenzymes found in?
LD1 and LD2 are found in greatest abundance in heart, RBCs, and kidney, with a significantly greater proportion of LD1 than LD2. LD3 is found in lung, spleen, lymphocytes, and pancreas. LD4 and LD5 are found in liver and skeletal muscle.
LDH is found in numerous tissues and is traditionally separable into 5 isoenzymes by electrophoresis. What is the “sixth” LD?
LD6 is sometimes seen migrating cathodal to LD5. Its presence is thought to be a dire finding, indicative of hepatic vascular insufficiency (usually in the setting of cardiovascular collapse).
Put the 5 LDH isoenzymes into decreasing order of comparative concentrations in normal serum, as well as in normal CSF.
Serum: LD2>LD1>LD3>LD4>LD5. CSF: LD1>LD2>LD3>LD4>LD5.
What is the best test to confirm that an elevated alkaline phosphatase is from the biliary tree?
GGT is found in the biliary epithelial cell, particularly those that line the small interlobular bile ducts and bile ductules. (it is also present in hepatocytes) Thus, it is exquisitely sensitive to biliary injury. 5’-nucleotidase (biliary epithelium is the main source) is another test to confirm that an elevated alk phos is due to hepatobiliary disease, but its relatively low sensitivity has kept its utility below that of GGT.
GGT is found to be __x the upper limit of normal in heavy drinkers.
GGT is found to be ~2-3x the upper limit of normal in heavy drinkers. It returns to normal in ~3 weeks with abstinence, and can then be followed as a marker of alcohol consumption.
GGT is present in smooth endoplasmic reticulum of hepatocytes (it is also present in biliary epithelial cells), and whenever there is induction due to an excess toxin, GGT levels increase. What toxins are classic for increased GGT?
Warfarin, barbiturates, dilantin, valproate, methotrexate, and alcohol. GGT is found to be ~2-3x the upper limit of normal in heavy drinkers. It returns to normal in ~3 weeks with abstinence, and can then be followed as a marker of alcohol consumption.
True or false. In Paget disease of bone, serum calcium is elevated.
False. Total serum alkaline phosphatase is elevated in over 85% of patients, and may be useful in monitoring the disease. Serum calcium, phosphorus, and aminotransferase are normal in Paget disease of bone.
The genes PRSS-1, PST1, and CFTR have all been implicated as causes of recurrent ___.
The genes PRSS-1 (cationic trypsinogen), PSTI (pancreatic secretory trypsin inhibitor), and CFTR (cystic fibrosis transmembrane conductance regulator) have all been implicated as causes of recurrent pancreatitis.
What test is most specific and sensitive for pancreatic exocrine function?
Elastase-1. Fecal fat, chymotrypsin, and elastase-1 are all sensitive and specific, but with elastase being the most so.
What patterns of amylase, CEA, and CA 19-9 are seen in pancreatic pseudocyst, serous cystadenoma, mucinous cystadenoma, intraductal papillary mucinous neoplasm, and solid-pseudopapillary tumor?
Pancreatic pseudocyst: high amylase, low CEA, high CA 19-9. Serous cystadenoma: low amylase, low CEA, low CA 19-9. Mucinous cystadenoma: low amylase, high CEA, normal to high CA 19-9. Intraductal papillary mucinous neoplasm: high amylase, high CEA, normal to high CA 19-9. Solid-pseudopapillary tumor: low amylase, low CEA, low CA 19-9.
What CK isoenzyme is the most widely distributed in the body?
CK-BB. It is found in nearly all tissues of the body, though found primarily in the brain.
Which CK isozyme migrates slightly slower than MM and whose appearance is associated with disseminated malignancies and poor prognosis?
Mitochondrial CK. If seen on electrophoresis, it appears as a faint band migrating slightly slower than CK-MM. Another abnormal CK is macro-CK, which is composed of a complex of antibody-bound CK.
The gene that encodes troponin __ is expressed in both cardiac and skeletal muscle, whereas troponin __ and troponin __ have separate cardiac and skeletal muscle genes.
The gene that encodes troponin C is expressed in both cardiac and skeletal muscle, whereas troponin T and troponin I have separate cardiac and skeletal muscle genes. cTnI is marginally more cardiac-specific than cTnT.
What is the earliest serum marker of AMI?
Usually within moments of an AMI, there is a elevation of myoglobin. However, myoglobin is very nonspecific and can be elevated due to a number of causes.
What is ischemia-modified albumin?
The amino terminus of albumin is modified with exposure to a number of conditions, such as acidosis, hypoxemia, and free radicals. The modification decreases the ability of albumin to bind cobalt. It can be used to measure myocardial ischemia, since ischemia-modified albumin rises within minutes of ischemic damage and returns to baseline within a few hours. The assay measures the amount of unbound cobalt, which reflects the level of ischemia-modified albumin.
Brain (B-type) natriuretic peptide (BNP) is released in response to increased ventricular filling pressure. It is released as an inactive pro-BNP peptide, which when cleaved releases ___ and ___.
Brain (B-type) natriuretic peptide (BNP) is released in response to increased ventricular filling pressure. It is released as an inactive pro-BNP peptide, which when cleaved releases an active BNP as well as the regulatory N-terminal pro-BNP, a very stable molecule which provides the most longitudinal information about CHF.
What is the purpose of serial measurements of elevated troponins in suspected AMI?
Increased sensitivity. The specificity of a single elevated troponin is very high; serial measurements don’t change that. However, a mildly elevated troponin may not be sensitive enough to detect AMI. Serial measurements of troponin increase the sensitivity, but don’t change the ability of the test to change its negative predictive value.
What is the purpose of measuring CK-MB in the presence of elevated troponin in a patient with a suspected AMI?
Troponin is not helpful in determining the time course of AMI. Troponin rises more slowly and stays elevated longer than CK-MB. If CK-MB continues to rise, it may indicate an acute event or an extension of an existing infarction, while a downward trend of CK-MB may indicate resolution of an infarction.
In the quantitation of protein by the Kjedahl technique, what is actually measured?
Ammonium nitrogen released by acid digestion. The Kjedahl technique is cumbersome and makes assumptions about average nitrogen content. Colorimetric assays are preferred for the measurement of protein, and all involve formation of a colored precipitate under alkaline or acidic conditions and then measuring the absorbance at the appropriate wavelength. Refractometry is used but has many interferences. Dye-binding is limited by uneven dye uptake by proteins.
What is the usual net charge on proteins and toward which pole do they migrate?
Most proteins bear a net negative charge at physiologic pH and as such migrate toward the anode or positive pole when subject to an electromotive force. Remember, anions have negative charges and are attracted to the positive pole or anode. Cations bear positive charges and are attracted to the negative pole or cathode.
Anions have negative charges and are attracted to the (positive/negative) pole. Cations bear positive charges and are attracted to the (positive/negative) pole.
Anions have negative charges and are attracted to the positive pole or anode. Cations bear positive charges and are attracted to the negative pole or cathode.
What is the fastest migrating band on standard serum protein electrophoresis performed at pH 8.6?
Albumin accounts for the majority of normal serum protein and is the fastest migrating major protein followed by the alpha, beta, then gamma region proteins.
True or false. Pre-albumin helps in maintenance of serum osmotic pressure.
False. Albumin, rather than pre-albumin, is responsible for maintenance of serum osmotic presure. Pre-albumin does function in the capacity to bind thyroid hormone and vitamin A. TTR misfolding and aggregation is known to be associated with the amyloid diseases senile systemic amyloidosis (SSA), familial amyloid polyneuropathy (FAP), and familial amyloid cardiomyopathy (FAC).
What is the origin of the word “transthyretin”?
Transthyretin (TTR) is a serum and cerebrospinal fluid carrier of the thyroid hormone thyroxine (T4) and retinol binding protein bound to retinol. This is how transthyretin gained its name, TRANSports THYroxine and RETINol. It is synthesized in the liver, choroid plexus and retinal pigment epithelium for secretion into the bloodstream, cerebrospinal fluid and the eye, respectively. TTR was originally called prealbumin (or Thyroxine-binding prealbumin) because it ran faster than albumin on electrophoresis gels.
What are 3 thyroid hormone (T4) binding proteins in serum, their relative binding affinities, and relative plasma concentrations?
Thyroxine binding globulin has highest binding affinity and lowest plasma concentrations. Transthryetin has mid-range binding affinity and mid-range plasma concentrations. Albumin has lowest binding affinity and highest plasma concentrations.
TSH levels are (higher/lower) in elderly people. TSH levels are (higher/lower) in obese people. TSH levels are (higher/lower) in pregnant women.
TSH levels are higher in elderly people. TSH levels are higher in obese people. TSH levels are lower in pregnant women.
What is a potential problem with TSH immunoassays in regards to levels and reference intervals?
Immunoassays employ monoclonal antibodies, which have limited and varying specificity to detect the epitopes of TSH. Which TSH molecules an assay detects will depend on the monoclonal antibody selected. Circulating TSH is heterogeneous, especially with respect to glycosylation. Because of heterogeneity in glycosylation, not all molecular forms of TSH are bioactive. In particular, although the upper limit of TSH is higher in older individuals, not all TSH in older persons may be bioactive. Commercial immunoassays for thyroid function testing may have up to a two-fold divergence between highest and lowest methods for T4 and T3. Ultrafiltration (or equilibrium dialysis) and tandem mass spectrometry are methods that have a better correlation between free T4 and log TSH. Immunoassays may also overestimate levels of T3 and free T3.
Transferrin may be elevated with iron deficiency and resemble an M-spike on serum protein electrophoresis. Where does the transferrin band migrate?
Transferrin is the predominant beta-1 protein. On standard serum electrophoresis, beta does not resolve into beta-1 and beta-2, but can on high-resolution electrophoresis. In the beta-2 region migrates IgA. C-reactive protein can be in beta-2 or gamma-2. The predominant alpha-1 band is alpha-1-antitrypsin; alpha-2 has haptoglobin and ceruloplasmin.
Why does alpha-2-macroglobulin concentration increase with nephrotic syndrome?
Due to its large size, alpha-2-macroglobulin is typically not lost with nephrotic syndrome. As a result of the loss of other smaller proteins and fluid, the alpha-2-macroglobulin concentration increases.
Why is there typically a double transferrin peak in CSF electrophoresis?
Both the unmodified and asialated forms can cross the blood-brain barrier through active transport.
What is the clinical significance of a twin albumin band?
Bisaslbuminemia is a normal variant seen in heterozygotes for different albumin allotypes. There is no clinical significance.
With cirrhosis, what characteristic finding is seen on serum electrophoresis?
Predominantly due to increased IgA, beta-gamma bridging is seen with cirrhosis. Cirrhosis can also show hypoalbuminemia with blunted alpha-1 and alpha-2 peaks.
What are some features of normal CSF protein electrophoresis that are different from normal serum protein electrophoresis?
Prominent pre-albumin band. Double beta-transferrin band. Dim albumin band. Dim alpha-2 band.
Oligoclonal bands seen in CSF protein electrophoresis but not in a concurrent serum protein electrophoresis support a diagnosis of ___.
Multiple sclerosis.
What type of cryoglobulin is most commonly associated with multiple myeloma or Waldenstrom macroglobulinemia?
Type I. Type I cryoglobulins are monoclonal and associated with monoclonal gammopathies, such as multiple myeloma or Waldenstrom macroglobulinemia. Type II is a mixture of polyclonal IgG and monoclonal IgM. Type III is a mixture of two or more polyclonal antibodies.
What is the most common cause of mixed cryoglobulinemia?
Hepatitis C.
What are the most common adverse events seen when correcting hyponatremia too slowly and when correcting hyponatremia too rapidly?
When correcting hyponatremia too slowly: cerebral edema. When correcting hyponatremia too rapidly: central pontine myelinolysis.
Hypovolemic hyponatremia is often due to ___. Euvolemic hyponatremia is often due to ___. Hypervolemic hyponatremia is often due to ___.
Hypovolemic hyponatremia is often due to water loss either through the kidneys or GI tract. Euvolemic hyponatremia is often due to drugs. Hypervolemic hyponatremia is often due to CHF, nephrotic syndrome, or cirrhosis.
Type I renal tubular acidosis is due to ___. Type II renal tubular acidosis is due to ___. Type IV renal tubular acidosis is due to ___.
Type I, or distal renal tubular acidosis is due to the inability to produce an acid urine. Associated with hypokalemia. Type II, or proximal renal tubular acidosis is due to bicarbonate wasting. Associated with hypokalemia. Type IV renal tubular acidosis is due to aldosterone deficiency. Associated with hyperkalemia.
In which renal tubular acidosis is hyperkalemia seen?
Type IV. Type I and type II are associated with hypokalemia.
Most common causes of primary hyperparathyroidism?
Parathyroid adenoma > parathyroid hyperplasia > parathyroid carcinoma.
80-90% of hypercalcemia is due to what 2 conditions?
Hyperparathyroidism or malignancy (often with the paraneoplastic expression of PTHrP).
List phlebotomy pre-collection variables.
Physiologic factors, including diurnal variation, exercise, fasting, diet, ethanol consumption, tobacco smoking, drug ingestion, and posture. Also, common interferences include tobacco smoking (in vivo) and collection-associated variables (in vitro).
List lab tests affected by diurnal variation, posture, and stress.
Cortisol: Peaks 4–6 AM; lowest 8 PM–12 AM; 50% lower at 8 PM than at 8 AM; increased with stress. ACTH: lower at night; increased with stress. Plasma renin activity: Lower at night; higher standing than supine. Aldosterone: Lower at night. Insulin: Lower at night. GH: Higher in afternoon and evening. Acid phosphatase: Higher in afternoon and evening. Thyroxine: Increases with exercise. Prolactin: Higher with stress; higher levels at 4 and 8 AM and at 8 and 10 PM. Iron: Peaks early to late morning; decreases up to 30% during the day. Calcium: 4% decrease supine. Hyperventilation such as from stress affects acid-base balance and elevates leukocyte counts, lactate, and free fatty acids. Upright position increases hydrostatic pressure, causing a reduction of plasma volume and increased concentration of proteins such as albumin.
What laboratory tests does exercise have an effect on?
Physical activity has transient and long-term effects on laboratory determinations. Transient changes may include an initial decrease followed by an increase in free fatty acids, and lactate may increase by as much as 300%. Exercise may elevate creatine phosphokinase (CK), aspartate aminotransferase (AST), and lactate dehydrogenase (LD), and may activate coagulation, fibrinolysis, and platelets. These changes are related to increased metabolic activities for energy purposes and usually return to preexercise levels soon after exercise cessation. Long-term effects of exercise may increase CK, aldolase, AST, and LD values. Chronic aerobic exercise is associated with lesser increases in plasma concentration of muscle enzymes such as CK, AST, alanine aminotransferase (ALT), and LD. Decreased levels of serum gonadotropin and sex steroid concentrations are seen in long-distance athletes while prolactin levels are elevated.
In what ways can an individual’s diet affect lab test results (specifically, glucose, triglycerides, bilirubin, potassium, alk phos, 5-HIAA, stool occult blood tests)?
An individual’s diet can greatly affect laboratory test results. The effect is transient and is easily controlled. Glucose and triglycerides, absorbed from food, increase after eating. After 48 hours of fasting, serum bilirubin concentrations may increase. Eating a meal, depending on fat content, may elevate plasma potassium, triglycerides, alkaline phosphatase, and 5-hydroxyindoleacetic acid (5-HIAA). Stool occult blood tests, which detect heme, are affected by the intake of meat, fish, iron, and horseradish, a source of peroxidase, causing a false-positive occult blood reaction.
What changes in lab test results can be seen with vegetarian diet, meat/protein-rich diet, Atkins diet, caffeine, ethanol ingestion and chronic alcohol abuse?
Certain foods or diet regimens may affect serum or urine constituents. Long-time vegetarian diets are reported to cause decreased concentrations of LDLs, VLDLs, total lipids, phospholipids, cholesterol, and triglycerides. Vitamin B12 deficiency can also occur, unless supplements are taken. A high meat or other protein-rich diet may increase serum urea, ammonia, and urate levels. High protein, low carbohydrate diets, such as the Atkins diet, greatly increase ketones in the urine and increase the serum BUN. Foods with a high unsaturated-to-saturated fatty acid ratio may show decreased serum cholesterol, while a diet rich in purines will show an increased urate value. Foods such as bananas, pineapples, tomatoes, and avocados are rich in serotonin. When ingested, elevated urine excretion of 5-HIAA may be observed. Beverages rich in caffeine elevate plasma free fatty acids and cause catecholamine release from the adrenal medulla and brain tissue. Ethanol ingestion increases plasma lactate, urate, and triglyceride concentrations. Elevated HDL cholesterol, GGT, urate, and MCV have been associated with chronic alcohol abuse.
Using a tourniquet to collect blood to determine lactate concentration may result in falsely increased values. Prolonged tourniquet application may also increase ___, ___, and ___ as the result of hemoconcentration when plasma water leaves the vein because of back pressure.
Using a tourniquet to collect blood to determine lactate concentration may result in falsely increased values. Prolonged tourniquet application may also increase serum enzymes, proteins, and protein-bound substances, including cholesterol, calcium, and triglycerides, as the result of hemoconcentration when plasma water leaves the vein because of back pressure.
How does patient age affect lab test values in the pediatric population (specifically, for Hb, bilirubin, glucose, alk phos, Cr, uric acid)?
Age of the patient has an effect on serum constituents. In the newborn, much of the Hb is Hb F, not Hb A, as seen in the adult. Bilirubin concentration rises after birth and peaks at about 5 days. In cases of hemolytic disease of the fetus and newborn (HDFN), bilirubin levels continue to rise. This often causes difficulty in distinguishing between physiologic jaundice and HDFN. Infants have a lower glucose level than adults because of their low glycogen reserve. With skeletal growth and muscle development, serum alkaline phosphatase and creatinine levels, respectively, also increase. The high uric acid level seen in a newborn decreases for the first 10 years of life, then increases, especially in boys, until the age of 16.
How does patient age affect lab test values in the adult population (specifically, cholesterol, triglycerides, uric acid, triiodothyronine, PTH, aldosterone, cortisol, testosterone, pituitary gonadotropins)?
Most serum constituents remain constant during adult life until the onset of menopause in women and middle age in men. Increases of about 2 mg/dL (0.05 mmol/L) per year in total cholesterol and 2 mg/dL (0.02 mmol/L) per year in triglycerides until midlife have been reported. The increase in cholesterol seen in postmenopausal women has been attributed to a decrease in estrogen levels. Uric acid levels peak in men in their 20s but do not peak in women until middle age. The elderly secrete less triiodothyronine, parathyroid hormone, aldosterone, and cortisol. After age 50, men experience a decrease in secretion rate and concentration of testosterone and women have an increase in pituitary gonadotropins, especially follicle-stimulating hormone.
Which lab test values are affected by gender?
After puberty, men generally have higher alkaline phosphatase, aminotransferase, creatine kinase, and aldolase levels than women; this is due to the larger muscle mass of men. Women have lower levels of magnesium, calcium, albumin, Hb, serum iron, and ferritin. Menstrual blood loss contributes to the lower iron values.
How are lab values different in a smoker compared to a nonsmoker?
Tobacco smokers have high blood carboxyhemoglobin levels, plasma catecholamines, and serum cortisol. Changes in these hormones often result in decreased numbers of eosinophils, while neutrophils, monocytes, and plasma fatty free acids increase. Chronic effects of smoking lead to increased Hb concentration, RBC count, MCV, and WBC count. Increased plasma levels of lactate, insulin, epinephrine, and growth hormone and urinary secretion of 5-HIAA are also seen. Vitamin B12 levels may be substantially decreased and have been reported to be inversely proportional to serum thiocyanate levels. Smoking also affects the body’s immune response. Immunoglobulin (Ig)A, IgG, and IgM are lower in smokers, and IgE levels are higher. Decreased sperm counts and motility and increased abnormal morphology have been reported in male smokers when compared with nonsmokers.
What blood constituents can be falsely elevated due to hemolysis?
Hemolysis can falsely increase blood constituents such as potassium, magnesium, iron, LD, phosphorus, ammonium, and total protein.
How can pseudohyperkalemia or pseudohypokalemia be seen in blood drawn from leukemic patients?
Pseudohyperkalemia can occur in patients with extremely high blast counts in acute or accelerated phase leukemias. Those blasts can be fragile and may lyse during standard phlebotomy, releasing potassium. In contrast, specimens with very high WBC counts that are collected gently can show pseudohypokalemia when potassium is taken up by highly metabolically active leukemic cells along with glucose; such specimens can be transported on ice to slow this enzymatically mediated uptake.
Which normally has a slightly higher value of potassium - serum or plasma?
Normally platelets release potassium during clotting, so serum has a slightly higher value of potassium than plasma from the same individual; this difference is accentuated when the platelet count is extremely elevated.
When serum bilirubin approaches 430 mmol/L (25 mg/L), interference may be observed in what chemistry assays?
When serum bilirubin approaches 430 mmol/L (25 mg/L), interference may be observed in assays for albumin (4-hydroxyazobenzene-2-carboxylic acid [HABA] procedure), cholesterol (using ferric chloride reagents), and total protein (Biuret procedure).
Lipemia may cause inhibition of what chemistry assays?
Lipemia occurs when serum triglyceride levels exceed 4.6 mmol/L (400 mg/dL). Inhibition of assays for amylase, urate, urea, CK, bilirubin, and total protein may be observed.
Overview of blood collection tubes.
