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600 Cards in this Set

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Most common method of hemoglobin measurement?
Cyanohemoglobin method
In cyanohemoglobin method, what substance is measured by spectrophotometry?
Hemiglobin cyanide (HiCN)

What is the intermediate produced during this reaction?
Hemiglobin

(Dissolution of hemoglobin in Potassium ferricyanide and potassium cyanide oxidizes it to hemiglobin, which is then complexed to cyanide)
At What wavelength is HiCN absorbed?
540 nm

What types of hemoglobin can be detected by HiCN method?
All except sulfhemoglobin (SHb)

(Includes Hb, HbO2, Hi, HbCO)
Centrifuging a tube of whole blood can be performed to calculate what parameter?
Hematocrit

What are the units of measurement for hematocrit?
None! The units cancel out during calculation!
Calculate MCV:
MCV= (Hct x 1000) / RBC

where RBC equals number of RBCs
Calculate MCHC:
MCHC = Hb / Hct
Which RBC parameters are typically measured by automated techniques?
red cell count (RBC), MCV, RDW, hemoglobin

Other parameters are calculated
What are the three methods that automated instruments employ to count cells?
Impedence (size), Optical: forward and side scatter, or combination of these two
What electrical measurement is being analyzed in the impedence method?
Voltage (V = current x resistance)

generated when cell passes through a charged aperture. The cell increases resistance.
Which cells are often included in the red blood cell count if impedence-only methodology is used?
Leukocytes (due to similar size), but typically the number is low enough to not matter
What is RDW a measure of?
The variation in size of RBCs, due to measuring the width of the Gaussian curve of red blood cell size.
Calculate Hematocrit:
Hct = MCV x RBC
Calculate MCHC:
MCHC = Hb / (Hct x 100)
Can leukocytes be counted by impedence?
Yes

What extra step must occur to do so?
Lysis of RBCs
In producing a leukocyte differential, what three measurements help make the scatterplot?
Impedence, Conductivity, and Side Scatter

What are these parameters measuring?
Respectively:

Volume/size, cell complexity, cytoplasmic granularity
Can platelets be measured by impedence?
Yes

What size is usually targeted for platelets?
2-20 fL
3 Causes of erroneously platelet counts in automated techniques?
Clotted specimens, large platelets, clumped platelets

What are 2 causes of clumped platelets?
Heparin

EDTA-Ab interactions
What are all techniques for reticulocyte count based on?
The measurement of ribosomal RNA, which is in abundance in retics
Three ways to measure retics:
Light microscopy, optical light scatter, flow cytometry

What step must be performed for the first two methods?
Stain with supravital dye (methylene blue, azure B) highlighting rRNA.
Which is more sensitive for measuring reticulocytes, automated or manual methods?
Automated

And which methods are the automated ones?
Optical light scatter and flow cytometry
High reticulocyte counts can increase which RBC parameter?
MCV

(They are larger than mature RBCs)
Calculate absolute reticulocyte count:
RBC count x percent reticulocytes
What is the corrected reticulocyte count?
Takes into account spurious increases in retics due to low Hct.

Calculate corrected reticulocyte count:
CRC = %retics x Hct/45
What is the RPI?
Reticulocyte production index: takes into account that in anemia, retics are released earlier than normal and take longer to mature in the circulation.

Calculate RPI:
RPI = CRC x 1/correction factor

Correction factors:
1 Hct normal
2 Hct is 30
3 Hct is 15
Calculate absolute retic count
Absolute retic count = % retics x RBC count
What is hemoglobin solubility test used to detect?
Insoluble forms of hemoglobin, like sickling hemoglobins

How is it performed?
Red cells are lysed with sodium dithionate and saponin. Turbidity is a positive result.
What does the sickling test detect?
Sickling hemoglobins

What is another name for this test?
Metabisulfite test
How is sickling test performed?
Whole blood is exposed to metabisulfite, causing HbS cells to sickle. A smear is then performed.
Which types of hemoglobin may be detected by a metabisulfite test?
SS, SA, SC, S-other, C harlem

Shortcomings of the sickling test?
need at least 10% HbS to work

(may be false neg if heavy transfused, neonates)
What is used to detect fetal hemoglobin?
Acid elution test

How is it performed?
HbA elutes from cells in acid buffer, but HbF does not. Cells that stay pink on staining after acid buffer exposure are HbF.
What are the two patterns of eosinophilia that may be seen with performance of the acid elution test?
Heterocellular (some cells) and Pancellular (all cells)

When are these seen?
Heterocellular-thalassemias, other causes of HbF

Pancellular-Hereditary persistance of HbF
How is the quantitative test for HbF performed?
HbF is resistant to alkali denaturation. HbA is precipitated out, then the optical density of remaining hemoglobin reflects HbF

Additional technique that can be used?
High pressure liquid chromatography
Where is hemoglobin A found on alkaline electrophoretic gel?
Near anode (fast moving)
What is the band near the the origin of alkaline electrophoresis?
Carbonic anhydrase
What is a "fast hemoglobin?"
One that migrates past HbA

What might it be?
HbH or HbBarts
What material is the gel made of in Alkaline electrophoresis?
Cellulose acetate

What is the pH?
8.6
What are the order of the bands on an alkaline electrophoresis gel?
- C S F A +
What bands can appear in the S region?
D, G, Lepore, S

Help in confirmation of HbS can be attained with:
Sickle screen test (metabisulfite)
What changes are seen on a gel with thalassemia?
None!

This is a quantitative disease. Gel looks normal!

What changes in a CBC could indicate thal?
Low MCV, increased RBC count
What is acid electrophoresis?
Gel electrophoresis performed at pH of 6.2 on citrate agar.

What is the order of bands on the gel?
- F A S C +
What is high pressure liquid chromatography used for in regards to variant hemoglobins?
Further resolution of hemoglobin types and quantitation
Mobile phase of HPLC?
Sample itself that will be pushed through column

What is the stationary phase of HPLC?
Numerous small particles packed in the column that will help elute out the variants
What can we do if a variant Hb cannot be resolved by electrophoresis or HPLC?
PCR for genetic evaluation
What is a Wright stain composed of?
Methylene blue dye with eosin and alcohol.

How do the components of cells dye?
Basic components (hemoglobin) attract eosin

Acid components (nucleic acids) attract methylene blue
Why must we use a buffer when performing Wright stain?
If too acidic, turns pinkish and can't see nuclear details. If too basic, the nucleus stains too darkly.
What does MPO stain?
Primary granules indicative of granulocytic differentiation
What is Sudan black B?
stains lipid material in granulocytic and monocytic cells
What is chloroacetate esterase?
Found in granulocytic series
What is non-specific esterase?
Stain monocytic series, also megakaryocytes, lymphocytic, granulocytic, and erythroid lineages to a lesser extent.
Examples of non-specific esterases?
alpha naphthyl acetate esterase and alpha naphythyl butyrate esterase.
What inhibits monocyte NSE activity?
sodium fluoride
When is PAS positive in hematolymphoid cells?
Most lymphoid and some myeloid blasts.
What is the pattern of PAS staining in ALL?
Block positivity, encircling the nucleus like rosary beads
What is the pattern of PAS staining in AML (if positive)?
diffuse and granular
What is a funny stain you can use in L3 ALL (Burkitts) to stain blasts?
Oil red O
What is Leukocyte Alkaline phosphatase mechanism that we exploit?
hydrolyzes naphthol AS-bisphophonate to form a colored product
What are we looking for with a Leukocyte Alkaline Phosphatase score?
CML
How many cells are counted?
100
Normal adults vs. CML LAP scores
Normal adults have higher numbers (more active granulocytes), while CML have cells with improper response to LAP and thus low scores
Other causes of low LAP scores?
PNH, MDS, Hypophosphatasia, neonatal sepsis (paradoxical--in adults, this elevates the LAP score)
What are causes of high LAP scores?
Leukemoid reactions, non-CML myeloproliferative reactions, steroid use, late pregnancy
What test do we use to detect fetal red cells?
Acid elution (Kleihauer-Betke)
What principle is at work in the Kleimauer-Betke test?
HbA is eluted out in acid. Hemoglobin F remains and can be stained with eosin.
In fetomaternal hemorrhage, is the staining pattern pancellular or heterocellular?
Heterocellular (some stain, some don't)
Granulocytic series staining patterns:

MPO, Sudan black B, CAE, NSE, PAS
MPO: +
SBB: +
CAE: +
NSE: variable, but less
PAS: if positive, then diffuse granular
Lymphoid series staining pattern:

MPO, SBB, CAE, NSE, PAS
MPO: neg
SBB: neg
CAE: neg
NSE: variable, but less
PAS: in ALL, rosary pattern around nucleus
Monocytic series staining pattern:

MPO, SBB, CAE, NSE, PAS
MPO: neg, occasionally very fine positive
SBB: pos
CAE: neg
NSE: pos
PAS: neg
Which types of AML show Auer rods--FAB classifications?
M1, M2, M3 ***, M4, M6
What does forward scatter tell us on flow cytometry?
Size
What does side scatter tell us on flow cytometry?
Nuclear complexity/cytoplasmic granularity
What is a fluorochrome?
Substance that absorbs light
What is the Stokes shift?
The difference in absorbed light (for telling size/complexity) and the emitted light (fluorochrome associated)
What is FITC?
fluorescein isothiocyanate
What is PE (in regards to flow cytometry)?
Phycoerythrin (a fluorochrome)
Are all antibodies used in fresh tissue flow cytometry available for tissue immunohistochemistry?
No
Place in order of CD45 expression (from dim to brightest):

Lymphocytes, Monocytes, Granulocytes, Stem cells (blasts), Erythrocytes
Erythrocytes (almost none) -> Blasts -> Granulocytes -> Lymphocytes/Monocytes (both bright)
What is TdT?
A marker of early lymphoid development

(Terminal Deoxynucleotide Transferase)

Expressed in what cells?
Precursor B and T cells
List Lymphoid stem cell flow markers:
CD34+, TdT+, HLA-DR +
List Pro-B cell flow markers:
CD34+, TdT+, HLA-DR+, CD19+, CD10+, Ig Heavy chain rearranging
List Pre-B cell flow markers:
CD34 lost, TdT lost, HLA-DR+, CD19+, CD20+, cytoplasmic mu heavy chain and Ig light chain rearranging
List B cell flow markers:
CD34-, TdT-, HLA-DR+, CD19+, CD20+, Cd21+, CD22+, surface Ig +
List Plasma cell markers:
CD34-, TdT-, HLA-DR+, CD19 lost, CD20 lost, cytoplasmic (not surface) Ig, CD38+, CD138+, often CD79a+
What is the normal blood/tissue T-cell:B-cell ratio?
2-6 : 1
The expression of surface CD3 coincides with the expression of what other surface T-cell marker?
surface T-cell receptor (TCR)
The loss of CD4 or CD8 on T-cells is associated with the loss of what other antigen?
CD1
The normal CD4:CD8 ratio is:
2:1
When is CD38 re-expressed by T-cells?
T-cell activation
Which are more prevalent overall: Alpha-beta light chain T-cells or gamma-delta light chain T-cells?
Alpha-beta

But where are there higher number of gamma-delta T-cells?
Dermal, Intestinal, Splenic T-cell populations have larger gamma-delta populations
What flow markers are seen with Natural Killer cells (NK cells)?
No TCR, No Immunoglobulin, No CD3 (on flow, may be there on IHC), CD16+, CD56+

What is CD16?
The receptor for the Fc portion of gamma heavy chains
Myeloblasts express what flow markers?
CD34, HLA-DR, CD38, CD117, CD13, and CD33 are common
Promyelocytes express what flow cytometric phenotype?
CD34-, HLA-DR-, CD13+, CD33+, CD15+
Metamyelocytes pick up what flow marker?
CD11b
Promonocytes express what flow cyometric phenotype?
CD34-, CD11b+, CD15+

