Nicole Gillain-Martin

Immunology, Human Biology

23.11

Questions and Answers

  • Nicole Gillain-Martin added an answer in EDTA:
    Is there any method to convert EDTA-plasma to serum?

    We have harvested a lot of discarded plasma samples from blood donor archive, which is containing EDTA as anticoagulant. Is there any method to convert EDTA-plasma to serum? Any idea is welcome. Thanks a lot! 

    Nicole Gillain-Martin · CHR Citadelle Hospital

    I agree with Mr Banerjee. Obviously, this will only be possible if the component you want assayed does not depend on the presence of calcium (or Mg)  or is not complexed by EDTA

  • Nicole Gillain-Martin added an answer in Clinical Diagnostics:
    A red crystal-like substance in urine. What can this be?

    I am sure this question is going to be one of the more peculiar ones ever asked here, so please forgive the strange atmosphere of the question! Being that I own my own laboratory and I wanted to learn more about urine (and it's components), I let curiosity get the better of me and decided to test whether or not i could separate urine from any of its components by gravity just for fun. Long story short, I collected my urine in a 50ml centrifuge tube and centrifuged it at various paramaters. Nothing happened during this time, however I was called suddenly away from the lab, and in a hurry, I mistakenly forgot to dispose of the urine in the tube before leaving! After returning about two and a half weeks later, I removed the centrifuge tube containing my urine from the rotor to find that there is a red crystal like growth on the wall of the tube.

    As I said, I do not know much about urine and I wanted to know if this was normal or not. What could the red crystal-like substance be? The centrifuge tube was sealed by the way and was never opened foe the entire time that it contained urine. The growth is quite visible and has the appearance almost identical to a naturally growing quarts or Amethyst crystal(but it is dark red). Any ideas what this is? Is is normal? Thanks!

    Nicole Gillain-Martin · CHR Citadelle Hospital

    I would have also thought of urate that occur naturally in many urine stored in the refrigerator. Your urine has reached room temperature, so it is certainly alkalized.

    Think of ammonium urate and also to phosphate crystals

    To reassure you, ask a specialist to quickly carry out a review on a morning urine sample given in the shortest possible time in the laboratory!

  • Nicole Gillain-Martin added an answer in Valproic Acid:
    What is the next test you would order for a patient with isolated ALP elevation?

    One of my patients, brought me an ALP=669 in an exam order by a psychiatrist because of the use of valproic acid (ALT, AST, GGT and Bilirubin are normal). I have already ordered serum Calcium, serum total protein and albumin, TSH and PTH. I wander if it is essential to order a bone scan.

    Nicole Gillain-Martin · CHR Citadelle Hospital

    I fully agree with Mrs Poggiali,: it is essential to achieve electrophoresis of iso-PAL will specify hepatic or bone origine. Macromolecular could be present, but should not be responsible for such an increase without being accompanied by an increase of GGT.

    Still think a transient hyperphosphatasemia (very rare in adults) and check the persistence of high value

    This patient is not suffering from kidney failure, I suppose

  • Nicole Gillain-Martin added an answer in Antibody Generation:
    Are serum levels of IgG, IgA or IgM antibodies generally known to change with age?

    In healthy individuals, do serum levels of any of the above immunoglobulin isotypes tend to increase or decrease? I can't seem to find any reference to them changing in a set pattern, but I may be looking in the wrong place/searching the wrong words..? Many thanks in anticipation..

    Nicole Gillain-Martin · CHR Citadelle Hospital

    It is usual to consider that the immunoglobulins increases with age, but the data are sometimes conflicting. This is likely due to the observed differences based on race (white or black) and gender. Some factors must be considered such as smoking and alcoholism. The recruitment of the reference population can have a great influence on the conclusions of the study. Read about it: A. Gonzales-Quintela & coll: serum levels of immunoglogulins (IgG,IgA,IgM) in a general adult population and their Relationship with alcohol consumption, smoking and common  metabolic abnormalities, Clinical and Experimental Immunology, 2007, 151:42-50.

    In practice, immunosenescence does not cause a reduction of the production of immunoglobulins, but modifications of the "quality" of these immunoglobulins (lack of affinity, antibody activity)

  • Nicole Gillain-Martin added an answer in Metformin:
    What is the cause of low hemoglobibh in 65 year old male with diabete?

