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Thalassemia is best thought of as a group of disorders rather than a single disease. They demonstrate a hemoglobin synthesis disorder in which there exists a defect in the rate of production of one or more of the globin chains. This defect results from either a heterozygous or homozygous deletion or inactivation of a globin chain gene.Thalassemias are named according to the affected gene or globin chain which is showing reduced or absent synthesis. Globin chain gene loci are found on the following chromosome locations:Chromosome 11 (Beta, Delta, Epsilon, and Gamma)Chromosome 16 (Alpha, and Zeta)

As mentioned previously, gene deletions that cause alpha thalassemia can be homozygous or heterozygous deletions. Homozygous alpha thalassemia (alpha thalassemia major), also known as hydrops fetalis, is a lethal hemoglobin disorder which usually results in stillborn infants. In this condition, both alpha chain loci on each chromosome of the pair are deleted, resulting in a total absence of alpha chains. These chains are needed for all normal hemoglobins. If born live, infants with alpha thalassemia major exhibit hepatosplenomegaly, ascites, edema, low birth weight and die within a few hours. Ethnic groups most commonly associated with this form of alpha thalassemia include those of primarily Southeast Asian and occasionally Mediterranean decent.

In the homozygous state (-α/-α), both parents contribute one missing locus.(drawing modified from Harmening, 1999)

Look at the phase in which reactions are occurring. Reactions at immediate spin (IS) usually are not clinically significant. Reactions at AHG are clinically significant. Check for a match in the reactivity pattern by comparing sample reactions and individual antibody reactions Varying strengths of reactions could indicate dosage. Dosage means that there are two "doses" of the same antigen present on the red cells . Antibodies that exhibit dosage react more strongly with homozygous cells (e.g., Jkb Jkb ) than with heterozygous cells (e.g., Jka Jkb) .

Cells 4 and 9 may be used for rule outs due to negative sample reaction. Screen cell I may be used for rule outs due to negative sample reaction. Look at the antigens present on cells 4 and 9 that are in the homozygous state (highlighted in green). Remember the 3 to rule in and 3 to rule out procedure. Antibodies ruled out (with 3 reactions): e, k, Kpb, Jsb, Jka, Leb, P1, Lub. A selected panel should be set up to rule out (with 3 reactions) the remaining clinically significant antibodies (E, D, C,c, K, Fya, Fyb, Lea, M,N, S, and s).

In this example the patient's plasma tests positive with both screening cells at a strength of 4+. In the panel below, reaction patterns show varying strengths, 2+ to 4+ (highlighted in green).4+ could indicate one strong antibody or a combination of several antibodies that increases the strength of the reaction.3+ could indicate the presence of just one strong antibody.2+ could indicate a weaker reaction of an antibody that commonly exhibits dosage if the panel cell is in the heterozygous state.Since Cw, Kpa, Jsa, Lua are not present on the testing cells, they are probably not causing these reactions. Perform rule outs using panel cells 5 and 7 (sample had no reaction in any phase with these panel cells)Antibodies that can probably be ruled out at this point because the corresponding antigens are present on cell 5 and/or 7: C, c, e, k, Kpb, Jsb, Fya, Jkb, Lea, M, N, s, P1, LubAntibodies that could not be ruled out with this panel: D,E, K, Fyb, Jka, Leb, SPredominant pattern of 4+ in panel cells 1,2,4,10 matches anti-D Varying strengths in reactions indicates a possible second antibody so selected cells should be picked to aid in identificationFind panel cells that do not contain D (antibody you suspect) and are homozygous positive for the antibodies you are trying to rule out.

The selected cells should be antigen-negative for the antibody that you think is present and antigen-positive (homozygous) for what you are trying to rule out. You are designing a panel that addresses your testing needs. Example: JkbIf you suspect that your patient has an anti-Jkb and further rule out cells are needed, then those rule out cells should be negative for Jkb. In the table below, donor cells 1,2, 4, 6, 9 and 10 may be used when creating a select panel to test the patient and help rule out the remaining possible antibodies. The homozygous rule applies when choosing which cells to use for testing (antigens highlighted in light-yellow).Example: Picking cells to rule out CUse panel cell 1 and panel cell 2 (C is in the homozygous state). Explanation: Panel cells 1 and 2 do not contain the antigen Jkb (signified by "0" on cell panel). If these cells are tested with the patient's plasma and the reaction is negative, it can be assumed that the patient does not have an antibody to C. C is now ruled out because there would be a total of 3 negative patient reactions with C positive cells (These two reactions and screen cell I from the antibody screen, shown again below). This should be done for all clinically significant antibodies that you were unable to rule out on the first panel.