Blood collection tubes have color-coded stoppers that distinguish the presence of a specific anticoagulant or additive, how the tube is chemically cleaned (e.g., for lead or iron determinations), or if the tube does not contain any additives. Tubes also come in various sizes for adult and pediatric patient populations. Draw volume is determined by the internal vacuum within the sealed tubes (e.g., 3.5, 4.0, 4.5, or 8.5 mL). The use of anticoagulants allows for analysis of whole blood specimens or plasma constituents obtained by centrifugation and separation of the plasma.
How does switching from glass to plastic blood collection tubes affect the order of draw?
Many laboratories have converted from glass to plastic collection tubes to minimize exposure to biohazardous material and broken glass; to lower biohazard waste disposal costs; and to comply with OSHA guidelines mandating substitution. This change from glass to plastic has required a modification in the order of draw. Whereas glass is a natural clot activator, plastic is not. So in order for blood to clot in plastic tubes, manufacturers coat the inside of the tube with a substance to facilitate clotting. However, if the tube following the clot activator tube remained the coat tube, as was the recommended order prior to 2003, carryover threatens the coag results. Glass or plastic tubes with additives, including gel tubes, are drawn after the citrate tube (blue top) to avoid interference with coagulation measurements. Glass or plastic serum tubes, without a clot activator or gel separator, may be drawn before the coagulation tubes are drawn, consistent with National Committee on Clinical Laboratory Standards (NCCLS) guidelines.
List blood collection tube details for stopper color, anticoagulant/additive, specimen type/use, and mechanism of action.
Red (glass); None; Serum/chemistry and serology; N/A. Red (plastic/Hemogard); Clot activator; Serum/chemistry and serology; Silica clot activator. Lavender (glass); K3EDTA in liquid form; Whole blood/hematology; Chelates (binds) calcium. Lavender (plastic); K2EDTA/spray-dried; Whole blood/hematology; Chelates (binds) calcium. Pink; Spray-dried K2EDTA; Whole blood/blood bank and molecular diagnostics; Chelates (binds) calcium. White; EDTA and gel; Plasma/molecular diagnostics; Chelates (binds) calcium. Light blue; Sodium citrate; Plasma/coagulation; Chelates (binds) calcium. Light blue; Thrombin and soybean trypsin inhibitor; Plasma/coagulation; Fibrin degradation products. Black; Sodium citrate ; Plasma/sed rates—hematology; Chelates (binds) calcium. Light green/black; Lithium heparin and gel; Plasma/chemistry; Inhibits thrombin formation. Green; Sodium heparin, lithium heparin; Plasma/chemistry; Inhibits thrombin formation. Royal blue; Sodium heparin, K2EDTA; Plasma/chemistry/toxicology; Heparin inhibits thrombin formation, Na2EDTA binds calcium. Gray; Sodium fluoride/potassium oxalate; Plasma/glucose testing; Inhibits glycolysis. Yellow; Sterile containing sodium polyanetholesulfonate; Serum/microbiology culture; Aids in bacterial recovery by inhibiting complement, phagocytes, and certain antibiotics. Yellow; Acid citrate dextrose; Plasma/blood bank, HLA phenotyping, and paternity testing; WBC preservative. Tan (glass); Sodium heparin; Plasma/lead testing; Inhibits thrombin formation. Tan (plastic); K2EDTA; Plasma/lead testing; Chelates (binds) calcium. Yellow/gray and orange; Thrombin; Serum/chemistry; Clot activator. Red/gray and gold; Clot activator separation gel; Serum/chemistry; Silica clot activator.
List the 5 most common used blood collection tubes (stopper color), the additive, function of the additive, and common lab tests run on them.
Light blue; 3.2% sodium citrate; prevents blood from clotting by binding calcium; coagulation. Red or gold (mottled or “tiger” top used with some tubes); serum tube with or without clot activator or gel; clot activator promotes blood clotting with glass or silica particles, gel separates serum from cells; chemistry, serology, immunology. Green; sodium or lithium heparin with or without gel; prevents clotting by inhibiting thrombin and thromboplastin; stat and routine chemistry. Lavender or pink; potassium ETDA; prevents clotting by binding calcium; hematology and blood bank. Gray; sodium fluoride, and sodium or potassium oxalate; fluoride inhibits glycolysis, and oxalate prevents clotting by precipitating calcium; glucose (especially when testing will be delayed), blood alcohol, lactic acid.
In what order should blood collection tubes be drawn?
- Blood-culture tubes (yellow). 2. Coagulation sodium citrate tube (blue stopper). 3. Serum tubes with or without clot activator or gel separator. 4. Heparin tubes with or without gel (green stopper). 5. Ethylenediaminetetraacetic acid tubes (lavender stopper). 6. Glycolytic inhibitor tubes (gray stopper).
Pink-top blood collection tubes are used in immunohematology for ABO grouping, Rh typing, and antibody screening. These tubes have a special cross-match label for information required by ___.
Pink tubes are used in immunohematology for ABO grouping, Rh typing, and antibody screening. These tubes have a special cross-match label for information required by the American Association of Blood Banks (AABB) and approved by the U.S. Food and Drug Administration (FDA) for blood bank collections.
What are advantages and disadvantages of heparin-containing blood collection tubes vs. EDTA-containing blood collection tubes?
Heparin has an advantage over EDTA as an anticoagulant, as it does not affect levels of ions such as calcium. However, heparin can interfere with some immunoassays. Heparin should not be used for coagulation or hematology testing. Heparinized plasma is preferred for potassium measurements to avoid an elevation due to the release of potassium from platelets as the blood clots. Lithium heparin may be used for most chemistry tests except for lithium and folate levels; for lithium, a serum specimen can be used instead. Sodium heparin cannot be used for assays measuring sodium levels, but it is recommended for trace elements, leads, and toxicology. Sodium heparin is the injectable form used for anticoagulant therapy.
Gray-top blood collection tubes are generally used for glucose measurements because they contain a preservative or antiglycolytic agent, such as sodium fluoride, which prevents glycolysis for ___ days.
Gray-top blood collection tubes are generally used for glucose measurements because they contain a preservative or antiglycolytic agent, such as sodium fluoride, which prevents glycolysis for 3 days. In bacterial septicemia, fluoride inhibition of glycolysis is neither adequate nor effective in preserving glucose concentration.
Therapeutic drug monitoring specimens should not be collected in blood collection tubes that contain gel separators because ___.
Red/gray- and gold-top tubes contain a clot activator and a separation gel. These tubes are referred to as serum separator tubes (SSTs) and are used most often for chemistry tests. Therapeutic drug monitoring specimens should not be collected in tubes that contain gel separators, as some gels absorb certain drugs, causing a falsely lowered result. Significant decreases in phenytoin, phenobarbital, lidocaine, quinidine, and carbamazepine have been reported. Also, tubes containing gels are not used in the blood bank or for immunologic testing, as the gel may interfere with the immunologic reactions.
What are clotting times for blood collection tubes with gel separators, tubes with clot activators, and tubes with no additives?
Clotting time for tubes using gel separators is approximately 30 minutes, and tubes that have clot activators, such as thrombin, will clot in 5 minutes. Plain red-stoppered tubes with no additives take about 60 minutes to clot completely.
Why can’t blood anticoagulated with oxalate be used to measure hematocrit?
Oxalate anticoagulants may shrink red cells; thus blood anticoagulated with oxalate cannot be used to measure hematocrit. Combined ammonium/potassium oxalate does not have the same effect of shrinking cells.
During storage, the concentration of a blood constituent in the specimen may change as a result of various processes, including ___.
During storage, the concentration of a blood constituent in the specimen may change as a result of various processes, including adsorption to glass or plastic tubes, protein denaturation, evaporation of volatile compounds, water movement into cells resulting in hemoconcentration of serum and plasma, and continuing metabolic activities of leukocytes and erythrocytes. These changes occur, although to varying degrees, at ambient temperature and during refrigeration or freezing. Storage requirements vary widely by analyte. Stability studies have shown that clinically significant analyte changes occur if serum or plasma remains in prolonged contact with blood cells. After separation from blood cells, analytes have the same stability in plasma and serum when stored under the same conditions.
After obtaining an arterial puncture sample for blood gases, why is the tube placed in ice water (or other coolant that will maintain a temperature of 1°–5° C)?
To minimize leukocyte consumption of oxygen.
What specific steps must be performed to obtain blood from central venous access lines?
Blood specimens drawn from catheters may be contaminated with whatever was administered or infused via the catheter. The solution (usually heparin) used to maintain patency of the vein must be cleared before blood for analysis is collected. Sufficient blood (minimum of 2–5 mL) must be withdrawn to clear the line (and then discarded), so laboratory data are reliable. In a separate syringe, the amount of blood required for the requested laboratory procedure(s) is then drawn. Strict aseptic technique must be followed to avoid site and/or catheter contamination. Coagulation measurements such as prothrombin time (PT), activated partial thromboplastin time (APTT), and thrombin time (TT) are extremely sensitive to heparin interference, so that even larger volumes of presample blood must be withdrawn before laboratory results are acceptable for these tests. The appropriate volume to be discarded should be established by each laboratory. The laboratory is sometimes asked to perform blood culture studies on blood drawn from indwelling catheters. Because the indwelling catheters are in place for a few days, this procedure is not recommended because organisms that grow on the walls of the catheter can contaminate the blood specimen.
What changes can be seen in urine characteristics and analytes with delayed testing?
Changes in color (Breakdown or alteration of chromogen or other urine constituent (e.g., hemoglobin, melanin, homogentisic acid, porphyrins)). Changes in odor (bacterial growth, decomposition). Increased turbidity (Increased bacteria, crystal formation, precipitation of amorphous material). Falsely low pH (Glucose converted to acids and alcohols by bacteria producing ammonia. Carbon dioxide (CO2) lost). Falsely elevated pH (Breakdown of urea by bacteria, forming ammonia). False-negative glucose (Utilization by bacteria (glycolysis)). False-negative ketone (Volatilization of acetone; breakdown of acetoacetate by bacteria). False-negative bilirubin (Destroyed by light; oxidation to biliverdin). False-negative urobilinogen (Destroyed by light). False-positive nitrite (Nitrite produced by bacteria after specimen is voided). False-negative nitrite (Nitrite converts to nitrogen and evaporates). Increased bacteriuria (Bacteria multiply in specimen before analysis). Disintegration of cells/casts (Unstable environment, especially in alkaline urine, hypotonic urine, or both).
On 24 hour urine collection specimens, what analytes are tested for with the following preservatives: 10 g boric acid, 10 mL 6N HCl, and 0.5 g sodium fluoride.
10 g boric acid: aldosterone, cortisol. 10 mL 6N HCl: catecholamines, cystine, homovanillic acid, hydroxyproline, metanephrines, oxalate, VMA. 0.5 g sodium fluoride: glucose.
On 24 hour urine collection specimens, what analytes are tested for using urine with no added preservatives (refrigerated)?
Amino acids, amylase, calcium, citrate, chloride, copper, creatinine, delta ALA, glucose, 5-HIAA, heavy metals (arsenic, lead, mercury), histamine, immunoelectrophoresis, lysozyme, magnesium, methylmalonic acid, microalbumin, mucopolysaccharides, phosphorus, porphobilinogen, porphyrins, potassium, protein, protein electrophoresis, sodium, urea, uric acid, xylose tolerance.
How is synovial fluid different from the other serous fluids?
Synovial fluid found in the joint cavities is an ultrafiltrate of plasma that is passed through fenestrations of the subsynovial capillary endothelium into the synovial cavity. Once in the cavity, it is combined with hyaluronic acid, a glycosaminoglycan secreted by the synovial lining cells. Synovial fluid differs from the other serous fluids in that it contains hyaluronic acid (mucin) and may contain crystals.
Recentrifugation of gel separator tubes has been associated with pseudo___.
Recentrifugation of gel separator tubes has been associated with pseudohyperkalemia. One study demonstrated that after initial centrifugation, a new serum layer will develop under the gel within the cellular layer. During storage, potassium leaks from the cellular layer into the new serum layer, creating hyperkalemia in this layer. When the tube is recentrifuged, the new serum layer will move above the gel layer and cause a pseudohyperkalemia in the serum for analysis. The same authors also demonstrated that a pseudonormokalemia in patients with true hypokalemia may be erroneously reported after recentrifugation.
What are ultracentrifuges?
Ultracentrifuges are high speed and capable of reaching a centrifugal force of 165,000 times gravity. These centrifuges require refrigeration chambers to compensate for the considerable heat produced. Ultracentrifuges are used to clear serum of chylomicrons, which is necessary to avoid interference with clinical testing.
Why can’t blood collected in tubes containing EDTA be used to measure calcium and magnesium?
The appropriate tube should always be used for collection of blood specimens because of interferences from some anticoagulants such as EDTA, which chelates both calcium and magnesium, thereby lowering the measurement of those ions and inhibiting some enzymes that require divalent cations (e.g., alkaline phosphatase by also chelating zinc).
Electrolyte measurements are not valid in blood collection tubes with ___.
Electrolyte measurements are not valid in blood collection tubes with potassium EDTA or sodium citrate.
What does excess citrate in plasma from insufficient blood volume collection into the tube do to clotting times?
Small volumes of blood may be acceptable for some chemistry tests or cell counts; however, coagulation tests require a minimal volume of blood in the collection tube to achieve correct balance with citrate anticoagulant, which also chelates calcium. Excess citrate in plasma from insufficient blood volume leads to falsely elevated clotting times. This interference is also important in polycythemia, when the hematocrit is abnormally high and plasma volume in which the citrate distributes is small. Thus, even in an apparently correctly filled tube, polycythemia can lead to falsely prolonged PT and PTT unless the amount of citrate anticoagulant in the tube is reduced proportionally to the decrease in plasma volume in that patient.
A widely used unit for wavelength in the visible spectrum is the ___. Electromagnetic radiation in the x-rays or gamma region may be expressed in terms of ___. Finally, because of its much longer wavelength, EMR in the infrared region may have units corresponding to the ___.
A widely used unit for wavelength in the visible spectrum is the nanometer, nm (10−9 m). Electromagnetic radiation in the x-rays or gamma region may be expressed in terms of angstrom units, Å (10−10 m). Finally, because of its much longer wavelength, EMR in the infrared region may have units corresponding to the micrometer, µm (10−6 m). 1 nm = 10−9 m = 10−7 cm; other units sometimes used include the following: 1 µm = 10−6 m = 10−4 cm, 1 Å = 10−10 m = 10−8 cm.
Describe reflectometry.
Measurement of analytes in biologic fluids using reflectometry has been used for decades. Two clinical applications include urine dipstick analysis and dry slide chemical analysis. A reflectometer is a filter photometer that measures the quantity of light reflected by a liquid sample that has been dispensed onto a grainy or fibrous solid support.
Describe nephelometry and turbidimetry.
Nephelometry and turbidimetry are based on the scattering of radiation by particles in suspension. Nephelometry and turbidimetry are used to measure the concentrations of large particles (such as antigen–antibody complexes, prealbumin, and other serum proteins) that because of their size cannot be measured by absorption spectroscopy. Nephelometry detects light that is scattered at various angles; scattered light yields a small signal that must be amplified. In contrast, turbidimetry measures a reduction in light transmission due to particle formation; thus, it detects a small decrease in a large signal.
What is osmometry?
Osmometry is the measurement of the osmolality of an aqueous solution such as serum, plasma, or urine. As osmotically active particles (e.g., glucose, urea nitrogen, sodium) are added to a solution, causing its osmolality to increase, four other properties of the solution are also affected: osmotic pressure, boiling point, freezing point, and vapor pressure. They are called colligative properties of the solution because they can be related to each other and to the osmolality. Osmometry is based on measuring changes in the colligative properties of solutions that occur owing to variations in particle concentration. Freezing-point depression osmometry is the most commonly used method for measuring the changes in colligative properties of a solution. It is based on the principle that addition of solute molecules lowers the temperature at which a solution freezes.
Flow cytometry is used to count and sort cells, as well as ___.
Flow cytometry is used to count and sort cells, as well as viral particles, DNA fragments, bacteria, and latex beads. Particles must be in suspension as single cells to be analyzed. If not, they can be made suitable for flow cytometry by the use of mechanical disruption or enzymatic digestion. Size restrictions also apply; cells or particles must be from 1–30 µm in diameter. Specialized flow cytometers are designed to handle smaller particles such as DNA fragments or bacteria.
What is a fluorescence-activated cell sorter?
FACS, an acronym for fluorescence-activated cell sorter, describes the ability of a flow cytometer to physically sort cells in a liquid suspension. To do so, the instrument design has to be modified to electrically charge cells of interest. This is done by first vibrating the sheath stream to break it into drops. The stream of drops flows past two charge (high-voltage) plates where cells of interest are electrically charged with a voltage pulse. Then the flow stream enters an electrical field where charged cells are deflected into suitable collection containers. Unwanted cells are not charged and are not deflected upon passing through the field.
Coulometry is used to measure ___ ion in serum, plasma, CSF, and sweat samples.
Coulometry is used to measure chloride ion in serum, plasma, CSF, and sweat samples. In measuring chloride with coulometry, a constant current is applied across the two silver electrodes, which liberate silver ions into the specimen at a constant rate. Chloride ions in the sample combine with released silver ions to produce insoluble silver chloride. A pair of indicator and reference electrodes senses the excess silver ions and stops the titration. The number of silver ions released by ionization, which is exactly equal to that of chloride ions in the sample, can be calculated from Faraday’s law.
What is anodic stripping voltammetry, and what is it used to measure?
Anodic stripping voltammetry is an electrochemical technique used to measure heavy metals such as lead. The measurement of lead in blood occurs in basically two steps. First, free lead is electroplated (deposited) onto the platinum cathodes at a characteristic voltage. Second, the lead is stripped off of the platinum cathode, and the current is monitored over time. The current value is proportional to the amount of lead in the blood sample. This technique allows the sample to be preconcentrated at the electrode, which enables the method to detect very low analyte levels.
What techniques are used in the laboratory that utilize the principles of conductance?
Electrolytic conductivity is a measure of the ability of a solution to carry an electrical current. The principles of conductance have several applications associated with clinical laboratory procedures. Examples include monitoring water purity, measuring analytes in blood such as urea, and serving as components of detectors used in high-performance liquid chromatography (HPLC), gas chromatography (GC), cell counters, and capillary electrophoresis.
For what primary purpose/analytic tests are the principles of impedance used in the laboratory?
Electrical impedance measurement is based on the change in electrical resistance across an aperture when a particle in conductive liquid passes through this aperture. Electrical impedance is used primarily in the hematology laboratory to enumerate leukocytes, erythrocytes, and platelets. In a typical electrical impedance instrument by Coulter, aspirated blood is divided into two separate volumes for measurements. One volume is mixed with diluent and is delivered to the cell bath, where erythrocyte and platelet counts are performed. As a cell passes through the aperture, partially occluding it, the electrical impedance increases, producing a voltage pulse, the size of which is proportional to the cell size. The number of pulses is directly related to the cell count. Particles measuring between 2 and 20 fL are counted as platelets, whereas those measuring greater than 36 fL are counted as erythrocytes. The other blood volume is mixed with diluent and a cytochemical-lytic reagent that lyses only the red blood cells. A leukocyte count is performed as the remaining cells pass through an aperture. Particles greater than 35 fL are recorded as leukocytes.
What is electrophoresis?
Electrophoresis is the separation of charged compounds based on their electrical charge. When a voltage is applied to a salt solution (usually sodium chloride), an electrical current is produced by the flow of ions: cations toward the cathode, and anions toward the anode. Conductivity of a solution increases with its total ionic concentration. The greater the net charges of a dissolved compound, the faster it moves through the solution toward the oppositely charged electrode. The net charge of a compound, in turn, depends on the solution pH. Electrophoresis separations often require high voltages (50–200 V DC); therefore, the power supply should supply a constant DC voltage at these levels. The buffer solution must have a carefully controlled ionic strength. A dilute buffer causes heat to be generated in the cell, and a high ionic strength does not allow good separation of the fractions. Common support media for electrophoresis in clinical work include cellulose acetate, agarose, and polyacrylamide gels. Total volume of specimen applied depends on the sensitivity of the detection method. For clinical work, 1 µL of serum may be applied. Once the electrophoresis is completed, the support medium is treated with a dye to identify the separated fractions. The most common dyes used for the visualization step include Amido Black, Ponceau S, Fat Red 7B, and Sudan Black B. To obtain a quantitative profile of the separated fractions, densitometry is performed on the stained support medium.
What is densiometry?
Electrophoresis is the separation of charged compounds based on their electrical charge. Then, to obtain a quantitative profile of the separated fractions, densitometry is performed on the stained support medium. A densitometer measures the absorbance of the stain on a support medium. The basic components of a densitometer include a light source, a monochromator and a movable carriage to scan the medium over the entire area, an optical system, and a photodetector. Signals detected by the photodetector are related to the absorbance of the sample stain on the support, which is proportional to the specimen concentration. The support medium is moved through the light beam at a fixed rate, so that a graph may be constructed that represents multiple density readings taken at different points. Most densitometers have a built-in integrator to find the area under the curve, so that all sample fractions can be quantified.
What is isoelectric focusing, and what clinical uses does it have?
