SIP

Subject Title: BLOOD BANK
Topic: Blood banking processes

yep, I was attached to the Blood Bank Laboratory for the whole of 20 weeks. There are 4 stations in the lab, 1) Requisitor 2) Screener 3) Depatching & Crossmatch 4) Issuer. I learnt quite a number of things through observations and some hands-on under supervision. I was not allowed to handle patients' samples yet as results had to be very accurate or else the consequences of wrong results can lead to adverse transfusion reaction.

- Sorting out labels that are returned to the lab after transfusion
- Handling of blood products received from CTM
-> Scan 1) Unit no. 2) Expiry date 3) Product code 4) Blood type into the LIS to document all the stock that are received.
-> Store in refrigerator under pending ABO rechecks for blood units.
-> Store in freezer at -60 degrees celsius for FFP (Fresh Frozen Plasma)
- Retrieval of transfusion records from filing cabinet
- Preparation of ABO tubes for screening and recheck
- ABO recheck of blood units

Purpose: To ensure correct blood type in the blood pack, preventing errors
E.g: Recheck of O+ve blood group
Procedures:
1) Paste the donor blood ID sticker on the "rechecking of donor blood form". Tally the unit no. of the blood pack with the label to prevent errors. (Every blood pack comes with a label which is used to fill in the particulars of the patient, date of transfusion, then issue out together with the blood unit and return back to the lab after transfusion.)
2) Add 1 drop of Anti-AB into a test tube.
3) Add saline into another tube.
4) Remove a segment from the blood pack. Draw the blood sample using a pipette. Dilute the blood in saline.
5) Add 1 drop of blood to the tube containing Anti-AB.
6) Centrifuge for 15 seconds at 3000 rpm.
7) Check for agglutination, record results under "rechecking of donor blood form".

Results: Negative. As O red cells lack of A and B antigens, thus there is no agglutination present with mixed with Anti-AB.

Blood samples are sent to Blood Bank Laboratory for ABO/Rh grouping and cross-match.

Role of a requisitor
When the blood samples are received, tally the doctor's signature/name, patient's NRIC no. and name on the request form with the label on the tube.

- Check with the LIS if the patient requires T1 or T2.
T1- requires ABO/Rh grouping only

T2- requires ABO/Rh grouping & Ab screening (15th day from the last Ab screening or 4th day onwards from the last blood transfusion which is just after last Ab screening.

- Centrifuge the blood tubes to separate the red cells and the serum/plasma.
Serum- top portion of the clotted blood (red tube)
Plasma- top portion of EDTA blood (lavender tube)

- Place the tubes according to the last digit of the patient's NRIC no. in the specimen rack for the screener.

Role of screener
1) Set up controls and calibrations, do daily quality control of working reagents to ensure the accuracy of results for all tests. These reagents are quality controlled daily.

• ABO cells recheck with Anti-A, Anti-B, Anti-AB to ensure that the correct cells are in the respective bottles
  
• Rh-ve & Rh+ve recheck with Anti-D

• Test if the Anti-Human globulin (AHG) reagent is working properly with *check cells & saline
  
  *Check cells are pools of group O RBCs sensitised with IgG
  
  Positive control: 1 drop of check cells with AHG reagent
  Negative control: 1 drop of check cells with 2 drops with of saline
  
  Interpretation of results
  Positive: Agglutination of RBCs
  Negative: No agglutination of RBCs


• Panel (SPO) cells recheck (SI, SII, SIII)
  Using gel card technique
  
  
  - Add 50uL of SI, SII & SIII into each 3 different microtube respectively.
  -Add 25uL of positive control cells (serum diluted with saline which contains the respective Ab) into the first 3 microtubes. Add 25uL of negative control cells (with no Ab) into the last 3 microtubes.
  - Incubate for 15 minutes
  - Centrifuge at 3000rpm for 15 seconds.
  - The results should be like the photo shown below:

*with permission from supervisor

Test: Antibody screening (Microtyping card gel)
Purpose: Screen for clinically significant blood group red cell antibodies in the pretransfusion samples, thus able to select suitabe blood units that will not cause harm to the patient, preventing adverse transfusion reactions. E.g: If the patient has Anti-Mia in the serum/plasma, blood units with the absence of Mia antigens must be issued to prevent transfusion reaction.

We used the ID LISS/Coombs Gel technique instead of the tube method we used in school.
The ID-card "LISS/Coombs" is a antibody screening panel which consists of 6 microtubes containing polyspecific AHG within the gel matrix, Rh-hr, Duffy, Kidd, Lewis, MNS, P, Kell and Mia antigens.


