DAVAO DOCTORS COLLEGE

MEDICAL LABORATORY SCIENCE DEPARTMENT

STUDENT NOTES: CLINCHEM1
Prepared by: Merryl Louise C. Matus, RMT

PREFINALS EDTA
ACTIVITY 10-A: NPN DETERMINATION Phosphate buffer (pH <13)
Reagent 2
BLOOD UREA NITROGEN Hypochlorite
Enzyme Concentration Urease
Urea: 80 mg/dL or
 Urea is a non-protein nitrogenous substance that is present
13.3 mmol/L
in highest concentration in the blood Standard
Equivalent to BUN:
 It is the major excretory product of protein metabolism 37.28 mg/dL or 6.2 mmol/L
 Most of the urea is excreted in the kidneys and/or through the
GI and skin Principle: Modified Berthelot’s reaction
 Only 40% of urea is reabsorbed  ammonium ions react with hypochlorite and salicylate to form
 Historic assays for urea were based on measurement of a green dye
nitrogen, thus the term, Blood Urea Nitrogen (BUN)  measured spectrophotometrically at 570-600nm
 Only the nitrogen part of urea is quantitated since it reflects  the increase in absorbance is proportional to BUN
the actual concentration of the entire urea compound concentration

BUN measurement Pipetting Scheme and Procedure

Enzymatic methods are most commonly used in the
 0.01 mL Patient’s Serum + 1.0 mL Reagent 1
measurement of urea
 Mix and incubate for 5 minutes at 37°C
 Add 1.0 mL Reagent 2
 Mix and incubate for 10 minutes at 37°C
 Measure the absorbance of the standard and the sample
against the reagent blank within 60 minutes

Calculation of Urea and BUN Concentration

UREA
For serum/plasma mg/dL mmol/L
Sample x
80 13.3
Standard
For urine mg/dL mmol/L
Sample x
80.8 1343
Standard
BUN
For serum/plasma mg/dL mmol/L
Sample x
37.28 6.2
Standard
For urine mg/dL mmol/L
Sample x
37.65 626.2
Standard

Conversion Factor
BUN Concentration = 0.466 x Urea Concentration
Urea Concentration = 2.14 x BUN Concentration

Reagent Composition

Reagent 1 Phosphate buffer (pH 7.0)

Sodium salicylate
Sodium nitroprusside
 PREFINALS
ACTIVITY 10-B: NPN DETERMINATION Reagent composition
CREATININE Picric Acid
Reagent 1 Sodium Hydroxide
 Creatinine is a product of muscle metabolism Sodium nitroprusside
 it is formed from the interaction between creatine and ATP Creatinine:
which is converted to creatine phosphate and ADP with the Standard
2 mg/dL or 176.8 µmol/L

Principle: Jaffe reaction

 forms in alkaline solution an orange-red colored complex with
picric acid
Creatinine + Picric Acid à Creatinine-picrate complex
 measured spectrophotometrically at 520nm
 the increase in absorbance is proportional to creatinine
concentration

Pipetting Scheme and Procedure

 0.01 mL Patient’s Serum + 1.0 mL Reagent 1
 Mix. After 30 seconds, read A1.
 After 2 minutes, read A2.
 A2-A1 = ΔA
help of the enzyme, creatine kinase.
Creatine is synthesized primarily in the liver from arginine, Calculation of Creatinine Concentration
glycine, and methionine. It is then transported to other
tissues, such as muscle, wherein it will serve as a high- Serum or Plasma
energy source. Conc = 2.0 x ΔA sample (mg/dL) Conc = 176.8 x ΔA sample (µmol/L)
ΔA STD ΔA STD
 Creatine phosphate loses phosphoric acid and creatine loses
Urine
water to form the cyclic compound, creatinine, which diffuses Conc = 10 x ΔA sample (mg/dL)
into the plasma ΔA STD
 about 99% is excreted in the urine 24-hr Urine
 Plasma creatinine is inversely related to glomerular filtration Conc = mg/dL x mL Conc = mg/24h x 0.00884
rate (GFR) urine/24 hr x 0.01 (mmol/24h)
 it is commonly used to assess renal filtration function (mg/24hr)

