Gently getting rid of red blood cells and debris would be of definite advantage to flow cytometry and image stream applications.  If it is automatable and maintains a healthy live cell population, that could be really useful.

Gentle separation is usually indicated if one wants to perform cellular functions or expression of certain activation markers of leucocytes/platelets. After all, you don't want to induce artefactual changes to the functional markers.

Depends on the nature of your research.  If you are looking to count or perform non-0functional tests on platelets, most common separation technologies are acceptable.. If you want the platelets to be viable for functional testing ,e.g., platelet aggregation, follow CLSI Approved Guideline H 58A for specimen collection and sample processing

I hope these métodos which we use in my lab are your utility:

Flow cytometry

The resolution of leukocyte subpopulations by light scattering is hampered when unlysed blood sample or full flow cytometric analyzes. This is due to the fact that red blood cells (RBCs) present in the sample interfere with measurements of light scattering and obscure signals other cell types. Interference is particularly large for forward scatter, causing large errors in the analysis of lymphocytes. For this reason, researchers typically lyse red blood cells in a sample when you want to analyze leukocytes. For lysing erythrocytes various reagents that are useful in the invention (for example, water, saponin and other reagents subsequently exposed lysate) are known in the art. The most commonly used is buffered ammonium chloride. Although generally can get a proper separation of debris from lymphocytes using a partitioning of the dispersion in ammonium chloride solutions, the absence of a fastening means of the biological sample can cause a biohazard. However, the addition of a fixative such as paraformaldehyde for solutions lysate ammonium chloride is unsatisfactory due to the generation of hydrogen ions to decrease the pH of the sample and reduce the fluorescein fluorescence, thus hindering analysis immunofluorescence.

Reagents lysate comprising paraformaldehyde, a salt of a weak acid and a polyhydric alcohol are also known in the reagent .This technique lysate is available from Becton Dickinson Immunocytometry Systems, San Jose, California, under the name lysing solution FACS and comprises diethylene glycol, heparin, citrate buffer and formaldehyde, pH 7.2. Because the presence of formaldehyde, FACS lysing solution provides the advantage of reducing biological hazards of the biological samples. However, its presence in a sample being analyzed by flow cytometry causes cell shrinkage and may lead to unsatisfactory levels of debris that can obscure the cell population to be compartmentalised, particularly lymphocytes. Other reagents known in the art lysate is described hereinafter in relation to methods of sample preparation "no wash".

Therefore, it is desirable to have methods available for the preparation of lysed blood samples that do not require separation of the lysate solution and washing before flow cytometric analysis. Such sample preparation methods "no wash" for flow cytometry are known in the art. Caldwell et al (AJCP 86: 600-607 (1986)) describe a method for washing without an immunophenotypic analysis of cells by flow cytometry in which lymphocytes are isolated from peripheral blood samples by fractionation on density gradients, avoiding the need for erythrocyte lysis. After immunofluorescent staining, the samples are subjected to flow cytometric analysis without washing, leaving the unbound fluorescent marker in the sample. Lymphocytes are identified and parameters compartmentalized using front and side light scatter.

US Patent # 5,030,554 describes the treatment of a blood sample with a lytic agent comprising a water soluble organic carboxylic acid in water having a pK value of greater than 3.0, a pH of about 2.6 to 4.0 and a counterion that does not materially alter the ionic strength of the sample. Separate stages staining lysate and inactivation are performed in a single vessel without separating unreacted dye and / or unconsumed reagents from the sample before analysis. Leukocyte subpopulations are identified and addressed in the traditional manner, using parameters forward scatter and side light scatter light. This washing method is not commercially available at the company Coulter Corporation under the name Q-PREP. The "Ortho-Diaagnostic" systems have also published a protocol for sample preparation without washing using ammonium chloride indicating that if the analysis of cells in the lysing solution not be performed within two hours, the cells must be centrifuged , washed, placed resuspended in phosphate buffered saline and analyzed in 8 hours.

