BOC Hematology Review

High Performance Liquid Chromatography (HPLC)

A form of chromatography in which a small sample is put into a column that can be manipulated with sophisticated solvent gradients to allow very refined separation and characterization; formerly called high-pressure liquid chromatography. Separates hemoglobin types in a cation exchange column and usually requires only one sample injection. Unlike electrophoresis, HPLC can identify and quantify low levels of Hb A2 and Hb F, but comigration of Hb A2 and Hb E occurs. Therefore HPLC is best used in the diagnosis of thalassemias rather than hemoglobinopathies because quantification of low levels of normal and abnormal hemoglobin levels is necessary to distinguish thalassemias.

Polycythemia vera (PV)

A neoplastic clonal MPN that results in an increases in erythrocytes, granulocytes, and platelets in the peripheral blood. Splenomegaly is common. In this disorder, stem cells are hypersensitive to, or function independently of, erythropoietin for cell growth. The diagnosis of PV requires that three major criteria and one minor criterion be met or that the patient meet the previous higher Hb level of 18.5g/dL in men. The three major criteria include: 1. Elevated Hb, Hct, or red cell mass level (Hb > 16.5 g/dL in men and women or Hct > 49% in men and Hct > 48% in women or RCM > 25% of the mean normal). 2. Bone marrow showing hypercellularity for age with trilineage growth (panmyelosis) including prominent erythroid, granulocytic proliferation with pleomorphic, mature megakaryocytes 3. Identification of the JAK2 V617F mutation or the JAK2 exon 12 mutation. The one minor criteria is low serum erythropoietin. Presenting symptoms are associated with hyperviscosity and hyperproliferation and include headache, weakness, pruritis, weight loss, and fatigue. Pruritis (itchiness) has been associated with elevated blood histamine and is often not associated with a visible rash. About half of PV patients have thrombocytosis and one- third experience thrombotic or hemorrhagic episodes. Hemoglobin, Hematocrit, Red blood cell volume, Total white blood cells, Granulocytes, Platelets = Increased Erythrocyte morphology = Normocytic / normochromic Leukocyte alkaline phosphatase = Normal or increased

AML with recurrent genetic abnormalities

"AML with recurrent genetic abnormalities" is a classification based on the cytogenetic and molecular mutations observed. - Translocation between the long arms of chromosomes 8 and 21, t(8;21)(q22;q22), which is representative of AML with maturation. - Acute promyelocytic leukemia is associated with a translocation between the long arms of chromosomes 15 and 17, t(15;17)(q24;q21) - A pericentric inversion of chromosome 16, inv(16)(p13.1q22), is seen in AML with increased eosinophils

Type 3 von Willebrand disease

"Nullallele"VWF gene translation or deletion mutations that may occur anywhere on the gene produce severe mucocutaneous and anatomic hemorrhage in compound heterozygotes or, in consanguinity, homozygotes. This is the most rare form of VWD, where the VWF concentration measured by immunoassay or by activity assay is less than 10%. Factor VIII is proportionally diminished or absent, and primary and secondary hemostasis is impaired.

Hb E

Asian Non-sickling beta globin chain variant in which lysine is substituted for glutamic acid in position 26. Mild unless associated with Hb S or Thalassemia. Microcytes and target cells. RBC survival time is shortened. The Hb E mutation is both a qualitative defect (because of the amino acid substitution in the globin chain) and a quantitative defect with a b-thalassemia phenotype (because of decreased production of the mutated globin chain). Hb E-b-thalassemia is most severe coinheritance.

Chediak-Higashi syndrome (CHS)

Autosomal recessive disease where there is a mutation into CHS1 LYST gene on chromosome 1q42.1-2. Clinical manifestations begin in infancy with partial albinism and severe recurrent life threatening bacterial infections. Hematological findings include giant lysosomal granules in granulocytes, monocytes, and lymphocytes; fused granules result in leukocyte dysfunction. Patients have bleeding issues due to dense granules in platelets. Pseudo-Chediak-Higashi granules are reported in patients with acute myeloid leukemia, chronic myeloid leukemia, and MSD

Type C NP

Autosomal recessive lipidosis in which mutations in NPC1 or NP2 gene (95% and 5% of cases, respectively) causes impaired cellular trafficking and homeostasis of cholesterol. The result is buildup of unesterified cholesterol in lysosomes. The clinical presentation in type C NP is heterogeneous with regard to age of onset and type and severity of neurologic and psychiatric symptoms, as well as visceral involvement. Early diagnosis is important because substrate reduction therapy can slow the progression of neurologic damage; however, because of its heterogeneous presentation, diagnosis is often challenging. The prognosis in type C NP is poor, with most patients dying before the age of 25 years.

Automated Reticulocyte count

Available automated reticulocyte analyzers include flow cytometry systems such as the FACS system from Becton, Dickinson or the Coulter EPICS system. All these analyzers evaluate reticulocytes based on optical scatter or fluorescence after the RBCs are treated with fluorescent dyes or nucleic acid stains to stain residual RNA in the reticulocytes. The Sysmex R-3000/3500 is a stand-alone reticulocyte analyzer that uses auramine O, a supravital fluorescent dye, and measures forward scatter and side fluorescence as the cells, in a sheath-stream, pass through a flow cell by an argon laser. - The signals are plotted on a scattergram with forward scatter intensity, which correlates with volume, plotted against fluorescence intensity, which is proportional to RNA content. - Automatic discrimination separates the populations into mature RBCs and reticulocytes.

Sickle solubility

Capitalizes on the decreased solubility of deoxygenated Hb S in solution, producing turbidity. Blood is added to a buffered salt solution containing a reducing agent, such as sodium hydrosulfite (dithionite), and a detergent-based lysing agent (saponin). Saponin dissolves membrane lipids, causing release of hemoglobin from RBCs, and dithionite reduces iron from the ferrous to the ferric oxidation state. Ferric iron is unable to bind oxygen, converting hemoglobin to the deoxygenated form. Deoxygenated Hb S polymerizes in solution, which renders it turbid, whereas solutions containing nonsickling hemoglobins remain clear. False-positive - hyperlipidemia - too much blood is added to the test solution - few rare hemoglobinopathies [Hb C-Harlem, Hb C-Ziguinchor, Hb S-Memphis, Hb S-Travis, Hb S-Antilles, Hb S-Providence, Hb S-Oman, Hb Alexander, and Hb Porte-Alegre] False-negative - infants younger than 6 months - low hematocrits

Chronic myeloid leukemia (CML)

Caused by a mutation in the stem cells produced by the bone marrow. The mutation causes the stem cells to produce too many underdeveloped white blood cells. It also leads to a reduction in the number of other blood cells, such as red blood cells. The clinical features are frequent infection, anemia, bleeding, and splenomegaly, all secondary to massive pathologic accumulation of myeloid progenitor cells in bone marrow, peripheral blood, and extramedullary tissues. Neutrophilia with all maturational stages present, basophilia, eosinophilia, and often thrombocytosis are noted in peripheral blood. Dramatic left shift with promyelocytes and a few blasts in peripheral blood. Platelets are normal or increased, and some may exhibit abnormal morphology.

megaloblastic anemia

Caused by deficiency of Vitmain B9 and B12 (folate). Anemia that results from inhibition of DNA synthesis during red blood cell production. Pernicious anemia is one cause of B12 deficiency while pregnancy leads to increased requirements which causes more folate to be needed. Nuclear maturation lags behind cytoplasmic development as a result of the impaired DNA synthesis, this results in larger cells. - this type is characterized by oval macrocytes and hypersegmented neutrophils in peripheral blood and by megaloblasts or large nucleated erythroid precursors in the bone marrow

