CLS review pointer guidelines EXAM 2 LAB

Analyte Details

 

Calcium

DYE BINDING

Analyte Details

Calcium

Principle of the reaction:Dye binding. We said o-Cresolphthalein. This one uses

{ Calcium+Arsenazo→alkaline →Ca-arsenazo complex}

purple color.

Reagent components.Arsenazo III (dye) and

8-hydroxyquinoline sulfonate (Mg++   will bind to this and not the dye, this corrects the Mg++ interference problem)

Appropriate sample:10 microliters.

Reference ranges:8.5-10.4 adults

Interferents: Mg++,glass tubes,lipemia.

wavelength: 600-670

Analyte Details

 

 

Magnesium

DYE BINDING

Analyte Details

Magnesium

Principle of the reaction:Dye binding Xylidyl Blue

serum Mg ions react with Xylidyl Blue in an alkaline solution to produce a red complex.

Reagent components:Xylidyl Blue (dye) , EGTA,(cleates Ca.), Surfactant (deproteinates),

Appropriate sample:10 microliters

Reference ranges:1.6-3.0 mg/dL adults

Interferents: hemolysis, lipemic, icteric, a number of drugs.

wavelength: 550

Analyte Details

 

Bilirubin (total)

colormetric, kinetic, end point assay-not linked.

Analyte Details

Bilirubin (total)

 Principle of the reaction:colormetric, kinetic, end point assay-not linked.

Sulfanillic acid reacts with sodiun nitrite to produce diazotized sulfanillic acid. Direct and indirect bilirubin couple with diazo to produce azobilirubin. In the presence of dimethyl sulfoxide (DMSO). The intensity of the color produced that is produced is directly proportional to the amount of total bilirubin concentration present in the sample.

Reagent components:Sulfanillic acid, sodium nitrite, hydrocloric acid and DMSO.

Appropriate sample: 0.05 microliter

Reference ranges:0.2-1.2 mg/dL

Interferents:a number of drugs

light labile, hemoglobin

wavelength 540-560

Analyte Details

 

Bilirubin (direct)

 COLORMETRIC

Analyte Details

Bilirubin (direct)

 Principle of the reaction:Sulfanillic acid reacts with sodiun nitrite to produce diazotized sulfanillic acid(diazo). Direct bilirubin couples with diazo to produce azobilirubin.The intensity of the color produced is directly proportional to the amount of direct bilirubin present in the sample.

Reagent components:Sufanillic acid in hydrocloric acid, sodium nitrite reagent.

Appropriate sample:0.10 microliters.

Reference ranges:0-0.5 mg/dL

Interferents:a number of drugs, protect from light.

Wavelength: (555) 540-560 

Analyte Details

URIC ACID

Enzymatic endpoint assay utilizes URICASE in a linked trinder reaction. 

Analyte Details

URIC ACID

 Principle of the reaction:Enzymatic endpoint assay utilizes URICASE in a linked trinder reaction.

Uric acid+O2+2H2O→uricase→Allantoin+CO2+H2O2

2H2O2+4-Aminoantipyrine+TBHBA→POD→Chromagen+4H2O.

Uric acid is oxidized by URICASE to allntoin and hydrogen peroxide. TBHBA + 4-aminoantipyrine+ hydrogen peroxide in the presence of preoxidase produces a colored chromogen that is measured at 520. The color intensity at 520 is proportional to the concentration of uric acid in the sample.

Reagent components:uric acid reagent TBHBA, uricase(enzyme), peroxidase (for trinder)

Appropriate sample:25 microliter

Reference ranges:2.5-7.7

Interferents:bilirubin, ascorbic acid, lipemic,

wavelength :520

Analyte Details

BUN(urea)

 ENZYMATIC KINETIC LINKED

Analyte Details

BUN(urea)

 Principle of the reaction:Enzymatic (urease) linked kinetic (NADH).

Urea+H2O→Urease→2NH3+CO2

NH3+∂-Ketoglutarate+NADH+H+GD→L-glutmate +NAD+ +H2O

Reagent components:NADH, Urease,Glutamate dehydrogenase, Ketogluterate

Appropriate sample:10 microliters

Reference ranges: 7-18mg/dL

Interferents:ammonia, fluoride (kills urease),some drugs.