WO 88/07187 describes document a reagent for red blood cell lysis comprising a carboxylic acid of low molecular weight, a sulfonic acid or an activated phenol. The lysed sample is directly analyzed by flow cytometry to identify leukocyte subpopulations based on front and angular light scattering. It is stated that the red cell fragments do not interfere or have an adverse effect on the photometric differentiation of leukocyte subpopulations using this lysing reagent.

In the case study Photometric

1-Stage freeze / thaw

Transfer factor is obtained from leukocyte concentrates units. Derived units are frozen and subsequently undergo five stages of freezing / thawing. In this regard, leukocyte concentrates packages are grouped to form batches of 20 leukocyte concentrates in plastic bags. freezing cycles storing lots at -20 ° C for one week start. Following completion of the thawing week packages leukocyte concentrate placing these in a sink under running water it is performed. Being completely defrosted is reinserted at -20 ° C and stored for a week. So on, until meet five cycles of freeze / thaw.

Example 2. Stage dialysis

The process begins by cutting a dialysis membrane 12 KDa to a 1 meter long 40 cm. This membrane is placed in a beaker containing 4 liters 2.5 liters of water Elix and boil for 10 minutes. Then, the dialysis membrane is removed and placed in another beaker containing 4 liters 2.5 liters of water Elix and boil for 10 minutes. Subsequently the dialysis membrane is removed and placed in another beaker containing 4 liters 2.5 liters of water Elix and sterilize for 15 minutes at 121 ° C. The dialysis membrane is maintained within the sterile water until use.

Once prepared the dialysis membrane, filling said membrane proceeds with leukocyte extract subjected to processes of freezing / thawing. This process begins by cleaning the bags containing white blood cell concentrate with 70% alcohol; subsequently cut one end of the bag using sterile surgical scissors. It is emptied and the contents decanted leukocyte concentrate is measured into a graduated cylinder. After the contents of the cylinder into a glass jar 1L is empty. steps with the various bags are repeated to have a volume of 1,100 liters of white blood cell concentrate into the bottle. Subsequently they measured pisa 800 milliliters of water in a test tube, and this volume is passed into a sterile 2 liter bottle. Using sterile gloves one end of the dialysis membrane is removed and make a knot 10 cm from the end, a second knot to 7 cm from the end is performed and tied surgical thread between the two knots. Then, the other end of the dialysis membrane is removed and a finger is inserted into the membrane, the dialysis membrane is pushed to form a concertina. The finger is removed and instead the neck of a funnel is placed, taking care not to break the membrane. the membrane is removed and the end with knots in a sterile bottle 2 liters, taking care to leave the surgical thread out of the bottle is placed. Then the end of the surgical thread is outside the flask with surgical forceps and emptied by decanting the leukocyte concentrate flask to the funnel to pour all within the dialysis membrane, taking care to put the membrane is subject gradually within 2 liter flask. Subsequently, the funnel end of the dialysis membrane and make double knot leaving 3 cm apart is removed. sterile forceps (clip) between two nodes is placed and a lid is placed aluminum sterile dialyzing and leave for 20 hours. Upon fulfillment of the 20 hours of dialysis samples for their analysis they are taken. After this is poured 'dialysis product in a glass 4 liter sterile by decantation, ensuring that the liquid decanted touch as little as possible the ends of the dialysis membrane. It is filtered through 0.22 pm, it is collected in a sterile bottle 2 liter volume as obtained dialysate is measured, and stored at -20 ° C until start tangential ultrafiltration.

