Cell separation in the buffy coat

One of the most rapid methods to determine cell counts in whole blood is by way of layer thickness measurements of the buffy coat. The purpose of this study was to determine the separation and purity of blood cells in the different layers of the buffy coat. Blood samples were centrifuged at 10,000 g in microhematocrit tubes with an inserted float to expand the buffy coat region. Whole blood from normal laboratory individuals separates by density into four regions: platelets, a layer of lymphocyte and monocytes, granulocytes and erythrocytes. A thin band of highly swollen red cells was discovered between the buffy coat layers of many normal volunteers and patients. Stereological analysis of electron micrographs showed that mixing of formed elements within the layers is less than 2% with the exception of some erythrocytes, which can make up a higher volume fraction in the lymphocyte/monocyte and granulocyte layers. The red cell column contains about 95.7% erythrocytes and is depleted of platelets and leukocytes. In approximately 5% of hospital blood samples, the granulocyte-erythrocyte interface was feathered and undetectable, and a significantly higher volume fraction of red cells was found among the granulocytes. Cell mass density determinations indicate that the erythrocytes in these abnormal granulocyte layers have a lowered mass density, overlapping with that of the granulocytes.     

A Practical and Novel Method to Extract Genomic DNA from Blood Collection Kits for Plasma Protein Preservation

Laboratory tests can be done on the cellular or fluid portions of the blood. The use of different blood collection tubes determines the portion of the blood that can be analyzed (whole blood, plasma or serum). Laboratories involved in studying the genetic basis of human disorders rely on anticoagulated whole blood collected in EDTA-containing vacutainer as the source of DNA for genetic / genomic analysis. Because most clinical laboratories perform biochemical, serologic and viral testing as a first step in phenotypic outcome investigation, anticoagulated blood is also collected in heparin-containing tube (plasma tube). Therefore when DNA and plasma are needed for simultaneous and parallel analyses of both genomic and proteomic data, it is customary to collect blood in both EDTA and heparin tubes. If blood could be collected in a single tube and serve as a source for both plasma and DNA, that method would be considered an advancement to existing methods. The use of the compacted blood after plasma extraction represents an alternative source for genomic DNA, thus minimizing the amount of blood samples processed and reducing the number of samples required from each patient. This would ultimately save time and resources.The BD P100 blood collection system for plasma protein preservation were created as an improved method over previous plasma or serum collection tubes¹, to stabilize the protein content of blood, enabling better protein biomarker discovery and proteomics experimentation from human blood. The BD P100 tubes contain 15.8 ml of spray-dried K2EDTA and a lyophilized proprietary broad spectrum cocktail of protease inhibitors to prevent coagulation and stabilize the plasma proteins. They also include a mechanical separator, which provides a physical barrier between plasma and cell pellets after centrifugation. Few methods have been devised to extract DNA from clotted blood samples collected in old plasma tubes^2-4. Challenges from these methods were mainly associated with the type of separator inside the tubes (gel separator) and included difficulty in recovering the clotted blood, the inconvenience of fragmenting or dispersing the clot, and obstruction of the clot extraction by the separation gel.We present the first method that extracts and purifies genomic DNA from blood drawn in the new BD P100 tubes. We compare the quality of the DNA sample from P100 tubes to that from EDTA tubes. Our approach is simple and efficient. It involves four major steps as follows: 1) the use of a plasma BD P100 (BD Diagnostics, Sparks, MD, USA) tube with mechanical separator for blood collection, 2) the removal of the mechanical separator using a combination of sucrose and a sterile paperclip metallic hook, 3) the separation of the buffy coat layer containing the white cells and 4) the isolation of the genomic DNA from the buffy coat using a regular commercial DNA extraction kit or a similar standard protocol.     

Rapid diagnosis of Brugia malayi and Wuchereria bancrofti filariasis by an acridine orange/microhematocrit tube technique.

A microhematocrit tube technique for diagnosis of human filariasis has been previously described. A system incorporating heparin, EDTA, and acridine orange into a microhematocrit tube (Quantitative Blood Count, QBC) has been commercially developed for the quantitation of blood counts and has been used for the diagnosis of malaria. We evaluated this test for its usefulness in the diagnosis of filariasis. Upon centrifugation, the parasites were concentrated in the area of the buffy coat and could be observed through the wall of the tube. The parasites were concentrated further by a plastic float that expands the buffy coat and confines the parasites to the periphery of the tube. Acridine orange stains the DNA of the parasite, and morphologic characteristics can be examined by fluorescence microscopy. The terminal and subterminal nuclei and long cephalic space of Brugia malayi, as well as the short cephalic space and caudal nuclei of Wuchereria bancrofti, were easily recognized and differentiated from each other. Microfilariae were detected in samples diluted to a level of approximately 50/ml.     

