ABORh and screening

Hello. For patients that may require a transfusion or require a pre-admission testing, ABORh grouping and antibody screening is done.

For ABORh grouping:

This step if done manually, comprises of the following reagents : anti-A, anti-B, anti-A,B, anti-D, A cells, B cells and O cells. These reagents are already added into the glass tubes by the staff before. This is because we receive a lot of orders everyday we cant simply wait for the specimens to arrive before dripping the reagents. After receiving the EDTA specimen, we'll centrifuge them.

Since the plasma layer is on top, we'll drip 2 drops of serum into each of the reverse grouping tubes, i.e. the A, B and O cells tube. The purpose of this step is to not disrupt the red cells below. Then, we'll pick up some red cell, dilute it in saline and add 1 drop of the 3-5% RBC suspension into each of the 4 forward grouping tubes, i.e. anti-A........ After this step is done, we'll give the 7 tubes a good shake and mix and then centrifuge them at 3,000rpm for 15seconds. Then, we gently agitate the cell button and record the results.

Procedures:

1) Spin specimen in a table top centrifuge at 3,500rpm for 7minutes.
2)Clearly label 7 glass tubes for forward (anti-A, anti-B, anti-A,B) and reverse grouping(A, B, O cells)
3)Into each corresponding tube, add 1 drop of the correct reagent.
4)Using a clean pipette, add 2 drops of test serum into each of the 3 reverse grouping tube.
5)Using the same pipette, prepare a 3-5% red cell suspension of the test cells in 0.9% saline.
6)Add 1 drop of the 3-5% suspension of cells into each of the 3 tubes containing anti-sera.
7)Give them a good mix.
8)Immediately spin them at 3000rpm for 15seconds.
9)Gently agitate and record results

If you don't mix the reagent and test cells/serum before centrifugation, you may not be able to get the correct results. So, it's very important we give the tubes a good mix before centrifugation, especially the reverse grouping tubes.

The next step we do will be antibody screening. There are 2 ways to screen for antibodies, the first will be using the tube method and the second will be using the microtyping Gel cards . I found that the Microtyping Gel cards give a more accurate results. There was this once, i took a specimen which had a 2+ reading using the machine. Then i tried to do manual screening, i ended up getting a negative result. Then i approached one of the staff(tp graduate). She repeated the manual testing with me, results obtained was still negative. Then she did the gel card method again. Tadum! 2+. Moral of the story: Gel Card > Tube. Anyway, the tube method is no longer in use, at least over here.

For gel card method, here is the procedures we take:

1)Identify the appropriate microtubes of the ID-Card"LISS/Coombs"
2)Remove the aluminium foil from as many microtubes as needed. 3)Pipette 50µl of SPO cells I, II and III into the appropriate microtubes marked with the corresponding test cells.
4) Add 25µl of the patient's plasma to each microtubes. 5)Incubate the ID-Card for 15minutes at 37 degrees in the ID incubator. 6)Centrifuge the ID card for 10 minutes in the ID centrifuge 7)Grade and record the results in the appropriate worksheet

The SPO cells are panel cells that contains some antigens, each of the 3 panel cell suspension contains different combinations of a some antigens. These antigens are blood group antigens like C, C, S, Mia etc...

So after test serum is added in, the cards are incubated at 37 deg for 15minutes. This allows agglutination to occur. If agglutination occurs, the agglutinated cells cannot pass through the gel while the nonagglutinated cells can pass through the gel.

A strong positive is seen as a line on the surface while weaker positives are distributed through the gel. To have an idea of how it looks like, click on the 2 links below.

https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEgm0t3RmarwNG2vWfNvlkLg7Tzlhp7nkONEbqjSOeSSamrxZXsreQktRBkOpjjM5hRljWvnbIkHcVokqxHcgeE8GnU6au9dsllIMMYkzknmxKlXnyvBntC_dVIURSG3uRHC6B68i2Sf7np_/s320/230720071167.jpg

This link shows the card that we use here. See the green part of the label, there are 3 lines, normally we'll write in I, II and III. As for the white portion, we'll write in the patient's ID. A patient only requires 3 microtubes, so 1 Gel card can be used for 2 patients.

http://www.cardinalhealth.com/us/en/distributedproducts/images/S/SPMTS4024.jpg

This link shows 6 microtubes, the reactions are 4+. 3+, 2+, 1+, neg and neg respectively.