Example 3. Stage tangential ultrafiltration

To perform ultrafiltration of the product according to the following: the cartridge 10 is sampled KDa and determine the amount of endotoxin present. system pressures (10 psi supply in the port and 5 psi in the retentate) are verified. Subsequently, a hose is connected to the power port install it on the head of the peristaltic pump and insert the other end into the jug containing the product dialysate to 12 KDa. Connect a second hose to permeate port of the filter unit and into clean 20 L carboy labeled as permeate 1. Connect a third hose to port retained and place in a third carboy labeled 20L RETAINED 1 (note : Prepare a more rETAINED labeled 1, 1 jug because two carboys retentate is obtained). Switch on the peristaltic pump and programmed to 1 L / min. Ultrafiltering the entire product. Measure 2 L beaker with the total amount of each product obtained. Recirculating retentate RETAINED 1 and 1, 1 to zero volume as follows: enter the supply hose on one of the carboys containing retentate (RETAINED RETAINED 1 or 1.1), introduce hose retained in the same garrafón that the supply hose (RETAINED 1 to RETAINED 1, 1), insert the hose permeate into another carboy identifying it as permeate 2 (note: as 2 garrafones permeate will be obtained another carboy identifying it as permeate 2 was prepared, 1. Turn the peristaltic pump and programmed to 1 L / min. ultrafiltering the retentate to a volume of zero. Measure specimen 2L with the total volume of the products obtained. the product of the three carboys is homogenized with permeate (permeate 1, permeate pERMEATE 2 and 2.1) as follows: introduce a hose to jug containing 2.1 permeate product install the hose in a peristaltic pump head and insert the other end into the jug containing the product pERMEATE 1, program the pump to 1 L / min and moving half the amount contained in the jug permeate permeate 2.1 to 1. Repeat steps a and b to move the other half of the quantity of permeate 2,1 the jug containing the permeate 2, leaving two demijohns with permeate two hoses are introduced into the jug pERMEATE 1 are installed on the heads of two peristaltic pumps (one pump for each hose) and introducing the other ends in the jug pERMEATE 2. Set the peristaltic pumps in opposite directions (one turn to the left and the other right turn) at a rate of 13 L / min. Initiate operation of both pumps and hold for 20 min. Identify both carboys as permeate TOTAL. 2L graduated cylinder measure the total volume obtained and quantify proteins. Performed according to the directions above ultrafiltration using a cartridge 1 KDa.

Example 4. Stage identification and quantification by liquid chromatography ultra-resolution by gel

This step is performed in conditions for qualitative and quantitative analysis by SEC-UPLC Acquity UPLC System in Class H system using gel filtration column Acquity pm BEH200 1.7 4.6 x 150 mm. Separation of the peptides is performed with a 50mM phosphate buffer solution with 50 mM sodium chloride at pH 7.0 and a isocratic flow 0.2 mL / min with a total elution time of 15 min. Chromatographic conditions listed above were used to obtain a calibration curve for quantitative determinations. Chromatographic profiles of transfer factor are obtained. These 11 peaks elute in a time interval of between 8.5 and 13.5 RETENTION min. . This chromatographic method allows us to make a qualitative analysis to detect the 11 characteristic peaks of transfer factor in a range of retention time of 8.5 to 13.5 min. For the approximate molecular weight range of the population of peptides transfer factor the molecular weight markers from Biorad (1.35 - 670 kDa) are used and Tryptophan (62 Daltons), so we can infer that the peptides factor transfer have less at 17 kDa corresponding to the myoglobin Biorad molecular weight standard.

Example 5. Stage validation biological in vitro

The MG-63 cell line (ATCC CRL-1427) are human osteosarcoma cells. MG-63 cells are seeded in culture dishes CORNING 12 well plates at a density of 1x104 cells per well in 500 medium EMEM culture Di (GIBCO cat no. 30-2003) supplemented with 10% FBS (GIBCO Cat No. 16000-044), stimulated cells are treated with transfer factor a concentration of 100 Dg / ml, a control proliferation, cells in which no stimulus is placed is placed. Cells are incubated for 24, 48 and 72 hours. The experiment was performed in triplicate for each condition. Determination of proliferation by trypan blue exclusion. After each incubation time the number of cells and cell viability is determined by trypan exclusion test 0.4% blue dye (SIGMA Cat No. T8124). The cells are detached by trypsinization (Cat No. 12563 Triple GIBCO) and are centrifuged at 125 X g for 5 min, then the account is done in a Neubauer chamber

