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At the end of this practical you should be able to:

 To hypothesise a likely mechanism of reversible enzyme inhibition

To suggest suitable substrate and inhibitor concentration ranges in order to measure the enzyme inhibitor constant.

To calculate required volumes of stock and buffer solutions in order to make up solutions of desired concentrations.

Matrix Characteristics

The attributes of quality control materials should include the following; the characteristic of indistinguishable matrix as for the blood specimen of the patient, which include turbidity, consistency, color and composition. For instance, a technique that assays serum will tests should be controlled using another based control of a human serum. Quality control material needs to be simple and easy to be used on the grounds that complicated reconstitution methods increase more venerability chances of errors. The condition in which fluids are not in a position to be reconstituted makes the control of fluid to be more advantageous compared lyophilized controls. Controls should have negligible vial to vial inconstancy; this is because changeability could be misjudged as efficient error in the instruments or methods. Quality control materials should be steadier for significant period of time. Controls with short live spans of usability will require frequent restructuring and validation against the outgoing materials, making more superfluous work. Quality control material should be accessible in sufficiently quantitative amounts in order to last for at least one year. Acquiring an extensive clump diminishes the circumstances that ranges of control must be built up.

Controls ought to have target esteems that are near restorative choice focuses. Quantitative tests ought to incorporate at least one control with an objective incentive in the sound individual reference interim while a second control with an objective esteem that might be found in a debilitated patient. Cases incorporate sodium controls of 140 and 115 mEq/L and glucose controls of 75 and 225 mg/dL. On the off chance that three control levels are run, a strangely low patient range ought to be incorporated. Quality control levels for restorative medication observing should reflect remedial, lethal, and trough esteems. On the off chance that a test is subjective, giving either positive or negative outcomes, a negative control and a frail positive control with a focus at the least perceivable level are suggested. Semi-quantitative tests ought to have controls at each review level - follow, 1+, 2+, and so on.

Both assays and unassay control material are accessible. Tested controls are estimated by a reference technique and sold with distributed target esteems. They are more costly than unassay controls and are not financially savvy for routine quality control in a healing center or reference lab. Tested controls are suggested for doctor office research centers. Unassay controls must be broke down by the research center to decide the objective esteem and adequate range. Comparison thinks about should be kept running between the flows and new unassay control materials. In the event that the new control material is from a similar maker, just five examples of the new control material should be hurried to build up a mean. On the off chance that the mean is near the mean of the active quality control material, the new control material can be acknowledged. No information focuses ought to be barred except if they are known to be aftereffect of operational mistakes. The standard deviation of the active controls is received for use until the point when enough information focuses are gathered for figuring.

  1. Running the patient and quality control samples in duplicate, set up reactions in tubes as follows,:

Simplicity of Use

Tube

0.9% NaCl

Serum

Biuret Reagent

Saline blank

2.5 ml

N/A

3.0 ml

Test

2.4 ml

0.1 ml

3.0 ml

  1. Mix thoroughly. Leave the colour to develop for 30 minutes and then measure the absorbance at 550nm. Use the saline blank to adjust the Spectrophotometer to 0.

Albumin by Bromcresol Green Binding

  1. Running the patient and quality control samples in duplicate, set up reactions in tubes as follows:

Tube

BCG solution

Serum

Blank

4.0 ml

N/A

Test

4.0 ml

20 μl

  1. Mix thoroughly and incubate for 10 minutes. Set the spectrophotometer to 628nm and adjust to 0 with blank.

Bilirubin – Jendrassik & Grof method

  1. Running the patient and quality control samples in duplicate, set up reactions in tubes as follows. Add the reagents in the order shown:

Tube

d.H2O

Serum

Diazo reagent

Caffeine benzoate

Test blank

0.2 ml

0.05 ml

N/A

N/A

Test direct

0.2 ml

0.05 ml

0.125 ml

N/A

Test total

0.2 ml

0.05 ml

0.125 ml

0.5 ml

  1. Mix thoroughly and incubate at room temperature for 10 minutes. Adding the

 Reagents in the order shown, add reagents to the same tubes as follows.