Here are the reasons why Microtyping Card Gel does better than the tube method. For the ID-Card gel system, labour intensive washing procedures are eliminated, this is because the panel cells are added to the microtubes before the plasma and serum is, thus, creates a barrier over the gel suspension. This step can prevent neutralization of AHG (present in card gel) by Serum IgG protein.

This antibody screening result is valid up to 14 days unless the patient has been recently transfused or is pregnant within the past 3 months. For a patient who is has received a transusion or is pregnant within the last 3 months, antibody detection must be performed on a specimen obtained within 3 days of the next scheduled transfusion. This is because these patient may have been sensitized and they may be producing new antibody that was not previously identified.

If the samples tested positive for antibody screening (a positive in any of the 3 microtubes) but has no known antibody on record, we'll repeat the screening and then sent the specimen down to Blood Services Group (BSG), aka CTM for antibody identification.

Yanhong / TG01 / 0703979E

Freezing of Red Blood Cells Admendments and Questions

Hey guys, thanks for the questions.

I have some amendments to my first post. First of all, not all the blood that is going to be frozen has to be quarantined. The blood that I was dealing with was malaria blood. Hence that is why they froze it. The donor may come from malaria countries and that may want to do a retest in the future. Hence they decided to freeze it while pending tests in the future. Normal red blood cell that you are going to freeze does not have to be quarantine. The computer system will record the freezing process once you do transformation. Secondly, the blood group has actually been written on the bag. What is missing is the blood group in the recipient card. So you have to actually record the blood group on the recipient card, as the card will be kept somewhere else. Thirdly, I was confused about the plasma extractor. I thought that it was a specific term that was used only when you want to extract plasma. After I was posted to the freezing lab, I was next posted to the centrifuge bay lab. In the centrifuge bay lab, the plasma will be removed from the whole blood and additive solution called SAGM (saline-adenine-glucose-mannitol) has been added to the packed cells. So when you are doing freezing, what am I actually squeezing out is the SAGM and the buffy coat. Fourthly, I would like to emphasize that the HCT has to be between 70 to 80 percent to pass. And lastly, during the addition of glycerolyte solution, the first pause is when you added 50ml for 5 mins and second pause is for another 50ml for 2 mins. When you done the second spin, you have to squeeze out 320 ml of glycerolyte solution out of the primary bag into the transfer bag. Lastly, after the addition of glycerolyte, the freezer bag is left standing for about 15 mins. This is to allow more time for the glycerol to replace the water inside the cells. I am truly sorry for the mistakes that I did in my earlier post. I doubled check what I did with my trainer and he pointed out some of my mistakes and misconceptions.

And now for the questions…

Hi Indah,
After adding the 100ml of the glycerolyte solution, why must the flow be stop? is it not possible to add the total volume at one go? also, what is the purpose of the shaker? Thanks!

zi shuang
0703383J

Thanks Zi Shuang for the question. The shaker is to mix the red cells with the glycerolyte solution as the solution flows into the freezer bag. The faster the speed of the shaker, the faster the flow of the solution out of the Glycerolyte 57 Solution bottle and into the freezer bag. It should be added in a stepwise fashion. So not too fast shaking. The pauses are to allow time for gradual equilibration between the red cells and the glycerol. Hence time is given for the glycerol to enter the cells and replace the water that is present in the red cells.

Hi Indah,

What is the purpose of the Glycerolyte 57 solution?

Qingling
0703433C

Thanks Qing Ling for the question. This solution is basically glycerol. It is a cryoprotective agent. During freezing, the water present in the cells can become ice crystals and cause injury to the cell membrane. This can lead to cell lysis. The glycerol is able to protect the cell from freezing injury and hence enhance cell recovery during thawing.

Hi Indah,
May I know what will happen to the blood that does not complete the freezing process within 4 hours?

