Lab Heme/Coagulation Flashcards
Within the general category of hemostatic disorders, what are some vascular causes of bleeding?
Hereditary hemorrhagic telangiectasia (Osler-Weber-Rendu syndrome). Ehlers-Danlos syndrome. Scurvy.
In the general category of hemorrhagic disorders, what substances can be specifically affected in primary, secondary, and tertiary hemostatis stages?
Primary hemostasis: platelets and vWF. Secondary hemostasis: clotting factors (deficiency, dysfunction, or inhibition). Tertiary hemostasis: factor XIII (failure to normally crosslink fibrin forming polymers of fibrin and excessive fibrinolysis).
Is the ristocetin-induced platelet aggregation normal or abnormal in Bernard-Soulier syndrome?
Abnormal. There is a deficiency of the platelet GPIb, which is needed to crosslink the platelets via vWF. Platelet aggregometry with ADP, collagen, and epinephrin is normal, though.
Is the ristocetin-induced platelet aggregation normal or abnormal in Glanzmann thrombasthemia?
Normal. There is a deficiency of the platelet GPIIb/IIIa, which is needed to crosslink the platelets via fibrinogen. Platelet aggregometry with ADP, collagen, and epinephrin is abnormal, though.
What factor is deficient in hemophilia A, B, and C?
Hemophilia A: factor VIII. Hemophilia B: factor IX. Hemophilia C: factor XI.
Elevations in PTT with normal PT and TT indicate deficiency, dysfunction, or inhibiton of what factors? What about elevation of PT with normal PTT and TT? What about elevation of PT and PTT with normal TT?
Elevations in PTT with normal PT and TT - factors VIII, IX, XI, or XII. Elevation of PT with normal PTT and TT - factor VII. Elevation of PT and PTT with normal TT - factors II, V, or X.
How is severity of hemophilia A (factor VIII deficiency) classified?
Severe (5% activity). 70% of cases are severe, 15% are moderate, and 15% are mild.
__% of patients with hemophilia A develop a factor VIII inhibitor with factor VIII replacement therapy.
25% of patients with hemophilia A develop a factor VIII inhibitor with factor VIII replacement therapy.
Hemoglobin makes up approximately (#)% of the cytoplasmic protein in a RBC. Hemoglobin is composed of (#) alpha-globins and (#) non-alpha-globins that are conjugated to (#) heme molecules that each contain (#) iron atom. Each hemoglobin molecule is capable of reversibly binding and transporting (#) oxygen molecues.
Hemoglobin makes up approximately 98% of the cytoplasmic protein in a RBC. Hemoglobin is composed of 2 alpha-globins and 2 non-alpha-globins (usually beta-globin in adults) that are conjugated to 4 heme molecules that each contain 1 iron atom. Each hemoglobin molecule is capable of reversibly binding and transporting 4 oxygen molecues.
Sideroblastic anemia.
The sideroblastic anemias are characterized by abnormal iron metabolism and heme synthesis within RBCs. In these disorders, iron is sequestered in erythroid cell mitochondria, making it unavailable for heme synthesis. The iron deposition swells and distorts the mitochondria. Because the mitochondria in a developing bone marrow erythrocyte are found in a perinuclear distribution, iron stains in these disorders show a characteristic pattern of iron staining with siderotic granules distributed around the RBC precursor nucleus, forming ringed sideroblasts. Sideroblastic anemias may be hereditary (either autosomal or X-linked) or acquired later in life. Acquired sideroblastic anemias arise from idiopathic causes (usually as a part of a myelodysplastic disorder) and toxic insults (lead, drugs, or alcohol).
Sideroblastic anemias are due to disordered ___ synthesis, and thalassemias are due to disordered ___ synthesis.
Sideroblastic anemias are due to disordered heme synthesis, and thalassemias are due to disordered globin synthesis.
What are Weibel-Palade bodies?
Weibel-Palade bodies are characteristic inclusions of endothelium measuring up to 3 um in maximum dimension. These membrane-bound structures contain up to 25 parallel tubular arrays. These bodies are the site of storage of von Willebrand factor.
What is the LAP score?
Alkaline phosphatase activity is found in the cytoplasm of neutrophils, osteoblasts, vascular endothelial cells, and some lymphocytes. The alk phos level of peripheral blood neutrophils is quantitated by the leukocyte alkaline phosphatase (LAP) score and is a useful screening test to differentiate chronic myelogenous leukemia from leukemoid reactions and other myeloproliferative disorders. The LAP score is usually performed using the Kaplow procedure. The LAP score is determined by evaluation of the staining intensity (ranging from 0 to 4+) of 100 counted neutrophils or bands. Normal LAP scores range from 15 to 130, but there may be variation in these ranges between laboratories. Entities with a low LAP score (130): Infections, growth factor therapy, myeloproliferative disorders other than CML, inflammatory disorders, pregnancy, oral contraceptives, stress, drugs (lithium, corticosteroids, estrogen). There is rapid loss of alk phos activity in samples drawn in EDTA anticoagulant. The test is optimally performed on fresh capillary blood fingerstick smears or on blood anticoagulated with heparin and should be performed within 48 hours after collection of the sample. The blood smears may be held in the freezer for 2-3 weeks with little loss of activity.
Defect in what RBC structural protein causes hereditary spherocytosis?
HS can be caused by any one of several defects in cytoskeletal proteins, including band 3, protein 4.2, spectrin (alpha and beta), and ankyrin. A deficiency in any of these components can lead to the cytoskeletal instability that underlies HS.
Defect in what RBC structural protein causes hereditary elliptocytosis?
The most common genetic defect (present in ~60^ of all cases of HE) is in spectrin (alpha or beta). Other mutations that cause HE are in band 4.1, band 3, and glycophorin C.
What % of elliptocytes are necessary for the diagnosis of hereditary elliptocytosis?
> 25% of all RBCs.
Defect in what RBC protein causes hereditary stomatocytosis?
Stomatin (causes defect in RBC sodium and potassium channels).
What are the formulas for calculating MCV, MCHC, and Hct?
MCV = Hct x 1000 / RBC. MCHC = Hb / Hct x 100. Hct = MCV x RBC.
What are the formulas for calculating absolute reticulocyte count, corrected reticulocyte count, and reticulocyte production index?
ARC = % retics x RBC. CRC = % retics x Hct / 45. The CRC takes into account spuriously increased reticulocyte percentages due to a low Hct. RPI = CRC x 1 / correction factor. The RPI reflects the fact that in anemia, reticulocytes are released earlier from the marrow and therefore have a longer maturation time than normal to mature into red cells. The correction factor is 1.0 when the Hct is normal, 2.0 when Hct is 30, and 3.0 when Hct is 15.
Most commonly, hemoglobin is measured by the ___ method.
Most commonly, hemoglobin is measured by the cyanohemoglobin (hemiglobin cyanide) method, in which hemoglobin is converted to hemiglobin cyanide (HiCN), whose concentration is measured by spectrophotometry. To carry out this conversion, blood is dissolved in a solution of potassium ferricyanide and potassium cyanide which oxidizes the hemoglobin to hemiglobin (Hi; methemoglobin) and then converts it to hemiglobin cyanide (HiCN). The solutions’ absorbance at 540 nm reflects the amount of hemoglobin originally present. This methods detects all forms of hemoglobin (Hb, HbO2, Hi, HbCO) except sulfhemoglobin (SHb).
What do MCHC and MCH measure? When is MCH useful?
MCH is derived from the ratio of total hemoglobin expressed as g/L and the erythrocyte count expressed as x 10^12/L, the results are presented in picograms (10^-12 g). Therefore, MCH provides an absolute gravimetric measurement of the hemoglobin in the average erythrocyte. MCHC, however, provides the ratio of the hemoglobin to the volume of the average cell. MCHC is useful in many conditions, such as normochromic normocytic anemias, where the amount of hemoglobin produced parallels changes in the MCV. However, when evaluating conditions with a decreased eruthrocyte volume, such as thalassemia or iron deficiency, or increased absolute erythrocyte hemoglobin such as hemochromatosis, changes in erythrocyte volumes create problems in interpretation if the MCV or MCHC are used. For example, as erythocytes age, the MCV will increase and consequently the MCHC will decrease, yet the absolute amount of MCH does not change. Such constancy of the MCH is why decreased MCH is used as a tool for evaluating possible thalassemia, iron deficiency, or hemochromatosis.
What laboratory monitoring tests are used for the following anticoagulants: Coumadin, Heparin, LMW heparin, Fondaparinux, Rivaroxaban, Argatroban, Lepirudin, Bivalirudin, Dabigatran.
Coumadin: INR. Heparin: PTT, anti-factor Xa. LMW heparin: Anti-factor Xa if needed. Fondaparinux: Anti-factor Xa if needed. Rivaroxaban: Anti-factor Xa if needed. Argatroban: PTT. Lepirudin: PTT. Bivalirudin: ACT. Dabigatran: Thrombin time to test for residual drug. Dilute thrombin time to assess extent of anticoagulation or test for residual drug.
Coumadin reduces the synthesis of what coagulation factors?
Factors II, VII, IX, and X, as well as protein C and protein S.
