exam 3 Flashcards
Precursor B lymphoblastic leukemia/ lymphoma
a neoplasm of lymphoblast’s committed to the B - cell lineage
B-LBL vs B-ALL
B-LBL: lymphoma mass, with or without minimal blood and BM involvement
Precursor B lymphoblastic leukemia epidemiology
B-LBL: 10% of lymphoblastic lymphoma, 75% of cases in patients under 18 years old
B-ALL: 75% of cases in children under 6 years old
Clinical features of B-ALL and B-LBL
B-ALL: WBC decreased, normal or markedly elevated, Anemia, thrombocytopenia, lymphadenopathy, hepatosplenomegaly, bone pain, arthralgias
B-LBL: skin, bone, soft tissue, and lymph node
Sites of involvement and morphology in Precursor B LL
B-ALL: blood and bone marrow in all cases
B-LBL: skin, bone, soft tissue, and lymph node, rare in mediastinal mass
Immunophenotype in Precursor BLL
TdT, HLA-DR
CD19, CD79a, CD10, CD24
t(4;11)(q21;q23) cases are typically negative for CD10 and CD24
Variably positive for CD20 and CD22
CD45 may be negative
Cytoplasmic Mu chain in pre-B ALL
Precursor BLL genetics
t(9;22)(q34;q11.2): 3-4% of cases, in most childhood cases associated with 190 kd, BCR/ABL fusion tyrosine kinase, unfavorable prognosis
t(4;11)(q21;q23): associated with AF4/MLL, 2-3% of cases, unfavorable prognosis
t(1;19)(q23;p11.3): associated with PBX/E2A. 6% of cases( 25% of pre - B ALL), unfavorable prognosis
t(12;21)(p13;q22): associated with TEL/AML-1, 16-29% of cases, favorable prognosis
Hyperdiploidy vs. Hypodiploidy
Hyperdiploidy: 20-25% of cases, favorable prognosis (>50)
Hypoploidy: 5% of cases, unfavorable prognosis (<50)
B - LL Differential diagnosis
Precursor T-ALL and AML: resolved with immunophenotyping
Hematogones ( normal B precursors): mimic blasts morphology, positive for TdT, CD34, CD10, pan B markers, in very young patients or in older patients with neuroblastoma, post - chemo, iron deficiency, ITP
Burkitt lymphoma
Blastoid variant of mantle cell lymphoma (positive for bcl-1, negative for TdT
Prognosis of B-LL
B-ALL: good prognosis in the pediatric group, 80% of patients cured, poorer diagnosis in adult group with more unfavorable genetic results
B-LBL: median survival of 60 months
Precursor T lymphoblastic leukemia/lymphoma
A neoplasm of lymphoblast committed to the T-cell lineage
lymphoma vs. lymphoblastic leukemia
Lymphoma: mass, without or minimal blood and BM involvement
Lymphoblastic leukemia: extensive BM and blood involvement (>25% BM cells)
Precursor T-LL epidemiology
15% of childhood ALL
25% of adult ALL
80-95% of lymphoblastic lymphoma
More in adolescents and males
Clinical features of T-LL
Leukemia: high WBC
Lymphoma: large mediastinal mass ( or other tissue mass), rapid growth, pleural fluid involvement
T-LL sites of involvement
Leukemia: blood and BM, all cases
Lymphoma: mediastinal mass: 50%, others include LN< skin, liver, spleen, Waldeyers ring, CNS, gonads
Morphology of T-LL
Similar to B-ALL/ LBL
High number of mitotic figures
A small number of LBL cases wiht eosinophilia, myeloid hyperplasia, some associated with t(8;13)(p11.2;q11-22), some of them developed myeloid malignancy
Cytochemistry of T-LL
acid phosphatase, TdT, PAS: nuclear
T-LL immunophenotype
TdT positive, cCD3 (the only lineage specific marker), CD4,8: double negative or positive, Variable surface: CD1a,2,3,4,7,10,79a,13,33,117( rare), TCR: may have rearrangement, not lineage specific
T-LL genetics
TCR loci ( 1/3 of T-ALL): 14q11.2 (alpha, delta), 7q35( beta), 7p(14-15)(gamma)
Genes:MYC (8q24.1): TAL (1p32), RBTN1(LMO1)(11p15), RBTN2(LMO2)(11p13), HOX11(10q24), LCK(1p34.3-35)
T-LL differential diagnosis
B-ALL and AML(M0): by immunophenotyping, not morphology
Hematogones: mimic blasts
Burkitt
Blastoid variant of mantle cell lymphoma
T-LL prognosis
survival comparable to B-ALL with current treatment
Fetal RBS analysis by Flow Cytometry
test performed in order to quantitate circulating fetal red cells in maternal blood, test indications are placental injury from trauma or rh incompatibility, usually performed to be a quantitative confirmation for the fetal screen done in blood bank.