Procedure:
Allow the test cell reagents & samples to reach room temperature before use.
1) Identify the appropriate microtubes of the ID-card "LISS/Coombs" with the patient's name.
2) Remove the aluminium foil as many microtubes as needed.
3) Pipette 50 uL of *SPO cells (SI, SII, SIII) to the appropriate microtubes
*SPO cells - Specific pooled O+ cells with C, e, N antigens, etc
4) Add 25uL of the patient's serum/plasma to each microtube.
5) Incubate the ID-card for 15 minutes in the ID-incubator at 37 degrees celsius.
6) Centrifuge the ID-card for 10 minutes in the ID-centrifuge.
7) Grade & record the results in appropriate worksheet.
8) Leave the gel card & corresponding worksheet for counterchecking by second medical technologist.

Positive results: If the patient's serum/plasma contains abnormal Ab specific to the antigens present in the SPO cells, it forms clumps with the panel cells, resulting in agglutination. Thus, it is unable to pass through the gel and gets trapped at the top portion of the tube. Strong +ve are seen as a line at the gel/fluid interface. Weaker +ve are seen distributed throughout the gel.

Positive samples will be sent to CTM for investigations using the antibody identification panel.

Negative results: If there's absence of abnormal Ab in the patient's serum/plasma, agglutination will not occur. Thus, the non-clump cells are able to pass through the gel and settle at the bottom of the tube. -ve are seen as a red cell button at the bottom of the tube.

*All results are counterchecked by second medical technologist for verification, ensuring accuracy of results & preventing analytical errors. (internal quality control)

Abbreviated crossmatch (Compatibilty testing)

Purpose: To test whether the donor's blood is compatible with the patient's serum, thus able to promote safe transfusion of blood and prevent transfusion reactions.

Procedures:
1) Take the requested blood unit from the refrigerator according to the patient's blood type.
2) Pull out a segment from the blood pack. Draw the blood sample using a pipette. Dilute the blood in saline.
3) Centrifuge at 3000rpm for 15 seconds to wash the cells.
4) Pour away the suspension into the waste bucket and add saline.
5) Add 3 drops of patient's serum into a tube.
6) Add 1 drop of the donor's blood into the same tube.
7) Centrifuge at 3000 rpm for 40 seconds.
8) Read the results under the microscope.

Positive results: Presence of agglutination
Negative results: Donor's RBC is compatible with the patient's (recipient's) serum/plasma, therefore able to proceed with the issuing of blood unit.

Crossmatch specimens are valid for 3 days from the day of receiving after which new specimens are required. (In case of disputes, for investigation & legal cases. The red cells may lyse after that period.) Samples are placed in the specimen racks according to the last digit of the patients' NRIC no. Day 1 samples are placed on the workbench at room temperature for easy retrieval during crossmatching. Day 2 & 3 samples are stored in the refrigerator. At end of day 3, they are bundled in yellow biohazard bags.

Some of the blood products that are issued by Blood Bank:

- Packed cells (without plasma & platelets)
Function: To raise the Hb level up faster as it's more concentrated with no plasma.
Life span: 45 days

- Whole blood
Function: To produce greater expansion of blood volume, especially for patients with massive blood loss
Life span: 36 days

- Platelets (random/apheresis)
Function: To prevent or to treat bleeding due to severely low platelet counts (thrombocytopenia)
Life span: 9 days

- FFP (Fresh Frozen Plasma) & cryoprecipitate
Function: For patients with abnormal or low levels of blood clotting proteins, such as hemophilia.
Life span: when frozen, can be kept for around 1 year. when thawed, had to be used within 24 hours.

- IvIg (Intravenous Gamma Globulin)
Function: To increase patient's immunity against infections

- Human albumin (5%/20%)
Function: For restoration and maintenance of circulating blood volume

- Factor VIII
Function: For treatment of acquired Factor VIII deficiency or hemophilia A

- Autologous blood

- Leco-reduced/ deglycerolysed RBC

Leco-reduced RBC: Where the leukocytes from red cells are filtered off or removed
Deglycerolised RBC: For patients who are sensitised with to IgA protein or to leukocyte or platelet antigens

- Washed cells (freezed with glycerol to preserve the cells)
They're used in cases of emergency when the stock is low. The cells are defrozen & washed to remove the preservatives. The disadvantage of washed cells is that their life span is very short.

yep, that's about it. Free free to ask any questions. I'll try my best to answer them all. Continue enjoy the rest of the SIP period! (: take care

Lastly, wanna share something.
my colleague told me this,
With skills comes good work.
With passion comes artistic work.
With passion & skills will produce a masterpiece.
yep, passion & skills are essential in excelling in your work. =)

Png En Hui Dorothy
0503239F
Tg01

SIP

Subject Title: Histopathology
Topic: Tissue processing

Hi all. I had been attached to a histopathology lab. My SIP consist of 5 components, they are: tissue processing, embedding, microtomy, special staining and cytology. I am posted to the tissue processing station for the first month of my SIP. In this station, my main task is to receive specimen, label specimen and assist the pathologist in recording and labelling of cassettes.