The effect of transfer factor on the cell line was determined using the test bromide 3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide (MTT, for its acronym in English. Briefly, 1 * I04 cells / well were treated with 100 Dg / ml. After 24, 48 and 72 hours of incubation the cells were washed twice with saline phosphate (PBS, for its acronym in English) and TT (0.5 mg / mL PBS ) is added to each well and incubated at 37 ° C for 30 minutes. the formazan crystals formed are dissolved by adding dimethyl sulfoxide (100 pL / well), and absorbance is read at 570 nm using a microplate reader ( Model 3550, BIO-RAD, Richmond, USA) the reduction in cell viability after treatment with transfer factor is expressed in terms of control cells (untreated) percentages of cell survival was calculated as follows:%.. cell survival = (absorbance of treated cells / absorbance of cells with solvent vehicle) X 100. the half inhibitory concentration (IC50) is calculated from the dose-response curve obtained by plotting the percentage of cell survival versus the concentration factor transfer.

Determination of proliferation by trypan blue exclusion

. After each incubation time the number of cells and cell viability was determined by exclusion test trypan blue dye 0.4% (SIGMA Cat No. T8124) .The cells are centrifuged at 125 X g for 5 min, then it is performed account in a Neubauer chamber .The peripheral blood leukocytes PBLs are spleen and lymph nodes a mixture of various cell populations, which have been separated according to their biological characteristics (antigenosreceptores, enzymes), biophysical (adherent, nonadherent, density, electrical surface charge, etc.). T lymphocytes and today there are sophisticated technological advances, such as flow cytometry which separates the cells electronically given volume B lymphocytes: From the point of view inmunol6gico two groups studied. one 0 two molecular membrane markers simultaneously.

maximum security techniques required in asepsis and quickly to ensure optimal cell viability. In this paper a simplified method for the isolation of peripheral blood leukocytes, PBL, with design and construction of a sterilizable chamber to remove by centrifugation erythrocytes immediately method shown, leaving leukocytes at the interface plasma / ficoll-hypaque easily separable.

The physical separation of different types of cells is a biornedica sangufneas practice that requires prompt and skill to ensure greater cell viability. This is possible with the protocol described here as it allows after obtaining the blood sample, it goes immediately to be centrifuged.

The input speed is determined by these cells two elements: a) the centrifugal force, which in turn depends on the angular rotation speed (1000- 2500 rpm), and the radius of gyration. For practical purposes the relative strength centrffuga as Caleula: FCR = K x R x N 'x 10' 5; where: K = 1,118; R = turning radius in centimeters; N = rpm. During centrifugation at maximum speed (2,500 rpm), the FCR for the top layer of the blood is 5162xG, and deep red blood cells have a value of 12.537xG. b) The gradual entry of erythrocytes to the camera is isochoric and induces the displacement of Ficoll-Hypaque into the syringe 10 which in turn causes go changing medium density separator. Erythrocytes drag associated with their 128-160 microns square cell surface solutes / solvents PLASMA, which can not be removed by the turbulence during the centrifugaci6n hidrodinarnica. . EI leukocyte volume is in static equilibrium at the interface, enlre the Ficoll-Hypaque and the plasma / platelet.

The density specifies that each cell type has, due to the physical relationship between the volume V, and its weight P, 0 is D = P / V. These two physical factors, the hydrodynamic properties between the topography of the plasma membranes (external) and the liquid medium (viscous turbulence) are added, distinguishing hydrophobic interactions, hydrophilic interactions electrical salt, etc., all of which allows physically differentiate a group of cells of another.

We must remember that the viscosity of the blood follows a hydrodynamic non-Newtonian behavior and therefore does not follow the laws described by the equation Poiseille. This is due to the presence of the phenomenon and the vast Rouleaux f1exibilidad erythrocytes.