Creatinine measurement Conversion Factor

mg/dL x 88.402 = µmol/L
µmol/L x 0.0113 = mg/dL
 PREFINALS 8 mg/dL or 476 µmol/L
ACTIVITY 10-C: NPN DETERMINATION
SERUM URIC ACID Principle: Uricase method
 uric acid is converted to allantoin and hydrogen peroxide in
 Uric acid is the product of catabolism of the purine nucleic the presence of uricase
acids  H2O2 reacts with 3,5-dichloro-2-hydroxybenzenesulfonic acid
 Although it is filtered by the glomerulus and secreted by the (DCHBS) and 4-aminophenazone (PAP) to give a red-violet
distal tubules into the urine, most uric acid is reabsorbed in quinoneimine dye
the proximal tubules and only <1% is excreted Uric Acid + O2 + 2 H2O2 -----uricaseàallantoin + CO2 +
H2O2
2 H2O2 + DCHBS + PAP -----peroxidaseàquinonemine + HCl
+ 4 H2O
 measured spectrophotometrically at 520nm
 the increase in absorbance is proportional to SUA
concentration

Pipetting Scheme and Procedure

 0.02 mL Patient’s Serum + 1.0 mL Reagent 1
 Mix and incubate for 10 minutes at 37°C
 Measure the absorbance of standard and the sample
against reagent blank within 15 minutes
 Uric acid is relatively insoluble in plasma and, at high
concentrations, can be deposited in the joints and tissue, Calculation of SUA Concentration
causing painful inflammation
Serum or plasma
 Although uric acid measurement may assess kidney function,
Conc = 8 x _Abs sample (mg/dL)
uric acid is not as reliable as urea and creatinine
Abs STD
Conc = 476 x _Abs sample (µmol/L)
Serum Uric Acid measurement Abs STD
Urine
Conc = 88 x ΔA sample (mg/dL)
ΔA STD
Conc = 5235 x ΔA sample (µmol/L)
ΔA STD

Reagent composition

Phospate buffer (pH 7.0)

4-aminophenazone
Reagent 1 DCHBS
Uricase
Peroxidase
Standard Uric Acid:
 PREFINALS  measured at 546nm
ACTIVITY 11: BILIRUBIN DETERMINATION  indirect bilirubin will only react with DSA in the presence of an
accelerator
 Bilirubin is the end product of hemoglobin catabolism  absorbance is directly proportional to the bilirubin
 Bilirubin determination is used to assess the liver’s excretory concentration in the sample
and/or secretory function Sulphanilic acid + sodium nitrite à DSA
 Most patients with liver problems present with a yellowish Bilirubin + DSA à DIRECT BILIRUBIN
discoloration of the skin and sclera of the eyes or jaundice Bilirubin + DSA + accelerator à TOTAL BILIRUBIN
 Jaundice is not noticeable to the naked eye until bilirubin Total bilirubin – Direct bilirubin = INDIRECT BILIRUBIN
levels reach 3.0-5.0 mg/dL
Two types of bilirubin: Pipetting Scheme and Procedure
 Conjugated bilirubin (B1) For TOTAL bilirubin
water insoluble, bound to albumin, indirectly measured, not  1.0 mL TBR + 1 drop TNR
filtered by the kidneys, not present in urine  mix and incubate for 5 minutes
 Unconjugated bilirubin (B2)  add 0.1 mL Patient’s serum
water soluble, not bound to albumin, directly measured,  mix and incubate at room temperature for 10-30 minutes
filtered by the kidneys, present in urine  measure the absorbance of the sample against the sample
blank ΔA546
For DIRECT bilirubin
 1.0 mL DBR + 1 drop DNR
 mix and incubate for 2 minutes
 add 0.1 mL Patient’s serum
 mix and incubate at room temperature at exactly 5 minutes
 measure the absorbance of the sample against the sample
blank ΔA546