How are heparin and the LMW heparins different in mechanism of action?
Heparin inhibits the coagulation cascade at multiple sites; all of the activated coagulation factors, except factor III, are targets. The LMW heparins inhibit the action of factors II and X, and the different LMW heparins may inhibit them to different extents.
How is INR calculated?
INR = [Patient PT / Mean of normal PT range] ^ ISI. ISI = International Sensitivity Index for thromboplastin, used for PT determination. A patient with high ISI (ISI = ~2) will have low sensitivity to factor deficiencies. A patient with low ISI (ISI = ~1) will have high sensitivity to factor deficiencies.
What is the algorithm for treating a high INR in a patient on warfarin (above 2.0-3.0 or 2.5-3.5, depending on underlying condition)?
If there is significant bleeding, discontinue warfarin, give vitamin K (most likely IV or sub-Q), and give plasma to replace the missing coagulation factors. If there is no significant bleeding, and there is a desire to regain the anticoagulated state as soon as possible OR the INR is less than 10, discontinue warfarin and allow INR to decline. If there is no significant bleeding, and there is no desire to regain the anticoagulated state as soon as possible OR the INR is greater than 10, discontinue warfarin and administer vitamin K (most likely oral).
How does protamine sulfate work to neutralize heparin?
Heparin is a very negatively charged compound, and protamine sulfate is a positively charged compound. They bind together and heparin is neutralized.
In what situations should heparin (via PTT or anti-factor Xa) be monitored in patients?
If it is LMWH used for prophylaxis or LMWH therapy in normal weight, normal renal function, non-pregnant patients, no monitoring is needed. If unfractionated heparin is used, target PTT to >2.0x mean of normal range (varies widely depending on reagents and instruments).
What are nonmalignant medical conditions associated with reactive eosinophilia?
Allergic reactions (drug reactions, asthma), parasitic infections (strongyloidiasis), schistosomiasis, filariasis, toxocariasis), metabolic abnormalities (adrenal insufficiency), humoral immunodeficiency (hyperimmunoglobulin E syndrome (Job syndrome), Wiskott-Aldrich syndrome, hyperimmunoglobulin M syndrome, immunoglobulin A deficiency), pulmonary eosinophilias (eosinophilic granulomatosis with polyangiitis (formerly Churg-Strauss syndrome), allergic bronchopulmonary aspergillosis, chronic and acute idiopathic eosinophilic pneumonias), autoimmune blistering skin diseases (dermatitis herpetiformis, bullous pemphigoid).
Most eosinophilias are reactive, polyclonal processes. In all these reactive processes, the increase in AEC appears to be mediated by cytokines, principally ___, which promotes proliferation of eosinophils and their precursors. Reactive eosinophilias may also involve overproduction of additional cytokines, such as ___ and ___, which can lead to concomitant elevation of IgE in reactive eosinophilias.
Most eosinophilias are reactive, polyclonal processes. In all these reactive processes, the increase in AEC appears to be mediated by cytokines, principally IL-5, which promotes proliferation of eosinophils and their precursors. Reactive eosinophilias may also involve overproduction of additional cytokines, such as IL-3 and IL-4, which can lead to concomitant elevation of IgE in reactive eosinophilias.
Why do many patients with Addison disease/adrenal insufficiency have blood eosinophilia?
This association exists because glucocorticoids inhibit eosinophil proliferation and survival, an effect that is exploited clinically when steroids are used as treatment of reactive eosinophilias. In adrenal insufficiency, the converse occurs; low glucocorticoid levels allow increased eosinophil proliferation and survival. This scenario is especially important in critically ill patients, in whom eosinophilia due to adrenal insufficiency is common. Given the inverse relationship between glucocorticoid levels and eosinophil count, the standard evaluation of eosinophilia should include attention to clinical signs of adrenal insufficiency (orthostatic hypotension, skin discoloration); routine chemistries, which may be abnormal in Addison disease; and, in some cases, morning cortisol levels.
Among the reactive eosinophilias in the developed world, allergic reactions are most common, comprising ___% of cases, and parasitic infections represent the second most-common cause, comprising ___% of cases.
Among the reactive eosinophilias in the developed world, allergic reactions are most common, comprising ~80% of cases, and parasitic infections represent the second most-common cause, comprising ~8% of cases.
Whereas autosomal-dominant hyperimmunoglobulin E syndrome is caused by mutations in (gene), (gene) and (gene) mutations are implicated in the comparatively rare autosomal-recessive forms of the disease.
Whereas autosomal-dominant hyperimmunoglobulin E syndrome is caused by mutations in STAT3, DOCK8 and TYK2 mutations are implicated in the comparatively rare autosomal-recessive forms of the disease.
Paraneoplastic eosinophilia is frequently observed in association with solid organ malignancies, with reported incidences ranging from ___ to ___%. The phenomenon is not specific to any particular tumor type, and a broad spectrum of primary tumors have been implicated. Instead, paraneoplastic eosinophilia appears to be most closely related to ___. Various mechanisms have been proposed to explain this association.
Paraneoplastic eosinophilia is frequently observed in association with solid organ malignancies, with reported incidences ranging from 0.5% to 7%. The phenomenon is not specific to any particular tumor type, and a broad spectrum of primary tumors have been implicated. Instead, paraneoplastic eosinophilia appears to be most closely related to tumor stage. It has been noted that blood eosinophilia is a poor prognostic sign and, in almost all cases, is associated with advanced metastatic disease. Various mechanisms have been proposed to explain this association. The eosinophilia may disappear after surgical excision and reappear with recurrence.
Reactive eosinophilia is frequently observed in patients with lymphoma, often because of increased production of growth factors by the malignant cell population. In Hodgkin lymphoma, the reported incidence of blood eosinophilia is ___%. Bone marrow and tissue eosinophilia are also common, although surprisingly, there is no strong correlation with peripheral eosinophil counts. The incidence of tissue eosinophilia varies between types of Hodgkin lymphoma and is most common in the ___ form and the ___ form of the disease. As with many reactive eosinophilias, the mechanism appears dependent on increased ___ levels, and possibly IgE levels, both which may be produced directly by Reed-Sternberg cells.
Reactive eosinophilia is frequently observed in patients with lymphoma, often because of increased production of growth factors by the malignant cell population. In Hodgkin lymphoma, the reported incidence of blood eosinophilia is 15%. Bone marrow and tissue eosinophilia are also common, although surprisingly, there is no strong correlation with peripheral eosinophil counts. The incidence of tissue eosinophilia varies between types of Hodgkin lymphoma and is most common in the mixed cellularity form and the nodular sclerosing form of the disease. As with many reactive eosinophilias, the mechanism appears dependent on increased IL-5 levels, and possibly IgE levels, both which may be produced directly by Reed-Sternberg cells.
Eosinophilia may be associated with a variety of B-cell and T-cell neoplasms. B-cell lymphoblastic leukemia (BLL) with eosinophilia is one such example. It is often associated with a characteristic cytogenetic abnormality ___, which juxtaposes the IL3 gene on chromosome __ to the immunoglobulin heavy chain locus (IgH) on chromosome __. These cases are recognized in the 2008 WHO classification as B lymphoblastic leukemia with ___; IL3-IgH.” This rearrangement implies a “reactive” eosinophilia, in which increased IL-3 expression by malignant, lymphoblastic clones provides a paracrine signal, driving polyclonal expansion of the eosinophils.
Eosinophilia may be associated with a variety of B-cell and T-cell neoplasms. B-cell lymphoblastic leukemia (BLL) with eosinophilia is one such example. It is often associated with a characteristic cytogenetic abnormality t(5;14), which juxtaposes the IL3 gene on chromosome 5 to the immunoglobulin heavy chain locus (IgH) on chromosome 14. These cases are recognized in the 2008 WHO classification as B lymphoblastic leukemia with t(5;14); IL3-IgH.” This rearrangement implies a “reactive” eosinophilia, in which increased IL-3 expression by malignant, lymphoblastic clones provides a paracrine signal, driving polyclonal expansion of the eosinophils.
What two conditions are absolute contraindications for platelet transfusion, and what condition is a strong relative contraindication?
Absolute contraindications: TTP and HIT. Immune thrombocytopenia (idiopathic thrombocytopenic purpura and post-transfusion purpura) are strong relative contraindications; Do not transfuse unless severe thrombocytopenia with life-threatening bleeding; Large numbers of units may be needed due to rapid immune destruction.
Are FFP, FP24, and 5-day cold plasma considered equivalent blood products?
Generally yes, except that FP24 and 5-day cold plasma should not be used for replacement of Factor V or Factor VIII. However, selective Factor V deficiency is rare, occuring in ~1/million and the general population and is not associated with a bleeding tendency; also, Factor VIII concentrate is an available product, and cryoprecipitate can be used if factor concentrate is unavailable. So FFP, FP24, and 5-day cold plasma are basically equivalent blood products.
Which coagulation factor has the shortest half-life?
Factor VII, at 4-7 hours. Factor I is 72-120 hours, factor II is 48-120 hours, factor V is 12-36 hours, factor VIII is 8-12 hours, factor IX is 18-24 hours, factor X is 24-48 hours, factor XI is 40-84 hours, factor XII is 48-52 hours, and factor XIII is 9-12 days.
