The following frequently asked questions are intended to provide initial help in case of technical questions for some of our products. For more detailed information, please contact support@buhlmannlabs.ch

Gastroenterology

Quantum Blue®

Cellular Allergy

Clinical Chemistry

Neuroimmunology

Gastroenterology – Stool extraction

Is there a preferred sampling time for stool?
Morning stool is preferred as it reflects best the current status of the intestine.
Can saline or water be added to hard or dry stool samples prior to extraction?
Nothing should ever be added to samples before extracting with any BÜHLMANN extraction devices.
How to extract liquid stool samples in the lab?

CALEX® Cap: Add 10 µl liquid stool in CALEX® body and mix.

Smart-Prep (Roche): Add 80 µl of liquid stool sample in cup or prefilled tube and mix with 4 ml of B-CAL-EX.

Quick-Prep (ScheBo): Add 26 µl of liquid stool in Quick-Prep Tube (1.3 ml) and mix.

Is there an optimal collection procedure?
Collect stool from at least 3-5 different positions in order to completely fill the grooves of the dosing tip.
Stool samples do not properly dissolve during extraction.
Prolong the incubation time and if possible vortex the sample from time to time.
How long are stool samples or extracts stable?

Stool samples: at least 8 month at -20°C or at least 6 days at 2-8°C.

Extracts: at least 24 month at –20°C or at least 7 days at 2-8°C.

 

Gastroenterology – fCAL ELISA

Does the dilution of samples have to be considered also for calibrators in order to calculate calprotectin concentrations?
No, the actual concentrations of the calibrators A-E are 4, 12, 40, 120 and 240 ng/ml. Therefore, the calibrators are ready to use in both, normal and extended range ELISA procedure.
Is there any high dose hook effect?
No decrease of the optical density can be observed at concentrations higher than the saturation of the system. Therefore, no high dose hook effect could be observed.
Do you recommend any technical precautions for washing steps?
Ensure for every washing a minimal incubation of at least 20 seconds. By using automated wash systems all strips must be filled before emptying begins in order to guarantee the minimal incubation time. Use “plate mode” in automated washer systems.
How was the repeatability tested? What results were obtained? (Intra-assay Precision)
3 different (low, medium and high calprotectin) samples were tested 20 times. Mean, SD and % CV was calculated accordingly.
How was the reproducibility tested? What results were obtained? (inter-assay Precision)
The inter-assay precision of the ELISA was calculated from 5 extracted stool samples. The aliquots were tested according to the assay procedure in 10 different runs by three technicians using 2 kit lots in two different labs.
Given the information that chronic intake of NSAID can give falsely elevated values for calprotectin, how long before a calprotectin test should patients stop taking NSAIDs?
Due to varying half-life time of NSAIDs, we recommend to stop the intake 1-2 weeks before the test.
Does blood in stool samples interfere with calprotectin measurement?
Blood in stool samples does not interfere. Normally, the amount of blood in stool is rather low compared to the amount of calprotectin found in stool.


Gastroenterology – Quantum Blue®

What about the calibrator curve of the Quantum Blue® assays?
The lot-specific calibrator is obtained from the RFID chip card. The Calprotectin concentrations of samples are directly calculated by the Quantum Blue® reader.
Are there any controls for the lateral flow calprotectin assays available?
Yes, they can be ordered separately and contain a ready-to-use low and high control.
Does the test cassette have to be read within 1 minute or can I wait longer?
We highly recommend reading the test cassettes within 1 minute, since the reading process of Quantum Blue® is optimized for 1 minute.
What are the required dilutions of stool extracts for the different Quantum Blue® assays?

For LF-CAL25 it is 1:1.6 for CALEX® Cap and 1:16 for Smart Prep and ScheBo.

For LF-CALE25 it is without further dilutions for CALEX® Cap and 1:10 for Smart Prep and ScheBo

For LF-CHR25 it is 1:15 for CALEX® Cap and 1:150 for Smart Prep and ScheBo.

 

Quantum Blue® – General Issues

What is a RFID card?
Each kit box contains a lot-specific RFID card, giving you lot-specific calibration curve data as no calibrators are within the kit.
Calibration is not OK or out of tolerance.
Consult the troubleshooting chapter in the Quantum Blue® reader manual or consult our support.
The reader does not connect to the computer although the USB cable is connected.
Try installing the appropriate driver found on the CD-ROM delivered with the Quantum Blue® reader.


Cellular Allergy – General Issues 

What is the impact of anti-allergic drugs on CAST® assays?
Interactions are possible with systemically administered anti-inflammatory drugs. However, no impact of anti-histamines has been shown. Nevertheless, we recommend to stop administration of such drugs according to our instruction for use. 
In case of performing a skin prick test or oral challenge, when does the CAST® assay has to be performed?
Blood samples for the assay have to be taken before any in vivo tests such as provocation test or skin test. 
How are blood samples handled before assay procedure?
Blood samples should not be centrifuged nor frozen before the assay.
Why does the assays contain two positive controls?
The anti-Fcepsilon receptor I antibody serves as positive control for IgE-dependent basophil degranulation. The fMLP is used as IgE-independent control for cell degranulation.
What are non-responders?
Non-responders are characterized by a low reactivity to anti-Fcepsilon receptor I or fMLP (<10% CD63 positive cells). 6.1% out of n=98 samples were non-responders to Fcepsilon receptor I and 4.9% out of n=61 were non-responders to fMLP in an internal study.