    A 65 male patient with diabete has the following serum biochemistry results.

    blood glucose 120 mg/dl and taking glyclzide metformine and pitoz-Hemoglobin is 12g/dl which is low-serum iron is normal-serum ferritin is normal-RBC is 4.4,vitamine B12 is normal.What is the cause of Low hemoglobin.What is your suggestion for treatment for low hemoglobin?

    Nicole Gillain-Martin · CHR Citadelle Hospital

    Does the patient have a chronic inflammatory syndrome, even discreet?
    (sedimentation rate, electrophoresis ...).

    Normal ferritin may correspond to an iron deficiency masked by this inflammation that will raise ferritin.

    It is also important to know the corpuscular volume and mean corpuscular hemoglobin concentration and the reticulocyte count.

    The patient's hemoglobin is slightly reduced and that is really worrying if you see in him a drop compared to previous values. Then seek occult blood loss

  • Nicole Gillain-Martin added an answer in LDH:
    How do you separate LDH iosenzymes?

    Which method is the best to separate LDH isoenymes in med lab?

    Nicole Gillain-Martin · CHR Citadelle Hospital

    You may use the Sebia kit for Hydrasys. It is semi-automatised.

    www.sebia-usa.com/products/ISO-LDH.html

    For more manual, use Helena Laboratories

    www.helena.com/Procedures/Pro077Rev3.pd

  • Nicole Gillain-Martin added an answer in Creatinine:
    Does anyone have knowledge on serum creatinine reference values in vegetarians?

    I just find one publication (1989) but I don't understand the serum creatinine values.

    Nicole Gillain-Martin · CHR Citadelle Hospital

    your reference provides information on serum creatinine conducted by the Jaffé technique (warning before the publication date of the new criteria of standardization of this technique)

    in reference population: 8.1 (M) and 7.6 (W) mg/L and in vegetarians 7.7 (M) and 7.8 (W) mg/L. What is surprising is the highest value in women vegetarians.

    to convert mg/L to mmol/L, multiply by 8.85

  • Nicole Gillain-Martin added an answer in Uric Acid:
    How long can I store on ice a blood sample which was frozen and unfrozen?

    People! I collected blood samples, separated the plasma and frozen it, but when the samples were transported to my laboratory, they unfrozen.. I don't know how long they remain in this situation (unfrozen). Do you think I can use them to analyse lipids, triglycerides, uric acid, cholesterol, lactate? I can't lose them it's for my mastership!

    Nicole Gillain-Martin · CHR Citadelle Hospital

    The components that you measure are quite stable except lactate (it is preferable to take it on oxalate/fluoride and centrifuge as soon as possible). Are the samples sented immediately or are the samples stored frozen for several days before sending? If samples are sent the same day and treated after 24 hours, not frozen samples in transport box for blood and ice pack seems sufficient. Make a simulation of the treatment of your samples to see if it has an impact on results.

  • Nicole Gillain-Martin added an answer in beta 2-Microglobulin:
    Any suggestions about a huge inter-assay variation with beta-2-microglobulin ELISA test?

    I am trying to measure Beta-2-Microglobulin concentration in urine samples from patient with renal disease, so the concentration is expected to be high. I first used abcam SimpleStep ELISA kit then R&D Quantikine to confirm. But the inter-assay variation is so big (>100%) leaving me no idea which is the right result. Can anyone suggest how to make these result more reproducible and reliable? Thanks

    Attached is some of the results on a spreadsheet.

    Nicole Gillain-Martin · CHR Citadelle Hospital

    I never had any problems with the assay of urine beta2microglobuline, for low values or high values. But I used a nephelometric technique (BNII Siemens) and a reagent consisting of latex particles + antibodies to increase sensitivity. Previously some laboratories used RIA methods to ensure sufficient sensitivity.

    Are you sure that the ELISA method is sensitive enough?

    As said Mr. Goutam Pandey, it is important to centrifuge the urine to avoid any  interference

     

  • Nicole Gillain-Martin added an answer in Bladder Cancer:
    Where does this protein come from?

    We are checking a protein in the urines in order to investigate its role as a marker for bladder cancer but, since its molecular weight is 45 Kda, how can I be sure that this protein come from the epithelium of the bladder or from somewhere else (passing through the glomerular barrier)? Thank you in advance for your help.