Once an antibody hypothesis is generated, most laboratories will select additional cells to rule-out any other commonly encountered antibodies that could not be ruled-out with the initial antibody screen and panel. Cells should be selected that are negative for the antigen(s) that correspond to the hypothesized antibody and positive for the antigen (s) to commonly encountered antibodies that have not been ruled out. If not ruled-out most laboratories will select cells for at least the following: anti-D, anti-C, anti-c, anti-E, anti-e, anti-K, anti-k, anti-Fya, anti-Fyb, Anti-Jka, anti-Jkb, anti-Lea, anti-Leb, anti-P1, anti-M, anti-N, anti-S, and anti-s. Antibodies to antigens of very low incidence (for example, anti-Jsa) are generally not eliminated in initial testing, but in most settings it is not feasible to try and find rule-out cells. In these cases, it is important for the technologist to understand that these antibodies HAVE NOT been ruled-out due to limitations in the test system.

Purpose: To design a set of panel cells that may help you to rule out additional antibodies and lead to the identification of the antibody that is present in the patient's plasma.Benefit of running selected cell panel: Decreases the use of reagents and specimen. How to choose selected panel cells: If you suspect that a specific antibody is present, the cells you choose for the select panel should be negative for that antigen and positive for the antigen you are trying to rule out (homozygous state).

Rule-out (also referred to as exclusion or cross-out) is a process by which antibodies are identified as being unlikely in a given sample because of the absence of an expected antigen-antibody reaction. In other words, the absence of a reaction is noted with a cell that is positive for the corresponding antigen. Rule-out, while very useful, can lead to error. Ruling out an antibody should be combined with other supporting data to increase confidence in the solution; the more data collected, the higher the probability that the final solution is correct.Non-reactive cells are selected for rule-out. To be classified as non-reactive, a cell must NOT have reacted in any phase of testing in a given panel or screen. In the case of cold antibodies: if reactions are only occurring at immediate spin and are negative in the AHG phase, then that panel cell can be used as a rule out cell for IgG reactive antibodies but not for antibodies that react at immediate spin (IgM).If there is no reaction with a panel cell then it is possible that antibodies to the antigens on that cell are not present in the sample being tested.

Thalassemias are part of a group of quantitative hemoglobin synthesis disorders in which a defect exists in the rate of production of one or more of the globin chains. This defect results from either a heterozygous or homozygous deletion or inactivation of a globin chain gene.Thalassemias are named according to the affected gene or the globin chain that is showing reduced or absent synthesis.Globin chain loci are found on:chromosome 11 (beta, delta, epsilon, and gamma)chromosome 16 (alpha, and zeta)

Delta-beta thalassemia exists in both heterozygous and homozygous forms. The symptoms are mild to moderate depending on the severity of the disease and can include mild, hypochromic anemia, slight hepatomegaly and/or splenomegaly and occasional bone changes due to the erythroid hyperplasia. Patients rarely require treatment, but blood transfusions may be necessary in certain cases. In this condition, the body compensates for the lack of beta and delta chain production by increasing the production of gamma globin chains, leading to an increased hemoglobin F level. This form of beta thalassemia can be found in many ethnic groups, but is most common in persons from Greece, Africa, and Italy.

Advantages of utilization of HbA1C over blood glucose measurement include: Fasting is not required Greater specimen stability Less fluctuations in day-to-day levels caused by stress and illness Disadvantages of utilization of HbA1C over blood glucose measurement include: Cost per test is higher than blood glucose. Conditions that shorten red blood cell (RBC) survival e.g., hemolytic anemia, homozygous sickle cell trait, pregnancy, or recent significant blood loss, will reduce exposure of RBCs to glucose, thereby lowering the HbA1C test value. Specimens with >10% fetal hemoglobin (HbF) may have a falsely decreased HbA1C test result. If onset of diabetes is rapid, blood glucose levels will more correctly reflect glycemia than HbA1C levels.

Sickle cell anemia is a qualitative hemoglobin synthesis disorder known as a hemoglobinopathy. Sickle hemoglobin (HbS) is a structural disorder caused by valine replacing glutamic acid in the sixth position on the beta chain. The heterozygous state, HbSA, is known as sickle cell trait, while the homozygous state, HbSS, is sickle cell anemia or sickle cell disease. A double heterozygous condition known as Hemoglobin SC disease also exists where one beta chain carries the mutation for HbS and the other beta chain carries the mutation for HbC. In addition, HbS can be present with thalassemia.Sickle cell anemia can also demonstrate hereditary persistance of fetal hemoglobin (HbS/HPFH).Other HbS combinations are very rare and include HbS/HbE, HbS/HbD LosAngeles, HbS/HbG-Philadelphia, and HbS/HbO Arab.

