ACDA is what is commonly used in Blood Bank.

I use ACD-A for washed platelet and I use citrate for PRP. But it seems to be efficient to use ACD-A for the PRP because the first step of preparing washed platelet is to prepare PRP.

In my hospital ,Antico-agulant citrate dextrose-A (ACD-A) is preferred but Citrate Phosphate Dextrose (CPD) is also useful for PRP . It is similar to ACD-A but has fewer supportive ingredients and, is 10% less effective inmaintaining platelet viability.

Our hospital Blood Bank Department has always been using ACD-A platelet pheresis unit to prepare PRP (we called it as plasma reduced platelets) by centrifuging down 10 min. at 22 C for 10 min, and then expressing out the unwanted plasma to final net volume of 65 ml.  We have never used 3.8% sodium citrate platelets for patient's transfusion needs.

For ex vivo platelet aggregation studies, you need to use 3.8% sodium citrate. If you use ACD in PRP, the pH will be too low to allow platelets to function correctly.  You can use the ACD for washed platelet experiments, but here, you re-suspend the platelets in a buffer that will re-adjust the pH to 7.4 after centrifuging the platelets. 

For PLT aggregation studies, we use 3.8% sodium citrate.

ACD-A is the standard anticoagulant used in transfusion medicine services.

We have always used ACDA for PRP and for washed platelets.

For preparing platelet rich plasma for platelet aggregation studies you can use 3.2% sodium citrate solution.  However, it is important to note the proportion of blood to sodium citrate solution.  You have to mix 1 part of 3.2% sodium citrate to 9 parts of whole blood.  So if you take 1 ml of sodium citrate then you have to mix with 9 ml of whole blood.  It is also important to note the accurate technique of blood draw.  One has to follow a double syringe technique of blood draw.  What it simply means that once you puncture the initial 1 to 2 ml of blood drawn should be discarded for possible contamination with tissue factor.  Once you have discarded the initial 2 ml then you change the syringe and then draw the blood for platelet aggregation.  Once you mix with the sodium citrate homogeneously, you have to centrifuge the blood at 800 rpm for 20 minutes at room temperature.  Avoid refrigerated centrifuges because the platelets are affected by cold temperatures.

It really depends on which application your platelet-rich-plasma is going to be used.

For standard assessment of platelet function using light transmission aggregometry, microplate adhesion or impedance aggregometry it is adviced to use 3,2% sodium citrate as an anticoagulant. Reason being that upon activation, calcium ions will be released from platelets and overcome the ionic equilibirum promoted by the citrate as a chelator.

For isolating platelets it is advised to replace the standard sodium citrate for Acid Citrate Dextrose (ACD-A) as the isolation procedure requires multiple washing steps. Platelets are more stable at 37C during spinning, but spinning them at room temperature (25 C) also works fine. Reducing the pH through ACD-A (it gets near 6.5) helps to impair activation of residual thrombin traces in the platelet tubes, and contributes to the overall maintenance of platelet morphology while shifting function to a minima. Normally you need to resuspend platelets to a proper Tyrode Buffer (pH 7.4) in order to recover their functionality. ACD has many advantages when it comes to preserve platelets, but keep in mind that platelets need extreme calibrated conditions for optimal preservation. Upon collection, use them as quick as possible. If time is a concern keep the solution in a water bath (37 C) and use within 1h.

I am adding one of two great papers on this subject. Will try to find another great paper comparing the morphology and function of human platelets collected in each anticoagulants. I think everyone will benefit from it.

Best regards, folks

Andre

Article Preparation of Washed Platelet Suspensions From Human and Rodent Blood

Nonactivated versus thrombin-activated platelets on wound healing and fibroblast-to-myofibroblast differentiation in vivo and in vitro.

Scherer SS1, Tobalem M, Vigato E, Heit Y, Modarressi A, Hinz B, Pittet B, Pietramaggiori G.

Author information

Abstract

BACKGROUND:

Platelet preparations for tissue healing are usually preactivated before application to deliver concentrated growth factors. In this study, the authors investigated the differences between nonactivated and thrombin-activated platelets in wound healing.

METHODS:

The healing effects (i.e., wound closure, myofibroblast formation, and angiogenesis) of nonactivated and thrombin-activated platelets were compared in experimental wounds in diabetic (db/db) animals. In vitro, fibroblast phenotype and function were tested in response to platelets and activated platelets. No treatment served as a negative control.