    Nicole Gillain-Martin · CHR Citadelle Hospital

    Usually the proteins present in the urine following a tubular or glomerular disease do not have this MW. It should however remember that light chain immunoglobulins can polymerize and have at that time a MW near 45 

  • Nicole Gillain-Martin added an answer in Aging:
    What is difference between the terms aging, old age, and elderly?
    see above
    Nicole Gillain-Martin · CHR Citadelle Hospital

    I found this interesting paper on Google:

    Ageism in Language

    The American Psychological Association (1) states in Section 2.17 Age, (p 69):

    “Elderly is not acceptable as a noun and is considered pejorative by some as an adjective. Older person is preferred. Age groups may also be described with adjectives: gerontologists may prefer to use combination terms for older age groups (young-old, old-old, very old, and oldest old), which should be used only as adjectives. Dementia is preferred to senility; senile dementia of the Alzheimer’s type is an accepted term.

    The American Medical Association states in Inclusive Language Section, 9.10.3 (p 268):

    Age.--Discrimination based on age is ageism, usually relevant to older persons. Avoid using age descriptors as nouns because of the tendency to stereotype a particular group as having a common set of characteristics. While in general the phrase the elderly should be avoided, use of the elderly may be appropriate (as in the impact of Medicare cuts on the elderly, for example). Otherwise terms such as older person, older people, elderly patients, geriatric patients, older patients, aging adult, or the older population are preferred.

    Note: In studies that involve humans, age should always be given specifically. Researchers in geriatrics may use defined terms for older age groups, eg, young-old (usually defined as 60 or 65 to 70 or so years) and old-old (80 years and older).

    In an ASA Connection newsletter (3), the following responses were reported for the prior month’s Question: What terms do you think are appropriate when referring to people ages 65-plus? Older adults, 80%; elders, 41%; seniors, 33%; senior citizens, 11%; elderly, 10%. Note: Total is >100% because respondents could select >1 answer. ASA Connection provides updates on events in aging, research and policy developments, and innovative practices. The newsletter is distributed monthly to members of the American Society on Aging and other professionals in the field of aging.

    “Senior” is considered passé, especially by today’s 78 million baby boomers. Recently some YMCA organizations have begun to refer to the older adults who participate in their fitness or physical activity programs as AOAs, ie, Active Older Adults.

     References:

    1.    American Psychological Association. Publication Manual of the American Psychological Association, 5th ed. Washington, DC: APA; 2001.

    2.    American Medical Association. American Medical Association Manual of Style, 9th ed. Baltimore, MD: Williams & Wilkins; 1998.

    3.    ASA Connection. June 2007. Available at www.asaging.org/ .

    may 2007

    National Resource Center on Nutrition, Plhysical Activity & Aging. Florida International University: http://nutritionandaging.fiu.edu.

  • Nicole Gillain-Martin added an answer in Polycythemia:
    What is the normal blood concentration of Erythropoietin (Epo)?

    What is the normal blood concentration of Erythropoietin (Epo)? What is the minimum level reported in medical conditions like Chronic Renal Failure etc.? What is the maximum reported concentration in conditions like Polycythemia etc.?

    Kindly provide references.

    Nicole Gillain-Martin · CHR Citadelle Hospital

    Some clinicians give great importance to the relationship between the concentration of erythropoietin observed and the value that should be that of the patient according to the degree of its anemia
    Some laboratories provide reference values according to the hematocrit; below that of the laboratory of the University Hospital of Liège

    Hématocrite                     20

    Moyenne et extrêmes  370 (100- >1000)


                                           25
                                           170 (70- 600)


                                           30
                                           80 (30-230)


                                           35

                                          30 (15-50)


                                         40

                                         15 (11-22)


                                       45

                                        12. 2 (<8-20)


                                         50
                                        10 (<8-20)
     


     

  • Nicole Gillain-Martin asked a question in Aspirin:
    Why is Aspirin used for decades abandoned in favor of another AINS?

    despite its anti-inflammatory properties and its role in the prevention of cardiovascular disease

  • Nicole Gillain-Martin added an answer in IgG:
    In ELISA IgG standards curve, why are my OD giving me a non linear increase but when i include concentration I don't get the sigmoid shape?

    I have done dilution series of the total IgG standards 12 times, when I check the od readings from different dilution series (1:2; 1:3; 1:10) they give a sigma shape graph, but when I align with the concentration (500ng) going down, it does not align well hence i loose the shape.

    The two graphs are attached.

    Nicole Gillain-Martin · CHR Citadelle Hospital

    Sorry for the late reply. You have acceptable calibration lines in some areas of concentrations (in yellow): Do not work with too high or too low concentrations
    The answer is the same in dilution and in absorbance (see example from your data)
    I find that variations in DO (or absorbance) is low.

