Bloodbanking - Rouleaux formation
Sorry for posting late =P haha!! Well anyway this time i'll be sharing more regarding the formation of rouleaux. As all of you guys should know, we always hear during the lectures that rouleaux formation is characterized by the formation of "coin like stacks" of RBCs. Indeed, it does look like coins stacking together. It was really an eye opening opportunity to be able to see the formation of these rouleaux in one of the patient sample.
It is very important to be able to identify that there is rouleaux formation as it may be misinterpreted as false positive. For instance in ABO grouping, etc.
Causes of Rouleaux Formation
The formation of rouleaux is actually caused by abnormal proteins in the serum. It is often observed in the serum of patients diagnosed with multiple myeloma or other dysproteinemias. Patients receiving intravenous therapy with high molecular weight fluids such as fibrinogens, dextran, or hydroxyethyl starch has been associated with the causation of rouleaux formation.
Resolution of rouleaux formation
How we can identify that a positive result was not a resultant of rouleaux formation is through
(i) Addition of saline to the slide containing the patient RBC, and then view microscopically to observe. If after addition of saline, free cells are observed, it indicates that rouleaux formation has occured. A true positive result is indicated by agglutination even after addition of saline.
(ii)Saline replacement technique (using test tube)- firstly, the serum and cells of the same patient are added together in the tube and centrifuge. As such, Ag-Ab reaction can take place should reactivity be present. Subsequently, the patient serum can then be removed and replace with saline. The mixture is then subjected to recentrifugation and examine for agglutination. Using this method, rouleaux can be dispersed but at the same time, the true agglutination will not be affected. This technique actually removes the abnormal proteins present.
Monday, November 06, 2006
Shi Ling
6:35 PM
Reply to comments: Advantages and Disadvantages of Gel Card Column Technique
Advantages of the technique:
(i) The reaction produced is relatively stable, thus results can be interpreted over several days. In fact, the results will remain as produced initially but the red cells added will eventually start to dry up at the end of thrid or fourth day.
(ii) Unlike in tube technique, the washing step could be omitted when using Gel cards since the AHG reagent is already present in the microtubes prior to test. This would inturn slightly shortens the time required for results to be produced.
(iii) A lower volume of sample (serum) is required as compare to tube technique.
Only 25uL of serum is required as compare to tube technique that may require 2-3drops. This would also shows that the tube technique is more sensitive than the tube technque.
Disadvantages of the technique:
(i) Presence of clots /fibrin strands / particulate materials that entrap cells and prevent them from moving through the gel may be misinterpretated as positive result.
(ii) Using Gel cards would cost more than using tube technique.
(iii) Neutralization of the AHG reagent could occur if serum was accidentally pipetted and settled into the AHG solution. This could lead to false negative results.
Monday, September 11, 2006
Shi Ling
11:20 AM
Blood Banking – Crossmatch laboratory
Hello everyone~!! Haha I found out something new that I could share with you guys. Well do you guys still remember the Antibody screening (ABSC) tests that we carried out in school using test tubes?? Over here at my organization, this is carried out using a Dia-Med ID Micro Typing System aka Gel Column technique. Each of this Gel cards consist of 6 columns of microtubes containing a gel solution with polyspecific AHG.
The processes involved in ABSC using the Gel card includes:
(i) To write the name of the patient in which ABSC is carried out for, on the GEL card.
[This allows identification of the patient whose sample is tested, as usually multiple different samples are run at the same time.]
(ii) Addition of 50 uL of Screening cells I / II / III to the first three columns respectively.
(iii) Addition of 25uL of serum / plasma to each of the three columns respectively
(iv) Incubate the Gel card at 37 degrees Celsius for 15 minutes
[The Gel card is used to screen IgG that is clinically significance at LISS phase followed by AHG]
(v) Centrifuge using Dia-Med ID centrifuge at a speed of 1175rpm for 10 minutes.
(vi) After addition of samples and centrifugation of the Gel cards, agglutinated cells are completely separated from the non-agglutinated cells as the agglutinated cells.