The receiving chamber has been designed cells to accommodate all three erythrocytes present in blood peri- pheric ml of normal hematocrit donor (X = 43% v / v). If a donor has a VE greater than 43.0% should be decreased blood volume Vs, so that the VE does not exceed the volume Vi of the chamber b3. Interface IE eritrocitaria should never exceed the level 1.

We distinguish two experimental protocols to isolate the various cell subpopulations either peripheral blood, lymphoid organs, spleen, bone marrow aspirates, etc .: separating the cells and their fragments by density gradients for biochemical / chemical diagnostic purposes, and separating the cells for in vitro culture isologos auto transplants logos. It should clarify the physiological properties of separation and washing solutions as needs to be optimal cell viability. For these protocols exist several chemical compounds such as sucrose, glycerol, gelatin, etc., which in varying degrees can alter cell viability. EI use of ficoll-hypaque and recently the percol, has brought considerable technical progress, since they can better adjust the ionic strength, pH, osmotic pressure and viscosity 10 close as possible to the physiological conditions of each cell type in cad asf to ensure that species and cell viability is close to 100%, observed average torque of a vital dye such as trypan blue.

Another possibility would leukocyte labeling with Tc HM-PAO is a technique often used in Nuclear Medicine since it was established by Peters in 1986. Later It was adapted by other authors with some modifications and variations as volume

of blood removed, settling time, gravities (g) used for obtaining

Leukocyte button and centrifugation times,

among other. In our service, in order to shorten marking time, we are using a method of obtaining leukocyte rich plasma (LRP)

consisting centrifuge at low g blood drawn ACD-A, together with the thickener agent

1) determining the number of g which provides most

leukocyte recovery; 2) analysis of the composition PRL cell obtained by sedimentation or by centrifugation; 3) the fixation of 99mTc HM-PAO

cellular blood elements, and 4) the comparison of the markings made by leukocyte

sedimentation and centrifugation, mainly.

The goodness of these results endorse the use This alternative method that significantly reducesmarking time regarding the usual sedimentation

a variable time (30-90 minutes) proposedmost authors.

Once determined the g that give us greater recovery of leukocyte and blood counts

initially drawn blood and of PRL, obtained by sedimentation (1XG) or centrifugation

(5xg) were determined recovery leukocytes and contamination of other cellular elements (Erythrocytes and platelets).

DISTRIBUTION activity DIFFERENT ELEMENTS OF CELLULAR

BLOOD

The determination of the activity attached to the different cellular elements of the blood was carried out centrifuging and resuspended labeled BLC

in 5 ml of PLC in density gradients. The following gradients were used:

HISTOPAQUE 1,077 / 1,119 (SIGMA). They are placed in a falcon tube 15 ml, 3 ml reagent density 1,119 and above it, without mixing, other

3 ml of density 1,077. Resuspension leukocyte It is carefully placed over the layers and centrifuged at 700xg for 30 min. After the

centrifugation, mononuclear cells and platelets They are retained in the top layer

density 1.077, granulocytes between the two layers and erythrocytes in the bottom of the tube. Nycoprep 1.063 (Nycomed). They are placed in a Falcon 15 ml tube, 3 ml of reagent density 1,063. Resuspension placed leukocyte carefully over layers and centrifuged at 350xg for 15 min. After centrifugation, approximately 70% of the platelets are retained on top of the interface between the sample and Nycoprep 1,063.

After centrifugation for 15 minutes quantifies The radioactivity of each layer of the gradient a gamma camera and the activity was determined 99m HM-PAO incorporated into cellular elements

blood.

The highest percentage of recovery It is obtained by centrifuging at 5xg which in our case is the minimum speed attainable by centrifuge. McAfee, recovered almost

100% of the leucocytes by centrifuging at 20xg for 14 minutes using a mixture of a part of 1% methylcellulose four parts of blood- ACD-A.

It is noted that no contaminat platelets,...............................................................................................................

Many thanks for the good feedback. Any additional applications?