Which coagulation factor has the longest half-life?
Factor XIII, at 9-12 days. Factor I is 72-120 hours, factor II is 48-120 hours, factor V is 12-36 hours, factor VII is 4-7 hours, factor VIII is 8-12 hours, factor IX is 18-24 hours, factor X is 24-48 hours, factor XI is 40-84 hours, factor XII is 48-52 hours.
What hematologic abnormalities may patients with hepatic angiosarcoma present?
Thrombocytopenia (secondary to entrapment of platelets in the tumor/Kasabach-Meritt syndrome). Microangiopathic hemolytic anemia (due to red cell fragmentation in the tumor). Disseminated intravascular coagulation.
POC PT/INR devices are approved for __. POC PT/INR devices have not been approved for __.
POC PT/INR devices are approved for warfarin monitoring in the stabilized patient. Once the patient’s dose requirements and anticoagulation effects have been stabilized for 1-2 weeks, using a central laboratory, testing can be moved to a POC device. POC PT/INR devices have not been approved for coagulation screening, testing for DIC, or use in the trauma/ED setting.
Hantavirus pulmonary syndrome. During the HPS prodrome, thrombocytopenia is the only dependable finding. Once pulmonary edema is established, however, there is a highly reproducible pentad of findings:_____.
Hantavirus pulmonary syndrome. During the HPS prodrome, thrombocytopenia is the only dependable finding. Once pulmonary edema is established, however, there is a highly reproducible pentad of findings: thrombocytopenia, left-shifted neutrophilia, neutrophils with lack of significant toxic granulation, increased hemoglobin concentration (hemoconcentration), and >10% of lymphocytes having immunoblastic morphology. Having 4 of these 5 has a high sensitivity and specificity for HPS.
Of the cytokines, IL-__ is the most specific for the eosinophil lineage.
Of the cytokines, IL-5 is the most specific for the eosinophil lineage. It is responsible for the selective differentiation of eosinophils and release of eosinophils from bone marrow.
Several named syndromes are associated with eosinophilia and eosinophilic infiltration of specific organs: eosinophilic cellulitis (__ syndrome), eosinophilic pneumonia (__ syndrome), eosinophilic fasciitis (__ syndrome), and eosinophilic vasculitis (__ syndrome).
Several named syndromes are associated with eosinophilia and eosinophilic infiltration of specific organs: eosinophilic cellulitis (Well syndrome), eosinophilic pneumonia (Loeffler syndrome), eosinophilic fasciitis (Shulman syndrome), and eosinophilic vasculitis (Churg-Strauss syndrome).
What hematologic abnormality is a serious potential complication of antithyroid drugs (carbimazole, methimazole, and propylthiouracil)?
Agranulocytosis. The effect is not dose-dependent. The incidence is 0.05-0.5% of all users. In most patients, the agranulocytosis occurs within the first 3 months of treatment. This often presents with pharyngitis, and patients are instructed to seek attention if they develop sore throat, fever, an oral ulcer, or other symptoms of infection (UTI, PNA). Minor depressions in the neutrophil count are much more common than true neutropenia.
List the constitutional neutropenias.
Cyclic/congenital (ELA2-related) neutropenia. Kostmann syndrome. Schwachman-Diamond syndrome. Chediak-Higashi syndrome. Fanconi anemia. Dyskeratosis congenita. Benign familial neutropenias. Glycogen storage disease type Ib. WHIM syndrome (myelokathexis). Reticular dysgenesis. Wiskott-Aldrich syndrome.
Separate these constitutional neutropenias into an isolated neutropenia category and a with other cytopenias category: Cyclic/congenital (ELA2-related) neutropenia. Kostmann syndrome. Schwachman-Diamond syndrome. Chediak-Higashi syndrome. Fanconi anemia. Dyskeratosis congenita. Glycogen storage disease type Ib. WHIM syndrome (myelokathexis). Reticular dysgenesis. Common variable immunodeficiency. Congenital amegakaryocytic thrombocytopenia.
Isolated neutropenia: Kostmann syndrome. Cyclic neutropenia. Glycogen storage disease type Ib. WHIM (myelokathexis) syndrome. Chediak-Higashi syndrome. Reticular dysgenesis. With other cytopenias: Shwachman-Diamond syndrome. Dyskeratosis congenita. Fanconi anemia. Common variable immunodeficiency. Congenital amegakaryocytic thrombocytopenia.
Isolated lymphopenia is uncommon but may be seen in what conditions (6)?
Isolated lymphopenia is uncommon but may be seen in SLE, HIV, SARS, anti-CD20 (rituxan) therapy, steroid therapy, and certain congenital immunodeficiencies (Bruton, SCID, DiGeorge, CVI).
With an isolated monocytopenia, consider what 2 conditions/situations?
With an isolated monocytopenia, consider hairy cell leukemia or steroid therapy. In patients undergoing chemotherapy, monocytopenia heralds the onset of neutropenia.
What are the 4 hereditary types of vWD?
The four hereditary types of vWD described are type 1, type 2, type 3, and platelet-type. Type 1 is most common, at 60-80% of all vWD cases. Type 2 has four subtypes: 2A, 2B, 2M and 2N (Normandy). Type 3 is the most severe form of vWD. Platelet-type vWD, also known as pseudo-vWD, is an autosomal dominant genetic defect of the platelets; The von Willebrand factor is qualitatively normal and genetic testing of the von Willebrand gene and vWF protein reveals no mutational alteration.
What are hematological indicators of poor prognosis in severe malaria?
Malaria is the single most important disease hazard from travel to developing countries. Worldwide, there are over 200 million annual cases with over 600,000 annual deaths. Hematological indicators of poor prognosis in severe malaria include a platelet count < 50 x 10E9/L, prolonged PT >3 seconds, prolonged APTT, fibrinogen < 200mg/dl, hyperparasitemia > 100,00/uL (high mortality if >500,000/uL), > 20% parasites and > 5% neutrophils containing visible malarial pigment. Early recognition and prompt management are important.
What is a hemophilic pseudotumor?
A serious but rare (<2% incidence) lesion that occurs in the bones and deep soft tissues of patients with hemophilia, which can be confused with simple bone cyst, aneurysmal bone cyst, osteosarcoma, or angiosarcoma. Recurrent hemorrhages with reparative changes are responsible for the clinicopathologic manifestations. In bone, they may be intraosseous or sub-periosteal and usually develop in the femur, pelvis, and tibia in adults and the small bones of the hands in children. Histologically, areas of extensive hemorrhage, thrombus formation, bone destruction, and florid new bone formation characterize this lesion and, taken out of context, can be misinterpreted as malignant. Juxtacortical and intramuscular lesions exhibit central masses of organizing blood encased by a fibrous capsule that may be composed of 3 distinct layers: an inner hemosiderin-rich collagenous layer, a central layer of dense fibrous tissue, and an outer layer rich in elastin.
How can pseudohyperkalemia or pseudohypokalemia be seen in blood drawn from leukemic patients?
Pseudohyperkalemia can occur in patients with extremely high blast counts in acute or accelerated phase leukemias. Those blasts can be fragile and may lyse during standard phlebotomy, releasing potassium. In contrast, specimens with very high WBC counts that are collected gently can show pseudohypokalemia when potassium is taken up by highly metabolically active leukemic cells along with glucose; such specimens can be transported on ice to slow this enzymatically mediated uptake.
Which normally has a slightly higher value of potassium - serum or plasma?
Normally platelets release potassium during clotting, so serum has a slightly higher value of potassium than plasma from the same individual; this difference is accentuated when the platelet count is extremely elevated.
Why can’t blood anticoagulated with oxalate be used to measure hematocrit?
Oxalate anticoagulants may shrink red cells; thus blood anticoagulated with oxalate cannot be used to measure hematocrit. Combined ammonium/potassium oxalate does not have the same effect of shrinking cells.
For what primary purpose/analytic tests are the principles of impedance used in the laboratory?
Electrical impedance measurement is based on the change in electrical resistance across an aperture when a particle in conductive liquid passes through this aperture. Electrical impedance is used primarily in the hematology laboratory to enumerate leukocytes, erythrocytes, and platelets. In a typical electrical impedance instrument by Coulter, aspirated blood is divided into two separate volumes for measurements. One volume is mixed with diluent and is delivered to the cell bath, where erythrocyte and platelet counts are performed. As a cell passes through the aperture, partially occluding it, the electrical impedance increases, producing a voltage pulse, the size of which is proportional to the cell size. The number of pulses is directly related to the cell count. Particles measuring between 2 and 20 fL are counted as platelets, whereas those measuring greater than 36 fL are counted as erythrocytes. The other blood volume is mixed with diluent and a cytochemical-lytic reagent that lyses only the red blood cells. A leukocyte count is performed as the remaining cells pass through an aperture. Particles greater than 35 fL are recorded as leukocytes.
Practically, the best indicator for anemia is ___.
