HOSP #		WARD	False Bay Hospital Casualties
CONSULTANT		DOB/AGE	33y Female

Abnormal Result

Urea 0.8 mmol/L; Creatinine 10 umol/L; Sodium 154 mmol/L; Potassium 5.4 mmol/L

Presenting Complaint

Above results do not make sense for a 33 year old female, except if muscle weight is extremely low.

History

Examination

N/A

Laboratory Investigations

Inspection of the sample:

Other Investigations

Lactate on analyser as done on serum sample: >22 mmol/L, dilution made: 1 in 10 revealed a lactate of 26.8 mmol/L in the sample. This could explain that Ringers Lactate was the contaminant in the sample, but can only be hypothesized with the available evidence.

Hematology results:

Final Diagnosis

Take Home Messages

Download File

Lactate cannot be measured in SST (serum separator tubes) without taking into account the fact that the red cells will continue to metabolize the glucose in the sample to lactate via anaerobic metabolism through glycolysis.

Lactate concentration increases linearly over time, in whole blood. Factors affecting the rate of production are, among others:

• Temperature
• Glucose concentration
• Additives in the blood tubes such as NaF

NaF inhibits enolase, an enzyme acting late in the glycolytic pathway, and has no effect on enzymes that act early in the glycolytic pathway. … Glycolysis is instantly inhibited in erythrocytes, leukocytes and platelets when the blood pH is maintained between 5.3 and 5.9 with a citrate buffer.

Sage Journals: Ann Clin Biochem 2013;50: 3–5. DOI: 10.1258/acb.2012.012135

Download File

Practical Documentation:

Aims

To perform serum albumin determinations on samples, explain the principle behind the Bromocresol Green method for albumin measurement and to list the factors that will cause interference with this method.

Principle

Albumin is known for its ability to bind many types of organic compounds, including organic dyes. When albumin selectively binds with Bromcresol Green (BCG) it causes a change in the absorbance maximum of BCG. The intense blue-green complex that is formed has an absorbance max of 670nm. Bromocresol reagent at pH 4.3 is negatively charged. The pI of albumin is 4.7.

For all spectrometric assays, always use a Reagent
blank. It usually contains all diluents and reagent in the reaction
solution, but no sample. Some reagent blanks do contain the sample as well, but
they lack one crucial reagent component needed to produce a colour-yielding
reaction. This is different from the water used to zero a spectrophotometer
(set 100% T).

Materials:

1. Albumin Stock Solution (100g/L)
   or Cobas calibrator
2. Serum patient samples (record
   the albumin results off track)
3. Cobas albumin controls
4. Bromocresol green reagent (BCG)
   – obtain Cobas reagent or make up as follows:
5. 7.5mg of
   BCG
6. 250mg
   EDTA
7. 250µL
   Tween 20
8. Bring up
   to 25mL with water

Methods:

1. Generate a calibration curve of
   at least 8 standards (0- 80 g/L) by diluting the Albumin Stock (100 g/L).
2. In labelled tubes, set up a
   calibration, controls and test samples as follows:

To set up a standard curve with more points along the usable range, I chose more points in the concentration range 20 – 40 g/L, which in my experience constitutes the bulk of albumin measurements.

3. Mix well read immediately at 630nm and record absorbances

4. Plot a standard curve and determine experimental concentrations of controls and serum samples

The figure below illustrates the formula used to determine the unknown concentrations.

Unknown concentrations were calculated as follows:

Compare results to expected results and comment on any differences between the manual BCG vs automated BCP assays.

A haemolysed sample is brought to the laboratory for albumin analysis. Can the sample be used? Discuss.

Yes. Hemoglobin does not absorb light at 670 nm, therefor will not interfere significantly with the analysis. See figure below:

It does however interfere in the following manner:

Hemoglobin decreases the apparent albumin concentration by 1 g/L for each 100 g/L added. Blanking does not correct this interference, and the negative bias is therefore caused by interference with the dye binding rather than hemoglobin color. For the BCP method, a blank correction is required on icteric sera and on grossly hemolyzed and grossly lipemic sera to correct for an underestimation of albumin caused by these agents. Heparin causes a positive interference with BCP and BCG methods. This interference can be eliminated by the addition of hexadimethrine bromide to a concentration of 50 mg/L in the BCP reagent

Kaplan’s Methods

Why is it not desirable to incubate the reaction before measuring the absorbance?