Liyana
0703827F

Thanks Liyana for the question. The 4-hour rule is incorporated based on the requirement of the American Association of Blood Banks (AABB). Packed cells and whole blood should not be left out in the room temperature for very long as it must be maintained at temperature of 2-6°C. I am not too sure what are the procedures if you cannot complete the freezing process within 4 hours. One lab technician can only handle 6 blood bags at a time. There is only one centrifuge in which 6 blood bags can fit. Haha. So should be okay.

Hey Indah!
very detailed explanation. although i tink if you have some photos would be better? but i can imagine the primary and the freezer bag together and all that. haha.
ok. question:

1) after transferring the blood to the freezer bag, how come there's a need to also centrifuge the primary bag as well since it has to be discarded eventually?

2)i was just wondering, since the blood are all frozen, when the blood is needed, what is done to de-froze (like the procedures) especially in the case of emergency?

thank you indah!

Janice Yeh :) Grp 6
0701885F

Thanks Janice for the questions. For the 1st question, you are referring to the 2nd spin. The primary bag is not discarded as afterwards you have to transfer 320ml of the glycerol (top layer) out of the freezer bag. For the 2nd question, the process is thawing and deglycerolisation. I did not get a chance to view this as I was practicing on the freezing part. I have only 2 days in this lab before being rotated. so ya. Perhaps, if I have sometime during my major project, I can request to view the process and put it as one of my post.

Indah!
Come up and join me for lunch someday okay.
For the blood, we're only getting the packed cells frozen right?
And erm. This process is quite lengthy and wouldn't be cheap i guess. So, what are the blood types that you guys will freeze? Is it by patient's request? Or just some rare blood group?

Poh Yan Hong
0703979E / TG01

Hey babe… thanks for the question. I don’t have the time to eat with you guys cos the eating place is far away. I made my mom to cook food for me early in the morning. Haha. And I eat with the rest of the staff there at the pantry. But if I managed to get out early for lunch then I will msg you.
Ok, back to the question. Lengthy, yes. Cheap, I don’t know. I guess you just need to buy boxes of glycerolyte solution bottles and transfer bags. About the types of blood that we froze, im not too sure if there are any preference. I guess if we are to stockpile blood, then blood group O is very popular as it is the second choice for transfusion of packed cells as it is universal donor and can be used as emergency blood. I think we also freeze rare blood groups such as Bombay blood.

Hi Indah, :)

I'm just curious why do we need to place the bag into the water bath for 30 mins and at 37 degree C? What is the bag and its purpose?

Rachel Gan :)

Thanks Rachel for the question. The bag is the primary bag. If you are referring to the type of bag, I think it is the Baxter bag, 450ml. There are other types of bag as well such as Terumo bag. The purpose of placing the bag in the water bath before adding the glycerol is to let the cells warm up and the membrane more fluid so that later it is easier for the glycerol to enter the cells and the water to go out of the cells. The timing is important. We have a stopwatch alarm to time for exactly 30 mins.

Hello Indah,

Hope you are well =)

Are you meaning to say that the process involves removing the plasma and the buffy coat ? Also, why must some plasma be added back into the primary bag until the weight is increased by 25g ?

Thanks,

Ng Tze Yang Justin
0703747F

Hey Justin. I’m very well. I apologise for the plasma part. It is actually SAGM – an additive solution you added to the packed cells as explained in my amendments. Why you add back 25 g? I was told that it is method for the HCT testing. A failed HCT is retested again. A repeated HCT failure means you have to do the entire process again. The HCT may be too low indicating your cell recovery during thawing will likely to be low. I’m guessing that too high HCT could be due to centrifuging effects where the cells are really packed together. To get an accurate HCT as possible, it is best to add back 25g.

Freezing of Red Blood Cells

Hi. I’m Indah and I’m attached to a blood bank laboratory section.

There are many different laboratories and I was rotated from one laboratory to another. One of the laboratory sections that I was posted to is the freezing of blood. Blood can be frozen and stored for up to 10 years. This can be applied in situations like rare blood groups or autologous blood for future surgery. I am going to bring you through the process that I have done.

