Evaluasi Validitas Human Cortisol Enzyme-Linked Immunosorbent Assay (ELISA) Kit dan Waktu Sentrifugasi Sampel Darah untuk Pengukuran Konsentrasi Hormon Kortisol pada Kambing Kacang

ABSTRAK. Penggunaan human cortisol enzyme-linked immunosorbent assay (ELISA) kit untuk pengukuran hormon kortisol pada hewan dan keterlambatan waktu sentrifugasi sampel darah untuk analisis hormon perlu dievaluasi. Penelitian ini bertujuan untuk mengevaluasi validitas human cortisol ELISA kit (EIA-1887, DRG Instruments GmbH, Germany) untuk pengukuran konsentrasi kortisol dan menguji pengaruh keterlambatan waktu sentrifugasi sampel darah terhadap stabilitas konsentrasi kortisol pada kambing kacang. Sampel darah dikoleksi dari delapan ekor kambing kacang. Uji validitas kit EIA-1887 dilakukan secara: a) analitik (uji parallelism, akurasi, dan presisi), dan b) biologis (pengukuran kortisol sebelum dan setelah transportasi). Uji keterlambatan waktu sentrifugasi terhadap stabilitas konsentrasi kortisol dilakukan dengan 5 perlakuan yaitu disentrifugasi kurang dari 1 jam (P1/kontrol), 6 jam (P6), 12 jam (P12), 18 jam (P18),dan 24 jam (P24) setelah darah dikoleksi. Data uji parallelism dianalisis dengan uji persamaan kemiringan, uji presisi dihitung % CV (coefficient variation) intra-assay dan inter-assay, uji akurasi dihitung % recovery, uji T untuk validasi biologis, dan uji ragam (One Way Anova) untuk pengaruh waktu sentrifugasi. Hasil uji parallelism menunjukkan kurva sampel kambing kacang sejajar/parallel dengan kurva standar kortisol. Akurasi kit EIA-1887 adalah 103,43±7,85%, dan % CV intra-assay dan inter-assay adalah 10%. Konsentrasi kortisol setelah transportasi secara signifikan lebih tinggi daripada sebelum transportasi (p0,05). Adanya penurunan secara nyata konsentrasi kortisol pada darah yang disentrifugasi 24 jam (P24) setelah koleksi (p0,05). Kesimpulan, human cortisol ELISA kit (EIA-1887) memiliki validitas yang baik secara analitik dan biologis untuk digunakan dalam pengukuran konsentrasi kortisol pada kambing kacang. Keterlambatan waktu sentrifugasi selama 24 jam berpengaruh terhadap konsentrasi kortisol. (Evaluation the Validity of Human Cortisol Enzyme-Linked Immunosorbent Assay (ELISA) Kit and Centrifugation Time of Blood Sample for Measuring the Concentration of Cortisol in Kacang Goats) ABSTRACT. The use of human cortisol ELISA kit for measuring cortisol in animals and delayed to blood centrifugation time for hormone measurement need to be evaluated. This study aimed to evaluate the validity of human cortisol ELISA kit (EIA-1887, DRG Instruments GmbH, Germany) for cortisol measurement and effect of delayed to blood centrifugation time on cortisol concentrations in kacang goats. Blood was collected from eight kacang goats. Validation test of EIA-1887 kit was performed through: a) analytical (parallelism, accuracy, and precision tests), and b) biological validations (measuring cortisol concentrations before and after transportation). Five treatments were performed to test delayed to centrifugation time: blood centrifuged at 1 h (control, P1), 6 h (P6), 12 h (P12), 18 h (P18), and 24 h (P24) after collection. Parallelism data were analyzed by slope equality test, precision and accuracy calculated by % CV of intra-and inter-assay, and % recovery, respectively. Data of biological validation and centrifugation time effects were analyzed by Student t-test, and one way ANOVA, respectively. Results of parallelism showed that serial dilution curve of kacang goat plasma was parallel with cortisol standard curves. Accuracy of EIA-1887 kit was 103.43±7.85%, and % CV of intra-and inter-assay were 10%. Concentration of cortisol after transportation was significantly higher than before transportation (p0.05). Concentration of cortisol was significantly decreased when blood was centrifuged at 24 h after collection (P0.05). In conclusion, human cortisol ELISA kit (EIA-1887) is a reliable assay for measuring cortisol in plasma of kacang goat. Delayed to blood centrifugation time affect cortisol concentrations.

Stability of freeze-dried plasma rich in growth factors eye drops stored for 3 months at different temperature conditions

Purpose: The purpose of this study was to analyze the biological content and activity of freeze-dried plasma rich in growth factors eye drops after their storage at 4°C and at room temperature for 3 months with respect to fresh samples (time 0). Methods: Plasma rich in growth factors was obtained after blood centrifugation from three healthy donors. After platelet activation, the obtained plasma rich in growth factors eye drops were lyophilized alone or in combination with lyoprotectant (trehalose), then they were stored for 3 months at room temperature or at 4°C. Several growth factors were analyzed at each storage time and condition. Furthermore, the proliferative and migratory potential of freeze-dried plasma rich in growth factors eye drops kept for 3 months at different temperature conditions was evaluated on primary human keratocytes. Results: The different growth factors analyzed maintained their levels at each time and storage condition. Freeze-dried plasma rich in growth factors eye drops stored at room temperature or 4°C for 3 months showed no significant differences on the proliferative activity of keratocytes in comparison with fresh samples. However, the number of migratory human keratocytes increased significantly after treatment with lyophilized plasma rich in growth factors eye drops kept for 3 months compared to those obtained at time 0. No significant differences were observed between the freeze-dried plasma rich in growth factors eye drops whether mixed or not with lyoprotectant. Conclusion: Freeze-dried plasma rich in growth factors eye drops preserve the main growth factors and their biological activity after storage at room temperature or 4°C for up to 3 months. Lyophilized plasma rich in growth factors eye drops conserve their biological features even without the use of lyoprotectants for at least 3 months.