Practically, the best indicator for anemia is a low RBC count or number of RBCs per volume of whole blood. Although the reference range for the red cell count varies with age, sex, and population, it encompasses values from around 4–6 × 106 RBCs per cubic millimeter (cu mm) or microliter. This range may change somewhat depending on the population. RBC counts below the lower limit of the reference range suggest the presence of anemia.
The chromicity of the red cells (i.e., the intensity of the red color of the cells due to intracellular Hb) is measured quantitatively by ___.
The chromicity of the red cells (i.e., the intensity of the red color of the cells due to intracellular Hb) is measured quantitatively by the mean corpuscular hemoglobin concentration, or MCHC.
Moderate to severe iron deficiency anemia is associated with a (decreased/normal/increased) RDW, while thalassemia and anemia of chronic disease are associated with a (decreased/normal/increased) RDW.
Moderate to severe iron deficiency anemia is associated with a increased RDW, while thalassemia and anemia of chronic disease are associated with a normal RDW.
What is RPI?
Peripheral blood reticulocytosis is a measure of bone marrow response in the face of anemia. A similar measure, RPI, corrects the reticulocyte count with respect to (1) the proportion of reticulocytes present in a patient without anemia, and (2) the premature release of reticulocytes into the peripheral circulation. Bone marrow response to anemia may be appropriate (hyperproliferative) with an RPI >3, generally indicating marrow red cell hyperproliferation; however, the anemia may be due to defective RBC production or marrow failure (hypoproliferative), which is generally indicated by an RPI <2.
What are some microcytic anemias?
Common microcytic anemias include IDA and the thalassemias. Some hemoglobinopathies and ACD may also be microcytic.
What conditions is ACD associated with?
ACD appears to be due to defective iron utilization/metabolism and is associated with chronic nonhematologic disorders such as chronic infections, connective tissue disorders, malignancy, and renal, thyroid, and pituitary disorders.
How can ferritin distinguish between IDA and ACD?
Normally, there is an equilibrium between intracellular and extracellular ferritin. The lower the stored iron becomes, the lower is the intracellular ferritin, and, consequently, the lower the extracellular ferritin becomes. The level of extracellular ferritin can be directly measured by determining the serum ferritin level, which is readily and accurately assessed on serum aliquots, using ELISA techniques. Therefore, serum ferritin levels give an excellent measure of available iron stores, noninvasively. Because iron stores in ACD are abundant, serum ferritin levels are characteristically normal to elevated. In contrast, in IDA, in which iron stores become depleted, serum ferritin levels are characteristically decreased. Thus serum ferritin level is one assay that can be used in differentiating IDA from ACD.
Iron binding capacity is a direct measure of the protein ___.
Iron binding capacity is a direct measure of the protein transferrin, which transports iron from the gut to iron storage sites in bone marrow.
Considerable overlap has been noted between serum levels of iron and iron-binding capacity in IDA and ACD. A somewhat more reliable discriminating measure of IDA is ___.
Considerable overlap has been noted between serum levels of iron and iron-binding capacity in IDA and ACD. A somewhat more reliable discriminating measure of IDA is the ratio of serum iron to IBC, known as the transferrin saturation. This ratio is around 1 : 3 for normal individuals, and in IDA it is significantly reduced to values of around 1 : 5 or lower. Again, considerable overlap is seen even in this ratio for patients with IDA and ACD, so the values should always be interpreted with care.
IDA and ACD are both hypoproliferative, microcytic anemias. What are some analyte abnormalities commonly seen for each?
IDA: low ferritin, increased IBC, decreased serum iron, reduced Fe/IBC ratio, generally increased RDW. ACD: generally high ferritin, normal IBC, decreased serum iron, normal Fe/IBC ratio, generally normal RDW.
Hemolytic anemia is a hyperproliferative, normocytic anemia. What are some analyte abnormalities commonly seen?
Schistocytosis, increased reticulocytes, low haptoglobin, elevated carboxyhemoglobin, elevated LD, elevated indirect bilirubin, generally increased RDW.
Aplastic anemia and renal failure both have hypoproliferative, normocytic anemia. What are some analyte abnormalities commonly seen for each?
Aplastic anemia: leukopenia, thrombocytopenia, hypocellular bone marrow, generally normal RDW. Renal failure: elevated BUN and Cr, low erythropoietin, burr cells may be present, generally normal RDW.
B12 and/or folate deficiency and hypothyroidism both have hypoproliferative, macrocytic anemia. What are some analyte abnormalities commonly seen for each?
B12 and/or folate deficiency: megaloblastic, low B12 and/or folate, hyperlobulated PMNs, macroovalocytes, increased RDW. Hypothyroidism: nonmegaloblastic, elevated TSH, normal RDW.
What is the relationship between hemolysis and haptoglobin?
After erythrocyte membrane breakdown, Hb is extruded. Extruded Hb becomes bound to the α-2 fraction protein haptoglobin. The Hb–haptoglobin complex becomes catabolized by macrophages that engulf these complexes by receptor-mediated endocytosis. Thus an excellent laboratory test for hemolytic anemia is a low haptoglobin value. Extremely sensitive and rapid ELISA assays for haptoglobin are available for this purpose.
Why do carbon monoxide and unconjugated bilirubin become elevated in blood in hemolytic anemia?
When Hb is extruded, large amounts of it become oxidized to methemoglobin. The heme portion dissociates and then becomes oxidized ultimately to bilirubin. The first step in this process is the oxidative opening of the porphyrin ring of heme with attendant liberation of CO. CO may be measured easily by gas chromatographic techniques or even more conveniently by cooximetry, based on spectrophotometry, as carboxyhemoglobin. Elevated CO levels in normochromic, normocytic anemias are an excellent indicator of hemolytic anemia. Because production of bilirubin, which is unconjugated, is increased, at least a transient elevation of serum indirect bilirubin occurs. This elevation, in the presence of normal liver function, will be modest.
Why are RBC and WBC precursors seen in the peripheral blood in microangiopathic hemolytic anemia?
Because MHA results from traumatic destruction of newly formed RBCs in the microvasculature, where both red and white blood cell (WBC) precursors are being formed, often both RBC and WBC precursors are released into the circulation. Thus, all findings of hemolytic anemia are present, in addition to which a significant number of precursor cells such as nucleated RBCs, myelocytes, and metamyelocytes are seen in the peripheral blood—a pattern termed the leukoerythroblastic picture.
How does microangiopathic hemolytic anemia occur?
Schistocytes may be present on peripheral blood films as the result of mechanical (prosthetic heart valve) or thermal (severe burns) destruction. Mechanical rupture of red cells within the microvasculature may also occur by physical damage to red cells in the microvasculature of bone marrow. This may be due to space-occupying lesions, such as metastatic tumors or leukemia or lymphoma, and also myelofibrosis. Besides space-occupying lesions, other causes of this type of anemia include disease states in which fibrin is deposited on endothelial surfaces, also resulting in shearing and fragmentation of newly synthesized red blood cells, as in DIC. In this condition, an abnormal activation of the coagulation process occurs, in which fibrin–platelet clots form intravascularly and embolize to virtually any tissue. These clots block the microvasculature of tissues, including that of bone marrow, resulting in the destruction of newly synthesized red cells. In addition, other disease states may give rise to MHA that may have an immunologic component (i.e., antibodies to determinants on endothelial cells or on other structures in the microvasculature), resulting in immune complex deposits with or without fibrin deposits. These states include TTP and HUS. MHA may also occur with other immune-mediated disorders (e.g., connective tissue disorders such as disseminated lupus erythematosus), where, again, endothelial damage from the attachment of immune complexes and complement produces fibrin deposition on endothelial surfaces.
How does renal failure cause anemia?
Another normocytic, hypoproliferative anemia is the anemia of chronic renal failure. Loss of the kidneys’ excretory function produces an increase in BUN and creatinine, as discussed later, as well as a buildup of metabolic byproducts. The resulting uremia appears to be responsible for changes in red cell shape, with burr cells (echinocytes) and ellipsoidal cells commonly present on peripheral blood films. Identification of burr cells on peripheral blood films during the course of illness may signal the development of renal dysfunction. In addition to decreased excretory function, the kidneys’ ability to produce erythropoietin is decreased, resulting in impaired erythropoiesis, such that the marrow’s response to hypoxia becomes inadequate. In contrast to aplastic anemia, white cell and platelet counts usually remain within normal limits.
Concomitant elevations of potassium and LD in serum should be taken as indications of ___.
Concomitant elevations of potassium and LD in serum should be taken as indications of hemolysis that occurs artifactually after a blood sample has been taken from the patient or, less commonly, as hemolysis that results from an underlying hemolytic condition.
What does it mean when a consistently low anion gap is seen?
Consistently low anion gaps, typically in the range of 1–3 mEq/L, signify the presence of high levels of basic protein, often a monoclonal paraprotein as occurs in plasma cell dyscrasias. Basic protein contains ammonium ions, the counter-ions for which are chloride. Now the “invisible” ion is ammonium, and a measurable increase in chloride ion occurs. This tends to decrease the anion gap. Persistently low anion gaps are a serious sign of possible malignancy (e.g., multiple myeloma).
