Chemistry lab exam 2

Serum protein electrophoresis (SPE)

What it does

Separates charged serum proteins in an electric field at approx pH 8.6

SEPERATION TECHNIQUE NOT DEFINITIVE DIAGNOSES!!!!

Serum protein electrophoresis (SPE)

Migration

(Fast to slow)

Albumin → a1 → a2 → B → y

Nonuniform migration: investigate voltage, gel condition, and overheating

Proteins migrate differently due to net charge, size (molecular weight), and shape

Normal pattern protein electrophoresis

Monoclonal gammopathy

Sharp spike in gamma region

Narrow M - spike

Often in Y or B region

Suggests: Monoclonal Gammopathy of Undetermined Significance (MGUS), Multiple myeloma, and Waldenstrom macroglobulinemia

(NOT PROOF OF IT THOUGH!!)

Next step: immunofixation to characterize the immunoglobulin heavy/light chain

Nephrotic syndrome

↓ albumin and y bands | ↑ a2 band

(Low albumin with prominent a2 region)

Suggests: “selective proteinuria” smaller proteins (albumin and y) excreted and only larger proteins (a2) retained

Active cirrhosis

B - Y bridging (decrease in albumin from liver disease)

Suggests: most common cause is cirrhosis; also seen in RA, SLE, chronic infections

Polyclonal gammopathy

y elevation prominent (Domed broad increase)

broad y region increase

Increased antibodies in blood (can mean multiple things)

Acute phase reactions

↑ in both a1 and a2 bands

↑ in B globins C3, C4, and C reactive protein,

a1: a1 antitrypsin and a1 glycoprotein

a2: ceruloplasmin and haptoglobin

Suggests: infection, malignancy, RA, hepatitis, surgery, trauma, burns, and heart attack

Total protein

6.0 - 8.3

Albumin

3.5 - 5.5

Hypoalbuminemia

breakdown from tissue damage and inflammation

Hyperalbuminemia

reflects dehydration

Calcium

8.6 - 10.3

Hypocalcemia

low albumin, chronic liver disease, and nephrotic syndrome

Hypercalcemia

less excretion in kidneys & increase absorption in gut

(stones, bones, abdominal groans, and mental moans)

Creatinine

Serum:

♂: 0.72 - 1.18 mg/dL

♀: 0.55 - 1.02 mg/dL

Spot urine:

♂: 20 - 320 mg/dL

♀: 20 - 275 mg/dL

↑ creatinine = ↓ GFR

↓ creatinine = malnutrition, low muscle mass, liver disease

Enzyme kinetics

LAG phase: Mixing, stabilization, and reagent equilibration

LINEAR phase: constant reaction rate; measure activity here

Depletion: rate falls as substrate becomes limiting

Enzyme kinetics

Substrate in excess + controlled pH, temperature and cofactors → comparable rates

How analyzer reports enzyme activity

Kinetic measurement

Collect absorbance values in the linear window

Activity (U/L) = ΔAbs/min × stored factor

The factor depends on reaction volumes, path length and molar absorptivity

How analyzer reports enzyme activity

Troubleshooting

Excessive absorbance change before the

measurement window can indicate an

overly rapid reaction or substrate depletion

A factor is method/analyzer specific

Coupled reactions: ALT

Pyruvate coupled with LDH to convert NADH to NAD+

measured signal: NADH ↓ at 340 nm

****More liver specific****

Coupled reactions: AST

Oxaloacetate is coupled with MDH to convert NADH to NAD+

measured signal: NADH ↓ at 340 nm

ALP method

substrate: p-nitrophenyl phosphate (pNPP) +H2O

Product: p-nitrophenol + phosphate

ALP releases a yellow p-nitrophenolate product measured kinetically near 405–408 nm

Zinc and magnesium support enzyme activity

AMP buffer accepts phosphate so it doesn’t inhibit ALP

Clinical interpretation (ALP and GGT)

ALP may originate from liver, bone, and other tissues

ALP ↑ with GGT ↑ supports a hepatobiliary source

(Extrahepatic obstruction)

ALP ↑ normal GGT may indicate bone disease

Bilirubin method: Total bilirubin

Diazo method; solubilizer enables indirect fraction to react

0.2 - 1.0 mg/dL

(React with diazotized sulfanilic acid)

Indirect is solubilized by:

Jendrassik Grof = sodium benzoate and caffeine

Evelyn Malloy = methanol

DMSO (Walters Gerarde) = dimethyl sulfoxide

Bilirubin method: Direct bilirubin

Reacts with diazo reagent without solubilizer

(Reacts directly with sulfanilic acid; no solubilization needed)

0.0 - 0.2 mg/dL

Bilirubin method: Indirect bilirubin

total - direct = calculated

NOT DIRECTLY MEASURED

0.2 - 0.8 mg/dL

total and direct bilirubin method

Both total and direct bilirubin methods form azobilirubin complex that’s measured around 554 - 560 nm