Blood that is going to be frozen has to be quarantined in the blood bank computer system and it will not be allowed for issue to the hospitals for transfusion. For every blood and its derived blood components that are quarantined, a unique quarantine number will be issued and is recorded in the quarantine logbook. The blood will be stored in the pending cold room. On the day that the blood will be frozen, it will be transfer to a refrigerator in the freezing laboratory, where it will be stored temporarily at 2 to 8°C. Blood that will have to be frozen are usually closed to its expiry date and freezing has to be done before expiration. I did freezing on red blood cells. The entire freezing process will have to done within 4 hours.

First of all, write down the blood group on the bag that will be frozen. If it is a single bag, the blood will need to be joined to a transfer bag to become a double bag. This can be done using a connector machine that will connect the two bags via the middle tubing. Tie a knot on the connected tubing to stop blood from flowing from the primary bag to the transfer bag. The double bag is then fold to fit in a centrifuge bucket for centrifugation. This is known as the first spin at 3200rpm for 4 minutes at 20°C. While waiting, we will do some documentation such as the recording the date of freezing, the blood donation number from the bag, the blood group, the quarantine number and the lot number of the Glycerolyte 57 solution bottle used.

After the first spin, carefully remove the blood bags from the centrifuge and place the primary bag on a plasma extractor. Untie the knot in the tubing and the plasma and the buffy coat (i.e. the top layer) will be squeezed out into the transfer bag. Then, while stopping the flow of blood from the two bags using your fingers, place the primary bag on a weighing scale. Fold the transfer bag in such a way that the buffy coat will settle to the bottom and add some plasma back into the primary bag until the weight is increased by 25g. Seal the tubing in between the two bags and dispose the transfer bag. Next, strip the tubing of the primary bag, ensuring no bubbles. Seal to form a segment and remove the segment from the rest of the tubing. Paste a donation number label on a test tube. Cut the separated segment and pour the blood into the test tube. Do a 1:1 dilution with NaCl. Test for the HCT using the automated ADIVA 120 machine. A pass is above 70% and a report is printed out. After that, weigh the blood again. Make sure to tare first with an empty bag. Record the volume of the blood (Volume = Mass/Density).

Place the bag inside a water bath for 30 minutes at 37°C. In the meantime, prepare the set up for the transfer of blood from the primary bag to the freezer bag (2000ml) and the glycerolyte solution. Connect the tubing from the glycerolyte bottle to the freezer bag but do not allow the flow of glycerolyte to the freezer bag by locking the clips. After 30 minutes, remove the bag from the water bath. Clean the needle of the freezer bag with methylated spirit and poke the needle to connect to the primary bag. Hang the primary bag upside down and allow the blood to drain to the freezer bag. After all the blood has been drained, transfer the whole set up to a shaker. Hang the glycerolyte bottle and unlock the clips, allowing glycerolyte solution to flow into the freezer bag and mix with the blood. After adding 100 ml, pause the flow by locking the clips and stop the shaker for 5 minutes. After adding another 50ml, pause again for 2 minutes. Transfer the rest of the glycerolyte solution, giving a total volume of 400ml. Lock all clips so that blood will not flow out of the freezer bag. Seal the tubing between the bottle and the freezer bag and discard the bottle.

Fold the freezer bag into a plastic bag and, together with the primary bag, they are centrifuged at 2500rpm for 10 minutes at 20°C. This is known as the second spin. Again carefully place the freezer bag onto the plasma extractor and squeeze out 50ml of the glycerolyte solution (i.e. top layer). Seal the tubing and discard the primary bag. Strip the freezer bag tubing.

Transform the blood product in the computer system into frozen red blood cells. (i.e. the transformation is done by a freezing laboratory staff as I do not have access to the computer system). Label the blood card with a frozen red blood cells label. Fold the bag in such a way that the tubing will not overlap with each other and place in a metal plate that is labelled with the donation number, blood group, quarantine number and date of freezing. Place the metal plate in the freezer at –80°C, laying it flat so that the blood will not sink to one side.

Some of the difficulties I faced while doing this were tying a knot, stripping the tubing and transferring the wanted volume from one bag into the other as the weight of the weighing scale keep on fluctuating.

Indah
0705361D

Cytogenetics

Hi everyone, I’m Justin here and I’m posted to cytogenetics lab for 20 weeks. The things I learnt are quite interesting as most of the stuff is not taught at school. For week 1 and 2, I’ve been observing procedures; reading documents; and practicing how to pair and arrange chromosomes. This week, I started performing some procedures.