Everyday in the morning, once we received the specimens, we will label the specimens and the request form with a bar coded biopsy number. The sample will then be carried to a room where trimming and examination of specimen is performed. Small specimens, such as appendix, gall bladder, bone marrow core, tonsils, cornea or gastric biopsies will be trimmed or passed into a cassette by a medical technologist. The large and complex specimens such as liver, kidney, intestine, breast, uterus, eye ball or femoral head will be examined and trimmed by a pathologist.

Lab personnel who are in charge of trimming or passing of small specimens into cassettes must make sure that the bar code and the specimen name on the bottle are matched with the request form before commencement of any procedure. Once confirmed, details of the specimen such as number of tissues in the bottle and type of tissue will be recorded. Measurements will also be taken. Specimens will then be trimmed into a smaller pieces using a surgical blade and pass into a cassette. For bone specimen, a bone saw is used to cut the specimen into smaller pieces and is then decalcify before processing. For prostate, whole mount is necessary (a bigger version of tissue block), as the whole entire piece of prostate will be dissected and submitted for investigation. For specimens such as gastric biopsy (about 0.1cm x 0.1cm in size), which are very small, will be coloured with ink and place in a filter paper. The purpose of using ink is to allow the person who is doing embedding to see the tissue clearly so that he/she can transfer the tissue into the mould. The filter paper will prevent lost of tissue during processing. Inks are also used by pathologist to indicate the orientation of the tissue. After trimming, if there is excess tissues, the excess tissues will be set aside for storage and indicate on the requisition form as tissues with reserve.

After passing of tissue into cassettes, the cassettes containing the tissue will be fixed in 10% neutral buffered formalin until all other tissues are ready to be processed. The pH of the formalin will be check daily to ensure that the pH is around 6.8-7.2 as neutral pH will minimize formalin precipitation. New formalin is changed at least twice a week. The principle of fixation is to stop autolysis. Fixatives form cross linkages between protein, thus preserved the tissue's in vivo structure.

The lab used an enclose tissue processor for tissue processing. An enclosed system means that tissues will remains stationary in a closed chamber and processed until the procedure is completed. Tissue processing consists of 3 components:
1) Dehydration: remove water from tissue using graded alcohol
2) Clearing: replace alcohol with an agent that is miscible with wax. Xylene is most commonly used, but some new machine use isopropanol instead of xylene, which make the working environment better.
3) Impregnation: tissue infiltrated with embedding medium.

Different types of tissue will have different processing time. For small fragment of tissues or biopsies, the processing time is usually 2-3 hours. For tissues of normal sizes, the processing time is usually set at 16 hours.

Apart from processing, I had also learnt some safety aspects in a lab, such as how to deal with chemical spillage, and the protocol as follow:
1) Isolate the area of spillage
2) Wear personal protective equipments, such as glove and mask
3) Choose an appropriate spillage kit available in the lab
4) Circulate the spillage with the spillage kit and max them
5) Scoop the spillage, and place the mixture in a bag and tighten it.

I think I will stop here first. Enjoy your SIP!

Wing Fat
TG01

Table

Hi all

Few of you asked about the difference in reference range. I made a table to compare the different parameters.

	Unit	Man	Women	Horse
White Cell Count	10^9/L	3.5-	15.0	6-10
Neutrophil Count	10^9/L	1.9-	9.0	2.5-7.0
Lymphocyte Count	10^9/L	0.9-	4.5	1.2-3.7
Monocyte Count	10^9/L	0.1-	3.1	0.2-0.7
Eosinophil Count	10^9/L	0.0-	0.5	0.0-0.2
Basophil Count	10^9/L	0.0-	0.2	0.0-0.2
Red Cell Count	10^12/L	4.5-5.5	3.8-4.8	7-10
Haemoglobin	g/dL	12.0-18.0	11.0-16.0	12-18
Pack Cell Volume(PCV) or Haematocrit(Hct)	%	40-50	36-46	32-46
Mean Cell Volume(MCV)	fL	80-99	78-97	44-54
Mean Cell Haemoglobin(MCH)	pg	27.0-32.0	25.5-31.5	15-18
Mean Cell Haemoglobin Concentration(MCHC)	g/dL	31.0-	35.9	31-39
RBC Distribution Width(RDW)	%	10.0-	14.5	11.6-14.8
Platelet Count	10^9/L	150-	450	6-210
Fibrinogen	g/L	1.7-	4.0	1-4

Douglas
TG01

SIP

Subject Title: Clinical Chemistry
Topic: Blood Gas Testing

Heys..I have been attached to clinical chemistry lab for the whole of my SIP. In this lab, there are 5 workstations. I had been posted to blood gas station for 2 weeks. I had learnt to use Roche Omni Cobas b 221 system for testing blood gases. This analyzer can measure analytes such as pH, pO2, pCO2, electrolyte ISE(e.g.Ca2+),total haemoglobin, oxyhaemoglobin, carboxyhaemoglobin, methemoglobin and O2 saturation. It consists of reagents such as S1 Rinse Solution, S2 Fluid Pack Solution and waste blood container.

