LFTs Flashcards
Calculate globulin gap
Total protein - albumin
Albumin method in your lab?
Bromocresol green (dye binding method) at pH 4.2, measured at 596/694 nm
Critically discuss dye binding methods for albumin
Two methods used in most labs: BCG and BCP, based on greater affinity of albumin for these dyes compared to other proteins. BCP slightly more specific, and results are slightly lower than BCG results (BCG reacts with globulins as well), particularly in patients with kidney failure. This has implications for reference intervals for albumin AND for parameters derived from albumin eg corrected calcium, globulin gap, calculated free testosterone. Dye-binding assays also tend to be less accurate when serum/plasma protein composition is abnormal. The many ligands of albumin do not typically affect dye-binding assays of serum/plasma.
Causes of decreased albumin
- Decreased synthesis
- malnutrition
- malabsorption
- chronic liver failure - Increased catabolism
- sepsis
- Malignancy
- other catabolic states - Increased plasma volume
- excess water intake - Redistribution
- ascites
- oedema
- sepsis - Increased loss
- protein losing enteropathy
- nephrotic syndrome
- burns causing plasma loss - Physiologic
- pregnancy
What is P-5’-P? What role does it play in the measurement of aminotransferases?
Vitamin B6, P-5’-P or pyridoxal-5’-phosphate acts as an amino group acceptor, and in its amino form - pyridoxamine-5’-phosphate - acts as an amino group donor.
P-5’-P is a coenzyme for aspartate and alanine aminotransferase. It acts as the amino group acceptor from alpha-ketoglutarate/pyruvate and amino group donor to form glutamate in the reactions catalysed by AST and ALT respectively.
Why is ALT preferred over AST as a marker of parenchymal liver injury?
ALT is more specific than AST and increased activity of ALT persists longer than AST. Thus the incremental benefit of routine determination of AST in addition to ALT may be limited
Bilirubin is conjugated by what enzyme? What is the gene encoding this enzyme?
Uridine diphosphate glucuronyltransferase 1A1
UGT1A1
What is delta-bilirubin and how is it formed?
Bilirubin monoglucuronide can be transported from the hepatocyte into the blood via ABCC3 transporters. In the blood, bilirubin monoglucuronide binds to albumin and forms delta-bilirubin
How is bilirubin transported in blood, prior to conjugation?
Bound to albumin
4 fractions of bilirubin
- Unconjugated
- Monoconjucated
- Diconjugated
- Delta-bilirubin
What is a bilirubinometer?
Point of care screening device for measuring bilirubin in neonates to determine if blood draw required. Transcutaneous spectrophotometric detection at 437nm (max absorbance of bilirubin)
Pre-analytical factors in bilirubin collection
Sensitive to light (causes photolysis, decreasing concentration)
Haemolysis
Some drugs (method dependent)
How is albumin bound bilirubin taken up by hepatocytes?
By OATP transporters
methods for bilirubin determination
- Diazo (reference)
- Enzymatic
- HPLC
- Spectrophotometric (transcutaneous bilirubinometer)
- Dry chemistry
Reference method for bilirubin
Diazo method
Describe the Diazo method for bilirubin
Reaction of diazotised sulfanilic acid (diazo reagent) to produce 2 coloured azodipyrroles that are measured spectrophotometrically, either at 530nm or after addition of alkaline tartrate, at 530 nm. Accelerated by alcohol/variety of other accelerators eg sodium benzoate. Accelerator causes dissociation of unconjugated bilirubin from albumin, so that both unconjugated and conjugated bilirubin can be measured. To prevent unconjugated bilirubin from reacting in direct methods, pH maintained ~1.0.
Bilirubin method in your lab
Enzymatic. Bilirubin is converted to biliverdin by bilirubin oxidase. The decrease in absorbance at 435/460nm is measured by spectrophotometry. Detection of total bilirubin occurs at pH 8. To detect direct bilirubin, p is 3.7-4.5. At this pH range, the enzyme oxidizes bilirubin conjugates and delta-bili but not unconjugated bili. At pH 10, bilirubin oxidase selectively oidizes the 2 glucuronides.
Liver disease causes an increase in which fraction of bilirubin?
Conjugated
Significance of urine bilirubin
Only appears in disease and reflects an increase in plasma conjugated bilirubin
In which organs is ALP present?
Liver, kidney, bone, intestine, placenta
Describe the multiple forms of ALP. A diagram may be helpful
True isoenzymes
Chromosome 1 - tissue nonspecific - bone, liver, kidney. Encoded by same gene but differ in CHO content (sialic acid chains)
Chromosome 2 - germ cell, placental, intestinal and fetal intestinal.
Activators of the ALP enzyme?
Mg2+, Co2+, Mn2+
T or F: Zn2+ is an activator of ALP
False. Zn2+ is a constituent metal ion
Inhibitors of ALP activity?
Phosphate, borate, oxalate, cyanide
T or F: ALP increase is greater in extrahepatic than intrahepatic obstruction
True. ALP increase can exceed greater than 4xURL in extrahepatic obstruction (eg stone, head of pancreas ca). Increase is proportional to the degree of obstruction.
Apart from obstruction, what other pathologies can cause extreme increases in ALP? (up to 10-12x URL?)
Cancer - advanced HCC or widespread liver mets
Name one prognostic use of ALP
In patients with primary biliary cirrhosis, ALP >2xURL predicts liver Tx/death
Describe the expected change in ALP with pregnancy
A rise of up to 2-3xURL by third trimester due to placental ALP
Describe the malignancy forms of ALP
Regan isoenzyme - essentially identical to placental ALP, heat stable to 65C
Kasahara isoenzyme - modified form of nonplacental isoenzymes
Nagao isoenzyme - germ cell ALP
Does ALP activity increase or decrease with refrigeration?
Increase
You want to measure ALP on a frozen sample, what preparation is needed?
Allow to thaw for 18-34 hours to allow full enzyme reactivation
50% of isolated increases in liver enzymes are caused by which 5 aetiologies?
- Drugs
- Alcohol
- NAFLD
- HBV
- HCV
What wavelength is albumin measured at?
BCG 628nm
BCP 600nm
Most common cause of isolated increase in unconjugated bilirubin?
Gilbert’s syndrome
Causes of hepatitic picture
- Viral
- Alcoholic
- NAFLD
- Toxic
- Ischaemic
- Drug induced
- Autoimmune
- Rare - Wilson’s, AAT deficiency, haemochromatosis
Causes of chronic hepatitis
HBV
HCV
AIH type 1
AIH type 2
Wilson’s
Drug
AAT deficiency
NAFLD
Idiopathic
Tests for AIH type 1
ANA, anti-SMA
Tests for AIH type 2
SLA, anti-LKM1