RESULTS:

Wounds treated with platelets reached 90 percent closure after 15 days, faster than activated platelets (26 days), and with higher levels of myofibroblasts and angiogenesis. In vitro, platelets enhanced cell migration and induced two-fold higher myofibroblast differentiation and contraction compared with activated platelets.

CONCLUSIONS:

Platelets stimulate wound healing more efficiently compared with activated platelets by enhancing fibroblast differentiation and contractile function. Similar levels of growth factors may induce different biological effects when delivered "on demand" rather than in an initial bolus.

The recommended anticoagulant (CLSI (Guideline H 58 A), BCSH, etc is 0.11 M sodium citrate (3.2%)  ACD is acceptable but not preferred for in vitro work.

ACD-A is recommended for PRP preparation and used for therapeutic purposes. I wanted to know from where can i get USP grade ACD-A vials in India for PRP. ?

acd-a  is recommended in the USA but this is largely because of work related to blood transfusions where one is storing the blood for several days.

It has been shown that after several hours platelets begin to activate spontaneously with simple citrate, but in my opinion this is largely irrelevant where one is using the PRP immediately after collection/centrifugation

acd-a has the problem that it is not comfortable to inject into tissues because of its acidity. I use simple citrate in 3.8% I know that there is a body of opinion that suggests 3.2% but this is largely when it is used for anticoagulation studies. For obvious reasons the amount of blood taken  has to match the amount of anticoagulant.

1ml citrate with 9 ml blood is fine, but in reality 0.5ml citrate with 9ml blood works well too

The more citrate you use the more calcium you have to add later to reverse the anticoagulant effect of the citrate - so called "activation"

In the US ACD-A is used as the only solution as far as I know.

Sergio

Can I use EDTA for PRP therapy as it gave with me a high platelet count in comparison to citrate ?

The evidence I have seen is that the platelets are partly activated by EDTA but that the Calcium is so completely chelated that the activation is suspended.

One of the big plus points about citrate is that it is completely natural, and from my own perspective it is better to avoid injecting something like EDTA when it can be avoided. Separately it is unclear why the platelet count is lower with citrate than EDTA, but this has been noted before. It is not just due to the diluting effect of the citrate volume.

I think the really important thing is to have a fresh sample of platelets that is as completely unactivated as possible. Platelets are activated by the mechanical shear forces of precipitation into the buffy layer during the centrifugation, so centrifuge speed is a vitally important element, probably far less important than the choice of anticoagulant

Hello Chris. Our research is focused on the quantitative analysis of the morphological signs of platelet activation. Just recently, we have compared morphometric features of platelets from blood anticoagulated either with 3,2% sodium citrate or ACD-A. PRP was prepared within 60 minutes after blood withdrawal by centrifugation for 12 min at 160 g and platelets were activated on glass slides for 30 min, 6 h and 12 h. The slides were analyzed by the use of standardized robotic microscopy and morphological parameters as area, perimeter, circularity and fractal dimension of the platelet outlines were measured. While the morphometric change of the platelet outlines between baseline, 6 and 12 h was comparable for both anitcoagulants, platelets from ACD-A anticoagulated blood showed significantly stronger morphometric signs of activation at all timepoints as compared to sodium citrate (Fig. 1).

Hi Max-Joseph

Thank you for your interesting research. I have some observations and questions.

1. I'd be interested to know how much citrate you use (whether in the form of ACD-A or plain citrate) to anticoagulate the blood. One commercial preparation kit uses 0.5ml 3.8% citrate per 9ml blood and this anticoagulates the blood effectively for at least 12 hours. On the basis that all the work on anticoagulation is historical by blood transfusion services and relates to the amount necessary for anticoagulating blood for extended periods, I tried different quantities of plain citrate and anything much less than 0.5ml 3.8% (possibly 3.2%) resulted in coagulation of the sample. The logic is that less anticoagulant volume =higher platelet concentration. However is there an increase in potential platelet activation with lower anticoagulant quantities?

2. In the majority of clinical situations it should be possible to prepare and use the PRP within 1 hour

3. Is mean circumference a totally accurate way of estimating platelet activation?

4. Research from other labs suggest that after 12 hours ACD-A is more effective at reducing platelet activation when measured by ELISA methods.

5. Do you have any ides to explain why the platelet count is reduced compared to EDTA samples? It appears this effect is reversible.