As they mature, what else is added?
CD64+ and CD14+
ALK is expressed in
anaplastic large cell lymphomas (ALCL) that are t(2;5)+.
Bcl-1 is expressed in
mantle cell lymphoma
Bcl-2 is normally expressed in
mantle cells and the small number of mantle cells that normally permeate the follicle center
the 2 diagnostic uses of Bcl-2
1.Bcl-2 is useful in distinguishing neoplastic from reactive germinal centers
2. separate Burkitt from Burkitt-like lymphomas
3. not useful to distinguish FL from other B-cell lymphomas.
1. + in neoplasms
2. - in Burkitt
3. use CD10 (for Follicular Lymphoma) for this purpose
Bcl-6 is normally expressed in
germinal center B-cells
Bcl-6 is expressed in these 4 lymphomas
Burkitt lymphoma,
follicular lymphoma (FL),
nodular lymphocyte predominant Hodgkin lymphoma (NLPHL),
a subset of diffuse large B-cell lymphoma (DLBCL).
cytogenetics for Bcl-6 overexpression
translocation involving the bcl-6 gene on chromosome 3q and the IgH gene on 14q.
CD1a is normally expressed by what 4 types of cells
Langerhans cells,
dendritic reticulum cells,
interdigitating reticulum cells,
cortical thymocytes.
CD2 is expressed by T-lymphocytes and NK cells (T/F)
TRUE
CD3 is expressed by T-lymphocytes and NK cells (T/F)
False, not by NK cells
question regarding the expression pattern (cytoplasmic vs. surface) in NK cells
Is CD3 surface or cytoplasmic?
Cytoplasmic expression precedes surface expression and may be detectable by immunohistochemistry (eg, in immature T cells and NK cells)
CD4 is normally expressed in what T-cells?
T helper
normal CD4:CD8 ratio
2-6:1
CD4+/CD8+ cells normally found in
thymic cortex
CD4−/CD8− cells found in
neoplasms
The majority of T-cell lymphomas and leukemias are CD4+ or CD8+?
CD4+
CD5 co-expression in B cells (2)
1. small lymphocytic lymphoma/chronic lymphocytic leukemia (SLL/CLL)
2. mantle cells lymphoma (MCL).
CD7 is expressed in NK cells (T/F)
TRUE
CD10 is expressed in
both normal and neoplastic follicle center cells.
CD11b (MAC-1) is normally expressed on
monocytes and granulocytes
neoplastic B cells expressing Cd11b
Hairy Cell Leukemia
CD11c (C3r) in neoplastic B cells
CLL/SLL and HCL.
CD13 is expressed in
granulocytic cells and precursors.
CD15 in anaplastic large cell lymphoma
negative
CD19 in neoplastic plasma cells
negative
CD20 expression is reduced or diminished in these B cells
1. plasma cells,
2. Reed-Sternberg cells (except in NLPHD),
3. anaplastic large cell lymphoma (a CD20+, CD30+ lymphoma is classified as a diffuse large cell lymphoma according to WHO).
4. Reduced in CLL/SLL
What is FMC-7
FMC-7 is an antibody that recognizes a particular conformation of CD20.
The significance of CD22
CD22 is expressed by normal and neoplastic B cells; this being the latest antigen acquired in B cell maturation, CD22 is seen in only a small percentage of B-ALL.
CD30 is normally found on (3)
plasma cells, immunoblasts (such as in viral (EBV) lymphadenopathy) and NK cells.
CD30 is abnormally found on (3)
Reed-Sternberg cells; anaplastic large cell lymphoma (ALCL) and embryonal carcinoma.
CD33 is expressed on
normal and neoplastic myeloid and monocytic cells
CD34 is expressed in what heme cells?
immature myeloid cells
CD41/CD61 are expressed in
normal and neoplastic megakaryocytic cells.
CD43 is normally expressed in
T cells
CD43 on B cells
MCL, SLL, and some cases of MZL (not commonly seen in FCL).
CD45 can be lost on these leukocytes (3)
myeloblast, plasma cells, Reed-Sternberg cells (except NLPHD).
CD45-RO expressed on
normal and neoplastic T cells.
CD56 is normally expressed in
NK cells, plasma cells, and neural cells
CD57 is expressed in
normal and neoplastic NK cells and most neural cells
CD59 expression is decreased in
paroxysmal nocturnal hemoglobinuria
CD79a is expressed in plasma cells (T/F)
T
besides PNET/EWING, CD99 is also expressed in (5)
lymphoblastic lymphomas,
granulosa cell tumors,
synovial sarcoma,
rhabdomyosarcoma,
solitary fibrous tumor
CD103 is a marker for
hairy cell leukemia
CD117 is expressed in (5)
GIST, melanocytes (particularly junctional), seminomas, progenitor myeloid cells (AML, CML) and mast cells (mastocytomas).
CD138 is only expressed on plasma cells (T/F)
FALSE, also in epithelial and mesenchymal cells
clusterin expression pattern in anaplastic large cell lymphoma
golgi
clusterin expression pattern in megakaryocytes
strong cytoplasmic
karyotypes are observed in what stage of the mitosis?
metaphase
chromosomal location and segments for the Ig heavy chain
chromosome 4, V, D, J, C
chromosomal location and segments for the Kappa light chain
chromosome 2, V, J, C
chromosomal location and segments for the lamda light chain
chromosome 22, V, J, C
the order of Ig gene arrangements (Kappa, Lamda, heavy chain)
heavy, kappa, lamda
The finding of only germline genes with no clonal rearrangements is indicative of
(i) a benign lymphoid proliferation, (ii) a lymphoid neoplasm composed of very early lymphoid cells, eg, some cases of ALCL, or (iii) nonlymphoid neoplasm.
compare the sensitivity and specificity of Soutern Blotting and PCR
PCR more specific, Southern Blot more sensitive
hallmarks fo the Hereditary spherocytosis
chronic hemolysis, jaundice, and splenomegaly
incidence of Hereditary spherocytosis
3.513888889
name defective cytoskeletal proteins in Hereditary spherocytosis
band 3, protein 4.2, spectrin, and ankyrin
The most characteristic CBC abnormality in Hereditary spherocytosis
increased MCHC
labs in hemolysis
increased LDH, bilirubin and reticulocytes
what are the lab tests for Hereditary spherocytosis
osmotic fragility test ; autohemolysis test
differential diagnosis for spherocytes in the a peripheral smear
immune-mediated hemolysis and HS. Usually a direct antiglobulin test (DAT) and low MCV will identify the former.
cytogenetics of Acute myelogenous leukemia (AML-M1)
t(9;22), 20% of cases
cytogenetics of Acute promyelocytic leukemia (APML)
t(15;17)(q22lq12-21)
Retinoic acid receptor (17) translocated to PML gene (15q); 90%
cytogenetics of Chronic myelogenous leukemia (CML)
t(9;22)(q34;q11)
Breakpoint cluster region (bcr) on 22; abl on 9; makes abnormal tyrosine kinase
cytogenetics of Chronic myelomoncytic leukemia (CMML)
t(5;10)(q33;q21)
Minority of cases
cytogenetics of Chronic eosinophilic leukemia (CEL)
T(5;12)(q33,p13)
PDGFRβ, and TEL
cytogenetics of Chronic lymphocytic leukemia (CLL)
del(13q), del(11q), +12, del(17p)
Only about 20% have a normal karyotype
cytogenetics of Lymphoplasmacytic leukemia (LPL)
t(9;14)(p13;q32)
Pax-5 and IgH
cytogenetics of MALT lymphoma
t(11;18)(q21;q21)
API2 and MLT
cytogenetics of Mantle cell lymphoma (MCL)
t(11;14)(q13;q32)
bcl-1 (PRAD-1; Cyclin D1) gene (11) translocated to Ig gene (14)
cytogenetics of Diffuse large B-cell lymphoma
t(3;14)(q27;q32)
bcl-6/IgH
cytogenetics of Follicular lymphoma (FL)
t(14;18)(q32;q21)
bcl-2 gene (18) translocated to Ig gene (14)
cytogenetics of Anaplastic large cell lymphoma (ALCL)
t(2;5)(p23;q35)
ALK and NPM
cytogenetics of Burkitt lymphoma
t(8;14)(q24;q32), t(2;8), and t(8;22)
c-myc gene (8) translocated to Ig gene (14), κ gene (2), or λ gene (22)
cytogenetics of β-globin chains (β, δ, γ)
11
2 copies of each per cell
cytogenetics of α-globin chains (α, ζ)
16
4 copies of each per cell
cytogenetics of Myelodysplasia
−5, −5q, −7, −7q, or −8
Most commonly seen are complex abnormalities involving one or more
elliptocytes can be seen in
Hereditary elliptocytosis (>25%), iron deficiency anemia, megaloblastic anemia, myelodysplasia, and myelophthisis
inheritance pattern of Hereditary elliptocytosis
autosomal dominant
protein defect in Hereditary elliptocytosis
Spectrin or band 3
three types of Hereditary elliptocytosis
1. common type
2. spherocytic type
3. stomatocytic type
1. in African Americans
2. double heterozygosity for HS and HE.
3. common finding in Malaysia. It is due to a band 3 protein defect and confers protection against P. vivax malaria.
the inheritance pattern of Hereditary stomatocytosis
autosomal dominant
two types of Hereditary stomatocytosis
1. a more severe hydrocytotic (overhydrated) type in which red cells take on extra water
2. a less severe xerocytotic type in which they lose water.
1. Significant stomatocytosis, macrocytosis, moderate to severe hemolysis, and a low (24–30%) MCHC characterize the hydrocytosis syndromes. The membrane protein Stomatin is decreased.
2. The xerocytosis syndromes have normocytic red cells characterized morphologically by spiculated dessicocytes, mild stomatocytosis, and target cells. As seen in HS, the MCHC is increased. The gene for xerocytic HSt has been mapped to 16q23-q24.
Hereditary stomatocytosis and splenectomy
HSt syndromes have a marked tendency towards thrombosis following splenectomy, so therapeutic splenectomy is generally avoided (and usually unnecessary).
Heinz bodies are associated with
G6PD deficiency
sources of oxidative stress in G6PD deficiency
medications; fava beans, infections
the inheritance pattern of G6PD deficiency
X-linked recessive
the two G6PD deficiency mutations and their enzyme activity in RBCs
G6PDmed and G6PDA-
10% and 20-60%
the peripheral smear of G6PD deficiency
poikilocytosis, spherocytosis, Heinz bodies (with supravital dyes such as methyl violet, crystal violet, or brilliant cresyl blue), bite cells, and blister cells
Tests for G6PD deficiency
1. The ascorbate cyanide test is performed by adding ascorbate cyanide to the patient's red cells. G6PD deficient red cells are more sensitive to this sort of oxidant stress than normal cells.
2. The fluorescent spot test is performed by incubating red cells with NADP and G6P and measuring the production of NADPH (which fluoresces).
during what time that the G6PD activity in deficient individuals can be normal?
In the days following a hemolytic crisis, the surviving red cells have normal G6PD activity; therefore, testing during this time may yield falsely negative (normal) results. Repeat testing in >3 months should confirm the diagnosis in these individuals
acquired Pyruvate kinase (PK) deficiency can be observed in
myelodysplastic syndrome or acute myeloid leukemia
biochemistry of Pyruvate kinase (PK) deficiency
PK deficiency leads to ATP depletion, impaired ion pumps, red cell dehydration, and finally hemolysis.
peripheral smear associated with Pyruvate kinase (PK) deficiency
Echinocytes
tests for Pyruvate kinase (PK) deficiency
1. The autohemolysis test is positive, but unlike HS, does not correct with the addition of glucose. It does normalize with the addition of ATP.
2. A fluorescent spot test is performed in which red cells are incubated with NADH (which fluoresces) to check for conversion to NAD (which does not).
Congenital dyserythropoietic anemia (CDA)
1. most common type?
2. inheritance?
3. smear findings?
4. serum findings?
5. alternate name?
1. type II
2. autosomal recessive
3. multinucleate erythroid precursors
4. positive acidified serum (Ham)
5. hereditary erythroblast multinuclearity with positive acidifed serum (HEMPAS)
Difference between positive Ham test in CDA type II and PNH?
CDA type II lysis with heterologous serum ONLY (due to abnormally high RBC i antigen)
PNH lysis with autologous and heterolgous serum
Paroxysmal nocturnal hematuria (PNH)
1. inheritance?
2. level of defect?
3. molecular defect?
4. associated gene mutation?
1. Acquired clonal disorder
2. hematopoietic stem cell
3. single defect, decreased glycosyl phosphatidyl inositol (GPI) anchors
4. PIG-A
T/F PNH only affects red cell populations.
False- defective hematopoietic stem cell clone dominates red cell population and eventually variable proportions of white cells and platelets
Glycosyl phosphatidyl inositol anchors
1. function?
2. gene encoded on?
3. gene location?
1. attaches array of proteins to cell surface, many of which deflect immune system destruction
2. on phosphatidyl inositol glycan class A (PIG-A) gene, initial step of synthesis
3. X chromosome
PNH
1. classic clinical presentation
2. finding of red cell indices?
3. long term complications?
1. episodic hemolysis, especially at night (classic, not usual)
2. chronic normocytic, normochromic anemia
3. thrombo- and leukopenias, eventually may evolve to aplastic anemia and/or AML
What are the characteristic abnormalities of RBCs in PNH?
diminished cell-surface decay-accelerating factor (DAF, CD55), decreased membrane inhibitor of reactive lysis (MIRL, CD59), decreased acetylcholinesterase (AchE), decreased CD16, and decreased CD48
Phenotype of RBCs in PNH?
hypersensitivity to complement mediated lysis
1. How is sucrose hemolysis test performed?
2. Positive test indicates?
1. incubate patient's RBCs in serum and isotonic sucrose (promotes complement binding)
2. increased hemolysis (positive test) compared to control indicates PNH
1. How is acidified serum (Ham) test performed?
2. positive test indicates?
1. incubate RBCs in heterologous and homologous serum that's been acidified (activating complement)
2. enhanced hemolysis in both sera consistent with PNH (heterologous only indicates CDA type II)
Flow cytometry findings in PNH?
decreased CD59 and CD55 on RBCs, leukocytes and platelets. Serial studies show expansion of abnormal populations with disease progression
PNH
1. T/F leukocyte alkaline phosphatase is decreased
1. True
What are bone marrow findings in PNH?
early in disease hypercellular, evolution to aplastic anemia and/or AML is common
Name group of disorders unified by anemia and ringed sideroblasts in the bone marrow aspirate?
Sideroblastic anemia
What are typical peripheral smear findings in sideroblastic anemia?
hypochromic anemia (maybe micro-, normo-, or macrocytic)
basophilic stippling may be present (due to overstaining of iron-containing Pappenheimer bodies)
Difference in red cell indices for acquired vs. hereditary sideroblastic anemia?
Acquired more likely macrocytic
Inherited more likely microcytic
T/F Classic finding in sideroblastic anemia is a bimodal red cell volume distribution
TRUE
What are the characteristic iron studies in sideroblastic anemia?
Elevated serum iron, high transferrin percent saturation, high ferritin
Why is there often hyperbilirubinemia, high LDH, and decreased serum haptoglobin in sideroblastic anemia?
Intramedullary hemolysis associated with ineffective erythropoiesis
Causes of acquired sideroblastic anemia?
-Clonal stem cell defects/myelodysplasia (refractory anemia with ringed sideroblasts) VAST MAJORITY OF CASES
-Meds (isoniazid, chloramphenicol, chemo)
-irradiation
-alcohol
-copper deficiency (rare)
What is Pearson syndrome?
sideroblastic anemia with pancreatic insufficiency This is a rare form of acquired sideroblastic anemia
Typical presentation of acquired sideroblastic anemia?
Older adults with macrocytic, hypochromic anemia. Marrow aspirate shows >15% ringed sideroblasts. Cytogenetic anomalies in 25-50% of cases
Inherited sideroblastic anemia
1. mode of inheritance?
2. responsible gene?
3. typical presentation?
1. X linked recessive
2. ALAS2, large number of mutations found
3. childhood, organ dysfunction due to iron overload, low MCV, high RDW (may have bimodal distribution)
2 causes of acquired pure red cell aplasia?
thymoma and parvo B19 infection
Cause of congenital pure red cell aplasia?
Blackfan-Diamond syndrome
Name disorder that causes transient arrest in RBC production, lasts about 2 weeks, usually barely noticed in healthy children and adults but can be catastrophic in those with chronic hemolytic anemia?
Parvo B19 infection
Mechanism of parvo B19 infection and marrow findings?
Infects erythroid progenitor cells, marrow exam shows numerous giant pronormoblasts, reduced mature forms, and viral nuclear inclusions
At what stage are erythroid precursors arrested when infected with parvo B19?
pronormoblast stage
Transient erythrocytopenia of childhood (TEC)
1. Define
2. peripheral smear findings
3. marrow findings