[Due to the gel solution, agglutinated cells gets trapped as they are unable to migrate down the gel while unbound Cells sediments to the bottom of the microtube.]
Note:
It is important to ensure that when pipetting the Screening cells into the microtubes, we do not pipette it directly into the gel as it could cause misinterpretation of results as positive (Production of false positive result). Instead, we should aspirate the cells with the micropipette tip facing the sides of the microtubes.
This is an example of the gel card that the organisation was using. However, it was not use to carry out ABO grouping. This pic was the only one that i found in the internet haha. Anyway as you can see there is a gel solution at the bottom of the microtubes that i was saying. Strong postivie samples are those whose agglutinated cells are trapped right on the top level of the gel like what is shown from the 4th column from the left while strong negative are those whose cells will sediments at the bottom like what is shown on the 5th and 6th column in the pic.
The results are reported by grading them in scores e.g 3+ / 2+, etc
Tuesday, August 22, 2006
Shi Ling
8:44 AM
Comment
Actually from what i can see from other's blog on Blood Banking, i find that in school normally we used a marker and labelled shortform on the centrifuge tube. However when come to the real working life in the BB lab, they got colour cap to represent a certain test of lab. I guess it is the same as in the histo lab where different colour represent different batches of tissue specimen that are received at different time and trimmed by pathologists or technologist and whether is fatty tissues or not. They are lot of indication in terms of colour for us to identify different specimens, tests or from certain labs.
Sunday, August 20, 2006
*=-Agnes-=* 채혜민
8:07 PM
Blood Group Testing Laboratory (BGT)
In this laboratory, we basically carry out Blood Group Testing on all blood donated via blood samples collected in sample tubes.
Preparation of Blood samples:
When each individual donate blood, blood is collected into 4 extra tubes encoded by different coloured caps. Below is a protocol of how each sample are then handled or prepared:
(i) K3EDTA Anti-coagulated sample 6ml – Centrifuge at 2000rpm for 5 minutes (Light purple coloured cap representing for Blood Group Testing Lab)
(ii) K2EDTA Anti-coagulated sample 6ml – Centrifuge at 2000rpm for 10 minutes (Dark purple coloured cap representing for Nucleic acid Testing Lab)
(iii) Plain Clotted Sample Tube 4ml – Centrifuge at 2000rpm for 20 minutes (Red Cap for Syphilis Testing Lab)
(iv) Plain Clotted Sample Tube 6ml – Centrifuge at 2000rpm for 20 minutes (Red Cap for Viral Serology Lab that tests for HIV, Hepatitis B and C)
Anti-Coagulated samples are required in BGT lab as there is a need to use both the plasma and red cells for testing. Thus, this is why the samples are place in anti-coagulant and not spun harshly. On the other hand, as serum are used for the testing involved in the Syphilis testing lab and Viral Serology Lab, blood samples are clotted in plain clotted sample tube without any anti-coagulants added.
All blood samples must be tested with tests including ABO Grouping (Forward and Reverse Grouping), Rhesus Typing, and Antibody Screening for presence of any clinically significant Antibody/Antibodies. These tests are carried out automatically using an automated machine termed “Olympus Pre-Tranfus PK7200”. These tests help to determine the Blood grouping of the individual donor and detect any possible clinically significant antibody/antibodies that causes hemolytic transfusion reaction. In order to tests for the presence of clinically significant Antibody (Ab), the 3 screening cells (Screening Cells I/II/III) used by the laboratory must make up to contain the following Antigens (Ag): D, C, E, c, e, K, k, Lea, Leb, Jka, Jkb, Fya, Fyb, P, M, N, S and s.
When is manual testing carried out?
(i) In cases where the blood samples (Red cells / Plasma / Red cells and Plasma) in the tube are insufficient for the probes of the machine to pick up.