Micro-Buffy Coats of Whole Blood: A Method for the Electron Microscopic Study of Mononuclear Cells

A method for the electron microscopic study of human peripheral lymphocytes by which very small buffy coats are obtained through centrifugation of heparinized whole blood in glass or plastic microhematocrit tubes is presented. This method is time saving and efficient, yielding well preserved material and a comparatively large number of mononuclear cells (mainly lymphocytes) in each thin section.     

Micro-buffy coats of whole blood: a method for the electron microscopic study of mononuclear cells

A method for the electron microscopic study of human peripheral lymphocytes by which very small buffy coats are obtained through centrifugation of heparinized whole blood in glass or plastic microhematocrit tubes is presented. This method is time saving and efficient, yielding well preserved material and a comparatively large number of mononuclear cells (mainly lymphocytes) in each thin section.     

Comparative methodology for the detection and differentiation of circulating microfilariae of Dirofilaria immitis in the dog

The sensitivities of the Knott's test (four 20-microl sediment aliquots), quantitative buffy coat capillary tube method (QBC tube, 111 microl of whole blood) and direct blood smear (DBS, 20 microl of whole blood) were evaluated for the detection of microfilaraemia in dogs. Undiluted whole blood samples taken from 70 Dirofilaria immitis antigen-positive dogs and 10 serially diluted microfilaraemic blood samples at concentrations of 400, 200, 100, 50, 25 and 12 microfilariae (mff) ml(-1) were examined. For filarial speciation, the buffy coat of QBC tubes was mixed with one drop of methylene blue-formalin solution and examined as a direct smear. In 52/70 microfilaraemic blood samples, the number of mff ranged from 12 to 321987 ml(-1) (median: 3199 ml(-1)). The diagnostic sensitivity of the Knott's test, QBC tube method and DBS in undiluted blood samples attained the 100%, 98% and 92.3% levels, respectively. Eighteen dogs tested amicrofilaraemic by all three methods. At concentrations of 400 mff ml(-1), a 100% sensitivity was found by all three methods, while at 200 mff ml(-1) the Knott's test, QBC tube and DBS were 100%, 100% and 90% sensitive, respectively. The relevant figures at 100 mff ml(-1) were 100%, 100% and 80%, at 50 mff ml(-1) 100%, 100% and 50%, at 25 mff ml(-1) 100%, 100% and 10% and at 12 mff ml(-1) 80%, 50% and 10%. At 50 and 25 mff ml(-1), the DBS was less sensitive compared to the other two methods, while at 12 mff ml(-1), only to the Knott's test. A significant correlation was found between the QBC tube method and Knott's test regarding mff speciation. Therefore, the QBC method may be considered a reliable alternative to the Knott's test for both the detection and speciation of mff in the dog.     

Survival in vivo of platelets stored for 48 hours in the buffycoat at 4 degrees C compared to platelet rich plasma stored at 22 degrees C

High speed centrifugation allows separation of whole blood into cell free plasma, a buffy coat and leukocyte poor red cells. The buffy coat can be used for the preparation of platelet concentrates. High lactate production at 22 degrees C requires storage of the buffy coat at 4 degrees C. Survival in vivo of platelet concentrates prepared from buffy coats stored at 4 degrees C for 48 h (BC-PC) was compared with the survival in vivo of platelet concentrates from platelet rich plasma stored at 22 degrees C for 48 h (PRP-PC). Both methods were studied in the same healthy volunteers (n = 8) using 51Cr labeled autologous platelets. The mean +/- SD recovery 15 min after reinfusion of the BC-PC was 30.5% +/- 13.3% and for PRP-PC 41.4% +/- 7.9% (p less than 0.0001). The survival in vivo for BC-PC was 2.4 days +/- 0.4 days and for PRP-PC 7.0 days +/- 1.4 days (p less than 0.0001). Since the survival in vivo is significantly less for platelets derived from the buffy coat stored at 4 degrees C, we advocate storage of platelets at 22 degrees C.     