1. Self limiting disorder arising in kids 1-4 years old previously healthy, temporary arrest of erythropoiesis
2. reticulocytopenia, normochromic, normocytic anemia
3. hypocellular with erythroid hypoplasia
1. What is Blackfan-Diamond syndrome?
2. T/F platelets and leukocytes are also affected?
3. marrow findings
4. treatment
1. -congenital pure red cell aplasia
-rare, constitutional disease
-usually evident by age 5
2. False
3. Erythroid precursors low or absent
4. corticosteroids (~75% response rate)
1. What antigen is overexpressed on red cells in Blackfan-Diamond syndrome?
i antigen
T/F Fetal hemoglobin is decreased in Blackfan-Diamond syndrome?
False - it is increased (in contrast to transient erythrocytopenia of childhood)
Acquired pure red cell aplasia
1. Affects what population?
2. Over half associated with what condition?
3. Other associated conditions?
1. Usually adults
2. Thymoma (esp. spindle cell/medullary type)
3. collagen vascular disease, lymphoproliferative disorders of large granular lymphocytes, meds
Acquired pure red cell aplasia increasingly seen with what kind of medical therapy?
Erythropoietin therapy, anti-Epo antibodies detected in many cases
Name 6 inherited disorders associated that may lead to aplastic anemia
Fanconi anemia, dyskeratosis congenita, Schwachman-Diamond syndrome, reticular dysgenesis, Down syndrome, and familial aplastic anemia
Name 3 inherited disorders associated with aplasia of single cell lines and the associated cell line
Kostman syndrome (neutropenia), Blackfan-Diamond syndrome (anemia), and thrombocytopenia-absent radii (TAR) syndrome (thrombocytopenia)
Fanconi anemia (FA)
1. mode of inheritance?
2. type of chromosomal disorder?
1. autosomal recessive
2. chromosomal breakage
What congenital anomalies are associated with Fanconia anemia?
They are (often first manifestations): abnormal skin pigmentation, skeletal anomalies, renal anomalies, short stature, micophthalmia, and mental retardation.
Up to 1/3 display none of these
What skin pigmentation disorders are associated with Fanconi anemia?
cafe au lait spots, hypo- and hyperpigmentation
What skeletal abnormalities are associated with Fanconi anemia?
Abnormal radii, hypoplastic thumb, scoliosis
What renal disorders are associated with Fanconi anemia?
horseshoe kidney
What's the initial clinical course of most patients with Fanconi anemia?
aplastic anemia develops in most patients by age 10. Often isolated anemia (or thrombocytopenia) before aplastic anemia
What are long term complications of Fanconi anemia?
Marrow failure or clonal hematopoietic defects including AML (M4 or M5) and MDS
What epithelial malignancies have an increased incidence in Fanconi anemia?
Cutaneous malignancies, hepatocellular carcinoma, gastric carcinoma and others
What are the diagnostic cytogenetic findings in Fanconi anemia?
-increased propensity for spontaneous chromosomal breakage
- particularly hypersensitive to clastogenic agents such as diepoxybutane and mitomycin C, requires altered preconditioning prior to bone marrow transplant
Name 5 chromosomal breakage syndromes
Fanconi anemia, xeroderma pigmentosum (XP), ataxia telangiectasia (TA), Bloom syndrome (BS), and Cockayne syndrome
Hemoglobin S
1. defect?
2. found in parts of the world where what infection is prevalent?
3. prevalence of trait among African Americans?
1. beta6 glu --> by valine
2. falciparum malaria
3. about 10%
What hemoglobin disorders have similar clinical manifestations to sickle cell disease?
sickle cell beta 0 thalassemia, SC disease (HbSC), and sickle cell beta + thalassemia
1. What causes red cells to sickle?
2. Average life span of a sickled RBC?
1. abnormal polymerization of deoxygenated hemoglobin S
2. 17 days (normal is 120 days)
What are the hemoglobin electrophoresis findings in sickle cell disease?
>80% HbS, 1-20% HbF, 1-4% HbA2, and 0% HbA
What disorders show sickled red cell on peripheral smear?
Hgb SS, S- beta thalassemia, S-C, S-D, and C Harlem
What test is based on the principle that metabisulfate promotes Hb deoxygenation?
metabisulfite sickling test, positive in hemoglobin SS disease
What's the mechanism of the Sickledex test?
aka dithionate solubility test, lysed RBCs incubated with dithionate that precipates HbS, positive in SS disease
At what age is sickle cell disease usually clinically apparent and why?
Usually at 6 months of age when HbF decreases and HbS levels increase beyond 50%
What hemoglobin disorder is characterized by chronic hemolytic anemia and recurrent crises?
sickle cell disease
What coagulation abnormality contributes to the clinical expression of sickle cell?
Thrombosis
Aplastic crises in sickle cell
1. etiology
2. what are the clinical characteristics?
3. duration?
4. level of anemia?
1. transient arrest of erythropoiesis
2. abrupt drop in Hgb, reticulocytes, and red cell precursors in the marrow
3. usually only a few days
4. can be severe
What infection is responsible for nearly 70% of aplastic crises in children with sickle cell?
Parvovirus B19
Splenic sequestration in sickle cell
1. clinical presentation?
2. often associated with?
3. who is most susceptible?
1. enlarged, tender spleen
2. viral illness
3. children and adults with SC disease or sickle cell beta + thalassemia
What may cause worsening of anemia in SS patients that progresses more slowly than aplastic crisis or splenic sequestration?
Progressive renal insufficiency (with decreasing erythropoietin) or supervening iron/folate/B12 deficiency
What is a hyperhemolytic crisis?
Sudden exacerbation anemia in SS with profound reticulocytosis and hyperbilirubinemia. Has been associated with G6PD deficiency.
Acute pain crises in SS
1. etiology
2. possible precipitating factors?
1. vasooclusive events within the bone
2. exposure to cold, dehydration, infection, or alcohol consumption
Acute chest syndrome in SS
1. clinical presentation?
2. possible etiologies?
1. dyspnea, cough, chest pain, and fever. May find tachypnea, leukocytosis, pulmonary infiltrate on chest xray, and progressive hypoxia
2. vasoocclusive events or bacterial pneumonia
Why are infections a major source of morbidity and mortality in sickle cell disease and what infections are most common?
Functional asplenia makes infections worse. S. pneumoniae infections are the most common overall, other common infections include Salmonella, Haemophilus and M. pneumoniae
Neurologic complications are frequent in SS disease. What are common manifestations?
TIAs, cerebral infarcts, cerebral hemorrhage, cord infarction, sensorineural hearing loss, and meningitis
1/3 of patient with SS disease will have angiographic appearance of what disease due to neurologic complications?
Moyamoya disease (segmental arterial stenoses with 'puff of smoke' collaterals
Common presentation of acute hepatic crisis in sickle cell disease?
aka right upper quadrant syndrome, presents as progressive jaundice, elevated LFTs, and a tender enlarged liver, usually resolves within 2 weeks but may progress to liver failure
Common chronic liver problems seen in SS disease?
Chronic nonspecific hepatomegaly and liver dysfunction (probably related to centrilobular congestion), chronic Hep C, pigmented gallstones
What are some pregnancy related complications in SS disease?
Increased rate of maternal and fetal deaths, increased preeclampsia, increased IUGR, intrauterine fetal demise and prematurity
Name the 7 classic sickle cell nephropathies
gross hematuria, papillary necrosis, nephrotic syndrome, renal infarction, isosthenuria, pyelonephritis, and renal medullary carcinoma (also increased in sickle cell trait and SC disease)
T/F Occular complications (proliferative retinopathy) occurs with same frequency in SS, SC and sickle beta + thalassemia
False, greater rates in SC and sickle beta + thalassemia than SS
What is a common complication of SS that may affect the vertebrae, hands, feet, and femoral and humeral heads ?
osteonecrosis
Sickle cell trait (SA)
1. clinical presentation?
2. peripheral smear findings?
3. characteristic hemoglobin electrophoresis findings?
4. screening test results?
1. usually asymptomatic
2. no sickle cells
3. 35 to 45% HbS, 1% HbF, 1-3% HbA2, 50-55% HbA
4. positive metabisulfite and dithionate tests
S-alpha-thalassemia
1. what is it?
2. HbS is increased or decreased and why?
1. when alpha thalassemia is coinherited with sickle cell trait
2. HbS is decreased, degree to which relative to number of alpha globin genes deleted
S-beta-thalassemia
1. what is it?
2. HbS is increased or decreased?
3. mild, intermediate, or severe disease?
1. when beta thalassemia is coinherited with sickle cell trait
2. increased (usually >50%)
3. severe depending upon type of beta thalassemia defect
Hemoglobin SC disease
1. what is it?
2. HbS levels?
3. mild, intermediate, or severe disease?
1. heterozygosity for HbS and HbC
2. ~50%
3. intermediate severity between SS and SA
Hemoglobin SC disease
1. average red cell life span?
2. which complications occur at about same rate as in SS disease?
3. peripheral smear findings?
1. 27 days
2. avascular necrosis of bone and proliferative retinopathy (others about half as frequent
3. mild sickling and abundant target cells
Hemoglobin A2 prime (HbA2')
1. what is it?
2. T/F it is best detected by gel electrophoresis
1. clinically insignficant delta-chain variant, occurs in 1-2% of African Americans
2. False, best detected by HPLC
1. Why can HbA2' lead to underdiagnosis of beta thalassemia trait?
2. How can calculations be adjusted to account for the answer to 1?
1. A2' barely detectable on gel electrophoresis
2. HbA2 and HbA2' levels must be added
HbC
1. mutation?
2. hemoglobin findings in trait (heterozygous AC)?
3. clinical presentation?
4. peripheral smear findings
1. beta6 glu --> lys
2. 40-50% Hgb in the C band (HbA2 = HbC
3. generally asymptomatic
4. scattered target cells
Hemoglobin C disease (homozygous CC)
1. hemoglobin findings?
2. clinical presentation?
3. peripheral smear findings?
1. 90% HbC, 7% HbF, 3% HbA2, 0% HbA
2. mild hemolytic anemia, splenomegaly
3. numerous target cells, hexagonal or rod shaped crystals may be found in red cells, esp after splenectomy
HbE
1. point mutation?
2. geographical prevalence?
3. CBC findings?
4. peripheral smear findings?
1. beta26 glu --> lys
2. Southeast Asia
3. CBC shows thalessmic indices
4. numerous target cells
HbD & G
1. clinical presentation?
2. gel findings on cellulose vs. citrate?
3. how to distinguish the 2?
1. clinically normal
2. cellulose acetate = band runs with HbS, citrate = band runs with A
3. HbG is an alpha chain defect thus may produce 2 HbA2 bands.
HbD is a beta chain defect
Hb Lepore
1. geographic prevalence?
2. What Hgb findings should raise suspicion for this?
3. gene abnormality?
4. location on gel electrophoresis?
5. T/F HbF is low
1. Mediterranean, especially Italy
2. whenever less than 30% (usually 15%) HbS present
3. Fusion between delta and beta genes
4. runs with HbS on cellulose (only 8-15% of total Hb)
5. False may be as high as 20%
Hb Constant Springs (Hb CS)
1. T/F doesn't affect Hgb indices
2. mutation?
3. what hemoglobins are produced in heterozygous adults and newborns?
1. False, causes thalassemic indices
2. mutation in alpha gene stop codon, producing abnormally and inefficient long transcript that produces thalassemia
3. Adults = HbA, HbCS, HbA2, and alphaCS-delta (4 band bands on cellulose gel)
Newborns = HbF and alphaCS-gamma also seen in addition to above
High oxygen affinity hemoglobins
1. characteristic O2 dissociation curve finding?
2. Findings on Hgb studies?
1. left shift (diagnostic)
2. Can't be resolved on gel or HPLC, key is erythrocytosis on CBC
Unstable hemoglobins
1. characteristic peripheral smear findings?
2. what physical state may precipitate hemolytic crisis?
3. How is screening for them carried out?
4. Examples?
1. Heinz bodies and bite cells
2. oxidative stress
3. incubating lysedred cells with 17% isopropanol, causes precipitation of unstable hemoglobins
4. Hbs Hasharon, Koln & Zurich only Hb Hammersmith associated with severe hemolysis
Methemoglobin (Hi)
1. how is different from normal hemoglobin?
2. what causes these changes?
3. T/F Hi is capable of combining with oxygen
1. The iron in the Hb molecule is in the oxidzied ferric (Fe+++) state instead the usual ferrous (Fe++) form
2. often due to oxidation of hemoglobin
3. False, Hi is unable to combine with oxygen
1. T/F methemogobin (Hi) is normally present in the body
2. How is Hi normally metabolized/
1. True, normally a small degree of Hb oxidation, up to 1.5% of Hb is Hi
2. reduced in erythrocytes by NADH-dependent methemoglobin reductase system
1. What's the threshold of methemoglobin (Hi) before causing cyanosis?
2. Gross appearance of blood?
1. When Hi reaches 10% of total Hb (around 1.5 g/dL) cyanoss results
2. Chocolate brown color
Which of the following is capable of detecting methemoglobin?
A. co-oximeter
B. pulse oximeter
C. arterial blood gas analyzer
A. CORRECT
B&C. INCORRECT, both estimate oxygen level by emitting red light (wavelength 660 nm) absorbed by oxyhemoglobin and infrared light (940 nm). Methemoglobin absorbs both of these wavelengths so it isn't detected
What causes hereditary methemoglobinemia?
Either deficiency in the reductase system or abnormal hemoglobins (HbM) upon which this enzyme cannot act
What is HbM?
A group of abnormal hemoglobins that prefer the ferric (methemoglobin) state due to various amino acid substitutions
At what age is hereditary methemoglobinemia clinically apparent?
Cyanosis appears at 6 months of age, unless there's M fetal Hb. If so cyanosis abates at about 6 months
Where are most M hemoglobins found on routine gels?
they run with A
Common causes of acquired methemoglobinemia?
Exposure to drugs or chemicals that increase the formation of Hi, common examples include: nitrites, quinones, phenacetin, and sulfonamides
Why are nitrites part of the treatment for cyanide toxicity?
They produce methemoglobin which has a high affinity for cyanide and chelates it
What's the treatment for methemoglobinemia?
Methylene blue, which reduces Hi to Hb
Sulfhemoglobin (SHb)
1. How is it formed?
2. What are Heinz bodies?
3. T/F SHb can't transport oxygen
1. when Hb is oxidized in the presence of sulfur
2. precipitate of SHb when it's further oxidized
3. True
T/F Unlike Hi, SHb can't be reduced to Hb
TRUE
At what level is SHb normally present and at what level does it cause cyanosis?
Normally present at >1% of total Hb, cyanosis manifests at around 3-4% or 0.5 g/dL
What conditions may increase sulfhemoglobin?
Presence of sulfonamides and C perfringens bacteremia (eneterogenous cyanosis)
Where is thalassemia most prevalent?
Mediterranean, Africa, Southeast Asia. Parallels the prevalence of malaria.
On what genes are the α- and β- genes located?
α-genes are located on chromosome 16. β-genes are located on chromosome 11p15.5.
How many copies of the β gene are on each chromosome?
One, for a total of two productive genes in normal cells.
How many copies of the α gene are on each chromosome?
Two, for a total of four α chain producing gene loci in each normal cell.
What type of mutations are most common in β-thalassemia?
Point mutations
What type of mutations are most common in α-thalassemia?
Large deletions
What is the most common genotype in African Americans with thalassemia?
(-α/). The is a genotype in which chromosome 16 has one normal and one deleted α gene.
What are the typical CBC findings in thalassemias?
Elevated RBC count (>5.5 x 10^12 in men, >5.0 x 10^12 in women) Low MCV (65-75 fL in α-thal; 55-65 fL in β-thal) Low hematocrit Normal to slightly elevated RDW
What MCV/RBC count favors thalassemia?
An MCV/RBC count ratio < 13 favors thalassemia, while a ratio > 15 favors iron deficiency.
What are typical peripheral smear findings in thalassemia?