Method to obtain platelet-rich plasma from rabbits (Oryctolagus cuniculus )

Platelet-rich plasma (PRP) is a product easy and inxpesnsive, and stands out to for its growth factors in tissue repair. To obtain PRP, centrifugation of whole blood is made with specific time and gravitational forces. Thus, the present work aimed to study a method of double centrifugation to obtain PRP in order to evaluate the effective increase of platelet concentration in the final product, the preparation of PRP gel, and to optimize preparation time of the final sample. Fifteen female White New Zealand rabbits underwent blood sampling for the preparation of PRP. Samples were separated in two sterile tubes containing sodium citrate. Tubes were submitted to the double centrifugation protocol, with lid closed and 1600 revolutions per minute (rpm) for 10 minutes, resulting in the separation of red blood cells, plasma with platelets and leucocytes. After were opened and plasma was pipetted and transferred into another sterile tube. Plasma was centrifuged again at 2000rpm for 10 minutes; as a result it was split into two parts: on the top, consisting of platelet-poor plasma (PPP) and at the bottom of the platelet button. Part of the PPP was discarded so that only 1ml remained in the tube along with the platelet button. This material was gently agitated to promote platelets resuspension and activated when added 0.3ml of calcium gluconate, resulting in PRP gel. Double centrifugation protocol was able to make platelet concentration 3 times higher in relation to the initial blood sample. The volume of calcium gluconate used for platelet activation was 0.3ml, and was sufficient to coagulate the sample. Coagulation time ranged from 8 to 20 minutes, with an average of 17.6 minutes. Therefore, time of blood centrifugation until to obtain PRP gel took only 40 minutes. It was concluded that PRP was successfully obtained by double centrifugation protocol, which is able to increase the platelet concentration in the sample compared with whole blood, allowing its use in surgical procedures. Furthermore, the preparation time is appropriate to obtain PRP in just 40 minutes, and calcium gluconate is able to promote the activation of platelets.

First record of Trypanosoma sp. (Protozoa: Kinetoplastida) in tuvira (Gymnotus aff. inaequilabiatus) in the Pantanal wetland, Mato Grosso do Sul State, Brazil

The blood infection by Trypanosoma sp. in tuvira (Gymnotus aff. inaequilabiatus) from the Pantanal wetland was reported in this study. Ten fish from the Paraguay River in the Pantanal were evaluated for the presence of hemoflagellates. Trypomastigotes of Trypanosoma sp. were observed in blood smears from three fish (30% prevalence) and some forms were seen to be undergoing division. Using the diagnostic methods of fresh examination and blood centrifugation in hematocrit capillary tubes, the prevalence rate was 80%. This is the first report of Trypanosoma sp. in tuvira in Brazil.

Bovine Trypanosomosis in three districts of East Gojjam Zone bordering the Blue Nile River in Ethiopia

Background: Bovine trypanosomosis is a serious constraint to agricultural production in extensive areas of Ethiopia. Methodology: A cross-sectional study was conducted to determine the prevalence of bovine infection with trypanosomes and to identify the prevailed trypanosome species in three districts of the East Gojjam zone bordering the Blue Nile River from March 2005 to February 2006. Cattle from 9 different localities were checked using microscopical examination of wet blood smears, thin and stained bloodsmears, and by blood centrifugation followed by the examination of the resultant buffy coats. Result: Of the total 3,360 cattle investigated, 8.2% (3.5%, 11.6% and 9.4% from Dejen, Machakel and Baso-Liben districts respectively) were found to be infected with trypanosomes. Of the total 275 positive animals, 249 (90.5%) appeared to be infected with Trypanosoma vivax; 11 (4%) were infected with T. congolense; and 15 (5.5%) were infected with mixed infection of T. vivax and T. congolense. The prevalence of infection with T. vivax was significantly higher than that of T. congolense (P

Determination of the Presence of Bovine Immunoglobulin G in Liquid or Spray-Dried Porcine Plasma and Whole Blood by Agar Gel Immunodiffusion

There is currently urgent interest in identifying the species of origin of the components of different animal by-products. In Europe, this interest is expected to increase with authorization of the re-introduction of these proteins into animal feed formulations. The number of validated methods to differentiate the species of origin for most of these products is limited. An easy, inexpensive, and accurate test was developed to determine the cross-contamination of bovine blood or plasma in porcine whole blood and plasma, both before and after spray drying. Agar gel immunodiffusion (AGID), the studied technique, detected the presence of bovine immunoglobulin G (IgG) in porcine plasma and in whole blood at inclusion levels >0.5% (v/v) in all cases. However, detectability was lower in liquid plasma (0.3%, v/v) and in liquid whole blood (0.5%, v/v). No differences were found when cross-contamination was simulated before or after whole blood centrifugation. The method described is reliable and inexpensive, and the samples are easy to prepare. Both minimal laboratory equipment and expertise are required for detection of bovine IgG in porcine blood products at inclusion levels of >0.5% (v/v).