Systemic lupus erythematosus; early cytologic diagnosis

The diagnosis of systemic lupus erythematosus-a relatively common disease-is difficult because of the variable nature of the symptoms, which resemble those of many other conditions. The finding of the characteristic cells is pathognomonic, although failure to find them does not rule out the diagnosis. If the diagnosis is suspected the "L.E." cell test should be performed on two samples of blood from the veins and one from the bone marrow. After separation of a haparinized sample by centrifuge, a drop from the buffy coat is Wright-stained on a slide and examined for rouleaux formation and for a hematoxylinstaining material sometimes seen in intercellular bodies (which may be surrounded by a rosette of leukocytes) and sometimes seen as ingested by a leukocyte. Only the last finding is positively diagnostic of lupus erythematosus.A statistical analysis of 62 cases treated at the Los Angeles County General Hospital is given. Because of the frequency of rheumatoidlike arthritic changes in the disease, all patients with this form of arthritis should be given the test. Spontaneous remission and then relapse after a long asymptomatic interval occurred in many cases. With early diagnosis and vigorous treatment with cortisone and corticotropin, many patients can be relieved of symptoms.     

Novel method for continuous cell separation by density gradient centrifugation: evaluation of a miniature separation column

A compact bench-top model of the centrifuge enables continuous cell separation based on density differences. The apparatus holds a small separation disk equipped with a circular channel (8 mL capacity) separated by a septum. A set of isotonic Percoll media with different densities is continuously introduced at one terminal and collected from the other. Under a centrifugal force field, cell suspension introduced into the proximal portion of the channel results in continuous separation of cells according to their densities. The performance of the apparatus was demonstrated with the separation of human buffy coat containing nucleated cells (>10(8)) among a large population (10(10)) of RBC. The results indicated that the method is capable of separating a large number of nucleated cells, with minimum damage, for a few hours of operation wherein neutrophils are well resolved from lymphocytes. The method may be applied to other types of samples including cord blood, blood from small animals, cultured cells, pancreatic beta cell islets, malaria parasites, sperm cells, etc.     

SYSTEMIC LUPUS ERYTHEMATOSUS—Early Cytologic Diagnosis

The diagnosis of systemic lupus erythematosus—a relatively common disease—is difficult because of the variable nature of the symptoms, which resemble those of many other conditions. The finding of the characteristic cells is pathognomonic, although failure to find them does not rule out the diagnosis.If the diagnosis is suspected the “L.E.” cell test should be performed on two samples of blood from the veins and one from the bone marrow. After separation of a haparinized sample by centrifuge, a drop from the buffy coat is Wright-stained on a slide and examined for rouleaux formation and for a hematoxylinstaining material sometimes seen in intercellular bodies (which may be surrounded by a rosette of leukocytes) and sometimes seen as ingested by a leukocyte. Only the last finding is positively diagnostic of lupus erythematosus.A statistical analysis of 62 cases treated at the Los Angeles County General Hospital is given. Because of the frequency of rheumatoidlike arthritic changes in the disease, all patients with this form of arthritis should be given the test. Spontaneous remission and then relapse after a long asymptomatic interval occurred in many cases. With early diagnosis and vigorous treatment with cortisone and corticotropin, many patients can be relieved of symptoms.     

Value of examining buffy coats for intragranulocytic micro-organisms in patients with fever.

To determine the significance of the presence of intragranulocytic micro-organisms in the blood buffy coat in patients with suspected infection, buffy coat examination and blood cultures were simultaneously performed in 455 consecutive patients with fever. There was no general correlation between the finding of intragranulocytic micro-organisms in the buffy coat and positive blood cultures. Patients with persistent bacteraemia and sterile blood cultures were, however, shown to have persistently positive buffy coat findings on repeated examination. These patients, who had culture-negative endocarditis or catheter-associated infections, had sterile blood cultures because of antibiotic treatment. Repeated positive findings in the buffy coat may therefore be valuable in detecting patients with persistent bacteraemia, but sporadic findings of micro-organisms in the buffy coats of acutely ill patients seem to have little diagnostic value.     

Preparation and Pathogen Inactivation of Double Dose Buffy Coat Platelet Products using the INTERCEPT Blood System

Blood centers are faced with many challenges including maximizing production yield from the blood product donations they receive as well as ensuring the highest possible level of safety for transfusion patients, including protection from transfusion transmitted diseases. This must be accomplished in a fiscally responsible manner which minimizes operating expenses including consumables, equipment, waste, and personnel costs, among others.Several methods are available to produce platelet concentrates for transfusion. One of the most common is the buffy coat method in which a single therapeutic platelet unit (≥ 2.0 x10¹¹ platelets per unit or per local regulations) is prepared by pooling the buffy coat layer from up to six whole blood donations. A procedure for producing "double dose" whole blood derived platelets has only recently been developed.Presented here is a novel method for preparing double dose whole blood derived platelet concentrates from pools of 7 buffy coats and subsequently treating the double dose units with the INTERCEPT Blood System for pathogen inactivation. INTERCEPT was developed to inactivate viruses, bacteria, parasites, and contaminating donor white cells which may be present in donated blood. Pairing INTERCEPT with the double dose buffy coat method by utilizing the INTERCEPT Processing Set with Dual Storage Containers (the "DS set"), allows blood centers to treat each of their double dose units in a single pathogen inactivation processing set, thereby maximizing patient safety while minimizing costs. The double dose buffy coat method requires fewer buffy coats and reduces the use of consumables by up to 50% (e.g. pooling sets, filter sets, platelet additive solution, and sterile connection wafers) compared to preparation and treatment of single dose buffy coat platelet units. Other cost savings include less waste, less equipment maintenance, lower power requirements, reduced personnel time, and lower collection cost compared to the apheresis technique.     