Microcytic hypochromic anemia with occasional target cells (more in β-thal) and basophilic stippling.
In what populations is α-thalassemia most common?
Sub-saharan Africans and southeast Asians. The α thal 1 gene is prevalent only in Asians, and it is they who are at risk for the very severe kinds of α-thalassemia (Hg Bart and Hg H diseases). The α thal 2 gene is most prevalent in blacks.
What type of symptoms do persons with single gene deletion α-thalassemia have?
None. They are entirely asyptomatic, have normal CBC, and normal electrophoresis.
Persons with α-thalassemia trait (2 gene deletions) manifest what clinically?
A CBC with thalassemic indices and electrophoresis with normal A and A2 bands. A2 is not increased. In the absence of iron deficiency, this can be interpreted as consistent with α-thal trait.
In what populations β-thalassemia most common?
Mediterranean populations.
At what age do manifestations of β-thalassemia become evident?
6 to 9 months.
Beta thal minor typically result from inheritance of how many abnormal β genes?
One. Either β+ or β⁰.
What is the most common electrophoretic pattern in beta thal minor?
High HbA2 (over 2.5%, usually 4-8%and normal HbF. The second most common shows a normal A2. This is due to simultaneous iron deficiency, and may be erroneously interpreted as consistent with α-thalassemia.
In δ-β thal (deletion of both the δ and β genes), what is the electrophoretic pattern?
Normal A2 and elevated HbF (5-20%).
In heterozygous Hb Lepore (fusion of δ and β), what is the electrophoretic pattern?
Normal A2, slightly elevated HbF, and a band in the S region comprising 6-15% (Hb Lepore).
Beta thal major results from inheritance of how many abnormal genes?
Two, such as β⁰ β⁰, β+med β+med, or β⁰ β+med.
What is the most common cause of death in childhood in patients with beta thal major?
Individuals are not anemic at birth but develop anemia within one year. The most common cause of death in childhood is infection.
What is the electrophoretic pattern in beta thal major?
Increased HbF (50-95%), normal to elevated A2, and little to no HbA.
What is the clinically distinguishing factor between beta thal intermidia and beta thal major?
Beta thal major is transfusion dependent.
Warm autoimmune hemolytic anemia is usually mediated by what type of antibody?
A warm-reacting IgG antibody.
In warm autoimmune hemolytic anemia, does the responsible antibody usually have a broad or narrow reactivity with red cell antigens?
Usually a broad reactivity, especially Rh antigens. Uncommonly, the antibody has a narrow specificity; eg, for a specific Rh antigen, Kell, Kidd, etc.
What is the crucial test in diagnosing autoimmune hemolytic anemia?
The DAT (antiglobulin test, Coombs test). It is positive in nearly all cases of warm autoimmune hemolytic anemia.
What type of reactivity can be seen in the DAT test in a patient with warm autoimmune hemolytic anemia?
Usually polyspecific and anti-IgG reagents, sometimes with both anti-IgG and anti-C3, and uncommonly with anti-C3 only. Infrequently, the DAT may be falsely negative, due to very rapid intravascular destruction of RBCs or very low titer Ab.
How are cells destroyed in warm autoimmune hemolytic anemia?
The Ab binds to Ags on the red cell surface. In most cases, the bound Ab acts as an opsonin that provoked RBC destruction by splenic macrophages (extravascular hemolysis). In this instance, some escape and are seen on smear as spherocytes. In other cases, the Ab activates complement and results in either intravascular hemolysis (through formation of the MAC), or opsonization (cascade arrested at C3b).
How do patients with warm autoimmune hemolytic anemia present?
Patients present with variable severity. Some have abrupt onset and severe symptomatic anemia, while others have a chronic low grade hemolysis.
Is warm autoimmune hemolytic anemia a primary or secondary disease?
Can be both. Secondary comprises about 70% of cases, and occurs in hematolymphoid neoplasms, inherited autoimmunity (common variable immunodeficiency, IgA deficiency, Bruton’s), collagen vascular disease, thymoma). Stem cell transplantation has also been associated.
What type of antibodies are cold agglutinins?
IgM
What is the specificity of cold agglutinins?
Cold agglutinins are IgM antibodies with specificity that is most commonly anti-I. Others include anti-i, anti-H, anti-Pr, and anti-IH.
What are the most important laboratory features in predicting pathogenicity of cold agglutinins?
Titer and thermal range (thermal amplitude).
At what temperature do nonpathologic cold agglutinins react?
Nonpathologic cold agglutinins react most strongly at 4⁰ C, but have variably wide thermal amplitudes and may react at up to 22⁰C.
What is the titer of most nonpathologic cold agglutinins?
The titer of benign cold agglutinins is usually <64 at 4⁰C.
What is the only reliable CBC index in the presence of cold agglutinins?
Hemoglobin.
At what temperature do pathologic cold agglutinins react?
They are reactive over a broad range, up to 32-37⁰C and cause spontaneous autoagglutination in anticoagulated blood at room temperature.
What is the titer of pathologic cold agglutinins?
The titer is often >1000 at 4⁰C.
What is the predominant clinical scenario in idiopathic cold autoimmune hemolytic anemia?
A chronic condition found predominantly older individuals complaining of acrocyanosis and Reynaud phenomenon with a moderate hemolytic anemia.
What is the clinical scenario in secondary cold autoimmune hemolytic anemia?
A transient condition often associated with infection. M pneumonia infection is associated with anti-I, and EBV-associated infectious mononucleosis is associated with anti-i.
for beta-globin, what are definitions and causes of βº, β+, silent and complex alleles?
1. βº: complete absence of β-chain production
2. β+: diminished β-chain production
3. silent: almost no impact on chain production
4. fusion δ-β- and γ-δ-β- chains resulting from deletion of noncoding intervening segments of the HBB gene cluster
in α–thalassemia, β4 and γ4 tetramers form?
Continued synthesis of normal amounts of the unaffected chain leads to its relative abundance and precipitation of these chains in the red cell, reducing the cell's lifespan.
what are the genotypes, CBC and electrophoresis findings of the five thalassemia syndromes?
Syndrome, Genotype, CBC Electrophoresis
1. normal, αα/αα, normal, normal
2. silent carrier, −α/αα, normal, normal
3. α-thal trait, −α/−α or −−/αα, thalassemic, normal
4. HbH disease, −−/−α or −−/αCSα, thalassemic Heinz bodies, fast-migrating Hb H, Hb H = β4 tetramers.
5. Hb Bart disease (hydrops fetalis), −−/−−, hypochromia nRBCs, fast-migrating Hb Barts. Hb Barts = γ4 tetramers.
α thalassemia is most common in which two populations and their prevalent alleles
sub-Saharan African and southeast Asian descent
Acquired hemoglobin H might be seen in (3)
myeloproliferative disorders, and myelodysplastic syndromes.
When does the direct Coombs test give false positives and false negatives?
Infrequently, the DAT may be falsely negative, due to very rapid intravascular destruction of erythrocytes or very low titer antibody. Furthermore, a small percentage of healthy people have a positive DAT.
When does the secondary autoimmune hemolytic anemia occurr? (5)
hematolymphoid neoplasms (especially CLL/SLL), inherited autoimmunity (especially antibody deficiency—common variable immunodeficiency, IgA deficiency, Bruton agammaglobulinemia), collagen vascular disease, and thymoma and Stem cell transplantation
anti-I is associated with what infection?
M pneumoniae
anti-I is associated with what infection?
EBV
How to avoid the false screening and crossmatch results caused by cold autoantibodies?
As with warm autoantibodies, the task is to look beyond the cold-reacting antibody for masked alloantibodies. Options include a prewarmed screen or crossmatch, using serum from a cold autoadsorption or adsorption with rabbit erythrocyte stroma (REST), or serum pretreated with DTT or 2-ME (disrupt IgM sulfhydryl bonds).
The reagents for Identifying Cold Agglutinins include Type O cord blood, Type O adult blood, Type A1 adult blood, Type A2 adult blood and Saliva. What antigen(s) do they possess?
Cells, I antigen, i antigen, H antigen, IH, Pr
Type O cord blood: −, +++, +++, +/-, +++
Type O adult blood: +++, −, +++, +++, +++
Type A1 adult blood: ++, −, −, -/+, +/-, +++
Type A2 adult blood: ++, −, +, +++, +++
Saliva: −, −, +
Paroxysmal cold hemoglobinuria is often seen in children associated with which morbility?
viral illnesses such as measles, mumps, chickenpox, and infectious mononucleosis
Definition of Paroxysmal cold hemoglobinuria
paroxysmal episodes of hemoglobinuria associated with cold exposure
treatment of Paroxysmal cold hemoglobinuria
keeping the patient warm and transfusing as necessary
Donath-Landsteiner test for Paroxysmal cold hemoglobinuria
A positive test is obtained if only incubation of the patient's red cells at 4°C then 37°C leads to hemolysis.
Cryoglobulins
immunoglobulins that precipitate reversibly at low temperatures.
1. How is sucrose hemolysis test performed?
2. Positive test indicates?
1. incubate patient's RBCs in serum and isotonic sucrose (promotes complement binding)
2. increased hemolysis (positive test) compared to control indicates PNH
1. How is acidified serum (Ham) test performed?
2. positive test indicates?
1. incubate RBCs in heterologous and homologous serum that's been acidified (activating complement)
2. enhanced hemolysis in both sera consistent with PNH (heterologous only indicates CDA type II)
Flow cytometry findings in PNH?
decreased CD59 and CD55 on RBCs, leukocytes and platelets. Serial studies show expansion of abnormal populations with disease progression
PNH
1. T/F leukocyte alkaline phosphatase is decreased
1. True
What are bone marrow findings in PNH?
early in disease hypercellular, evolution to aplastic anemia and/or AML is common
Name group of disorders unified by anemia and ringed sideroblasts in the bone marrow aspirate?
Sideroblastic anemia
What are typical peripheral smear findings in sideroblastic anemia?
hypochromic anemia (maybe micro-, normo-, or macrocytic)
basophilic stippling may be present (due to overstaining of iron-containing Pappenheimer bodies)
Difference in red cell indices for acquired vs. hereditary sideroblastic anemia?
Acquired more likely macrocytic
Inherited more likely microcytic
T/F Classic finding in sideroblastic anemia is a bimodal red cell volume distribution
TRUE
What are the characteristic iron studies in sideroblastic anemia?
Elevated serum iron, high transferrin percent saturation, high ferritin
Why is there often hyperbilirubinemia, high LDH, and decreased serum haptoglobin in sideroblastic anemia?
Intramedullary hemolysis associated with ineffective erythropoiesis
Causes of acquired sideroblastic anemia?
-Clonal stem cell defects/myelodysplasia (refractory anemia with ringed sideroblasts) VAST MAJORITY OF CASES
-Meds (isoniazid, chloramphenicol, chemo)
-irradiation
-alcohol
-copper deficiency (rare)
What is Pearson syndrome?
sideroblastic anemia with pancreatic insufficiency This is a rare form of acquired sideroblastic anemia
Typical presentation of acquired sideroblastic anemia?
Older adults with macrocytic, hypochromic anemia. Marrow aspirate shows >15% ringed sideroblasts. Cytogenetic anomalies in 25-50% of cases
Inherited sideroblastic anemia
1. mode of inheritance?
2. responsible gene?
3. typical presentation?
1. X linked recessive
2. ALAS2, large number of mutations found
3. childhood, organ dysfunction due to iron overload, low MCV, high RDW (may have bimodal distribution)
2 causes of acquired pure red cell aplasia?
thymoma and parvo B19 infection
Cause of congenital pure red cell aplasia?
Blackfan-Diamond syndrome
Name disorder that causes transient arrest in RBC production, lasts about 2 weeks, usually barely noticed in healthy children and adults but can be catastrophic in those with chronic hemolytic anemia?
Parvo B19 infection
Mechanism of parvo B19 infection and marrow findings?
Infects erythroid progenitor cells, marrow exam shows numerous giant pronormoblasts, reduced mature forms, and viral nuclear inclusions
At what stage are erythroid precursors arrested when infected with parvo B19?
pronormoblast stage
Transient erythrocytopenia of childhood (TEC)
1. Define
2. peripheral smear findings
3. marrow findings
1. Self limiting disorder arising in kids 1-4 years old previously healthy, temporary arrest of erythropoiesis
2. reticulocytopenia, normochromic, normocytic anemia
3. hypocellular with erythroid hypoplasia
1. What is Blackfan-Diamond syndrome?
2. T/F platelets and leukocytes are also affected?
3. marrow findings
4. treatment
1. -congenital pure red cell aplasia
-rare, constitutional disease
-usually evident by age 5
2. False
3. Erythroid precursors low or absent
4. corticosteroids (~75% response rate)
1. What antigen is overexpressed on red cells in Blackfan-Diamond syndrome?
i antigen
T/F Fetal hemoglobin is decreased in Blackfan-Diamond syndrome?
False - it is increased (in contrast to transient erythrocytopenia of childhood)
Acquired pure red cell aplasia
1. Affects what population?
2. Over half associated with what condition?
3. Other associated conditions?
1. Usually adults
2. Thymoma (esp. spindle cell/medullary type)
3. collagen vascular disease, lymphoproliferative disorders of large granular lymphocytes, meds
Acquired pure red cell aplasia increasingly seen with what kind of medical therapy?
Erythropoietin therapy, anti-Epo antibodies detected in many cases
Name 6 inherited disorders associated that may lead to aplastic anemia
Fanconi anemia, dyskeratosis congenita, Schwachman-Diamond syndrome, reticular dysgenesis, Down syndrome, and familial aplastic anemia
Name 3 inherited disorders associated with aplasia of single cell lines and the associated cell line
Kostman syndrome (neutropenia), Blackfan-Diamond syndrome (anemia), and thrombocytopenia-absent radii (TAR) syndrome (thrombocytopenia)
Fanconi anemia (FA)
1. mode of inheritance?
2. type of chromosomal disorder?
1. autosomal recessive
2. chromosomal breakage
What congenital anomalies are associated with Fanconia anemia?
They are (often first manifestations): abnormal skin pigmentation, skeletal anomalies, renal anomalies, short stature, micophthalmia, and mental retardation.
Up to 1/3 display none of these
What skin pigmentation disorders are associated with Fanconi anemia?
cafe au lait spots, hypo- and hyperpigmentation
What skeletal abnormalities are associated with Fanconi anemia?
Abnormal radii, hypoplastic thumb, scoliosis
What renal disorders are associated with Fanconi anemia?
horseshoe kidney
What's the initial clinical course of most patients with Fanconi anemia?
aplastic anemia develops in most patients by age 10. Often isolated anemia (or thrombocytopenia) before aplastic anemia
What are long term complications of Fanconi anemia?
Marrow failure or clonal hematopoietic defects including AML (M4 or M5) and MDS
What epithelial malignancies have an increased incidence in Fanconi anemia?
Cutaneous malignancies, hepatocellular carcinoma, gastric carcinoma and others
What are the diagnostic cytogenetic findings in Fanconi anemia?
-increased propensity for spontaneous chromosomal breakage
- particularly hypersensitive to clastogenic agents such as diepoxybutane and mitomycin C, requires altered preconditioning prior to bone marrow transplant
Name 5 chromosomal breakage syndromes
Fanconi anemia, xeroderma pigmentosum (XP), ataxia telangiectasia (TA), Bloom syndrome (BS), and Cockayne syndrome
Hemoglobin S
1. defect?
2. found in parts of the world where what infection is prevalent?
3. prevalence of trait among African Americans?
1. beta6 glu --> by valine
2. falciparum malaria
3. about 10%
What hemoglobin disorders have similar clinical manifestations to sickle cell disease?
