Hematocrit

Most POCT glucose methods measure plasma glucose and not whole blood glucose.  Therefore, test will be affected by hematocrit extremes

Note:  When using a POCT device, a low systolic BP (must be > 80 mm hg) will yield interstitial fluid so glucose will be falsely low

What are the glucose values diagnostic for diabetes..

Fasting

2 hr. postprandial

Random

Fasting                    ≥ 126

2 hr. postprandial     ≥ 200

Random                   ≥ 200    

By testing the patient's blood sample one hour after an oral 50 gram glucose load.  

Positive if the glucose is > 140 mg/dL...

glucose oxidase

hexokinase method

Gray top tubes have sodium fluoride which prevents glycolysis.  

It is best to use grey top tubes is testing will be delayed

After an intravascular hemolytic episode, which of the following would normally be decreased?

a.  transferrin

b.  troponin

c.  haptoglobin

d.  hemosiderin

c

Haptoglobin is used to screen and monitor INTRAvascular hemolysis.  In intravascular hemolysis, haptoglobin will bind free hemoglobin released from cells.

In EXTRAvascular hemolysis, the reticuloendothelial system (splenic monocytes) phagocytize the RBCs therefore hemoglobin is not released into circulation.  Haptoglobin levels are NORMAL in extravascular hemolysis

After a severe intravascular hemolytic episode, which of the following would be expected?

a.  increased unconjugated bilirubin, increased haptoglobin

b.  increased unconjugated bilirubin, decreased haptoglobin

c.  decreased unconjugated bilirubin, no change in haptoglobin

d.  decreased unconjugated bilirubin, decreased haptoglobin

b

deficiency of glucuronyl transferase

Most infants develop visible jaundice due to elevation of unconjugated bilirubin concentration during their first week. This common condition is called physiological jaundice. 

Mechanism involved in physiological jaundice are mainly:

• Relatively low activity of the enzyme glucuronosyltransferase which normally converts unconjugated bilirubin to conjugated bilirubin that can be excreted into the gastrointestinal tract.
• Before birth, this enzyme is actively down-regulated, since bilirubin needs to remain unconjugated in order to cross the placenta to avoid being accumulated in the fetus. After birth, it takes some time for this enzyme to gain function.
• Shorter life span of fetal red blood cells, being approximately 80 to 90 days in a full term infant, compared to 100 to 120 days in adults.
• Relatively low conversion of bilirubin to urobilinogen by the intestinal flora, resulting in relatively high absorption of bilirubin back into the circulation.

Urobilinogen is elevated in:

a.  hemolytic anemia

b.  severe dehydration

c.  malnutrition

d.  biliary obstructin

obstruction of bile ducts

Clinical Significance

Urinary urobilinogen may be increased in the presence of a hemolytic process such as hemolytic anemia. It may also be increased with infectious hepatitis, or with cirrhosis. Comparing the urinary bilirubin result with the urobilinogen result may assist in distinguishing between red cell hemolysis, hepatic disease, and biliary obstruction.

Urobilinogen is increased in hemolytic disease and urine bilirubin is negative. Urobilinogen is increased in hepatic disease, and urine bilirubin may be positive or negative.

Urobilinogen is low with biliary obstruction, and urine bilirubin is positive. Reagent strips methods however, cannot distinguish normal urobilinogen from absent urobilinogen, as might be seen in complete biliary obstruction.

List first to last enzymes that increase following an MI (myocardial infarction)

a.  AST, CK, LD

b.  LD, CK, AST

c.  LD, AST, CK

d.  CK, AST, LD

D

CKMB  - least specific for MI

LD1 > LD2 - slowest to rise after MI

Myoglobin - fastest to rise after MI (negative results can rule out MI)

Cardiac troponin - most specific for MI and lasts longest n the blood

Which enzyme is most often elevated in pernicious anemia?

LD

Lactate dehydrogenase (LD) activity is present in all cells of the body with highest concentrations in heart, liver, muscle, kidney, lung, and erythrocytes. Serum LD is elevated in a number of clinical conditions.