Evidence-based diagnostic algorithm for visceral leishmaniasis in Bangladesh

Evidence-based diagnostic algorithm is highly recommended for the visceral leishmaniasis (VL). This cross-sectional study was performed in Bangladesh to evaluate VL diagnostic tools including serology, buffy coat smear microscopy for LD body and various DNA-based techniques using buffy coat in 100 confirmed VL cases and 100 controls. The performance of tools against spleen smear (gold standard) was evaluated using kappa coefficient. Diagnostic precision and other inherent indicators were considered for index scoring (IS) of performance of tools using factor analysis. A diagnostic algorithm was formulated based on the IS and availability of the tools at different health care facilities of Bangladesh. A high level of agreement (kappa ≥  0.80) was observed for all the diagnostic tools. The highest kappa coefficients were found for rK39 RDT and rK39 ELISA (0.95), followed by ssuRNA-PCR (0.94), Buffy coat smear (0.93), rK28 ELISA (0.92), rK28 RDT (0.89), LAMP (0.89), Mini-exon PCR (0.86), ITS1 (0.85), and ITS2 PCR (0.80). rK39 RDT was found to be the best diagnostic test (IS: 1.7) followed by rK28 RDT (IS: 1.5), buffy coat smear microscopy (IS: 0.5), rK39 & rK28 ELISA (IS: 0.3), ssuRNA-PCR (IS: ‐0.7) and LAMP, Mini-exon, ITS1, & ITS2 PCR (IS: ‐0.9). rK39 RDT has been proposed as the best option for primary health care facilities, while buffy coat smear microscopy was found to be a good adjunct for confirmation of serology-positive cases and proposed for secondary and tertiary facilities. ssuRNA-PCR or LAMP can be an alternate confirmation tool only applicable to the tertiary facilities.     

A New Procedure for Quantitative Measurements of Virus Particles in Crude Preparations

A new method is described for the quantitative measurement of virus concentration in crude preparations by density gradient centrifugation and electron microscopy. The centrifugation is carried out in a specially designed centrifuge tube which permits separation and sedimentation of virus particles at different levels according to their sedimentation velocity. The gradient of a mixture of heavy and normal water (D(2)O-H(2)O) is designed to sediment the virus particles with constant velocity so that the optimal time of centrifugation can easily be calculated. The virus particles are collected on carbon-coated nickel grids floating on mercury at the bottom of the centrifuge tube and are counted by means of electron microscopy. The efficiency of the method is demonstrated with a crude plant extract of tobacco mosaic virus.     

An immunogold-silver staining method for detection of cell surface antigens in cell smears

We developed an indirect immunogold-silver staining method for detection of leukocyte cell surface antigens in cell smears. Air-dried and fixed cytocentrifuge preparations or smears of peripheral blood leukocytes were incubated with monoclonal antibodies (MAb) and colloidal gold-labeled secondary antibodies. The preparations were post-fixed and silver enhancement was performed. The smears were counterstained with May-Grunwald-Giemsa and examined in brightfield light microscopy. The morphology of the cells was well preserved. Leukocytes reacting with the MAb showed black granules on their surface membranes. The intense immunostaining and the low background allowed a rapid enumeration of the positive cells. The labeling could be detected with high sensitivity by epipolarization microscopy. This immunogold-silver staining method was used to quantify T- and B-lymphocytes and natural killer cells in buffy coat smears of normal adult blood. These lymphocyte subsets correlated well with those obtained in smears with the alkaline phosphatase-anti-alkaline phosphatase (APAAP) method and with those found by labeling of mononuclear cells in suspension with immunogold-silver staining. This immunogold-silver staining method forms a good alternative to immunoenzyme methods for study of hematologic cells. In addition, it could be a general procedure for detection of cell surface antigens in all kinds of cell smears.     