Plasma transfusion is generally indicated for what conditions/situations?
Plasma transfusion is generally indicated for coagulation factor deficiency, disseminated intravascular coagulation, dilutional coagulopathy, urgent warfarin reversal, and thrombotic thrombocytopenic purpura.
Anaplasma phagocytophilum. What is the old name? What disease does it cause?
Anaplasma phagocytophilum (formerly Ehrlichia phagocytophilum) is a gram-negative rickettsial bacterium that is unusual in its tropism to neutrophils. It causes anaplasmosis in sheep and cattle, also known as tick-borne fever and pasture fever, and also causes the zoonotic disease human granulocytic anaplasmosis.
Platelet transfusion in ITP is (not/relatively/absolutely) contraindicated.
The platelet transfusion is relatively contraindicated in immune thrombocytopenic purpura. In this setting, posttransfusion platelet survival is extremely brief, and platelet transfusion is indicated only if there is severe hemorrhage.
Standard coagulation tests such as PT and APTT are commonly used to assess the need for plasma transfusion; however, these tests are poorly predictive of bleeding risk. In general, if the PT and APTT are less than ___ times the midpoint of the reference range, no benefit will be obtained from plasma transfusion. If the INR is used, in general, no benefit will be derived from plasma transfusion if it is less than ___, although for nervous system and retinal hemorrhage, plasma may be reasonably used unless the INR is less than ___.
Standard coagulation tests such as PT and APTT are commonly used to assess the need for plasma transfusion; however, these tests are poorly predictive of bleeding risk. In general, if the PT and APTT are less than 1.5 times the midpoint of the reference range, no benefit will be obtained from plasma transfusion. If the INR is used, in general, no benefit will be derived from plasma transfusion if it is less than 1.5, although for nervous system and retinal hemorrhage, plasma may be reasonably used unless the INR is less than 1.3.
What are D-dimers?
The coagulation cascade concludes as thrombin converts fibrinogen into fibrin monomer. Fibrin monomers self-assemble into fibers, with growth of fibrin via end-to-end and side-to-side association of molecules. They are then covalently cross-linked by factor XIIIa at the outer D domains of adjacent fibrin monomers and the central E domain of a third fibrin monomer molecule. The structure developed within fibrin in which 2 D domains are covalently linked is referred to as D-dimer.
What are the clinical conditions associated with elevated levels of D-dimer?
The clinical conditions associated with elevated levels of D-dimer are numerous. Some of these include thrombosis (arterial or venous), pulmonary embolism, venous thrombosis, disseminated intravascular coagulation, myocardial infarction, stroke, postoperative state, liver disease, malignancy, and pregnancy.
What are the 2 different types of units used to represent D-dimer?
D-dimer is reported in mass units and, as these assays have evolved, 2 different types of units have been used to represent D-dimer: the fibrinogen equivalent unit (FEU) at 340 kDa and the D-dimer unit (DDU) at 195 kDa. Fibrin monomer polymerizes end to end and side to side forming a fiber, fibrin, in a process that is not enzymatically driven. The polymerization of fibrin monomer occurs with the association of the D domains of 2 fibrin monomers (referred to as D-dimer) with the E domain of a third. Soluble fragments of fibrin (fibrin degradation products) that contain variable numbers of the D-D domains (D-dimer) are produced. When assayed, the fragments are quantified in 2 different types of units, the DDU and the FEU. The mass of the FEU, related to the mass of fibrinogen, is about 1.7 times greater than the mass of the DDU. Because the D-dimer is reported in mass units, the type of units involved is very important in setting the threshold for the exclusion of venous thromboembolism and for interpretation of reported results. Adding to the complexity of reporting these values is the variability in the magnitude of units reported, for example, ng/mL, μg/mL, and μg/L. Variability in the type and magnitude of units has led to confusion in some laboratories as they attempt to use the assay, especially as they set a threshold for the exclusion of VTE.
In the P blood group system, what is auto-anti-P?
An autoantibody with anti-P specificity is seen in patients with paroxysmal cold hemoglobinuria, a clinical syndrome that may occur in children following viral infection. In PCH, autoanti-P is an IgG, biphasic hemolysin capable of binding RBCs at colder temperatures, followed by intravascular hemolysis at body temperature. This characteristic can be demonstrated in vitro in the Donath-Landsteiner test.
An autoantibody with anti-___ specificity is seen in patients with paroxysmal cold hemoglobinuria, a clinical syndrome that may occur in children following viral infection.
An autoantibody with anti-P specificity is seen in patients with paroxysmal cold hemoglobinuria, a clinical syndrome that may occur in children following viral infection.
What can cause additional reactivity on reverse typing of blood?
Extra antibodies. Transfusion (plasma components not type-specific to patient; IV Ig/albumin). Transplantation. Subgroup of a major blood type (especially subgroup of A (i.e. A2). Rouleaux (multiple myeloma; chronic inflammatory disorders).
An acquired Donull phenotype can be observed in what condition?
An acquired Donull phenotype can be observed in paroxysmal nocturnal hemoglobinuria type III (PNH-III). A hematopoietic stem cell disorder, PNH III is characterized by chronic hemolysis due to an absence of all GPI-linked glycoproteins, including Dombrock, Cromer, and Cartwright antigens.
The Donull or Gy(a−) phenotype is a rare, autosomal recessive phenotype of the Dombrock blood group system. An acquired Donull phenotype can be observed in paroxysmal nocturnal hemoglobinuria type III (PNH-III). A hematopoietic stem cell disorder, PNH III is characterized by chronic hemolysis due to an absence of all GPI-linked glycoproteins, including Dombrock, ___, and ___ antigens.
The Donull or Gy(a−) phenotype is a rare, autosomal recessive phenotype of the Dombrock blood group system. An acquired Donull phenotype can be observed in paroxysmal nocturnal hemoglobinuria type III (PNH-III). A hematopoietic stem cell disorder, PNH III is characterized by chronic hemolysis due to an absence of all GPI-linked glycoproteins, including Dombrock, Cromer, and Cartwright antigens.
Gerbich antigens are decreased in patients with ___ due to protein 4.1 (an RBC cytoskeletal protein) deficiency.
Gerbich antigens are decreased in patients with hereditary elliptocytosis due to protein 4.1 (an RBC cytoskeletal protein) deficiency.
By what physiologic mechanisms does hydrops fetalis occur?
HDFN is the destruction of fetal or newborn RBCs by maternal alloantibodies specific for inherited paternal RBC antigen(s). The maternal IgG is transported across the placenta into the fetal circulation where it binds to the corresponding RBC antigen, targeting the antibody-coated RBCs for destruction by macrophages in the fetal spleen. The fetal marrow initially responds by increasing erythropoiesis and releases many of the newly produced RBCs into the circulation prematurely as nucleated precursors, leading to the term “erythroblastosis fetalis.” With worsening anemia, erythropoiesis expands to the liver and spleen, causing organ enlargement and portal hypertension. A resulting decrease in liver production of albumin leads to reduced plasma colloid osmotic pressure, generalized edema, ascites, and effusions known as “hydrops fetalis.”
Do RBCs have HLA class I antigens?
The immature nucleated red cell also has class I antigens on its surface, but as the RBC matures the expression of class I antigens is diminished. Remnants of class I HLA antigens on red blood cells are also known as Bennet-Goodspeed (Bg) antigens. The antigens are listed with their corresponding class I HLA designation: Bga – HLA-B7, Bgb – HLA-B17, Bgc – HLA –A28.
On what cell types are HLA class II antigens expressed?
HLA class II antigens are selectively expressed by dendritic cells, macrophages, B-lymphocytes, and activated T-cells that function in antigen processing and self versus non-self recognition.
The i antigen from the I blood group system is normally present only in children. In what conditions/situations do adults have increased i antigen?
The iadult phenotype is a rare, autosomal recessive phenotype found in <1/10,000 donors. In Asia, the iadult phenotype can be associated with congenital cataracts. i antigen is also observed on cord RBCs and reticulocytes and in megaloblastic anemia, leukemia, and chronic hemolytic states as a sign of stressed erythropoiesis. Elevated i antigen is also observed in HEMPAS (hereditary erythroblastic multinuclearity with positive acidified-serum test), a congenital dyserythropoietic anemia.
Antibodies to the I blood group system. What Ig isotype? Auto and alloantibodies?
Anti-I and anti-i are antibodies of IgM isotype, reactive at room temperature. Autoantibodies to I are relatively common and are usually low-titered cold agglutinins. Some anti-I can have IH specificity, reacting stronger with group O and A2 RBC. Although generally benign, hemolysis secondary to high-titered anti-I is observed in cold autoimmune hemolytic anemia (CAIHA). CAIHA can occur in the setting of malignancy and occasionally infection (e.g., Mycoplasma pneumoniae). These antibodies display high thermal amplitude, often agglutinating RBCs at temperatures of 30°–34° C. In contrast, alloanti-I is relatively rare and is found as a naturally occurring antibody in iadult individuals.
Rouleaux formation or “pseudoagglutination.” In what conditions/situations is it seen and how do they cause the rouleaux formation?