Incubating the sample can give rise to other non-specific binding of analytes in the sample to the chromophore dye and a falsely elevated reading can be obtained.

Some analyzers can measure the absorbance of the BCG reaction within 30 seconds after adding sample. Does this tend to increase the specificity of the reaction?

Yes. The less time there is for other interfering substances to bind to BCG, potentially the more specific it will be to albumin, as albumin is the more specific binding to BCG.

Practical 3 :
PROTEIN ASSAY- BRADFORD
Total /100

INTRODUCTION
The Bradford protein assay, is a spectrophotometric assay that is more popular for protein concentration determination than other known protein assay such as the Lowry assay. The Bradford assay is simple, more sensitive and faster than other protein assays (Kruger, 2009). This assay is an example of a dye-binding assay and the dye used is Coomassie Brilliant Blue G-250 (Bradford, 1976; Becker, Caldwell and Zachgo, 1996; Kruger, 2009; Nouroozi and Noroozi, Moulood Valipour Ahmadizadeh, 2015). The principle of this assay relies on the physical interaction of the dye and protein in solution and results in an observable change in colour. The colour change is as follows; red (Amax 465 nm), when not bound to proteins and blue (Amax 595 nm) form of the dye carries a (-) charge and interacts with (+) charges on proteins to form a complex (Becker, Caldwell and Zachgo, 1996).

OBJECTIVES
2.1. To prepare standards through a series of dilutions
2.2. To measure the unknown concentration of a protein in solution via a spectrophotometer
2.3. To analyse, interpret results about CV%, SD, LOD and LOQ.

PROCEDURES
A. PREPARATION BEFORE THE PRACTICAL
Complete the following BEFORE your practical session:
• You would need to do some extra reading on the Bradford protein assay and spectrophotometer principles (i.e. Beer Lambert law) in preparation for your practical and test.
• Find and print SDS’s for the following chemicals; Tris (trisaminomethane), Hidrochloric acid (HCL), ethanol, Phosphoric acid and Coomassie Brilliant Blue G-250.
• Prepare a practical plan for your experiment that you will be conducting today.

B. PRACTICAL SESSION (Total 50)
Complete the following DURING your practical session:
(1) Complete the practical test.
(2) Using the Bradford Assay determine as follows:
Materials provided:
• Tris buffer: 10 mM Tris-HCl (pH 7.0)]
• Bovine serum albumin (BSA) stock solution: [2 mg/ml BSA in Tris buffer (pH 7)]
• Bradford reagent; [0.01% (w/v) Coomassie Brilliant Blue G-250, 4.7% (w/v) ethanol, 8.5% (w/v) phosphoric acid]
• Unknown protein sample

Method:
a) In Eppendorf tubes, prepare a series of BSA solutions of varying concentration by diluting the
2 mg/ml BSA stock solution with Tris-HCL buffer (You will need ~200μL of each dilution) to set up a calibration curve (at least 7 concentrations to be used).

Serial dilutions were made in 1.5mL Eppendorf tubes:

b) Add 2.5 mL of Bradford reagent to a separate cuvette for each of your samples and label them appropriately. Consider the value of determining the concentration of one or more dilutions of your unknown sample as well as the undiluted (“neat”) unknown sample.

To save cuvettes, I have used an old refurbished microtitre plate with 10x less volume, hence 250uL

c) Prepare your samples by adding 50μL of each protein sample (diluted standard or unknown) separately to the Bradford reagent in the appropriately labelled tube. Mix the tubes by gentle inversion several times, and let the colour develop for 5 min. Observe and record the colour change of your standard samples as a function of protein concentration. A blank sample is prepared by mixing 50μL Tris buffer with 2.5 ml Bradford reagent.

