1. In platelet count if you found, in the hematological report or results, the platelet count is up to 100 - 500 ´ 103, less than the normal range ( 700 to 1100 ´ 103 ). How to correct it in the next project?
Answer: In such a case, collect the blood in 1.5
mg/ml of EDTA solution, shake it slowly and tap it with a finger, repeat the
experiment with fresh blood (This condition mainly occurs due to the instant
blood clot, due to this the blood sample gives a different result every time you take the reading. In this condition, repeat the reading immediately
two to three times and compare it with the normal range.) Many times the blood sipper of the hematology analyzer sips only the clotted area in the Eppendorf, thus giving less likely platelet counts. To increase or get to the exact result make the Eppendorf
2. Function Battery Test (Irwine Test): It
has three major areas:
Neurobehavioral: It includes Landing Foot Splay (30cm 10 to 9 cm), Air
writing reflex (30cm, dorsal/ if not came dorsal side then problem), Grip
strength, and Auditory finger responses.
Motor: Use
of an Actimeter or an Open-field, to check skeleton muscles by Fast and slow
movement, Stereotype movement that includes grooming, itching, and rearing- that
you have observed manually, it also includes tonic and colonic movement.
Autonomous: All body secretions- lacrimal, salivary, mucus, eye color, fur
appearance, pupil dilatation, eye blinking response.
3. What you will do if, in your data, the blood glucose level is reduced than the
normal range?
Answer: If the blood glucose level is reduced than the normal range, it means
before the blood withdrawal animal has fasted for more than 12 hrs. Also, standardize
the blood glucose level by glucometer or standardize your blood glucose level in your lab animals. Many time serum glucose is reduced just because of a less
effective centrifuge to produce serum, sometime RBCs remains in serum those RBCs consume the glucose from the blood, which can be blocked by glycolysis by the use of Sodium fluoride.
4. Clinical Biochemistry:
If possible complete the biochemistry same day, if the not possible same day, preserve
the serum at -80 C only, and complete the biochemistry sample reading the very next
day. (Preserving the serum at -80 for a long time, may reduce the protein
level).
- During blood withdrawal give the proper handling and complete anesthesia to all animals to remove the errors in SGOT and SGPT values.
- Use lithium heparin for serum (immediately centrifuge) preparation, it will help give more amount of serum than the without lithium heparin. Neither uses Heparin sodium nor EDTA, and both may change the electrolyte (Sodium and calcium respectively) results.
5. Vaginal Smear
(Optional in 407, and mandatory in 408). Explained in detail below video.
6. Reticulocytes: For
reticulocyte count generally 6-part Hematology Analyzer is required, many times
you find variations in the final result. That can be overcome, by performing the
same parameter manually in the lab and comparing it with the hematology analyzer’s
report. Make a slide smear with EDTA mixed blood, and stain the slides with Methylene
blue. Finally, count the reticulocyte number per 1000 RBC. The normal range
is 1.7 to 4.7 % (or 4 to 40 per 1000 RBC).
7. Organs that are separated
during Gross Necropsy in OECD 407 and 408:
|
Sr. No: |
Organs |
Sections |
Slides |
|
1 |
Liver |
3 |
1 |
|
2 |
Kidney |
2 |
1 |
|
3 |
Adrenals |
2 |
1 |
|
4 |
Spleen |
1 |
|
|
5 |
Brain |
3 |
1 |
|
6 |
Pituitary |
1 |
1 |
|
7 |
Aorta |
1 |
|
|
8 |
Heart |
1 |
1 |
|
9 |
Thymus |
1 |
1 |
|
10 |
Urinary Bladder |
1 |
|
|
11 |
Testes |
2 |
1 |
|
12 |
Ovaries |
2 |
1 |
|
13 |
Uterine horn |
2 |
|
|
14 |
Uterine Cervix |
1 |
1 |
|
15 |
Vagina |
1 |
|
|
16 |
Epididymis |
2 |
1 |
|
17 |
Prostate |
1 |
1 |
|
18 |
Seminal Vesicles with
coagulation glands |
2 |
1 |
|
19 |
Thyroid |
1 |
1 |
|
20 |
Trachea |
1 |
|
|
21 |
Lungs |
3 |
1 |
|
22 |
Stomach |
3 |
1 |
|
23 |
Pancreas |
1 |
1 |
|
24 |
Duodenum |
1 |
1 |
|
25 |
Jejunum |
1 |
|
|
26 |
Ileum |
1 |
|
|
27 |
Caecum |
1 |
1 |
|
28 |
Colon |
1 |
|
|
29 |
Rectum |
1 |
|
|
30 |
Sciatic Nerve |
1 |
1 |
|
31 |
Skin with mammary
gland |
1 |
|
|
32 |
Eye |
2 |
1 |
|
33 |
Skeletal Muscle |
2 |
1 |
|
34 |
Mesenteric lymph node |
1 |
1 |
|
35 |
Femur with bone marrow |
1 |
1 |
|
36 |
Spinal Cord |
3 |
1 |
|
37 |
Salivary Gland |
1 |
1 |
|
38 |
Mandibular Lymph node |
1 |
1 |
Note: In the table, it has mentioned the number of sections and slides
needed as per organ size.
8. Eyes and Testes are preserved in modified Davison fixative (For
100 ml composition: 50 ml distilled water + 30 ml formaldehyde + 5 ml glacial
acid +15 ml ethanol).
9. 10 % Normal buffer formalin (For 1000 ml composition: 900 ml
distilled water +100 ml formalin + 4 g diphosphate buffer + 6 g Monophosphate
buffer).
10. Dissection: Blood withdrawals are done using isoflurane and 150
mg thiopental sodium is used for euthanizing the animals by intraperitoneal injection.
11. Hormones like T3, T4, and TSH are only evaluated in OECD 408, by using
blood serum in a biochemistry analyzer or the Elisa method.
12. In ophthalmic observation retinal damage, corneal opacity, and blood vessels aneurysm are checked thrice throughout the experiment, Initially after quarantine for animal allocation in the group, the second time before Day 28 day in the 4th week, and finally before day 42 in 6th week for recovery animals.
13. Coagulation (Fibrinogen, PT, APPT): We have to prepare a 3.2 % Sodium citrate anticoagulant in plasma for Coagulation analysis. EDTA causes chelate with calcium thus 3.2 % tri-sodium citrate is generally used as an anticoagulation agent.
END OF DOCUMENTS
You may like to read these links:
1. Check Out the Tricks and Tips with Some Trouble Points While Conducting in OECD 407 and 408
2. List of OECD Guidelines or Toxicological Studies
3. List of Guidelines Used for Medical Devices Testing
4. What are Bradford Hill's criteria for any chemical identification?
5. Animal Facility Design - Small Laboratory Animals (Rat, Mice, Rabbit, Guinea Pig)
6. List of Chemicals Needed for In-vivo Laboratory
7. OECD Test Guidelines 425 detailedOutlines| AOT425StatPgm Software Installation LD50 Calculation
8. List of Guidelines for Toxicology Animal Studies
9. Checklist for Toxicology (OECD-407) study preparation for Histopathology
10. Source: Research Needs YouTube channel video: OECD Test Guidelines 407 and 408 detailed descriptions in Hindi and OECD 407 and 408 Study Troubleshooting
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