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Chem Unit6Ex 11 He Me Pigments

Chem Unit6Ex 11 He Me Pigments

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MLAB 2401 - Clinical Chemistry Lab Manual
w
1
UNIT: Heme Pigments – Iron/TIBC, dry lab
Ex#10heme.wpd
TaskSerum determination of iron and iron-binding capacity.Objectives After completion of this exercise, the student will be able to:1.Determining serum total iron concentration.2.Determine serum unsaturated iron binding capacity.3.Calculate TIBC.4.Calculate % saturation.5.State normal values for the above mentioned procedures.6.State the purpose(s) for determining serum iron, TIBC, and % saturation.7.Identify and state the purpose of significant reagent(s) used in the serum iron and UIBCprocedures.Supplies and Equipment1.Spectrophotometer capable of measuring transmitted light at 560 nm2.Iron-free test tubes, and Iron-free deionized wate3.Pipets (conventional or automatic)a.0.05 mlc.2.0 mle. 1.0 mlb.0.5 mld.5.0 ml4.37°C water bath5.Sigma Iron and Total Iron-binding capacity kit reagents (procedure No. 565)ReferencesKaplan, et al. Clinical chemistry (3rd ed.). Chapter 12, pp. 329-335.Sigma Diagnostics product insert, procedure No. 565, Iron and Total Iron-binding CapacityPurposeIron is an essential element to most living organisms. It is a vital component of a group of hemeproteins that function in oxygen transport. Iron also participates in a number of body chemicalreactions including enzyme reactions. Unlike other trace minerals, iron is regulated by its absorptionand not its excretion. It is believed that intestinal mucosal cells contain special proteins necessaryfor the absorption of iron, and to some degree, these proteins regulate its absorption.During the normal breakdown process of RBCs, the released iron is picked up in the plasma bytransferrin to be recycled and there is virtually no loss of the iron in the urine. Yet, iron deficiencystates are wide spread, occurring most commonly in women (with extensive iron losses duringpregnancy and during menstrual bleeding) and in nursing infants (who have virtually no iron intake).Factors considered to be primarily responsible for iron deficiency include: insufficient dietary intake,impaired absorption, and acute or chronic blood loss. Early diagnosis of iron deficiency is difficult andmay require the evaluation of a number of chemistry tests including: serum iron level, TIBC, serumferritin concentration, and zinc protoporphyrin/heme ration (ZPP/H).
 
UNIT: Heme Pigments (continued)
MLAB 2401 - Clinical Chemistry Lab Manual
w
2Principle
Serum Iron
– At acid pH, and in the presence of suitable reducing agent, transferrin-bound serumiron dissociates to form ferrous ions. These react with ferrozine to produce a magenta coloredcomplex with an absorption maximum near 560 nm. The difference in color intensity at thiswavelength, before and after addition of ferrozine, is proportional to serum iron concentration.
UIBC
– At alkaline pH, ferrous ions added to serum bind specifically with transferrin at unsaturatediron-binding sites. Remaining unbound ferrous ions are measured with the ferrozine reaction. Thedifference between the amount of unbound iron and the total amount added to serum is equivalentto the quantity bound to transferrin. This is the UIBC.
TIBC
– The serum TIBC equals the total iron plus the UIBC.SpecimenBlood should be collected using materials that are iron-free. As soon as blood clots, separate serum. Although occult hemoglobin does not interfere, only clear non-hemolyzed serum (not plasma) issuitable for assay. Each mg of hemoglobin contains 3.4
:
g iron. Serum iron reportedly is stable for at least four days stored at room temperature or one week in the refrigerator.Procedure ISerum Total Iron1.To test tubes labeled Blank, Standard, Test, and add 2.5 mL Iron Buffer Reagent.2.a.To Blank, add 0.5 mL iron-free water.b.To Standard, add 0.5 mL Iron Standard.c.To Test, add 0.5 mL serum.Mix each test tube thoroughly.3.a.After setting the spectrophotometer wavelength to 560 nm, zero the instrument using theBlank (for double beam spectrophotometers, read Standard and Tests against the Blank).b.Read and record absorbance (A) of Standard and Tests. This is the Initial A.4.To each test tube add 0.05 mL Iron Color Reagent. Mix thoroughly and place in water bath at37°C for 10 minutes.5.Read and record absorbance of Test and Standard vs. Blank as reference at 560 nm. This isthe Final A.6.CalculationsSerum Total Iron (
:
g/dL) =
 
UNIT: Heme Pigments (continued)
MLAB 2401 - Clinical Chemistry Lab Manual
w
3*Concentration (
:
g/dL) of Iron Standard
EXAMPLE:
A serum gave the following absorbance values:
Test
Final A = 0.130
Test
Initial A = 0.090
Standard
Final A = 0.375
Standard
Initial A = 0.000
Serum Total Iron (
:
g/dL) =
NOTE:
If serum total iron value is greater than 500
:
g/dL, dilute sample with equal volume of saline, repeat assay and multiply result by 2.Procedure IISerum Unsaturated Iron-Binding Capacity (UIBC)1.To test tubes labeled Blank, Standard, Test, add 2.0 mL UIBC Buffer Reagent.2.a.To Blank, add 1.0 mL iron-free water.b.To Standard, add 0.5 mL iron-free water and 0.5 mL Iron Standard.c.To Test, add 0.5 mL serum and 0.5 mL Iron Standard.Mix each test tube thoroughly.3.Read and record absorbance (A) of Test and Standard vs. Blank as reference at 560 nm. Thisis the Initial A.4.To each test tube, add 0.05 mL Iron Color Reagent. Mix thoroughly and place in 37°C water bath for 10 minutes.5.Read and record absorbance of Test and Standard versus Blank as reference at 560 nm. Thisis the Final A.
TestTest
NOTE:
Occasionally, the difference between Initial A and Final A may be very smallbecause of high degree of unsaturation of transferrin with iron. The sample should then bediluted (1 part serum and 1 part iron-free water) and test repeated. The result is then multipliedby 2.6.Calculations

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