Bilirubin sample

Protect bilirubin specimens from light, exposure can falsely lower measured bilirubin

Total protein: colorimetric assay

Biuret reaction

Cupric ions react with peptide bonds under alkaline pH to form a violet complex measure at 544nm

Albumin (Colorimetric assay)

Bromcresol green (BOG)

BCG binds albumin to form a blue green complex measured at 629 nm. Signal proportional to albumin concentration

Jaundice patterns: Prehepatic

0.2 - 0.8 mg/dL

Indirect bilirubin ↑

Due to excess RBC destruction

Ex.) hemolytic disease, hereditary spherocytosis, sickle cell anemia

Jaundice patterns: Hepatic

Direct and/or indirect ↑

Due to impaired uptake, conjugation, or secretion

Ex.) hepatitis, cirrhosis

Impaired: Gilbert (lack enzyme to conjugate bilirubin effectively)

Conjugation: Crigler - Najjar (liver cant conjugate bilirubin)

Secretion: Dubin johnson (liver can’t pump conjuagted out of cells)

Jaundice patterns: Post hepatic

Direct bilirubin ↑

Due to blockage preventing elimination of conjugated bilirubin

Ex.) gallstones, tumors, cholestasis

Hepatocellular

35 & 0- 35 U/L

AST & ALT ↑ ↑ ↑

ALT may exceed AST

Viral, toxic, ischemic injury

(bilirubin ↑ with injury)

Cholestatic

33 - 98 & 6 - 45

ALP & GGT ↑ ↑ ↑

Dark tea like urine and pale stool

(bilirubin ↑)

Alcohol associated

35 & 0- 35 U/L

AST ↑ ↑ ↑ (significant) > ALT

GGT ↑

AST is commonly 2x of ALT

Advanced liver disease

AST & ALT (variable)

ALP & GGT (variable)

3.5 - 5.5

Albumin ↓↓ and/or PT prolonged

Bilirubin consistency check

Total bilirubin 0.3 mg/dL

Direct bilirubin 0.4 mg/dL

Physiologically inconsistent fractions. Check units, transcription, QC, interferences and repeat as appropriate.

Above analytical range

If AST/ALT exceed the validated analytical measuring range: perform an approved dilution, rerun and multiply by the appropriate dilution factor

Dilution calculations

Dilution calculations

RF latex agglutiantion: On latex particle

Human gamma globulin (IgG), including the Fc region recognized by rheumatoid factor.

Passive/indirect latex agglutination assay: antigen - coated particles detects patient antibody

RF latex agglutiantion: in patient serum

Rheumatoid factor: usually IgM autoantibody against the Fc of IgG

Positive RF ≠ diagnosis of RA

Negative RF ≠ exclusion of RA

Rheumatoid factor (RF): what are we detecting?

Rheumatoid factor is an autoantibody (commonly IgM) that binds with the Fc portion of IgG

Kit uses latex particles coated with human gamma globulin (IgG)

RF in patient serum cross links the coated latex particles → visible agglutination

RF IS NOT DISEASE SPECIFIC

RF titer: where does reactivity stop?

Last reactive dilution = 1:16

Infectious mononucleosis assay

What it detects

Heterophile antibodies associated with IM, NOT EBV antigen or EBV-specific antibody

Guinea pig kidney antigen absorbs many non-IM heterophile antibodies

Infectious mononucleosis assay

Indicator and limits

Treated horse RBCs agglutinate when relevant antibodies remain

Early infection and younger children may test negative despite EBV infection

Agglutination: visual result and controls

Reactive appearance

RF latex: Visible particle clumps

Color slide II IM: Dark clumps on blue green field

Agglutination: visual result and controls

Non reactive appearance

RF latex: Smooth suspension

Color slide II IM: Uniform tan/brown ; fine granularity may occur

Agglutination: visual result and controls

Decision rule

If positive or negative control fails, do not report patient results.

Timing, mixing, reagent volumes and drying can change appearances

Lipid methods: from analyte to signal

Total cholesterol

Cholesterol esterase hydrolyzes esters.

Cholesterol oxidase generates H₂O₂.

Peroxidase uses H₂O₂ in a colored dye reaction

Lipid methods: from analyte to signal

Triglycerides

Lipase releases glycerol.

Enzymatic reactions ultimately generate H₂O₂, which drives a measurable chromogenic reaction

Calculated LDL - C: work the numbers

Total cholesterol - HDL-C - (triglycerides / 5)

If triglycerides are over 400 then the equation is invalid; alternative equations may be used

An AST result flags “above linearity.” What is the best laboratory response?

Dilute according to validated procedure and repeat

An IM slide shows fine brown granularity but no distinct dark clumps. Both

controls passed. How should you interpret it?

Nonreactive in this assay