(ii) Production of a positive result for antibody screening by the machine. Confirmatory tests are done manually using Screening cells I/II/III with the serum of the donor. Should a positive result be produced again, the blood sample is sent to the Red Cell Reference lab to determine the exact Ab present. The reason why serum is used instead of plasma is because plasma contains anti-coagulant that could possibly interfere with the detection of some complement activating antibodies.
(iii) Donor is Rhesus negative. A repeated manual test involving the usage of Anti-D and plasma of donor is carried out. When a positive result is produced, a confirmatory test is further carried out, Du testing, to detect weak D antigen. On top of that, CDE Ag typing is carried out.
(iv) Presence of undetermined blood group samples e.g. subgroups.
In the BGT Lab, the laboratory staffs prepare certain reagents manually. This includes A and B cells used for reverse grouping, and Screening cells used in antibody screening.
(i) Undiluted cells are poured to fill half of a tube
(ii) Saline is added then added to fill the tube
(iii) The tube is then centrifuge at 3000rpm for 7 minutes
(iv) After which, saline is removed using a transfer pipette. This step is repeated for three times.
(v) Finally, celpresol, a red cell preservative is added.
Purpose of washing these red cells prior to used:
(a) Removal of plasma present with the red cells. This is because presence of plasma could lead to formation of clots when the red cells are mixed with serum that contains residual thrombin.
(b) Plasma tends to cause rouleaux formation that could interfere with agglutination formation.
(c) Plasma contains anti-coagulant that may interfere with detection of complement binding antibodies
Monday, August 07, 2006
Shi Ling
6:24 PM
Medical Microbiology (Comments)
Actually in our biochemistry lab we also do Grams stain for urine samples (urine phase contrast) suspected to contain microorganisms. We are suppose to observe and note down the morphology of the microorganisms like gram negative or positive, rods or cocci. If there are signs of a significant microbial infection, we will report it and the microbiology lab will be informed and biochemical tests would be conducted by them. The same applies for CSF specimens (cerebrospinal fluid).
Sunday, July 23, 2006
rAinE
5:22 PM
REPLY TO COMMENTS
Uses of Blood Products
1)
Red packed cells could be used in patients with cases of anaemia. This helps to increase the level of Hb and thus delivery of oxygen to tissues.
2)
Whole blood is able to provide oxygen carrying capacity (due to increase in Hb level) and blood volume expansion. It is thus useful for bleeding patients who have lost 20% of total blood volume.
3)
Platelets are required for blood clotting processes. It is prepared from individual units of whole blood by warm centrifugation at a temperature of about 20 to 24 degrees celcius. It is usually used for the treatment of patients with leukemia, low platelet count(e.g thrombocytopenia) or abnormal platelet function (e.g chemotherapy), in order to control bleeding. As platelet concentrates contain leukocytes which may cause graft versus host disease and febrile transfusion reactions, the leukocytes could be reduced/removed to prevent the above consequences. Platelets concentrates with leukocyte reduced/removed are thus term as Leukocyte reduced platelets.
4)
Plasma - Plasma is prepared from a whole unit of blood via centrifugation. There are two types of plasma that could be issued for used. The first type is Fresh frozen plasma while the other type of plasma is cryosupernatant. The difference between the content of the two plasma is that cryosupernatant is depleted of cryoprecipitate. Which means the cryosupernatant, would have half of its fibrinogen, Clotting factor VIII and fibronectin removed as cryoprecipitate. It is also depleted of the largest multimers of von Willebrand factor that sediment in the cryoprecipitate fraction..
Fresh Frozen Plasma (FFP) is separated and frozen within 8 hours of whole blood collection. It contains plasma proteins (e.g albumin, globulins, fibrinogen) and all the coagulation factors. A unit of FFP contains about 200 units of Factor VIII plus the other labile plasma coagulation factor, Factor V. FFP is used mainly to provide replacement coagulation factors when concentrate is not available or appropriate. It provides normal levels of all clotting factors and is used for patients with thrombotic thrombocytopenic purpura (TTP), antithrombin III deficiency, immediate hemostasis, reversal of the warfarin effect, and for massive transfusion with coagulopathy.
