Evaluation and additional recommendations for preparing a whole blood control material

OBJECTIVE: The assessment of an easy to prepare and low cost control material for Hematology, available for manual and automated methods. MATERIAL AND METHOD: Aliquots of stabilized whole blood were prepared by partial fixation with aldehydes; the stability at different temperatures (4. 20 and 37 °C) during periods of up to 8-9 weeks and aliquot variability with both methods were controlled. RESULTS: Aliquot variability with automated methods at day 1, expressed as CV% (coefficient of variation) was: white blood cells (WBC) 2.7, red blood cells (RBC) 0.7, hemoglobin (Hb) 0.6, hematocrit (Hct) 0.7, mean cell volume (MCV) 0.3, mean cell hemoglobin (MCH) 0.6, mean cell hemoglobin concentration (MCHC) 0.7, and platelets (PLT) 4.6. The CV (coefficient of variation) percentages obtained with manual methods in one of the batches were: WBC 23, Hct 2.8, Hb 4.5, MCHC 5.9, PLT 41. Samples stored at 4ºC and 20ºC showed good stability, only a very low initial hemolysis being observed, whereas those stored at 37ºC deteriobed a rapidly (metahemoglobin formation, aggregation of WBC and platelets, as well as alteration of erythrocyte indexes). CONCLUSIONS: It was confirmed that, as long as there is no exposure to high temperatures during distribution, this material is stable, allowing assessment, both esternal and internal, for control purposes, with acceptable reproductivity, both for manual and auttomatic methods.

Dapsone hypersensitivity syndrome in an adolescent during treatment during of leprosy

A 12 y old girl was admitted 24 days after start a WHO multidrug therapy scheme for multibacillary leprosy (dapsone, clofazimine and rifampicin) with intense jaundice, generalized lymphadenopathy, hepatoesplenomegaly, oral erosions, conjunctivitis, morbiliform rash and edema of face, ankles and hands. The main laboratory data on admission included: hemoglobin, 8.4 g/dL; WBC, 15,710 cells/mm³; platelet count, 100,000 cells/mm³; INR = 1.49; increased serum levels of aspartate and alanine aminotransferases, gamma-glutamyl transpeptidase, alkaline phosphatase, direct and indirect bilirubin. Following, the clinical conditions had deteriorated, developing exfoliative dermatitis, shock, generalized edema, acute renal and hepatic failure, pancytopenia, intestinal bleeding, pneumonia, urinary tract infection and bacteremia, needing adrenergic drugs, replacement of fluids and blood product components, and antibiotics. Ten days after admission she started to improve, and was discharged to home at day 39th, after start new supervised treatment for leprosy with clofazimine and rifampicin, without adverse effects. This presentation fulfils the criteria for the diagnosis of dapsone hypersensitivity syndrome (fever, generalized lymphadenopathy, exfoliative rash, anemia and liver involvement with mixed hepatocellular and cholestatic features). Physicians, mainly in geographical areas with high prevalence rates of leprosy, should be aware to this severe, and probably not so rare, hypersensitivity reaction to dapsone.

Human infection by Angiostrongylus costaricensis in Venezuela: first report of a confirmed case

A proven case of human infection caused by Angiostrongylus costaricensis is reported for the first time in Venezuela. The patient was a 57-year-old female surgically operated because of signs of peritonitis with a palpable mass at the lower right quadrant of the abdomen. WBC count reported 16,600 cells/mm³, with 46% eosinophils. The tumoral aspect of ileocolic area and peritoneal lymph nodes prompted the resection of a large area of the terminal ileum, cecum, part of the ascending colon and a small part of the jejunum, where a small lesion was found. The pathology showed thickened areas of the intestinal wall with areas of hemorrhage and a perforation of the cecum. Histology showed intense eosinophil infiltration of the whole intestinal wall, granulomas with giant cells and eosinophils. Some of the granuloma surrounded round or oval eggs with content characterized by a large empty area, cells or embryo in the center, and sometimes nematode larvae. A cross section of an adult nematode worm was observed inside a branch of mesenteric artery. The intestinal affected area, the characteristics of the lesions, the presence of eggs in the submucosa with nematode larvae inside, and the observation of a nematode inside a mesenteric artery, makes sufficient criteria for the diagnosis of an infection by Angiostrongylus costaricensis.