Patients with multiple myeloma, Waldenström’s macroglobulinemia, and hyperviscosity syndromes have high concentrations of abnormal serum proteins that change the net surface charge on the RBC membrane. The cells thus cluster together in clumps that resemble macroscopic hemagglutination. Plasma expanders, such as dextran and hydroxyethyl starch, as well as some intravenous X-ray contrast materials can also cause rouleaux formation.
Patients with multiple myeloma, Waldenström’s macroglobulinemia, and hyperviscosity syndromes have high concentrations of abnormal serum proteins that change the net surface charge on the RBC membrane that can result in rouleaux formation or “pseudoagglutination”. How can the rouleaux formation be differentiated from true agglutination?
Rouleaux can be differentiated from true agglutination by direct microscopy (1) by the classical “stacked-coin” formation in rouleaux, and (2) by the loss of rouleaux after washing and resuspension in saline.
What is the primary biologic function of haptoglobin?
Haptoglobin binds free hemoglobin to prevent iron loss and kidney damage during hemolysis. Any hemolytic process (immune, mechanical, environmental) can result in free hemoglobin entering the vascular system.
Is haptoglobin a positive or negative acute phase reactant?
Positive acute phase reactant.
Where is haptoglobin made? What basic biochemical structure does it have?
Haptoglobin is made in the liver as a single polypeptide chain that is cleaved post-translationally to generate alpha and beta subunits. These polypeptide chains can then be linked via disulfide bonds into four peptide chains in two pairs (alpha-beta)2 confirmation, much like hemoglobin.
In the blood bank, what is an antibody identification technique performed on serum that is useful in cases of WAIHA?
Serum adsorption. Adsorb serum antibodies with autologous cells, selected allogeneic cells, or rabbit erythrocyte stroma (REST) at appropriate temperature for reactivity of antibody. This physically removes broadly reactive autoantibodies or alloantibodies from serum by reacting with absorbing cells to allow for detection of underlying IgG antibodies—particularly useful in cases of WAIHA.
What technique can be used in the blood bank to harvest autologous red cells (excluding transfused donor red cells) in patients with sickle cell disease? Because sickle cell patients are regularly transfused, this technique is useful for facilities that provide antigen-matched red cells for their sickle cell patients.
Hypotonic cell lysis. The method is based on the resistance of Hg S red cells to hypotonic saline. The patient sample is repeatedly washed with 0.3% hypotonic saline to lyse normal donor red cells until gross hemolysis is no longer present. The cells are then washed and resuspended in 0.9% saline for antigen typing.
Individuals with warm reactive autoantibodies account for approximately __%–__% of cases of AIHA.
Individuals with warm reactive autoantibodies account for approximately 70%–80% of cases of AIHA.
In what conditions can secondary WAIHA be seen?
Secondary WAIHA is often observed in autoimmune diseases (frequently systemic lupus erythematosus), in lymphoproliferative disorders, and following viral infections.
Autoantibodies in WAIHA are usually Ig__ and (mono/poly)clonal in nature, showing optimal in vitro reactivity at __° C. The DAT will be positive in approximately __% of cases.
Autoantibodies in WAIHA are usually IgG and polyclonal in nature, showing optimal in vitro reactivity at 37° C. The DAT will be positive in approximately 80% of cases.
In WAIHA, the DAT will be positive in approximately 80% of cases. What can be done if the DAT is negative?
In those instances where the DAT is negative, the use of more sensitive techniques, such as radioisotope or enzyme-labeled DAT, can demonstrate low levels of IgG or, less frequently, IgM and IgA on red cells.
In WAIHA, what IgG subtype(s) is involved? Is hemolysis intravascular or extravascular?
In WAIHA, causative antibodies are predominantly IgG1 and/or IgG3. Despite the ability of these subclasses to activate complement, immune destruction of the patient’s autologous cells occurs largely through the spleen and liver via extravascular pathways.
Are the autoantibodies in WAIHA narrowly reactive or broadly reactive?
In general, warm autoantibodies display a broad specificity, reacting with all normal RBCs. Relatively few individuals with WAIHA will show a single simple specificity.
In WAIHA, what serologic testing can be done beyond the DAT?
Transfusion is usually avoided for as long as possible in patients with WAIHA. If transfusion is necessary, it is important to characterize the autoantibody and exclude the presence of RBC alloantibodies. Typical investigation strategies should always include adsorption of the serum (Because a strong, broadly reactive serum autoantibody can mask the presence of significant alloantibodies, adsorption studies should be performed in these patients), followed by antibody detection and identification on the adsorbed serum. Subsequent testing can also be performed on eluted antibody, although it seldom yields further useful information unless the serum is nonreactive.
Brief overview of cold autoantibodies/CAIHA.
Cold autoantibodies may be detected in the serum of many normal individuals if tested under the right conditions. However, most of these antibodies are benign cold agglutinins that show optimal reactivity at 4° C and little or no reactivity at 37° C. Cold agglutinins are often a nuisance that can interfere with ABO/Rh typing, antibody detection, and crossmatching when polyspecific AHG is used. Although usually ignored as clinically insignificant, cold agglutinins may become pathologic by virtue of expanded thermal amplitude and a significant increase in titer, frequently in association with certain disease states. DAT, serum titration, and characterization of thermal amplitude are the most important serologic tests in evaluating a possible diagnosis of CAIHA.
What are the 3 most important serologic tests used in evaluation of a possible diagnosis of CAIHA?
DAT, serum titration, and characterization of thermal amplitude are the most important serologic tests in evaluating a possible diagnosis of CAIHA.
Cold agglutinins are often merely a nuisance that can interfere with ABO/Rh typing, antibody detection, and crossmatching when polyspecific AHG is used. When can cold agglutinins become pathologic?
Although usually ignored as clinically insignificant, cold agglutinins may become pathologic by virtue of expanded thermal amplitude and a significant increase in titer, frequently in association with certain disease states.
Cold agglutinin disease (CAD) accounts for about __% of total cases of AIHA.
Cold agglutinin disease (CAD) accounts for about 20% of total cases of AIHA. As with WAIHA, CAD may be idiopathic or secondary following infection or malignancy.
In contrast to the anti-I cold agglutinins found in normal sera (usually less than 1 : 64 when tested at 4° C), anti-I in CAD is often of very high titer, with ranges of 1 : ___ to 1 : ___.
In contrast to the anti-I cold agglutinins found in normal sera (usually less than 1 : 64 when tested at 4° C), anti-I in CAD is often of very high titer, with ranges of 1 : 10,000 to 1 : 1,000,000. The antibody also has expanded thermal amplitude, reacting with RBCs at temperatures in the range of 30°–34° C in vitro, especially in tests with albumin-suspended RBCs. The DAT is typically positive with polyspecific and anti-C3 reagents.
In addition to laboratory findings of intravascular hemolysis, PCH patients may have ___ on peripheral blood smear.
In addition to laboratory findings of intravascular hemolysis, PCH patients may have erythrophagocytosis on peripheral blood smear.
What is the most common causative antibody in paroxysmal cold hemoglobinuria?
Autoanti-P, also known as the Donath-Landsteiner (DL) antibody, is the most common causative antibody in PCH. It is an IgG, biphasic autohemolysin capable of binding to RBCs at cold temperatures and causing intravascular hemolysis of those cells at body temperature.
When performing the Donath-Landsteiner test to detect the biphasic hemolysin characteristic of paroxysmal cold hemoglobinuria, why is a fresh blood sample needed, and why should it not be drawn into an EDTA tube?
The DL test requires that a fresh blood sample be used to ensure that an adequate supply of complement is available, because complement is relatively unstable and deteriorates during storage. The sample should be drawn and immediately stored at 37° C until clot formation. It is also important not to draw the blood into an anticoagulant such as EDTA because chelation of calcium ions will prevent complement activation and thus in vitro hemolysis.
What is a “mixed” AIHA?
A “mixed” AIHA occurs in s serum will react in all phases of testing (at room temperature and at 37° C) in the IAT. The cold-reactive antibody is typically anti-I or anti-i, and the warm-reactive antibody is typically a 37° C reactive IgG autoantibody. To adsorb autoantibody in a mixed AIHA, the sample is sequentially adsorbed at 37° C to adsorb warm-reactive autoantibody, followed by incubation in an ice bath for 30 minutes to adsorb cold-reactive antibody. Clinically, mixed AIHA usually is similar to WAIHA, although some patients may have symptoms of both WAIHA and CAD.
Historically, drug-induced hemolytic anemia has been subclassified based on its serologic characteristics into what four types?
Drug-independent antibodies. Drug-dependent antibodies are subclassified into two types: Drug-dependent reactivity with drug-coated cells and Drug-dependent reactivity with uncoated cells. Nonimmunologic protein adsorption. There are also combination mechanisms.
Historically, drug-induced hemolytic anemia has been subclassified based on its serologic characteristics into four types: Drug-independent antibodies. Drug-dependent antibodies (further subclassified into two types): Drug-dependent reactivity with drug-coated cells and Drug-dependent reactivity with uncoated cells. Nonimmunologic protein adsorption. How do they work?
