Hemoconcentration

Hemoconcentration is the rise in cellular and protein concentration in the blood due to loss of blood plasma. This can be effected by many things including exercise. The loss of plasma in exercise can be atrributed to two things:

1. Increased blood pressure causes an increase in hydrostatic pressure in the capillaries thus forcing plasma from vasculture to intestinal compartment.

2.As metabolic waste products rise in the active muscles, intramuscular osmotic pressure increases wich attracts fluid to te muscles.

A prolonged tourniquet time may lead to blood pooling at the venipuncture site, a condition called hemoconcentration.Hemoconcentration can cause falsely elevated results for glucose, potassium, and protein-based analytes such as cholesterol.

Ideally, the tourniquet should be in place no longer than one minute to prevent hemoconcentration. If the phlebotomist takes longer than one minute to assess and locate vein of choice for venipuncture, it is best practice to release the tourniquet, assemble supplies and reapply tourniquet immediately before needle insertion.

Hemoconcentration: An increased concentration of larger molecules and formed elements in the blood may be due to several factors:

·         Prolonged tourniquet application (no more than 1 minute)

·         Massaging, squeezing, or probing a site

·         Long-term IV therapy

·         Sclerosed or occluded veins

Prolonged Tourniquet Application:

·         Primary effect is hemoconcentration of non-filterable elements (i.e. proteins). The hydrostatic pressure causes some water and filterable elements to leave the extracellular space.

·         Significant increases can be found in total protein, aspartate aminotransferase (AST), total lipids, cholesterol, iron

·         Affects packed cell volume and other cellular elements

Assignment 1 Clinical imunohaematology iii SBD 0523

Assignment 1

Clinical imunohaematology iii

SBD 0523

1.    Method to perform absorptions (cold and warm) and 4 applications

Antibody Adsorption

•         Definition: The removal of antibody from serum by combining a serum sample with appropriate RBCs under optimal conditions.      

•         Antibody can be removed from a serum by adsorption to red cells carrying the corresponding antigen.

•         Serum and cells are mixed. After the antibody attaches to the membrane bound antigens the serum and cells are separated, the antibody remains attached to the RBCs.

•         Mix serum with antibody with red cells with corresponding antigen. Incubate at appropriate temperature. Anti-e antibodies have been adsorbed out of the serum and onto the RBCs.

Antibody Adsorption: Application

• Separating multiple antibodies present in a single serum.
• Removing auto antibody activity to permit detection of underlying allo antibodies.
• Removing unwanted antibody from a serum that contains an antibody suitable for reagent use.
• Confirming the presence of specific antigens on red cells through their ability to remove antibody of corresponding specificity from previously characterized serum.

Adsorption:
Separating Multiple Antibodies

•         Patient red cell phenotype is very helpful: Pheno typing the patient can help in choosing a red cell phenotype that will give the most information. If none of the antibodies have been identified then a weak reacting cell can be used assuming that only one antibody may be reacting with it

Separating Multiple Antibodies

•         If one or more of the antibodies have been identified, then red cells lacking those antigens are usually chosen so that only one antibody is removed. Large volumes of red cells are required so Reagent vials are insufficient. Donor blood or staff members end up being the most convenient.

Positive DAT

•         Auto adsorption is simply mixing patient serum with patient red cells to remove the auto antibody remaining in the serum under optimal conditions. If it is a warm auto antibody then adsorb at warm temps, if it is a cold auto antibody then adsorb at cold temps .Redo test serum to determine degree of removal of auto antibody. May require second adsorption. Test panel with adsorbed serum to check for presence of allo antibody that was masked by the auto antibody.

Sometimes, patients with autoantiobodies require red cell transfusion. The most important technical issue faced by the transfusion service is determining if the patient's serum contains alloantibodies in addition to the autoantibody. Between 15 and 40% of patients with autoimmune hemolytic anemia have alloantibodies. The most common alloantibodies detected in the sera of patients with AIHA in descending order are: anti-E, K, C, Fya, Jka, and c. the most frequently used method for detecting alloantibodies in the presence of a broadly reactive autoantibody is the warm autoadsorption procedure. Autoantibody is removed from autologous red cells by heat or chemical treatment and then the red cells are treated with an enzyme to enhance autoantibody adsorption. The most popular technique is to treat red cells with ZZAP, which is a combinination of dithiothreitol and papain. Autoantibody is then adsobed from plasma with these treated autologous red cells. Several adsorptions may be necessary to remove all of the autoantibody. If no antibody is detected, the adsorbed plasma can be used for crossmatching donor units. If alloantibody is present, it must be identified and antigen negative units selected for crossmatching.   The warm autoadsorption procedure is not useful in patients who have been transfused within the past 30 days because even a small percentage of transfused cells may adsorb alloantibody, producing a falsely negative result. In this situation, allogeneic adsorptions may be required. By selecting two or three samples of red cells of varying phenotypes, almost all clinically significant alloantibodies can be detected. For example, adsorbing a serum containing an autoantibody and an anti-Jka alloantibody with Jka negative red cells will remove the autoantibody but not the anti-Jka . Once the autoantibody is removed, the remaining alloantibodies can be identified using a panel. This procedure is usually performed at a blood center reference laboratory instead of a hospital transfusion