Isoelectric focusing (IEF) techniques are performed similarly to other electrophoresis methods, except that the separating molecules migrate through a pH gradient. This pH gradient is created by adding acid to the anodic area of the electrolyte cell and adding base to the cathode area. A solution of ampholytes (mixtures of small amphoteric ions with different pIs) is placed between the two electrodes. These ampholytes have high buffering capacity at their respective isoelectric points. The ampholytes close to the anode carry a net positive charge, and those close to the cathode carry a net negative charge. When an electrical voltage is applied, each ampholyte will rapidly migrate to the area where the pH is equal to its isoelectric point. With their high buffering capacity, the ampholytes create stable pH zones for the more slowly migrating proteins. The advantage of isoelectric focusing techniques lies in their ability to resolve mixtures of proteins. Using narrow-range ampholytes, macromolecules differing in isoelectric point by only 0.02 pH units can be identified. Isoelectric focusing has been useful in measuring serum acid phosphatase isoenzymes. Its application has also been extended to detect oligoclonal immunoglobulin bands in CSF and isoenzymes of creatine kinase and alkaline phosphatase in serum.
Are chromatographic techniques classified according to their mobile phase or their stationary phase?
Their mobile phase (gas chromatography, liquid chromatography).
What types of gasses are used in gas chromatography?
Carrier gases (mobile phase), which must be chemically inert, include helium, hydrogen, and nitrogen. Other substances used as mobile phases include steam and supercritical fluids. Examples of these are carbon dioxide, nitrous oxide, and ammonia. The carrier gas should be of high purity, and the flow must be tightly controlled to ensure optimum column efficiency and reproducibility of test results.
What can be done in gas chromatography if the molecule of interest is not volatile enough for direct injection?
Samples are manually introduced into the GC using a syringe pipet or an automated syringe pipet system. A pipet tip pierces a plastic septum located in the injector port. Each injection port is heated to very high temperatures. Samples are vaporized and swept onto the column. If the molecule of interest is not volatile enough for direct injection, it is necessary to derivatize it into a more volatile form. Most derivatization reactions belong to one of three groups: silylation, alkylation, and acylation. Silylation is the most common technique that replaces active hydrogens on the compounds with alkylsilyl groups. This substitution results in a more volatile form that is also less polar and more thermally stable.
What types of detectors can be used in gas chromatography?
Examples of detectors used in GC include a flame ionization detector, a thermal conductivity detector, a nitrogen–phosphorus detector, an electron capture detector, a flame photometric detector, and a mass spectrometric detector. Flame ionization detectors (FIDs) are commonly used and are capable of detecting a wide variety of organic compounds and many inorganic compounds. This type of detector measures the ions produced by the compounds when burned in a hydrogen–air flame. An electrode collects the ions, and the magnitude of the resulting electrical current is proportional to the amount of substance. FIDs can be modified to make them especially sensitive to molecules, including many drugs that contain either nitrogen or phosphorus (the so-called nitrogen–phosphorus detector, or NPD).
What are advantages of liquid chromatography over gas chromatography?
GC as a separation technique has some restrictions that make liquid chromatography a suitable alternative. Many organic compounds are too unstable or are insufficiently volatile to be assayed by GC without prior chemical derivatization. Liquid chromatography techniques use lower temperatures for separation, thereby achieving better separation of thermolabile compounds. These two factors allow liquid chromatography to separate compounds that cannot be separated by GC. Finally, it is easier to recover a sample in liquid chromatography than in gas chromatography. The mobile phase can be removed, and the sample can be processed further or reanalyzed under different conditions.
What major steps are involved in mass spectrometry?
Mass spectrometry typically involves the following major steps: (1) conversion of the parent molecule into a stream of ions (usually singly charged positive ions); (2) separation of the ions by mass/charge ratio (m/z), where m is the mass of the ion in atomic mass units and z is its charge; and (3) counting of the number of ions of each type or measurement of current produced when the ions strike a transducer. Because most of the ions formed in mass spectrometers are singly charged, the m/z is often shortened to the more convenient term mass. Strictly speaking, this abbreviation is incorrect, but it is widely used in mass spectrometry literature. This term also represents the x-axis for MS spectrums of molecules plotted against their relative abundance (y-axis).
What are the 3 basic components of mass spectrometers?
All mass spectrometers have three basic components: an ion source, a mass analyzer, and an ion detector. The inlet unit admits samples to the mass spectrometer. When the instrument is part of a GC/MS arrangement, the inlet unit must be heated to maintain the volatile compounds in the vapor state upon coming into the ion source unit. It must also strip away most of the carrier gas to adapt to the high-vacuum condition required for mass spectrometry operation.
What are MALDI and SELDI?
The matrix-assisted laser desorption ionization (MALDI) source consists of a solid mixture of analyte and matrix (including organic chromophore) on a sample plate, along with a laser light and ion optics. When the chromophore absorbs the laser light, it vaporizes and lifts the analyte ions from the surface into a gas phase directly above the target plate and into the analyzer. The MALDI technique is considered an offline ionization technique because the sample is purified, deposited, and dried on the sample plate before analysis. Surface-enhanced laser desorption ionization (SELDI) is a technique that measures proteins from complex biologic specimens such as serum, plasma, intestinal fluids, urine, cell lysates, and cellular secretion products. Proteins are captured by adsorption, partition, electrostatic interaction, or affinity chromatography on a solid-phase protein chip surface. A laser ionizes samples that have been co-crystallized with a matrix on a target surface.
Describe SELDI and its protein chip arrays.
Surface-enhanced laser desorption ionization (SELDI) is a technique that measures proteins from complex biologic specimens. Proteins are captured by adsorption, partition, electrostatic interaction, or affinity chromatography on a solid-phase protein chip surface. A laser ionizes samples that have been co-crystallized with a matrix on a target surface. The protein chip chromatographic surfaces in SELDI are uniquely designed to retain proteins from complex mixtures according to their specific properties. After the addition of a matrix solution, proteins can be ionized with a nitrogen laser and their molecular masses measured by time-of-flight (TOF) MS. The protein chip arrays are the heart of the SELDI-TOF MS technology and distinguish it from other MS-based systems. Each array is composed of different chromatographic surfaces that, unlike HPLC or GC, are designed to retain, not elute, proteins of interest. The protein chip arrays have an aluminum base with several spots composed of a chemical (anionic, cationic, hydrophobic, hydrophilic, or metal ion) or biochemical (immobilized antibody, receptor, DNA, enzymes, etc.) active surface. Each surface is designed to retain proteins according to a general or specific physicochemical property of the proteins. Chemically active surfaces retain whole classes of proteins, and surfaces to which a biochemical agent, such as an antibody or other type of affinity reagent, is coupled are designed to interact specifically with a single target protein.
What is a scintillation counter?
Scintillations are flashes of light that occur when gamma rays or charged particles interact with matter. Chemicals used to convert their energy into light energy are called scintillators. If gamma rays or ionizing particles are absorbed in a scintillator, some energy absorbed by the scintillator is emitted as a pulse of visible light or near-UV radiation. A photomultiplier tube detects light directly or through an internally reflecting optic fiber. A scintillation counter is an instrument that detects scintillations using a photomultiplier tube and counts the electrical impulses produced by the scintillations. An application for scintillation counting is radioimmunoassay (RIA). Two types of scintillation methods exist: crystal scintillation and liquid scintillation. Crystal scintillation generally is used to detect gamma radiation, while liquid scintillation is primarily used to count radionuclides that emit beta particles.
What is capillary electrophoresis and what are its applications?
Capillary electrophoresis is a separation technique where a sample introduced into a fused silica capillary is separated by electro-osmotic flow by application of a high voltage across the capillary ends. Applications of capillary electrophoresis include separation of serum proteins and hemoglobin variants.
Laboratory results must undergo a two-step postanalytic review for ___ and ___.
Laboratory results must undergo a two-step postanalytic review for analytic correctness (using delta checks, linearity ranges, etc.) and for clinical significance for the patient (applying critical values, reference ranges, pretest and posttest probability, etc.).
Reference intervals are most commonly defined as ___.
Reference intervals are most commonly defined as the range of values into which 95% of nondiseased individuals will fall; this definition implies that 5% of nondiseased individuals can have laboratory results outside the reference range. In most settings, reference ranges are determined by the laboratory, with varying degrees of input from the clinical staff.
Interfering substances in laboratory specimens are most commonly lipids (lipemia), hemoglobin (Hb) (hemolysis), paraproteins (gammopathies), or bilirubin (icterus). The mechanism for this interference is dependent on the substance and the analytic method. What is the mechanism for how lipids interfere with spectrophotometric assays and with assays using ion-specific electrodes?
In spectrophotometric assays, lipids interfere mainly by increasing light scatter (turbidity); in assays using ion-specific electrodes for measurement, lipids will affect results by solvent exclusion.
What are delta checks?
Delta checks are defined as comparing a current laboratory result with results obtained on a previous specimen from the same patient. Parameters chosen for delta checks should not be subject to large intraindividual variations; for example, many laboratories have delta checks in place for the mean corpuscular volume of red cells.
What is a ROC curve?
Because diagnostic tests are not perfect, there is always a tradeoff between sensitivity and specificity, making it difficult to determine an optimal cutoff. The receiver operator characteristic (ROC) curve is a useful tool for identifying the optimal cutoff for a diagnostic test by calculating the sensitivity and specificity combinations across the entire range of cutoff values. In addition, the ROC curves of two or more tests can be compared to identify the one with the greatest discriminating ability.
How are ROC curves constructed?
The receiver operator characteristic (ROC) curve is a useful tool for identifying the optimal cutoff for a diagnostic test by calculating the sensitivity and specificity combinations across the entire range of cutoff values. An ROC curve is constructed by calculating sensitivity and specificity across the entire range of cutoffs for the diagnostic test being evaluated. Sensitivity is plotted on the y-axis and 1− specificity (or the FP rate) on the x-axis; construction of the curve shows the tradeoff between sensitivity and specificity at each cutoff. The ROC curve for a test with no ability to predict disease (i.e., random chance) is a 45-degree line drawn through the origin. As the discriminatory ability of a test increases, the curve progresses outward toward the upper left-hand corner. The area under the ROC curve (AUC) is a single measure of the overall discriminating ability of a test; the minimum AUC is 0.5, which is the area under the 45-degree line, and the maximum AUC is 1.0 for a perfect diagnostic test.
What is AUC?
The ROC curve for a test with no ability to predict disease (i.e., random chance) is a 45-degree line drawn through the origin. As the discriminatory ability of a test increases, the curve progresses outward toward the upper left-hand corner. The area under the ROC curve (AUC) is a single measure of the overall discriminating ability of a test; the minimum AUC is 0.5, which is the area under the 45-degree line, and the maximum AUC is 1.0 for a perfect diagnostic test. Because diagnostic tests are not absolutely perfect, the AUC will range from 0.5 to 1.0; the higher the AUC, the greater is the overall discriminating ability of the test. In general, an AUC that is greater than 0.8 suggests that the diagnostic test has good discriminatory power.
How can you compare two ROC curves (ie two diagnostic tests) with their AUC?
The receiver operator characteristic (ROC) curve is a useful tool for identifying the optimal cutoff for a diagnostic test by calculating the sensitivity and specificity combinations across the entire range of cutoff values. The optimal cutoff is identified by the coordinate that maximizes discriminatory power, the location on the ROC curve with the largest distance perpendicular from the 45-degree line. The second useful application for the ROC curve is to compare two or more diagnostic methods by calculating the area under the curve. If the area under the curve is similar, then no difference is observed between the two tests. However, if one ROC curve has a greater AUC than a comparison test, it has better sensitivity and specificity at all cutoffs. However, curves may cross, indicating that performance depends on the desired use of the test (i.e., whether a higher sensitivity or specificity is required for clinical use). If test A has a lower FP rate than test B at high sensitivity, test A is a better screening test while test B would be the better confirmatory test because it has greater sensitivity at high specificities.
What is the positivity criterion?
The likelihood ratio may be used to select the optimal cutoff for an assay that best separates disease from nondisease. The LR, like sensitivity and specificity, varies with the cutoff. To select the optimal discriminatory cutoff, one might simply pick the cutoff resulting in the greatest LR for a positive test. However, this approach does not take into account the clinical impact of the test result. The clinical consequences of a FP test (e.g., unnecessary surgery) or a FN test (e.g., a missed chance to treat) need to be weighed when the optimal cutoff is determined. The positivity criterion is a method that allows one to assess the optimal cutoff with numeric estimates for clinical impact, or consequences, of test results. In determining which cutoff will serve as the positivity criterion, a finite list of possible cutoffs is generated, and an LR for a positive test is calculated for each cutoff value. Next, the consequences of each of the four possible testing outcomes (TP, TN, FP, and FN) are assigned numeric estimates with respect to some outcome (e.g., morbidity, mortality, cost). Values for the estimated consequences, as well as prevalence estimates of the disease, are then used in the LRthreshold equation, which gives the optimal assay cutoff. From the list of possible analyte concentrations, one can now select the cutoff that most closely approximates the LRthreshold. In this way, one may select a test cutoff that not only optimizes the discriminatory power of the assay, but also fits the clinical need that the test is meant to address.
What is Osler’s rule?
Especially if the patient is younger than 60 years of age, try to attribute all abnormal laboratory findings to a single cause. Only if there is no possible way to correlate all abnormal findings should the possibility of multiple diagnoses be entertained.
The chromicity of the red cells (i.e., the intensity of the red color of the cells due to intracellular Hb) is measured quantitatively by ___.
The chromicity of the red cells (i.e., the intensity of the red color of the cells due to intracellular Hb) is measured quantitatively by the mean corpuscular hemoglobin concentration, or MCHC.
Iron binding capacity is a direct measure of the protein ___.
Iron binding capacity is a direct measure of the protein transferrin, which transports iron from the gut to iron storage sites in bone marrow.
Considerable overlap has been noted between serum levels of iron and iron-binding capacity in IDA and ACD. A somewhat more reliable discriminating measure of IDA is ___.
Considerable overlap has been noted between serum levels of iron and iron-binding capacity in IDA and ACD. A somewhat more reliable discriminating measure of IDA is the ratio of serum iron to IBC, known as the transferrin saturation. This ratio is around 1 : 3 for normal individuals, and in IDA it is significantly reduced to values of around 1 : 5 or lower. Again, considerable overlap is seen even in this ratio for patients with IDA and ACD, so the values should always be interpreted with care.
IDA and ACD are both hypoproliferative, microcytic anemias. What are some analyte abnormalities commonly seen for each?
IDA: low ferritin, increased IBC, decreased serum iron, reduced Fe/IBC ratio, generally increased RDW.
ACD: generally high ferritin, normal IBC, decreased serum iron, normal Fe/IBC ratio, generally normal RDW.
Hemolytic anemia is a hyperproliferative, normocytic anemia. What are some analyte abnormalities commonly seen?
Schistocytosis, increased reticulocytes, low haptoglobin, elevated carboxyhemoglobin, elevated LD, elevated indirect bilirubin, generally increased RDW.
Aplastic anemia and renal failure both have hypoproliferative, normocytic anemia. What are some analyte abnormalities commonly seen for each?
Aplastic anemia: leukopenia, thrombocytopenia, hypocellular bone marrow, generally normal RDW. Renal failure: elevated BUN and Cr, low erythropoietin, burr cells may be present, generally normal RDW.
B12 and/or folate deficiency and hypothyroidism both have hypoproliferative, macrocytic anemia. What are some analyte abnormalities commonly seen for each?
B12 and/or folate deficiency: megaloblastic, low B12 and/or folate, hyperlobulated PMNs, macroovalocytes, increased RDW. Hypothyroidism: nonmegaloblastic, elevated TSH, normal RDW.
What is the relationship between hemolysis and haptoglobin?
After erythrocyte membrane breakdown, Hb is extruded. Extruded Hb becomes bound to the α-2 fraction protein haptoglobin. The Hb–haptoglobin complex becomes catabolized by macrophages that engulf these complexes by receptor-mediated endocytosis. Thus an excellent laboratory test for hemolytic anemia is a low haptoglobin value. Extremely sensitive and rapid ELISA assays for haptoglobin are available for this purpose.
Why do carbon monoxide and unconjugated bilirubin become elevated in blood in hemolytic anemia?
When Hb is extruded, large amounts of it become oxidized to methemoglobin. The heme portion dissociates and then becomes oxidized ultimately to bilirubin. The first step in this process is the oxidative opening of the porphyrin ring of heme with attendant liberation of CO. CO may be measured easily by gas chromatographic techniques or even more conveniently by cooximetry, based on spectrophotometry, as carboxyhemoglobin. Elevated CO levels in normochromic, normocytic anemias are an excellent indicator of hemolytic anemia. Because production of bilirubin, which is unconjugated, is increased, at least a transient elevation of serum indirect bilirubin occurs. This elevation, in the presence of normal liver function, will be modest.
How does renal failure cause anemia?
Another normocytic, hypoproliferative anemia is the anemia of chronic renal failure. Loss of the kidneys’ excretory function produces an increase in BUN and creatinine, as discussed later, as well as a buildup of metabolic byproducts. The resulting uremia appears to be responsible for changes in red cell shape, with burr cells (echinocytes) and ellipsoidal cells commonly present on peripheral blood films. Identification of burr cells on peripheral blood films during the course of illness may signal the development of renal dysfunction. In addition to decreased excretory function, the kidneys’ ability to produce erythropoietin is decreased, resulting in impaired erythropoiesis, such that the marrow’s response to hypoxia becomes inadequate. In contrast to aplastic anemia, white cell and platelet counts usually remain within normal limits.
70% of the total filtered sodium is reabsorbed in what part of the nephron?
Proximal convoluted tubule.
Which causes higher urinary sodium and urine osmolality, SIADH or aldosterone deficit?
SIADH.
In all forms of hyponatremia, the ___ ion concentration is generally low because it is the chief counter-ion for sodium.
In all forms of hyponatremia, the chloride ion concentration is generally low because it is the chief counter-ion for sodium.
For these causes of hyponatremia (overhydration, diuretics, SIADH, adrenal failure, Bartter’s syndrome, diabetic hyperosmolarity) give as low, normal, or high the urine Na, urine osmolality, serum K, and 24 hour urine Na.
Overhydration: low urine Na, low urine osmolality, normal or low serum K, low 24 hour urine Na. Diuretics: low urine Na, low urine osmolality, low serum K, high 24 hour urine Na. SIADH: high urine Na, high urine osmolality, normal or low serum K, high 24 hour urine Na. Adrenal failure: mildly elevated urine Na, normal urine osmolality, high serum K, high 24 hour urine Na. Bartter’s syndrome: low urine Na, low urine osmolality, low serum K, high 24 hour urine Na. Diabetic hyperosmolarity: normal urine Na, normal urine osmolality, high serum K, normal 24 hour urine Na.
Roughly, for every 100 mg/dL increase in serum glucose, a ___ mEq/L decrease in serum Na concentration is seen.
Roughly, for every 100 mg/dL increase in serum glucose, a 1.6 mEq/L decrease in serum Na concentration is seen.
Does using ion-selective electrodes eliminate the pseudohyponatremia artifact?
This condition is usually caused by the presence of excess lipids in serum. No sodium ions are dissolved in lipids, which can take up a considerable volume of serum. If the absolute amount of sodium in a given volume of serum is determined, as with such methods of sodium determination as flame photometry, this value is divided by the sample volume to get the concentration. But part of this volume is lipid that has no sodium. So a falsely low value of sodium can be obtained. This artifact is eliminated by the use of ion-selective electrodes that directly determine the concentration of sodium and do not depend upon knowledge of the volume of serum. Note that although most modern, high throughput chemistry analyzers measure serum sodium using ion-selective electrodes, they perform a predilution of the specimen (so-called indirect potentiometry), and hence the measurement is relative to volume and is susceptible to pseudohyponatremia.
For these causes of hypernatremia (dehydration, diabetes insipidus, Cushing’s disease or syndrome) give as low, normal, or high the urine Na, urine osmolality, serum K, and 24 hour urine Na.
Dehydration: high urine Na, high urine osmolality, normal serum K, 24 hour urine Na varies. Diabetes insipidus: low urine Na, low urine osmolality, normal serum K, low 24 hour urine Na. Cushing’s disease or syndrome: low urine Na, normal urine osmolality, low serum K, low 24 hour urine Na.
Many of the causes of hypokalemia, including overhydration, use of loop diuretics, SIADH, and Bartter’s syndrome, overlap with those of hyponatremia. In addition to overlap of these causes with those of hyponatremia, what 3 states lead uniquely to hypokalemia?
- Infusion of insulin to diabetic individuals. This results in rather large influxes of potassium into cells, which lower it in serum. 2. Alkalosis. RBCs are themselves excellent buffers. They are capable of exchanging potassium for hydrogen ions. Thus, in acidosis, H+ ions enter red cells in exchange for K+ ions. Conversely, in alkalosis, H+ ions leave red cells (to neutralize excess base), and K+ ions enter the red cells. 3. Vomiting. The major loss consists of both H+ and K+ from the stomach. Loss of K+ in gastric fluid may be less important than the overall fluid loss, which causes activation of aldosterone and renal wasting of K+.
Concomitant elevations of potassium and LD in serum should be taken as indications of ___.
Concomitant elevations of potassium and LD in serum should be taken as indications of hemolysis that occurs artifactually after a blood sample has been taken from the patient or, less commonly, as hemolysis that results from an underlying hemolytic condition.
Urea is excreted by the renal tubules at a rate that is (proportional to/not proportional to) the glomerular filtration rate.
Urea is excreted by the renal tubules at a rate that is roughly proportional to the GFR. Note, therefore, that the retained urea (i.e., plasma or serum urea or BUN) is approximately inversely proportional to the GFR.
What is the formula for GFR?