Removal of Trypanosoma cruzi by white cell-reduction filters: an electronmicroscopic study

White cell (WBC)-reduction filters have been shown to be effective in removing infectious agents from infected blood products. In this study, the mechanisms of Trypanosoma cruzi (T. cruzi) retention by WBC-reduction filters were assessed. Human packed red blood cell (PRBC) and platelet concentrate (PC) samples were contaminated with T. cruzi organisms (Y strain; 3.4 x 10(6)/ml), and then filtered using WBC-reduction experimental filters that provided about 3 log10 WBC removal. Transmission electron microscopy sections showed that T. cruzi parasites were removed from contaminated PRBC and PC samples primarily by mechanical mechanism without interacting with filter fibbers or blood cells. In addition, we found that T. cruzi parasites were also removed by a direct fibber adhesion. These data indicate that T. cruzi parasites are removed from infected blood not only by mechanical mechanism but also by biological mechanism probably mediated by parasite surface proteins.

Production of cytokine and chemokines by human mononuclear cells and whole blood cells after infection with Trypanosoma cruzi

INTRODUCTION: The innate immune response is the first mechanism of protection against Trypanosoma cruzi, and the interaction of inflammatory cells with parasite molecules may activate this response and modulate the adaptive immune system. This study aimed to analyze the levels of cytokines and chemokines synthesized by the whole blood cells (WBC) and peripheral blood mononuclear cells (PBMC) of individuals seronegative for Chagas disease after interaction with live T. cruzi trypomastigotes. METHODS: IL-12, IL-10, TNF-α, TGF-β, CCL-5, CCL-2, CCL-3, and CXCL-9 were measured by ELISA. Nitrite was determined by the Griess method. RESULTS: IL-10 was produced at high levels by WBC compared with PBMC, even after incubation with live trypomastigotes. Production of TNF-α by both PBMC and WBC was significantly higher after stimulation with trypomastigotes. Only PBMC produced significantly higher levels of IL-12 after parasite stimulation. Stimulation of cultures with trypomastigotes induced an increase of CXCL-9 levels produced by WBC. Nitrite levels produced by PBMC increased after the addition of parasites to the culture. CONCLUSIONS: Surface molecules of T. cruzi may induce the production of cytokines and chemokines by cells of the innate immune system through the activation of specific receptors not evaluated in this experiment. The ability to induce IL-12 and TNF-α contributes to shift the adaptive response towards a Th1 profile.

Methodological limitations of counting total leukocytes and thrombocytes in reptiles (Amazon turtle, Podocnemis expansa): an analysis and discussion

The aim of this paper is to compare three different methods for counting white blood cells [WBC] (Natt and Herrick method, estimation with 1,000 and 2,000 erythrocytes) and three methods for counting total thrombocytes [TT] (Wojtaszek method, estimation with 1,000 and 2,000 erythrocytes) in a South American freshwater turtle species, Podocnemis expansa, Schweigger 1812 (Reptilia, Pelomedusidae). Direct WBC counts using the Natt and Herrick method showed limitations, which are discussed here. The WBC and TT counts using 1,000 erythrocytes from blood smears are not recommended for Amazon turtles nor other reptilian species, since wide variation in counts can be observed. Estimation methods for determining WBC and TT based on 2,000 erythrocytes of blood smears were most acceptable because they allow a differentiation between leukocytes and thrombocytes and also had a smaller variation. The methods investigated here for the Amazon turtle, which have been widely used in other reptile species, provided evidence that the most acceptable method is not that of using diluted stains and a hemocytometer.

Evaluation of buffy coat 16S rRNA PCR, buffy coat culture and whole blood PCR for detection of bacteraemia

The use of Gram type-specific PCR on buffy coat from clinical specimens for the detection of bacteraemia was evaluated for the first time using whole blood culture as the gold standard. In addition, the established buffy coat culture and whole blood PCR were also compared. Gram-positive bacteria belonging to six species and Gram-negative bacteria from 10 species were isolated and identified by culture and detected using broad-range 16S rDNA primers and Gram-specific primers. Data from the three methods all conferred very high sensitivity, specificity, positive and negative predictive values when compared to whole blood culture. The Kappa coefficients of agreement were 0.9819 (buffy coat PCR), 0.9458 (whole blood PCR) and 1.0 (buffy coat culture), which establishes their validity as alternative methods to routine blood culture in detecting bacteraemia. In addition, results showed that there was a direct correlation of WBC counts greater than 12,000 cells per mm³ to the occurrence of bacteraemia as detected by the four methods (p < 0.05).