Cold antibodies

Most are IgM and not clinically significant.May interfere with detection of clinically significant antibodies if they react at AHG phase.Screen cells and panel cells will have positive reactions in IS phase and strength will diminish or antibody will not be detected at AHG phase.Auto control will be positive if the cold antibody is an autoantibody.Binding of antibody to antigen occurs at room or colder temperatures and may start to disassociate from the red cell membrane at warmer temperatures. Reactions will appear weaker or be negative at warmer temperatures. (Example: 4+ at IS phase and W (weak)+ at AHG phase.)

Prewarming

If a non specific cold antibody or cold agglutinin is suspected, warm the sample and testing reagents, including saline, to 37° C. Only do reaction readings at AHG; bypassing the optimum reaction temperature prevents activation and binding of the cold antibody.

Definition: a method designed to remove antibody from serum by incubating it with red cells positive for the corresponding antigen.

Applications

1.    Removing autoantibody activity to permit detection of possible coexisting alloantibodies. For example, if a patient has an autoanti-1, an autoadsorption at 4°C can be done to remove it, and the autoadsorbed serum can be run against a panel to identify any possible alloantibodies present.

2.    Reagent preparation: removing anti-A or anti-B or other unwanted antibodies from serum that contains an antibody suitable for reagent use, e.g., serum from a group A donor containing anti-D can be adsorbed with group B Rh(D) negative blood cells: the anti-B will be adsorbed, but the anti-D will not.

3.    Separating multiple antibodies to aid in identification: for example, if you suspect a serum contains anti-c and other unidentified antibodies that react with R₁R₁ cells, you could adsorb the serum with R₁R₁ cells. Anti-c would remain free in the supernatant and could be confirmed by testing the supernatant with panel cells. The antibodies which attached to the R₁R₁ cells could be eluted and identified.

Note: adsorptions are often done in conjunction with elutions

4.    Confirmation of the presence of a weak antigen on red cells: this can be done by proving their ability to adsorb and remove specific antibody. For example, supposing a patient's ABO group gave the following results:

anti-A	anti-B	A1 cells	B cells
--	--	--	4+

5.    The patient may be a group A with a very weak A antigen (weak subgroup of A). His red cells could be incubated with anti-A at 4°C for an increased time (e.g., 4 - 12 hours) and then an elution could be prepared. If the eluate contain anti-A, it is possible to conclude the A antigen must have been on the red cells.

6.    Confirmation of antibody specificity: if you suspect a serum contains an antibody (e.g., anti-c) plus one or more antibodies (e.g., anti-K and anti-Fy^a), you can confirm the anti-c by doing an adsorption followed by an elution, e.g., incubate serum with red cells that are Fy^a-, K^-, but c positive, then do an elution of the adsorbed cells and test it with panel cells to confirm it contains anti-c.

2. Antibody elution methods

Elution is the removal of antibodies bound to red cell membrane. The objective is to recover the antibody in a usable form. In other words, we want it still functional so it can be identified.

Methods

–        Heat or Freeze thaw: used for ABO HDN

–        Acid or Organic solvent: warm reactive auto and allo antibodies

Elution is the process of removal of antibody off of red cell membrane. Often, the red cell is destroyed in the process while the antibody is left free in the eluate.

Antibody Elution: Application

Investigation of Positive DAT

–        Hemolytic Disease of the Newborn (H DN), Autoimmune hemolytic anemia, etc.

Concentration and purification of antibody, the detection of weakly expressed antigens, and identification of multiple antibody specificities. Used in conjunction with adsorption technique. Preparation of antibody free red blood cells for use in phenotyping  or autologous adsorption studies.