Creatinine is secreted but is also reabsorbed to an approximately equal extent, so that the net effect is that the amount filtered is the amount excreted. The total amount of creatinine filtered then is its urinary concentration, Ucr × volume of urine (V), over a given time. The total plasma that delivered this quantity of creatinine to the glomerulus is the total amount of creatinine filtered divided by the plasma concentration, Pcr. This quantity is also the creatinine clearance (Ccr). So GFR = Ccr = Ucr x V/Pcr.
What is BUN/Cr for pre-renal disease?
Pre-renal disease is where renal plasma flow is reduced from such lesions as renal artery stenosis, renal vein thrombosis, and the like. This causes a reduction in the GFR and a rise in BUN. However, the serum creatinine levels, with a reference range of about 0.5–1.0 mg/dL, generally will remain within normal limits or may be mildly elevated because low GFR will result in lower urine flow. Pcr and Ucr generally will remain within normal limits. Thus a disproportionate rise in BUN over creatinine will be noted. The normal BUN/creatinine ratio is 10–20 : 1, and in pre-renal disease, it rises to well above 20 : 1.
What is BUN/Cr for renal and post-renal disease?
As in pre-renal disease, there will be a rise in BUN due to low GFR. Now, however, creatinine filtration will be compromised, so that its serum level will rise correspondingly. Thus, in true renal disease, both BUN and creatinine rise together, maintaining the BUN/creatinine ratio at 10–20 : 1. This pattern also occurs in so-called post-renal disease.
In renal failure, how can you tell if the lesion is tubular or glomerular?
If a patient is on a fluid-restricted diet, the osmolality of urine (Uosm) should be significantly higher than the osmolality of plasma (Posm). In fact, Uosm/Posm is >1.2 for normal individuals. If a 24-hour urine specimen collection from the above patient on a fluid-restricted diet is measured for Uosm, we can determine where the lesion has occurred. If Uosm/Posm <1.2, then the urine is not being concentrated, so a tubular lesion must be present. On the other hand, if a normal ratio is found, then, by exclusion, the lesion must be glomerular.
What are the acronyms CHARD and CHIMPS?
CHARD is for causes of hypocalcemia: Calcitonin, Hypoparathyroidism, Alkalosis, Renal failure, vitamin D deficit. CHIMPS is for causes of hypercalcemia: Cancer, Hyperthyroidism, Iatrogenic causes, Multiple myeloma, Hyperparathyroidism, Sarcoidosis (and also acidosis).
About half of the calcium circulating in blood is bound to serum protein. Where is the remainder?
About half of the calcium circulating in blood is bound to serum protein, mainly albumin; the remainder may be chelated in tight complexes with ions such as citrate and oxalate or may be found in ionic complexes with counter-ions such as chloride—so-called free calcium.
What are components of blood gas measurements?
Blood gas determinations refer to the quantitative measurement of the pH of arterial blood, the partial pressure of carbon dioxide (pCO2), the bicarbonate, the partial pressure of oxygen (pO2), oxygen saturation, and base excess. Three of these quantities are interdependent on one another, that is, the PCO2, the bicarbonate, and the pH, by the Henderson-Hasselbalch equation.
For metabolic acidosis, metabolic alkalosis, respiratory acidosis, and respiratory alkalosis, give the bicarbonate and pCO2 as high or low, and list typical causes.
Metabolic acidosis: low bicarb, low pCO2; typical causes are diabetic ketoacidosis, lactic acidosis (as from gram neg sepsis), renal failure. Metabolic alkalosis: high bicarb, high pCO2; typical causes are vomiting (loss of HCl). Respiratory acidosis: high bicarb, high pCO2; typical causes are COPD, PNA, paralysis of respiratory muscles, CNS diseases affecting the brainstem in areas involved in respiratory control. Respiratory alkalosis: low bicarb, low pCO2; typical causes are anxiety or acute pain (hyperventilation).
What are approximate normal levels for serum sodium, chloride, and bicarbonate?
Normal serum sodium is about 140 mEq/L, chloride is usually around 100 mEq/L, and bicarbonate around 2 mEq/L.
How is the anion gap defined, and what is normal?
The anion gap is then defined as Na+ − (Cl− + HCO3−), which for normal individuals is around 16. This 16 mEq/L really accounts for the other counter-ions that neutralize sodium but are not measured in serum.
The presence of a widened anion gap signifies the presence of a metabolic acidosis due to ___.
The presence of a widened anion gap signifies the presence of a metabolic acidosis due to a non–chloride-containing acid. If metabolic acidosis is due to the presence of an acid whose counter-ion is not Cl−, such as acetoacetic acid (in diabetic acidosis) or lactic acid as in sepsis or hypoperfusion, then bicarbonate is reduced, as above, but no corresponding increase in Cl− occurs. Therefore, there is an increase in the anion gap, which can reach values of 25–30 mEq/L.
What does it mean when a consistently low anion gap is seen?
Consistently low anion gaps, typically in the range of 1–3 mEq/L, signify the presence of high levels of basic protein, often a monoclonal paraprotein as occurs in plasma cell dyscrasias. Basic protein contains ammonium ions, the counter-ions for which are chloride. Now the “invisible” ion is ammonium, and a measurable increase in chloride ion occurs. This tends to decrease the anion gap. Persistently low anion gaps are a serious sign of possible malignancy (e.g., multiple myeloma).
Does acidosis shift the oxygen-hemoglobin dissociation curve to the right or left?
To the right.
Patients with nonketotic, hyperosmolar state may have a hyperosmolar serum, hyperglycemia, (hypokalemia/hyperkalemia), and (hyponatremia/hypernatremia).
In nonketotic, hyperosmolar coma, RBC and WBC water flows from the cells into the vascular volume, tending to dilute analytes such as sodium. Thus, the patient may have a hyperosmolar serum, hyperglycemia, hyperkalemia, and hyponatremia. In ketotic states, the patient will have, additionally, a metabolic acidosis and a large anion gap.
For hepatitis, cirrhosis, biliary obstruction, space-occupying lesion, passive congestion, and fulminant failure, list the AST, ALT, LD, alk phos, total protein, albumin, bilirubin, and ammonia as L, N, or H.
Hepatitis: H, H, H, H, N, N, H, N. Cirrhosis: N, N, N, N to sl-H, L, L, H, H. Biliary obstruction: N, N, N, H, N, N, H, N. Space-occupying lesion: N or H, N or H, H, H, N, N, N-H, N. Passive congestion: sl-H, sl-H, sl-H, N to sl-H, N, N, N to sl-H, N. Fulminant failure: very H, H, H, H, L, L, H, H.
What 3 enzymes are elevated with biliary tract injury?
AP, GGT, and 5’-N.
Space-occupying lesions of the liver are characterized, for reasons that are not well understood, by isolated elevations of the enzymes ___ and ___.
Space-occupying lesions of the liver are characterized, for reasons that are not well understood, by isolated elevations of the enzymes AP and LD. The most common cause of this condition is metastatic carcinoma to the liver.
How are skeletal TnI and cardiac TnI different?
Different genes encode TnI in skeletal and cardiac muscle, giving rise to isoforms that differ significantly in sequence. Cardiac TnI contains an additional 31 amino acid residues on its N-terminal.
What are acute phase reactants?
Increase in concentration of acute phase reactants (APR) comprises a major pathophysiologic phenomenon that accompanies inflammation and tissue injury. With the acute phase response, normal homeostatic mechanisms are replaced by new set points that presumably contribute to defensive or adaptive capabilities. Despite its name, APR accompany both acute and chronic inflammatory states. They can occur in association with a wide variety of disorders, including infection, trauma, infarction, inflammatory arthritides, and various neoplasms. Acute phase proteins are defined as those proteins whose serum concentrations increase or decrease by at least 25 percent during inflammatory states. Such proteins are termed either positive or negative APR, respectively. Changes in the levels of APR largely reflect altered production by hepatocytes.
List postive acute phase reactants and negative acute phase reactants.
Acute phase proteins are defined as those proteins whose serum concentrations increase or decrease by at least 25 percent during inflammatory states. Such proteins are termed either positive or negative APR, respectively. Increases in APR can vary from approximately 50 percent for ceruloplasmin and several components of the complement cascade to 1000-fold or more for CRP and serum amyloid A. Other positive APR include fibrinogen, alpha-1 antitrypsin, haptoglobin, alpha-1 acid glycoprotein (orosomucoid), IL-1 receptor antagonist, hepcidin, ferritin, procalcitonin, and others. Transferrin is decreased with acute inflammation and increased with chronic inflammation. Negative APR include albumin and transthyretin. The ESR, a nonprotein APR, reflects plasma viscosity and the presence of acute phase proteins.
What is a dexamethasone suppression test, when is it used, and what are expected results?
In cases of hypercortisolism, especially when ACTH levels may remain in the reference range or have “borderline” high or low values, it is often desirable to perform the dexamethasone suppression test. Dexamethasone is a potent glucocorticoid that strongly suppresses normal pituitary ACTH secretion. This can be accomplished with low-dose dexamethasone. If low-dose dexamethasone causes diminished serum cortisol levels and low values for urinary free cortisol, pituitary function is most likely normal while the adrenals are hypersecreting cortisol (i.e., primary hyperadrenalism). This test may be used further to distinguish the possible source of primary hyperadrenalism (i.e., hyperplasia vs. adenoma or carcinoma). High-dose dexamethasone will generally lower serum cortisol levels in adrenal hyperplasia, but it will have no effect in adrenal adenoma or carcinoma.
How is vitamin D produced in the body?
Vitamin D induces absorption of calcium from the gut by acting on vitamin D receptors in the cells lining the gut. It is produced from 7-dehydrocholesterol in skin by ultraviolet light that induces the synthesis of cholecalciferol. In the liver, cholecalciferol is converted to 25-hydroxycholecalciferol, which has a half-life of several weeks. This form is further hydroxylated in the kidneys to 1,25-dihydroxycholecalciferol, the active form of vitamin D. The half-life of active vitamin D is on the order of hours.
Which form of vitamin D is measured in serum assays, and why?
7-dehydrocholesterol in skin is converted by ultraviolet light into cholecalciferol. In the liver, cholecalciferol is converted to 25-hydroxycholecalciferol, which has a half-life of several weeks. This form is further hydroxylated in the kidneys to 1,25-dihydroxycholecalciferol, the active form of vitamin D. The half-life of active vitamin D is on the order of hours. Thus, in serum assays for vitamin D, the analyte whose serum level best reflects vitamin D levels is the precursor, 25-hydroxycholecalciferol. This may give falsely elevated levels of vitamin D in cases of renal failure where conversion of the precursor to active vitamin D is impaired. However, 25-hydroxycholecalciferol itself has some effects on calcium reabsorption, although its affinity for the vitamin D receptor is only about one-one thousandth of that for the fully active form.
Define the following single-rule QC Westgard rules: 13s, 12s, 22s, R4s , 41s , 10x.
13s refers to a control rule that is commonly used with a Levey-Jennings chart when the control limits are set as the mean plus 3s and the mean minus 3s. A run is rejected when a single control measurement exceeds the mean plus 3s or the mean minus 3s control limit. 12srefers to the control rule that is commonly used with a Levey-Jennings chart when the control limits are set as the mean plus/minus 2s. In the original Westgard multirule QC procedure, this rule is used as a warning rule to trigger careful inspection of the control data by the following rejection rules. 22s - reject when 2 consecutive control measurements exceed the same mean plus 2s or the same mean minus 2s control limit. R4s - reject when 1 control measurement in a group exceeds the mean plus 2s and another exceeds the mean minus 2s.
41s - reject when 4 consecutive control measurements exceed the same mean plus 1s or the same mean minus 1s control limit. 10x - reject when 10 consecutive control measurements fall on one side of the mean. In addition, you will sometimes see some modifications of this last rule to make it fit more easily with Ns of 4: the 8x and 12x rules.
Why are the Westgard rules 12S and 10x not recommended for use?
The 12S rule has a good probability of detecting errors (e.g. almost 90% probability of detecting a 2.5 SD bias) but has a high false alert rate. Because of this high false alert rate, it is not recommended to use a 12S rule. The 10x (or 10m) rules are also not recommended because of an excessive false alert rate. Many contemporary analyzers are very stable and may produce QC results with a small standard deviation interval (SDI) on one side of the target value for extended periods of time. Consequently, a 10x rule generally is not recommended, as this condition does not infer a problem with clinical interpretation of patient results when the magnitude of the difference is small. In contrast, the 13S rule has a low false alert rate, but a lower probability to detect an error (e.g., a 55% probability to detect a 2.5 SD bias). It is recommended to improve the efficiency of QC interpretive rules by combining two or more rules and applying them simultaneously as multi-rule criteria.
What are some points to be aware of when changing reagent lots and changing method calibrator lots and their effect on QC?
Because the matrix-related interaction between a QC material and a reagent can change with a different reagent lot, QC results may not be a reliable indicator of a method’s performance for patient samples following a reagent lot change. For this reason, it is necessary to use clinical patient samples to verify the consistency of results between old and new lots of reagents. When a new lot of calibrator is used, with no change in reagents, there is no change in matrix interaction between the QC material and the reagents. In this situation, QC results provide a reliable indication of calibration status with the new lot of calibrator. If the QC results indicate a bias following use of a new lot of calibrator, the calibration has changed and needs to be corrected to ensure consistent results for patient samples.
Some types of laboratory errors can be identified by comparing a patient’s current test result against a previous result for the same analyte. This comparison is called a “delta check.” Delta checks can detect analytic errors; however, their main purpose is to ___.
Some types of laboratory errors can be identified by comparing a patient’s current test result against a previous result for the same analyte. This comparison is called a “delta check.” The previous result is taken from a specified time interval in the past during which the result is not likely to have changed physiologically. This limitation restricts the analytes that can be effectively monitored with a delta check. Delta checks can detect analytic errors; however, their main purpose is to detect mislabeled samples and samples altered by dilution with IV fluid. Consequently, an effective delta check process can be established using a limited number of analytes.
What are “average of normals” or “moving average” techniques used in statistical QC?
Automated approaches to determine the mean (or median) for groups of sequential patient results used as a continuous process control parameter have been described. These methods are called “average of normals” (AON) or “moving average” techniques and are suitable for use in higher volume assays in chemistry and hematology. In general, these approaches evaluate sequential patient results over time intervals such as several hours to 1 or more days. For some analytes, patients may need to be partitioned to obtain subgroups whose results are expected to be homogeneous. Consideration of influences for partitioning includes age, gender, ethnicity, and disease conditions.
Certain lab tests that are performed together (i.e., as a panel) must be coded correctly. Ten panels have been approved by the AMA. What are these 10?
The 10 AMA organ or disease-oriented panels (CPT, 2009) are: basic metabolic panel (calcium, ionized), basic metabolic panel (calcium, total), general health panel, electrolyte panel, comprehensive metabolic panel, obstetric panel, lipid panel, renal function panel, acute hepatitis panel, and hepatic function panel. When these panels are performed, they must be coded with the unique panel code and not by each individual test’s CPT code. Reimbursement is much lower for a panel than it is for the sum total reimbursement of each test. Coding for each individual test component as opposed to one panel code is considered “unbundling,” which is a fraudulent billing practice.
What are the components of a basic metabolic panel and a comprehensive metabolic panel?
Basic metabolic panels can include ionized calcium under one CPT code, or total calcium under another CPT code. Including the calcium, a basic metabolic panel includes carbon dioxide, chloride, creatinine, glucose, potassium, sodium, and BUN. A comprehensive metabolic panel includes albumin, total bilirubin, calcium, carbon dioxide, chloride, creatinine, glucose, alkaline phosphatase, potassium, total protein, sodium, ALT, AST, and BUN.
What are the 8 categories that governments classify chemical weapons into? Also give examples in each category.
Nerve agents (sarin, tabun, soman, VX, malathion, carbaryl (Sevin)). Blood agents (hydrogen cyanide, cyanogen chloride). Blister agents (nitrogen and sulfur mustards, lewisite). Heavy metals (arsenic). Pulmonary agents (chlorine gas, phosgene). Dioxins (tetrachlorodibenzodioxine - a component of Agent Orange). Incapacitating or psychotomimetic agents (quinuclidinylbenzylate, phencyclidine). Corrosive acids and bases (sulfuric acid, sodium hydroxide).
What is the role of the Level A laboratory in the case of an act of chemical or nuclear terrorism?
First responder teams and HAZMAT teams have portable devices that detect and identify hazardous chemicals. First responder teams, HAZMAT teams, and ERs have portable devices to detect radiation. The role of the Level A laboratory is secondary; mainly triage and supportive care of patients affected by the incident (especially after nuclear terrorism). In the case of chemical terrorism, labs can provide some useful analytic data, such as: detecting some nerve toxins by ELISA, PCR, or MS; measuring increased cholinesterase activity such as would be seen in patients exposed to nerve agents; and detecting metabolites of vesicants such as mustard gas or Lewisite by GC-MS.
True or false. All Ig classes (M, G, A, D, and E) can have kappa or lambda light chains.
True.
What are the valencies (Ag binding) for the 5 Ig classes?
IgG: 2. IgM: 5 (valency of 10 can be seen with very small haptens). IgA: 2-4. IgD: 1. IgE: 1.
What are the in vivo half-lives in days, serum concentration in mg/mL, percentage of total serum Ig, and extravascular distribution of the 5 Ig classes?
IgM: 10 days, 0.5-2 mg/mL, 6%, fluids. IgG: 21 days, 8-16 mg/mL, 80%, tissue. IgA: 6 days, 1.4-4 mg/mL, 13%, secretions. IgD: 3 days, 0-0.04 mg/mL, 0-1%, -. IgE: 2 days, trace concentration, -, secretions.
Which Ig class has the longest in vivo half-life, and which has the shortest?
IgG - 21 days, IgM - 10 days, IgA - 6 days, IgD - 3 days, IgE - 2 days.
Which Ig class(es): is inactivated by sulfhydryl reagents; crosses the placenta; induce agglutination; fix complement; bind to Fc gamma receptors?
IgM is ++++ inactivated by sulfhydryl reagents; IgA is +/-; IgG is -. Only IgG crosses the placenta. IgM is ++++ at inducing agglutination; IgA is ++, and IgG is +. IgM fixes complement +++ (classical); IgG ++ (classical); IgA + (alternative). IgG has +++ binding to Fc gamma receptors; IgA and IgE are +; IgM and IgD are -.
For the 4 subclasses of IgG: IgG1, IgG2, IgG3, and IgG4, list the in vivo half-life in days, relative serum concentration in %, ability of placental transfer, ability of complement fixation, and ability of macrophage binding.
In vivo half-life in days: 21, 21, 7, 21. Relative serum concentration in %: 64-70, 23-28, 4-7, 3-4. Ability of placental transfer: ++, +/-, ++, ++. Ability of complement fixation: +++, ++, ++++, +/-. Ability of macrophage binding: +++, +, +++, +/-.
What is a paratope?
The combining site of an antibody, where it is in physical contact with an antigenic determinant or epitope, is called the paratope. Binding involves formation of multiple noncovalent bonds between the antigen and amino acids of the paratope.
What are the 2 different types of units used to represent D-dimer?
D-dimer is reported in mass units and, as these assays have evolved, 2 different types of units have been used to represent D-dimer: the fibrinogen equivalent unit (FEU) at 340 kDa and the D-dimer unit (DDU) at 195 kDa. Fibrin monomer polymerizes end to end and side to side forming a fiber, fibrin, in a process that is not enzymatically driven. The polymerization of fibrin monomer occurs with the association of the D domains of 2 fibrin monomers (referred to as D-dimer) with the E domain of a third. Soluble fragments of fibrin (fibrin degradation products) that contain variable numbers of the D-D domains (D-dimer) are produced. When assayed, the fragments are quantified in 2 different types of units, the DDU and the FEU. The mass of the FEU, related to the mass of fibrinogen, is about 1.7 times greater than the mass of the DDU. Because the D-dimer is reported in mass units, the type of units involved is very important in setting the threshold for the exclusion of venous thromboembolism and for interpretation of reported results. Adding to the complexity of reporting these values is the variability in the magnitude of units reported, for example, ng/mL, μg/mL, and μg/L. Variability in the type and magnitude of units has led to confusion in some laboratories as they attempt to use the assay, especially as they set a threshold for the exclusion of VTE.
What is the primary biologic function of haptoglobin?
Haptoglobin binds free hemoglobin to prevent iron loss and kidney damage during hemolysis. Any hemolytic process (immune, mechanical, environmental) can result in free hemoglobin entering the vascular system.
Is haptoglobin a positive or negative acute phase reactant?
Positive acute phase reactant.
Where is haptoglobin made? What basic biochemical structure does it have?
Haptoglobin is made in the liver as a single polypeptide chain that is cleaved post-translationally to generate alpha and beta subunits. These polypeptide chains can then be linked via disulfide bonds into four peptide chains in two pairs (alpha-beta)2 confirmation, much like hemoglobin.
What proteins are in the beta peak on SPEP?
The beta zone contains transferrin, LDL, and C3.
Familial dyslipidemias: Types I, IIa, IIb, III, IV, and V. Which one(s) will have visible lipemia in the blood collection tube (assuming the person is truly fasting and not postprandial)?
Types I and V. But they have no increase in risk for atherosclerosis.
Familial dyslipidemias: Types I, IIa, IIb, III, IV, and V. Which one is the most common? Which one(s) has the highest risk of CAD? Which one(s) can be treated with diet changes only?
Type IIb is the most common. Types IIa and IIb have the highest risk of CAD. Type I can be treated with diet changes only.