Analyte Details

Creatinine

 COLORMETRIC

Analyte Details

Creatinine

 Principle of the reaction:Colormetric (picrate acid/modified jaffe reaction) kinetic assay

Creatinine + Sodium picrate →Alkaline→ Creatinine-picrate complex

Yellow-orange

Reagent components:alkaline buffer, picric acid

Appropriate sample:50 microliters

Reference ranges:0.40-1.40 mg/dL

Interferents: dyes such as bromsulfophthalein and phenolsophthalein (they bind picrate) can use zinc sample pretreatment to remove these dyes before assay.

Analyte Details

Total Cholesterol

ENZYMATIC ENDPOINT 

TRINDER

→Analyte Details

Total Cholesterol

 Principle of the reaction:

Cholesterol Esterase→C Esters→Cholesterol+Fatty acids

Cholesterol+O2→C. Oxidase→Cholesterol-3-one+H2O2

2H2O2+ 4-Aminoantipyrine+p-HBS→Peroxidase→Quinoneimine+2H2O

(trinder)

Reagent components:4-Aminoantipyrine, Sodium Cholate, Cholesterol esterase,cholesterol oxidase, Horseradish Peroxidase, P-Hydroxybenzene Sulfonate, Surfactant,

Appropriate sample:10 microliters

Reference ranges:<200mg/dL

Interferents:

Wavelenght 520

Analyte Details

HDL Cholesterol

precipitation

 

Analyte Details

HDL Cholesterol

precipitation

 Principle of the reaction:When serum is combined with reagent dextran sulfate and magnesium ions precipitate the LDL and VDRL fractions, leaving the HDL fraction in solution. The HDL cholesterol is then determined using enzymatic cholesterol assays

Reagent components:HDL cholesterol Precipitating reagents, dextran sulfate, Magnesium ions, sodium.

Appropriate sample:50 microliters

Reference ranges:27-78

Interferents: Hemolysis, icteric, ascorbic acid

Analyte Details

HDL Cholesterol

Direct

 

 SEPARATION

ENZYMATIC

Analyte Details

HDL Cholesterol

Direct

 Principle of the reaction:The liquid auto HDL Cholesterol assay is a homogeneous method for directly measuring serum HDL-C levels without the need for any off-time pretreatment or centrafugation steps. The method is a two reagent format. The first reagent contains a-clyclodextran and dextran sulfate to stabilize LDL, VLDL and cholymicrons. (so they don’t react with the second reagent). The second reagent contains PEG modified enzymes that selectively react with the cholesterol present in the HDL particles. Consequently, only the HDL cholesterol is subject to cholesterol measurement.

Reagent components:clyclodextran, PEG

Appropriate sample:4 microliters

;

;Analyte Details

LDL;Cholesterol

Calculated

friedwald formula

friedwald formula

;

LDL-C=Total Cholesterol -HDL-C – Trig/5

;Analyte Details

;

Triglycerides;

;

ENZYMATIC

TRINDER

Analyte Details

Triglycerides;

;Principle of the reaction:linked pyruvate kinase enzymatic ultra violet.Triglycerides in the sample are hydrolyzed by by lipase to glycerol and fatty acids. The glycerol is then phosphorylated by adenosine-5-triphosphate (ATP) to glucerol 3 phosphate(G3P) and adenosine-3-diphosphate in a reaction catalyzed by glycerol kinase(GK). Glycerol3 phosphate is then converted to dihydroxyacetone phosphate (DAP)and hydrogen peroxide by glycerol phosphate oxidase(GPO). The hydrogen peroxide then reacts with 4-aminoantipyrine (4-AAP) and 3-hydroxy-2-4-6 tribrombenzoic acid (TBHB) in a reaction catalyzed by peroxidase to yeald a red colored quinoneimine dye. The intensity of the color produced is directly proportional to the concentration of triglycerides in the sample when measured at 540.