Adsorption-Elution Techniques

•         Adsorb and Elute antibodies in a multiple antibody serum. Used to isolate individual antibodies.

•         Serum should be Adsorbed until no more antibody reactivity is detected.

•         Elution of adsorbed red blood cells is performed. A Panel is then tested with the eluate to determine the specificity of the eluted antibody.

Advantage and disadvantage of elution method.

It can detect the un identify the causative agent on the immune system, it is simple test,cheap,no special equipment and chemical needed,presecnecs in kit easy to carry. This method is destroy the red cell causing them rendering no longer available for testing the fragment of the rbc will affect the result that will be released.

3. Pre warm technique

B.General- Prewarm technique can be used to prevent cold-reactive alloantibodies or autoantibodies from reacting in the IAT phase. Specifically, prewarm technique prevents cold antibodies from binding complement at RT (as opposed to 37^o C) and subsequently being detected by anti-C3 in the IAT by polyspecific AHG serum. This assumes that pretransfusion testing is done

• using clotted specimens (C1qrs requires free Ca⁺⁺ ions; EDTA, for example, chelates Ca⁺⁺ and stops complement binding at C1q)
• Using polyspecific AHG containing both anti-IgG and anti-C3.

If these pre transfusion test conditions apply, an antibody-antigen reaction occurring at RT, before tests are incubated at 37^o C can be detected in the IAT phase. What happens is that the antibody reacts at RT and binds complement to C3. The clinically insignificant cold antibodies then elute off the red cells at 37^o C , but any C3 bound at RT will remain on the cells and be detected by the anti-C3 in polyspecific AHG serum.Prewarm technique is somewhat controversial, because some transfusion services use it to determine the clinical significance of antibodies, something that it was never intended to do.

Method

The prewarm method is briefly outlined below. A detailed method can be found in the AABB Technical Manual and similar references.

1. Prewarm serum and red cells to 37^o C for approximately 10 minutes prior to doing the pretransfusion test.
2. Also prewarm a bottle of saline and a pipette to 37^o C .
3. Add serum to cells using a prewarmed pipette.
4. Ensure that the tests remain at 37^o C before they are read. This includes washing the antiglobulin tests using saline prewarmed to 37^o C and using a centrifuge kept inside a 37^o C dry-air incubator.

There is no need to identify cold antibodies solely reactive at RT or lower unless they cause an ABO discrepancy. In this case, once the antibody is identified and the ABO discrepancy is resolved, it is not necessary to antigen type donors. Crossmatch-compatible blood (using prewarm technique) can be issued.

Note: If clotted specimens are used for pretransfusion tests, to prevent detection of C3 that may be bound at RT by cold antibodies, rather than doing prewarm technique, some labs prevent the problem by using monospecific anti-IgG antiglobulin serum.   

C.Purpose and Applications To Sera Containing Cold Autoantibody And Cold Alloantibodies

 Evidence is presented for the existence in human sera of autoantibodies of different specificities. It is shown that cold autoantibodies parallel in their behavior the natural isoagglutinins, and therefore are probably directed against the nucleus of the A-B-O substance. The autoantibodies found in typical cases of acquired hemolytic anemia parallel the behavior of Rh-Hr antibodies, and are presumably directed against the nucleus of the Rh-Hr substance. A serum is also described containing an autoantibody which mimics the behavior of M-N antibodies, and therefore is presumably directed against the M-N-S substance. The possibility of the existence of autoantibodies of still other specificities must also be considered. The hypothesis presented here, while not fully established, is useful since it suggests new avenues of approach to the problem of autosensitization.

D. INCOMPLICATION FOR THE NEED TO ANTIGEN TYPE DONOR BLOOD ????

Everyone who gets a blood transfusion is exposed to foreign red cell antigens (unless they receive their own blood, or blood from an identical twin). Yet not everyone makes red cell antibodies. Only 1 - 2% of hospitalized patients have irregular alloantibodies (5% or more if multitransfused or multiparous). Factors which affect whether or not antibodies will be made include these:

Immunogenicity: Only a few red cell antigens are very immunogenic (e.g., ABH in the ABO system, D in the Rh system, and Kk in the Kell system). Other Rh antigens (CcEe) and antigens in the Kidd and Duffy systems are not nearly as immunogenic. The strong immunogenicity for the D antigen is shown by the fact that approximately 70% of Rh(D) negative persons produce anti-D upon exposure to the D antigen. After the D antigen, the K antigen appears to be the most immunogenic.