Why should you not use a sample from a heparin tube if you are going to run PCR?
Heparin inhibits DNA polymerase.
How can you calculate the corrected calcium from measured total calcium and albumin?
For each 1 g/dL the albumin drops below the “normal” level of 4 g/dL, add 0.8 mg/dL to the total calcium.
What is the most common cause of secondary hyperparathyroidism?
Secondary hyperparathyroidism is due to increased physiological PTH as a response to increased resistance to PTH activity, most commonly in end-stage renal disease.
Is % oxygen saturation (SaO2) directly measured or calculated by arterial blood gas analyzers?
Calculated. SaO2 is calculated from the hematocrit and the PaO2 plotted on a standard hemoglobin oxygen dissociation curve.
PaO2, PaCO2, SaO2, pH. Which of these are directly measured and which are calculated on an arterial blood gas analyzer?
PaO2, PaCO2, and pH are directly measured. SaO2 is calculated from the hematocrit and the PaO2 plotted on a standard hemoglobin oxygen dissociation curve.
Arterial blood gas analyzer, pulse oximeter, co-oximeter, peripheral blood smear. Which of these techniques is best at detecting carbon monoxide poisoning?
The co-oximeter, which has the ability to directly measure the proportions of several hemoglobin subtypes. The other techniques, if they possess the ability to do so, calculate the proportions rather than measure them. In addition, multiple potential confounding elements are present in some of the techniques, such as poor peripheral perfusion or recent transfusion.
How do you calculate the anion gap?
[Na+] - ( [Cl-] + [HCO3-] )
Which type of azotemia (prerenal, renal, or postrenal) is most likely when the BUN/Cr ratio is maintained but the levels of both are elevated?
Renal azotemia. A maintained but elevated ratio is suggestive of an intrinsic renal defect, most commonly glomerulonephritis or tubulointerstitial nephritis. Deranged ratios, such as when BUN increases more than Cr, is suggestive of impaired renal perfusion, such as prerenal (insufficient volume to the kidney) and postrenal (insufficient volume out of the kidney).
What is the most common cause of renal failure in cirrhotic patients?
Spontaneous bacterial peritonitis.
At what wavelength light is bilirubin absorbance maximal?
450 nm.
What are the 4 main sources of alkaline phosphatase in the body?
Bile ducts, bone, placenta, intestine.
Put the 4 alkaline phosphatase isoenzymes into order of anodal mobility on electrophoresis.
Anodal mobility 1 = biliary. Anodal mobility 2 = bone. Anodal mobility 3 = placenta. Anodal mobility 4 = intestinal.
Is bone alkaline phosphatase produced by osteoblasts or osteoclasts?
Bone alkaline phosphatase is produced by osteoblasts and reflects bone-forming activity.
Some of the highest alkaline phosphatase levels are seen in what disease?
Paget disease of bone.
What is the Regan isoenzyme?
AKA carcino-placental alkaline phosphatase. Appears to be mostly identical to placental alk phos (same anodal mobility, same degree of inhibition by L-phenylalanine, slightly less degree of heat/urea inhibition). The Regan isoenzyme is observed in ~5% of individuals with carcinoma.
For the 4 alkaline phosphatase isoenzymes, list how strongly each is inhibited by L-phenylalanine and by heat/urea (sensitivity to heating parallels sensitivity to urea incubation, which is why they are grouped).
Biliary: -, +. Bone: -/+++. Placenta: +++/-. Intestinal: +++/+. For sensitivity to heat/urea, think “bone burns, placenta persists.”
Are GGT levels higher in men or women? In African Americans?
GGT levels are considerably higher in men and African Americans.
Hyperammonemia is nearly always due to ___.
Hyperammonemia is nearly always due to liver failure. However, particularly in children, it should raise suspicion for an inborn error of metabolism (especially urea cycle enzyme deficiencies).
Bilirubinuria indicates (conjugated/unconjugated) hyperbilirubinemia.
Bilirubinuria indicates conjugated hyperbilirubinemia, because unconjugated bilirubin, even when quite elevated, does not appear in urine.
Does direct spectrophotometry measure direct or indirect bilirubin?
Both, at the same time. This method is only capable of measuring total bilirubin.
In unconjugated hyperbilirubinemia… If the step affected is heme conversion to unconjugated bilirubin, the pathologic process is ___. If the step affected is delivery of unconjugated bilirubin to liver, the potential pathologic processes are ___. If the step affected is uptake of unconjugated bilirubin into hepatocyte, the potential pathologic processes are ___. If the step affected is conjugation of bilirubin in hepatocyte, the potential pathologic processes are ___.
In unconjugated hyperbilirubinemia… If the step affected is heme conversion to unconjugated bilirubin (increased production), the pathologic process is extravascular hemolysis. If the step affected is delivery of unconjugated bilirubin to liver, the potential pathologic processes are blood shunting (cirrhosis) or right heart failure. If the step affected is uptake of unconjugated bilirubin into hepatocyte, the potential pathologic processes are Gilbert syndrome and drugs such as rifampin. If the step affected is conjugation of bilirubin in hepatocyte, the potential pathologic processes are Crigler-Najjar syndrome and hypothyroidism.
In conjugated hyperbilirubinemia… If the step affected is transmembrane secretion of conjugated bilirubin into canaliculus (hepatocellular jaundice), the potential pathologic processes are ___. If the step affected is flow of conjugated bilirubin through canaliculi and bile ducts (cholestatic jaundice), the potential pathologic process is ___.
In conjugated hyperbilirubinemia… If the step affected is transmembrane secretion of conjugated bilirubin into canaliculus (hepatocellular jaundice), the potential pathologic processes are Dubin-Johnson syndrome, hepatitis, endotoxin (sepsis), pregnancy (estrogen), and drugs such as estrogen and cyclosporine. If the step affected is flow of conjugated bilirubin through canaliculi and bile ducts (cholestatic jaundice), the potential pathologic process is mechanical obstruction such as from PBC, PSC, tumor, stricture, or stone.
What is delta bilirubin?
If conjugated hyperbilirubinemia is prolonged, conjugated bilirubin can become covalently linked to serum albumin, resulting in a moiety known as delta bilirubin. Both liver and kidney are incapable of excreting delta bilirubin; thus, even after resolution of the underlying cause, conjugated hyperbilirubinemia (in the form of delta bilirubin) may persist for some time.
How can you distinguish hepatocellular jaundice from cholestatic jaundice based on alkaline phosphatase, transaminases, serum cholesterol, and pruritis?
Hepatocellular jaundice: alk phos 3x upper limit of normal, cholesterol normal, and pruritis absent. Cholestatic jaundice: alk phos >3x upper limit of normal, transaminases <3x upper limit of normal, cholesterol increased, and pruritis present.
In autoimmune hepatitis there is a polyclonal increase in Ig__, while in primary biliary cirrhosis there is a polyclonal increase in Ig__.
In autoimmune hepatitis there is a polyclonal increase in IgG, while in primary biliary cirrhosis there is a polyclonal increase in IgM.
What are 6 general causes of unconjugated neonatal hyperbilirubinemia?
Physiologic jaundice. Breast milk jaundice. Polycythemia. Hemolysis (HDN, hemoglobinopathies, inherited membrane or enzyme defects). Bowel obstruction (Hirschprung disease, cystic fibrosis, ileal atresia). Inherited disorders of bilirubin metabolism (Gilbert syndrome, Crigler-Najjar syndrome).
What are 6 general causes of conjugated neonatal hyperbilirubinemia?
Biliary obstruction (extrahepatic biliary atresia). Sepsis or TORCH infection. Neonatal hepatitis (idiopathic, Wilson disease, alpha-1 antitrypsin deficiency). Metabolic disorders (galactosemia, hereditary fructose intolerance, glycogen storage disease). Inherited disorders of bilirubin transport (Dubin-Johnson syndrome, Rotor syndrome). Parenteral alimentation.
What areas of the brain specifically are affected in kernicterus?
Bilirubin has a special affinity for the globus pallidus, the hippocampus, and the subthalamic nucleus. Many other structures, including the striatum, thalamus, cranial nerve nuclei, inferior olives and dentate nuclei of the cerebellum are less frequently affected.
Is phototherapy effective for unconjugated or conjugated hyperbilirubinemia?
Unconjugated hyperbilirubinemia. Phototherapy converts unconjugated bilirubin into a molecule that can be excreted without conjugation; phototherapy is not useful for conjugated hyperbilirubinemia.
What are the characteristics of physiologic jaundice in neonates?
Usually noted between days 2-3 of neonatal life and rarely rises at a rate greater than 5 mg/dL/day. Usually peaks by days 4-5 and rarely exceeds 5-6 mg/dL.
In a healthy term infant, phototherapy should be considered when bilirubin exceeds __ mg/dL before 12 hours of age, __ mg/dL before 18 hours of age, and __ mg/dL before 24 hours of age.
In a healthy term infant, phototherapy should be considered when bilirubin exceeds 10 mg/dL before 12 hours of age, 12 mg/dL before 18 hours of age, and 14 mg/dL before 24 hours of age.
Out of viral, toxic, ischemic, and alcoholic hepatitis, which tend to have AST:ALT over 1?
The AST:ALT is over 2 in 80% of patients with toxic, ischemic, and alcoholic hepatitis. It is usually <1 in viral hepatitis.
Out of alcoholic hepatitis, acute hepatitis A, acute hepatitis B, and acute hepatitis C, in which conditions does jaundice occur most frequently?
Jaundice occurs in 70% of patients with alcoholic hepatitis and acute hepatitis A, and occurs in 20-30% of patients with acute hepatitis B and acute hepatitis C.
What is the best indicator of prognosis in acute hepatic injury?
The PT is probably the best indicator of prognosis In acute hepatic injury, with PT prolongation >4 seconds indicating severe liver injury and an unfavorable prognosis. But bilirubin >15 mg/dL is also indicative of severe liver injury and an unfavorable prognosis.
What are the 2 isoenzymes of serum amylase?
Pancreatic and salivary. But when subjected to electrophoresis, 6 bands result, with the first three being salivary and the slowest three being pancreatic.
How many bands form when serum amylase is subjected to electrophoresis?
When serum amylase is subjected to electrophoresis, 6 bands result, with the first three being salivary and the slowest three being pancreatic.
Other than electrophoresis and monoclonal antibody assays, how can salivary and pancreatic amylase be differentiated?
An inhibition test: Salivary amylase is sensitive to inhibition by the wheat germ lectin, triticum vulgaris.
Serum amylase rises within __ to __ hours of the onset of acute pancreatitis and returns to normal in __ to __ days.
Serum amylase rises within 2 to 24 hours of the onset of acute pancreatitis and returns to normal in 2 to 3 days.
How can renal insufficiency spuriously elevate serum amylase?
Amylase is primarily cleared by glomerular filtration.
Up to 10% of cases of acute pancreatitis are associated with normal levels of amylase, with this finding most common in people with hypertriglyceridemia-associated acute pancreatitis. Why?
Triglycerides competitively interfere with the amylase assay.
List non-pancreatic causes of hyperamylasemia.
Diabetic ketoacidosis, peptic ulcer disease, acute cholecystitis, ectopic pregnancy, salpingitis, bowel ischemia, intestinal obstruction, renal insufficiency, salivary gland pathology, and macroamylasemia. Amylase may also be raised somewhat by the administration of opioid analgesics, due presumably to contracture of the sphincter of Oddi.
How much overlap is seen in the range of levels of amylase for acute pancreatitis vs other causes?
The degree of elevation tends to be higher in acute pancreatitis, but there is considerable overlap in the ranges. Pancreatitis causes amylase in the range of 250-1000 Somogyi units, while other causes are 200-500.
What is macroamylasemia?
A benign acquired condition (associated with celiac disease, lymphoma, HIV infection, monoclonal gammopathy, rheumatoid arthritis, and ulcerative colitis) with an incidence of ~1%, in which apparently healthy individuals have markedly elevated serum amylase levels (with low urine amylase levels), due to Ig-amylase complexes.
Amylase is spuriously elevated in renal insufficiency. Is lipase?
Yes. But unlike amylase, hypertriglyceridemia does not interfere with lipase measurement.
What are 2 additional markers of acute pancreatitis other than amylase and lipase?
Serum and urine trypsinogen-2 and elastase-1.
What tests are available for measuring pancreatic exocrine function?
Invasive: Secretin-CCK/secretin-pancreozymin test. Noninvasive: Fecal fat. Fecal elastase-1. Fecal chymotrypsin (bentiromide).
What is the secretin-CCK test?
An endoscope is introduced, and the duodenal concentrations of pancreatic exocrine products (bicarbonate, amylase, lipase, trypsin) are measured after IV administration of secretin and CCK.
A fecal fat test (72 hour fecal fat quantitation) is positive in pancreatic exocrine dysfunction. What other conditions will also result in a positive fecal fat test?
Severe ileal diseases (such as Crohn disease) or ileal resection.
What does the D-xylose test measure?
Small bowel mucosal absorptive capacity.
Pancreatic cysts: pseudocyst, serous cystadenoma, mucinous cystadenoma (mucinous cystic neoplasm), intraductal papillary mucinous tumor, solid-pseudopapillary tumor. What will the patterns for amylase, CEA, and CA 19-9 be for each?
Pseudocyst: inc, dec, inc. Serous cystadenoma: dec, dec, dec. Mucinous cystadenoma (mucinous cystic neoplasm): dec, inc, nl to inc. Intraductal papillary mucinous tumor: inc, inc, nl to inc. Solid-pseudopapillary tumor: dec, dec, dec.
Of the 3 CK isoenzymes, which is the fastest migrating and which is the slowest migrating?
The fastest is CK-BB (CK1). The slowest is CK-MM (CK3).
CK-BB (CK1) is found primarily in the brain, with lesser amounts found in what other locations?
Stomach, bladder, and prostate.
CK-MM is found in skeletal and cardiac muscle. Skeletal muscle is ~__% MM, and cardiac muscle is ~__% MM.
CK-MM is found in skeletal and cardiac muscle. Skeletal muscle is ~99% MM, and cardiac muscle is ~70% MM.
In normal subjects, serum CK is composed almost exclusively of which isoenzyme?
In normal subjects serum CK is almost 100% CK-MM isoenzyme, mostly from skeletal muscle.
CK-MB is found in skeletal and cardiac muscle. Skeletal muscle is ~__% MB, and cardiac muscle is ~__% MB.
CK-MB is found in skeletal and cardiac muscle. Skeletal muscle is ~1% MB, and cardiac muscle is ~30% MB.
The ratio of CK-MB to total CK is called the “relative index.” What is its utility?
The relative index adds to the ability of either assay alone to distinguish myocardial infarction. A relative index of about 2% is often the chosen cut-off.
What is macro-CK, and where is it seen?
There are 2 types. Macro-CK type 1 is a CK-Ig complex, with CK-BB complexing to IgG or IgA. On electrophoresis it migrates between MM and MB. It is found in healthy elderly women. Macro-CK type 2 is a polymeric form of mitochondrial CK. On electrophoresis it migrates cathodal to CK-MM. It is associated with malignancies.
What are CK isoforms AKA CK subforms?
Metabolic breakdown products of CK. With high-resolution electrophoresis, CK-MB resolves into 2 bands and CK-MM resolves into 3 bands. CK-MB2 and CK-MM3 are the tissue forms that are released from damaged myocardium. CK-MB1 is the plasma form and arises as a result of cleavage of a terminal lysine from the M peptide. CK-MM1and CK-MM2 are also cleavage products. A relative increase of tissue forms indicates a recent enzyme leak.
What are troponins?
Troponins are a group of enzymes consisting of TnT, TnI, and TnC that are involved in mediating the actin-myosin interactions that result in muscle contraction.
TnT, TnI, and TnC all have cardiac and skeletal muscle forms. Which are encoded by the same genes and which are encoded by different genes?
While the same gene encodes cardiac and skeletal TnC, separate genes encode TnI and TnT in cardiac and skeletal muscle.
Is cardiac muscle troponin present mostly as a free or a bound form in the cell?
A small proportion of cardiac muscle troponin is free in the cytoplasm but the vast majority of it is bound to actin and myosin. Thus, there is both an immediate release of cytoplasmic troponin by infarcted cardiac muscle (within 4-8 hours) and a sustained release of bound troponin (over 10-14 days).
What is the Altered Cobalt Binding (ACB) assay?
A measured quantity of cobalt is added to patient plasma and the amount of unbound cobalt is measured. The quantity of unbound cobalt is a measure of ischemia-modified albumin (rises within minutes of myocardial ischemia and returns to baseline within 6 hours due to rapid hepatic clearance).
In what locations are the natriuretic peptides ANP, BNP, and CNP produced?
ANP is synthesized mainly by atrial myocytes (but synthesis of ANP also takes place in the ventricles, brain, kidney, and adrenals). ANP is stored in granules and stimulation results in a rapid response. BNP is synthesized by ventricular myocytes (predominantly) and brain, as well as atrial myocytes under some conditions. BNP is secreted by a constitutive mechanism; only small amounts are stored and cells are dependent upon activation of the BNP gene when secretion is needed. CNP is synthesized by brain and endothelium.
Serial measurements improve the sensitivity of both troponin and CK-MB for AMI to almost 100%. How do the 2 compare for specificity?
The specificity of CK-MB cannot equal that of troponin, because of false-positives from non-ischemic cardiac injury (pericarditis, myocarditis) and skeletal muscle disease (rhabdomyolysis, exercise). However, troponin is not useful to determine temporal stage of the MI (it remains elevated for up to 2 weeks following AMI), so serial CK-MB is still useful to see if the infarct is acute or resolving, or if there is a complication of MI such as extension of the infarct.
What is the “washout phenomenon” in cardiac reperfusion?
An indication of successful reperfusion following thrombolysis is the washout phenomenon. In successful reperfusion, all of the cardiac markers peak earlier than normal; although, their sequence of peaks parallels that of normal MI. In addition, their peak concentrations may be higher than they would have been with unreperfused MI.
Can methods that exploit the capacity of solutes to alter light transmitted through a solvent, such as refractometry, be used for protein quantitation?
Refractive index can be accurate for measuring serum protein concentration as dissolved solute for levels above 2.5 g/dL. However, there are many potential interferences and hemolysis, lipemia, icterus, and azotemia produce erroneously high results. Refractive index cannot be used for urine protein measurements because of excess amounts of solutes in relation to the protein.
What technique is the gold standard for protein quantitation?
Measurement of nitrogen content. The Kjeldahl technique consists of acid digestion to release ammonium ions from nitrogen-containing compounds. The ammonium can then be quantitated by conversion to ammonia gas and titration as a base, or by nesslerization, in which double iodides (potassium and mercuric) form a colored complex with ammonia in alkali. Although determination of nitrogen content can be extremely precise, its use for calculation of protein concentration depends on the exact protein composition of a sample, because each protein has a somewhat different nitrogen content according to amino acid composition. For a sample of a purified protein, nitrogen content is highly accurate for estimating protein concentration when the nitrogen content on a molar basis is already known for that purified protein. Because clinical samples consist of unpredictable mixtures of different proteins, and measurement of nitrogen content is not a simple procedure, it is not commonly used in clinical laboratories.
What is the Biuret technique?
A colorimetric technique highly specific for proteins and peptides is the Biuret method, by which copper salts in alkaline solution form a purple complex with substances containing two or more peptide bonds. The absorbance of the resulting chelate at 540 nm is proportional to total protein concentration. Interferences are minimal, although ammonium ion may acidify the reaction, while hemoglobin and bilirubin absorb in the same region as the Biuret complex (540–560 nm). The Biuret method is extensively used in clinical laboratories, particularly in automated analyzers in which protein concentration can be measured down to 10 or 15 mg/dL.
What are electrochemical cells?
Electrochemical cells are contained systems in which a redox reaction occurs. There are two types of electrochemical cells: galvanic cells (AKA voltaic cells) and electrolytic cells. Spontaneous reactions occur in galvanic cells, and nonspontaneous reactions in electrolytic cells. Both types contain electrodes at which oxidation and reduction occur. For all electrochemical cells, the electrode at which oxidation occurs is called the anode, and the electrode where reduction occurs is called the cathode.
Match: anode/cathode; positive/negative.
Trick question. The anode of an electrolytic cell is considered positive, since it is attached to the positive pole of the battery and so attracts anions from the solution. The anode of a galvanic cell is considered negative because the spontaneous oxidation reaction that takes place at the galvanic cell’s anode is the original source of that cell’s negative charge (i.e. Is the source of electrons). In spite of this difference in designating charge, oxidation takes place at the anode in both types of cells, and electrons always flow through the wire from the anode to the cathode.
What 2 forces act on proteins during electrophoresis?
Electromotive and endosmotic. When a charge is applied (from a power source), this creates an electromotive force. The solid support has a slight negative charge and is drawn toward the positive pole (anode), but, being a solid support, it cannot move. There is instead a compensatory flow of the fluid buffer towards the negative pole (cathode). This buffer flow is called endosmosis and has the capacity to carry with it substances suspended within the medium. Since most proteins bear a net negative charge, electromotive force tends to pull them to the anode (positive pole), whereas endosmosis pulls them toward the cathode (negative pole). In most proteins, the electromotive force exceeds the endosmotic force, and they move to variable extents towards the anode. However, gamma globulins have a weak net negative charge and the endosmotic force displaces them toward the cathode.
Immunoelectrophoresis, immunofixation electrophoresis, and immunotyping (immunosubtraction) are methods used for ___.