  • Nicole Gillain-Martin asked a question in Chronic Renal Failure:
    What importance do you attribute to the type of protein responsible for proteinuria?

    The intensity of proteinuria is an important element to define the degree of chronic renal failure. As a clinician, what importance do you attribute to the type of protein (low/high molecular weight) detected?

  • Nicole Gillain-Martin added an answer in IgG:
    In ELISA IgG standards curve, why are my OD giving me a non linear increase but when i include concentration I don't get the sigmoid shape?

    I have done dilution series of the total IgG standards 12 times, when I check the od readings from different dilution series (1:2; 1:3; 1:10) they give a sigma shape graph, but when I align with the concentration (500ng) going down, it does not align well hence i loose the shape.

    The two graphs are attached.

    Nicole Gillain-Martin · CHR Citadelle Hospital

    I do not understand: the ordinate should be the same in the two graphs: the absorbance (as in 2)
    on 2; how concentration (abscissa) can it be zero?

    send me your numbers

  • Nicole Gillain-Martin added an answer in Vitamin D-Binding Protein:
    Which 'one' biomarker should I consider to detect nephrotoxicity induced by Herbicides in general?

    KIM-1, NGAL, NAG, alpha-1 microglobulin, cystatin-c or vitamin-D binding protein.

    Of above all, which 'one' biomarker should I consider to indicate an overall kidney damage in the patients exposed to higher levels of herbicides.

    364      
    Nicole Gillain-Martin · CHR Citadelle Hospital

    Thank you to Carl Wesolowski for this information about  this new approach to the determination of GFR (unknown to me)
    Isotopic techniques are unfortunately not always accessible

  • Nicole Gillain-Martin added an answer in Vitamin D-Binding Protein:
    Which 'one' biomarker should I consider to detect nephrotoxicity induced by Herbicides in general?

    KIM-1, NGAL, NAG, alpha-1 microglobulin, cystatin-c or vitamin-D binding protein.

    Of above all, which 'one' biomarker should I consider to indicate an overall kidney damage in the patients exposed to higher levels of herbicides.

    364      
    Nicole Gillain-Martin · CHR Citadelle Hospital

    NAG has the advantage of being easily measurable by colorimetry and easily automated.

    Consider also the low MW protein assay as alpha1microglobuline. It increases in urine soon after aggression and is also easy to measure. You can also assaying beta2microglobuline, but it is less sensitive. Urine cystatin is not a sensitive marker. 

    About NGAL, this marker is used primarily in the context of AKI, no personal opinion on the utility to detect a toxic effect in the short or long term
     

  • Nicole Gillain-Martin added an answer in Biomarkers:
    Which biomarkers could need a better assay?

    Hi everyone!

    We have found that a mass spectrometry based technology we are working on can do really fast measurements of targeted proteins.

    We would like to demonstrate the technology with established biomarkers that have known troubles with conventional assays (e.g. poor selectivity with exiting ELISA approaches etc).

    But being the dumb chromatography person that I am, I am not too familiar with which markers that are notoriously in need of better assays.

    Therefore: Do you have any suggestions for cool biomarkers to test a proof of concept for a novel MS tech?

    I wish you all a great weekend!

    Nicole Gillain-Martin · CHR Citadelle Hospital

    There are many difficulties with the assay of sclerostin with current kits

  • Nicole Gillain-Martin added an answer in Urolithiasis:
    What is the best method for collection of 24 hours urine samples for evaluation of promoters and inhibitors of urinary stone formation?

    medical management of urolithiasis in recurrent stone formation requires estimation of promoters and inhibitors of stone formation in 24 hours urinary and blood evaluation. Can any one give us the best method in collection of 24 hours urinary sample.

    Nicole Gillain-Martin · CHR Citadelle Hospital

    You are asking a question that I do not think anyone really resolved. Indeed, the suggested additive varies depending on the constituent analyzed. The urine stored at room temperature becomes alkaline which may alter the dosage of phosphate and calcium. For the determination of oxalic acid also it is advisable to acidify the urine. But this acidification  can be harmful as it will promote the precipitation of uric acid. In addition, acidification may alter some assays performed with enzymes. In practice, one does not use an additive. The urine should ideally be stored in the refrigerator or in a cool place during the 24H of the collection which is not always possible for the patient. So, if possible, the patient after placing the urine of the night in the container shall be to the laboratory as soon as possible so that the urine may be analyzed the same day. Do not exceed 24 hours. Say to the patient to bring all the bottles he had collected. This is the lab that must collect and homogenize them!