Chronic inflammation

Causes iron to be sequestered away from the blood in order to protect against invading pathogens mediated by iron binding proteins called transferring. This results in diminished heme synthesis

Niemann-Pick disease (NP)

Characterized by an accumulation of fat in cellular lysosomes of vital organs, which impairs their function, leading to a range of clinical findings. NP has three subtypes. Types A and B are caused by recessive mutations in the SMPD1 gene located within the chromosomal region 11p15.4. This results in a deficiency of lysosomal hydrolase enzyme acid sphingomyelinase (ASM) and a subsequent buildup of the substrate sphingomyelin in the liver, spleen, and lungs. Foam cells and sea-blue histiocytes can be seen in the bone marrow. - Foam cells are macrophages with cytoplasm packed with lipid-filled lysosomes that appear as small vacuoles (foam) after staining - Sea blue histiocytes are macrophages with lipofuscin-, glycophospholipid-, and sphingomyelin contained in -cytoplasmic granules

Myeloproliferative neoplasms (MPNs)

Clonal hematopoietic disorders caused by genetic mutations in the hematopoietic stem cells (HSCs) that result in expansion, excessive production, and accumulation of mature erythrocytes, granulocytes, and platelets. The MPNs have the propensity to transform into other MPNs or progress into acute leukemias (ALs). WHO has classified MPNs into four predominant disorders: 1. Chronic myeloid leukemia (CML) 2. Polycythemia vera (PV), also known as polycythemia rubra vera; 3. Essential (primary) thrombocythemia (ET) 4. Primary myelofibrosis (PMF), formerly known as agnogenic myelofibrosis with myeloid metaplasia and chronic idiopathic myelofibrosis.

Recurring cytogenetic abnormalities

In hematologic neoplasias specific structural rearrangements are associated with distinct subtypes of leukemia that have characteristic morphologic and clinical features. In chronic myeloid leukemia the primary aberration is the Philadelphia chromosome resulting from a translocation between the long arms of chromosomes 9 and 22, t(9;22)(q34;q11.2). A sideline of this clone would include secondary abnormalities, such as trisomy for chromosome 8, written as 18,t(9;22)(q34;q11.2). Leukemias are clonal proliferations of malignant leukocytes that arise initially in the bone marrow before disseminating to the peripheral blood, lymph nodes, and other organs. The four main leukemia categories are acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), and chronic myeloid leukemia (CML). The World Health Organization (WHO) classification: AML with recurrent genetic abnormalities

Type B NP

In type B (the non-neuronopathic form) there is approximately 10% to 20% normal enzyme activity. Type B NP is more common in individuals of Northern African descent and presents in the first decade to adulthood with a variable clinical course. - Although there is no neurocognitive impairment, patients experience massive hepatosplenomegaly, heart disease, and pulmonary insufficiency.

Type 1 von Willebrand disease

Most common & most mild Type 1 VWD is a quantitative VWF deficiency caused by one of several autosomal dominant frameshifts, nonsense mutations, or deletions that may occur anywhere in the VWF gene There is mild to moderate systemic bleeding, usually after a hemostatic challenge such as dental extraction or surgery. In women, menorrhagia, which predicts postpartum hemorrhage, is a common complaint that leads to the diagnosis of VWD. However, because mucocutaneous bleeding symptoms occur in normal people to varying degrees, diagnosis requires scrupulous laboratory testing.

Bruton tyrosine kinase (BTK) deficiency

antibody deficiency, X-linked agammaglobulinemia Characterized by reductions in all serum immunoglobulin isotypes and profoundly decreased or absent B cells. Caused by a mutation in the gene encoding Bruton tyrosine kinase, resulting in decreased production of BTK, which is important for B cell development, differentiation, and signaling. - Without BTK lymphocyte fail to fully mature, leading to severe hypogammaglobulinemia and an inability to produce specific antibodies. Infants with BTK deficiency display symptoms between 4 and 6 months, once maternal antibodies have cleared. Recurring life-threatening bacterial infections ensue. Risk of fungal and viral (except enterovirus) infection is low because of normal T cell function.

Pelger-Huët anomaly (PHA)

benign inherited condition characterized by hyposegmentation of the neutrophil's nucleus and excessive chromatin clumping.

erythrocyte sedimentation rate (ESR)

measures time it takes for erythrocytes to settle to the bottom of a test tube Ordered with other tests to detect and monitor the course of inflammatory conditions such as rheumatoid arthritis, infections, or certain malignancies. It is also useful in the diagnosis of temporal arteritis and polymyalgia rheumatica. ESR is not a specific test for inflammatory diseases and is elevated in many other conditions such as plasma cell myeloma, pregnancy, anemia, and older age. It is also prone to technical errors that can falsely elevate or decrease the sedimentation rate. Because of its low specificity and sensitivity, the ESR is not recommended as a screening test to detect inflammatory conditions in asymptomatic individuals. C-reactive protein level = more predictable and reliable test to monitor inflammation

Acute lymphoblastic leukemia (ALL)

most common presentation of lymphoblastic leukemia/lymphoma Primarily a disease of childhood and adolescence, accounting for 25% of childhood cancers and up to 75% of childhood leukemia. Patients with B cell ALL typically present with fatigue (caused by anemia), fever (caused by neutropenia and infection), and mucocutaneous bleeding (caused by thrombocytopenia). - Enlargement of the spleen and liver may be seen In T cell ALL, there may be a large mass in the mediastinum leading to compromise of regional anatomic structures. Similar to B-ALL, T-ALL may present with anemia, thrombocytopenia, organomegaly, and bone pain, although the degree of leukopenia is often less severe.

automated cell counting

most rely on only two basic principles of operation: electronic impedance (resistance) and optical scatter. Electronic impedance, or low-voltage direct current (DC) resistance, was developed by Coulter, it is the the most common methodology used. Optical scatter, using both laser and nonlaser light, is often employed in today's hematology instrumentation. Automated Reticulocyte Counts - evaluate reticulocytes using optical scatter or fluorescence after the RBCs are treated with fluorescent dyes or nucleic acid stains to stain residual RNA in the reticulocytes.

Plasma cell dyscrasias

neoplasms of *immunoglobulin secreting cells* (plasma cells) that *mostly arise in bone* (general) and result in the secretion of a single immunoglobin produced by the tumor plasma cells - they are malignant disorders of terminally differentiated B cells Most common example is multiple myeloma which is generally a disease of the elderly. MM is a bone marrow-based disease with extramedullary extension to bone or soft tissue. Characterized by hypercalcemia, renal failure, anemia, and osteolytic lesions. - rouleaux formation of the red blood cells occurs secondary to the presence of the paraprotein

Manual Cell Counts

performed using a hemacytometer, or counting chamber, and manual dilutions made with calibrated, automated pipettes and diluents Hemacytometer (counting chamber) = must meet the specifications of the National Bureau of Standards Total count = [cells counted / dilution factor] / [area (mm2) x depth (0.1)] Or Total count = [cells counted x dilution factor x 10] / [area (mm2)] WBC count Whole blood anticoagulated with EDTA or blood from a skin puncture is diluted with 1% buffered ammonium oxalate or a weak acid solution. The diluting fluid lyses the nonnucleated RBCs in the specimen to prevent their interference in the count. The typical dilution of blood for the WBC count is 1:20. Manual RBC counts are rarely performed because of the inaccuracy of the count and questionable necessity. Reticulocyte count Whole blood, anticoagulated with EDTA, is stained with a supravital stain, such as new methylene blue. Any nonnucleated RBC that contains two or more particles of blue-stained granulofilamentous material after new methylene blue staining is defined as a reticulocyte. Reticulocytes (%) = number of reticulocytes x 100 / 1000 (RBCs counted)