Laparoscopic versus open splenectomy in dogs

In the last few years, the use of laparoscopy in veterinary medicine has expanded and consequently so was the need for studies that establish the advantages, disadvantages and possible complications of each procedure. The purpose of the current study was to describe a laparoscopic splenectomy technique and the alterations due to this access, and compare it to the open procedure in dogs. A total of 15 healthy female mongrel dogs were used, with mean weight of 17.4±2.5kg. The animals were distributed into three groups: Group IA of open splenectomy (laparotomy) using double ligation of the vessels of the splenic hilum with poliglicolic acid, Group IB of open splenectomy (laparotomy) with bipolar electrocoagulation of the splenic hilum, and Group II of laparoscopic access with bipolar electrocoagulation of the splenic hilum. Operative time, blood loss, size of incisions, complications during and after surgery were evaluated. Other parameters included pain scores, white blood cell (WBC) counts and postoperative serum concentrations of alanine aminotransferase (ALT), alkaline phosphatase (ALP), creatine kinase (CK), C-reactive protein (CRP), glucose and cortisol. No differences were found in the evaluation of parameters between both open splenectomy techniques employed. Laparoscopic access presented significant differences (p<0,05) when compared with open surgery: Longer operative time, smaller abdominal access, decrease in blood loss, lower concentrations of CRP, higher levels of CK and ALP, and lower scores in the pain scale. Laparoscopic surgery showed fewer complications of the surgical wound. No significant differences were observed between groups in the postoperative temperature, WBC, ALT, cortisol and glucose concentrations. In conclusion, the laparoscopic technique is useful for splenectomy in dogs, being advantageous in terms of blood loss, surgical stress and surgical wounds. However, it expends more operative time and causes transitory increase in hepatic and muscular enzymes.

Hematologic, serum biochemistry and urinary values for captive Crab-eating Fox (Cerdocyon thous) in São Paulo state, Brazil

The importance of studies with hematological, serum biochemistry and urinary values of Crab-eating Fox (Cerdocyon thous) is based on the need for health care and maintenance of those populations. This paper has the objective to investigate hematological, serum biochemistry and urinary physiological parameters of the Crab-eating fox, comparing gender and age differences. Blood samples were collected in 2003 from 52 animals of different Zoos in São Paulo state, Brazil; 7mL of blood was used to obtain a complete blood cell count (CBC) and the profile of the serum biochemistry. Moreover, 5mL of urine were collected for analysis. There was no difference in values for male and female animals, as for the CBC and serum biochemistry. Some hematological and serum biochemical parameters were influenced by age, showing significant differences. Urinalysis results were just demonstrated in a descriptive form. The studied values were, RBC 4.35±0.73 x 10(6) /µL, WBC 7.72±3.66 x 10³ /µL (predominance of segmented neutrophils), platelets 227.06±111.58 x 10³ /µL, urea 43.06±14.28mg/dL and creatinine 1.03±0.24mg/dL. Hematological, serum biochemistry and urinary values obtained in this study can be used as physiological values of the captive Crab-eating Fox. It is possible to conclude that wild species need their own reference values, differentiating animals in captivity from free-ranging animals.

Effect of sex and seasons of the year on hematologic and serum biochemical variables of captive brown brocket deer (Mazama gouazoubira)