Immunoelectrophoresis, immunofixation electrophoresis, and immunotyping (immunosubtraction) are methods used for characterizing a suspected monoclonal band observed on SPEP or UPEP.
Albumin constitutes ~__ of total plasma protein and has a half-life of ___.
Albumin constitutes ~2/3 of total plasma protein and has a half-life of 17 days.
What is analbuminemia? What are clinical and laboratory manifestations?
Analbuminemia is a rare AR disorder manifested by the absence or severe reduction of circulating serum albumin. Analbuminemic individuals usually have surprisingly minimal findings, since the absence of albumin is partially compensated for by an increase of other serum proteins, such as globulins, transferrin, and coagulation factors. The main clinical symptoms include mild edema and
occasionally chronic fatigue or lipodystrophy, especially in women, while the most common
biochemical signs are gross hyperlipidemia with hypercholesterolemia and elevated LDL-cholesterol
levels. The finding of a low albumin concentration during routine SPEP, with normal liver function and no GI or renal protein loss, suggests the clinical diagnosis. This needs to be confirmed by the molecular diagnosis, based on the identification of the causative mutation within the albumin (ALB) gene by DNA sequence analysis.
What conditions result in the greatest decrements of serum albumin?
Hepatic synthetic function is poorly reflected in the albumin, since only in end-stage liver disease is serum albumin noticeably decreased. The greatest decrements in serum albumin are seen in protein-losing conditions such as protein-losing enteropathy and nephrotic syndrome.
In non-diabetic individuals, up to __% of albumin is normally glycosylated nonenzymatically in the serum. In diabetics with poor control, up to __% becomes glycosylated.
In non-diabetic individuals, up to 8% of albumin is normally glycosylated nonenzymatically in the serum. In diabetics with poor control, up to 25% becomes glycosylated.
What is the fastest migrating protein on SPEP?
Prealbumin. Due to its sparseness, however, it is not normally seen on traditional SPEP and seen only faintly on high-resolution SPEP.
T4 and T3 circulate in the blood bound to what 3 different binding proteins?
T4 and T3 circulate in the blood bound to 3 different binding proteins: thyroxine-binding globulin (TBG), prealbumin (AKA thyroxine-binding prealbumin (TBPA) AKA transthyretin (TTR)), and albumin. TBG carries 70% of the circulating T4 and T3 due to its high affinity. Prealbumin binds to ~10-15% of the hormones (mostly T4), and albumin binds to the remaining 10-15%.
What is the half-life of prealbumin?
48 hours.
Why is prealbumin a relatively prominent component of CSF protein and a sharp prealbumin band a hallmark of CSF protein electrophoresis?
Prealbumin crosses the blood-brain barrier and is actively secreted into the CSF by the choroid plexus.
What is the prominent “prealbumin” band that is often seen in the SPEP of heparinized patients?
The prominent “prealbumin” band that is often seen in heparinized patients results from an alteration in beta-lipoprotein such that it migrates in the prealbumin range.
A prominent “prealbumin” band that is often seen in heparinized patients results from an alteration in beta-lipoprotein such that it migrates in the prealbumin range. In what conditions/situations are true elevations in prealbumin seen?
Alcohol use, renal dysfunction and drugs (corticosteroids and other hormones, high-dose NSAIDs). .
In high-resolution SPEP, where are alpha-1-lipoprotein and beta-lipoprotein found?
Alpha-1-lipoprotein (a constituent of HDL) is found at the albumin/alpha-1 interface. Beta-lipoprotein (a constituent of LDL) is found at the beta-1/beta-2 interface.
Is alpha-1-antitrypsin deficiency detectable with SPEP?
SPEP can be used to screen for AAT deficiency, in which the serum will display a markedly diminished alpha-1 band.
What are the constituents of the alpha-1 band in high resolution SPEP?
Alpha-1 antitrypsin is usually the major component of the alpha-1 band. Alpha-1 acid glycoprotein (orosomucoid) is usually a minor component of the alpha-1 band but a major component of the increased alpha-1 band seen in acute inflammation.
What are causes of increased or decreased serum ceruloplasmin levels?
A falsely normal or increased ceruloplasmin may be seen in inflammatory states (ceruloplasmin is an acute phase reactant) or pregnancy. Decreased ceruloplasmin is seen with Wilson disease, hepatic failure, malnutrition, and Menke syndrome.
What are constituents of the alpha-2 band in high resolution SPEP?
Alpha-2 macroglobulin, haptoglobin, and ceruloplasmin.
What is seen at the alpha-2/beta interface in high resolution SPEP?
This area is usually empty, but hemoglobin, usually absent from serum, may be present here when there is a hemolysis, giving a possible pseudo-M-spike.
Does haptoglobin bind myoglobin?
No. Haptoglobin is a protein that binds free hemoglobin.
What are the 3 possible haptoglobin phenotypes? Is there a disease association with any particular phenotype?
The haptoglobin genetic locus at 16q22 is polymorphic with two classes of alleles: type 1 and type 2. Based on these 2 alleles, the 3 haptoglobin phenotypes/protein products are: Hp 1-1, Hp 2-1, and Hp 2-2. The 2-2 phenotype is an independent risk factor for CV disease in DM.
What is the half-life of haptoglobin? What is the half-life of haptoglobin-hemoglobin complex? How is the haptoglobin-hemoglobin complex removed from circulation?
The half-life of haptoglobin is 3.5 days, and the half-life of the haptoglobin-hemoglobin complex is ~10 minutes. The haptoglobin-hemoglobin complex is removed by binding to the CD163 receptors on the surface of macrophages and monocytes.
What is the major biologic function of haptoglobin?
The major biologic function of haptoglobin is to
bind hemoglobin in an equimolar ratio with very high affinity to prevent hemoglobin-mediated renal parenchymal injury and loss of iron following intravascular hemolysis. In addition, haptoglobin can inhibit prostaglandin synthesis and is believed to have anti-inflammatory and antioxidant properties
in the body.
What are constituents of the beta-1 band, beta-1/beta-2 interface, and beta-2 band in high-resolution SPEP?
Transferrin is the major beta globulin and makes up the beta-1 band. Transferrin may be high in iron deficiency, giving a possible pseudo-M-spike. Beta-lipoprotein, a constituent of LDL, is seen at the beta-1/beta-2 interface. The beta-2 band is made up of IgA (runs beta-2 to gamma), CRP (runs beta-2 to gamma), and complement. Also, fibrinogen, usually absent from serum but may be present with incomplete clotting, may straddle the beta/gamma interface, giving a possible pseudo-M-spike.
When can fibrinogen appear in SPEP, and where does it appear?
Fibrinogen is supposed to be absent from serum, most of it having been consumed in the clot. However, if the specimen clots incompletely (such as in a heparinized patient), fibrinogen may be seen in the SPEP straddling the beta/gamma interface and misinterpreted as an M protein. Other causes of fibrinogen in serum include patients with dysfibrinogenemia, APL/APLS syndrome, liver disease, or vitamin K deficiency.
Fibrinogen can sometimes be present in serum (heparinized patient with incompletely clotted specimen, dysfibrinogenemia, APL syndrome, liver disease, or vitamin K deficiency) and appear as a pseudo-M-spike on SPEP. What can be done to remedy this interference?
Absolute ethanol can be used in vitro to selectively precipitate fibrinogen and obviate this interference.
What can produce pseudo-M-spikes on SPEP?
Hemoglobin, usually absent from serum, may be present at the alpha-2/beta interface when there is hemolysis, Transferrin (beta-1 band) can be increased in iron deficiency. Fibrinogen, usually absent from serum, may be present in the beta-2 band when there is incomplete clotting.
What 2 acute phase reactants are decreased in acute inflammation then increased in chronic inflammation?
Transferrin is decreased in acute inflammation, then increased in chronic inflammation. Gamma globulings are normal to decreased in acute inflammation then increased in chronic inflammation.
In what conditions/situations is transferrin increased?
Chronic inflammatory state, iron deficiency, pregnancy, estrogen therapy.
The blood-brain barrier transports transferrin into the CSF, but not before modifying a percentage of it to make ___.
The blood-brain barrier transports transferrin into the CSF, but not before modifying a percentage of it to make asialated transferrin (so-called Tau protein), while the rest of it is unmodified. Thus, the double transferrin peak that is one of the hallmarks of CSF protein electrophoresis.
Where is C-reactive protein produced? What does its name derive from?
CRP is produced in the liver. Its name derives from its reaction with Streptococcal capsular (C) polysaccharide.
Formerly, assays for CRP had an analytical sensitivity of ~5mg/L. This was appropriate, as the CRP test then was used to support such diagnoses as bacterial endocarditis, appendicitis, active collagen vascular disease, etc, in which the CRP tended to be well over 10mg/L. Now, high-sensitivity CRP assays are available, that have an analytical sensitivity of ___.
What is the physiologic role of C-reactive protein?
The physiological role of CRP is to bind to phosphocholine expressed on the surface of dead or dying cells (and some types of bacteria) in order to activate the complement system. CRP binds to phosphocholine on microbes and damaged cells and enhances phagocytosis by macrophages. Thus, CRP participates in the clearance of necrotic and apoptotic cells.
CRP levels are broken into 3 categories: normal CRP level, low-level CRP elevation, and high-level CRP elevation. What are the numbers for each?
Normal CRP level: 10 mg/L (usually indicative of active inflammation).
What is the pattern for normal serum seen on SPEP?
Normal serum has a nearly invisible pre-albumin band and a very large albumin band. This is followed by a small peaked alpha-1 band, an alpha-2 band, a bimodal beta, and a broad gamma.
What is the pattern for nephrotic syndrome seen on SPEP?
In nephrotic syndrome due to minimal change disease, there is an especially selective loss of albumin (selective proteinuria). In other forms of nephrotic syndrome, nearly all proteins are lost, including gamma globulins. However, in all types of nephrotic syndrome, larger protein molecules are retained. The result is dimming of all the electrophoretic bands, most prominently the albumin band, with the conspicuous exception of the alpha-2 band which contains the large protein alpha-2 macroglobulin.
What is the pattern for acute inflammation seen on SPEP?
The acute phase reactants account for an increase in the alpha-1 and alpha-2 bands. Albumin is slightly decreased. Initially, the gamma globulins are unchanged, but with prolonged inflammation, these are polyclonally increased.
What is the pattern for cirrhosis seen on SPEP?
Beta-gamma bridging (attributed mainly to increased serum IgA) is the hallmark of cirrhosis. Additional features include hypoalbuminemi and blunted alpha-1 and alpha-2 bands.
In ~__% of sera with an M-protein, two M-proteins are present (biclonal gammopathy).
In ~5% of sera with an M-protein, two M-proteins are present (biclonal gammopathy). Biclonal gammopathies are typically detected when two different heavy or light chain classes of M-proteins are detected on immunofixation (eg, an IgA kappa M-protein and an IgM lambda M-protein in the same patient). Sometimes, the presence of monomers and polymers of a single M-protein may produce two spikes on the SPEP and can resemble a biclonal gammopathy (this should be considered monoclonal rather than biclonal).
With a monoclonal gammopathy, SPEP shows an M-spike in what region?
Usually within the gamma region, but may be in beta-gamma, beta, or alpha-2.
What are monoclonal gammopathies? What are M-proteins?
The monoclonal gammopathies (paraproteinemias or dysproteinemias) are a group of disorders characterized by the proliferation of a single clone of plasma cells, which produces an immunologically homogeneous immunoglobulin commonly referred to as a paraprotein or monoclonal protein (M-protein or M-component, where the “M” stands for monoclonal). The M-protein can be an intact immunoglobulin (ie, containing both heavy and light chains) or can be composed of only light chains or (rarely) only heavy chains.
What are causes of hypogammaglobulinemia?
Hypogammaglobulinemia may be congenital, sex-linked, and/or part of a combined immunodeficiency state. It may also be acquired, as in multiple myeloma (~10% of patients), primary amyloidosis (~20% of patients), chronic lymphocytic leukemia, lymphoma, corticosteroids, or nephrotic syndrome.
Panhypogammaglobulinemia occurs in ~__% of patients with multiple myeloma, and in ~__% of patients with primary amyloidosis.
Panhypogammaglobulinemia occurs in ~10% of patients with multiple myeloma. Most of these patients have a Bence Jones protein (monoclonal free kappa or lambda light chains) in the urine, but lack intact immunoglobulins in the serum. Panhypogammaglobulinemia occurs in ~20% of patients with primary amyloidosis, often associated with a nephrotic pattern (mainly albumin with nonselective globulin loss) in the urine.
What is the most common cause of hyperviscosity?
Waldenström macroglobulinemia, with increased concentrations of IgM, is the most common cause of hyperviscosity, but hyperviscosity can also occur in patients with high concentrations of monoclonal IgA or IgG. Serum viscosity should be determined whenever the monoclonal IgM protein spike is >4 g/dL or the IgA or IgG protein spike is >6 g/dL.
Serum viscosity is normally in the range of __ to __ centipoise (cp). Many patients have symptoms when the viscosity is >__ cp and most have symptoms when the viscosity reaches __ to __ cp.
Serum viscosity is normally in the range of 1.5 to 1.8 centipoise (cp). Many patients have symptoms when the viscosity is >4 cp and most have symptoms when the viscosity reaches 6 to 7 cp.
How does electrophoresis of CSF differ from that of serum?
The CSF normally contains essentially all the proteins present in serum, although in smaller quantities. The most characteristic features of the normal CSF electrophoresis are a prominent pre-albumin band and a double beta (transferrin) band. Additional subtler features are a dim albumin and alpha-2 band.
How is CSF electrophoresis used to diagnose multiple sclerosis?
A diagnosis of multiple sclerosis is supported by finding oligoclonal bands (several distinct bands in the gamma region reflective of several clonal immunoglobulins). These bands should be absent from the patient’s serum, run concurrently, to be specific.
What diseases/conditions can cause oligoclonal bands in CSF?
Elevations in oligoclonally expanded immunoglobulin concentrations in the CSF, termed oligoclonal bands, may occur in any disorder that disrupts the blood-brain barrier. Oligoclonal bands may also be caused by intrathecal production of IgG, and the presence of such bands is a diagnostic criterion for multiple sclerosis. Other diseases that can cause oligoclonal bands in the CSF include infections (eg, nervous system Lyme disease), autoimmune diseases, brain tumors, and lymphoproliferative diseases.
In UPEP, what is seen in a glomerular proteinuria pattern?
Glomerular proteinuria is due to increased filtration of macromolecules (such as albumin) across the glomerular capillary wall. On UPEP there are strong albumin, alpha-1 and beta bands. Very large proteins, due to the persistence of some filtering function, and very small proteins due to tubular absorption, do not make their way into urine. This leaves the protein in between these, most notably albumin, AAT, and transferrin.
In UPEP, what is seen in a tubular proteinuria paattern?
Weak albumin band and strong alpha-1 and beta bands. This pattern results from the impaired tubular reabsorption of low molecular weight proteins normally filtered freely by the glomerulus and almost completely reabsorbed in the proximal tubules, such as alpha-2-macroglobulin, beta-2-microglobulin, and Ig light chains.
In UPEP, what is seen in an overflow proteinuria pattern?
Increased excretion of LMW proteins can occur with marked overproduction of a particular protein, leading to increased glomerular filtration and excretion. This is almost always due to immunoglobulin light chains in multiple myeloma but may also be due to lysozyme (in acute myelomonocytic leukemia), myoglobin (in rhabdomyolysis), or free hemoglobin (in intravascular hemolysis) that is not bound to haptoglobin. In these settings, the filtered load is increased to a level that exceeds the normal proximal reabsorptive capacity.
Precipitation of blood proteins at temperatures lower than 37ºC is referred to as cryoprecipitation. What are the two types of cryoprecipitates?
Cryoglobulin is present when proteins precipitate from an individual’s serum and plasma, and cryofibrinogen refers to the precipitate from plasma only. Cryoglobulins are either immunoglobulins or a mixture of immunoglobulins and complement components. Cryofibrinogen is typically composed of a mixture of fibrinogen, fibrin, fibronectin, and fibrin split products.
In the Brouet classification, what is type I cryoglobulin?
The presence of isolated monoclonal Ig (typically IgG or IgM, less commonly IgA or free immunoglobulin light chains) is the criterion for classification as a type I CG. The proportion of patients with a type I CG accounts for 5-25% of cases. The hematologic diagnoses are typically Waldenström’s macroglobulinemia or multiple myeloma.
In the Brouet classification, what is type II cryoglobulin?
A mixture of polyclonal Ig in association with a monoclonal Ig, typically IgM or IgA, with rheumatoid factor activity defines type II CG. This type of CG, also called essential mixed cryoglobulinemia, accounts for 40-60% of cases. Type II CGs are often due to persistent viral infections, particularly hepatitic C and human immunodeficiency virus infections.
In the Brouet classification, what is type III cryoglobulin?
Mixed cryoglobulins consisting of polyclonal immunoglobulins characterize type III CGs. 40-50% of all CG cases are type III, and these are often secondary to connective tissue diseases.
What is the most common type of cryoglobulin?
Type II.
What is the name of the naturally occuring anti-diuretic hormone in humans? Where is it synthesized?
Arginine vasopressin (AVP), the naturally occurring ADH in humans, is an octapeptide similar in structure to oxytocin. AVP is synthesized in the cell bodies of neurons in the supraoptic and paraventricular nuclei of the anterior hypothalamus and travels along the supraopticohypophyseal tract into the posterior pituitary. Here, it is stored in secretory granules in association with a carrier protein, neurophysin, in the terminal dilatations of secretory neurons that rest against blood vessels.
Anti-diuretic hormone (ADH)/arginine vasopressin (AVP). What are the 2 major stimuli for its secretion?
The major stimuli for AVP secretion are hyperosmolality and effective circulating volume depletion, which are sensed by osmoreceptors and baroreceptors, respectively. Osmoreceptors are specialized cells in the hypothalamus that perceive changes in the extracellular fluid (ECF) osmolality. Baroreceptors are located in the carotid sinus, aortic arch, and left atrium; these receptors participate in the nonosmolar control of AVP release by responding to a change in effective circulating volume.
What are the features of SIADH, and how is it diagnosed?
SIADH consists of hyponatremia, inappropriately elevated urine osmolality (>100 mOsm/kg), and decreased serum osmolality in a euvolemic patient. There is continued renal excretion of sodium. The hyponatremia is corrected by fluid restriction. SIADH should be diagnosed when these findings occur in the setting of otherwise normal cardiac, renal, adrenal, hepatic, and thyroid function; in the absence of diuretic therapy; and in absence of other factors known to stimulate ADH secretion, such as hypotension, severe pain, nausea, and stress.
DDx of hyponatremia.
Pseudohyponatremia (from hyperglycemia, hyperlipidemia, hyperproteinemia). True hyponatremia with hypovolemia: renal losses (diuretics, medullary renal disease, Addison’s disease, RTA type I) or extrarenal losses (GI losses, third spacing). True hyponatremia with euvolemia: SIADH, psychogenic polydipsia, drugs with ADH-like effect. True hyponatremia with hypervolemia: CHF, cirrhosis, nephrotic syndrome.
What are causes of hypokalemia?
Inadequate potassium intake. Increased potassium excretion (mineralocorticoid excess (endogenous or exogenous), hyperreninism (renal artery stenosis), osmotic diuresis (mannitol, hyperglycemia), increased gastrointestinal losses, drugs, genetic disorders). Transcellular shifts (alkalosis, correction of DKA, refeeding, hypothermia).
What genetic disorders can cause hypokalemia?
Congenital adrenal hyperplasia (11-beta hydroxylase or 17-alpha hydroxylase deficiency). Glucocorticoid-remediable hypertension. Bartter syndrome. Gitelman syndrome. Liddle syndrome. Gullner syndrome. Glucocorticoid receptor deficiency. Hypokalemic periodic paralysis. Thyrotoxic periodic paralysis (TTPP). Seizures, sensorineural deafness, ataxia, mental retardation, and electrolyte imbalance (SeSAME syndrome).
Nearly all cases of acidosis are associated with hyperkalemia. What are some exceptions?
The most common cause for metabolic acidosis with hypokalemia is GI loss (eg, diarrhea, laxative use). Other less common etiologies include renal loss of potassium secondary to renal tubular acidosis (types I and II?) or salt-wasting nephropathy.
How is serum ethanol level converted to BAC?
To convert serum ethanol level to BAC, move the decimal point 3 places to the left. Example, a 100 mg/dL serum ethanol level is equivalent to a 0.10 (g/dL) BAC, or 0.10% (weight/volume). This means that one tenth of a percent of a person’s blood volume is alcohol or that a person has 1 part alcohol per 1000 parts blood.
Blood alcohol concentration in serum or plasma is __x higher than in whole blood.
Blood alcohol concentration in serum or plasma is 1.2x higher than in whole blood.
Where is ethanol mostly absorbed from in the body? How is it eliminated?
Ethanol is absorbed primarily in the small intestine and secondarily in the stomach. It is eliminated mostly by metabolism in the liver, with the remainder excreted through urine, exhaled breath, and sweat.
More than 90% of ethanol is oxidized in the liver primarily by what 2 enzymes? What are the metabolites?
Alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH). Metabolism at high alcohol concentrations follows zero order kinetics (independent of time and concentration of the drug). In the initial metabolic pathway, ADH breaks down alcohol into the toxic metabolite acetaldehyde. Small amounts of ADH are also found in other organs such as the stomach. This metabolite is further broken down to acetate by ALDH. Acetate is fed into the Krebs cycle and eventually broken down to water and carbon dioxide.