Interpretation 

• Marked elevations in lactate dehydrogenase (LD) activity can be observed in megaloblastic anemia, untreated pernicious anemia, Hodgkin's disease, abdominal and lung cancers, severe shock, and hypoxia.
•  Moderate to slight increases in LD levels are seen in myocardial infarction (MI), pulmonary infarction, pulmonary embolism, leukemia, hemolytic anemia, infectious mononucleosis, progressive muscular dystrophy (especially in the early and middle stages of the disease), liver disease, and renal disease.  
• In liver disease, elevations of LD are not as great as the increases in aspartate amino transferase (AST) and alanine aminotransferase (ALT).  
• Increased levels of the enzyme are found in about one third of patients with renal disease, especially those with tubular necrosis or pyelonephritis. However, these elevations do not correlate well with proteinuria or other parameters of renal disease.

T or F

A normal level of CK (creatine kinase) may be found in hepatitis patients

lactate dehydrogenase (LD)

LD-I appears elevated in the serum about 24 to 48 hours after a myocardial infarction (MI), but is generally not as useful as troponin or creatine kinase-MB (CK-MB) for detection of MI, unless the MI occurred at least 24 hours prior to testing. Normally, LD-II is greater than LD-I; however, when a MI has occurred, there is a "flip" in the usual ratio of LD-I/LD-II from <1 to >1 (or at least >0.9). Use of the ratio for evaluation of patients with possible cardiovascular injury has largely been replaced by TPNT / Troponin T, Serum.

In which of the following is alkaline phosphatase elevated (ALP)?

a.  muscular dystrophy

b.  acute pancreatitis

c.  insect poisoning

d.  osteogenic bone cancer

d.

CK is elevated in muscular dystrophy

ALP is elevated in Paget's disease as well.  Additionally, children will have elevted ALP due to growth spurts

ALP

Biliary tract obstruction - elevations in ALP AND GGT.  GGT is also used to indicate alcoholism

Two tests most useful in routine clinical evaluation of dyslipidemias

a.  fasting and nonfasting lipids

b.  cholesterol esters

c.  total and HDL cholesterol

d.  cold flotation of chylomicrons

c.  total cholesterol and HDL cholesterol

heparin sulfate-manganese precipitation

(method for direct HDL)

( total cholesterol - HDL) - (triglycerides/5)

Triglyceride levels over 400 would invalidate results

phosphatidyl glycerol (PG)

The lungs are the last of the organ systems to mature sufficiently to support extrauterine life. Fetal lung immaturity, or the respiratory distress syndrome (RDS), occurs most often when insufficient lung surfactant is present.

Lecithin-Sphingomyelin Ratio

• The first widely accepted direct test for assessment of fetal pulmonary status was the L/S ratio. This test evaluates a change in the relative amounts of lecithin (phosphatidyl choline) and sphingomyelin (a phospholipid of unknown origin) in amniotic fluid samples as gestational age increases. 
• Determination of the L/S ratio involves thin-layer chromatography after organic extraction. 

Phosphatidylglycerol (PG)

• Phosphatidylglycerol is a minor constituent of surfactant that generally appears several weeks after the increase in lecithin concentration. Because PG enhances the spread of phospholipids on the alveolar surface, its presence indicates an advanced state of fetal pulmonary maturity.

• Phosphatidylglycerol determination can be accomplished by either thin-layer chromatography or by slide agglutination.

Which of the following would best distinguish primary from secondary hypothyroidism

a.  T3

b.  free T4

c.  TBG

d.  TSH

d

Hypothyroidism is divided in primary, caused by failure of thyroid function and secondary (central) due to the failure of adequate thyroid-stimulating hormone (TSH) secretion from the pituitary gland or thyrotrophin-releasing hormone (TRH) from the hypothalamus.

Secondary hypothyroidism can be differentiated in pituitary and hypothalamic by the use of TRH test.

The purpose of using a monoclonal beta hCG test is:

a.  detect both FSH and hCG

b.  eliminate false negative reactions

c. detect hCG specificity

d.  neutralize luteinizing hormone

C

Pregnancy tests are widely used both by the public and by healthcare professionals. All tests depend on the measurement of human chorionic gonadotrophin (HCG) in urine. Other pregnancy-specific materials have been proposed as pregnancy tests but none can better the sensitivity and convenience offered by immunoassay of HCG. Ultrasound detection is also not as sensitive as HCG measurement. The current generation of tests is based on monoclonal antibodies to the beta-subunit of HCG; these virtually eliminate the possibility of cross-reaction with pituitary luteinizing hormone (LH) and it is this feature which permits the high sensitivity. 