Antigen Volume: Exposure to a very small volume of antigen may result in immune tolerance. Similarly, exposure to a large volume of foreign antigens simultaneously may produce immune paralysis. In general, the greater the volume of foreign antigen, the more likely it is that antibody will be produced.

Immune response genes: Antibody production seems partially to be under the genetic control of immune response (IR) genes that are closely linked to the HLA-D region on chromosome #6. IR genes can control whether or not someone is a good or a poor antibody responder. They can also be specific in their control of antibody production. For example, approximately 30% of Rh(D) negative people fail to produce anti-D no matter how much Rh(D) positive blood they receive. Perhaps these people have IR genes that make them incapable of producing anti-D.

4. Reason why test grade – to 4 Grading Agglutination Reactions

PRINCIPLE

The degree of red cell agglutination observed in any blood bank test procedure is significant and should be recorded. The degree of agglutination gives an indication of the amount of antigen or antibody present. The manner in which the cell buttons are resuspended and reactions are read is critical for accurate results.

PROCEDURE

1. Read only one tube at a time, using the lighted agglutination viewer.
2. Before resuspending, observe the supernate for hemolysis. If hemolysis is observed, this is a positive reaction and should be noted on the worksheet.
3. Grasp the tube lightly at the top, and hold vertically so that the cell button is facing toward you when viewed in the mirror.
4. VERY GENTLY shake the tube (shake from the wrist) to dislodge the cell button. Continuously observe the cells as they come off the cell button.
5. When the cell button is about one-half broken up, gently tip the tube horizontally and observe for agglutination.
6. Return the tube to the upright position and continue gently shaking until the entire cell button is dislodged.
7. Tilt the tube once more and immediately grade and record the reaction. Consult the reaction grading scheme below.

 

NOTE:

• Overshaking can break up fragile agglutinates, resulting in a false negative reaction.
• Failure to completely resuspend the cell button may result in a mixed-field reaction being missed.

GRADING SYSTEM FOR REACTIONS

Macroscopic:

4+ One solid aggregate or clump of cells.

3+ Several large aggregates, clear background.

2+ Small to medium sized aggregates, clear background.

1+ Small aggregates, turbid reddish background.

+W Tiny aggregates, turbid reddish background.

MF Mixed Field – Any degree of agglutination in a sea of unagglutinated cells.

Hem - Hemolysis is interpreted as a positive reaction and may be graded as complete or partial. Both hemolysis and agglutination may be recorded on the same tube.

Ø Negative - no agglutination, smooth reddish background.

Microscopic:

+ Positive - aggregates of at least 3-5 cells.

Ø Negative - no agglutination.

GRADING REACTIONS

Grading agglutination reactions gives an indication of the relative amount of antigen or antibody present. All tubes tests should be graded. The technique used in the resuspension of the cells will affect the grading of the reaction. The correct procedure for resuspending and grading reactions follow

Resuspension Procedure

• use lighted agglutination viewer
• read only one tube at a time
• hold tube upright
• position cell button so it is facing you in the mirror

very gently shake the tube and observe how the cells come off the cell button

Grading reactions

·         Swiling off-negative

·         Coming off in chucks-positive

·         Continue shaking till all cell resuspends

·         Tilt tube.read and grade reaction.

Grading system:

 4+-solid clump

3+ -several large clumps

2+-small to medium sized clumps;clear background

1+-small clumps;cloudy background

+w- tiny aggregate;cloudy background

+micro – positive upon microscope examination only.

Mf – mixed field .small clumps amidst many unagglutinated cell.can be confused with 1+.

Hem – hemolyzed (a positive reaction)

Neg –negative ,no agglutination ,

 

Washed cell suspension

3 % red blood cell concentration is saline

Between 2-5% cell suspensions provided optimum antigen concentration for the tube method for red blood cell typing.to make sure your suspension is within this ranges use reagent red cell for comparison.

Washing Red Blood Cells Before Making the 3% Suspension

The purpose of washing the red blood cells is to remove plasma, which contains substance that may interfere with antigen-antibody reaction.  The following may be in the plasma and may interfere with testing:

• Soluble antigens such as A and B may be present and neutralize your reagent.
• Interfering proteins such as Wharton's jelly that is seen in newborn cord blood, cold-acting autoimmune antibodies, and increased levels of immunoglobulins that may cause either agglutination or rouleaux..
• Hemolyzed red blood cells due to a difficult draw will interfere in your grading interpretation of hemolysis
• Fibrinogen can result in fibrin strands forming that makes grading reactions difficult.