Physiopathology of cytomegalovirus infections

Since more than fifteen years ago, the occurrence of Cytomegalovirus (CMV) infections in transfused patients has incriminated blood as a potential vehicle for CMV transmission, resulting in post-transfusion mononucleosis (PTM). Although only Diosi and al. (1969) reported viraemia in two of 35 asymptomatic blood donors, CMV was not recovered from any leukocyte specimen of numerous healthy blood bank donors, assayed by cocultivation of undisrupted buffy coat with a virus sensitive monolayer of human fibroblasts. Nevertheless numerous experimental studies have shown that CMV might persist in very low titer or in a latent state in mononuclear leukocytes in monocytes and macrophages and in lymphocytes. A better knowledge of the site an mechanisms of latency should help to control CMV transmission by fresh and bank blood or transfusion of viable leukocytes.     

Cryopreservation of human granulocytes.

Granulocyte preservation was undertaken using hydroxyethylstarch for both sedimentation of red cells and cryopreservation of buffy coat white cells from CPD whole blood. Buffy coats were mixed with HES to a final concentration of 4% (w/v) and hematocrit of 30%, and sedimented in inverted plastic syringes. The leukocyte enriched (100–500×) supernatant was frozen at 2.0 °C/min to ?80 °C (and stored frozen up to 3 months). Alternatively, sedimented leukocytes were frozen after a slow addition of 10% DMSO to 5%. Tubes were thawed at 37 °C, and DMSO was removed by dilution with Hank's solution containing CPD and centrifugation. The pellets of granulocytes were resuspended in Normosol.Buffy coat from 10 units yielded 60 ± 9.7% of the available whole blood leukocytes, of which 43 ± 14% were recovered after sedimentation in HES. Freezing in DMSO yielded all, 101% of the prefrozen leukocytes. Postthawed viability of granulocytes was estimated morphologically and by their ability to inhibit the rate of growth of E. coli. Complete inhibition was observed at a ratio of one E. coli to one granulocyte. Postthawed granulocytes were characterized by high myeloperoxidase activity and exclusion of trypan blue. Approximately 25% of the total available granulocytes in CPD whole blood were recovered.     

A simple method for human peripheral blood monocyte isolation

We describe a simple method using percoll gradient for isolation of highly enriched human monocytes. High numbers of fully functional cells are obtained from whole blood or buffy coat cells. The use of simple laboratory equipment and a relatively cheap reagent makes the described method a convenient approach to obtaining human monocytes.     

Role of the eosinophil in serum-mediated adherence of equine leukocytes to infective larvae of Strongylus vulgaris.

The adherence of equine leukocytes to Strongylus vulgaris infective larvae (L3) in the presence of normal and immune sera was examined in vitro. Immune sera promoted adherence of buffy coat cells from ponies with S. vulgaris-induced eosinophilia (eosinophilic ponies) to S. vulgaris L3. However, eosinophils in the buffy coat cells were the predominant adherent cell type. Studies using leukocyte populations enriched for eosinophils, neutrophils, and mononuclear cells from eosinophilic ponies support the observations using buffy coat cells that eosinophils were the main effector cells. Adherent eosinophils from eosinophilic ponies immobilized L3. Neutrophils were less adherent and did not immobilize L3. Mononuclear cells failed to adhere. Normal eosinophils from strongly-naive ponies did not immobilize S. vulgaris L3 in the presence of immune serum, suggesting the in vivo activation of eosinophils in eosinophilic animals. Immune serum promoted less adherence of buffy coat cells to Strongylus edentatus or mixed species of Cyathostominae L3, suggesting that the serum-mediated cellular adherence phenomenon was species-specific. Normal serum promoted less cellular adherence to S. vulgaris L3 than immune serum. The adherence mediated by normal serum was removed by heat inactivation, suggesting that this nonspecific phenomenon was a complement-mediated reaction. Immune globulins promoted reactions similar to that seen using heat-inactivated immune serum, whereas normal globulins did not promote adherence. Immune globulins absorbed with pieces of S. vulgaris adult worms did not promote the adherence of buffy coat cells to S. vulgaris L3, suggesting that adult and L3 stages share antigens important in this phenomenon that resulted in the removal of specific adherence antibody during absorption.     

Preparation of leukocyte-poor platelet concentrates from buffy coats by a new type of quadruple bag

A new quadruple blood bag system for collection of platelet concentrates is described. The principle modification consists of a smaller (150 ml) buffy coat bag in conjunction with SAG-mannitol for red cell preservation. Platelet yield and function were comparable to conventional techniques, leukocyte contamination was lower (1.7 +/- 1.3 x 10(7) per unit). The system is recommended for specialized institutions with a high demand of blood components.