Platelets - Science topic

I suppose you mean `high on-treatment platelet reactivity'. Several tests are used for diagnosis, thus the definition of 'normal values' and the cut-off for positive diagnosis depend on assay used. Generally, the 50% or more inhibition of platelet reactivity is considered as 'adequate' response to clopidogrel.

Hello Silvia Clapauch ,

Good to know! Thank you for all the information.

All best,

Alexandra

Also, kindly check:

I might be a bit late, but could you try activating the platelets, add a fixation step of 15 min in paraformaldehyde (1% final concentration) to prevent platelet activation during further steps and then add Ca2+ to allow annexin V labeling?

Functional platelet studies after a period of high interest are currently less frequent because attempts to correlate in-vitro outcomes with the in-vivo platelet function have not been encouraging. So, thromboelastography and aggregometry with ADP, ristocetin and other agents are practically abandoned. The in vivo studies, such as the bleeding time or the vascular fragility test, are burdened by the lack of standardization and, in any case, they are global tests that investigate different phases of the platelet vascular response. Ultimately, only generic automated tests such as total and reticulated platelet counts provide partial information that is not always easily interpretable.

I think it carries very little sensitivity.

Dear Fang Yafei!

You should add calcium chloride or calcium gluconate. It can resolve your problem.

Sincerely yours,

Artem

Not sure to fully capture the question about the Fc fragment but it may bind to many cells - from lymphocytes, basophils, ..., platelets. So you may have non specific or false positive signals when performing your flow cytometry. Best is to use blocking reagents (IgG, anti-Fc receptor antibodies) to saturate the receptors before staining your cells with labeled antibodies.

Red blood cells should be eliminated. Lysis would help to clear some confounding factors and to sort your flow cytometry deliverables.

Most of the time : Ice-cold ammonium chloride lysis buffer : NH4Cl, NaHCO3, EDTA, and washed several times along the centrifugation with PBS.

There are gels to remove albumine from serum samples for proteonics studies, check whether these can be used for exosome preparations. Alternatively, spin the exosomes through a sucrose cushion, soluble proteins should stay in the sample zone. With metrizamide such cushions can be even isotonic, but it is much more expensive.

you can wash the platelet in tyrod buffer and then assay the protein content

You can see how we did test mice over time:

Dear O. P. Strakhova,

I still believe that Gilbert's syndrome has to be excluded as a causes for the mildly elevated indirect hyperbilrubinaema and bradycardia.

Regards.

You can check it on hemocytometer using Trypan blue dye at 20X objective and look for any round bright dots

It would be better to use a line graph with markers of days on the abscissa and quantitative indicators of cell composition on the ordinate, on my opinion.

Dear Ma'am Savannah Free please read these articles, they might be helpful for your study.

You can identify the RBC quite nicely according to forward and sideward scatter. Platelets are much smaller and you can gate most of the WBC away. I would not necessarily freeze the RBC before flow cytometry. The type of staining you use, depends essentially on what you want to do research on.

Above both are correct

Thank you very much! So, if I unterstood well, you are using the whole blood, then stain and fix or fix and stain? I will probably follow fixation after staining. What did you mean by diluting with staining solution?

Some authors describe it as an excessive amount of protein in the serum. Take a look at "Morin S et al. A GroEL homologue from endosymbiotic bacteria of the whitefly Bemisia tabaciis implicated in the circulative transmission of tomato yellow leaf curl virus. Virology. 1999;256(1):75-84."

Completely agree with Waltraud C Schrottmaier comment.

The concentration of stimulant matter a lot. At higher concentration the stimulants become cytotoxic. You must titrate the amount of antibodies in different assays. Most of the antibodies works well in 1:10 to 1:50 dilution of recommended concentration.

Dear Kritika Ramchiary to you first question:

You can count the blood components by flowcytometry just by adding true count beads to the sample while acquiring.

Otherwise you will get the counts but not the concentration (numbers/uL). SO the true counts beads has a defined number in per microlitter volume. you add 10 microlitter beads to sample and take 900 beads/ ul is their defined value. so when your cytometer will count 900 beads in its column, it will consider it 1 ul volume and will give you value of L, M, N in per microlitter.