Which of the following tumor markers are frequently found in patients with primary liver cancer (hepatoma)?

a. alpha fetoprotein

b. beta hCG

c. ferritin

d. anti-hyaluronidase

The oncoprotein produced by a carcinoma of the colon that can be used to monitor response to treatment

a. alpha1 antitrypsin

b. alpha fetoprotein

c. carcinoembryonic antigen (CEA)

d. beta human chorionic gonadotropin

Quantification of the nitrogen content of protein

Although the Kjeldahl technique for the determination of protein nitrogen is too cumbersome for use in routine testing, it is considered to be the reference method of choice to validate

materials used with the biuret method. The Kjeldahl technique is based on the quantification of the nitrogen content of protein. It is estimated that the average nitrogen content of protein is 16% of the total weight. In the Kjeldahl technique, protein undergoes a digestion process with sulfuric acid through which the nitrogen

content of the protein is converted to ammonium ion. The ammonium ion in turn may be reacted with Nessler's reagent, forming a colored product that is read spectrophotometrically, or the ammonium ion may undergo distillation, liberating ammonia that is titrated

number of peptide bonds

The biuret reaction is based on the complexing of cupric ions in an alkaline solution with the peptide linkages of protein molecules. Because the amino acids of all proteins are joined together by peptide bonds, this method provides an accurate quantification of

the total protein content of serum. The greater the amount of protein in a specimen, the greater will be the number of available peptide bonds for reaction and the more intense the colored reaction will be. In the biuret reaction, the intensity of the reddish

violet color produced is proportional to the number of peptide bonds present.

Coomassie brilliant blue

The concentration of total protein in cerebrospinal fluid (CSF) is 15^5 mg/dL. Such a

low level of protein requires a method with sufficient sensitivity such as Coomassie brilliant

blue. Turbidimetric methods can also be used to

quantify protein in CSF. Neither biuret nor Ponceau S has the sensitivity needed, and bromcresol green measures only albumin and does not react with the globulins.

Bacterial meningitis

multiple sclerosis

cerebral infarction

CSF is an ultrafiltrate of blood plasma. It is made in the choroid plexus of the ventricles of the brain.

Protein quantification is among the tests generally ordered on CSF; other tests include glucose, culture and sensitivity, and differential

cell count.

CSF protein may be quantified using turbidimetric (e.g., sulfosalicylic

acid and benzethonium chloride) or dyebinding

methods (e.g., Coomassie brilliant blue).

Elevated levels of CSF protein are found

in such disorders as bacterial, viral, and fungal

meningitis; multiple sclerosis; neoplasm; disk

herniation; and cerebral infarction.

Low levels of CSF protein are found in hyperthyroidism and in CSF leakage from the central nervous system.

Haptoglobin

Increased serum concentrations of haptoglobin are seen in inflammatory conditions and tissue necrosis, whereas decreased levels are seen in hemolytic situations in which there is extensive red blood cell destruction. In the latter situation, haptoglobin binds with free hemoglobin to form a stable complex that may then be removed by the reticuloendothelial

system. Because of the size of the haptoglobin-hemoglobin complex, urinary excretion

of hemoglobin by the kidney is avoided,

thereby preventing the loss of iron by the kidney.

Bromcresol green and bromcresol purple

Bromcresol green (BCG) and bromcresol

purple (BCP) are anionic dyes that bind selectively with albumin without preliminary extraction of the globulins. The nature of the dyes is such that the color of the free dye is different from the color of the albumin-dye complex so that the color change is directly proportional to the concentration of albumin in the specimen. Although amido black, Ponceau S, and Coomassie brilliant blue are able to bind albumin, they also react with the globulins, thus prohibiting their use in a direct procedure for

quantification of serum albumin.