  • Nicole Gillain-Martin added an answer in Urolithiasis:
    What is the best method for collection of 24 hours urine samples for evaluation of promoters and inhibitors of urinary stone formation?

    medical management of urolithiasis in recurrent stone formation requires estimation of promoters and inhibitors of stone formation in 24 hours urinary and blood evaluation. Can any one give us the best method in collection of 24 hours urinary sample.

    Nicole Gillain-Martin · CHR Citadelle Hospital

    It is important to give the patient a suitable container to collect the urine of 24 hours, so a container that can hold at least 2 liters (diuresis may be more important in some patients) and if possible in which he can urinate directly. The patient should start collecting the morning at a definite time. The simplest is when patient rises. He urine as usual in the toilet, but from that moment, he must retain all its urines, absolutely all. If the patient urine once in the toilet, he will have to start all over again! He collects his urine  in the container before going to bed and get up at the same time as the previous day. He collects his urine into the container. He has a complete collection of 24H urine: day and night.
     

  • Nicole Gillain-Martin added an answer in Polycythemia:
    What is the normal blood concentration of Erythropoietin (Epo)?

    What is the normal blood concentration of Erythropoietin (Epo)? What is the minimum level reported in medical conditions like Chronic Renal Failure etc.? What is the maximum reported concentration in conditions like Polycythemia etc.?

    Kindly provide references.

    Nicole Gillain-Martin · CHR Citadelle Hospital

    The values observed in 170 healthy adults with normal hematocrit are: 3.3 to 23.4 mU/ml
    (from WE and WL Owen Roberts, Clinica Chimica Acta, 2011, 412: 480-482).

  • Nicole Gillain-Martin added an answer in EGFR:
    Is nutrition important in clinical treatment of severe reduction of eGFR?

    when  an  eGFR is 15 - 29 ml/min/1.73 mwhich part of treatment, either clinical action alone or Nutrition part- is really works to get back to 30-59 ml/min/1.73 m2 or the actions for preceding stages? or increase the ca absorption, lipoprotein activity or develop the metabolic consequences?

    Nicole Gillain-Martin · CHR Citadelle Hospital

    That's right, in current practice, nephrologists will not advise a restricted diet intake of proteins; according to them, the patients naturally eat less meat (natural disgust). This avoids denutrition. A restriction is only recommended if the patient has clearly a diet with excess proteins. The guidelines I gave you dated a few years, they are no longer applicable. Sorry for the misinformation.

  • Nicole Gillain-Martin added an answer in Buffer:
    Why is there no current during my electrophoresis experiment?

    During western blotting, there is no current flow (mA and Watt) during electrophoresis. i have checked the connection, ph buffer, make new buffer (1X, 2X and 5X), but still no flow of the current..can anybody help me with this kind of situation...TQ in advance.

    Really need help here.

    Nicole Gillain-Martin · CHR Citadelle Hospital

    I guess you place your gel on a plate to which are connected the electrodes. This plate should be wet before placing the gel on the plate (the manufacturer may recommend a product that promotes the flow of current)

  • Nicole Gillain-Martin added an answer in EGFR:
    Is nutrition important in clinical treatment of severe reduction of eGFR?

    when  an  eGFR is 15 - 29 ml/min/1.73 mwhich part of treatment, either clinical action alone or Nutrition part- is really works to get back to 30-59 ml/min/1.73 m2 or the actions for preceding stages? or increase the ca absorption, lipoprotein activity or develop the metabolic consequences?

    Nicole Gillain-Martin · CHR Citadelle Hospital

    Yes, nutrition is very important in renal failure, especially protein intake. Following P. Jungers (Necker Hospital, Paris), it must be of the order of 0.7 g / kg body weight / day if creatinine clearance is <20 ml/min/1.73m2,  of 0.8 if cl.creat. between 20 and 40, 1, if cl.creat is  40- 60 and < or = 1.2,  if cl. creat is > 60 ml/min/1.73m2. Not reduce protein intake too much especially in the elderly and ensure that abumine does not drop below 35 g / L. A supplement of vitamins, Ca, the control of the amount of water and ingested Na should be considered depending on the circumstances and the type of kidney disease. 

    For a recent development, see the clinical pratice guideline for evaluation and menagement of chronic kidney disease (KDIGO 2012)

    www.kidney-international.org

     

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