Type 2 von Willebrand disease

produces normal amounts of abnormal vWF VWF levels may be normal or moderately decreased, but VWF function is consistently reduced. Laboratory testing is essential to identifying and confirming type 2 subtypes because the diagnosis affects treatment choices. Subtype 2A = arises from well-characterized autosomal dominant point mutations in the A2 and D1 structural domains of the VWF molecule. These mutations render VWF susceptible to increased proteolysis by ADAMTS13, which leads to a predominance of small- molecular-weight plasma multimers. Patients with subtype 2A VWD have normal or slightly reduced VWF antigen levels as measured by immunoassay, with moderate to markedly reduced VWF activity as a result of the loss of the high-molecular-weight and intermediate-molecular-weight multimers essential for platelet adhesion Subtype 2B = mutations within the A1 domain raise the affinity of VWF for platelet GPIb/IX/V, its customary binding site; these are hence "gain-of-function" mutations. HMW-VWF multimers spontaneously bind resting platelets. Subtype 2B VWD may be confirmed using a specially designed reduced-concentration, ristocetin-induced (RIPA) platelet agglutination assay. Subtype 2M = qualitative VWF variant that possesses poor platelet receptor binding despite generating a normal multimeric distribution pattern in electrophoresis. The distinguishing feature of subtype 2M that separates it from type 1 is a discrepancy between the concentration of VWF:Ag and its activity as measured using the VWF ristocetin cofactor assay described later, despite a normal multimeric pattern. Subtype 2N = An autosomal VWF gene missense mutation in the D9 domain impairs the protein's factor VIII binding site function. The disorder is also known as autosomal hemophilia because its clinical symptoms are indistinguishable from the symptoms of hemophilia except that it affects both men and women. In boys or men, subtype 2N is suspected when a male patient misdiagnosed as a hemophilia A sufferer fails to respond to factor VIII concentrate therapy. The diagnosis of VWD subtype 2N is confirmed using a molecular assay that detects the specific mutation responsible for the abnormal FVIII binding function.

Wiskott-Aldrich syndrome

rare X-linked disease caused by one of more than 400 mutations in the WAS gene => decreased levels of WASp protein WASp is important in cytoskeletal remodeling and nuclear transcription in hematopoietic cells. T cells are decreased; B cells, T cells and NK cells, neutrophils and monocytes are dysfunctional which leads to bacterial, viral and fungal infections. - There is a risk of bleeding due to thrombocytopenia and small abnormal platelets

May-Hegglin anomaly

rare, autosomal dominant disorder characterized by variable thrombocytopenia, giant platelets, and large Döhle body-like inclusions in neutrophils, eosinophils, basophils, and monocytes. Caused by a mutation in the MYH9 gene on chromosome 22q12-13. There is an issue in the production of myosin heavy chain type IIA, which affects megakaryocyte maturation and platelet fragmentation when shedding from megakaryocytes. The basophilic Döhle body-like leukocyte inclusions in May-Hegglin anomaly are composed of precipitated myosin heavy chains. - True Döhle bodies consist of lamellar rows of rough endoplasmic reticulum. Most individuals with May Hegglin anomaly are asymptomatic, but a few have mild bleeding tendencies related to the degree of thrombocytopenia.

Lymphoid neoplasms

result from B and T lymphocyte precursor cells or their descendent cell types Example include lymphoma, myeloma, and lymphoid leukemia. They arise from the malignant transformation of normal lymphoid cells at various stages of differentiation.

Unstable hemoglobin disorder

result from genetic mutations to globin genes creating hemoglobin products that precipitate in vivo, producing Heinz bodies and causing a hemolytic anemia. - Most unstable hemoglobin variants have no clinical significance, although the majority have increased oxygen affinity. The instability of the hemoglobin molecule may be due to (1) substitution of a charged for an uncharged amino acid in the interior of the molecule (2) substitution of a polar for a nonpolar amino acid in the hydrophobic heme pocket (3) substitution of an amino acid in the A and B chains at the intersubunit contact points (4) replacement of an amino acid with proline in the a helix section of a chain (5) deletion or elongation of the primary structure Unstable hemoglobin disorder is usually detected in early childhood in patients with hemolytic anemia accompanied by jaundice and splenomegaly. Fever or ingestion of an oxidant exacerbates the hemolysis. Hemoglobin precipitates in RBCs as Heinz bodies.Heinz bodies can be trapped mechanically in the splenic sieve, which shortens RBC survival. Most common is Hb Köln. Others include Hb Hammersmith, Hb Zurich, and Hb Gun Hill. Laboratory Diagnosis - slight hypochromia - prominent basophilic stippling (excessive clumping of ribosomes) - hemoglobin levels range from 7 to 12 g/dL - Heinz bodies - excrete dark urine that contains dipyrrole Isopropanol precipitation test = unstable hemoglobins are present, rapid precipitation occurs in 5 minutes and heavy flocculation occurs after 20 minutes

Thrombotic Thrombocytopenic Purpura (TTP)

results from impaired funciton of ADAMTS13, a von-Willebrand factor cleaving protease (disintegrin). Thus, large vWF multimers build and cause a prothrombotic state resulting in microvascular thrombosis. - ADAMTS13 regulates the size of circulating von Willebrand factor (VWF) by cleaving ultralong VWF multimers (ULVWF) into shorter segments that have less hemostatic potential. Thrombocytopenia and hemolytic anemia with shistocyte formation occur as erythrocytes are sheared by platelet rich thrombi. Characterized by the abrupt appearance of microangiopathic hemolytic anemia, severe thrombocytopenia, and markedly elevated serum LD activity. Typical initial laboratory findings in all types of TTP include a hemoglobin level of 8 to 10 g/dL, a platelet count of 10 to 30 x 10^9/L, and schistocytes on the peripheral blood film.

Essential (primary) thrombocythemia (ET)

the body produces too many platelets for unknown reasons. This can cause abnormal blood clotting or bleeding. - Platelet count usually more than 600 x 10^9/L and sometimes even more than 1000 x 10^9/L Three mutations in particular, JAK2, MPL, and CALR, are considered driver mutations for ET. WHO group requires meeting all four major criteria or the first three major criteria and one minor criteria for the diagnosis of ET. Major diagnostic criteria include: 1. megakaryocyte proliferation with large and mature morphology, little to no granulocyte or erythroid proliferation 2. rarely, minor (grade 1)increase in reticulin fibers 3. must not meet any criteria for BCR-ABL1-positive CML, PV, PMF, MDS or other myeloid neoplasms 4. must demonstrate JAK2 V617F, CALR, or MPL mutations Minor criteria include presence of a clonal marker or absence of evidence of reactive thrombocytosis.

Absolute Erythrocytosis

the form where red cell mass is raised and hematocrit is elevated above prescribed limits. Causes = primarily when there is an intrinsic problem in the bone marrow; secondary when an event outside the bone marrow drives erythropoiesis. Example: renal artery stenosis which produces an area of hypoxia that increases EPO production

iron deficiency anemia

the most common cause of microcytic anemia Iron level is insufficient for maintaining normal erythropoiesis. Characterized by abnormal iron studies, and in the early stages of the disease the only sign is reduced iron stores

Acute Myeloid Leukemia (AML)

the most common form of leukemia in adults; develops when the bone marrow produces too many myeloblasts Most patients with AML have a total white blood cell (WBC) count between 5 and 30 x 10^9/L. Myeloblasts are present in the peripheral blood in 90% of patients. Anemia, thrombocytopenia, and neutropenia give rise to the clinical findings of pallor, fatigue, fever, bruising, and bleeding. In addition, disseminated intravascular coagulation and other bleeding abnormalities can be significant. Common abnormalities in laboratory test results include hyperuricemia (caused by increased cellular turnover), hyperphosphatemia (due to cell lysis), and hypocalcemia (the latter two are also involved in progressive bone destruction). Hypokalemia is also common at presentation.