Tube

Ascorbic acid

Diazo reagent

Caffeine benzoate

Tartrate

Test blank

0.025 ml

0.125 ml

0.5 ml

0.375 ml

Test direct

0.025 ml

N/A

0.5 ml

0.375 ml

Test total

0.025 ml

N/A

N/A

0.375 ml  

  1. Mix all tubes. Measure the absorbance at 607nm against water as 0 blank in the order shown in the table.
  2. Deduct the absorbance obtained for the blank from the absorbance obtained for the test.

Assay 1: Total Protein by Biuret

Sample

Concentration (g/L)

Absorbance 1

Absorbance 2

Standard blank

0.00

0.000

0.002

Standard 1

10.00

0.070

0.064

Standard 2

20.00

0.172

0.163

Standard 3

40.00

0.226

0.234

Standard 4

60.00

0.385

0.387

Standard 5

80.00

0.451

0.437

Standard 6

100.00

0.550

0.542

Patient 1

0.234

0.221

Quality control

0.275

0.263

Assay 2: Albumin by Bromocresol green

Sample

Concentration (g/L)

Absorbance 1

Absorbance 2

Standard blank

0.00

0.000

0.000

Standard 1

5.0

0.116

0.115

Standard 2

10.0

0.251

0.248

Standard 3

20.0

0.374

0.372

Standard 4

30.0

0.512

0.520

Standard 5

40.0

0.662

0.651

Standard 6

50.0

0.810

0.823

Standard 7

60.0

0.904

0.893

Standard 8

70.0

1.088

1.042

Patient 1

0.412

0.426

Quality control

0.521

0.518


Assay 3: Bilirubin by Jendrassik & Grof method

Sample

Concentration (µM)

Absorbance 1

Absorbance 2

Standard blank

0.000

0.000

0.001

Standard 1

10.0

0.030

0.032

Standard 2

20.0

0.055

0.049

Standard 3

35.0

0.092

0.097

Standard 4

55.0

0.158

0.162

Standard 5

75.0

0.202

0.200

Standard 6

90.0

0.266

0.252

Patient 1 blank

0.006

0.005

Patient 1 direct

0.015

0.016

Patient 1 total

0.053

0.049

QC blank

0.002

0.001

QC direct

0.068

0.061

QC total

0.211

0.198

Plot showing assay 1, (Total Protein by Biuret) results, of concentration in g/L against mean absorbance with R2 and equation of the line.

Therefore, we are able to work out the concentration of the patient sample and Quality control:

y=0.0054x+0.026

0.2275-0.026/0.0054 = 37.31 g/L

0.269-0.026/0.0054= 45g/L

Patient sample concentration is: 37.31 g/L

Quality Control Concentration is: 45g/L

Plot showing assay 2, (Albumin by Bromocresol green) results, of concentration in g/L against mean absorbance with R2 and equation of the line.

Using this plot the patient and quality control sample concentration are as follows:

y=0.0146x + 0.0587

0.419-0.0587/0.0146= 24.68 g/L

0.5195-0.0587/0.0416= 31.56 g/L

Patient sample Concentration: 24.68 g/L

Quality Control Concentration: 31.56 g/L

Plot showing assay 3, (Bilirubin by Jendrassik and Grof method) results, of concentration in µM against mean absorbance with R2 and equation of the line.

Using this plot the patient and quality control sample concentration are as follows:

y=0.0028x - 0.0007

0.0675-0.0007/0.0028= 23.86 g/L

0.09186-0.0007/0.0028= 32.56 g/L

Patient sample Concentration: 23.86 g/L

Quality Control Concentration: 32.56 g/L

The chemical analysis of blood test measure several components of plasma, and majorly serum will be considered to be used, this is because serum can easily be acquired from a clotted blood in absence of adding anticoagulant. The variations of the outcome of the concentrations of the chemical components of the blood will broadly indicate the presence of diseases. For example an increase in the concentration of bilirubin in the serum will indicate a disorder of liver and bile ducts or an increase in rate of destruction of hemoglobin.