Cryosupernatant is prepared from FFP. It requires firstly the thawing of FFP at a temperature of 1-6 degrees celsius for about 18 hours. Following which, it is then centrifuge. This allows the seperation of cryoprecipitate and cryosupernatant. Cryosupernatant could also be used for the treatment of TTP. It is also thought to be a more effective treatment than FFP.
5)
Cryoprecipitate. In each of cryoprecipitate prepared, it
contains an average of 80 or more units of Factor VIII (FVIII:C),minimum of 150 mg of fibrinogen, some Factor XIII originally present in the fresh plasma and von Willebrand Factor of Factor VIII molecule. Cryoprecipitate is used in replacement of fibrinogen and Factor XIII and in platelet functional defect (uremia). It is also used in the treatment of
von Willebrand disease (click link for details on von willebrand disease) and Factor VIII replacement when specific factor concentrates are unavailable
6)
Fibrin Glue/Sealant are made from blood clotting protein, fibrinogen. In surgery, it is applied topically to help control bleeding. For instance, it is used to stop oozing from small, sometimes inaccessible, blood vessels during surgery when conventional surgical techniques are not feasible.
7) Irradiated blood products: Irradiation of blood products inactivates any lymphocytes present. Severely immunosuppressed patients who may develop graft-versus-host disease after transfusion of viable immunocompetent lymphocytes should receive irradiated blood products.
8) Leukocytes reduced blood products: Blood product with leukocyte reduced helps prevent the risks of graft versus host disease and febrile transfusion reactions. Febrile transfusion reaction could occur as a result of formation of antibodies by the recipient’s against the White blood cells of donor.
Red Blood Cell StoragePacked RBC are prepared from a whole blood unit via centrifugation. During preparation of RBC, red cell preservative solution are added after the removal of plasma. This enable RBC to be kept in the "Cold room" at a temperature of 1-6 degrees celsius for a maximum of 42 days. However, if their expiry dates are near and they are still not used, they can then be kept for up to 10 years by freezing them. As such, they are then kept frozen. However, glycerol are added to prevent red cells damages such as loss od intracellular water and stress to cell membrane during freezing.
Tests done in Dispatched labIn the dispatched laboratory, no tests are carried out. The blood products that have arrived at the dispatched lab have already been tested by other laboratory in our organisation.
Some of the tests that are carried out before the blood product could be issued includes :
(i) ABO and Rh typing,
(ii) Screnning for unexpected red cell antibodies and,
(iii) Tests for infectious diseases such as HIV type I and II, Hepatits A and B, Syphilis.
Sunday, July 09, 2006
Shi Ling
2:21 PM
BLOOD BANKING (COMMENTS)Basically after reading my group mate's post and comments posted by other groups on the dispatched lab, i did a little research on Autologous Blood Transfusion.
Autologous Blood TransfusionIt is the collection and then re-infusion of the patient's own blood or blood components. Autologous blood donation can also be known as self-transfusion of blood. Although it is not completely risk free, autologous Blood is the safest form of Blood transfusion. This kind of blood transfusion eliminates reactions due to donor recipient incompatibility and precludes exposure to transfusion transmitted infection. Many of the antibodies in the donated blood, which develops when people are exposed to diseases, may be undetectable despite intensive testing, and these can cause transfusion reactions in patients receiving the blood. While Blood bank tests greatly reduce the risks of acquiring certain infectious diseases, these risks can not be eliminated entirely. Each individual's own blood is considered the safest blood for transfusion.
Over the recent years, there is increasing awareness of the diseases or blood transfusion reactions that are resulted by the usage of allogenic (blood from donor other than the patient) blood for transfusion. This resulted in the increase in autologous blood transfusion.
There are mainly 5 kinds of autologous catergories:
1) Preoperative autologous Blood donation, transfusion and storage (PABD): it is done by drawing units of blood from the patient usually starting (in short term case) three to five weeks before an elective surgical procedure and stored for transfusion during the time of the surgery.