Drug-independent antibodies do not require the addition of drug into the test system for detection, even though the drug is responsible for inducing hemolytic anemia. Drug-dependent antibodies, on the other hand, require the presence of the drug for serologic reactivity. Drug-dependent antibodies can be subclassified into two types, depending on whether antibody reactivity requires drug-coated red cells or simply the presence of drug in serum. Nonimmunologic protein adsorption proposes that a change in the red cell membrane causes immune globulins and other plasma proteins to be adsorbed nonspecifically to the red cell membrane. There are also combination mechanisms.
What are the 6 stages of maturation for granulocytes?
Myeloblast –> promyelocyte –> myelocyte –> metamyelocyte –> bands –> polymorphonuclear neutrophils.
The 6 stages of maturation for granulocytes are: Myeloblast –> promyelocyte –> myelocyte –> metamyelocyte –> bands –> polymorphonuclear neutrophils. Where is the line between the proliferative pool and the postmitotic pool?
Cells from the first 3 maturation stages constitute the proliferative pool, while cells from the last 3 maturation stages are postmitotic.
The term “large granular lymphocytes (LGLs)” usually refers to NK cells, because up to __% of LGLs function as NK cells.
The term “large granular lymphocytes (LGLs)” usually refers to NK cells, because up to 75% of LGLs function as NK cells.
What does the term “left shift” mean?
Left shift refers to an increased percentage of immature granulocyte forms in the peripheral blood, which may exhibit toxic granulations (prominent primary granules) and Dohle bodies (prominent secondary granules) in response to severe infections. The presence of myelocytes or even less mature granulocyte forms in peripheral blood should raise the question of an underlying hematologic malignancy or severe trauma.
What are leukemoid reactions?
Leukemoid reactions represent exaggerated leukocytosis (typically 50,000–100,000/mL) and may include in the peripheral blood all recognizable stages of neutrophil maturation, that is, from myeloblasts to mature granulocytes. Leukemoid reactions typically last hours to days and may be caused by either benign or malignant conditions. A leukoerythroblastic reaction caused by myelophthisis is similar (but the total WBC does not need to be high) and also includes nucleated RBCs.
What clinical situations can leukoerythroblastosis be seen in?
Leukoerythroblastosis indicates severe disruption of the marrow by overwhelming infection, myelofibrosis, or bone marrow invasion by cancer, and may be associated with extramedullary hematopoiesis. A leukoerythroblastic reaction in infants can occur with severe hemolytic anemia (eg, erythroblastosis fetalis) or the rare bone disorder, osteopetrosis, in which failure of osteoclasts to resorb bone causes loss of hematopoietic marrow space and resultant extramedullary hematopoiesis.
What does the term “hyperleukocytosis” refer to?
Hyperleukocytosis refers to a WBC count greater than 100,000/mL, and is seen almost exclusively in leukemias and myeloproliferative disorders.
What is leukostasis?
Leukostasis, or sludging of WBC in small vessels of the brain, lungs, and kidneys, is an oncologic emergency that may cause life-threatening cerebral infarcts, cerebral hemorrhage, or pulmonary insufficiency caused by impaired blood flow. It is most common with acute myelogenous leukemia.
In which leukemia is leukostasis seen most commonly in?
Leukostasis is more common in acute myelogenous leukemia than in acute lymphoblastic leukemia, because myeloblasts are larger and more adhesive than lymphoblasts; it is rarely seen in chronic leukemias, even with extremely high WBC counts.
How does asplenia result in an increased WBC count? How does splenomegaly result in a relative lymphocytosis?
Because the normal spleen retains a large number of leukocytes, asplenia is associated with an increased WBC count. Splenomegaly may cause relative lymphocytosis as a result of splenic sequestration of granulocytes.
How do corticosteroids result in an increased WBC count?
Corticosteroids, which demarginate granulocytes, decrease neutrophil release from the marrow, and reduce neutrophil egress from the circulation, frequently cause leukocytosis.
Which infectious processes can result in a leukemoid reaction, and which can result in leukopenia?
Some patients with Clostridium difficile infection or tuberculosis may manifest a leukemoid reaction with a WBC count greater than 50,000/mL. Conversely, typhoid fever, brucellosis, tularemia, rickettsial diseases, ehrlichiosis, leishmaniasis, and some cases of Staphylococcus aureus infection may be associated with leukopenia.
Infectious lymphocytosis (generally 20,000–50,000/mL small, mature-appearing lymphocytes) is mainly a disease of children. What infectious entities can cause it?
It may be related to coxsackievirus A or B6, echovirus, and adenovirus 12, and is rarely associated with splenomegaly or lymphadenopathy. Infection with EBV can cause atypical lymphocytosis (large and reactive lymphocytes with abundant basophilic cytoplasm) and lymphadenopathy. HTLV-1 may produce a transient lymphocytosis (usually <20,000/mL) with fever, rash, and lymphadenopathy. In contrast to most other bacterial infections, pertussis (whooping cough) is frequently accompanied by lymphocytosis.
Familial neutrophilia, an autosomal dominant disorder of prominent leukocytosis (>20,000/mL), splenomegaly, and widened diploe of the skull, is caused by a mutation in the ___ gene.
Familial neutrophilia, an autosomal dominant disorder of prominent leukocytosis (>20,000/mL), splenomegaly, and widened diploe of the skull, is caused by a mutation in the G-CSF receptor gene (CSF3R). Neutrophils in this disorder are functionally normal and the leukocytosis has no clinical consequences.
Pelger-Huet anomaly (PHA) is characterized by mature neutrophils with bilobed nuclei, rather than the characteristic multilobed nuclear morphology. PHA is caused by a mutation in the ___ gene.
Pelger-Huet anomaly (PHA) is characterized by mature neutrophils with bilobed nuclei, rather than the characteristic multilobed nuclear morphology. PHA is caused by a mutation in the lamin B receptor gene. Neutrophil function in PHA is normal, but automated cell counters may indicate a left-shifted WBC because they mistakenly classify the cells as immature granulocytes.
Transient myeloproliferative disorder (TMD) is seen in up to __% of patients with Down syndrome (trisomy 21). TMD may also be seen in patients with trisomy 21 mosaicism who are phenotypically normal. TMD is characterized by peripheral blood leukocytosis in early infancy, and may include circulating myeloblasts in association with an accumulation of megakaryoblasts in the blood, liver, and marrow. TMD typically persists for several weeks and resolves spontaneously in most patients, but up to __% of affected patients later develop acute megakaryoblastic leukemia.
Transient myeloproliferative disorder (TMD) is seen in up to 10% of patients with Down syndrome (trisomy 21). TMD may also be seen in
patients with trisomy 21 mosaicism who are phenotypically normal. TMD is characterized by peripheral blood leukocytosis in early infancy, and may include circulating myeloblasts in association with an accumulation of megakaryoblasts in the blood, liver, and marrow. TMD typically persists for several weeks and resolves spontaneously in most patients, but up to 30% of affected patients later develop acute megakaryoblastic leukemia.
What are reticulated platelets?
Reticulated platelets (retPLTs) are platelets that are newly released from bone marrow megakaryocytes and still contain RNA. They have a short lifespan in the circulation and therefore reflect current megakaryopoietic activity, exactly comparable with reticulocytes and erythropoiesis. Reticulated platelets have clinical utility because they can help distinguish bone marrow failure from peripheral destruction in thrombocytopenia. Furthermore, retPLTs are an early predictor of bone marrow recovery after chemotherapy and transplantation.
Dx of polycythemia vera can be made when JAK2 V617F or exon 12 mutation is detected, along with increased hemoglobin and low or normal levels of erythropoietin. Clinically, what differences are seen in those with V617F mutation and in those with exon 12 mutation?
Clinically, patients with exon 12 mutations typically present with isolated erythrocytosis and suppressed erythropoietin, in contrast to the trilineage hyperplasia characteristic of patients with V617F mutation. Bone marrow from patients with exon 12 mutation often exhibits nonspecific morphology, with isolated erythroid proliferation and absence of prominent megakaryocyte atypia and clustering. Demonstration of exon 12 mutation in these patients is particularly helpful for ruling out reactive erythrocytosis.
What 2 mutually exclusive mutations are seen in virtually all cases of polycythemia vera?
JAK2 V617F (~96%) or JAK2 exon 12 mutations (~3%).
True or false. JAK2 and MPL mutations are completely specific for MPN.
False. Other hematological neoplasms (eg, myelodysplastic syndrome, chronic myelomonocytic leukemia, acute myeloid leukemia, and acute lymphoblastic leukemia) may harbor JAK2 and MPL mutations in low frequencies. Because JAK2 and MPL mutations are not completely specific for MPN, finding of these mutations in isolation does not warrant a diagnosis of MPN.
JAK2 gene. Chromosome? What does it encode? What happens with mutation?
The JAK2 gene maps to chromosome band 9p24 and encodes a tyrosine kinase protein composed of 1132 amino acids. It contains three critical domains: JH1, JH2, and four-point-one, ezrin, radixin, moesin (FERM) homolog domains. JAK2 protein kinase activity is activated by phosphorylation of its kinase domain. Activation of JAK2 induces signal transduction from both type 1 and type 2 cytokine receptors. Constitutive activation of JAK2 by either point mutation or fusion protein causes activation of the JAK/STAT pathway. The activated JAK2 causes phosphorylation of STATs, which then dimerize and translocate to the nucleus, where they regulate gene transcription. The constitutive tyrosine phosphorylation activity promotes cytokine hypersensitivity.