sickle cell beta 0 thalassemia, SC disease (HbSC), and sickle cell beta + thalassemia
1. What causes red cells to sickle?
2. Average life span of a sickled RBC?
1. abnormal polymerization of deoxygenated hemoglobin S
2. 17 days (normal is 120 days)
What are the hemoglobin electrophoresis findings in sickle cell disease?
>80% HbS, 1-20% HbF, 1-4% HbA2, and 0% HbA
What disorders show sickled red cell on peripheral smear?
Hgb SS, S- beta thalassemia, S-C, S-D, and C Harlem
What test is based on the principle that metabisulfate promotes Hb deoxygenation?
metabisulfite sickling test, positive in hemoglobin SS disease
What's the mechanism of the Sickledex test?
aka dithionate solubility test, lysed RBCs incubated with dithionate that precipates HbS, positive in SS disease
At what age is sickle cell disease usually clinically apparent and why?
Usually at 6 months of age when HbF decreases and HbS levels increase beyond 50%
What hemoglobin disorder is characterized by chronic hemolytic anemia and recurrent crises?
sickle cell disease
What coagulation abnormality contributes to the clinical expression of sickle cell?
Thrombosis
Aplastic crises in sickle cell
1. etiology
2. what are the clinical characteristics?
3. duration?
4. level of anemia?
1. transient arrest of erythropoiesis
2. abrupt drop in Hgb, reticulocytes, and red cell precursors in the marrow
3. usually only a few days
4. can be severe
What infection is responsible for nearly 70% of aplastic crises in children with sickle cell?
Parvovirus B19
Splenic sequestration in sickle cell
1. clinical presentation?
2. often associated with?
3. who is most susceptible?
1. enlarged, tender spleen
2. viral illness
3. children and adults with SC disease or sickle cell beta + thalassemia
What may cause worsening of anemia in SS patients that progresses more slowly than aplastic crisis or splenic sequestration?
Progressive renal insufficiency (with decreasing erythropoietin) or supervening iron/folate/B12 deficiency
What is a hyperhemolytic crisis?
Sudden exacerbation anemia in SS with profound reticulocytosis and hyperbilirubinemia. Has been associated with G6PD deficiency.
Acute pain crises in SS
1. etiology
2. possible precipitating factors?
1. vasooclusive events within the bone
2. exposure to cold, dehydration, infection, or alcohol consumption
Acute chest syndrome in SS
1. clinical presentation?
2. possible etiologies?
1. dyspnea, cough, chest pain, and fever. May find tachypnea, leukocytosis, pulmonary infiltrate on chest xray, and progressive hypoxia
2. vasoocclusive events or bacterial pneumonia
Why are infections a major source of morbidity and mortality in sickle cell disease and what infections are most common?
Functional asplenia makes infections worse. S. pneumoniae infections are the most common overall, other common infections include Salmonella, Haemophilus and M. pneumoniae
Neurologic complications are frequent in SS disease. What are common manifestations?
TIAs, cerebral infarcts, cerebral hemorrhage, cord infarction, sensorineural hearing loss, and meningitis
1/3 of patient with SS disease will have angiographic appearance of what disease due to neurologic complications?
Moyamoya disease (segmental arterial stenoses with 'puff of smoke' collaterals
Common presentation of acute hepatic crisis in sickle cell disease?
aka right upper quadrant syndrome, presents as progressive jaundice, elevated LFTs, and a tender enlarged liver, usually resolves within 2 weeks but may progress to liver failure
Common chronic liver problems seen in SS disease?
Chronic nonspecific hepatomegaly and liver dysfunction (probably related to centrilobular congestion), chronic Hep C, pigmented gallstones
What are some pregnancy related complications in SS disease?
Increased rate of maternal and fetal deaths, increased preeclampsia, increased IUGR, intrauterine fetal demise and prematurity
Name the 7 classic sickle cell nephropathies
gross hematuria, papillary necrosis, nephrotic syndrome, renal infarction, isosthenuria, pyelonephritis, and renal medullary carcinoma (also increased in sickle cell trait and SC disease)
T/F Occular complications (proliferative retinopathy) occurs with same frequency in SS, SC and sickle beta + thalassemia
False, greater rates in SC and sickle beta + thalassemia than SS
What is a common complication of SS that may affect the vertebrae, hands, feet, and femoral and humeral heads ?
osteonecrosis
Sickle cell trait (SA)
1. clinical presentation?
2. peripheral smear findings?
3. characteristic hemoglobin electrophoresis findings?
4. screening test results?
1. usually asymptomatic
2. no sickle cells
3. 35 to 45% HbS, 1% HbF, 1-3% HbA2, 50-55% HbA
4. positive metabisulfite and dithionate tests
S-alpha-thalassemia
1. what is it?
2. HbS is increased or decreased and why?
1. when alpha thalassemia is coinherited with sickle cell trait
2. HbS is decreased, degree to which relative to number of alpha globin genes deleted
S-beta-thalassemia
1. what is it?
2. HbS is increased or decreased?
3. mild, intermediate, or severe disease?
1. when beta thalassemia is coinherited with sickle cell trait
2. increased (usually >50%)
3. severe depending upon type of beta thalassemia defect
Hemoglobin SC disease
1. what is it?
2. HbS levels?
3. mild, intermediate, or severe disease?
1. heterozygosity for HbS and HbC
2. ~50%
3. intermediate severity between SS and SA
Hemoglobin SC disease
1. average red cell life span?
2. which complications occur at about same rate as in SS disease?
3. peripheral smear findings?
1. 27 days
2. avascular necrosis of bone and proliferative retinopathy (others about half as frequent
3. mild sickling and abundant target cells
Hemoglobin A2 prime (HbA2')
1. what is it?
2. T/F it is best detected by gel electrophoresis
1. clinically insignficant delta-chain variant, occurs in 1-2% of African Americans
2. False, best detected by HPLC
1. Why can HbA2' lead to underdiagnosis of beta thalassemia trait?
2. How can calculations be adjusted to account for the answer to 1?
1. A2' barely detectable on gel electrophoresis
2. HbA2 and HbA2' levels must be added
HbC
1. mutation?
2. hemoglobin findings in trait (heterozygous AC)?
3. clinical presentation?
4. peripheral smear findings
1. beta6 glu --> lys
2. 40-50% Hgb in the C band (HbA2 = HbC
3. generally asymptomatic
4. scattered target cells
Hemoglobin C disease (homozygous CC)
1. hemoglobin findings?
2. clinical presentation?
3. peripheral smear findings?
1. 90% HbC, 7% HbF, 3% HbA2, 0% HbA
2. mild hemolytic anemia, splenomegaly
3. numerous target cells, hexagonal or rod shaped crystals may be found in red cells, esp after splenectomy
HbE
1. point mutation?
2. geographical prevalence?
3. CBC findings?
4. peripheral smear findings?
1. beta26 glu --> lys
2. Southeast Asia
3. CBC shows thalessmic indices
4. numerous target cells
HbD & G
1. clinical presentation?
2. gel findings on cellulose vs. citrate?
3. how to distinguish the 2?
1. clinically normal
2. cellulose acetate = band runs with HbS, citrate = band runs with A
3. HbG is an alpha chain defect thus may produce 2 HbA2 bands.
HbD is a beta chain defect
Hb Lepore
1. geographic prevalence?
2. What Hgb findings should raise suspicion for this?
3. gene abnormality?
4. location on gel electrophoresis?
5. T/F HbF is low
1. Mediterranean, especially Italy
2. whenever less than 30% (usually 15%) HbS present
3. Fusion between delta and beta genes
4. runs with HbS on cellulose (only 8-15% of total Hb)
5. False may be as high as 20%
Hb Constant Springs (Hb CS)
1. T/F doesn't affect Hgb indices
2. mutation?
3. what hemoglobins are produced in heterozygous adults and newborns?
1. False, causes thalassemic indices
2. mutation in alpha gene stop codon, producing abnormally and inefficient long transcript that produces thalassemia
3. Adults = HbA, HbCS, HbA2, and alphaCS-delta (4 band bands on cellulose gel)
Newborns = HbF and alphaCS-gamma also seen in addition to above
High oxygen affinity hemoglobins
1. characteristic O2 dissociation curve finding?
2. Findings on Hgb studies?
1. left shift (diagnostic)
2. Can't be resolved on gel or HPLC, key is erythrocytosis on CBC
Unstable hemoglobins
1. characteristic peripheral smear findings?
2. what physical state may precipitate hemolytic crisis?
3. How is screening for them carried out?
4. Examples?
1. Heinz bodies and bite cells
2. oxidative stress
3. incubating lysedred cells with 17% isopropanol, causes precipitation of unstable hemoglobins
4. Hbs Hasharon, Koln & Zurich only Hb Hammersmith associated with severe hemolysis
Methemoglobin (Hi)
1. how is different from normal hemoglobin?
2. what causes these changes?
3. T/F Hi is capable of combining with oxygen
1. The iron in the Hb molecule is in the oxidzied ferric (Fe+++) state instead the usual ferrous (Fe++) form
2. often due to oxidation of hemoglobin
3. False, Hi is unable to combine with oxygen
1. T/F methemogobin (Hi) is normally present in the body
2. How is Hi normally metabolized/
1. True, normally a small degree of Hb oxidation, up to 1.5% of Hb is Hi
2. reduced in erythrocytes by NADH-dependent methemoglobin reductase system
1. What's the threshold of methemoglobin (Hi) before causing cyanosis?
2. Gross appearance of blood?
1. When Hi reaches 10% of total Hb (around 1.5 g/dL) cyanoss results
2. Chocolate brown color
Which of the following is capable of detecting methemoglobin?
A. co-oximeter
B. pulse oximeter
C. arterial blood gas analyzer
A. CORRECT
B&C. INCORRECT, both estimate oxygen level by emitting red light (wavelength 660 nm) absorbed by oxyhemoglobin and infrared light (940 nm). Methemoglobin absorbs both of these wavelengths so it isn't detected
What causes hereditary methemoglobinemia?
Either deficiency in the reductase system or abnormal hemoglobins (HbM) upon which this enzyme cannot act
What is HbM?
A group of abnormal hemoglobins that prefer the ferric (methemoglobin) state due to various amino acid substitutions
At what age is hereditary methemoglobinemia clinically apparent?
Cyanosis appears at 6 months of age, unless there's M fetal Hb. If so cyanosis abates at about 6 months
Where are most M hemoglobins found on routine gels?
they run with A
Common causes of acquired methemoglobinemia?
Exposure to drugs or chemicals that increase the formation of Hi, common examples include: nitrites, quinones, phenacetin, and sulfonamides
Why are nitrites part of the treatment for cyanide toxicity?
They produce methemoglobin which has a high affinity for cyanide and chelates it
What's the treatment for methemoglobinemia?
Methylene blue, which reduces Hi to Hb
Sulfhemoglobin (SHb)
1. How is it formed?
2. What are Heinz bodies?
3. T/F SHb can't transport oxygen
1. when Hb is oxidized in the presence of sulfur
2. precipitate of SHb when it's further oxidized
3. True
T/F Unlike Hi, SHb can't be reduced to Hb
TRUE
At what level is SHb normally present and at what level does it cause cyanosis?
Normally present at >1% of total Hb, cyanosis manifests at around 3-4% or 0.5 g/dL
What conditions may increase sulfhemoglobin?
Presence of sulfonamides and C perfringens bacteremia (eneterogenous cyanosis)
Where is thalassemia most prevalent?
Mediterranean, Africa, Southeast Asia. Parallels the prevalence of malaria.
On what genes are the α- and β- genes located?
α-genes are located on chromosome 16. β-genes are located on chromosome 11p15.5.
How many copies of the β gene are on each chromosome?
One, for a total of two productive genes in normal cells.
How many copies of the α gene are on each chromosome?
Two, for a total of four α chain producing gene loci in each normal cell.
What type of mutations are most common in β-thalassemia?
Point mutations
What type of mutations are most common in α-thalassemia?
Large deletions
What is the most common genotype in African Americans with thalassemia?
(-α/). The is a genotype in which chromosome 16 has one normal and one deleted α gene.
What are the typical CBC findings in thalassemias?
Elevated RBC count (>5.5 x 10^12 in men, >5.0 x 10^12 in women) Low MCV (65-75 fL in α-thal; 55-65 fL in β-thal) Low hematocrit Normal to slightly elevated RDW
What MCV/RBC count favors thalassemia?
An MCV/RBC count ratio < 13 favors thalassemia, while a ratio > 15 favors iron deficiency.
What are typical peripheral smear findings in thalassemia?
Microcytic hypochromic anemia with occasional target cells (more in β-thal) and basophilic stippling.
In what populations is α-thalassemia most common?
Sub-saharan Africans and southeast Asians. The α thal 1 gene is prevalent only in Asians, and it is they who are at risk for the very severe kinds of α-thalassemia (Hg Bart and Hg H diseases). The α thal 2 gene is most prevalent in blacks.
What type of symptoms do persons with single gene deletion α-thalassemia have?
None. They are entirely asyptomatic, have normal CBC, and normal electrophoresis.
Persons with α-thalassemia trait (2 gene deletions) manifest what clinically?
A CBC with thalassemic indices and electrophoresis with normal A and A2 bands. A2 is not increased. In the absence of iron deficiency, this can be interpreted as consistent with α-thal trait.
In what populations β-thalassemia most common?
Mediterranean populations.
At what age do manifestations of β-thalassemia become evident?
6 to 9 months.
Beta thal minor typically result from inheritance of how many abnormal β genes?
One. Either β+ or β⁰.
What is the most common electrophoretic pattern in beta thal minor?
High HbA2 (over 2.5%, usually 4-8%and normal HbF. The second most common shows a normal A2. This is due to simultaneous iron deficiency, and may be erroneously interpreted as consistent with α-thalassemia.
In δ-β thal (deletion of both the δ and β genes), what is the electrophoretic pattern?
Normal A2 and elevated HbF (5-20%).
In heterozygous Hb Lepore (fusion of δ and β), what is the electrophoretic pattern?
Normal A2, slightly elevated HbF, and a band in the S region comprising 6-15% (Hb Lepore).
Beta thal major results from inheritance of how many abnormal genes?
Two, such as β⁰ β⁰, β+med β+med, or β⁰ β+med.
What is the most common cause of death in childhood in patients with beta thal major?
Individuals are not anemic at birth but develop anemia within one year. The most common cause of death in childhood is infection.
What is the electrophoretic pattern in beta thal major?
Increased HbF (50-95%), normal to elevated A2, and little to no HbA.
What is the clinically distinguishing factor between beta thal intermidia and beta thal major?
Beta thal major is transfusion dependent.
Warm autoimmune hemolytic anemia is usually mediated by what type of antibody?
A warm-reacting IgG antibody.
In warm autoimmune hemolytic anemia, does the responsible antibody usually have a broad or narrow reactivity with red cell antigens?
Usually a broad reactivity, especially Rh antigens. Uncommonly, the antibody has a narrow specificity; eg, for a specific Rh antigen, Kell, Kidd, etc.
What is the crucial test in diagnosing autoimmune hemolytic anemia?
The DAT (antiglobulin test, Coombs test). It is positive in nearly all cases of warm autoimmune hemolytic anemia.
What type of reactivity can be seen in the DAT test in a patient with warm autoimmune hemolytic anemia?
Usually polyspecific and anti-IgG reagents, sometimes with both anti-IgG and anti-C3, and uncommonly with anti-C3 only. Infrequently, the DAT may be falsely negative, due to very rapid intravascular destruction of RBCs or very low titer Ab.
How are cells destroyed in warm autoimmune hemolytic anemia?
The Ab binds to Ags on the red cell surface. In most cases, the bound Ab acts as an opsonin that provoked RBC destruction by splenic macrophages (extravascular hemolysis). In this instance, some escape and are seen on smear as spherocytes. In other cases, the Ab activates complement and results in either intravascular hemolysis (through formation of the MAC), or opsonization (cascade arrested at C3b).