Principle of Measurement
O2: use of clark measurement principle. It is the measurement of current generated by reduction of O2
CO2: measurement of pH change in electrode caused by CO2
pH and Ca2+: potentiometric electrodes based on the measurement of a potential under no current flow. Calculation of these require use of reference electrode
Total haemoglobin: light absorption in whole blood measured at different wavelengths

Calibration, QC, Maintenance
Calibration, quality control and maintenance need to be done before running any patient's samples. Everyday in the morning, calibration results, which include system cal, 1 point cal and 2 point cal, need to be printed out and filed for future audits. Calibrators are not required as the analyzer will automatically calibrate by itself in time intervals. This will plot out a calibration curve and to determine any errors in the measurement of the analyzer so that is adjusted to show true values. After that, quality controls need to be done to ensure the results are within the control limits. QC is performed daily at 9am and 4pm. The commercial control ampoules are stored at 2-8 degree celsius and are temperature sensitive. Hence, it has to "grip warm" in the palm for about 10min before running to achieve successful QC results. The results will also have to be filed. Lastly, maintenance has to be done to make sure it is in good condition. This includes:
- Checking thermal paper supply
- Checking reagents level
- Cleaning the fill port(where the blood is pumped in)with wet cotton swab for removing blood stains
Now, the analyzer will be ready to run patient's samples. These samples are collected in heparinzed syringe which contains anticoagulant to prevent clotting of blood

Procedures
1)Stamp time for the patient's sample
2)Ensure is sent in ice bag
3)Label barcode on both the syringe and request form
4)Order tests requested into LIS
5)Mix the syringe well and check for blood clots before pumping into analyzer
6)Check the analyzer is at ready mode and having green signal before pumping
7)Pump blood into analyzer
8)Scan barcode

Test Results
Analytes-------- Reference Range
pH--------------> 7.350-7.450
pCO2------------> 35.0-45.0 mmHg
pO2-------------> 75.0-100.0 mmHg
O2 saturation---> 95.0-100.0%
Ca2+------------> 1.140-1.320 mmol/L
Total haemoglobin---> 11.5-17.4 g/dL
Oxyhaemoglobin------> 95.0-99.0%
Carboxyhaemoglobin--> 0.5-2.5%
Methemoglobin-------> 0.0-1.5%

Clinical Interpretation
After the results are out, we have to check for any abnormal high or low levels which will be flagged red in the LIS. For example if the pH is less than 7.1 and pCO2 is more than 45 mmHg, we need to call the nurse to inform these panic values. This shows that the patient is having respiratory acidosis. If there is abnormal high pCO2, we have to check if blood sample appear dark and re-run the test. Always ensure pO2 is directly proportionally to O2 saturation. After verifying the results, we can file them into LIS. Therefore, blood gas testing is for diagnostic and therapeutic reasons

That's all for now. Enjoy your SIP!
Soong Ci Liang
0503333G
TG01

SIP

Subject Title: CCHEM/ HAEM/ LMQA/ MMIC
Topic : Bacteriology - Blood Culture

Definition:
A blood culture is a test to determine if microorganisms such as bacteria, mycobacteria, or fungus are present in the blood. A sample of blood is put in a special laboratory preparation and is incubated in a controlled environment for 1 to 7 days.

Principle:
Bacteria growing in a suitable broth causes turbidity of medium or gas production. The BACTEC Fluorescent series blood culture system, detects carbon dioxide (CO2) production by multiplying bacteria.

Materials:
Media contains Soybean-Casein Digest broth with resin and 0.05% w/v sodium polyanethol sulfonate (SPS). All bactec media are dispensed with added CO2. Anaerobic media are prereduced and dispensed with CO2 and N2.

Procedures:
1. Vials are arranged and labeled in racks.
2. If requisition form presents infectious endocarditis in its clinical diagnosis, a red ribbon is tied on the vials for indication.
3. Vials’ barcodes are scanned and incubated aerobically at 35ºC and are agitated throughout the routine 5-day protocol.
4. Negative vials are removed after few days while positive vials are removed from BACTEC machine for flagging, Gram Stain and subculture in appropriate plates.

False Negative Results:
Delay in transportation could lead to false negative results. Inoculated Bactec/F Plus vials can only be left at room temperature up to 48 hours before placing into BACTEC machine.

For photos on BACTEC system and procedures after flagging positive vials, please visit here. (Azhar's entry - linked with permission)


Boon Ching
TG01