Under low oxygen tension, homozygous hemoglobin S will polymerize, forming tactoids or fluid polymers. These polymers realign and cause the red blood cell to become deformed. While reoxygenation reverses the deformed shape, it is only temporary as the tactoids continue to grow during repeated passes through the sickling cycle as shown in image 1.Image 1This process is enhanced by a low pH, increased levels of 2,3-DPG, increased temperature (above 37oC), and a state of dehydration. As the cycle is repeated, a potential for occlusion exists as cells become more rigid and contribute to increased viscosity (see image 2).Image 2Repeated occurrence of sickling eventually leads to an irreversibly damaged cell, which is involved in occlusion or otherwise removed from circulation (see image 3).Image 3

The sickle cell gene is most prevalent in Africa, although it is also common in Mediterranean countries, India, and the Middle East. Less than 2% of African Americans are homozygous for HbS and 8 - 10 % are heterozygous.

The following list corresponds to this image of an acid hemoglobin electrophoresis.Lanes 1 and 2 contain controls ASC and AF (Remember, AF and ASC are labels and do not indicate the order of migration.)Lanes 3 and 4 (patient 1): Heterozygous Sickle Cell Trait (HbSA) Hb S is approximately 30%Lanes 5 and 6 (patient 2): Double Heterozygous HbSC Disease (HbSC)Lanes 7 and 8 (patient 3):Homozygous Sickle Cell Disease (HbSS)Hb S is 100%Lanes 9 and 10 (patient 4): Sickle Cell Hemoglobin with Hereditary Persistance of Fetal Hemoglobin. Hb S is approximately 70%; HbF 30%.

Investigative tests on the father depend on which maternal antibodies are present.1. Mother has anti-D ABO and Rh typing with anti-D, -C, -E, -c,-e to determine probable Rh genotype* to predict the chance the fetus has of being Rh positive and affected by HDFN; Test for weak D if initial Rh typing appears to be D-negative. * For D+ fathers, the probable Rh genotype can be determined using serologic tests, i.e., DCEce typing to determine if the father is probably homozygous or heterozygous for D.2. Other maternal clinically significant antibodies Phenotype father for the corresponding antigen and its antithetical antigen (e.g., K and k)

Rh Genotype (Father and Fetus)As noted, usually molecular typing of the father is done only if the mother has anti-D or an antibody to another antigen for which molecular methods exist. In the case of a mother with anti-D and a father who is D+ using serologic methods, molecular typing can determine the father's RHD heterozygosity or homozygosity*. If the father is homozygous for the RHD allele, all of his offspring will be Rh positive, negating the need for fetal D testing, but indicating that the fetus should be monitored for HDFN. If the father is heterozygous for RHD, the Rh type of the fetus should be determined to see if HDFN is possible. * For D+ fathers, the probable Rh genotype can be determined using serologic tests, i.e., DCEce typing to determine if the father is probably homozygous or heterozygous for D (see later).

The prevalence of common HFE mutations among persons with hereditary hemochromatosis (HH) has been reported in numerous studies conducted in the US, France, Australia, and other countries. Homozygous C282Y mutation (C282Y/C282Y) is present in 82% to 90% of Caucasian patients diagnosed with iron overload due to HH.(7) This suggests a strong link between the genotype and the phenotypic presentation of clinical iron overload. Much lower percentages of persons diagnosed with HH do not have two C282Y mutations. A small percentage of persons diagnosed with HH are compound heterozygotes for C282Y and H63D (C282Y/H63D), are homozygous for H63D (H63D/H63D), heterozygous for C282Y (C282Y/wild type) or for H63D (H63D/wild type), or carry S65C or other HFE mutations.It may be that symptomatic heterozygotes are actually HFE-compound heterozygotes with additional unidentified mutations modifying the expression of the more severe known mutation. It is quite possible that more mutations of HFE and elucidation of other gene mutations modifying HFE will be discovered in the future enabling scientists to better explain the phenotypic heterogeneity of this disorder.In the US, the C282Y mutation is most prevalent in the non-Hispanic white population. It is much less common among Hispanics and African Americans.