As above, to save reagent and test my pipetting skills, I have used 5uL as one set of additions and also made a 1:1 (2x) dilution of my standards to run another calibration curve. The unknown sample was also added as neat and a 2x dilution.

d) You will need to determine which portion of the UV/vis spectrum, specifically which wavelength will be useful for following the dye bound by protein. Take a full spectral scan of your Bradford reagent blank. When your standard samples have fully developed, take a full spectral scan of the most concentrated standard you prepared.

e) Based on your results, choose a single wavelength suitable to analyse the results of your dye-binding assay. Measure and record the absorbance of each standard and unknown sample at your chosen wavelengths using cuvettes.

From above wavelength scan it is evidenced that the maximum absorbance after colour development occurs at 595 as published widely in the literature for the Bradford assay. I am, however going to perform a slight variation also published before, by using the ratio of absorbance 595nm/465nm as the signal, as it has before been shown to be more sensitive. The rationale thereof is that there is reduction of absorbance intensity at 465nm and increase of intensity at 595nm, hence likely causing slight increases in sensitivity and arguably a more accurate assay.

f) At your determined wavelength, read your unknown sample at approximately every 10 minutes for 1 hour, you will use these readings to calculate the percentage (%) change over time.

g) Prepare your highest standard in five (5) cuvettes and read the absorbances of each cuvette two times, so in total you will have 10 readings. These results you will use to calculate the protein concentrations and then calculate the CV% and SD

h) Make sure your lab space is clean and disinfected.

C. AFTER PRACTICAL SESSION
Complete the following for submission before or during the next practical
Answer the following questions:

QUESTION 1 (Total 15)
Using the resulting equation for the calibration curve, determine the protein concentration of your unknown sample.

To determine the unknown:

Via AU595nm:

y=0.4694x + 0.6086

x= (y-0.6086)/0.4694

x= 1.34g/L

Via the 595/465nm ratio:

y = 1.2289x + 0.6072

x=(y-0.6072)/1.2289

x= 1.25g/L

QUESTION 2 (Total 10)
Calculate the percentage (%) change over time of your unknown sample and comment on the stability of your assay.

Referring to Fig. 2 – To calculate the %change over time is relatively simple by using the slope of the linear regression line: If using the orange line’s formula at 595nm only: slope = – 0.0081, which means that the absorbance values decreases in general by 0.0081 AU per minute. This is, however subject to variability as the reagent/protein complexes was observed as precipitating out and the final portion of the curve (after 50 min) is likely not fitting due to this phenomenon. Nevertheless, using a decrease of 0.0081AU per minute, means that at an absorbance value of 1.25 (top of the calibration curve),

0.0081/1.25*100 =

0.65% decrease in absorbance per minute.

The stability of the assay is likely around 15-20 minutes.

QUESTION 3 (Total 15)
Calculate the CV% and SD using your data.

QUESTION 4 (Total 10)
Calculate the LOD, LOQ, and comment on the linearity of your assay.

Using only the 595nm absorbance yielded a poor result (r=0.9784).

Using the 595/465 ratio, the linearity was much better (r = 0.9999)

SE of intercept: Excel Function: STEYX(X-values;Y-values)

LOD = 3.3 * (SD of intercept / slope)

LOQ = 10 * (SD of intercept / slope)

REFERENCES:
Becker, J. M., Caldwell, G. A. and Zachgo, E. A. (1996) ‘Protein Assays’, in Biotechnology. Elsevier, pp. 119–124. doi: 10.1016/b978-012084562-0/50069-2.
Bradford, M. M. (1976) A Rapid and Sensitive Method for the Quantitation of Microgram Quantities of Protein Utilizing the Principle of Protein-Dye Binding, ANALYTICAL BIOCHEMISTRY.
Kruger, N. J. (2009) ‘The Bradford Method For Protein Quantitation’, in The Protein Protocols Handbook. Humana Press, Totowa, NJ, pp. 17–24. doi: 10.1007/978-1-59745-198-7_4.
Nouroozi, R. V. and Noroozi, Moulood Valipour Ahmadizadeh, M. (2015) ‘Determination of Protein Concentration Using Bradford Microplate Protein Quantification Assay’, International Electronic Journal of Medicine, 4(1), pp. 11–17. doi: 10.31661/iejm158.