Good Technique when washing and making a 3% cell suspension involves the following:.

• Place 1 to 3 drops of blood in the tube
• Aim the tip of the saline bottle towards the center of the tube and forcibly squirt saline into the tube.
• Fill the tube 3/4 full of saline (there will be less splattering in the centrifuge)
• Centrifuge long enough spin to pull most of cells into a button in the bottom of the tube.
• Decant the saline completely
• Shake the tube to resuspend cell button before washing the cells again. It will depend on the procedure being done as to how many types of washing are going to be done.

F.TETS READ AS BOTH AGGLUTINATION AND HEMOLYSIS

because the rbc in the experiment can be happening to be agglutinate or hemolysis because of the reaction upon the serum patient and the donor cell,this reaction happen because the antigen and antibody reaction.so we must aware to read the agglutination and hemolysis in this experiment.we must not taking this step easily because it may show the life of the human.

g.explained why test are read macroscopically and microscopically?

Macroscopic observation is done by the naked eye by looking onto the tube,the hemolisys or the agglutination of the blood can be confirm between ranged 2-4 +,this because the appearances of the substances is clearly and can be confirm nicely,in the microscopically the observation is using the microscope.the blood specimen were taken and placed on the slide to be observed by using microscope.we can see the agglutination and hemolysis of the blood eventhough without using the staining.the observation by using the microscope can be check the grade between 1-2+ this because people can be miss numbering the grade of the hemolysis and the aggelutination.

h.explaine why the tets must be handle gently when reading them?

The shaking and strirng the test will make the reading is error this because we want to see the normal reaction of agglutination by itself that occur naturally by the blood reaction. If we not handle gently the test will not be evaluate with good numbering and we will also mistake to reading them in non absolute state, Over shaking can break up fragile agglutinates, resulting in a false negative reaction

i.explained the importance of making legible enteries on workshhets when recording tets,and why result are  recorde with pen,with correction initialed.

When recording the test.we must used the legible enteries on the worksheets to make sure the result that be recorded can be identify it result and not crumple tighter with other result,if the crumple of the information happen it may affect the test that making the test must be re –run again ,this will wasting the time and energy also the sample.it also making the result can be read by other mlt or scientific officer for the test run or checking the result is valide or not,if someone want to retrieve the information it can see the worksheet easily to get the information.the recorded result must be using the pen because the pen ink is permanent ,not affected by the eareser,cannot be delete if it is on the papper,we not using the pencil becaue the pencil can be changeable and the ink not permeant,it can be easily eareser by the eraser,the tip of the pencil also is easily broken and need to be  sharp again.but the pen not have to be sharp again and it also have much color compare to the pencil that is only black,if we are the pen pointer can be adjust from size 0.5-1.0 mm .

The coorection that need to be make we must using the initialed only to advoid any meesy stain on the papper by the liquid papper ,the liquid apper is wet and crusty ,it can make the report is messy and many white spot,if we initialed it the will be no messy and also we can retrieve back the information easily without any problem.

j.explained why microscope reading are made on approxiamately 4-6 fields.

The rbc agglutination is big and easily seen on low focus,that why we need only using the 4-6fields only ,we don’t want to see the rbc but we want to see the agglutination and the hemolysis of the rbc,the phenomena and the appearances of the rbc is easily discovered by the microcope.we can see the formation and the reaction that happen with the help of the microscope.

k.explained why a test result should be recorded immediately after reading?

When doing the experiment any result that have shown must be observed and recorded at that moment,this because the longer the time taken to write the result,the experiment will changed and become invalide becaue the result it changed due to time changing,we must expect this going to happe because it is the natural order of the nature,so we must take the result as soon as possible when getting it,this may help to obtain a good result.in the hemaglutination the longer the specimen keep away the specimen will be changed into other type of result.like when we getting the result on the frist minute we have the 1+ but after 20 minutes the result happen to be 3+,so if we taken at 20 minute our result will be invalid and must re-do the test .it also keeping the sop procedure are taken correctly.

l. discuss the importance of paying careful attention to clerical detail.

The clerical detail will give extra precaution and make the result is valid. So the result that we will make can be trusted and according to the iso protocol, as we know we must follow the protocol that used worldwide by the WHO. The clerical error must be follow because without it the result we made is not valid,