You can definitely count RBC as well by this method, but you know RBC are seen in Log scale while WBCs are seen on Linear scale in flowcytometer, so getting value of both the cell types need to be optimized. It might be done (does not seems impossible), if you want to do.

To your next question:

Yes RBC and WBCs can be differentiated by their scatter pattern as well as cell surface markers.

As far as the suggested methodology with hematocrit capillary, numerous unknown variables interfere with the supposed counting, PLT volume in primis. As an example, some thrombocytopenias having an increased MPV could have a falsely higher "counting" with that method

Hi Rh Rj, it depends on the platelet preparation methods that you use. I add PGI2 to prevent platelet aggregation during the centrifugation steps. PGI2 gets deactivated after 30 min. Therefore, I wait for at least 30 min (it is called resting) at RT on my bench, I do not agitate them during resting. Then you can start with the experiments.

Your washed platelets shouldn't have any extracellular fibrinogen so you can safely activate them with minimal aggregation. A simple procedure I have used to activate washed platelets and fix them is to:

Hi Rh Rj . Have you tried treating PRP with PGE1 before washing? Also platelets need constant rocking motion.

Dear Rh Rj , We usually keep our cells in 1% PFA for overnight at 4 degree C and treat at 4% PFA for 10 minutes at RT.

In one of our research on Monocyte-Platlet aggregation, we fixed the platlets as we all lysed RBC at once with RBC lysis buffer (BD Biosciences).

Lowering concentration of PFA need to be compensated with increasing time.

Dear Basak!

Yes, you can use MPO to study neutrophill-platelet aggregation

Freshly washed platelets (2×108 cells/ml) were incubated with gentle shaking in a phosphate buffered saline (PBS) (10 mM Na2HPO4/KH2PO4, 137 mM NaCl, 2.7 mM KCl, pH 7.4), containing 1 mM CaCl2 and 0.5 mM MgCl2 with or without MPO for 10 minutes at 37°C. In some experiments cell suspension was supplemented with sugars (α-methyl-D-mannoside, lactose or N-acetyl-D-glucosamine at the final concentrations of 60 mM) or either with 50% embryonic calf serum or platelet-poor plasma. Then cells were applied on poly-L-lysine-coated glass slides. After 30 minutes the samples were washed with PBS and fixed in 4% paraformaldehyde in PBS for 10 minutes, then again washed with PBS. To visualize MPO, cover slips with platelets were incubated for 1 hour in PBS containing anti-MPO antibody (1:5,000 dilution). After being washed with PBS three times to eliminate the excess of antibodies cover slips were kept for 1 hour in PBS containing anti-rat IgG-FITC antibodies (1:10,000 dilution). Then cells were again washed three times with PBS before mounting on slides. Images were acquired using laser scanning confocal microscope LSM 510 META (Carl Zeiss, Germany) with an immersion lens Plan Apochromat 63×/1.4 Oil (Carl Zeiss, Germany) and processed using LSM 510 software.

Platelet aggregation studies were performed using both optical and impedance aggregometry. Platelet aggregation was detected by recording changes in light transmission at 540 nm of cell suspensions at 37°C on a computerized aggregometer AP 2110 from SOLAR (Minsk, Belarus). 400 µl PRP (2.5×108 cells/ml) or washed platelets (2.5×108 cells/ml) in PBS, containing 1 mM CaCl2 and 0.5 mM MgCl2 were incubated at 37°C with stirring for 3 minutes (with or without MPO) before adding an agonist (ADP to PRP and thrombin to washed platelets). Aggregation curves were recorded for 10 minutes. To quantify agonist-induced aggregation we used the maximal rate of cell aggregation calculated automatically by aggregometer software. Electronic impedance aggregation measurements were performed using an impedance lumi-aggregometer Chrono-log 700 (USA). An aliquot of whole blood (0.5 ml) was diluted with an equal volume of prewarmed isotonic saline and incubated for 5 minutes at 37°C with or without MPO. Impedance of each sample was monitored in sequential 1-minute intervals until a stable baseline was established. Then ADP (5 µM) was added and aggregation was continuously monitored. Impedance aggregometry results are expressed as amplitude (or maximum aggregation) [ohm] at 6 minutes after reagent addition.