Raw data:

HOSP #		WARD	Surgical ICU
CONSULTANT	Dr. Heleen Vreede	DOB/AGE	23y Female

Abnormal Result

Fluid triglycerides were requested on three samples, without any clinical information.

Presenting Complaint

The laboratory history was explored, to find that the patient had three subsequent samplings daily from a pleural fluid cavity.

History

A month prior to presentation, the patient had a breached stillbirth.

The following was found on Histology:

EPISODE NUMBER:
SA03381462
CLINICAL DETAILS:
23 year old female. G02 P2-1. VDRL: negative. Smoking: 8/day. Alcohol: none. HIV: negative. 750g breech stillbirth at 28/40.
MACROSCOPY:
The plate measures 130 x 100 x 30mm and weighs 196.8g.
The membranes are complete without evidence of meconium staining.
The cord measures 300mm in length with an average diameter of 15mm. Three umbilical vessels are identified with 3 twists per 100mm. The cord insertion is off-centre, 20mm from the plate margin.
Congested blood vessels are identified on examination of the foetal surface.
Cut-sections of the plate show:
- Retroplacental clot.
MICROSCOPY:
General:
Placental weight for gestational age is below the 10th percentile at 196.8g. Partial autolysis is present throughout the specimen.
The umbilical cord and membranes:
The umbilical cord contains two arteries and one vein. There is no evidence of vasculitis or funisitis. Wharton's jelly and membranes do not show meconium uptake. The amniocytes are intact and show no evidence of vacuolation or hyperplasia.
Chorionic plate:
The chorionic plate vessels are focally dilated. There is no evidence of chorioamnionitis.
Villi and intervillous spaces:
The stem villous vessels show partial obliteration as well as stromal sclerosis. Distal villous hypoplasia as well as accelerated villous maturation is seen. Intervillous thrombi are seen as well as intraparenchymal extension of the retroplacental haematoma. There is no evidence of infarction, villitis or intervillositis.
Maternal surface:
There is evidence of a large retroplacental haematoma. No chronic deciduitis or untransformed blood vessels are seen.
PATHOLOGICAL DIAGNOSIS:
Placenta, examination:
Maternal vascular malperfusion.
Retroplacental haematoma.
Reported by: Dr M Du Toit

Clinicians were indeed querying a chylothorax. A thoracic duct injury was suspected.

Examination

Unfortunately little clinical information is known, as can be seen above. The following table shows the clinical info which has been captured on the respective episodes’ request forms:

      Clinical history for episodes 
< SA03215505 >      ?UTI IN PREGNANCY
< SA03381462 > 03/10/2019     PLACENTA
< SA03466495 > 07/11/2019     MEDIASTINITIS
< SA03466500 > 08/11/2019     MEDIASTINITIS
< SA03466533 > 08/11/2019     MEDIASTINITIS
< SA03467168 > 08/11/2019     MEDIASTINITIS
< XC00366131 >      ILLEG
< SA03467081 >      ILLEGIBLE
< SA03469305 >      NECK ABSCESS
< SA03469995 >      ILLEGIBLE
< SA03472064 >      MEDIASITINITIS
< SA03470738 >      NECK ABSCESS
< SA03476491 >      PUS FLUID + MEDIASTINITIS
< SA03476496 >      PUS / FLUID + MEDIASTINITIS
< SA03476502 >      PUS / FLUID + MEDIASTINITIS
< SA03476507 >      PUS / FLUID + MEDIASTINITIS
< SA03489396 >      ?CHYLOTHORAX
< SA03485823 >      SEPSIS
< SA03491179 >      NECK ABSCESS
< SA03493180 >      Neck abscess with sepsis.
< SA03494446 >      CVC TIP
< SA03509355 >      LOOSE STOOLS
< SA03513657 >      THORACIC SURGERY
< SA03531013 >      MEDIASTINITIS

Laboratory Investigations

ChylothoraxDownload

Other Investigations

Final Diagnosis

A pleural fluid triglyceride >1.24 is suggestive of chylothorax.