How do patients with warm autoimmune hemolytic anemia present?
Patients present with variable severity. Some have abrupt onset and severe symptomatic anemia, while others have a chronic low grade hemolysis.
Is warm autoimmune hemolytic anemia a primary or secondary disease?
Can be both. Secondary comprises about 70% of cases, and occurs in hematolymphoid neoplasms, inherited autoimmunity (common variable immunodeficiency, IgA deficiency, Bruton’s), collagen vascular disease, thymoma). Stem cell transplantation has also been associated.
What type of antibodies are cold agglutinins?
IgM
What is the specificity of cold agglutinins?
Cold agglutinins are IgM antibodies with specificity that is most commonly anti-I. Others include anti-i, anti-H, anti-Pr, and anti-IH.
What are the most important laboratory features in predicting pathogenicity of cold agglutinins?
Titer and thermal range (thermal amplitude).
At what temperature do nonpathologic cold agglutinins react?
Nonpathologic cold agglutinins react most strongly at 4⁰ C, but have variably wide thermal amplitudes and may react at up to 22⁰C.
What is the titer of most nonpathologic cold agglutinins?
The titer of benign cold agglutinins is usually <64 at 4⁰C.
What is the only reliable CBC index in the presence of cold agglutinins?
Hemoglobin.
At what temperature do pathologic cold agglutinins react?
They are reactive over a broad range, up to 32-37⁰C and cause spontaneous autoagglutination in anticoagulated blood at room temperature.
What is the titer of pathologic cold agglutinins?
The titer is often >1000 at 4⁰C.
What is the predominant clinical scenario in idiopathic cold autoimmune hemolytic anemia?
A chronic condition found predominantly older individuals complaining of acrocyanosis and Reynaud phenomenon with a moderate hemolytic anemia.
What is the clinical scenario in secondary cold autoimmune hemolytic anemia?
A transient condition often associated with infection. M pneumonia infection is associated with anti-I, and EBV-associated infectious mononucleosis is associated with anti-i.
for beta-globin, what are definitions and causes of βº, β+, silent and complex alleles?
1. βº: complete absence of β-chain production
2. β+: diminished β-chain production
3. silent: almost no impact on chain production
4. fusion δ-β- and γ-δ-β- chains resulting from deletion of noncoding intervening segments of the HBB gene cluster
in α–thalassemia, β4 and γ4 tetramers form?
Continued synthesis of normal amounts of the unaffected chain leads to its relative abundance and precipitation of these chains in the red cell, reducing the cell's lifespan.
what are the genotypes, CBC and electrophoresis findings of the five thalassemia syndromes?
Syndrome, Genotype, CBC Electrophoresis
1. normal, αα/αα, normal, normal
2. silent carrier, −α/αα, normal, normal
3. α-thal trait, −α/−α or −−/αα, thalassemic, normal
4. HbH disease, −−/−α or −−/αCSα, thalassemic Heinz bodies, fast-migrating Hb H, Hb H = β4 tetramers.
5. Hb Bart disease (hydrops fetalis), −−/−−, hypochromia nRBCs, fast-migrating Hb Barts. Hb Barts = γ4 tetramers.
α thalassemia is most common in which two populations and their prevalent alleles
sub-Saharan African and southeast Asian descent
Acquired hemoglobin H might be seen in (3)
myeloproliferative disorders, and myelodysplastic syndromes.
When does the direct Coombs test give false positives and false negatives?
Infrequently, the DAT may be falsely negative, due to very rapid intravascular destruction of erythrocytes or very low titer antibody. Furthermore, a small percentage of healthy people have a positive DAT.
When does the secondary autoimmune hemolytic anemia occurr? (5)
hematolymphoid neoplasms (especially CLL/SLL), inherited autoimmunity (especially antibody deficiency—common variable immunodeficiency, IgA deficiency, Bruton agammaglobulinemia), collagen vascular disease, and thymoma and Stem cell transplantation
anti-I is associated with what infection?
M pneumoniae
anti-I is associated with what infection?
EBV
How to avoid the false screening and crossmatch results caused by cold autoantibodies?
As with warm autoantibodies, the task is to look beyond the cold-reacting antibody for masked alloantibodies. Options include a prewarmed screen or crossmatch, using serum from a cold autoadsorption or adsorption with rabbit erythrocyte stroma (REST), or serum pretreated with DTT or 2-ME (disrupt IgM sulfhydryl bonds).
The reagents for Identifying Cold Agglutinins include Type O cord blood, Type O adult blood, Type A1 adult blood, Type A2 adult blood and Saliva. What antigen(s) do they possess?
Cells, I antigen, i antigen, H antigen, IH, Pr
Type O cord blood: −, +++, +++, +/-, +++
Type O adult blood: +++, −, +++, +++, +++
Type A1 adult blood: ++, −, −, -/+, +/-, +++
Type A2 adult blood: ++, −, +, +++, +++
Saliva: −, −, +
Paroxysmal cold hemoglobinuria is often seen in children associated with which morbility?
viral illnesses such as measles, mumps, chickenpox, and infectious mononucleosis
Definition of Paroxysmal cold hemoglobinuria
paroxysmal episodes of hemoglobinuria associated with cold exposure
treatment of Paroxysmal cold hemoglobinuria
keeping the patient warm and transfusing as necessary
Donath-Landsteiner test for Paroxysmal cold hemoglobinuria
A positive test is obtained if only incubation of the patient's red cells at 4°C then 37°C leads to hemolysis.
Cryoglobulins
immunoglobulins that precipitate reversibly at low temperatures.
how To detect cryoglobulins
blood is drawn and kept at 37°C until clotted. It is centrifuged at 37°, and the remaining serum is stored at 4°C for at least 3 days. It is then centrifuged at 4°. Any precipitate that forms is a cryoprecipitate which can be subjected to electrophoresis for characterization.
Three types of cryoglobulins
Type I cryoglobulins are monoclonal immunoglobulins found in association with multiple myeloma or Waldenstrom macroglobulinemia. Type II cryoglobulins are a mixture of a monoclonal IgM and a polyclonal IgG. The IgM has rheumatoid factor activity (anti-IgG). Type II is the most common type of cryoglobulin. Type III is a mixture of two polyclonal immunoglobulins.
most common cause of mixed cryoglobulinemia
HCV
the most common renal finding in cryoglobulinemia
membranoproliferative glomerulonephritis (MPGN)
peripheral smear for iron deficiency anemia
Microcytosis (↓ MCV)
Hypochromia (↓ MCH)
Anemia
Anisocytosis (↑ RDW)
Poikilocytosis (thin elliptocytes = pencil cells)
Thrombocytosis
Chemistries for iron deficiency anemia
↑ Zinc protoporphyrin (ZPP)
↓ Iron
↑ Total iron binding capacity (TIBC)
↓ Iron saturation
↓ Ferritin
the temporal progression in peripheral smear for iron deficiency
1. decrease in the serum ferritin is the earliest event, 2. followed by a decrease in the percent saturation of transferrin, decreased serum iron, and increased zinc protoporphyrin (ZPP).
3. then a decrease in the hemoglobin, followed by what is initially a normocytic, normochromic anemia.
4. The MCV progressively drops, eventually below 80, and the red cells become progressively hypochromic and poikilocytotic.
serum ferritin in iron deficiency
usually under 10 μg/L in established iron deficiency. Ferritin is an acute-phase reactant, however, and may be nonspecifically elevated in hepatic insufficiency (impaired clearance).
when is Serum soluble transferrin receptor elevated?
whenever there is a relative lack of iron (iron deficiency and in erythroid hyperplasia such as hemolytic anemia, hemorrhage, or polycythemia)
when are zinc protoporphyrin (ZPP) and free erythrocyte protoporphyrin (FEP) elevated?
in iron deficiency but also elevated in lead poisoning and anemia of chronic disease
1 mL of whole blood contains how much iron?
1 mL of whole blood contains 0.5 mg of iron (1 mL of packed RBCs contains 1 mg of iron).
Where in GI is iron absorbed?
duodenum
iron deficiency and lead levels
Children with elevated lead levels are more likely to have iron deficiency
chemistries for anemia of chronic disease
decreased iron, decreased TIBC, >15% iron saturation and normal to elevated ferritin
How is B12 absorbed?
B12 is ingested in animal products (mostly), bound to R factor in stomach, released from R factor in duodenum by pancreatic enzymes, and bound to gastric-derived intrinsic factor (IF). IF-bound B12 is absorbed in the ileum, bound to transcobalamin I & II (90%) in enterocytes, and exported to the blood stream.
How is folate absorbed?
Folate is ingested in green vegetables (mostly), absorbed in jejunum, and released from enterocytes as N5-methyl folate. After transport in blood stream, it is converted in target cells to THF by the B12-dependent methyltransferase.
peripheral smear for megaloblastic anemia
marked oval macrocytosis, hypersegmented neutrophils, and large platelets
bone marrow findings for megaloblastic anemia
1. hypercellular marrow.
2. Nuclear maturation arrest associated with essentially normal cytoplasmic maturation leads to the characteristic nuclear:cytoplasmic dyssynchrony
LDH and serum bilirubin in megaloblastic anemia
Many erythroblasts perish while still in the marrow. Thus megaloblastic anemia is in part a hemolytic anemia, and is commonly associated with a very high LDH and a mild to moderate elevation in serum bilirubin.
What cause falsely normal serum folate and falsely low RBC folate
1. One or several balanced meals can quickly normalize the serum folate, but the red blood cell folate is more stable over time.
2. Vitamin B12 deficiency can produce a falsely low RBC folate, but it does not affect the serum folate.
serum B12 in HIV patients
Serum B12 levels are often low in patients with HIV infection, but true B12 deficiency is very uncommon in this situation.
B12 level in myeloproliferative diseases
falsely elevated B12 level
Schilling test
The patient is given a parenteral dose of unlabeled B12 followed by an oral dose of radiolabeled vitamin B12. The purpose of the unlabeled dose is to fully saturate the body with B12 so that the radiolabeled dose will be quickly excreted in the urine. A 24-hour urine sample is then collected. A low level of urinary radioactivity confirms B12 malabsorption, but it does not identify the specific gastrointestinal defect. The second part of the Schilling test is then undertaken. The patient is given another oral dose of radiolabeled B12 in addition to oral intrinsic factor. Patients with pernicious anemia will demonstrate enhanced absorption (increased urinary radioactivity) in this second part of the test.
sprue, folate or B12 deficiency?
folate
pernicous anemia , folate or B12 deficiency?
B12
pancreatic insufficiency, folate or B12 deficiency?
B12
crohn disease, folate or B12 deficiency?
B12
Methotrexate, folate or B12 deficiency?
folate
Dilantin, folate or B12 deficiency?
B12
Transcobalamin II deficiency, folate or B12 deficiency?
B12
peripheral smear for anemia of chronic disease
normocytic and normochromic, but is microcytic in up to a third of cases
the diagnostic criteria for anemia of chronic disease
1. low reticulocyte count
2. normocytic or microcytic anemia
3. increased iron stores (normal to high serum ferritin or increased stainable iron in a bone marrow biopsy) and a low serum iron, low transferrin, and low total iron-binding capacity
What to do if ferritin is normal in a suspected iron deficiency?
test the soluble serum transferrin receptor
In Anemia usually accompanied by reticulocytosis, when is the reticulocyte count is misleadingly normal?
1. early treatment of iron, folate, or B12 deficiency
2. rare autoimmune hemolysis that targets not only mature erythrocytes but also maturing marrow erythroid precursors
Basophilic stippling
Small blue dots in red cells, due to clusters of ribosomes.
Hemolytic anemias, lead poisoning, thalassemia
Pappenheimer bodies
Larger, more irregular, and grayer than basophilic stippling, due to iron-containing mitochondria
Asplenia, sideroblastic anemia
Heinz bodies Bite cells
Heinz bodies: gray-black round inclusions, seen only with supravital stains (crystal violet). Bite cells: sharp bite-like defects in red cells where a Heinz body has been removed in the spleen. Both are due to denatured hemoglobin
Oxidative injury: G6PD deficiency or unstable hemoglobins
Howell-Jolly bodies Cabot rings
Howell-Jolly body: dot-like dark purple inclusion. Cabot ring: ring-shaped dark purple inclusion. Both represent a residual nuclear fragment
Asplenia
Target cells
Red cells with a dark circle within the central area of pallor, reflecting redundant membrane
Thalassemia, hemoglobin C, liver disease
Schistocytes
Fragmented red blood cells, taking shapes such as helmet-shaped cells, due to mechanical red cell fragmentation.
Microangiopathic hemolytic anemias (MHA): DIC, TTP, HUS, HELLP. Mechanical heart valves.
Dacrocytes (teardrop cells)
Teardrop or pear-shaped erythrocytes
Can be seen in relatively benign conditions (thalassemia, megaloblastic anemia), often seen in myelophthisis
Echinocytes (burr cells)
Red blood cells that have circumferential undulations or spiny projections with pointed tips
Uremia, gastric cancer, pyruvate kinase deficiency
Acanthocytes (spur cells)
Red blood cells that have circumferential blunt and spiny projections with bulbous tips
Liver disease, abetalipoproteinemia, Mcleod phenotype
Spherocytes
Red cells without central pallor due to decreased red cell membrane
Immune hemolytic anemia, hereditary spherocytosis
Elliptocytes
Red cells twice as long as they are wide
Iron deficiency, hereditary elliptocytosis
Stomatocytes
Red cells whose area of central pallor is elongated in a mouth-like shape
Alcohol, Dilantin, Rh null phenotype (absence of Rh antigens), hereditary stomatocytosis
EPO-secreting neoplasms
renal cell carcinoma,
cerebellar hemangioblastoma,
uterine leiomyomas, and
hepatocellular carcinoma
A transient neonatal polycythemia may be seen in
of diabeteic mothers (IDM) and Down syndrome
basophilia, thrombocytosis, and splenomegaly
myeloproliferative disorders
In Polycythemia Vera, describe changes in RBC mass, Pa02, Leukocytes & basophils, LAP score, Serum B12, EPO, Serum iron/stainable iron, Platelet aggregation studies.
↑ RBC mass, Normal Pa02, ↑ Leukocytes & basophils, ↑ LAP score, ↑ Serum B12, ↓ EPO, ↓ Serum iron/stainable iron, Platelet aggregation studies abnormal
Neutrophilia
-Reactive neutrophilia usually does not exceed 30 x 10^3
-commonly due to infection
-other causes include meds (steroids), trauma, burns, systemic inflammation, seizure, exercise, post-splenectomy, leukocyte adhesion defect, and pregnancy
When neutrophilia is persistent or without cause think Myeloproliferative disorder.
Accompanied by toxic granulation, Dohle bodies, and cytoplasmic vacuoles.
Features favoring Myeloproliferative disorders in persistent/unidentified cause are:
-Basophilia
-Lack of "toxic" morphology
-A 'myelocyte bulge' =(myelocytes present, often out numbering
In reactive neutrophilia the LAP score is
Increased.
It is also increased in non-CML forms of myelodysplastic disorders.
In CML the LAP score is
Decreased
GM-CSF causes
-marked lymphocytosis
-toxic granulation
- nRBCs
- abnormal nuclear segmentation
A type of reactive neutrophilia in which toxic changes are regularly absent?
Hantavirus pulmonary syndrome
During HPS prodrome, thrombocytopenia is the only dependable finding.
- once pulmonary edema sets in, so does left-shift, lack of toxic granulation, increased hemoglobin (hemoconcentration) and < 10% of lymphocytes having immunoblastic morphology. 4 of 5 is highly specific.
Causes of lymphocytosis includes:
-viral (EBV,CMV,HIV, etc.), toxoplasmosis, and medications
Reactive lymphocytic proliferations in blood and bone marrow are composed principally of:
T lymphocytes