Fetal RBC procedure
moms blood is drawn and a CBC to determine amount of blood to be used, blood and 3 levels of controls, RBC membranes are fixed using glutaraldehyde, cells are washed, holes are punched in the cell membrane using triton, cells are incubated with a cytoplasmic (intracellular) monoclonal antibody to fetal hemoglobin
Fetal RBC equations
% of fetal RBC’s = # of fetal cells counted/ # mom cells counted x 100
mls of bleed = % of fetal cells x 50
Rhogam
Given to any Rh - mom who has a Rh+ baby in order to prevent hemolytic disease of the newborn (HDN)
OSF gives 1 vial of Rhogam for every 30mls of fetal bleed and then we add one additional vial
Direct Immunofluorescence ( lymphocyte subsets)
direct immunofluorescence is used to diagnose or follow patients with immunodeficiency or states
done by using a small amount of blood with fluorochrome - labeled antibodies
performed on the Aquois CL
Performed on whole blood that must be kept at room temp as refrigeration falsely decreases CD4
Lymphocyte subsets
Human lymphocytes can be divided into 3 sub - populations: T cells, B cells, and NK cells, based on their function and cell surface antigens
T cells participate in antigen specific cell - mediated immunity and regulate immunoglobulin secretion by B cells
B - cell produce immunoglobulin
NK cells are in themselves cytotoxic
Test for 6 cel surface antigens and report the percentage and absolute values
CD3, CD4, CD8 ( T- cells)
CD19 (B-cells)
CD16, CD56 (NK cells)
Lymphocyte subset procedure
A small amount of whole blood and a monoclonal cocktail labeled with FITC, RD1, ECD, and PC5 fluorochromes: Tetra 1 and Tetra 2
Blood and monolconal antibody are added to a well on a well plate incubated
The RBCs are lysed but not washed using the A and B reagents
WBCs are analyzed via flow cytometry using light scatter, fluorescence, and EV
The aquois is closed, load and go system
Why lymphocyte subsets are performed
To determine a person’s immune status: HIV patients, oncology patients, patients taking immunosuppressive drugs, immunodeficiency disorders
CD4 most commonly monitored marker in HIV patients
When CD4 less than 14% and absolute less than 200 the HIV patients is then considered to have AIDS, IDPH also notified.
HLA-B27
MHC class 1 molecule, cell surface glycoprotein
Presence of HLA-B27 on surface of T- cells contributes to immune system dysfunction
Clinical significance ( inflammatory autoimmune disorders known as spondyloarthopathies)
Most studied B allele due to its association with these disorders
Fluorochrome labeled antibodies target a specefic cell surface antigen
HLA - B27 test performance
50 ul of whole blood is added to a 30 ul of antibody
controls used
Results interpreted as either positive, negative, or indeterminate
Any result within 10 channels of the decision marker line on the positive side will be sent for confirmation to rule out interference from another antigen.
Flow cytometry definition
technique used for counting, examining, and sorting microscopic particles suspended in a stream of fluid. It allows simultaneous multi- parametric analysis of the physical and chemical characteristics of single cells flowing through an optical and electronic detection apparatus.
measurement of cellular properties as they move in a fluid past a set of detectors, measures in fluorescence using a laser
Fluidics in flow cytometry
Sheath fluid: buffer saline is usually used
Specimen to be analyzedz: injected into the center of the sheath fluid, forced hydrodynamic focusing for passage of cells one by one past the laser.