Radiofrequency

used in conjunction with DC impedance The cell interior density is proportional to pulse height or change in the RF signal. Conductivity, as measured by this high-frequency electromagnetic probe, is attenuated by nucleus-to-cytoplasm ratio, nuclear density, and cytoplasmic granulation.

Esterases

used to differentiate myeloblasts and neutrophilic granulocytes from cells of monocytic origin Esterase stains can be used to distinguish acute leukemias that are granulocytic from leukemias that are primarily of monocytic origin. - When naphthol AS-D chloroacetate is used as a substrate, the reaction is positive in the granulocytic cells and negative to weak in the monocytic cells. - Chloroacetate esterase is present in the primary granules of neutrophils. Leukemic myeloblasts generally show a positive reaction. Auer rods also show positivity. a-Naphthyl acetate, in contrast to naphthol AS-D chloroacetate, reveals strong esterase activity in monocytes that can be inhibited with the addition of sodium fluoride. - Granulocytes and lymphoid cells generally show a negative result on nonspecific esterase staining

Hematocrit (Hct)

• Females 37-47% • Males 42-52% Volume percentage of red blood cells in blood. Decreased = insufficient supply of healthy red blood cells (anemia), large number of white blood cells due to long-term illness, infection or a white blood cell disorder such as leukemia or lymphoma, vitamin or mineral deficiencies Increased = Dehydration, carbon monoxide poisoning, congenital heart disease, bone marrow disease (polycythemia vera), erythrocytosis.

Spurious results

A common limitation of impedance methods is an instrument's inability to distinguish cells reliably from other particles or cell fragments of the same volume. Cell fragments may be counted as platelets in specimens from chemotherapy-treated patients with increased WBC fragility Schistocytes or small RBCs may interfere with the platelet count. Larger platelet clumps may be counted as WBCs, which results in a falsely decreased platelet count and potentially increases the WBC count. Micromegakaryocytes may be counted as nucleated RBCs or WBCs. RBCs containing variant hemoglobins such as Hb S or Hb C are often resistant to lysis, and the unlysed cells can be falsely counted as nucleated RBCs or WBCs and interfere in the hemoglobin reaction. Limitations resulting from inherent specimen problems include those related to the presence of cold agglutinins, icterus, and lipemia. Cold agglutinins manifest as a classic pattern of increased MCV (often greater than 130 fL), markedly decreased RBC count, and increased MCHC (often greater than 40 g/dL). Careful examination of the histograms or cytograms from the instruments may yield clues to this abnormality. Icterus and lipemia directly affect hemoglobin measurements and related indices. Specific problems with older specimens include increased WBC fragility, swelling and possible lysis of RBCs, and the deterioration of platelets.

Pseudothrombocytopenia

A condition in which the platelet count is falsely decreased Some patients' blood undergoes an in vitro phenomenon called platelet "satellitosis" when anticoagulated with EDTA as the platelets surround or adhere to neutrophils which causes low values when counted with automated methods. - eliminated by recollecting specimens in sodium citrate tubes (light blue top) and ensuring that the proper ratio of nine parts blood to one part anticoagulant is observed (a properly filled tube) platelet clumping or the presence of giant platelets can also cause this

Glucose-6-phosphate dehydrogenase (G6PD)

An enzyme that aids in the proper functioning of red blood cells; its deficiency, a genetic condition, leads to hemolytic anemia. G6PD provides the only means of generating NADPH for glutathione reduction, and in its absence erythrocytes are particularly vulnerable to oxidative damage. With normal G6PD activity, the HMP detoxifies oxidative compounds and safeguards hemoglobin, sulfhydryl-containing enzymes, and membrane thiols, allowing RBCs to safely carry O2. However, in G6PD deficiency, the most common inherited RBC enzyme deficiency worldwide, the ability to detoxify is hampered, resulting in hereditary nonspherocytic anemia.

Body Fluid Cell Counts

Automated Methods Must count on dedicated body fluid (BF) mode which optimizes technologies already present to account for the different cellular composition of body fluids. Some features of the BF mode include extended counting for low cell counts, sample dilution not required, and flagging capabilities all of which result in improvement of accuracy and turnaround times compared with manual methods. Manual Methods hemacytometer; but this method has several limitations as it is time consuming and labor intensive, has high interobserver variability and has poor reproducibility. Cell counts are performed with undiluted fluid if the fluid is clear. If the fluid is hazy or bloody, appropriate dilutions should be made to permit accurate counts of RBCs and WBC or TNC. The diluting fluid for RBCs is isotonic saline. Diluting fluids for WBCs include glacial acetic acid to lyse the RBCs, or Türk solution, which contains glacial acetic acid and methylene blue to stain the nuclei of the WBCs. Acetic acid cannot be used for synovial fluids because synovial fluid contains hyaluronic acid, which coagulates in acetic acid. A small amount of hyaluronidase powder or one drop of 0.05% hyaluronidase in phosphate buffer per milliliter of fluid should be added to the synovial fluid sample to liquefy it before performing manual cell counts or automated cell counts (on hematology analyzer) or preparing cytocentrifuge slides. Total count = cells counted x dilution factor / area (mm2) x depth (0.1)

Hemoglobins With Increased Oxygen Affinity

Autosomal dominant pattern of inheritance and most individuals have equal volumes of Hb A and the abnormal variant. - Exceptions to this are compound heterozygotes for Hb Abruzzo and B -thalassemia and for Hb Crete and B-thalassemia (>85% abnormal Hgb) These hemoglobins fail to release oxygen on demand, and hypoxia results. Kidneys sense the hypoxia and respond by increasing the release of erythropoietin, which leads to a compensatory erythrocytosis. They do not precipitate in vivo to produce hemolysis and there is no abnormal RBC morphology. Erythrocytosis is usually detected during routine examination because the patient generally has a high RBC count, hemoglobin, and hematocrit. The WBC count, platelet count, and peripheral blood film findings are generally normal. An abnormal band that separates from the A band is present on alkaline electrophoresis in some variants; however, if a band is not found, the diagnosis of increased oxygen affinity cannot be ruled out. In some cases the abnormal hemoglobin can be separated using citrate agar electrophoresis (pH 6.0). Measurement of oxygen affinity is required for definitive diagnosis.

Electronic Impedance

Based on the detection and measurement of changes in electrical resistance produced by cells as they traverse a small aperture. Cells suspended in an electrically conductive diluent such as saline are pulled through an aperture (orifice) in a glass tube. Electrical resistance between the two electrodes, or impedance in the current, occurs as the cells pass through the sensing aperture, causing voltage pulses that are measurable. The number of pulses is proportional to the number of cells counted. The height of the voltage pulse is directly proportional to the volume of the cell, which allows discrimination and counting of cells of specific volumes through the use of threshold circuits.