The additional finding of a thin white layer present on the top of the sample after centrifugation indicates the presence of chylomicrons in the sample, which further supports the diagnosis of chylothorax.

Take Home Messages

The primary role of the thoracic duct is to carry 60 – 70% of ingested fat at a concentration of 0.4 – 6 g/dl from the intestine to the circulatory system.

• Chyle contains large amounts of cholesterol, triglycerides, chylomicrons and fat soluble vitamins.
• Lymph is the other main constituent of chyle and is made up of
  • immunoglobulins
  • enzymes
  • between 400 and 6800 white blood cells/ml, the majority of which are lymphocytes.
• Chyle transportation is maximal after a high fat meal and minimal with starvation where flow is reduced to almost a trickle.

Classically, a chyloma, a collection of chyle below the pleura develops when the thoracic duct first leaks. Although rarely detected, it manifests itself as a swelling in the supraclavicular fossa which may be associated with severe chest pain, dyspnoea and tachycardia.

Chylomas can also manifest themselves at other sites of the pleura without causing supraclavicular swelling. Eventually the chyloma bursts through the pleura where the chyle accumulates in the pleural space.

Very rarely, the chyle leak may lead to chylomediastinum or chylopericardium.

Roughly 2.4 l of chyle is transported through the lymphatic system every day.

Damage to, or rupture of the thoracic duct can give rise to a large and rapid accumulation of fluid in the pleural space.

Causes of chylothorax can be classified as follows:

The theorized causes of chylothorax in this case could be one of the following:

1. The breached stillbirth a month prior to presentation could have caused rupture of the thoracic duct due to increased pressure due to extreme valsalva. Could this be dormant for 1 month and then present with the neck abscess with an anaerobic infection? Could anaerobic bacterial transmission from the gut into a chylous cyst in the neck be the cause? It is however unlikely that delivery of a 750g fetus causes that much trauma.
2. Iatrogenic chylothorax due to the thoracic surgery.
3. Left-sided placement of a Central Venous Catheter during delivery of the stillbirth, which accidentally damaged the thoracic duct. The placement of the CVP could also have caused a pneumothorax with subsequent stretching and damage to the thoracic duct.

Remember Chylomicrons float!

Abnormal result

Paracetamol 25ug/ml (163 umol/L)     Serum osmolarity 310mmol/L

Presenting Complaints

Brought to casualties with stupor from Mitchells Plein Hospital.

History

33 y female presented with stupor after ingestion of an unknown amount of pills.  Empty container of Amitriptiline and Paracetamol was found with her.

Examination

Non-specific neurologic signs, but delirium present. Patient did have an episode of vomiting.  No pathological signs on abdominal examination.

Laboratory Investigations

Paracetamol: The Paracetamol level was never repeated after admission.  Doing an in-house experiment with calibrator and spiking the calibrator samples with N-acetylcysteine correlating with therapeutic plasma levels, I demonstrated that our method on the Roche analyzer, with the enzymatic assay, causes a clinically significant negative interference in the measured paracetamol.                                                                 

The enzymatic assay principle:

_{arylacylamidase hydrolysis                       o-cresal + periodate catalyst}

Acetaminophen→     p-aminophenol+acetate →    indophenol (measured @600nm)

Other Investigations

Tricyclic antidepressant levels 58 ug/L ([TCA] in overdose patients range from 29-1732ug/L, but has not been found to correlate to clinical outcome, unless plasma level is more than 1000ug/L).

Final Diagnosis

Klebsiella Sepsis (confirmed on blood culture 1 day after death) DIC with marked renal failure.

Take Home Messages

• Paracetamol reporting units must be confirmed, we generally use ug/ml, but it has created confusion previously, as nomograms used in South Africa generally use ug/ml.
• N-acetyl cysteine may cause negative interference with the measurement of paracetamol in the enzymatic assay.  Sampling for Paracetamol levels should thus be done before an IV dose of NAC is given to eliminate this possible error.  National guidelines with toxicology will likely be amended.