Clinical Chemistry IMPC_CBC_003

Purpose

Clinical chemistry determines biochemical parameters in plasma including enzymatic activity, specific substrates and electrolytes.

 

Ontological description: MP:0001545 – blood physiology abnormalities.

Experimental Design

Minimum number of mutant animals: must maintain ≥ 7 size for male and female.

Age of animals: 17 weeks

Sexual dimorphism: Yes for some of the parameters.

Equipment

  1. Clinical chemistry analyser
  2. Vortex
  3. Refrigerated centrifuge
  4. Eppendorf tubes
  5. Pipettes (200-1000 ul)

Procedure

Set up the clinical chemistry analyser and perform QC analyses of the control reagents in accordance with the equipment guidelines.

  

Sample collection and preparation:

  1. Collect the appropriate volume of blood required (160-200μl of plasma), for the clinical chemistry analyser being used for assessment, in gel tube containing lithium Heparin with the relevant blood collection procedure (see IMPC protocol Blood collection by retro-orbital puncture). Time of day for collection is in the morning, starting no earlier than 07:30.
  2. Keep whole blood samples in a bag on wet ice until centrifugation. Centrifuge for 10 minutes at 5000 x g in a refrigerated centrifuge set at 8°C. If plasma samples cannot be analysed immediately, keep them in the fridge until analysis.
  3. Analysis of samples is optimally done on the day of collection. When not possible the plasma samples can be stored at 2-8°C. If samples require storage for > 48 hours, freeze plasma at -20 °C in single aliquots. All samples are allowed to come to room temperature prior to analysis.
  4. Use plasma samples undiluted or diluted to a ratio of 1:2 with deionised water if the volume is insufficient.
  5. Plasma samples that were frozen or stored in the fridge should be vortexed briefly and centrifuged again at ~5000 x g for 2-3 minutes immediately prior to analysis. If necessary, remove fibrin clots using a wooden applicator.

 

Analysis:

Samples that produce results that lie outside the linear range for a specific assay have to be re-tested. In some cases it may be necessary to dilute samples with water to bring test results into range.

Notes

Blood collection for Clinical Chemistry and Hematology is performed as a non-fasting, terminal procedure, with some mice being used for subsequent gross pathology and other clinic-specific parameters included in terminal assessments. Whole blood (for Hematology) and plasma (for Clinical Chemistry) require different collection tubes so two independent samples are required from each mouse.

The information about the date of the experiment, that is the date when the measurement is performed, is an important parameter which is to be submitted in the Experiment xml file (dateOfExperiment="2013-02-28").

Dilution. Dilution of blood is highly discouraged, but is allowed when the total necessary amount is not obtained. If dilution is necessary then the assays should be done in one run.

Hemolysis. Two fields currently exist to capture metadata information about the hemolysis status in the clinical chemistry plasma samples.The first is the LIH Hemolysis severity score which can only be performed by clinics who run one of the Beckman Coulter AU-series of analysers. Such clinics are encouraged to capture and submit the hemolysis score of the LIH test in this field. Clinics who do not have an AU analyser are encouraged to use the second/alternative field which is simply titled Hemolysis.  Simply enter "slight”, “moderate”, or “marked" based on whether the sample is visibly haemolysed or not. Provision of this information is not compulsory and it is suggested that any clinic completes at least one field or the other (not both).

Data QC

  1. Plasma samples must be free of Fibrin clots in order to be analysed.
  2. Badly haemolysed samples should be discarded.
  3. Each morning, all parameters are tested with control sera (see ESLIM_015_001_Annex_3: Controls for biochemistry on AU400). Some parameters are tested with control serum level 1 (Beckman Coulter System Reagent, ODC0003) and control serum level 2 (Beckman Coulter System Reagent, ODC0004), which consists of lyophilised human plasma with a normal and a pathological concentration. Other parameters are tested with specific controls from other suppliers.
  4. Controls are thawed and vortexed before utilisation and loaded according to the analyser’s display. Control values must lie within the acceptable range indicated by the manufacturer, otherwise the specific tests must be recalibrated and specific measurements repeated. Controls can be stored in 200μl aliquots at -20°C for up to 1 week.

Metadata and examples

 

Metadata

Example

Equipment ID

ID of the machine used when more than 1 is used  having same model and manufacturer. E.g. machine 1, machine 2, machine Minnie, machine Mickey Mouse, etc.

Equipment manufacturer

Manufacturer of the equipment. E.g. Olympus Diagnostics.

Equipment model

Model of the equipment. E.g.  AU400

Blood collection tubes

The tubes used for blood collection. E.g. Sarstedt Li-Heparin gel tubes or Kabe Labortechnik Lithium heparin coated tubes.

Anaesthesia used for blood collection

The drug used for anaesthesia during blood collection. E. g. Isofluorane.

Method of blood collection

Concise description of the method used for blood collection. E.g. retro-orbital puncture.

Anticoagulant

Anticoagulant drug used for blood collection. E.g. Li-Heparin.

Samples kept on ice between collection and analysis

Yes/No.

Storage temperature from blood collection till measurement

E.g. 2°C

Sample status

Indicate if the sample were frozen (analysis on the same day of collection not possible) or fresh (analysis on the same day of collection). E.g Fresh/Frozen.

Plasma dilution

Dilution is highly discouraged but if necessary indicate here. E.g. “No dilution” or 1:2. Note that results submitted to DCC are assumed to be already corrected for any dilutions made.

ID of blood collection SOP

ID of the protocol followed for blood collection. Can be a center specific protocol. E.g. ESLIM_024_001.

Date and time of blood collection

Time of day for collection is in the morning, starting no earlier than 07:30. E.g. Year, month, day, time.

Date of measurement

The day of blood analysis. Year, month, day.

Hemolysis status

If no AU analyser score is provided, indicate here the gauged degree of hemolysis. E.g. slight/moderate/marked.

Blood collection experimenter ID

An ID of any format to be used coherently both inside the same procedure and for all procedures indicating the experimenter who collected the blood. E.g. Harw_001, or 1/2/3.

Blood analysis experimenter ID An ID of any format to be used coherently both inside the same procedure and for all procedures indicating the experimenter who analyzed the blood. E.g. Harw_001, or 1/2/3.

Date equipment last calibrated

Most recent date in which the equipment (or any part of) used in the procedure was subject to a calibration event.

Date and time of sacrifice

The date and time when the mouse is sacrified.

 

 

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