2) Intraoperative hemodilution: Blood is collected at the start of surgery and the fluid volume lost is replaced with appropriate IV solutions, and then finally, stored Blood is reinfused after surgery.
3) Intraoperative Blood salvage: Blood is salvaged from the surgical area during the operation for re-infusion during or after the surgical procedure.
4) Postoperative Blood salvage: Blood is collected after the surgical procedure and is completed by draining of the operative area and re-infused.
5) Autologous self stored Blood: The patient's own blood is preserved in a frozen state for use at a later time, in case he/her need of a Blood transfusion arises. It is considered the safest blood he/she can receive. This process eliminates donor-transmitted diseases and if the patient have a very rare blood type, or contains rare components, this process is of great significance in emergency cases. Autologous Blood is of a perfect match. In addition to the groups of A, B, O and Rh types, there are as many as 100 or more sub-types. The chances of obtaining a transfusion from a donor other than the patient, where all sub-types match, is estimated to be less than 1 in 100,000.
Benefits of Autologous Blood Transfusion
1) Availability- no cross matching is required.
2) Safety- tjhere will be no risk of transfusion reactions due to incompatibility.
3) Purity- there will be no risk of transmitted disease, such as : HIV, Hepatitis B & C, HTLV 1&2, & Syphilis.
Lastly, i think that Autologous Blood Transfusion is a good method to prevent blood transfusion reactions and incompatibility. It is good in the short term such as for either post or pre operation but in the long run, considering the cost i think that it would be very expensive to store our own blood in the blood bank.
MEDICAL MICROBIOLOGY (COMMENTS)Actually after reading the other group's blog regarding the machines they use in the microbiology labs, i found out that the
BACTEC 9240 machine was used in most of the microbiology labs. So i was wondering if this machine is of great significance in the identification of bacteria in the blood. After reading some of the online articles, i agree with what one of the group has concluded that the Bactec 9240 plays an important role in the laboratory as it allows quick identification and then treatment of bacteria infection in the blood.
There is this website that shows an evaluation of the Bactec 9240 system for blood cultures.
The purpose of the study was to determine species of bacteria causing sepsis and the rate of false positives and negatives in the results obtained in the evaluation of blood cultures by the Bactec 9240 system.
For the period of one year, from September 1994 to September 1995, 8022 blood cultures which were taken by various clinics from patients with fever or sepsis were evaluated using the Bactec 9240 system. For this purpose, Bactec plus Aerobic/F and Bactec Peds plus/F vials were examined for 7 days. Then the identification of the bacteria was made using the Sceptor system.
Results show that out of the 8022 blood cultures, 1763 were positive with the Bactec 9240 system and of these, 2.05% were false positives. Also, the subculturing of the 6259 negative blood cultures in the Bactec system, various bacteria and fungi were isolated in 11 cultures which produce 0.18% false negatives. The research also show that majority of the bacteria isolated were Staphylococcus species,they made up 52.34% of the total microorganisms which grew in the cultures. In addition, out of the 1763 cultures that were positive with Bactec 9240, 19 (1.08%) were positive after the 5th day.
The conclusion of the report states that the time involved in the evaluation of blood cultures is shortened when the Bactec 9240 system is used and there is less contamination of the blood cultures since subculturing is done only once. (Ann Med Sci1997;6:53-56) These systems are considered more expensive than the conventional methods, particularly for developing countries. However, it shortens the growth period, lowers the rate of contamination, a lower rate of false positives and false negatives, and reduction of the workload. All of these factors contribute to continue use of this system for the evaluation of blood cultures.
Bibliography
1) A.Yaman, I.H. Dundar and P. Aksungur. (1997).
Evaluation of the Bactec 9240 System for Blood Cultures. Retrieved on 9 July 2006. On-Line:
http://ams.cu.edu.tr/January1997Vol6No1/evaluati.htm. Other articles related to Bactec 9240:
- http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=500674
- http://sepsis.researchtoday.net/archive/1/4/746.htm
- http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=15227618&dopt=Abstract
rAinE
4:22 AM