Flow cytometry Paroxysmal nocturnal hemoglobinuria (PNH)

Before the development of the flow cytometric assays, PNH diagnosis was based on detection of increased susceptibility of RBCs to lysis by the Ham or sucrose hemolysis tests, both of which showed inconsistent sensitivity. Flow cytometry significantly improved the sensitivity and specificity of PNH testing. The decreased expression of glycosylphosphatidylinositol-anchored proteins on RBCs, granulocytes, and monocytes of PNH patients can be measured by flow cytometry. The loss of these proteins is diagnostic of PNH and correlates with clinical symptoms. Typically, several markers are investigated simultaneously including CD59, CD24, CD14 and aerolysin.

Babies

By the 1st month of embryonic life primitive erythroblasts are the first blood cells, they are formed in the mesenchyme of the yolk sac. By the 6th week of embryonic life, the liver becomes the primary hematopoietic organ for making definitive erythroblasts, which turn into non nucleated RBCs By mid fetal life, the spleen and lymph nodes begin limited role as a secondary lymphoid organ. In the last half of fetal life, bone marrow hematopoiesis begins and the liver slowly stops hematopoiesis by last trimester. Shortly after birth, the liver stops hematopoiesis completely and the bone marrow hematopoiesis is the primary method of production for erythrocytes, granulocytes, monocytes, platelets, and B lymphocytes.

BCR-ABL

CML A common qualitative mutation is a translocation that results in a chimeric fusion gene, such as the t(9;22) translocation forming the BCR-ABL1 fusion gene in CML and in some cases of acute lymphoblastic leukemia. - constitutive and unregulated tyrosine kinase activity Philadelphia chromosome, t(9;22);BCR-ABL1, is a hallmark of chronic myeloid leukemia but also can occur in de novo pediatric and adult B-LL. Oncogene - fusion protein Tyrosine kinase, t(9;22), signal transducer CML and ALL (poor) Tx: imatinib

Hypoproliferative anemia

Defined as production inadequate number of erythrocytes to maintain homeostasis. Characterized by decreased reticulocyte count. Can be caused by nutritional deficiencies, chronic kidney disease or inflammatory disease.

Bone marrow hematopoiesis

Divided into 3 major cell types 1. Stem cells or pluripotential / multipotential cells retain ability to differentiate into any cell line. a) colony forming units - spleen (CFU-S) = can be either secondary multipotential stem cells which turn into primitive B/T lymphocytes or multipotential stem cells which give rise to non-lymphocytes 2. Progenitor (committed) cells or unipotential cells include BFU-E, CFU-E, CFU-MEG, and CFU-GM. 3. Precursor cells. Each type of unipotential cell will mature into blast form (ex: myeloblast, erythroblast, etc)

Glanzmann thrombasthenia (GT)

Due to a genetic GPIIb/IIIa deficiency; platelet aggregation is impaired - Genetic mutations are distributed widely over the ITGA2B and ITGB3 genes present on chromosome 17, which code for GP IIb/IIIa. Inherited as an autosomal recessive disorder and is seen most frequently in populations with a high degree of consanguinity. Heterozygotes are clinically normal, whereas homozygotes have serious bleeding problems. This rare disorder manifests itself clinically in the neonatal period or infancy, occasionally with bleeding after circumcision and frequently with epistaxis and gingival bleeding. Hemorrhagic manifestations include petechiae, purpura, menorrhagia, gastrointestinal bleeding, and hematuria. Historically, patients with GT have been designated as type 1 or type 2. Individuals with type 2 disease have more GP IIb/IIIa complexes (10% to 20% of normal) than those with type 1 disease (0% to 5% of normal), although there is considerable variability within each subdivision. Additionally, patients with type 2 disease are less affected by abnormal clot retraction and fibrinogen binding. Laboratory features - normal platelet count - normal platelet morphology, - lack of platelet aggregation in response to all platelet activating agents (ADP, collagen, thrombin, and epinephrine) - GT platelets are activated by thrombin to a lesser degree than normal platelets Treatment Treatment of bleeding episodes requires the transfusion of normal platelets. However, defective platelets may interfere with the normal transfused platelets, and it may be necessary to infuse more donor platelets than expected to control bleeding. It is recommended to use pre-storage, leukocyte-reduced apheresis platelets or HLA-matched donor platelets to prevent HLA immunization. Patients with GT should avoid anticoagulants and antiplatelet agents such as aspirin and nonsteroidal antiinflammatory drugs

Acute hemorrhage anemia

Due to abrupt drop in RBCs, most often by hemolysis or acute hemorrhage

Heparin-induced thrombocytopenia (HIT)

Effect of unfractionated heparin therapy in which IgG antibodies to heparin-platelet factor 4 complex develop. The antibody and target antigen complex bind platelet Fc receptors and activate platelets causing thrombocytopenia. Associated venous and arterial thrombosis can lead to amputation and death. C-serotonin release assay (SRA), demonstrates the ability of the anti-H:PF4 HIT antibody to activate platelets in the presence of heparin.

Electrophoresis and Hemoglobin

Electrophoresis is based on the separation of hemoglobin molecules in an electric field primarily as a result of differences in total molecular charge. In alkaline electrophoresis, hemoglobin molecules assume a negative charge and migrate toward the anode (positive pole). Performed on agarose medium. Because some hemoglobins have the same charge and therefore the same electrophoretic mobility patterns, hemoglobins that exhibit an abnormal electrophoretic pattern at an alkaline pH may be subjected to electrophoresis at an acid pH for definitive separation. In an acid pH some hemoglobins assume a negative charge and migrate toward the anode, whereas others are positively charged and migrate toward the cathode (negative pole). For example, Hb S migrates with Hb D and Hb G on alkaline electrophoresis but separates from Hb D and Hb G on acid electrophoresis. Hb D and Hb G are further differentiated from Hb S in that they produce a negative result on the hemoglobin solubility test. Similarly, Hb C migrates with Hb E and Hb O on alkaline electrophoresis but separates on acid electrophoresis.

Prussian blue

Fe7(CN)18 The gold standard for assessment of body iron stores. Staining is conducted routinely when bone marrow or liver biopsies are collected for other purposes. Although ferric iron reacts with the reagent, ferritin is not typically detected, likely because of the intact protein cage. In high concentration, it can appear as a diffuse cytoplasmic blueness. Hemosiderin stains readily, forming distinct dark blue granules

Heinz bodies

G6PD deficiency Composed of denatured precipitated Hb, stains with Supravital stains such as Brillians cresol blue or new methylene blue. They are not seen with Wright stain. Their presence can also indicate thalassemia (HbH) or unstable Hb

Production

Hematopoietic tissues is where blood cell production or regulation occurs. This includes fetal hematopoiesis, spleen, lymphatic tissue, and the bone marrow.

Hemoglobins With Decreased Oxygen Affinity

Hemoglobins with decreased oxygen affinity quickly release oxygen to the tissues, which results in normal to decreased hemoglobin concentration, slight anemia, and cyanosis (blue coloration). Best known is Hgb Kansas, which has an amino acid substitution of asparagine by threonine at position 102 of the b chain. These hemoglobins may be present when cyanosis and a normal arterial oxygen tension coexist, and most may be detected by starch gel electrophoresis, HPLC, or DNA-based globin gene analysis.

Hemaglobinopathies

Group of genetic diseases that are the most common type. All result from genetic mutation of 1 or more genes that affect hemoglobin synthesis. This effect is either qualitatively or quantitively. In quality = hemoglobin synthesis occurs at a normal rate but Hb molecule has an altered amino acid sequence in globin chains, thus altering its structure and function. In quantity = it is a thalassemia and the structure is normal but at a decreased rate, forcing the body to make other Hb that are not affected by the mutation to compensate for the anemia (ex: sickle cell)

Thalassemia

Groups of inherited autosomal recessive hematologic disorders that cause anemia because of the decreased or absent synthesis of a globin chain. Imbalance of globin causes hemolysis and impairs erythropoiesis. Common in those of Mediterranean, African American, and Southeast Asian descent.

