Artifactual changes in hematological variables in equine blood samples stored at different temperatures and various anticoagulants

The objective of this study was to determine the effect of storage time, temperature, and anticoagulant on hematologic parameters in equine blood samples. Blood samples were obtained from 50 clinically healthy warm-blooded horses at two major equestrian complexes in Tehran, Iran. The samples were collected in three different tubes containing EDTA, sodium citrate, or heparin and were analyzed within 4?h of collection. Blood samples collected into EDTA-containing tubes were stored at 4°C or 24°C. Each sample was analyzed again at 24, 48, and 72?h after collection. The statistically significant (P?<?0.05) alterations included decreased RBC count and increased hemoglobin concentration [Hgb] in blood samples stored at 24°C after 48 and 72?h; increased hematocrit in blood samples stored at 24°C after 24?h; decreased hematocrit in blood samples stored at 4°C after 72?h; decreased MCHC in blood samples stored at 4°C after 72?h; and decreased total WBC count in blood samples stored at 24°C after 48?h. Although there was no significant difference in hematologic analytes between heparinized and EDTA-anticoagulated blood samples, most of the hematologic analytes were decreased significantly (P?<?0.05) in sodium citrate-containing blood samples, compared with blood samples stored in EDTA. The results of this study suggest that within certain limitations for some hematologic analytes, equine blood samples stored in EDTA at 4°C for up to 72?h may be suitable for hematologic testing.     

The leukocyte composition of peripheral blood buffy coat

To assist in the interpretation of buffy coat smears from leukopenic patients, we determined the composition of the buffy coat of the blood of normal volunteers. Leukocytes were concentrated by centrifugation in microhematocrit tubes. Stained smears of these cells showed minor differences in the proportions of neutrophils, lymphocytes, and monocytes when compared to unconcentrated peripheral blood. Granulocytes of greater immaturity than the metamyelocyte were not encountered, and their presence should suggest the possibility of marrow dysfunction. Occasional young monocytoid cells and "stimulated" lymphocytes were seen.     

Cytomegalovirus DNA stability in EDTA anti-coagulated whole blood and plasma samples

Background

Cytomegalovirus (CMV) DNA viral load testing is routinely performed in centers that serve patients that are immunosuppressed from organ or hematopoietic stem cell transplantation. Clinical laboratories that offer this testing often face practical concerns about the storage of these specimens to ensure accurate measurement for patient care. The published studies that assess CMV DNA stability at 4 °C have done so only up to 72 h.

Objective

Our objective was to determine the stability of CMV DNA in whole blood and plasma for clinical viral load testing over a 14 day period.

Study design

Twenty-one plasma samples that were CMV-positive and twenty whole blood samples (including eleven CMV-negative whole blood samples spiked with CMV-positive plasma) were stored at 4 °C and underwent extraction and amplification at 3 time points: Day 0, Day 7, and Day 14.

Results

Log₁₀ values were calculated and t-test was performed on the values comparing Day 0 to Day 14 for plasma and whole blood. There was no statistically significant difference between Day 0 and Day 14 for both specimen types, including the CMV-negative whole blood specimens that were spiked with CMV-positive plasma.

Conclusions

CMV DNA in plasma and whole blood is stable for 14 days at a temperature of 4 °C.     

Detection of Pneumococcus from whole blood, buffy coat and serum samples by PCR during bacteremia in mice.

Whole blood, purified leukocyte fraction and serum were investigated as specimens for the detection of pneumococcal bacteremia by polymerase chain reaction (PCR) in mice. The PCR findings were compared to the blood culture results. Samples were taken from animals 3 and 12 h after intraperitoneal bacterial challenge. The pneumococcal culture was positive in 27% and 77% of blood samples at 3 and 12 h after challenge, respectively. All whole blood samples were positive by PCR at both time points. Of the buffy coat samples, two of the three pools were PCR-positive at 3 h and all pools at 12 h after bacterial challenge. In the serum sample group, only 40% of the sera were PCR-positive at 3 h, while at 12 h 90% of the samples were PCR-positive. According to these results, whole blood seems to be the best specimen for the detection of pneumococcal DNA by PCR in bacteremic mice.     