These take the form of atypical lymphocytes, they are larger with moderate to abundant cytoplasm and are often granulated.
Syndrome of persistent polyclonal B lymphocytosis
Young adults who smoke and are often HLA-DR7+
-Presents as mild absolute increase in lymphocytes with indented bilobed nuclei and abundant pale-staining cytoplasm.
- There is polyclonal IgM hypergammaglobulinemia, and no other cytopenias.
Reactive lymphocytosis in children with small mature lymphocytes with clefted nuclei (reider cells) is classically associated with?
Pertussis - whooping cough
Bordella pertussis
Gram negative, aerobic coccobacillus
Non-motile
Bordet-Gengou agar or Buffered charcoal yeast extract plate with cephalosporin to select for organism, mercury drop like colonies
Monocytosis is often reactive, but persistent monocytosis without other explanation is...
Think CMML (chronic myelomonocytic leukemia)
Causes of monocytosis
-collagen vascular diseases
-chronic infection (listeria and TB)
- malignancy
- neutropenia
A monocytic neoplasm is suggested by the presence of
promonocytes and/or splenomegaly
A serum measurment to clarify that a neoplastic process (ie AML) has monocytic differentiation?
Lysozyme
Eosinophilia is almost always an allergic reaction. Worldwide, helminthic infections. Other causes?
-collagen vascular diseases
-malignancy
-inflammatory bowel disease
-GM-CSF
What cytokine is specific for eosinophilic lineage
Interleukin-5
Responsible for selective differentiation of eos and release of eos from bone marrow
Eosinophilic cellulitis
Well syndrome
-Recurrent granulomatous dermatitis with eosinophilia
-mildly pruritic or tender cellulitis like eruption with edema, flame figures and marked eos in the dermis
Eosinophilic pneumonia
Loeffler syndrome
Eosinophilic fasciitis
Shulman syndrome
Thickened subq septa and deep fascia with variable inflammatory infiltrate with plasma cells and eos.
Eosinophilic vasculitis
Churg-Strauss
-Medium and small vessel vasculitis leading to necrosis.
-Markers include eos and granulomas
-P-anca positive
The normal number of blasts in the peripheral blood is?
NONE
To determine blast lineage one must
Get cytochemical and immunophenotypic studies done to properly classify.
When meloblasts are present the criteria for the diagnosis of AML in the peripheral blood is?
>20% blasts
If peripheral blood criteria is not met one must get a bone marrow.
How can one distinguish M3 AML (APML) from mon-M3 AMLs?
Decisive test for M3 is detection of the t(15,17) translocation, but time consuming.
Less definitive but more rapid approach is to use morphology, MPO, and the combined CD34-/CD13+/CD33+/HLA-DR- immunophenotype
If all the blasts are lymphoid in a patient what is the differential?
Burkitt
Precursor-B-ALL
Precursor-T-ALL
Infections that cause neutropenia include
Typhoid
Brucellosis
Tuleremia
Rickettsial
Overwhelming sepsis of any bacterial cause
Causes of neutrophil destruction include
Autoimmune(lupus, RA)
Splenomegaly
Medications
Infection
The most common cause of neutropenia?
Medications
Antibiotics
Anti-thyroids (agranulocytosis)
Anticonvulsant
Procainamide
NSAIDs
Felty syndrome
The triad of
RA
Neutropenia
Splenomegaly
Decreased production of isolated neutrophil cells can be caused by?
Meds
Granular lymphocyte leukemia
And the constitutional neutropenias
In neutropenia the peripheral smear shows compensatory?
Monocytosis
Lymphopenia
Uncommon seen in SLE, HIV, SARS, antiCD20 (rituxan) therapy, steroids and certain congenital immunodeficiency (Burton, SCID, DiGeorge, CVI)
Monocytopenia
Think hairy cell
In pts undergoing chemo, monocytopenia heralds the onset of neutropenia
A reactive follicular hyperplasia patter involves
Increase in lymphoid follicles usually due to an expansion of the germinal center. The mantle zone is intact. Polarization is manifested as a greater thickness of the mantle zone towards the capsule (or to the crypt-tonsil/towards serosa in intestine). Germinal centers have mitosis and tingible-body macrophages
In follicular lymphoma the germinal centers usually remain separate and vary in size.
In follicular hyperplasia staining with bcl-2 is
Weak or absent within the germinal center ( but strong in the surrounding mantle)
PCNA (Ki-67, proliferating cell nuclear antigen) has what kind of expression in follicular hyperplasia?
Strong
Follicular lymphoma
Follicles are naked (mantle obliterated) and confluent
Mitosis is rare
bcl-2 stain in strong
PCNA is weak
Causes of follicular pattern of hyperplasia includes:
-Unknown etiology
-Viruses (HIV)
-Rheumatoid arthritis and sjogren (often with inter follicular plasmacytosis
-syphilis (has plasmacytosis and capsular/trabecular thickening and capsular infiltration by plasma cells)
Castlemans - two types
Hyaline vascular type most common- in the mediastinum, not systemic.