Is it possible that some of the platelets have degranulated prior to fixation? What are you using for fixation and is it fresh? I would also recommend going back to your FSC and SSC plots and seeing if the phenotype segregation can be attributed to differences in size (FSC and /or granularity (SSC)

After viewing the images change of media not suggested

CM-H2DCFDA dye was used for analysis the oxidative stress of cancer cells after drug treat. In here we did not fix the cells. Analysis with FCM. In cell cycle analysis cells was fixed with 70% ethanol.

Thanks all for the responses. No easy answer it seems! I've ordered another set of tests to be run on plasmas that were collected at the same time as the serum to see if there is any difference.

@Jan - will look at the platelet count as you suggested. This is probably relevant to the pathology I'm interested in too!

hi Ayah Al Qaryoute

-- For microRNA work, go for Qiagen products, their all microRNA research products are the best in the market.

-- You can isolate total RNA from your samples using TriZol method. Then, you can use the Qiagen miscript-II-RT kit for cDNA synthesis. This kit will convert all miRNA present in the sample into miRNA-specific cDNA. Now, you can analyze your miRNA of interest by SYBr qRT-PCR using miRNA primers.

-- Even you can buy specific miRNA isolation kit and isolate miRNA. But ultimately cDNA synthesis will matter for further qRT-PCR.

Keep this point in a note that

-> All miRNA cDNA synthesis kit not works with SYBr, many only works with Taqman. So before buying check its requirements.

-> Prefer to use a pre-designed miRNA primer.

-> Use at least 3 endogenous control.

Now, miRNA primer has universal sequences. you will find the stem-loop & mature miRNA sequence in the miRDB database. Well, as I said, prefer predesigned primers of your interested miRNA from any standard company. Nowadays, Companies do a slight modification or say a technology, which enhances stability and avoids unwanted binding. Qiagen has LNA tech. based miRNA primers, those are readymade, pre-mix (FP+RP), available for most of the miRNAs. Just dissolve and use 1ul for per rxn.

Regards

Saurabh

Kanika Arora is correct; Annexin V will not bind without Ca+2 present at 5 mM. Diluting the Ca+2 leads to loss of signal. And there are frequent problems with staining permeablized cells. B-D does make a permeablization/fixation kit that seems to work well.

But I am curious at to why you are using Annexin V to light up your platelets. Annexin V binds to phosphatidyl ethanolamine [PE], which is on the inner cell membrane. It only migrates to the outer cell membrane during apoptosis due to loss of membrane polarity.

Wouldn't it be easier to visualize your platelets in FL1 with anti-CD41 FITC or anti-CD61 FITC?

Dear Dr. Ahmed T. Hussein , Thank you for this interesting question.

Let me share our experience.

In India, the people suffering from Dengue, used to take papaya leaf juice (as fresh) or papaya leaf tea ( papaya leaf used instead of tea leaf)- this is in addition to normal medical treatment.

Though the platelet count comes down, it never goes down to the extent of a life threatening situation.

Personally, I used to take once in a while - when I have body pain - it acts as a natural pain killer.

Report:

Source: https://www.ndtv.com/india-news/dengue-on-the-rise-does-drinking-papaya-leaf-juice-really-help-1715193

Thanks and warm regards

you might try passing the platelets through decreasing bore size (diameter) needles. Mechanical stimulation is the old man's trick to degranulate platelets. ADP sounds like a great, more physiological stimulator.

Thank you Muhammad Irfan I will check my fixation technique!

I have made considerable studies on neonatal thrombocytopenia in Nigeria and will gladly join your team to investigate more on newborn platelets. My PhD thesis was on aetiology and pattern of neonatal thrombocytopenia at birth in Delta State, Nigeria. Other similar publications include

1. prevalence of thrombocytopenia at birth among apparently healthy newborns in Delta State, Nigeria

2. Alterations in platelet-to-Lymphocyte ratio and some white blood cell indices in neonatal thrombocytopenia

3. Submitted for publication: Some maternal and neonatal risk factors associated with early on-set neonatal thrombocytopenia in Delta State, Nigeria

4. Submitted for review: Variation in some haematological parameters in the newborn at different locations in Delta State, Nigeria

I have over 12 other publications in reputation journals.

My research experience with add value to your Lab

For both questions, the answer is the same. Yes you can. The important thing is to anti-coagulate the blood.

