6. Clotted specimens should not be disturbed for

20–30 minutes.

7. Prolonged contact of serum or plasma with blood cells

should be avoided to minimize glycolysis and/or shift

of constituents from cells to serum or plasma.

8. If the patient is already on an I/V drip, withdraw blood

from the other arm.

9. Refrigeration of freshly collected blood specimen

before clotting has occurred or freezing of whole blood

before separation should be avoided.

10. Lactescence: Milky or lipemic plasma and serum are

frequently obtained with blood samples collected

1 to 2 hours after a fatty meal or patients with

hyperlipoproteinemia. It is associated with blood

neutral fat levels. This interferes with certain photometric measurements, e.g. uric acid and enzymes. It

also produces false elevations when serum is used in

final test mixture but not in the reagent blanks.

11. Concentration changes: Changes in drawn blood

samples from the original constituent concentrations

occur through dilution or evaporation.

12. Composition changes: The major sources of

composition alteration in blood specimens are

bacterial and enzymatic effect, loss of volatile

blood constituents by diffusion or evaporation and

interchange of substance between the liquid and

cellular components of blood. Protection from light

is essential for certain constituents (e.g. bilirubin).

13. Bacterial changes: These include ammonia formation

from urea and can be minimized by

a. Sterile handling of the blood samples wherever

possible

b. Prompt separation of cells from plasma or serum

c. Storage of the specimen at 4–6oC until analyzed

or freezing at –20oC (minus 20oC) when possible.

14. Enzymatic changes: Glycolysis is minimized by the same

measures as bacterial changes, except that sterility has

no effect unless an enzymatic method is employed.

Clinical Chemistry and Drug Interference

(See Appendix II)

Drug interference can occur in two ways:

Pharmacologic Interference

Whereupon some action of the drug or its metabolite can

cause an alteration (in vivo) in the concentration of the

substance being measured by the test, or

Chemical Interference

In which some physical or chemical property of the drug

can alter the analysis directly.

Control Sera

Before one proceeds for knowing the unknown, one must

have some known standards with which to compare and

obtain the results of the unknown. Most kits do provide

a standard solution of the substance to be measured but

the following are outstanding and are extremely useful for

preparation of calibration curves.

Normal Values Differ with Different Kits and

Manufactures. Always Consult the Product Insert

for Exact Method for a Particular Kit, Follow the

Manufacturer’s Instructions Strictly

(Kits of most tests mentioned in this and the next chapter

can be obtained from Coral Clinical Systems, Goa).

Control Sera from Boehringer

Product Standardises

1. Precibil Bilirubin

2. Preciflo (for Technicon systems) 23, chemical parameters

3. Precilip (normal range) 26, chemical parameters

4. Precilip EL (elevated range) 6 parameters (mainly lipids)

5. Precinorm E (normal range) 16 enzyme parameters

6. Precipath E (elevated range) 16 enzymic parameters

7. Precinorm S (normal range) 5 chemical parameters

8. Precipath S (elevated range) 15 chemical parameters

9. Precinorm U (normal range) 38 chemical and enzymic parameters. Suitable for both automated and manual procedures

10. Precipath U (abnormal range) -do11. Precinorm protein (normal range) For IgG, IgM and transferrin values

12. Special control serum for HDL cholesterol

(For all the above mentioned controls the exact assay values are provided by the manufacturers)

468 Concise Book of Medical Laboratory Technology: Methods and Interpretations Blood Urea Nitrogen (BUN)

Normal Values

SI units

Young adult < 40 5–18 mg/dL 1.8–6.5 mmol/L

Adult 5–20 mg/dL 1.8–7.1 mmol/L

Elderly > 60 8–21 mg/dL 2.9–7.5 mmol/L

Mild azotemia 20–50 mg/dL 7.1–17.7 mmol/L

Children

 Cord blood

Premature

21–40 mg/dL 7.5–14.3 mmol/L

  Infant, first 7 days 3–25 mg/dL 1.1–7.9 mmol/L

  Full-term Newborn 4–18 mg/dL 1.4–6.4 mmol/L

Infant 5–18 mg/dL 1.8–6.4 mmol/L

Child 5–18 mg/dL 1.8–6.4 mmol/L

Panic level > 100 mg/dL > 35.7 mmol/L

Urea (DAM Method)

(Courtesy: Tulip Group of Companies)

For the determination of urea in serum, plasma and urine

(for in vitro diagnostic use only).

Summary

Urea is the end product of the protein metabolism. It is

synthesized in the liver from the ammonia produced by

the catabolism of amino acids. It is transported by the

blood to the kidneys from where it is excreted. Increased

levels are found in renal diseases, urinary obstructions,

shock, congestive heart failure and burns. Decreased

levels are found in liver failure and pregnancy.

Principle

Urea in an acidic medium condenses with diacetyl

monoxime at 100°C to form a red colored complex. Intensity

of the color formed is directly proportional to the amount of

urea present in the sample.

 100°C

Urea + Diacetyl monoxime Red colored complex

Normal Reference Values

Serum, Plasma : 14–40 mg/dL

Urine : upto 20 g/L

It is recommended that each laboratory establish its

own normal range representing its patient population.

Contents 25 Tests 50 Tests

L1: Urea reagent 75 mL 150 mL

L2: Acid reagent 75 mL 150 mL

L3 : DAM reagent 75 mL 150 mL

S: Urea standard (40 mg/dL) 5 mL 5 mL

Storage/Stability

All reagents are stable at RT till the expiry mentioned on

the labels.

Reagent Preparation

Reagents are ready to use. Do not pipette with mouth.

Sample Material

Serum, plasma or urine. Urine should be of 24 hours

collection. Dilute the urine specimen 1:20 with distilled/

deionised water before the assay. Urea is reported to be

stable in serum for 5 days at 2–8°C.

Procedure

Wavelength/filter : 520 nm (Hg 546 nm)/Green

Temperature : 100°C

Light path : 1 cm

Pipette into clean dry test tubes labeled as blank (B),

standard (S), and test (T):

Addition

Sequence

B

(mL)

S

(mL)

T

(mL)

Urea reagent (L1) 1.0 1.0 1.0

Acid reagent (L2) 1.0 1.0 1.0

DAM reagent (L3) 1.0 1.0 1.0

Distilled water 0.01 — —

Urea standard (S) — 0.01 —

Sample — — 0.01

Mix well and keep the test tubes in boiling water (100°C)

for 10 minutes. Cool under running tap water and measure

the absorbance of the standard (Abs S), and test sample

(Abs T) against the blank.

Calculations

 Abs T Urea in mg/dL =........ _______ × 40 Abs S

 Abs T Urine Urea in g/L = _______ × 8

 Abs S

Urea nitrogen in mg/dL = Urea in mg/dL × 0.467.

Clinical Chemistry 469

Linearity

The procedure is linear upto 70 mg/dL. If values exceed this

limit, dilute the sample with distilled water and repeat the

assay, Calculate the value using the proper dilution factor.

Note

The presence of ammonia does not interfere in this test.

System Parameters

Reaction End point Interval

Wavelength : 520 nm Sample volume : 0.01 mL

Zero Setting :  Reagent

blank

Reagent volume : 3.00 mL

Incub temp. : 100°C Standard : 40 mg/dL

Incub time : 10 minutes Factor :

Delay time : React slope : Increasing

Read time : — Linearity : 70 mg/dL

No. of read : — Units : mg/dL

Urea (Mod Berthelot Method)

(Courtesy: Tulip Group of Companies)