MPL (myeloproliferative leukemia virus oncogene) gene. Chromosome? What does it encode? What happens with mutation?
The MPL gene maps to chromosome band 1p34 and encodes the thrombopoietin receptor, which binds to thrombopoietin, the primary cytokine that regulates megakaryocyte development and platelet production, as well as hematopoietic stem cell homeostasis. Binding to thrombopoietin to MPL leads to activation of JAK2, which phosphorylates MPL and initiates a cascade of downstream signaling events that regulate cell survival, proliferation, and differentiation. The mutation W515L results in impaired function of the autoinhibitory region and subsequent ligand-independent thrombopoietin receptor activation. This then leads to subsequent activation of downstream tyrosine kinases and activation of transcription factors STAT3 and STAT5, which in turn leads to transformation of hematopoietic cells into cytokine-independent clones, resulting in megakaryocytic hyperplasia and marrow fibrosis. The mutation Y252H, which is located in the extracellular domain of MPL, confers hypersensitivity to thrombopoietin and increases the generation of megakaryocyte colonies in vitro and leads to increased thrombopoietin signaling and cell growth and survival.
In unconjugated hyperbilirubinemia… If the step affected is heme conversion to unconjugated bilirubin, the pathologic process is ___. If the step affected is delivery of unconjugated bilirubin to liver, the potential pathologic processes are ___. If the step affected is uptake of unconjugated bilirubin into hepatocyte, the potential pathologic processes are ___. If the step affected is conjugation of bilirubin in hepatocyte, the potential pathologic processes are ___.
In unconjugated hyperbilirubinemia… If the step affected is heme conversion to unconjugated bilirubin (increased production), the pathologic process is extravascular hemolysis. If the step affected is delivery of unconjugated bilirubin to liver, the potential pathologic processes are blood shunting (cirrhosis) or right heart failure. If the step affected is uptake of unconjugated bilirubin into hepatocyte, the potential pathologic processes are Gilbert syndrome and drugs such as rifampin. If the step affected is conjugation of bilirubin in hepatocyte, the potential pathologic processes are Crigler-Najjar syndrome and hypothyroidism.
What are 6 general causes of unconjugated neonatal hyperbilirubinemia?
Physiologic jaundice. Breast milk jaundice. Polycythemia. Hemolysis (HDN, hemoglobinopathies, inherited membrane or enzyme defects). Bowel obstruction (Hirschprung disease, cystic fibrosis, ileal atresia). Inherited disorders of bilirubin metabolism (Gilbert syndrome, Crigler-Najjar syndrome).
What is macroamylasemia?
A benign acquired condition (associated with celiac disease, lymphoma, HIV infection, monoclonal gammopathy, rheumatoid arthritis, and ulcerative colitis) with an incidence of ~1%, in which apparently healthy individuals have markedly elevated serum amylase levels (with low urine amylase levels), due to Ig-amylase complexes.
What 2 forces act on proteins during electrophoresis?
Electromotive and endosmotic. When a charge is applied (from a power source), this creates an electromotive force. The solid support has a slight negative charge and is drawn toward the positive pole (anode), but, being a solid support, it cannot move. There is instead a compensatory flow of the fluid buffer towards the negative pole (cathode). This buffer flow is called endosmosis and has the capacity to carry with it substances suspended within the medium. Since most proteins bear a net negative charge, electromotive force tends to pull them to the anode (positive pole), whereas endosmosis pulls them toward the cathode (negative pole). In most proteins, the electromotive force exceeds the endosmotic force, and they move to variable extents towards the anode. However, gamma globulins have a weak net negative charge and the endosmotic force displaces them toward the cathode.
What are the units for MCV, MCH, and MCHC?
MCV is the volume (in fL = 10^-9 uL = 10^-15 L) of the average circulating red blood cell. MCH is the hemoglobin content (in pg/red cell) of the average circulating red blood cell. MCHC is the hemoglobin concentration (in g/dL) within circulating red blood cells (grams of hemoglobin per 100 mL of packed red blood cells).
In impedance counting for red blood cell indices, what are size thresholds for RBCs, WBCs, and platelets?
Particles with a volume 36-360 fL are counted as RBCs; leukocytes are within this size range but their relative number is usually so small that the effect is negligible. For counting WBCs, the RBCs are lysed, and particles larger than 36 fL are counted as WBCs. Particles measuring 2-20 (or 30) fL are counted as platelets.
What techniques are available for enumerating reticulocytes?
Manual counting by light microscopy, optical light scatters, and flow cytometry. In the first two methods, red cells are stained with a supravital dye (such as new methylene blue or azure B) which highlight residual cytoplasmic RNA. In flow cytometry, the cells are stained with an RNA-specific fluorochrome (such as Auramine O or thiazole orange).
What are 2 tests for rapid detection of sicking hemoglobin?
The hemoglobin solubility (dithionate) test detects insoluble forms of hemoglobin withint a lysate of blood. The sickling (metabisulfite) test detects red cells with sickling hemoglobins.
Describe the hemoglobin solubility (dithionate) test.
The hemoglobin solubility (dithionate) test detects insoluble forms of hemoglobin withint a lysate of blood. In this test, red cells are lysed in sodium dithionate buffer with saponin. After several minutes, marked turbidity indicates a positive screen. Note that this test detects free hemoglobin with altered solubility. Like the sickling (metabisulfite) test, a positive result indicates sickling hemoglobin, but gives no information on genotype and may be positive in SS, SA, SC, SD, and C harlem.
Describe the sickling (metabisulfite) test.
The sickling (metabisulfite) test detects red cells with sickling hemoglobins. In this test, whole blood is subjected to metabisulfite, which encourages cells containing HbS to sickle. A smear is then examined microscopically for sickling. Like the hemoglobin solubility (dithionate) test, a positive result does not give genotypic information and may be positive in SS, SA, SC, S-other, and C harlem.
What can give false negative results for the hemoglobin solubility (dithionate) test and the sickling (metabisulfite) test?
The hemoglobin solubility (dithionate) test may be negative when the concentration of HbS is too small, such as in neonates. The sickling (metabisulfite) test requires at least 10% HbS to be positive, so may be negative in neonates or in those very aggressively transfused.
What are the following erythrocyte inclusions composed of: Howell-Jolly bodies, Heinz bodies, Pappenheimer bodies, basophilic stippling, hemoglobin H inclusion.
Howell-Jolly bodies - DNA fragments. Heinz bodies - Denatured, precipitated hemoglobin (visible only with supravital stain). Pappenheimer bodies - Iron-containing inclusions. Basophilic stippling - Ribosomal RNA. Hemoglobin H inclusion - Precipitated beta-hemoglobin chains (visible only with supravital stain).
What is seen at the alpha-2/beta interface in high resolution SPEP?
This area is usually empty, but hemoglobin, usually absent from serum, may be present here when there is a hemolysis - a possible pseudo-M-spike.
Does haptoglobin bind myoglobin?
No. Haptoglobin is a protein that binds free hemoglobin.
What are the 3 possible haptoglobin phenotypes? Is there a disease association with any particular phenotype?
The haptoglobin genetic locus at 16q22 is polymorphic with two classes of alleles: type 1 and type 2. Based on these 2 alleles, the 3 haptoglobin phenotypes/protein products are: Hp 1-1, Hp 2-1, and Hp 2-2. The 2-2 phenotype is an independent risk factor for CV disease in DM.
What is the half-life of haptoglobin? What is the half-life of haptoglobin-hemoglobin complex? How is the haptoglobin-hemoglobin complex removed from circulation?
The half-life of haptoglobin is 3.5 days, and the half-life of the haptoglobin-hemoglobin complex is ~10 minutes. The haptoglobin-hemoglobin complex is removed by binding to the CD163 receptors on the surface of macrophages and monocytes.
In ~__% of sera with an M-protein, two M-proteins are present (biclonal gammopathy).
In ~5% of sera with an M-protein, two M-proteins are present (biclonal gammopathy). Biclonal gammopathies are typically detected when two different heavy or light chain classes of M-proteins are detected on immunofixation (eg, an IgA kappa M-protein and an IgM lambda M-protein in the same patient). Sometimes, the presence of monomers and polymers of a single M-protein may produce two spikes on the SPEP and can resemble a biclonal gammopathy (this should be considered monoclonal rather than biclonal).
What are monoclonal gammopathies? What are M-proteins?
The monoclonal gammopathies (paraproteinemias or dysproteinemias) are a group of disorders characterized by the proliferation of a single clone of plasma cells, which produces an immunologically homogeneous immunoglobulin commonly referred to as a paraprotein or monoclonal protein (M-protein or M-component, where the “M” stands for monoclonal). The M-protein can be an intact immunoglobulin (ie, containing both heavy and light chains) or can be composed of only light chains or (rarely) only heavy chains.