We usually collect whole blood into acid- citrate-dextrose (ACD, 3.2%) sterile tubes. Depending on your read-out you can use this blood for flow cytometry, alternatively you can subsequently isolate the platelets.

Briefly, the whole blood needs to be first centrifuged at 150xg for 20min at room temperature (RT). The platelet-rich plasma (PRP) then needs to be removed and PGE1 was added to the PRP to prevent exogenous platelet activation. The PRP is then centrifuged at 400xg for 20min at RT. The platelet pellet was re-suspended in warmed (37oC) PIPES saline glucose (PSG).

More details can be found here:

PMID: 32573711

PMID: 31366617

Hi Liliana,

1. Make your titration with activated platelets, normally you will not see any positive result for CD62P on unactivated platelets. but because you are using PRP there will be low level of activation due to centrifugation and preparation phase.

2. Your dilutions seemed quite high to me for antibodies to be used on flow, are you using a primary antibody? In this case, our platelets will be even more stimulated while you are preparing for secondary antibody. You can decide through the amount that does not effect the staining index, best is to make a lineer graph to see where you obtain the same results (mean/median channel) with suggested quantity.

3. To use geometric mean or median depends on the cytometry you are measuring, but as long as you are using the same parameter for all your data in this research you will be fine. The best way to present your data would be following MiCyt Guidelines available at below links:

With best wishes,

Mouse platelets were most sensitive to both agonists. Unlike in man and dog the maximal response to ADP was greater than to thrombin in mouse, rat and guinea pig. P2Y(12) blockade was in all species equally effective as ADP removal in inhibiting thrombin-induced platelet activation whereas P2Y(1) blockade was almost ineffective.

GF biomarkers act on stimulation of PLT for its functions

Daer Asghar!

Please you look at this article:

The microtubule cytoskeleton

PLT shape and integrity are maintained by a well-defined and highly specialized cytoskeleton composed of an intricate system of molecular struts and girders that also regulate PLT size. Likewise, released PLTs contain 10- to 20-fold increased numbers of peripheral microtubule coils, relative to normal controls, that are often organized like balls of yarn rather than rings, which may explain why the majority of these cells have a spherical rather than the discoid form. The inability to further constrict proPLT tips before abscission resulting from bending energies determined by the cortical microtubule band could account for the increase in terminal PLT size.

Mega Platelets are pre mature one

enclosed

There are several articles on the use of antiplatelet therapy in acute coronary syndrome.

Thank you Joyleen Maryann Fernandes.

Dear @ Igor,

Please open the attached link below -

Hi Malcolm,

Thank you very much for the suggestion. I will look into this kit.

firstly, to minimize the platelet contamination from PBMCsw, best way is multiple washing with PBS at low speed for longer time.

And to cryo-preserve buffy coat, you should use 90%FBS+10% DMSO, but if you only going to extract nucleic acid from cells, Using 90% Complete medium (90% RPMI + 10% FBS) with 10% DMSO may also work.

Few people use lower concentration of DMSO as, DMSO hinders in the downstreaming process (DMSO is know inhibitor of PCR). SO, you may use as low as 7% DMSO.

All the best.

Please go through the article "Basics of Estimating Measurement Uncertainty" by

Graham H White.

This happens with every instrument based on laser, as lasers have their defined life (few thousand hours). So, at voltage "X", you get MFI "999" as the time ( consumption hour) passes, your laser weakens so you give "X-x" voltage, so it leads to decreased MFI.

With time, you need to increase voltages to make a constant incident light, which further will maintain the constant MFI.

There are certain beads (Flosser beads), which maintain the system for you. Once you run the beads and set the voltage, it set it for you.

Anything you find any changes in MFI (suspect of power of incident light), u need to run Flowset beads (according to manufacture protocol) to automatically set voltage to gain similar output.

Hope, it was helpful.

If the problem is the total volume in the tube, you could mix after the first centrifugation, when you take the "cloud" of PBMCs... All from the same person I suppose :D

A brief literature search will enable you to find papers describing methods for preparation of platelet membrane proteins. Here are a few examples:

I also have some PDW data in % and wish to convert to fL.

Both can be used. Heparin is batter

Gating from the CD41 or CD61-SSC dot plot, which can effectively remove the interference of debris and impurities.