The Brown brocket deer (Mazama gouazoubira) is the most common free-living and captive deer in South America, especially in Brazil, and has great ecological and scientific significance. However, data on hematological and biochemical parameters in brown brocket deer are scarce. The goal of this study was to establish reference ranges for hematological and biochemical parameters of Mazama gouazoubira, comparing differences during the seasons of the year and between sex. Blood samples from ten adult healthy brown brocket deer (6 female and 4 male) were collected during daytime, monthly, during 12 months. The animals were maintained in individual stable, protected from noise and fed ad libitum with commercial ration and green fodder. For blood collection, animals were submitted to physical restrain for no longer than 2 minutes. The following parameters were determined: red blood cell count (RBC), haemoglobin concentration, packed cell volume (PCV), mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH), mean corpuscular haemoglobin concentration (MCHC), white blood cell count (WBC), platelet count, enzyme activity of alanine aminotransferase (ALT), aspartate aminotransferase (AST) and gamma-glutamyl transferase (GGT) and serum levels of alkaline phosphatase (ALP), creatine kinase (CK), total protein (TP), albumin, cholesterol, total calcium, ionic calcium, sodium, potassium, magnesium, triglycerides, creatinine and urea. Values were compared according to season and sex. RBC count, WBC count and MCV suggested seasonal influence. Haemoglobin concentration, PCV and MCV were influenced by sex. Serum concentration of total calcium, ionic calcium, sodium, potassium and magnesium were influenced by season. Serum magnesium was also influenced by sex. The blood parameters herein reported may be useful as reference values for diagnostic and prognostic purposes in captive brown-brocket deer.

Analysis of hematologic and serum chemistry values of Spheniscus magellanicus with molecular detection of avian malarial parasites (Plasmodium spp.)

Magellanic penguins (Spheniscus magellanicus) routinely migrate from their breeding colonies to Southern Brazil often contracting diseases during this migration, notably avian malaria, which has been already reported in Brazil and throughout the world. Detection of Plasmodium spp. in blood smears is the routine diagnostic method of avian malaria, however it has a low sensitivity rate when compared to molecular methods. Considering the negative impact of avian malaria on penguins, the aim of this study was to detect the presence of Plasmodium spp. in Magellanic penguins using Polymerase Chain Reaction (PCR) and by verifying clinical, hematological, and biochemical alterations in blood samples as well as to verify the likely prognosis in response to infection. Blood samples were obtained from 75 penguins to determine packed cell volume (PCV), red blood cell (RBC) and white blood cell (WBC) counts, mean corpuscular volume (MCV), uric acid, total protein, albumin, globulin and aspartate aminotransferase (AST) activity levels. Whole blood samples were used for PCR assays. Plasmodium spp. was detected in 32.0% of the specimens using PCR and in 29.3% using microscopic analyses. Anorexia, diarrhea and neurological disorders were more frequent in penguins with malaria and a significant weight difference between infected and non-infected penguins was detected. PCV and MCV rates showed no significant difference. RBC and WBC counts were lower in animals with avian malaria and leukopenia was present in some penguins. Basophil and lymphocyte counts were lower in infected penguins along with high monocyte counts. There was no significant difference in AST activities between infected and non-infected animals. There was a significant increase in uric acid values, however a decrease in albumin values was observed in infected penguins. Based on this study, we concluded that Plasmodium spp. occurs in Magellanic penguins of rehabilitation centers in Southeastern Brazil, compromising the weight of infected animals with clinical alterations appearing in severe cases of this disease. It was also noted that, although the hematological abnormalities presented by these animals may not have been conclusive, leukopenia, monocytosis and the decrease of basophils and lymphocytes revealed an unfavorable prognosis, and Plasmodium spp. infections may progress with elevated uric acid concentration and low albumin levels.

Erythrocytes modulate cell cycle progression but not the baseline frequency of sister chromatid exchanges in pig lymphocytes

The effect of co-culturing varying concentrations of pig and human red blood cells (RBCs) on the baseline frequency of sister chromatid exchanges (SCEs) and cell-cycle progression in pig plasma (PLCs) and whole blood leukocyte cultures (WBCs) was studied. No variation in SCE frequency was observed between pig control WBC and PLC. Addition of pig and human RBCs to pig PLCs did not modify the baseline frequency of SCEs. On the other hand, cell proliferation was slower in PLCs than in WBCs. The addition of pig or human RBCs to PLCs accelerated the cell-cycle progression of pig lymphocytes. When RBCs were added to PLCs the concentration and time sequence of RBC incorporation affected the cell-cycle progression of swine lymphocytes. When doses of pig or human RBCs equivalent to those present in WBCs were added immediately after PLC stimulation, the cell-cycle kinetics were similar to those of WBCs. Shorter co-incubation periods or a reduction in the dose of RBCs made cell-cycle progression intermediate between PLC and WBC values. Thus, pig and human RBCs modulated the in vitro cell-cycle progression of pig lymphocytes in a time- and dose-dependent manner, and the low baseline frequency of SCEs of pig lymphocytes is independent of the presence or absence of erythrocytes in culture