Biuret

Biuret reagent is a combination of copper

sulfate, potassium iodide in sodium hydroxide,

and potassium sodium tartrate. The copper sulfate is the key to the reaction because it is the cupric ion that complexes with the peptide

bonds of protein. To keep the copper in solution

until its use, potassium sodium tartrate is

employed as a complexing agent, whereas the

autoreduction of copper is prevented by potassium

iodide.

a1-antritrypsin

a1 Antitrypsin is an acute-phase reactant protein whose concentration increases in response to inflammation, a1-Antitrypsin inhibits the self-destruction of one's own tissue by forming inactive complexes with proteolytic enzymes. In this way the enzymes are inhibited, and tissue destruction through self-digestion is avoided. 

Antitrypsin has been found to have the highest

concentration in serum of any of the plasma proteolytic inhibitors. It is an effective inhibitor of the enzymes chymotrypsin, plasmin, thrombin, collagenase, and elastase. The primary effect of aj-antitrypsin may be seen in the respiratory tract and the closed spaces of the body where physiological pH values are maintained, a1-Antitrypsin is least effective in the stomach and intestines.

Fetal fibronectin

Fibronectin is an adhesive glycoprotein that

functions with collagen to support cell adhesion.

It is a normal constituent in the placenta and

amniotic fluid. As labor begins, a change occurs

in cell adhesion that affects the placenta and

uterine wall. The level of fetal fibronectin

increases in the secretions of the cervix and

vagina. When this occurs prematurely, the

increase in fetal fibronectin is used to predict

risk of premature birth.

hyperalbuminemia

In multiple myeloma there is an abnormal

proliferation of plasma cells. These plasma cells

produce a homogeneous immunoglobulin protein that stains as a well-defined peak in the gamma region. Because of the presence of this monoclonal protein, the serum total protein will be elevated.

Bone destruction is commonly seen in this

disorder, with the plasma cells forming densely

packed groups in the lytic areas. Hypercalcemia

is primarily the result of bone destruction.

neural tube defect

spina bifada

fetal distress

(DECREASED a1-fetoprotein for down syndrome)

An

increased AFP level in maternal serum is associated with such disorders as neural tube defects, spina bifida, and fetal distress.

A decreased AFP level in maternal serum is characteristic of Down syndrome.

A1-fetoprotein

AFP is normally produced only by the fetus,

with blood levels disappearing shortly after

birth. However, in the adult, such conditions as

hepatoma or teratoma stimulate the production

of this primitive protein by the tumor cells. The

quantification of AFP may be used both diagnostically and as a monitor of chemotherapy.

urea

Constituents in the plasma that contain the

element nitrogen are categorized as being

protein- or nonprotein-nitrogen compounds. The

principal substances included among the nonprotein-nitrogen compounds are urea, amino acids, uric acid, creatinine, creatine, and ammonia.

Of these compounds, urea is present in the

plasma in the greatest concentration, comprising approximately 45% of the nonprotein-nitrogen fraction.

Sodium fluoride

In addition to the fact that sodium fluoride is

a weak anticoagulant, it also functions as an

antiglycolytic agent and is used as a preservative for glucose in blood specimens. With the urease reagent systems for the quantification of urea, the use of sodium fluoride must be avoided because of its inhibitory effect on this system.

Additionally, contamination from the use of

ammonium oxalate and ammonium heparin

must be avoided, because urease catalyzes the

production of ammonium carbonate from urea.

In several methods, the ammonium ion formed

reacts proportionally to the amount of urea originally present in the sample. Anticoagulants containing ammonium would contribute falsely to the urea result.

Of the two methods for determining urea: Chemical method (diacetyl method) and kinetic method (urease/glutamate dehydrogenase assay).

In the diacetyl method, what does diacetyle react with to form a yellow product?

Urea

In the diacetyl method, acidic diacetyl reacts

directly with urea to form a yellow-diazine

derivative. Thiosemicarbazide and ferric ions are

reagents used to intensify the color of the reaction.

Because urea is quantified directly, the

method does not suffer from interferences from ammonia contamination, as do some of the urea methods

ammonia

Adequate specificity is generally obtained

when using the urease/glutamate dehydrogenase method. Because urease hydrolyzes urea to ammonia and water, a positive interference from endogenous ammonia will occur with elevated blood levels of ammonia. Such interference may occur from use of aged blood specimens and in certain metabolic diseases.

urease and glutamate dehydrogenase

This is a kinetic method

Synthesis of urea from ammonia

The formation of urea and its excretion in the urine provide the principal means by which

the body is able to free itself of excess ammonia.