Hgb C

Lysine substituted for glutamic acid in 6th position of beta chain; almost exclusively in the African American population Hb C polymers form under high oxygen tension. The polymers in Hb C form a short, thick crystal within the RBCs and it crystalizes in the oxygenated state. Homozygous hemoglobin C disease (Hb CC) manifests as a milder disease compared with SCD. Mild splenomegaly and hemolysis may be present. In addition, vasoocclusive crises do not occur. Heterozygous hemoglobin C trait (Hb AC) is asymptomatic. Laboratory Diagnosis A mild to moderate, normochromic, normocytic anemia occurs in homozygous Hb C disease. Occasionally, some microcytosis and mild hypochromia may be present. There is a marked increase in the number of target cells and a slight to moderate increase in the number of reticulocytes (2% to 3%), and nucleated RBCs may be present in peripheral blood. Hexagonal crystals of Hb C form within RBCs and may be seen on the peripheral blood film Crystals are densely stained and vary in size and appear oblong with pyramid-shaped or pointed ends. These crystals may be seen on wet preparations by washing RBCs and resuspending them in a solution of sodium citrate or hypertonic saline.

Normocytic anemia

MCV 80-100 RBC must be examined previously to rule out dimorphic population. Some normocytic anemias develop as result of premature destruction and shortened survival of RBCs (hemolytic anemia) and they are characterized by increased reticulocyte counts. Other normocytic anemia are caused by decreased production of RBCs and are characterized by decreased reticulocyte counts.

macrocytic anemia

MCV > 100 with large RBCs Arise from conditions that result in megaloblastic or nonmegaloblastic red cell development in the bone marrow

Microcytic anemia

MCV less than 80 with small RBCs. Caused by condition that result in decreased hemoglobin synthesis. Heme synthesis is diminished in iron deficiency, iron sequestration (chronic inflammatory states), and defective protoporphyrin synthesis (sideroblastic anemia, lead poisoning). Globin chain synthesis is insufficient or defective in thalassemia and in Hb E disease.

Acquired hemolytic anemia

May be caused by an autoimmune response in which the immune system destroys its own cells. This is especially common in patients who already have an autoimmune disorder such as lupus. Medications can also be suspect, examples include : penicillin, quinine, methyldopa, and sulfonamides

Hereditary hemolytic anemia

May be caused by inherited enzyme deficiencies inside RBCs that then caused them to become fragile and subject to destruction. Common example of decreased enzymes causing this include pyruvate kinase or glucose-6-phosphate dehydrogenase Other examples of hemolytic anemia include sickle cell or thalassemia

MCH

Mean Cell Hemoglobin - average weight of hemoglobin in a RBC, expressed in picograms (pg), or 10^-12 g: Hb x 10 / RBC count (x 10^12 / L) = MCH Normal values = 26-32 Generally not considered in the classification of anemias

MCHC (chromic)

Mean cell hemoglobin concentration - average concentration of hemoglobin per red cell Hb (g/dl) x 100 / Hct = MCHC Normal values = 32-36 or normochromic

MCV (cytic)

Mean cell volume - average volume of RBC expressed in femtoliter (fL) or 10^-15 L. Hct x 10 / RBC count (x 10^12 / L) = MCV Normal values = 80-100 or normocytic

Benign leukocyte disorders

Not caused by clonal or neoplastic changes in hematopoietic precursor cells. These changes can be genetic or acquired and involve 1 or more lineages : neutrophil, lymphocyte, monocyte, eosinophil, and basophil. These changes also affect the number of circulating cells, morphology or both

Function

Of circulating lymphocytes, 75-85% are T while 10-15% are B A majority of oxygen is carried by hemoglobin (Hb) in the RBCs About 90% of CO2 is converted to bicarbonate and H+ ions.

Rule of three

Only for normocytic normochromic RBC. The value of hematocrit should be 3x the value of hemoglobin plus or minus 3.

Hbg S (Sickle cell)

Patients with SCD have inherited a sickle (S) gene from one parent and an S, C, or B-thalassemia gene from the other. Among patients with SCD, individuals who are homozygotes (Hb SS) have more severe disease than individuals who are compound heterozygotes for Hb S (Hb SC or Hb S-b-thal). Nonpolar (hydrophobic) valine is placed in the position that polar glutamic acid once held. Increased blood viscosity and sickle cell formation slow blood flow. In addition to a decrease in oxygen tension, there is a reduction in pH and an increase in 2,3-bisphosphoglycerate. - Reduced blood flow prolongs the exposure of Hb S-containing RBCs to a hypoxic environment, and the lower tissue pH decreases oxygen affinity, which further promotes sickling. The end result is occlusion of capillaries and arterioles by sickled RBCs and infarction of surrounding tissue. The peripheral blood film shows marked poikilocytosis and anisocytosis with normal RBCs, sickle cells, target cells, nucleated RBCs, along with a few spherocytes, basophilic stippling, Pappenheimer bodies, and Howell-Jolly bodies. - The presence of sickle cells and target cells is the hallmark of SCD. There is moderate to marked polychromasia with a reticulocyte count between 10% and 25%, corresponding with the hemolytic state and the resultant bone marrow response. Moderate leukocytosis is usually present with neutrophilia and a mild shift toward immature granulocytes. The leukocyte alkaline phosphatase score is not elevated when neutrophilia is caused by sickle cell crisis alone when no underlying infection is present. Thrombocytosis is usually present. The diagnosis of SCD is generally a two-step process begun by first demonstrating the insolubility of deoxygenated Hb S in solution followed by confirmation of its presence using hemoglobin electrophoresis, high-performance liquid chromatography (HPLC), or capillary electrophoresis.

Destruction

RBCs are filtered through the spleen which has pores that are 3 um in diameter. The average RBC is 7um and so they must squeeze through the pore. RBCs that are old or contain cytoplasmic inclusions do not have enough pliability to get through, and they are destroyed. The spleen acts as a reservoir for platelets and lymphocytes as up to 30% of circulating platelets are sequestered into the spleen by slow transit.

Acute leukemia

Rapid clinical proliferation in the bone marrow Specific causes are generally unknown, ex: toxins that lead to mutations, radiation and exposure to organic solvents (benzene). Mutations lead to leukemic stem cell that mitigate / proliferate and then sustain leukemia.

Alder-Reilly anomaly (AR)

Rare inherited disorder characterized by granulocytes (monocytes and lymphocytes less often) with large, darkly staining metachromatic cytoplasmic granules. The characteristic granulation, called Reilly bodies, is also found in the mucopolysaccharidoses (MPSs). The cytoplasmic granules contain partially digested mucopolysaccharides. Leukocyte function is not affected in AR. AR bodies in neutrophils may resemble heavy toxic granulation, however, Reilly bodies can also be present in monocytes and lymphocytes, whereas toxic granulation occurs only in neutrophils.

Myelodysplastic Syndrome (MDS)

Refractory anemia Characterized by persistent cytopenia (decreased mature blood cells). Mutation in TMPRSS6 gene causes iron refractory, iron deficiency anemia. One of a group of cancers in which immature blood cells in the bone marrow do not mature, so do not become healthy blood cells.

Kleihauer-Betke stain

Test for detecting fetal red blood cells in the maternal circulation. Blood films are immersed in an acid buffer, which causes adult hemoglobin (Hb A) to be eluted from the cells. The film is stained, and red blood cells that have fetal hemoglobin (Hb F) take up the stain.

