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6.1 What is the distinction between the following terms: white blood cells, granulocytes, and neutrophils?
White blood cells include all nucleated white blood cells, that is, lymphocytes and granulocytes. Granulocytes include neutrophils, basophils, and eosinophils. Neutrophils refer to multi-lobed granulocytes that account for ~70% of granulocytes.
6.2 What are the risks associated with amniocentesis or fetal blood sampling?
Spontaneous or delayed abortion, infection, further immunization of mother, errors in genetic diagnosis because of potential maternal contamination of foetal samples.
6.3 Why have laboratories concentrated on HPA-1a for free fetal maternal DNA typing?
Anti-HPA-1a is the most frequent cause of neonatal alloimmune thrombocytopaenia.
6.4 How would you distinguish between platelet-specific allo- and autoantibodies in a transfused patient?
Alloantibodies would have a HPA specifi city. The patient should type as homozygous for the corresponding HPA allele. Auto-antibodies will have a broader, glycoprotein specifi city. Confi rmation of autoimmunity will require a direct test for platelet associated immunoglobulins using platelets obtained from the patient at least ten days after the last platelet transfusion.
6.5 What interventions might be used to reduce the impact of platelet autoantibodies in patients with platelet transfusion refractoriness?
IvIgG is of proven benefit in autoimmune thrombocytopenia and PTP and has been shown reduce haemorrhage, if not improve platelet count, in platelet transfusion refractoriness.
6.6 Why are liver transplants from patients with autoimmune thrombocytopenia are more likely than other organs to be associated with the horizontal transfer of ITP?
Liver transplants can be associated with the horizontal transfer of autoantibodies.
6.7 What is a stereo-isomer?
Molecules can exist in two forms, mirror-images of one another D (dextrotatory) and L (levorotatory), D and L-forms of these molecules are termed stereoisomers.
6.8 What is meant by the term ‘degeneracy of the genetic code’?
This term refers to the redundancy in the triplet code in which a nucleotide substitution in the codon may translate into the same amino acid. An example of this occurs in FcRIIIB where there are five nucleotide differences between FcγRIIIBHNA-1a and FcγRIIIBHNA-1b but only four amino acid differences.
6.9 What is the difference between antibody avidity and affinity?
Affinity refers to the binding constant of an individual antibody binding site. Affinity is the culminant binding constant associated with multiple antibody binding sites, for example the affinity of IgM antibodies may be low but the avidity is high because there are multiple binding sites.
6.10 Why is there reactivity of the HNA-1b antiserum with granulocytes from the donor typed as FCGR3*01; FCGR3B*03 in image 6.9a iv? And why is the agglutination less than that observed with the FCGR3*02 donor in image 6.9a ii?
The FCGR3B*03 allele expresses some HNA-1b antigen, but not as much as the FCGR3B*02 allele.
6.11 Why is does the HNA-1d antiserum not react with the donor typed as FCGR3*01; FCGR3B*03 in image 6.9b iv?
HNA-1d is considered antithetical to HNA-1c, hence is not expressed by the FCGR3B*03 allele. It is only expressed by FCGR3*02 alleles, hence FCGR3*01 alleles will not express it.
6.12 What do you understand to be the major challenges in undertaking in vitro serological investigations with granulocytes using traditional techniques?
Relatively low circulating cell counts in normal adults, granulocytes cannot be stored, cell isolation is time consuming, cells are prone to clumping if centrifugation is too hard or there is bacterial contamination of buffers, donors are not routinely typed for HNA, presence of antigens on cell surface, for example HLA class I, FcR receptors on cell surface.
6.13 What would be the consequences of performing a sub-optimal number of washes in the PIFT or GIFT following incubation with patient serum?
Increased levels of serum immunoglobulin would consume antiglobulin reagents and reduce the apparent levels of antibody binding.
6.14 What problems might be encountered in using fluorescent microbeads coupled to HPA and HNA proteins/peptides for detecting platelet and granulocyte antibodies?
The coupled peptides/proteins may not be able to generate the HPA/HNA epitopes due to lack of conformation of the antigens. Thus they may not be able to detect specific platelet and granulocyte antibodies.
6.15 What are the main problems in the detection and identification of platelet and granulocyte antibodies?
Cells are metabolically active and will clump in presence of excess calcium (platelets) or bacterial contamination (granulocytes) or if centrifuged at an excessive g force (both cells). Presence of HLA class I and other antigens on cell surface (e.g. I on granulocytes and ABO on platelets).
6.16 Give some examples of red cell antigens that are targets for autoantibodies.
Rh specificities, P and I.
6.17 Why are irradiated platelets used for intrauterine transfusions?
This is a measure to prevent alloimmunisation to any leucocytes that may be present in the platelet preparation.
6.18 What is meant by the term zygosity?
This term is used to describe whether a person has inherited the same or different forms of an allelic characteristic from the parents. Typically, for a biallelic characteristic, an individual will be either homozygous, that is, have two copies of the same allelic form of the gene (one from each parent) or heterozygous, that is, have one copy of one allelic characteristic from one parent and another copy of the other allele from the other parent.
6.19 How might platelet autoantibodies be generated following blood transfusion in PTP?
In an individual previously immunized, for example to HPA-1a, it has been proposed that the strong secondary immune response (anamnestic response) to a HPA-1a(+) blood transfusion results in a breaking of tolerance of autoreactive B cell clones recognizing platelet specific antigens. The breaking of tolerance results in the transient production of platelet auto-antibodies and causes the patient’s platelet count to decrease. The prompt response to IvIgG indirectly supports this theory since IvIgG is a successful treatment for many patients with autoimmune thrombocytopenia.
6.20 What is leucodepletion?
This is the process of reducing leucocytes from blood during the blood collection process. It was introduced to reduce the risk of retro-viral infections.
6.21 How could leucodepletion have reduced the incidence of PTP?
The answer is not clear but one possibility lies in the fact that platelets are removed by leucodepletion filters as well as white cells. The reduced concentration of platelets in red cell concentrates may not provoke the required anamnestic response to initiate PTP.
6.22 What samples and which laboratory techniques would you use to investigate a suspected case of TRALI?
Stratified investigation as follows: 1. Tests for HNA, HLA class I and II antibodies in female and male transfused donors. 2. If 1) positive, HNA or HLA class I and II typing of recipient and implicated donor as indicated. 3. If specificity could not be determined undertake crossmatch between donor plasma and recipient granulocyte and lymphocytes. 4. Test untransfused males if clinical story is convincing