Cytogenetics is the study of chromosome number, morphology, and the abnormalities that can be correlated to diseases. The lab I’m in is classified into 3 areas. Namely, Prenatal, which handles amniotic fluid and chorionic vili for diagnosis of chromosomal disorders ; Bone marrow, which handles bone marrow, bone core, and peripheral blood samples for haematological disorders; and FISH(fluorescence in situ hybridization), which uses specific molecular probes linked to fluorescent dyes to detect conditions eg. deletion syndromes. I’m in the bone marrow area, and will talk about the workflow for producing a karyotype, with some bone marrow specific procedures.

Sample: Bone Marrow/ Bone Core

When the sample arrives and test requisition has been performed, the first part of the process is the culture set up. The samples will be centrifuged and the buffy layer is obtained. 2 cultures will be set up. The type of cultures set up (direct harvest; 24-hr; 48-hr; 72 hr with mitogens) depends on the suspected diagnosis of the patient. The reason is that certain abnormal cell clones can be detected immediately but others need a longer time to grow, hence the need for 2 cultures and disease specific durations. The addition of mitogens (growth factors) allows the stimulation of specific clones. For example, in suspected cases of CLL (chronic lymphocytic leukemia), the 2 cultures set up would be 72-hr with TPA and PHA. TPA and PHA are growth factors specific for B-cells and T-cells respectively. This is so that we can determine the exact cause of the CLL (whether it’s due to B or T cells). For direct harvest, the harvesting process is done immediately (see next step), while for 24, 48, and 72 hour cultures are prepared, by inoculating the buffy coat into complete RPMI 1640 media (used by my lab, contains nutrients for bone marrow cell culture), and incubating them for the respective durations in a 37ºC CO2 incubator. I’ll go into more specific details of culture durations and procedures in subsequent posts.

The next part is the harvesting, which are the processing steps so as to obtain metaphases for chromosomal analysis. The first step is the mitotic arrest. Cell cycle is arrested at metaphase using a reagent known as colcemid. Colcemid inhibits spindle fiber formation by depolymerising tubulin, thus preventing the progress of the cell cycle. The next step is hypotonic treatment. A hypotonic solution (potassium chloride is used in my lab) causes water to enter the cell. This allows cells to swell and allows the chromosomes to spread out for easier analysis. In my lab the colcemid and potassium chloride is incorporated into 1 solution-“direct-harvest media”. The third step is fixation. The fixative used is a modified Carnoy’s fixative (without the chlorofoam?), which consist of methanol and acetic acid in the ratio of 3:1. The purpose of fixation is to harden cells and chromosomes, making them resistant to changes, and allowing for staining. The fixative also destroys debris (of cell clots etc), thus allowing a clearer suspension for easier analysis.

After harvesting, fixed cells are dropped onto slides so that they can be analysed. The slides must be grease and dirt free as other particles may interfere with analysis. The slides are then baked in an oven at 90ºC for 2 hours. The purpose of baking as that it gives better contrast for staining and allows the chromosomes to be stained. For staining, the most common staining method for routine analysis is G banding by trypsin using Giemsa and Wright’s stain (GTG banding). This staining allows chromosomes to be individually identified. The enzyme trypsin allows the staining dyes to enter the chromosomes.

After staining, the slide is sent for analysis. 20 metaphases will be analysed. The use of 20 metaphases is to ensure that an extra chromosome in one metaphase is not due to some random error (eg. overspreading because hypotonic treatment too long). A minimum of 5 metaphases must be karyotyped for analysis. Karyotyping refers to the arranging and pairing of chromosomes in order (1-22, XX or XY) Karyotyping can be done through computer programs or manually, in which the photo of the metaphase is taken and chromosomes cut out by hand (I do that as part of training for identifying chromosomes).