Flow Cytometry Lymphoid Lineage

The B and T lymphocytes are derived from lymphoid progenitors that express CD34, TdT, and HLA-DR. The earliest B cell markers include CD19, cytoplasmic CD22, and cytoplasmic CD79. As B cell precursors mature, they acquire the CD10 antigen. The appearance of the mature B cell marker CD20 coincides with the decrease in the expression of CD10. Another specific immature B cell marker is the cytoplasmic m chain that eventually is transported to the surface and forms the B cell receptor. T cells express CD34 and TdT. The first markers associated with T cell lineage are CD2, CD7, and cytoplasmic CD3. CD2 and CD7 are also present in natural killer (NK) cells.

Thrombocytosis

an abnormal increase in the number of platelets in the circulating blood - Signal inflammation or trauma but convey modest intrinsic significance. Symptoms: Headache, Dizziness or lightheadedness, Chest pain, Weakness, Numbness or tingling of the hands and feet Reactive thrombocytosis = caused by an underlying medical problem, such as: Acute bleeding and blood loss, Cancer, Infections, Iron deficiency, Removal of your spleen, hemolytic anemia, Inflammatory disorders, such as rheumatoid arthritis, sarcoidosis or inflammatory bowel disease, and Surgery or other type of trauma

Monoclonal gammopathies

an uncontrolled proliferation of a single clone of plasma cells at the expense of other clones

Gaucher disease

The most common of the lysosomal lipid storage diseases. Autosomal recessive disorder caused by a defect or deficiency in the catabolic enzyme b- glucocerebrosidase, which is necessary for glycolipid metabolism. As a result, there is an accumulation of unmetabolized substrate sphingolipid glucocerebroside in macrophages throughout the body, including osteoclasts in bone and microglia in the brain. - At least 1 in 17 Ashkenazi Jews are carriers There are three types of Gaucher disease and type I is by far the most common. Neurologic symptoms are key factors to differentiate the subtypes. The phenotype is quite heterogeneous, with some patients being asymptomatic, whereas others experience a multitude of clinical problems. Bone marrow replacement by Gaucher cells contribute to anemia and thrombocytopenia, which are common findings in these patients. Gaucher cells are distinctive macrophages, single or in clusters, exhibiting abundant fibrillar blue-gray cytoplasm with a striated or wrinkled appearance (sometimes described as onion skin-like)

Nonmegaloblastic anemia

liver disease, alcoholism, reticulocytosis, metabolic disorders, drugs (5-FU, AZT, hydroxyurea) Large RBCs but this change is typically related to membrane changes caused by disruptions of the cholesterol to phospholipid ratio. These macrocytes are mostly round and erythroid precursors in the bone marrow do NOT show megaloblastic changes.

Decreased production of platelets

Thrombocytopenia caused by: - bone marrow disorder such as leukemia or an immune system problem - side effect of taking certain medications Thrombocytopenia signs and symptoms may include: Easy or excessive bruising (purpura) Superficial bleeding into the skin that appears as a rash of pinpoint-sized reddish-purple spots (petechiae), usually on the lower legs Prolonged bleeding from cuts Bleeding from your gums or nose Blood in urine or stools Unusually heavy menstrual flows Fatigue Enlarged spleen Factors that can decrease platelet production include: - Leukemia and other cancers - Some types of anemia - Viral infections, such as hepatitis C or HIV - Chemotherapy drugs and radiation therapy - Heavy alcohol consumption

hydrodynamic focusing

To be analyzed individually, cells must pass separately, one by one, through the illumination and detection system of a flow cytometer. This is accomplished by injecting a cell suspension into a stream of sheath fluid. This creates a central core of individually aligned cells surrounded by a sheath fluid. "a technique that enables users of flow cytometry cells to gauge the size of particles in a flow channel, whether they be blood cells, viruses or bacteria."

Type A NP

Type A (acute neuronopathic form) NP mostly affects Eastern European Jews. Type A presents in infancy and is associated with failure to thrive, lymphadenopathy, hepatosplenomegaly, vision problems, and rapid neurodegenerative decline that results in death, usually by 4 years of age. In type A, there is less than 5% of normal sphingomyelinase activity.

Flow cytometry

Used for analysis of cell lineage in acute leukemia or a detection of clonality in lymphoid populations but also makes it possible to discern abnormal populations in chronic myeloid neoplasms, quantitate minimal residual disease, and monitor immunodeficiency states. Prolonged transport or transport under inappropriate conditions may render a specimen unsuitable for analysis. Peripheral blood and bone marrow specimens should be processed within 24 to 48 hours from the time of collection, dependent on the anticoagulant. Certain specimens, such as body cavity fluids or samples from neoplasms with a high proliferative activity, may require even more rapid processing. To obtain a pure population of nucleated cells, red blood cells (RBCs) are lysed. Cellularity and viability of a specimen are routinely assessed before a sample is stained. Cell count can be obtained using automated cell counters or flow cytometry. A specimen is stained with propidium iodide or 7-amino actinomycin to test viability. The development of monoclonal antibodies made flow cytometry widespread.

Myeloperoxidase (MPO)

Used for differentiating the blasts of AML from those of ALL. An enzyme found in the primary granules of granulocytic cells (neutrophils, eosinophils, and, to a certain extent, monocytes). Lymphocytes do not exhibit MPO activity. In many cases of the AMLs (without maturation, with maturation, and promyelocytic leukemia), it has been found that more than 80% of the blasts show MPO activity. Auer rods found in leukemic blasts and promyelocytes test strongly MPO positive. In contrast, lymphoblasts in ALL and lymphoid cells are MPO negative. It is important that the reaction only in the blast cells be used as the determining factor for the differentiation of acute leukemias.

Sudan black B (SBB)

Used for the differentiation of AML from ALL. SBB stains cellular lipids. Granulocytes (neutrophils) show a positive reaction to SBB from the myeloblast through the maturation series. The staining becomes more intense as the cell matures as a result of the increase in the numbers of primary and secondary granules. Monocytic cells can demonstrate negative to weakly positive staining due to various changes that occur during differentiation. Lymphoid cells generally do not stain.

Flow cytometry leukemia

Uses: Distinguishing acute lymphoblastic leukemia (ALL) from acute myeloid leukemia (AML). Immunologic subtyping of ALL Recognizing AML with minimal morphologic or cytochemical evidence of differentiation Almost invariably, blasts are characterized by a low- density expression of CD45 antigen. In normal bone marrow a blast gate includes a relatively low number of cells showing the immature myeloid immunophenotype. - In acute myeloid and lymphoblastic leukemias, this region becomes densely populated by immature cells, which reflects the increased number of blasts seen in a bone marrow. The location of the immature population on the CD45/SS displays depends on the subtype of acute myeloid leukemia (AML).

Flow cytometry lymphoma

Uses: Distinguishing reactive lymphocytes and lymphoid hyperplasia from malignant lymphoma Distinguishing between malignant lymphoma and acute leukemia Similar to myeloid neoplasms, a diagnosis of lymphoid malignancies relies on the expression of lineage-associated markers corresponding to specific stages of lymphoid development. The sentinel feature of mature B and T cells is the presence of surface receptor complexes. A neoplastic lymphoid population is characterized by an expression of a monoclonal B or T cell receptor. In most cases a clonality confirms the malignant nature of lymphoid proliferation. In lymphoblastic (precursor-derived) neoplasms, an expansion of a population with homogeneous marker expression, rather than clonality, is diagnostic of malignancy.