Usefulness of quantitative buffy coat blood parasite detection system in diagnosis of malaria

A rapid test for diagnosis of malaria based on acridine orange staining of centrifuged blood samples in a microhematocrit tube (QBC) was compared with thick and thin peripheral blood smears in 2274 samples. Malaria was diagnosed in 239 (10.5%) patients by Leishman's staining technique and QBC method. The QBC method allowed detection of an additional 89 (3.9%) cases. Thus the prevalence rate of malaria during the study was 14.4%. In 1946 patients who were negative by the QBC technique, the Leishman's stained smears did not provide any help in malaria diagnosis. Analysis of the relative quantity of parasites in the specimens, in the QBC method, revealed that 80 out of 89 QBC positive but smear negative cases, had a very low parasite number (less than 10 parasites per QBC field). Although QBC method was superior to the smear for malarial parasite detection, species identification was not possible in 26 (7.9%) cases by this technique. In 95.7% (n = 314) QBC positive cases, the buffy coat in the QBC tube appeared pigmented (gray to black). The colour of the buffy coat was therefore considered by us as a predictor of positivity and could be taken as an indicator for a careful and more prolonged search for the parasites. Thus, the QBC technique has its advantages in terms of speed, sensitivity and ease, especially in an endemic area as ours, where the level of parasitaemia is low and more than 70 to 80 smears need to be examined per day. However, the age old Romanowsky stains still appear superior for species identification.     

Granulocyte enriched buffy coat transfusions in neutropenic patients

Twenty four courses of granulocyte enriched buffy coat transfusions were administered to 22 different infected neutropenic patients. Those patients who received an average of greater than or equal to 13 units per transfusion, which represented a mean of 1.02 X 10(10) granulocytes, had a survival rate of only 30% which was not significantly different from the 28.5% found among patients who received an average of less than or equal to 12 units per transfusion, which represented a mean of .63 X 10(10) granulocytes. In addition, no significant difference in survival rate was found between patients who received a course of greater than or equal to four transfusions and those who received a course of less than or equal to three transfusions. Finally, no significant difference in survival rate was found between patients with acute leukemia and those with other disorders or between patients with positive cultures and those whose cultures were negative. Given the poor clinical results associated with buffy coat transfusions, it is concluded that every effort should be made to recruit single leukapheresis donors for the support of infected neutropenic patients, rather than use granulocyte enriched buffy coats as they are presently produced.     

Comparative study of peripheral blood smear, quantitative buffy coat and modified centrifuged blood smear in malaria diagnosis

The present study was aimed at modifying the centrifuged blood smear (modified centrifuged blood smear or MCBS), to make it a feasible and standardized procedure. The results obtained were compared with the current diagnostic methods - peripheral blood smear (PBS) and quantitative buffy coat (QBC). Blood samples collected from 100 suspected malaria patients were subjected to all three tests. It was found that PBS had 86.79% sensitivity and was absolutely specific. QBC was 96.22% sensitive and 93.61% specific. The majority of variations occurred in PBS negative cases; cases with parasite count Plasmodium falciparum. It was seen that by the addition of centrifugation to the conventional smear technique (MCBS) improved its sensitivity from 86.79% to near 100%. QBC and MCBS were found superior to PBS. Since MCBS combines principles of both QBC and PBS, it is as sensitive as QBC, as specific as PBS, and above all, easily performed and affordable.     

Peripheral blood buffy coat smear: a promising tool for diagnosis of visceral leishmaniasis

Confirmative diagnosis of visceral leishmaniasis (VL) is still a challenge at the primary health care facilities in most of the rural areas of endemicity in the Indian subcontinent. Conventional methods for parasitological confirmation are risky and require skilled personnel, and hence they are unavailable to the poor people in the regions of endemicity. Buffy coat smear microscopy, as a minimally invasive, simple alternative for the parasitological diagnosis of VL, was evaluated in this prospective study. One hundred twelve VL patients were enrolled in this study. The buffy coat was separated from peripheral blood of all enrolled subjects using Histopaque-1119 solution. Leishman-stained buffy coat smears were examined for Leishmania donovani bodies, and buffy coat was also utilized for detection of parasite DNA by Leishmania nested PCR (LnPCR) for all cases. Concomitant splenic smears could be examined for L. donovani bodies in 66 cases, and the parasite load was graded on a scale of 1+ to 6+ for L. donovani-positive smears. All splenic smear-positive cases were also found to be positive by LnPCR. Of 112 enrolled VL cases, 103 (92%) were found to be positive for L. donovani bodies in buffy coat smear microscopy, which is promising as a confirmative diagnosis tool. We have also found a significant association of the buffy coat smear positivity with parasitic burden in the spleen smear. In this preliminary observation in Bangladesh, buffy coat smear microscopy has been found to be very simple, minimally invasive, and risk-free method of parasitological diagnosis of VL with a good diagnostic accuracy and potential for field use.     