An estimate for blood alcohol level would be each ounce of whisky, 12 ounces of beer, or 5 ounces of wine raises blood alcohol level by ___ mg/dL. Peak is reached anywhere between ___ to ___ after the last drink. Metabolism is ___ mg/dL/hour and depends upon a multitude of factors.
An estimate for blood alcohol level would be each ounce of whisky, 12 ounces of beer, or 5 ounces of wine raises blood alcohol level by 15-25 mg/dL. Peak is reached anywhere between 30 minutes (if consumption in fasting state) to 3 hours after the last drink. Metabolism is anywhere from 10-30 mg/dL/hour and depends upon a multitude of factors.
Can mesenchymal hamartoma of liver and infantile hemangioendothelioma cause elevated AFP levels?
Yes.
What are the Ranson criteria?
Clinical prediction rules for predicting the severity of (non-gallstone) acute pancreatitis. AT ADMISSION: Age in years >55. WBC count >16,000 cells/uL. Blood glucose >200 mg/dL. Serum AST >250 IU/L. Serum LDH >350 IU/L. WITHIN 48 HOURS: Hematocrit fall of 10% or more. BUN increase by 5 mg/dL or more in spite of fluids. Serum calcium level 6 L. pO2 4 mEq/L. INTERPRETATION: If score is 3 or more, severe pancreatitis is likely, and if score is less than 3, severe pancreatitis is unlikely. Alternatively, score of 0-2 = 2% mortality, 3-4 = 15% mortality, 5-6 = 40% mortality, and 7-8 = 100% mortality.
The effect of PTH on renal tubules causes (increased/decreased) calciium reabsorption and (increased/decreased) phosphate excretion.
The effect of PTH on renal tubules causes increased calciium reabsorption and increased phosphate excretion.
Primary hyperparathyroidism is characterized by increased calcium, decreased phosphate, increased chloride, and increased nephrogenous cAMP. The decreased phosphate is in contrast to many other forms of hypercalcemia, which often have increased phosphate. Why does this occur?
This is because PTH has a dual effect on renal tubules, where there is increased calcium reabsorption and increased phosphate excretion.
Primary hyperparathyroidism is caused by solitary parathyroid adenoma in __%, 4-gland hyperplasia in __%, and carcinoma in __%.
Primary hyperparathyroidism is caused by solitary parathyroid adenoma in 90%, 4-gland hyperplasia in 9%, and carcinoma in 1%.
The locus on chromosome 11 that, in germline form, leads to MEN1 has been found to contain somatic mutation in ~__% of sporadic parathyroid adenomas.
The locus on chromosome 11 that, in germline form, leads to MEN1 has been found to contain somatic mutation in ~25% of sporadic parathyroid adenomas.
Humoral hypercalcemia of malignancy (HHM) accounts for up to 80 percent of patients with hypercalcemia of malignancy. What is the most common mediator of this syndrome?
The most common mediator of this syndrome is PTHrP (parathyroid hormone-related protein).
A (decreased/increased) nephrogenous cAMP in the presence of (decreased/normal/increased) PTH is highly suggestive of humoral hypercalcemia of malignancy.
An increased nephrogenous cAMP in the presence of normal PTH is highly suggestive of humoral hypercalcemia of malignancy.
PTH and vitamin D. How do they differ in increasing/decreasing calcium and phosphate excretion/absorption by the kidneys?
PTH causes increased calcium reabsorption and increased phosphate excretion. Vitamin D causes increased calcium reabsorption and increased phosphate reabsorption.
What granulomatous disease can potentially cause hypercalcemia?
The histiocytes of sarcoidal granulomas appear to have the capacity to activate vitamin D to the active form (1,25 dihydroxy vitamin D). This phenomenon is rarely seen in other types of granulomatous disease.
What are the three major mechanisms by which hypercalcemia of malignancy can occur?
There are three major mechanisms by which hypercalcemia of malignancy can occur: osteolytic metastases with local release of cytokines (including osteoclast activating factors); tumor secretion of parathyroid hormone-related protein (PTHrP); and tumor production of 1,25-dihydroxyvitamin D (calcitriol). Additionally, some tumors cause ectopic secretion of PTH.
There are three major mechanisms by which hypercalcemia of malignancy can occur: osteolytic metastases with local release of cytokines (including osteoclast activating factors); tumor secretion of parathyroid hormone-related protein (PTHrP) (humoral hypercalcemia); and tumor production of 1,25-dihydroxyvitamin D (calcitriol). Additionally, some tumors cause ectopic secretion of PTH. List some malignancies that cause each of the three main types.
Osteolytic metastases: breast cancer, multiple myeloma, lymphoma, leukemia. Humoral hypercalcemia (PTHrP): squamous cell carcinoma (lung, head and neck), RCC, breast cancer, bladder carcinoma, ovarian carcinoma, non-Hodgkin lymphoma, CML, leukemia, lymphoma. Tumor production of calcitriol: lymphoma (Hodgkin, non-Hodgkin, lymphomatosis/granulomatosis), ovarian dysgerminoma.
There are three major mechanisms by which hypercalcemia of malignancy can occur: osteolytic metastases with local release of cytokines (including osteoclast activating factors); tumor secretion of parathyroid hormone-related protein (PTHrP) (humoral hypercalcemia); and tumor production of 1,25-dihydroxyvitamin D (calcitriol). What are the actual physiologic mechanisms by which these three processes occur?
Hypercalcemia in patients with osteolytic metastases is primarily due to increased bone resorption and release of calcium from bone (is mediated by osteoclasts and is not a direct effect of tumor cells). Hypercalcemia in patients with tumors secreting PTHrP is due to both increased bone resorption and distal renal tubular calcium reabsorption. In patients with tumoral production of 1,25-dihydroxyvitamin D, hypercalcemia is the result of a combination of increased bone resorption and intestinal calcium absorption.
Increased production of 1,25-dihydroxyvitamin D (calcitriol) is the cause of almost all cases of hypercalcemia in what type of lymphoma?
Increased production of 1,25-dihydroxyvitamin D (calcitriol) is the cause of almost all cases of hypercalcemia in Hodgkin lymphoma and approximately one-third of cases in non-Hodgkin lymphoma. An occasional patient with Hodgkin lymphoma, however, has hypercalcemia due to PTHrP (humoral hypercalcemia of malignancy), as do some with non-Hodgkin lymphoma.
Hypercalcemia can be produced by a variety of disorders, but primary hyperparathyroidism and malignancy account for most cases. The symptoms and signs associated with hypercalcemia are typically independent of the etiology. What symptoms are seen with mild, moderate, and severe hypercalcemia?
Patients with mild hypercalcemia (calcium 14 mg/dL [3.5 mmol/L]), there is often progression of these symptoms.
PTH synthesis. What is the initial form, and what is the biologically active form?
PTH is synthesized as a 115- amino acid polypeptide called pre-pro-PTH, which is cleaved within parathyroid cells at the N-terminal portion first to pro-PTH (90 amino acids) and then to PTH (84 amino acids). The latter is the major storage, secreted, and biologically active form of the hormone. Calcium regulates not only the release but also the synthesis and degradation of PTH, in all its molecular forms.
What forms/fragments of PTH are secreted in hypocalcemia and hypercalcemia?
In addition to intact PTH, some inactive C-terminal fragments and small amounts of active N-terminal fragments of PTH are present in the parathyroid glands. During hypocalcemia, intracellular degradation of PTH decreases, and mostly PTH 1-84 is secreted; in comparison, during hypercalcemia mostly biologically inactive C-terminal fragments of PTH are secreted.
Circulating immunoreactive PTH in normal subjects comprises: intact PTH (__-__%), C-terminal fragments (__-__%), and N-terminal fragments (__%).
Circulating immunoreactive PTH in normal subjects comprises: intact PTH (5-30%), C-terminal fragments (70-95%), and N-terminal fragments (small percentage).
For the 4 forms of PTH (intact PTH, N-terminal PTH, mid-region PTH, C-terminal PTH), give biologic activity as + or -, and give half-life as short or long.
Intact PTH: +, short (plasma half-life of 2-4 min). N-terminal PTH: +, short. Mid-region PTH: -, long. C-terminal PTH: -, long (plasma half-life 5-10x longer than intact PTH).
Does acidosis increase or decrease the proportion of free calcium?
Acidosis increases the proportion of free calcium by competing for binding sites on albumin. Likewise, alkalosis decreases free calcium.
If the total serum calcium is low but the ionized calcium is normal, it is called ___. If the total serum calcium is high in the setting of a normal ionized calcium, it is called ___.
If the total serum calcium is low but the ionized calcium is normal, it is called pseudohypocalcemia. If the total serum calcium is high in the setting of a normal ionized calcium, it is called pseudohypercalcemia.
The total serum calcium concentration consists of what three fractions? And what are their relative proportions?
~40% is bound to albumin in a ratio of 0.8 mg/dL (0.2 mmol/L or 0.4 meq/L) of calcium per 1.0 g/dL (10 g/L) of albumin. ~45% circulates as physiologically active ionized (or free) calcium. ~15% is bound to multiple organic and inorganic anions such as sulfate, phosphate, lactate, and citrate.
What is the correction for ionized calcium level in the setting of hypo/hyperalbuminemia?
The equation used to measure corrected calcium in cases of hypo/hyperalbuminemia is: Corrected (Ca) = Measured total (Ca) + (0.8 x [4.5 - (alb)]). Or, 0.8 mg/dL Ca per 1g/dL protein. While this formula often provides a good estimate of the ionized calcium, its use should be avoided in patients with acid-base disturbances, renal insufficiency, liver disease, and neonates.
In most cases of humoral hypercalcemia of malignancy, certain tumors secrete PTH-related protein (PTHrP), such as squamous cell carcinoma, breast cancer, and lymphomas. What normal epithelia secrete PTHrP?
Squamous epithelium and lactating breast epithelium.
What are causes of hypercalcemia?
PTH-mediated: primary hyperparathyroidism, familial (MEN-I and -IIa, familial isolated hyperparathyroidism, familial hypocalciuric hypercalcemia), tertiary hyperparathyroidism. PTH-independent: hypercalcemia of malignancy, vitamin D intoxication, chronic granulomatous disorders, medications (thiazide diuretics, lithium, teriparatide, theophylline toxicity, excessive vitamin A), miscellaneous (hyperthyroidism, acromegaly, pheochromocytoma, adrenal insufficiency, immobilization, parenteral nutrition, milk-alkali syndrome).
The CASR gene on 3q encodes the protein CaSR (Calcium-sensing receptor). CaSR is expressed in multiple tissues and senses small changes in the serum ionized calcium. What are some disorders caused by mutations in this gene?
Activating mutations: autosomal dominant hypocalcemia (also called autosomal dominant hypoparathyroidism) (ADH), ADH with features of Bartter’s syndrome. Inactivating mutations: familial hypocalciuric hypercalcemia (FHH1; also called familial benign hypercalcemia), neonatal (NHPT) and neonatal severe primary hyperparathyroidism (NSHPT), and occasionally familial isolated hyperparathyroidism.
What are causes of secondary hyperparathyroidism?
Renal failure (impaired calcitriol production, hyperphosphatemia). Decreased calcium intake. Calcium malabsorption (vitamin D deficiency, bariatric surgery, celiac disease, pancreatic disease (fat malabsorption)). Renal calcium loss (idiopathic hypercalciuria, loop diuretics). Inhibiton of bone resorption (bisphosphonates, hungry bone syndrome).
What are the 4 main categories of causes of hypocalcemia?
4 main categories: Low PTH (hypoparathyroidism). High PTH (secondary hyperparathyroidism in response to hypocalcemia). Drugs. Disorders of magnesium metabolism.
What are causes of hypocalcemia?
Low PTH (hypoparathyroidism): genetic disorders, post-surgical (thyroidectomy, parathyroidectomy, radical neck dissection), autoimmune, infiltration of the parathyroid gland (granulomatous, iron overload, metastases), radiation-induced destruction of parathyroid glands, hungry bone syndrome, HIV infection. High PTH (secondary hyperparathyroidism in response to hypocalcemia): vitamin D deficiency or resistance, parathyroid hormone resistance, renal disease, loss of calcium from the circulation (hyperphosphatemia, tumor lysis, acute pancreatitis, osteoblastic metastases, acute respiratory alkalosis, sepsis or acute severe illness. Drugs. Disorders of magnesium metabolism.
How does secondary hyperparathyroidism lead to renal osteodystrophy?
Secondary hyperparathyroidism (AKA pseudohypoparathyroidism) is due to peripheral resistance to the action of PTH. The PTH level is normal to high, calcium level is low, and phosphate is high. This persistent hyperparathyroid state produces marked activation of osteoclasts in bone, leading to so-called brown tumors of bone (renal osteodystrophy).
How does hypomagnesemia affect PTH secretion?
Mild transient deccreases in serum magnesium cause increased PTH secretion (an attempt on the part of the parathyroids to maintain the balance of divalent cations). However, persistent or marked hypomagnesemia causes PTH resistance (when the hypomagnesemia is milder) or inhibits PTH secretion (when the hypomagnesemia is more severe), leading to hypoparathyroidism.
Respiratory acidosis is due to too little elimination of CO2 by the lungs (hypoventilation); the primary change is in CO2. Respiratory alkalosis is due to too much elimination of CO2 by the lungs (hyperventilation); the primary change is in CO2. Compensation in primary respiratory disorders involves buffers and alterations in renal handling of (HCO3-). What are metabolic disorders due to, what is the primary change in, and what does compensation involve?
Metabolic disorders are due to excessive intake, excessive production, or too little renal elimination of an acid or base; the primary change is in (HCO3-). Compensation in primary metabolic disorders involves buffers and alterations in pulmonary handling of CO2.
What is the Henderson-Hasselbalch equation?
The Henderson–Hasselbalch equation describes the derivation of pH as a measure of acidity (using pKa, the negative log of the acid dissociation constant) in biological and chemical systems. pH = pKa + log(base/acid).
The Henderson-Hasselbalch equation is pH = pKa + log(base/acid). What are the values of the components of the equation in normal individuals?
pH = 7.4. pKa = 6.1. Base = [bicarb] = 24. Acid = dissolved PaCO2 = 0.03 x PaCO2 = 0.03 x 40 = 1.2. Substituting these into the equation, 7.4 = 6.1 + log(24/1.2) = 6.1 + 1.3 = 7.4. The body makes every attempt to maintain the validity of this equation. Since it must maintain a pH of 7.4 and the pK is a constant, it can only alter [bicarb] and PaCO2, and so the ratio of these must remain 20:1.
Arterial blood gas analyzers have electrodes to measure what variables directly?
ABG analyzers use a pH electrode, a PaCO2 electrode, and a PaO2 electrode to measure these variables directly. They usually also derive a % saturation of oxygen through a calculation that assums a normal hemoglobin oxygen affinity.
What does a pulse oximeter measure, and how does it work?
Pulse oximeters measure hemoglobin oxygen saturation directly by measuring the absorbance of transdermally transmitted light. It emits two wavelengths of light: one that deoxyhemoglobin absorbs, and one that oxyhemoglobin absorbs.
How does a co-oximeter work, and what can it measure?
The co-oximeter is a spectrophotometric dvice that measures absorbance of multiple wavelengths of light. It is thus able to measure (not calculate) the proportions of oxyhemoglobin, deoxyhemoglobin, methemoglobin, and carboxyhemoglobin. Oxygen saturation is then the ratio of oxyhemoglobin to the total.
Out of ABG analyzer, pulse oximeter, and co-oximeter, which is the method of choice for detecting carbon monoxide poisoning?
Co-oximetry.
Equation for calculating anion gap? What is normal?
Anion gap = [Na] - ([Cl] + [HCO3]). Normal = <12.
Anion gap = [Na] - ([Cl] + [HCO3]). But hypoalbuminemia can mask the anion gap. What is the formula for correcting this?
Corrected anion gap = [Na] - ([Cl] + [HCO3]) + 2.5(4-albumin).
Equation for calculating osmolal gap? What is normal?
Osmolal gap = osm measured - (2[Na] + [Glu]/18 + BUN/2.8). Normal = <10.
What is a delta-delta?
A comparison of the delta bicarb and the delta anion gap. These changes should be essentially 1:1 (within +/- 2) in simple acid-base disorders. A delta-delta greater than that is indicative of a complex disorder.
For metabolic acidosis, respiratory acidosis, metabolic alkalosis, and respiratory alkalosis, which directions do pH and [HCO3] go?
Metabolic acidosis: pH and [HCO3] go same direction (bicarb usually 44 mmHg). Metabolic alkalosis: pH and [HCO3] go same direction (bicarb usually >25 mEq/L). Respiratory alkalosis: pH and [HCO3] go opposite direction (pCO2 usually <40 mmHg).
How do you determine if compensation is adequate for metabolic acidosis, metabolic alkalosis, acute respiratory alkalosis or acidosis, and chronic respiratory alkalosis or acidosis?
Metabolic acidosis: for each 1.3 mEq fall in [HCO3], the pCO2 decreases by 1.0 mmHg. Metabolic alkalosis: for each 0.6 mEq rise in [HCO3], the pCO2 increases by 1.0 mmHg. Acute respiratory alkalosis or acidosis: for each 1 mmHg change in pCO2, the [HCO3] changes by 0.1 in the same direction. Chronic respiratory alkalosis or acidosis: for each 1 mmHg change in pCO2, the [HCO3] changes by 0.4 in the same direction.
Metabolic acidosis can be categorized by presence or absence of anion gap. List causes from each category.
With increased AG (>12): ketoacidosis (diabetic, starvation, EtOH-associated), lactic acidosis, D-lactic acidosis, ingestions (methanol, ethylene glycol, diethylene glycol, propylene glycol, salicylate, toluene (if early or if kidney function is impaired)), pyroglutamic acid (5-oxoproline), CKD/uremia. With normal AG (<12): diarrhea or other intestinal losses, ureteral diversion, ketoacidosis posttreatment, carbonic anhydrase inhibitors, type 1 (distal) RTA, type 2 (proximal) RTA, type 4 RTA (hypoaldosteronism), toluene ingestion (if late and if renal function is preserved - dut to excretion of sodium and potassium hippurate in the urine), CKD and tubular dysfunction (but relatively preserved GFR).
How can toluene ingestion cause metabolic acidosis with increased anion gap in some cases and metabolic acidosis with normal anion gap in other cases?
Toluene ingestion causes increased AG if early after ingestion or if kidney function is impaired. It causes normal AG if late after ingestion and if renal function is preserved - due to excretion of sodium and potassium hippurate in the urine.
Elevated plasma osmolal gap can be seen with or without metabolic acidosis. List causes of each category.
With anion gap metabolic acidosis: ingestion (ethylene glycol, methanol, formaldehyde, paraldehyde); lactic acidosis; diabetic ketoacidosis; alcoholic ketoacidosis; end-stage CKD (GFR <10 mL/min) without regular dialysis. Without metabolic acidosis: ingestion (ethanol, isopropyl alcohol, diethyl ether); infusion of nonconductive glycine, sorbitol, or mannitol solutions; severe hyperproteinemia; severe hyperlipidemia.
Metabolic alkalosis can be categorized by chloride responsive (U Cl 10). List causes of each.
Chloride responsive: diuretic therapy, vomiting, NGT suction, villous adenoma, carbenicillin, contraction alkalosis. Chloride resistant: hyperaldosteronism, Cushing syndrome, exogenous steroids, licorice (glyccrhizic acid), Bartter syndrome, milk-alkali syndrome.
Which are more often symptomatic: sympathetic (including pheochromocytomas) or parasympathetic paragangliomas?
Pheochromocytomas and most sympathetic paragangliomas are often associated with clinical symptoms, while only a small percentage of parasympathetic paragangliomas are symptomatic. Parasympathetic paragangliomas often lack tyrosine hydroxylase, the enzyme required for catecholamine synthesis, and are therefore usually nonfunctional.
At least 30% of paragangliomas and pheochromocytomas are associated with familial syndromes. Specific genotype-biochemical correlations highlight the importance of laboratory testing to characterize patterns of catecholamine excess. The biochemical profiles of tumors associated with mutations of genes encoding succinate dehydrogenase subunits, (collectively referred to as SDHx; x refers to all subunits, SDHA refers to subunit A, etc) are characterized by ___ and/or ___ production. VHL-related tumors are associated with ___ production. RET- and NF1-related tumors are associated with ___ production.
The biochemical profiles of tumors associated with mutations of genes encoding succinate dehydrogenase subunits, (collectively referred to as SDHx; x refers to all subunits, SDHA refers to subunit A, etc) are characterized by dopamine and/or norepinephrine production. VHL-related tumors are associated with norepinephrine production. RET- and NF1-related tumors are associated with epinephrine production.
In laboratory biochemical testing for pheochromocytomas and paragangliomas, why is measuring catecholamine metabolites superior to measurement of the parent catecholamine?
Catecholamines (dopamine, norepinephrine, and epinephrine) are not continuously secreted in normal conditions, and undergo intracellular methylation by the tumor cells the produce them. Biochemical testing for the O-methylated metabolites of dopamine, norepinephrine, and epinephrine (methoxytyramine, normetanephrine, and metanephrine, respectively) in plasma and/or urine is therefore superior to measurement of the parent catecholamines.
There are several nonprotein nitrogenous compounds in the blood, collectively termed nonprotein nitrogen (NPN). List.
Urea, individual amino acids, urate, creatinine, and ammonia. Urea is the largest component of NPN.
Does BUN underestimate or overestimate GFR?