RBC in PRP is a contamination and it should be free of RBC.

Hi Sreeja Narayanan,

Due to the small period of conservation (under 5 days) of the platelets, it's very difficult ( not impossible) to find them outside your place, as the colleagues propose, the best way to get them functional is by contacting the nearest blood bank in your place, however, when you obtain them ensure strict transport procedures like continues agitation in temperature between 21 to 22 C°, in the other side confirm whether you need the platelets functional or not, because as you mentioned above you need to study their membranes, in this case, I think you should do the study on both periods: under 5 days and above 5 days and compare. If the results are the same in this case you can easily get them after 5 days.

Good luck

Dear you can use other Haemtology autoanalysees to do it.

HI André Rapaille, this is very good compact equipment with 2 laser 4 fluorescent detectors. we have used this equipment and working on both the software (Research and clinical). Do let me know on parvindsingh1995@gmail.com for any specific query.

If you are going to inject them I believe the body will take care of necessary pH adjustments.

Hi, try preliminary cold plasma treatment with oxygen or air.

Thanks alot sir for precious suggestions!

many thanks Emmanuel (and sorry for the delay!)

Mononuclear cells sometimes get activated in thaw cycle and start pro coagulant activity. This paper is helpful.

Thanks Emmanuel Ifeanyi Obeagu, Dharmesh Chandra Sharma, Gayle Haider for your remarkable comments.

Hi Christian

There is a cheeky way to collect white cells including monocytes from platelet apheresis sample. If you have a leucocyte depletion filter for platelets, put the apheresis sample through it and then flush the white cells out from the filter using saline. You could then do ficol sedimentation to get the mononuclear cells. Good luck.

Siva

Part of the answer comes to how the PRP is prepared

Neutrophils phagocytose activated platelets so it is useful to exclude them from your PRP. However they also phagocytose bacteria.

PRP can be applied topically to the ulcer and will aid the healing process. It can also be applied to burns and will help stop secondary infection as well as speed up the healing process. It is one area of use where PRP that is prepared to include neutrophils has apparently been shown to be more effective since the beneficial effects outweigh the platelet phagocytosis effect

I have only heard this as I have not used it on ulcers/burns myself but it certainly makes sense!I cannot give you references

I have also heard that the PRP can be injected into the ulcer as well as applied topically

Dengue virus does not attack the blood platelets. The latter are destroyed due to autoimmune reaction induced as a result of infection by dengue virus.

First, I would try to make sure you still have platelets in your flow samples. We focus on PMNs and PBMCs and find platelets are so often washed off in sample prep (and aggregates filtered out) we don't even consider them. Concentrate your flow samples (after all filtration, etc.) and have a look with a light microscope to see if you have any platelets left. If your marker is working for platelets but also granulocytes, you should be able to separate those by FSC-SSC.

I'm sure you've seen this, but just in case...

http://www.bloodjournal.org/content/121/10/e70.long?sso-checked=true

Also, you can always try to find them by a process of exclusion - find a granulocyte specific marker, lymphocyte and monocyte specific marker and platelets should be what you have left.

Dear Bahare,

Platelets are "pieces" of megakaryocytes and have no nucleous. So, it is impossible to cultivate them.

Regards,

Nelder Gontijo

Thank you very much for your answer.

interested

Hi Jamie,

Isolation of extracellular vesicles can be a challenge. There are some factors in such a workflow that is important to pay attention to when designing the protocol. One such critical factor is the amount of beads used.

If the downstream application is flow cytometry it is important to use as few beads as possible. typically we use in the range of 40 µL Dynabeads (from 1x10e7 beads/mL vial) in a 100 µL sample volume. Those few beads will capture max number of exosomes/bead = strong signal in flow. However, given the low number of beads used - this is not isolation since most of the exosomes will be remaining in the sample.

For isolation and downstream applications such as western blot the use of beads is completely different. here, as many beads as possible is required in order to get a good WB signal (those beads will give a very low flow signal). Here we typically use 40 µL Dynabeads (from 1.3x10e8 beads/mL vial).

Other factors that is important to pay attention to is the incubation time. Extending the incubation time - even to over night increases the capture significantly.

Feel free to get in touch at ketil.pedersen@thermofisher.com

kind regards

ketil