6. I would certainly favour plain citrate since the pH of ACD-A makes it painful to inject

None of these observations are a criticism of your work for there are many unknowns. :)

Best

Chris

Hello Chris,

Thank you for sharing your experience. I answer to your comments.

ad 1: We use standard Becton Dickingson vacutainer tubes. For citrate anticoagulated blood we use the light blue tubes (Nr 367714) with a volume of 4,5 ml containing 0,105 M bufferd tri-sodium-citrate (equal to 3,2 % tri-sodium citrate as stated by BD [1]). For ACD-A we use the light yellow BD 8,5 ml standard vacutainer tubes (Nr 366645). As to BD (Page 22) [2] they contain 22,0 g/l sodium citrate, 24,5 g/l dextrose8,0 g/l citric acid and 0,15 g/l antimicotic agent. In earlier days, I was mixing citrate with the blood sample directly in the syringe at different concentrations. I did not have problems with premature coagulation of the sample. But i left this way totally, as for me it is important to reliably standardize the whole examination process as far as possible.

ad 2: It really depends on your aims. For the use of PRP as a therapeutic agent, I agree with you. For the use of PRP for diagnostic purposes (my approach), it would be preferable to have the PRP preparation at best several hours after blood withdrawal at a lab. But these are dreams of the future ...

ad 3: We have shown, that the activation status of a platelet directly correlates to its morphological change (Kraus et al, Platelets, 2013 [3]). The circumference of a blood platelet as seen in high resolution microscopy directly correlates to its activation state. But indeed, this is by most not the only parameter to morphometrically assess the activation status of a blood platelet. We currently measure about 30 different parameters to assess the morphological change of the platelet as for example the circularity, the fractal dimension, the area, the eccentricity, the number, width and length of the filopodia and so on. From these parameters we calculate a machine learning based score [4] of platelet activation and so on, bla, bla, bla. I thought that these details would not be to interesting for you. Therefore I had just uploaded the one graphic regarding the circumference, as it impressively shows, that there is a difference in the morphology of platelets depending on the anticoagulant used.

ad 4: there are so many unknowns … platelets are so sensible to so many different factors. This is, why we have just initiated a blinded and prospective study to morphometrically examine the influence of different preanalytic factors as for example the anticoagulant used. The data, I posted above are first unpublished data from this study. Many things remain to be understood and to be discussed.

ad 5: From my clinical practice, I know, that when EDTA is used as an anticoagulant platelet count is often reduced due to pseudothrombocytaemia. But, if I get you right, you experienced the situation exactly the other way round with higher platelet counts for EDTA as compared to citrate-anticoagulated blood? I have no explanation for this.

ad 6: I also prefer citrate as it seems to be more “natural” . I was quite surprised that from the first results of our study, ACD-A would be the more appropriate anticoagulant in our setting. This remains to be investigated.

[1] http://www.bd.com/resource.aspx?IDX=24959

[2] https://www.bd.com/resource.aspx?IDX=10155

[3] Article Fractal and Euclidean descriptors of platelet shape

[4] Conference Paper Active or Not -Machine-learning based Prediction of Platelet...

Hui, long answer ... May I ask you, do you also use PRP in a therapeutic approach or solely for aesthetic use ?

Best regards, Max

You should use ACD-A.

And what is your reason for saying this Emmanuel Ifeanyi Obeagu

:)

Any thoughts on using CPD solution for PRP preparation? The CPD formulation is 1.66g Sodium Citrate, 1.61g Dextrose, 188mg Citric Acid and 140mg Monobasic Sodium Phosphate.

CPD is used for anticoagulation of blood for extended periods of storage for eventual transfusion and provided some nutrients to keep the blood in good condition. I can see no reason why it would not be effective for PRP preparation. However would it add any extra benefits over Sodium Citrate?

Personally I cannot see that it necessarily would. The time from venesection to using the prepared PRP is in the order of 30-60mins, not enough time for the platelets to spontaneously activate when Sodium Citrate is used.

Use ACD-A is better because it keep the viability of platelet

ACD-A is better

Hi Alan Saheem and Suha Talal

What evidence do you have for your opinion?

First of all 3.8 citrate affects the result of coagulation tests as stated by many researchers and they prefer 3.2 concentrations instead

ACD-A is suggested as it will maintain the integrity, viability and shelf of Life of platetes better.

I do not agree with Emmanuel. ACD-A is useful for platelets that are going to be transfused - they do perform a bit better than older formulations. For platelet function tests, 0.105 M bufferred sodium citrate is the recommended anticoagulant. ACD-A does not work well if at all for murine species which we observed again just last week at a major academic center. See my previous response on that.

anybody could share the protocol for washing human platelets?