 

Cellular Allergy – Flow CAST®

What are the prerequisites on the flow cytometer?
Any cytometer that has a 488 nm argon laser can excite the fluorophores (FITC and PE) used in this test.
What has to be considered during blood collection?
The venipuncture tubes have to be filled to the dedicated volume with blood. Insufficiently filled tubes (< 50%) lead to higher EDTA concentration in the sample and may lead to false negative results.
Does the assay have to be performed immediately or can blood samples be stored overnight?
Blood samples should be used within 48 hours for allergens such as insect venoms, environmental, occupational, inhalants and food. Samples using drug allergens and food additives should be analysed within 24 hours.
T cells also express CCR3 at low levels. What is the impact of CCR3 positive T cells on the assay?
It has low impact. For 8 samples that were double stained using anti-CCR3 and anti-CD3 antibody, the mean % of CD3 positive cells within the population of double-stained cells was 3.9%.
What should be done if patient background is elevated (>5%)?
Basophils are very sensitive cells. Avoid shear stress. Always use deionized, double distilled water that is ultra-filtrated. Furthermore, make sure that no contamination occurs during the assay (e.g. through dust, pollen, etc).
What if the three main cell populations (lymphocytes, monocytes and granulocytes) do not appear on the FSC/SSC plot?
Check whether assay procedure has been followed according to the IFU (especially insufficient cell lysis may interfere since contamination of erythrocytes may occur), check instrument settings and adjust voltage.

 

Cellular Allergy – CAST® ELISA

Does the assay have to be performed immediately or can blood samples be stored overnight?
Perform the cell stimulation within 24 hours after blood collection. Store blood samples if necessary refrigerated at 2-8°C.
Which blood and how much is needed for the assay?
Use only EDTA whole blood and collect sufficient into EDTA venipuncture tubes. 200 µl of whole blood are needed per reaction tube. Calculate the amount of blood needed. For further information see also IFU on page 4.

 

Cellular Allergy – CAST® Allergens

Are the CAST® allergens CE-marked?
All BÜHLMANN allergens are CE-marked in combination with the CAST® assays.
What are the storage conditions of allergens?
CAST® Allergens can be stored unopened at – 20°C until expiration date. Reconstituted allergens should be used immediately. Reconstituted bee venom and wasp venom allergens are stable for 1 month at -20°C.
Are CAST® allergens from native source?
CAST® allergens are partly recombinant and partly from native source. Contact your local distributor for more information.
Are there any allergen included in the assay?
Allergens have to be ordered separately.


Clinical Chemistry – ACE kinetic

What are the interference limits of the ACE assay in lipemic serum samples?
Lipemic sera may be used up to a serum concentration of 250 mg/dl. 
What are the interference limits of the ACE assay in icteric serum samples?
Icteric sera may be used up to a serum concentration of ≤ 20 mg/dl
Can the assay be performed in hemolytic samples?
No, hemolytic samples cannot be used.
How is the ACE activity defined?
One unit of ACE activity is defined as the amount of enzyme required for release one µmol of hipuric acid per minute and per liter of serum at 37°C as determined by the colorimetric assay.
What is the functional sensitivity of the ACE assay?
The functional sensitivity is roughly 12 U/l. 
Can the BÜHLMANN ACE assay be used for estimating ACE levels in urine?
No, the intended use of the assay is for serum only.
What clinical analyzers can be used for BÜHLMANN ACE kinetic assays?
BÜHLMANN offers CE-marked settings for various clinical analyzers from all major manufacturers. Contact your local distributor for more information.

 

Clinical Chemistry – ACE high sensitive

How is the ACE activity defined?
One unit of ACE activity is defined as the amount of enzyme required for release one µmol of hipuric acid per minute and per liter of serum at 37°C as determined by the colorimetric assay.
How long can CSF samples be stored prior to the ACK assay?
CSF samples can be stored for 24 hours at 2-8°C or at -20°C for longer time periods.
What is the functional sensitivity of the ACE high sensitivity assay?
The intended use of the high sensitivity assay for ACE is to evaluate ACE levels in cerebrospinal fluid (CSF). The functional sensitivity is roughly 1.0 U/l.
Do I have to dilute the CSF samples prior to the ACE assay?
No, CSF samples have to be used undiluted.
What clinical analyzers can be used for BÜHLMANN ACE kinetic assays?
BÜHLMANN offers CE-marked settings for various clinical analyzers from all major manufacturers. Contact your local distributor for more information.

 

Clinical Chemistry – GHB

What are the interference limits of the assay in lipemic serum samples?
No interference is detected (further details see GHB IFU page 4).
What are the interference limits of the assay in icteric serum samples?
No interference is detected (further details see GHB IFU page 4).
What are the interference limits of the assay in hemolytic samples?
No interference is detected (further details see GHB IFU page 4).
What are the interference limits of the assay with ethanol?
Up to 3‰ the measured GHB concentration is below 10 mg/L. 1 g/L Ethanol raises the GHB value by 3 mg/L.


Neuroimmunology

What is the grey zone in the quantitative measurement of anti-ganglioside antibodies?
It reflects the dynamic properties of peripheral neuropathies. Patients within a grey zone should be considered for re-testing and further clinical investigations.
Why are several incubation steps performed at 4°C?
Our highly specific anti-ganglioside antibodies react with low affinity and are therefore performing best at lower temperatures.
What is meant by the two-step diagnostic approach?
The GanglioCombi Mag™ ELISA offers the possibility to screen in a first step using the IgM/IgG-mix, while in a second step the presence of antibodies using the specific IgM or IgG enzyme labels is confirmed.