Primary myelofibrosis (PMF)

a rare bone marrow disorder that is characterized by abnormalities in blood cell production (hematopoiesis) and scarring (formation of fibrous tissue) within the bone marrow PMF can form de novo or as an evolutionary consequence of PV or ET. The peripheral blood film exhibits immature granulocytes and normoblasts, dacryocytes (teardrop-shaped RBCs), and other bizarre RBC shapes. Caused by mutation in one of three driver genes: JAK2 (V617F), CALR, or MPL that affect the JAK-STAT pathway. Symptoms result from anemia, myeloproliferation, or splenomegaly and include fatigue, weakness, shortness of breath, palpitations, loss of appetite, weight loss, night sweats, pruritis, pain in the extremities and bones, bleeding, and discomfort or pain in the left upper quadrant associated with splenomegaly. In the early stages, anemia, leukocytosis with a left shift, and thrombocytosis are identified that are consistent with a MPN. However, as the fibrosis develops in the bone marrow, blood cell counts fall and pancytopenia eventually develops, along with leukoerythroblastosis, anisocytosis, and poikilocytosis.

Von Willebrand Disease

bleeding disorder caused by a deficiency of von Willebrand factor, a "sticky" protein that lines blood vessels and reacts with platelets to form a plug that leads to clot formation The most prevalent inherited mucocutaneous bleeding disorder. Any one of dozens of germline mutations may cause VWD as these mutations produce quantitative (type 1) or qualitative (functional, type 2) VWF abnormalities. - Both quantitative and functional abnormalities lead to decreased platelet adhesion to injured vessel walls, impairing primary hemostasis. VWD inheritance is autosomal dominant and affects both sexes. VWF is synthesized in the endoplasmic reticulum of endothelial cells and stored in their cytoplasmic Weibel-Palade bodies. It is also synthesized in megakaryocytes and stored in the a-granules of platelets. Weibel-Palade bodies and a-granules release VWF in response to a variety of hemostatic and inflammatory stimuli. Structural (qualitative) or quantitative VWF abnormalities reduce platelet adhesion, which leads to mucocutaneous hemorrhage of varying severity. Definitive diagnosis of VWD depends on the combination of a personal and family history of mucocutaneous bleeding and the laboratory demonstration of decreased VWF concentration or activity (function). - complete blood count - PT - PTT - quantitative VWF:Ag assay Corrected by the addition of normal plasma or cryoprecipitate.

Sideroblastic anemia

defect in mitochondrial heme synthesis producing ringed sideroblasts Caused by deficient protoporphyrin synthesis that can be either congenital by X linked or autosomal mutations or acquired by myelodysplastic syndrome. additional causes include Lead poisoning and Zinc overdose.

Relative Erythrocytosis

dehydration An increase in the RBC count without an increase in total RBC mass. Usually caused by loss of plasma volume in the resultant hemoconcentration as seen in diarrhea or vomiting that caused dehydration.

Idiopathic Thrombocytopenic Purpura (ITP)

disorder in which a deficiency of platelets results in abnormal blood clotting, marked by tiny purple bruises (purpura) that form under the skin Due to autoimmune antibodies to the VWF-cleaving protease ADAMTS13 causing a severe functional deficiency. Secondary TTP is associated with stem cell transplantation, disseminated cancer, pregnancy, and use of certain drugs. Most patients will respond favorably to plasma exchange therapy due to the removal of the offending ADAMTS13 autoantibody and infusion of replacement ADAMTS13 enzyme from donor plasma.

Waldenström's Macroglobulinemia

low-grade lymphoplasmacytic lymphoma associated with aberrant secretion of IgM. High levels of IgM can result in a hyperviscosity syndrome, requiring emergent plasmapheresis to alleviate symptoms. A distinct somatic mutation in the myeloid differentiation factor 88 (MYD88) gene, a member of the Toll-like receptor pathway, is found in over 90% of patients with WM and is a molecular marker for the disease and can differentiate it from other lymphomas that morphologically exhibit plasmacytic differentiation. Heavy chain disease is also a lymphoplasmacytic disorder. Symptoms are dependent on the heavy chain involved, but, as in MM, the paraprotein may cause significant damage to organs such as the kidney or liver.

Optical Scatter

flow cytometers A hydrodynamically focused sample stream is directed through a quartz flow cell past a focused light source. Laser light, termed monochromatic light because it is emitted at a single wavelength, differs from brightfield light in its intensity, its coherence (i.e., it travels in phase), and its low divergence or spread. - These characteristics allow for the detection of interference in the laser beam and enable enumeration and differentiation of cell types. As the cells pass through the sensing zone and interrupt the beam, light is scattered in all directions. Light scatter results from the interaction between the processes of absorption, diffraction (bending around corners or the surface of a cell), refraction (bending because of a change in speed), and reflection (backward scatter of rays caused by an obstruction). Forward-angle light scatter (0 degrees) = cell volume Orthogonal light scatter (90 degrees), or side scatter = inside the cell and correlates with degree of internal complexity Forward low-angle scatter (2 to 3 degrees) and forward high-angle scatter (5 to 15 degrees) = cell volume and refractive index or with internal complexity

Bernard-Soulier (Giant Platelet) Syndrome (BSS)

genetic GPIB deficiency; platelet ADHESION impaired, blood smear shows thrombocytopenia with enlarged platelets. enlarged because they are slightly more immature A rare disorder of platelet adhesion that usually manifests in infancy or childhood with hemorrhage characteristic of defective platelet function: ecchymoses, epistaxis, and gingival bleeding. BSS is inherited as an autosomal recessive disorder in which the GP Ib/IX/V complex is missing from the platelet surface or exhibits abnormal function. Inability to bind to VWF accounts for the inability of platelets to adhere to exposed subendothelium and the resultant bleeding characteristic of this disorder Heterozygotes who have about 50% of normal levels of GP Ib, GP V, and GP IX have normal or near-normal platelet function. Homozygotes have enlarged platelets, thrombocytopenia, and usually decreased platelet survival, which lead to a moderate to severe bleeding disorder. Laboratory features BSS platelets have normal aggregation responses to ADP, epinephrine, collagen, and arachidonic acid but do not respond to ristocetin and have diminished response to thrombin. The lack of response to ristocetin is due to the lack of GP Ib/IX/V complexes and the inability of BSS platelets to bind VWF. Treatment Antiplatelet therapy should be avoided. Platelet transfusions are the therapy of choice, but patients invariably develop alloantibodies, limiting further platelet transfusions. BSS patients tend to do better when apheresis platelets are used for transfusion because this limits the number of donors to which the patient is exposed and the rate of alloimmunization tends to be lower.

Hb C-Harlem (Hb C-Georgetown)

has a double substitution on the B chain. The substitution of valine for glutamic acid at position 6 of the B chain is identical to the Hb S substitution, and the substitution at position 73 of aspartic acid for asparagine is the same as the Hb Korle Bu mutation. It got its name as the abnormal hemoglobin migrates with Hb C on alkaline hemoglobin electrophoresis. Patients heterozygous for this anomaly are asymptomatic, but patients with compound heterozygosity for Hb S and Hb C-Harlem have crises similar to those with Hb SS disease. On alkaline hemoglobin electrophoresis, Hb C-Harlem migrates in the C position. Citrate agar electrophoresis at pH 6.2, however, shows migration of Hb C-Harlem in the S position.

Erythrocytosis

increase in the number of red blood cells making the blood thicker than usual, leading to an increased risk of blood clots and other complications

Quantitative Hemoglobinopathies

involve imbalance in the number of globin chains Quantitative globin defects result in thalassemias.