In terms of clinical significance, the use of bone marrow specimens allows for the diagnosis and prognosis of haematological disorders. This is because consistent chromosomal abnormalities will be associated with a particular disease. For example, in patients with Chronic Myelogenous Leukemia (CML), the 9-22 translocation will be observed (t(9;22)(q34;q11)) in abnormal cells. For prognostic information, a change in karyotype will show it. In patients with CML, if an extra chromosome 8; chromosome 19 or Philadelphia chromosome is observed, it indicates progression to the blast crisis of CML, whereby the prognosis is poor and the survival rate for patients is about 2-5 months (refer to MBIO lecture notes topic 5).

That’s all for now. I’ll go into more specific details of each stage in subsequent posts. Sorry for the long post. Enjoy your SIP !

Ng Tze Yang Justin
0703747F

Blood Banking

Hi, Liyana here :)

During my first week I was appointed to the blood banking section.

Once sample is received (in an EDTA tube), it needs to be checked for clotting, if there is clotting, the sample needs to be rejected. If no clotting is observed, the sample will be centrifuged to seperate the RBC portion from the plasma.

The lab uses an automated ABO/Rh typing and Ab screening. The analyzer is known as AUTOVUE Innova which uses the same principle as gel card technique. The sample would be loaded onto the analyzer. The analyzer uses a cassette for ABO/Rh typing, the cassette consists of forward and reverse grouping. The cassette has 6 columns consisting of:

Forward grouping (reagents are readily present in column):
1. anti-A
2. anti-B
3. anti-D

Reverse grouping (reagents will be added to the column):
1. A cells
2. B cells

(note: It does not use O cells)

The last coloumn is used as a control, whereby the probe would add patient's serum and RBC (initially diluted in saline).

The probe would initially suck up the plasma (this is done first to prevent disruption of the sepration between plasma and RBC) and add it to the columns containing A cells, B cells and the control. Subsequently, it would suck up the RBC and dilute it in saline before it is added to columns consisting of anti-A, anti-B, anti-D and control.

The cassette would then be transported to the centrifuge (located within the analyzer as well). It would undergo two type of centrifugation, low speed and high speed. The purpose of low speed is to allow the cells to intereact with the reagents. The high speed is to seperate the agglutinated cells from the non-agglutinated cells. The agglutinated cells would be large and will not be able to migrate to the bottom of the column, settles at the top. However, non-agglutinated cells are small enough to seep through the beads in the column and settle to the bottom.

As for Ab screening, the cassette consists of AHG for detection of IgG Ab. BLISS will be added to each column to lower zeta potential and enhance interaction between RBCs and Abs . Then, S1, S2 and S3 cells would be added to respective columns followed by plasma to each column (plasma is added later to prevent neutralization of AHG). Therefore, Ab screening only makes use of three columns though there are six columns in one cassette (the cassettes can run two different samples). Unlike, ABO/Rh typing, the cassette needs to be incubated for ten minutes before it undergoes centrifugation (both low and high speed). Interpretation of results is smilar to that of ABO/Rh typing.

The analyzer would read the cassettes and determine the patients ABO/Rh group and detect if there are clinically significant Ab present. However, Ab screening result would only appear as positive or negative, it would not be able to identify the Ab if result is positive.

Subsequently, results would be checked against the patient's history. If there are discrepancies or no history of the patient is found, the test would then be conducted again manually for rechecking. The lab uses tube method for ABO/Rh typing (similar to practicals done in school). However, it uses the gel card technique for Ab screening. A same cassette for Ab screening used by the analyzer is used for this method too. The steps for Ab screening using gel card technique are as follows:

1. S1, S2 and S3 cells are added to the three columns respectively.
2. Plasma is added to each column.
3. Incubation
4. Centrifugation
5. Read results; grade accordingly i.e.: 4+, 3+, 2+, 1+, 0

If a crossmatch needs to be conducted, the patient's Ab screening results will determine the type of crossmatch. If Ab screening result is negative, an abbreviated crossmatch is conducted whereby donor's RBC (diluted in saline) is reacted with patient's plasma. However, if Ab screening is positive, a full crossmatch (similar to practicals done in school) needs to be conducted whereby the three phases, i.e: saline, LISS and AHG, needs to be conducted using donor's RBC (diluted in saline) and patient's serum. In addition, if the results is negative in the AHG phase, Coomb's reagent needs to be added by acting as a control to ensure that the AHG is not neutralized by unbound Abs.

Liyana
0703827F
TG01