Flow Cytometry Optics
Laser light source: excitation of fluorescent antibody tag on or in the cell
Lenses, filters, and mirrors: separate light into different colors, send to photodetectors
Photodetectors: Photodiode: FALS - forward angle light scatter ( measure the size of the cell
- Photomultiplier: measures granularity or complexity of the cell
Electronics
Photoconductors convert photons of light to electronic signal ( voltage)
Impulse is related to intensity of light
Electrical response altered by: applying voltage across PMT, adjusting gain of amplifiers
Data stored and displayed from individual cells as events
Test specific quality control
Lymphocyte subsets - low and normal
Fetal RBC - negative, low and high
HLA-B27: positive and negative
Leukemia/lymphoma: check known positive and negative fluorescence for each antibody used that day and monthly
L/L specimen requirements
Blood, Bone marrow, tissue, body fluid (except urine), FNA (fine needle aspirate)
L/L specimen preparation
Slides of specimen are stained and analyzed, specimens are processed to leave only WBCs, viability count is performed (except blood), WBCs are stained and incubated with monoclonal antibodies, cells are washed, cells are re-suspended and run on the cytometer
L/L staining
monoclonal antibodies are added to the cell suspension to detect specific antigens on or in the cell.
Monoclonal antibodies are antibodies to cellular antigens covalently bonded to a fluorochrome
Fluorochromes are excited by the laser light and the attached antibody fluoresces
OSF uses 10 fluorochromes, FITC, PE,ECD,PC5.5,PC7, APC, APC-A700, APC - 750, PB, KrO
L/L specimen analysis
Cell suspension placed on cytometer, cells pass by laser one by one, electronic signals are received from photodetectors, points are plotted on a histogram, cells of interest are gated, results are interpreted by the hematopathologist
Clinical applications of flow cytometry
CD4+ enumeration, CD34+ enumeration, detection and enumeration of fetal RBC, enumeration of WBC in banked blood, tissue and RBC typing, Neutrophil oxidative burst/ detection CGD, Diagnosis of PNH, Diagnosis of mature lymphoid neoplasms, diagnosis of acute leukemia, diagnosis of mature myeloid neoplasms, DNA ploidy, endless things.
Paroxysmal Nocturnal Hemoglobinuria (PNH)
acquired hematologic disorder characterized by nocturnal hemoglobinuria, chronic hemolytic anemia, thrombosis, pancytopenia, and occasionally acute or chronic myeloid malignancies such as aplastic anemia.
Tri - lineage hematopoietic stem cell disorder meaning it affects all cell lines present in the bone marrow
The abnormal cells (clones) in PNH fully or partially lack glycosylphosphatidylinositol linked proteins.
Caused by a somatic mutation in the phosphatidylinositol glycan A gene, PIGA.
PNH testing
flow cytometry replaced the sucrose hemolysis test and the hams test:
- RBCs placed into a low ionic strength solution and observed for hemolysis. Flow is the gold standard and can detect the presence or absence of GPI - linked proteins on RBCs, WBCs and platelets
Patients with PNH are missing RBC GPI - anchored complement regulatory proteins such as CD55 and CD59 which cause increased suspectibility to complement mediated lysis.
Organization of immature and mature WBC
Lymphoid: mature - mature lymphoproliferative disease. immature - lymphoblast’s (ALL)
Myeloid: mature - MPN (CML, other MPNs), MDS, overlap syndrome. immature - myeloblasts (AML, excess blasts, accelerated phase)
Chronic myeloid leukemia
leukocytosis with absolute neutrophilia and left shift maturation, absolute basophilia and eosinophilia, thrombocytosis, bone marrow hypocellularity with granulocytic proliferation, cytogenic t(9;22)(q34;11), molecular products (BCR/ABL fusion gene, fusion mRNA)
Organization of mature lymph
B lineage: Hodgkin - classic and NLP hodgkin, Non - hodgkin: matue b - cell lymphoma/ leukemias, plasma cell neoplasms
T lineage: Hodgkin - 0, non - hodgkin - mature t cell lymphoma/leukemias