The effects of changes in haematocrit on the intrarenal distribution of blood flow in the dog's kidney

1. The effect of changes in the haematocrit of blood perfusing the kidney on its intrarenal distribution was studied in dogs.

2. Two types of preparations were employed. (i) In the isolated perfused kidney evidence is presented that flow in the autoregulating preparation represents predominantly cortical flow while flow in the `low flow non-autoregulating' kidney reflects medullary flow. (ii) In the intact kidney renal blood flow rate and its intrarenal distribution was studied by the injection of ¹³³Xe into the renal artery and measuring its clearance from the kidney by an external counter.

3. In both types of preparation cortical flow was found to be independent of changes in P.C.V. but medullary flow varied inversely with haematocrit.

4. A change in the haematocrit of the perfusing blood leads to alteration of its viscosity. It was argued that an increase in viscosity must lead to a reduction in the resistance of the cortical afferent arterioles but that medullary afferent arterioles were not able to respond in this manner.

5. These findings demonstrate that changes in total body haematocrit cause a redistribution of blood flow between renal cortex and medulla.

     

Studies of stress in farm animals

Major changes have taken place in animal production over the last three decades. Housing conditions have changed dramatically over this period, and there has been a striking increase in production. Agricultural animals try to cope with these highly demanding conditions (stressors) using behavioural and physiological stress responses aiming to restore homeostasis. When these responses are not successful or when they are thwarted, typical behavioural and physiological symptoms of chronic stress occur. In this situation, the welfare of the animal is clearly at stake. Moreover, chronic stress may seriously affect the efficiency of animal production and the quality of the product. Ultimately, detailed knowledge of stress responses in agricultural animals will allow the formulation of housing and management requirements, including handling by humans, which will benefit welfare and health of farm animals as well as production efficiency. This paper briefly addresses some important issues regarding the study of stress and welfare in farm animals, and discusses some recently developed experimental methods as well as relevant results obtained in our laboratory. Originally presented at ECCP 97.     

Fluorescence in situ hybridization (FISH) using snap frozen buffy coat

We report the successful application of interphase fluorescent in situ hybridization (FISH) using extracted nuclei from snap frozen buffy coat stored for the purpose of molecular analysis. Included in this study were 30 frozen buffy coat specimens, ranging from <1 day to 3 yr old. Thawing of the buffy coat at 37 degrees C was followed by lysing erythrocytes with lysing solution (LYSE S III DIFF, Coulter Corp.). Cells were fixed and slides were prepared according to manufacturer's FISH protocol. All specimens from the 30 frozen-thawed buffy coats were suitable for FISH evaluation, and signal intensities were not significantly related to the age of specimens. In conclusion, interphase FISH is feasible using frozen-thawed buffy coat; the technique will be useful for retrospective molecular cytogenetic analysis of hematologic malignancies.     

A comparison of peripheral blood and buffy coat smear examination for the prediction of bone marrow relapse of acute lymphoblastic leukaemia in childhood

In an attempt to see if buffy coat smear examination might be an alternative to bone marrow aspiration for predicting relapse, 98 consecutive bone marrow aspirates from 96 children with acute lymphoblastic leukaemia were examined blind with buffy coat and peripheral blood from the same patients. The 28 bone marrow aspirates from children no longer on treatment were all normal, and routine aspirates would appear unjustified in these patients. Eight of the remaining marrows showed relapse, but only three were not predicted from the peripheral blood and buffy coat. In no case was buffy coat superior to peripheral blood in the detection of bone marrow relapse. Routine bone marrow aspirates are an inefficient way of diagnosing relapse in acute lymphoblastic leukaemia in childhood, despite their precision, and a prospective study is needed to determine their value.