Reagent components: ATP, LIPASE, TBHB, Peroxidase, surfactant, (deproteinator)

Appropriate sample:10 microliters.

Reference ranges:36-165 mg/dL

Interferents:hemolysis glycerol, bilirubin, detergents, drugs

;;Analyte Details

Serum;Iron;

;

COLORMETRIC

Analyte Details

Serum;Iron;

;Principle of the reaction:Transferrin bound iron is released at acid pH and reduced from ferric to ferrous ion these ions react with ferrozine to form a violet colored complex

Reagent components: acid buffer and ferrozine

Appropriate sample: 0.5ml (500microliters)

Reference ranges: 60-150 ug/dL

Interferents:drugs testtubes, not hemoglobin

;Analyte Details

Serum;Iron;(TIBC)(UIBC)

;

COLORMETRIC

Analyte Details

Serum;Iron;(TIBC)(UIBC)

;Principle of the reaction:Transferrin bound iron is released at acid pH and reduced from ferric to ferrous ion these ions react with ferrozine to form a violet colored complex.

(TIBC) A known amount of ferrous ions are added to serum at an alkaline pH. The ferrous ions bind with transferrin. the additional unbound ferrous ions are measured using the ferrozine reaction.The difference between the amount of ferrous ions added and the unbound ions measured is the unsaturated iron binding capacity.The TIBC is equal to the serum iron concentration plus the UIBC.

Reagent components:alkaline and acid buffer and ferrozine

Appropriate sample:0.5ml (500microliters)

Reference ranges:60-150 ug/dL

Interferents:drugs testtubes, not hemoglobin

Sweat cloride

Method

Sweat cloride

Method

pilocarpine nitrate iontrophoresis

Collection of sweat and gavimetric measurement of collected sweat.

Sweat cloride

What is the gold standard of sweat analysis for CF ?

Based on the Cotlove Cloridometer measurement of cloride

Sweat cloride

Saftey considerations.

Can cause burns, current interupts heart.

Trained staff

Monitor electrodes

monitor electrical feed

time

do not cross chest.

Sweat cloride

Notes.

1. What preanalytical problems are there with collection of the sweat cloride?
Not proper volume, not properly wrapped, not properly packaged, volumes are critical.
2. What method will you use? cottlove cloridometer.
3. What about screening? conductivity.???
4. What is the difference. Other ions.
5.What is the name of the electrophoretic method? perodine;Iontrophrotic method.
6.What are some of the dangers with this method? You need a well trained person. You must have automatic shutoff devices and a controlled power source. You can cause a burn.;
7. Why do you not want to cross the electrodes on the chest? Because you can cause a heart attack.
8. How long is collection? 5-10 mins.
9. What is the;reportable values? 1-165
10.What is the cutoff value for CF? ;60 , Evaluate at ;40.
11. Do all CF patients have results;; 40? No a few can have lower than 40.
12. Is;greater than 165 possible? No greater than 165 is incompatible with life.
13. Do you ever see this high of a results? yes, improper collection, ;or allowing the sweat to evaporate.
14. In any one testing period how many test should be done?. 2
15. Why do you have to do 2? Quality control. The two should agree within a certian amount.
16. To confirm that someone has CF, ;how many times should testing be done? 2 times.
17. Why dont they just do DNA testing? Because there are too many variants.
18. Is there a minimum amount of sample that you need, that you should use? No less than 15 microliters.
19. What are some of the pathological changes with CF? infection, pancreatitis,
;
I did not get a very good recording (too far away) so I missed some points. (I think)

Sweat cloride

Cut-Off levels

What is the cutoff value for CF? ;60 , elaluate at ;40.
;Do all CF patients have results;; 40? No a few can have lower than 40.

Sweat cloride

Maximum physiological cloride concentration

Is;greater than 165 possible? No greater than 165 is incompatible with life.

Sweat cloride

Other screening testing methods

conductivity , osmolality

Sweat cloride

Reason and number of repititons to confirm CF.

2 for quality control.
What are the complexones for Ca?

Arsenazo

complexes with calcium to form a purple colored complex

;

and o-Cresophthalein

What are the complexones for Mg?
xyladil blue
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