The blood test can be carried out manually through individual procedures for every analysis, but if considering carrying out several numbers of chemical analyses in the laboratories then autoanalyzer is advisable to be used in order to save time.

Considering the autoanalyzer machine the serum will be automatically be drawn from the test tube, through a small diameter tubing, subdivision of the serum reagent is done and addition of appropriate reagent is done, the product determine is measured using photoelectric instruments, where the results from each serum concentration of numerous substance are read directly.

Control Stability

Firstly, it is observed from the calculation in assay 1 that the quality control concentration of total protein was 45 g/l which was less than the expected value of 47.7 g/l by a percentage of 5.66%, while in assay 2 the quality control concentration of Albumin was 31.56 g/l which was more than the expected value of 31 g/l by a percentage of 1.81% and finally in assay 3 the quality control concentration of Bilirubin was 32.56 g/l which was more than the expected value of 32 g/l by a percentage of 1.81%, this is a clear indication that all the three assay were valid since they fall on the limits of 10% of the expected values.

Secondly, the comparison of the valid assay on the patients sample concentration to their corresponding reference ranges showed that all the three assays could not fall within the reference ranges, as per the assay 1 and assay 2 the total protein and albumin decrease demonstrates that either the patient`s production of albumin or globulin proteins is impaired, through malnutrition or severe liver disorder or the patient is suffering from nephronic syndrome or the patient is having congestive heart failure.

Thirdly, the assay 3 showed the level of bilirubin increased this demonstrated that the patient is suffering from liver damage.

Finally, from the three assay it clear that the patient is having an abnormal health

Reference 

Matic, G.B., Chapman, E.S., Zaiss, M., Rothe, G. and Schmitz, G., 1998. Whole blood analysis of reticulated platelets: improvements of detection and assay stability. Cytometry: The Journal of the International Society for Analytical Cytology, 34(5), pp.229-234.

Wang, J.C., Doedens, M. and Dick, J.E., 1997. Primitive human hematopoietic cells are enriched in cord blood compared with adult bone marrow or mobilized peripheral blood as measured by the quantitative in vivo SCID-repopulating cell assay. Blood, 89(11), pp.3919-3924.

Najafian, N., Salama, A.D., Fedoseyeva, E.V., Benichou, G. and Sayegh, M.H., 2002. Enzyme-linked immunosorbent spot assay analysis of peripheral blood lymphocyte reactivity to donor HLA-DR peptides: potential novel assay for prediction of outcomes for renal transplant recipients. Journal of the American Society of Nephrology, 13(1), pp.252-259.

Omar, H., Chamberlin, A., Walker, V. and Wood, P.J., 2001. Immulite 2000 parathyroid hormone assay: stability of parathyroid hormone in EDTA blood kept at room temperature for 48 h. Annals of clinical biochemistry, 38(5), pp.561-563.

James, C.S. ed., 2013. Analytical chemistry of foods. Springer Science & Business Media.

Zhou, P. and Regenstein, J.M., 2006. Determination of total protein content in gelatin solutions with the Lowry or Biuret assay. Journal of food science, 71(8).

Ellidag, H.Y., Eren, E., Y?lmaz, N. and Cekin, Y., 2014. Oxidative stress and ischemia-modified albumin in chronic ischemic heart failure. Redox Report, 19(3), pp.118-123.

Webster, D., Bignell, A.H.C. and Attwood, E.C., 1974. An assessment of the suitability of bromocresol green for the determination of serum albumin. Clinica Chimica Acta, 53(1), pp.101-108.

Kazmierczak, S.C., Robertson, A.F., Briley, K.P., Kreamer, B. and Gourley, G.R., 2004. Transcutaneous measurement of bilirubin in newborns: comparison with an automated Jendrassik–Grof procedure and HPLC. Clinical chemistry, 50(2), pp.433-435.

Rolinski, B., Küster, H., Ugele, B., Gruber, R. and Horn, K., 2001. Total bilirubin measurement by photometry on a blood gas analyzer: potential for use in neonatal testing at the point of care. Clinical chemistry, 47(10), pp.1845-1847.

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