Calculation of Bilirubin Concentration

Bilirubin concentration = ΔA546 x 13.0 (mg/dL)

Bilirubin concentration = ΔA546 x 17.1 (µmol/L)
ΔA = A of sample blank – A of sample

BILIRUBIN MEASUREMENT
Reagent composition
Sulfanilic acid
Hydrochloric acid
TOTAL BILIRUBIN REAGENT
Caffeine (accelerator)
Sodium benzoate
TOTAL NITRITE REAGENT Sodium nitrite
DIRECT BILIRUBIN REAGENT Sulfanilic acid
Hydrochloric acid
DIRECT NITRITE REAGENT Sodium nitrite

Principle: Jendrassik and Grof method

 direct bilirubin reacts with diazotized sulphanilic acid to form
a red azo dye
 PREFINALS CA 125 Ovarian cancer
DISCUSSION ON TUMOR MARKERS

 Tumor: uncontrolled growth of cells that occurs in solid

tissues such as an organ, muscle, or bone causing the
formation of a solid mass
 Cancer: disease wherein there is uncontrolled growth of cells
 Neoplasia: general term for accelerated growth of tissue
 Benign tumor: “non-cancerous”; stays as it is and doesn’t
change
 Malignant tumor: “cancerous”; spreads and invades other
tissues or organs

Oncofetal antigens: normally expressed during fetal life;

BENIGN MALIGNANT when it reappears in adulthood, it could mean cancer
EPITHELIAL
Adenomas Carcinomas In childhood:
TISSUE
CONNECTIVE Abundant protein synthesized by the
Sarcomas fetal liver
TISSUE
Increased:
neural tube defects such as
ancephaly and spina bifida
Decreased: Down’s syndrome/
Alpha Fetoprotein trisomy 21
(AFP) In adulthood:
 Most common cancer in women: breast cancer AFP production increases after the
 Most common cancer in men: prostate cancer stimulus of liver diseases
NonCA: Viral hepatitis and cirrhosis
of the liver
Tumor Markers CA: also made by primary liver
 substances found in body tissues or fluids that help show the tumors such as heptacellular
presence or type of cancer carcinoma
 produced either by the cancer cells itself or by the body in
response to the presence of cancer In childhood:
 Detected in a solid tumor or in circulating tumor cells present produced in gastrointestinal tissue
in body fluids during fetal development
3 Ideal Characteristics of a Tumor Marker: In adulthood:
CEA was discovered in malignant
 tumor specific Carcinoembryonic
tumors of the GI and pancreas
 absent in healthy individuals antigen (CEA)
Non CA: liver disease, inflammatory
 readily detectable in body fluids bowel disease, pancreatitis, heavy
Tumor markers should be: smoking
 Highly sensitive CA: GI, breast, pancreatic or lung
 Highly specific cancer cells
 Correlate with the cancer stage or tumour mass
 Correlate with the prognosis
 Have a reliable prediction value

Carbohydrate antigens
CA 15-3, CA 25-29 Breast cancer
CA 19-9 Pancreatic cancer
Protein Tumor Markers
Serum M-Protein
Plasma cell cancer diagnosis and
Serum Free light
therapeutic monitoring
chains
Hematologic malignancy and/or
B2-Microglobulin lymphoproliferative disorders
prognosis

Receptor Tumor Markers

Estrogen Receptor
Breast cancer hormonal therapy
Progesterone
indicator
Receptor
Breast cancer
Her-2/neu Determine the type of therapy
Epidermal growth
Head, neck, ovarian and cervical CA
factor receptor
prognosis
(EGRF)

Enzyme Tumor Markers

ALP ACP LPS
NSA AMS GGT
CK 5’N TRYPSIN
PSA LDH

Endocrine Tumor Markers

HCG CALCITONIN ACTH
CATECHOLAMINES SEROTONIN 5-HIAA
ADH GASTRIN GLUCAGON
INSULIN PRL ANDROGENS
TSH GH PTH
C-PEPTIDE VMA HVA