Urea is freely filtered and partially reabsorbed by the nephron; this reabsorption has the consequence that BUN always slightly underestimates GFR.
Urea is freely filtered and partially reabsorbed by the nephron; this reabsorption has the consequence that BUN always slightly underestimates GFR. What happens in hypovolemia?
Reabsorption increases with hypovolemia; thus BUN underestimates GFR even more in hypovolemic states.
How is creatinine measured?
The creatinine concentration was classically determined, and is still often determined, by the Jaffe reaction - alkaline picrate forms a colored complex with creatinine. Alternative enzymatic (using creatinase) and colorimetric assays are available.
What is creatinine? How does the kidney handle it? Does it underestimate or overestimate GFR?
Creatinine is derived from the metabolism of creatine in skeletal muscle and from dietary meat intake. It is released into the circulation at a relatively constant rate. Creatinine is freely filtered across the glomerulus and is neither reabsorbed nor metabolized by the kidney. However, approximately 10 to 40 percent of urinary creatinine is derived from tubular secretion by the organic cation secretory pathways in the proximal tubule, with the quantity secreted increasing with increasing serum creatinine concentration. At all serum concentrations, creatinine slightly overestimates GFR.
BUN and Cr. Do they underestimate or overestimate GFR?
BUN underestimates GFR. Cr overestimates GFR.
The simplest way to calculate GFR is based on creatinine clearance. What is the equation?
ClCr = UCr x VUr / PCr. Where UCr is urine creatinine in mg/dL, VUr is volume of urine in mL/24 hrs, and PCr is plasma creatinine in mg/dL. But this formula is overly simplistic and the MDRD study equation is the most widely recommended formula now.
The simplest way to calculate GFR is based on creatinine clearance: ClCr = UCr x VUr / PCr. But what 2 factors weaken the suitability of this formula for clinical use?
Nonlinearity and nonglomerular influences upon creatinine. Nonlinearity: The inverse relationship between GFR and Cr is nonlinear. Mild to moderate degrees of GFR impairment do not cause appreciable increases in Cr concentration; only when GFR is about half of normal does creatinine rise precipitously and begin to linearly reflect changes in GFR. Nonglomerular influences upon creatinine: The serum Cr concentration is increased by muscle mass/activity/injury and protein intake; it is decreased by age; it is influences by race, age, sex, and numerous medications.
The Modification of Diet in Renal Disease Study equation is the most widely recommended formula for calculating eGFR. In what populations has it been studied and validated? What patient variables do you need to know?
The MDRD equation has been studied and validated in adult Caucasian and African American populations with impaired kidney function (GFR
The Modification of Diet in Renal Disease Study equation is the most widely recommended formula for calculating eGFR. How many variations of the equation are there?
4, based on if the lab reports serum Cr in mg/dL (conventional units) or umol/L (SI units), and whether the lab uses a Cr assay that has been (traceable) or has not been (original (non-traceable)) calibrated to be traceable to an isotope dilution mass spectometry (IDMS) method.
The result given by the Modification of Diet in Renal Disease Study equation is multiplied by 0.742 if the patient is ___ and by 1.210 is the patient is ___.
The result given by the Modification of Diet in Renal Disease Study equation is multiplied by 0.742 if the patient is female and by 1.210 is the patient is African American.
What units is eGFR given in?
mL/min/1.73m^2.
What is a normal BUN/Cr ratio? What does it suggest when BUN and Cr are elevated but the ratio is maintained? when the ratio is elevated? when the ratio is decreased?
When BUN and Cr are elevated but the ratio is maintained, it is suggestive of intra-renal disease (GN and TIN) - renal azotemia. When the ratio is elevated, is suggests prerenal azotemia (poor renal perfusion) or postrenal azotemia (renal obstruction). A decreased BUN/Cr is rare and may result from dietary protein insufficiency or severe liver disease.
What is cystatin C and how is it handled by the kidney?
Cystatin C is a cysteine protease inhibitor produced by nearly all cells in the body that is freely filtered by the glomerulus, not reabsorbed, and is metabolized by the proximal tubular epithelium.
Normal proteinuria does not exceed ___ mg/day and consists mainly of ___ and ___.
Normal proteinuria does not exceed 150 mg/day and consists mainly of Tamm-Horsfall protein and minute amounts of albumin.
The urine protein assay is sensitive to all kinds of protein (albumin, globulins, Bence-Jones), and its lower limit of detection is about ___ mg/dL.
The urine protein assay is sensitive to all kinds of protein (albumin, globulins, Bence-Jones), and its lower limit of detection is about 3 mg/dL.
The urine dipstick test is most sensitive to albumin (but not enough to detect microalbuminuria); it is not sensitive to globulins or even to very high levels of Bence-Jones protein. The lower limit of detection of the urine dipstick test for albumin is ___ mg/dL.
The urine dipstick test is most sensitive to albumin (but not enough to detect microalbuminuria); it is not sensitive to globulins or even to very high levels of Bence-Jones protein. The lower limit of detection of the urine dipstick test for albumin is 18 mg/dL.
What are the lower limits of detection of: the urine protein assay for protein, the urine dipstick test for albumin, and the microalbumin assay for albumin?
3 mg/dL, 18 mg/dL, and 0.3 mg/dL, respectively.
What units are the albumin:creatinine ratio and the protein:creatinine ratio reported in?
The albumin:creatinine ratio is reported in mg/g, and the protein:creatinine ratio is reported in mg/mg.
What 2 main entities are in the DDx of a high urine protein concentration with a negative microalbumin assay?
Bence-Jones protein and hook effect (a false negative result with certain immunoassays due to very high concentrations of a particular analyte).
What is the “hook effect”?
The hook effect or the prozone effect is a false negative result with certain immunoassays due to very high concentrations of a particular analyte. The hook effect mostly affects one-step immunometric assays, such as two-site immunoassays in which both the capture and detection antibody are added simultaneously, free analyte and analyte bound to labelled antibody compete for the limited number of antibody-binding sites and in the presence of higher analyte concentration will decrease rather than increase label bound to the solid phase.
The 2 general categories of interferences seen in immunoassays are 1. Interferences that alter the measurable analyte concentration in the sample and 2. Interferences that alter antibody binding. Give 3 examples of each.
Interferences that alter the measurable analyte concentration in the sample: Hormone binding proteins. Pre-analytical factors, e.g., anticoagulants, sample storage. Autoanalyte antibodies. Interferences that alter antibody binding: Heterophile antibodies. Human anti-animal antibodies. High-dose hook effect.
For the following types of interferences seen in immunoassays, give the assay types affected. Crossreactivity. Hook effect. Antibody interference. Signal interference. Matrix effects.
Crossreactivity: All, but primarily competitive assays. Hook effect: Nephelometric, turbidimetric, and immunometric assays. Antibody interference: All, but primarily immunometric assays. Signal interference: All. Matrix effects: All.
The presence of beta-2-microglobulin and lysozyme in urine suggests what type of kidney dysfunction?
Tubular dysfunction, since these proteins are freely filtered by the glomerulus then completely reabsorbed by the normally functioning proximal convoluted tubule.
How is chronic kidney disease defined?
CKD is defined as the presence of kidney damage (usually detected as urinary albumin excretion of ≥30 mg/day, or equivalent) OR decreased kidney function (defined as eGFR <60 mL/min per 1.73 m^2) for three or more months, irrespective of the cause. The persistence of the damage or decreased function for at least three months is necessary to distinguish CKD from acute kidney disease/injury.
How is chronic kidney disease staged?
The 2012 Kidney Disease: Improving Global Outcomes (KDIGO) guidelines stage according to: cause of disease, 6 categories of eGFR (G stages), and 3 categories of albuminuria (A stages).
Acute kidney injury can be classified into what 3 general categories?
Prerenal - As an adaptive response to severe volume depletion and hypotension, with structurally intact nephrons. Renal/Intrinsic - In response to cytotoxic, ischemic, or inflammatory insults to the kidney, with structural and functional damage. Postrenal - From obstruction to the passage of urine. While this classification is useful in establishing a differential diagnosis, many pathophysiologic features are shared among the different categories.
The most common cause of renal/intrinsic acute kidney injury is ___, the most common causes of which are ___ and ___.
The most common cause of renal/intrinsic acute kidney injury is ATN, the most common causes of which are ischemia and nephrotoxins.
The FENa is almost always low (<1%) in prerenal ARF, the exception being when the patient has been given diuretics or has glycosuria. What can be measured in place of FENa in these patients?
In these patients, fractional excretion of urea can be a useful alternative.
In renal failure, what features on urinalysis can suggest prerenal ARF, GN, ATN, and TIN?
Prerenal ARF: A lack of findings or isolated hyaline casts. GN: Dysmorphic RBCs and RBC casts. ATN: Pigmented casts. TIN: Leukocytes (in the absence of infection - sterile pyuria), WBC casts, and/or eosinophils.
At what weeks’ gestation do bilirubin levels peak in normal fetuses (unaffected by hemolytic disease of the newborn)?
Bilirubin levels normally peak at 23-25 weeks’ gestation in unaffected fetuses.
What is a Liley curve/chart?
It predicts the severity of hemolytic disease of the newborn based on amniotic fluid bilirubin levels. On semilog paper, absorbances are plotted against wavelength. A straight line is drawn from the point at 350 nm to the point at 550 nm. This line reflects the theoretical plot if there were no pigments in the fluid. The difference between the line and the actual absorbance at 450 nm is the delta OD450, which reflects the bilirubin concentration. The delta OD450 is plotted against the EGA on a Liley chart, and results fall into zones I, II, or III.
What do results in each of the 3 zones on a Liley curve indicate?
A result in Zone I indicates mild or no disease. Fetuses in zone I are usually followed with amniocentesis every 3 weeks. A result in zone II indicates intermediate disease. Fetuses in low Zone II are usually followed by amniocentesis every 1-2 weeks. A result above the middle of Zone II may require transfusion or delivery. Patients with results in zone I or low zone II can be allowed to proceed to term, at which point labor should be induced. In most cases, patients in the middle of zone II can progress to 36-38 weeks of gestation. If results are in zone III or rising in zone II, deliver immediately for EGA >36 wks, and consider intrauterine transfusion otherwise.
What is hCG? What are its subunits and which is measured in immunoassays? What units is hCG concentration expressed in?
hCG is a glycoprotein heterodimer composed of an alpha and a beta chain. The alpha subunit is identical to that found in TSH, FSH, and LH. The predominant methodologies involve immunoassay directed against the beta subunit. The concentration is expressed in mIU/mL and ng/mL (100 mIU = 8 ng).
When can false negative and false positive hCG measurements occur, and what can be done to remedy them?
False negative hCG can occur with urine testing, especially if the urine is dilute. When there is high suspicion, the negative urine hCG is confirmed with a serum hCG test. False positive hCG can occur due to heterophile antibody interference. The measurement can be repeated on a different analyzer, pretreat sample with a heterophile binding reagent, or run serial dilutions.
What is “phantom” hCG?
A false-positive hCG result that is a consequence of interference of heterophil antibodies with standard assays for hCG. A diagnosis of “phantom” hCG should be
suspected in patients with a negative urine and a positive serum hCG. Apart from pregnancy, heterophil bridging antibodies in immunometric hCG assays can cause falsely elevated levels in patients with gestational trophoblastic diseases, choriocarcinoma, testicular germ cell tumors
and IgA deficiencies.
What is the pattern of hCG levels in a normal gestation, from conception to postpartum?
hCG becomes detectable 6-8 days following conception (about the time of implantation), when levels are 10-50 mIU/mL. The hCG level doubles every 2 days until 1200 mIU/mL at 10 weeks. The level doubles every 3 days between 1200 to 6000 mIU/mL. The level doubles every 4 days above 6000 until it peaks near the end of the first trimester around 10,000 (800 ng/mL). After delivery, hCG normally remains detectable for 2 weeks.
When does hCG first become detectable in a normal gestation?
hCG becomes detectable 6-8 days following conception (about the time of implantation), when levels are 10-50 mIU/mL.
For how long after delivery is hCG detectable?
After delivery, hCG normally remains detectable for 2 weeks. The disappearance of hCG after term pregnancy is best viewed as triphasic, with rapid, medium, and slow half-lives of 3.6 hrs, 18 hrs, and 53 hrs.
What are some causes of abnormally elevated hCG in pregnancy?
Multiple gestations, polyhydramnios, eclampsia, erythroblastosis fetalis.
If a normal hCG dynamic is not seen, this suggests an abnormal pregnancy. Specifically, if serum hCG does not rise at least 66% in 48 hrs, or if the hCG falls during this time, this suggests an ectopic pregnancy or nonviable IUP. However, a normal rate of rise can be seen in __% of ectopics, and an abnormal rate of rise can be seen in __% of normal IUP.
If a normal hCG dynamic is not seen, this suggests an abnormal pregnancy. Specifically, if serum hCG does not rise at least 66% in 48 hrs, or if the hCG falls during this time, this suggests an ectopic pregnancy or nonviable IUP. However, a normal rate of rise can be seen in 15% of ectopics, and an abnormal rate of rise can be seen in 15% of normal IUP.
With transabdominal US, a gestational sac should be detectable when the hCG >__ mIU/mL. When the hCG is >__ mIU/mL, the absence of an IUP detectable by transvaginal US is 90% specific for an ectopic.
With transabdominal US, a gestational sac should be detectable when the hCG >6000 mIU/mL. When the hCG is >1400 mIU/mL, the absence of an IUP detectable by transvaginal US is 90% specific for an ectopic.
A serum progesterone level >__ ng/mL virtually assures an IUP. Levels <__ ng/mL are strongly (almost 100%) predictive of an abnormal pregnancy.
A serum progesterone level >25 ng/mL virtually assures an IUP. Levels <5 ng/mL are strongly (almost 100%) predictive of an abnormal pregnancy. However, most patients fall somewhere in between these thresholds.
For how long after a normal delivery is hCG detectable? For how long after removal of an ectopic pregnancy is hCG detectable? For how long after spontaneous abortion is hCG detectable? For how long after evacuation of an uncomplicated molar pregnancy is hCG detectable?
2 weeks. 4 weeks. 4-6 weeks. Up to 10 weeks.
After evacuation of a molar pregnancy, hCG levels must be monitored weekly until undetectable for __. It is then measured monthly for __.
After evacuation of a molar pregnancy, hCG levels must be monitored weekly until undetectable for 3 consecutive weeks. It is then measured monthly for 1 year.
What are the sensitivities of the following for Down syndrome? Triple screen, quad test, integrated screen (and then the addition of US nuchal fold thickness).
Triple screen - 70%, Quad test - 80%. Integrated screen - 85%. Integrated screen combined with US nuchal fold thickness - >90%.
What are the components of the following prenatal screening panels? Triple screen. Quad test. Integrated screen.
Triple screen: hCG, AFP, unconjugated estriol. Quad test: the components of the triple screen plus dimeric inhibin A. Integrated screen: PAPP-A and hCG are measured in the 1st trimester; AFP, unconjugated estriol, and dimeric inhibin A are measured in the 2nd trimester; then the data are combined.
What is the main reason for better performance of the Quad test over the triple screen for prenatal screening?
The improved performance of the Quad test comes mainly from amelioration of the effects of inaccurate gestational age which plague the triple screen.
How do diabetes and smoking affect the values of the prenatal triple screen?
In diabetic mothers, unconjugated estriol and hCG are mildly decreased. In mothers who smoke, unconjugated estriol and hCG are decreased, while AFP is increased.
Trisomy 18 (Edwards syndrome) usually shows a characteristic pattern on prenatal triple screen. What is it?
All 3 components (uE, hCG, AFP) are decreased.
Trisomy 21 (Down syndrome) shows a characteristic pattern on prenatal triple screen. What is it?
hCG increased, AFP and uE decreased. Also, dimeric inhibin A is increased.
Neural tube defects show a characteristic pattern on prenatal triple screen. What is it?
AFP increased, uE decreased, hCG normal. With increased AFP, if an US confirms gestational age and excludes overt anatomic abnormalities, multiple gestations, or fetal demise, then amniocentesis is performed to obtain amniotic fluid for AFP and AChE.
2-3% of maternal serum AFPs are elevated on prenatal screening; of these, __% are due to an actual NTD. The sensitivity of MSAFP screening is __%.
2-3% of maternal serum AFPs are elevated on prenatal screening; of these, 10% are due to an actual NTD. The sensitivity of MSAFP screening is 90% - the sensitivity is worse for multiple gestations (30%).
AFP is the principal plasma protein in the fetus. It is a fetal specific globulin, synthesized by the fetal yolk sac, gastrointestinal tract, and liver. What factors affect MSAFP level interpretation?
MSAFP rises progressively during the 1st and 2nd trimesters. Adjustments to the MSAFP interpretation are made for maternal weight, race, number of fetuses, and maternal diabetes. Increased maternal weight can have a dilutional effect on the AFP, providing a falsely low value. Levels are much higher in multiple gestations, and much lower in maternal diabetes.
MSAFP is >2.5 MOM in >80% of NTDs. How many MOM are considered abnormal in maternal diabetes, and in twin gestations?
> 2.0 MOM is considered abnormal in maternal diabetes, and >4.5 MOM is considered abnormal in twin gestations.
What conditions are associated with increased MSAFP?
NTDs. Omphalocele and gastroschisis. Renal anomalies. Sacrococcygeal teratoma. Cystic hygroma. Hydrops fetalis. Turner syndrome. Bowel obstruction. Twins. Wrong gestational age. Fetal demise. Fetal-maternal hemorrhage.
Maternal serum hCG is ~__x higher than normal in Down syndrome.
Maternal serum hCG is ~2x higher than normal in Down syndrome.
Unconjugated estriol is weakly sensitive to Down syndrome, but is a very good indicator of what other 3 conditions?
Trisomy 18 (Edward syndrome). Smith-Lemli-Optiz syndrome. Inherited (fetal) deficiencies of steroid sulfatase.
Dimeric inhibin A is a glycoprotein produced by the placenta. In a Down syndrome fetus, DIA is increased to an average of ___ MOM.
Dimeric inhibin A is a glycoprotein produced by the placenta. In a Down syndrome fetus, DIA is increased to an average of 1.9 MOM.
What is fetal fibronectin? Where is it found? How can it be used to determine preterm labor?
Fetal fibronectin is a protein found normally at the placental fetomaternal interface. Cervicovaginal fluid contains fetal fibronectin briefly during early gestation, after which time it is absent until just before labor. The absence of fetal fibronectin has very high NPV and can exclude impending preterm birth. A positive result suggests the onset of preterm labor, but the overall PPV is low.
Mature type II pneumocytes produce a mixture of phospholipids composed predominantly of lecithin. The vast majority of lecithin is ___. Lesser amounts of ___, ___, ___, and ___ also comprise lecithin.
Mature type II pneumocytes produce a mixture of phospholipids composed predominantly of lecithin. The vast majority of lecithin is disaturated phosphatidylcholine (DSPC). Lesser amounts of phosphatidylglycerol (PG), phosphatidylinositol (PI), phosphatidylethanolamine (PE), and sphingomyelin also comprise lecithin.
At what gestational ages is fetal lung maturity a concern?
It is an issue for gestation between 34 to 37 weeks. Prior to 34 weeks, fetal lung maturity is unlikely, and the risk of RDS is very high. After 37 weeks the risk of RDS is exceedingly low, and fetal lung maturity testing is generally not indicated, except in the presence of poorly controlled maternal diabetes.
What are tests that can be done to determine fetal lung maturity?
Lecithin/sphingomyelin ratio. Phosphatidylglycerol concentration. Foam stability. Lamellar body number density. Disaturated phosphatidylcholine concentration. Fluorescence polarization assay.
What is the rationale for determining lecithin/sphingomyelin ratio to evaluate fetal lung maturity?
Lecithin increases with gestational age, while sphingomyelin remains at a relatively constant 2% of total surfactant phospholipid. Until 26 wks, the ratio is 1:1. After that, the L:S ratio increases until 2:1 is reached around 35 wks. This ratio is generally taken to indicate fetal lung maturity. Above 2:1, 2% of premature infants will develop RDS. Below 2:1, nearly 60% will.
What are potential problems in evaluation of lecithin/sphingomyelin ratio to determine fetal lung maturity?
In DM, a ratio of 2:1 does not ensure FLM. The phosphatidylglycerol concentration is more reliable in this senario. The presence of meconium falsely decreases the L:S ratio. The presence of blood normalizes the L:S ratio to ~1.5. The CV of the L:S ratio is high.
What is a better test than L:S ratio to determine FLM in maternal DM?
Phosphatidylglycerol concentration.
How is/when is phosphtidylglycerol concentration used to determine FLM?
PG is first detected around 36 wks and its presence is indicative of FLM. Neither blood nor meconium interfere with PG determinations, making it the test of choice when the only available specimen is a contaminated one. PG can be measured by TLC or agglutination.
How is/when is the foam stability test used to determine FLM?
When pulmonary surfactant is present in amniotic fluid in sufficient concentration, the fluid is able to form a highly stable film that can support the structure of a foam. The amniotic fluid is serially diluted with ethanol, and the highest concentration of ethanol at which a complete ring of bubbles is seen is the foam stability index (FSI). An FSI greater than 0.47 is considered indicative of fetal lung maturity.
How is the lamellar body number density test used to determine FLM?
Surfactant lamellar bodies are about the size of platelets, and the platelet channel of a cell counter can be used to quantify them. An LBND greater than 50,000/mL is predictive of maturity.
How is the disaturated phosphatidylcholine concentration used to determine FLM?
An alternative to the L/S ratio is to determine