For reasons as yet unknown, not all individuals who are homozygous for the C282Y mutation display phenotypic features of HH, and persons with H63D polymorphisms rarely develop iron overload. The penetrance (percentage of individuals with a specific genotype who express the associated phenotype) of HFE mutations is generally considered to be low. Results of a recent meta analysis by the US Preventive Services Task Force conclude that 38% to 50% of all C282Y homozygotes develop some evidence of iron overload, but that only 10% to 33% develop clinical disease due to HH. (8) In other words, some individuals may have elevated iron test results such as transferrin saturation, but do not demonstrate significant organ damage. Estimates of penetrance in some studies have found it to be even lower. Penetrance of HFE mutations is currently a controversial subject among experts, and the significance of finding HFE mutations in a given individual is often unclear. The probability that a given individual with HFE mutations will develop clinical disease from iron overload cannot be determined at this time.

Homozygous "hh" individuals do not form H substance and thus have no way for late sugars to attach. The blood group resulting from the homozygous "hh" condition is called the Bombay blood group (Bombay phenotype). Due to the presence of anti-H in the serum of a person with the Bombay phenotype, only blood from another person with the Bombay phenotype may be transfused.

Single nucleotide polymorphisms (SNP) genotype analysis looks for small changes in sequences instead of simply identifying the amplicon. One of the best ways to detect SNPs is to compare melting curves. The use of real-time PCR to create melting curves can detect differences as small as one base pair. Melting curve analysis is an assessment of the disassociation characteristics of double stranded DNA when exposed to heat. The strength of the hydrogen bond of each piece of DNA is dependent upon several different factors: the length of the DNA segment, the amount of guanine and cytosine pairs, and the degree of compliment. In real-time PCR the fluorescence that is given off by the probes will decrease once the strand of DNA disassociates. After the DNA or RNA is amplified, the temperature of the sample is slowly increased while the fluorescence is recorded. The melting points should show up as peaks when plotting temperature against fluorescence. These peaks allow one to differentiate between homozygous wild type, heterozygous, and homozygous mutant alleles. One clinical use of this method is to detect both HIV-1 and HIV-2 in samples.

Sickle cells can be seen in the peripheral blood of patients who have homozygous sickle cell anemia; however other tests are needed to make the diagnosis. Most sickled cells can revert back to the discoid shape when oxygenated. About 10% of sickled cells are unable to revert back to their original shape after repeated sickling episodes.

FatherPolicies for Rh typing fathers vary widely and often Rh typing not done unless the mother develops anti-D.Some labs consider typing the father if paternity is certain. For example: ABO and Rh type father with anti-D, -C, -E, -c,-e to determine probable Rh genotype Test for weak D if initial Rh typing appears to be D-negative) If father is Rh negative, RhIg is not needed. The purpose of DCEce typing Rh positive fathers is to determine if the father is probably homozygous or heterozygous for D to predict the chance the fetus has of being Rh positive. For example: Rh Phenotype Results D C E c e + + –̶ + + For these results, the father could have one of three Rh genotypes: CDe/cde CDe/cDe cDe/Cde Because the most common is CDe/cde (R1r), the father would be assessed as probably heterozygous for D. The father's actual Rh genotype can be determined by molecular methods, if available.

Pelger-Huet anomaly is the inherited form of neutrophilic hyposegmentation. Its transmittance is autosomal dominant and the anomaly is present in about one out of every 6000 people. When present on a peripheral blood smear, more than 70% of the segmented neutrophils will have bi-lobed or mono-lobed nuclei. A bi-lobed nucleus will have two round segments of nearly equal size, connected by a thin chromatin strand. A mono-lobed nucleus may be peanut shaped, slightly indented, or round; chromatin appears fully mature and parachromatin is evident. Pelger-Huet anomaly in the homozygous state has an increased number of cells with singular round nuclei and decreased numbers of the bilobed forms. The main function of neutrophils is phagocytosis. This function is not affected in either the acquired or the inherited anomaly. Since inherited Pelger Huet anomaly is associated with functionally normal neutrophils, the neutrophils are considered a nonpathological variant.

Pelger-Huet anomaly is a congenitally acquired condition of nuclear segmentation that has no clinical significance. There is no loss of cellular function.The condition can be suspected if typical bilobed, "pince-nez" nuclei are observed (section A in the composite image). Band neutrophils usually have two distinct lobes, connected by a relatively short but thick bridge as illustrated in sections B and D. Monolobulated cells may also be encountered, especially if a homozygous inheritance is present, as illustrated in section C. If 70% or more of the segmented neutrophils on the differential possess these nuclear morphologies, the possibility of a homozygous Pelger-Huet should be considered.