Clinical Chemistry (CHE221) Professor Hicks Lecture 9

Transcription

1 Clinical Chemistry (CHE221) Professor Hicks Lecture 9 Pre-lab Lecture for Determination of Hemoglobin, Iron, and Total Iron- Binding Capacity

2 3 Procedures Today hemoglobin iron (in serum) unsaturated iron binding capacity (UIBC in serum) these two together can be used to calculate TIBC Total Iron Binding Capacity = UIBC + Iron

3 Procedure (Manual) 1. Dispense 3.0 ml of total hemoglobin reagent into test tubes labeled blank, control, and unknown 15 L (blank = water; control = blood with known hemoglobin; unknown = patient blood) 2. DO NOT MIX WITH ACIDS! Discard in designated waste bottles only

4 Hemoglobin Flowchart 3000 L total hemoglobin reagent 15 L blank (water) 15 L control (whole blood known hemoglobin) 15 L unknown (whole blood unknown hemoglobin) incubate 3 minutes room temperature 1) Mix by stretching a small piece of parafilm over top and gently inverting culture tube or cuvette 2) zero instrument with blank at 540 nm 3) measure absorbance at 540 nm Reagent oxidizes Fe 2+ in hemoglobin to Fe 3+ In this state it is called methemoglobin methemoglobin then reacts with CN- to form cyanmethemoglobin absorbance change at 540 nm oxidant and CN - together in one reagent Body has an enzyme (methemoglobin reductase) that reverses the oxidation to methemoglobin typically ~ 1% hemoglobin exists as methemoglobin

5 Calculation of Hemoglobin absorbance of unknown absorbance of standard concentration of standard = concentration of unknown A = bc bc(unknown) bc(standard) concentration of standard = concentration of unknown cancels because the compound absorbing the light is same in standard and sample b cancels because the pathlength is same if measured in same or similar cuvettes Your standard today has a concentration of g/dl

6 Test and Tap Water

7 Hemoglobin + acid Fe 2+ released into solution Ferrozine + Fe 2+ highly colored complex Ferrozine Fe 2+

8 Serum Iron flowchart add 2500 L Iron Buffer Reagent ph = L iron free water blank L iron standard standard L serum unknown iron test L tap water 1) Mix by stretching a small piece of parafilm over top and gently inverting culture tube or cuvette 2) Zero instrument with blank at 560 nm 3) Measure absorbance at 560 nm. Record as A 1 add 50 L Iron Color Reagent blank standard test tap water 1) Mix by stretching a small piece of parafilm over top and gently inverting culture tube or cuvette 2) incubate 37 C for 10 minutes 3) re-zero instrument with blank at 560 nm 4) measure absorbance at 560 nm. Record as A 2

9 Calculation of Iron Iron in test sample = A 2 (test) A 1 (test) A 2 (std) A 1 (std) Concentration Standard concentration of iron standard in Fe 2+ = 500 g/dl Iron in tap water = A 2 (tap water) A 1 (tap water) A 2 (std) A 1 (std) Conc Standard Why do we bother to measure the A 1 s? (absorbances before adding the ferrozine- iron color reagent) Serum is not colorless! The yellow color is not caused by iron and may be different for different serums. So each serum has this variable contribution to the absorbance removed by subtracting the A 1 value..

10

11 Unsaturated Iron Binding Capacity (UIBC) UIBC = Concentration of Standard - A2 (test) A1 (test) A2 (std) A1 (std) Concentration Standard concentration of iron standard in Fe 2+ = 500 g/dl UIBC = 500 g/dl - A 2 (test) A 1 (test) A 2 (std) A 1 (std) 500 g/dl UIBC = initial amount Fe 2+ free in solution - amount of Fe 2+ in solution that did not go into transferrin UIBC = amount of Fe 2+ from solution that did go into empty transferrin binding sites

12 UIBC flowchart add 2000 L UIBC Buffer L iron-free water blank L iron-free water L iron standard standard L sample L iron standard test 1) Mix by stretching a small piece of parafilm over top and gently inverting culture tube or cuvette 2) zero instrument with blank at 560 nm 3) measure absorbances at 560 nm. Record as A 1 add 50 L Iron Color Reagent 1) incubate 37 C for 10 minutes 2) Mix by stretching a small piece of parafilm over top and gently inverting culture tube or cuvette 3) re-zero instrument with blank at 560 nm 4) measure absorbances at 560 nm. Record as A 2

13 reminders about protocol WEAR GLOVES! ALL wastes in medical waste containers Before measurement 1) Be sure sample holder is aligned properly 2) Wipe off cuvettes with Kim wipes before measurement 3) Check to make sure no bubbles are present- cover and gently invert to remove air bubbles