-The germinal centers are atretic rather than hyperplastic.
-Characterized by one or two central hyalinized blood vessels.
-The mantle is often hyperplastic in an onion ring fashion
Plasma cell variant - multicentric, often assoc. with systemic manifestations like POEMS.
-resembles rheumatoid lymphadenitis with interfollicular plasmacytosis and hyperplastic or regressed germinal centers
-some cases caused by HHV-8 often arising in HIV. These show blurring of the germinal center-mantle boundary, atypical plasma cells (can be clonal), and increased immunoblasts
Interfollicular pattern is characterized by
Expansion of the interfollicular (paracortical) zones
- lymphoid follicles are small
- can be a variety of cells: small lymphocytes, transformed lymphocytes, immunoblasts, and plasma cells.

Differential diagnosis includes:
-viral (CMV, postvaccinal lymphadenitis)
- hypersensitivity reaction (Dilantin)
- kimura disease
Kimura Disease
-endemic in Asia
- young men
- presents as soft tissue mass of head/neck with cervical lymphadenopathy, peripheral eosinophilia, and increased IgE.
- recurrences frequent, but good prognosis
Lymph nodes are characterized by florid follicular hyperplasia with increased vascularity and proteinaceous deposits, paracortical eosinophilia, increased prominence of post capillary venules, and interfollicular viral-type immunoblasts.
Angiolymphoid hyperplasia with eosinophilia
Women effected more than men
- skin lesions
- lacks lymphadenopathy, eosinophilia, and hyper immunoglobulin E.

In contrast to kimura disease
Sinus pattern of hyperplasia is characterized by
Dilated sinuses with histiocytes

Differential diagnosis includes:
Sinus histiocytosis
Rosai-Dorfman
Lymphangiogram effect
Whipple Disease
Hemophagocytic syndrome
Detmatopathic lymphadenitis
Rosai-Dorfman
-Bilateral cervical lymphadenopathy
-sinuses filled with foamy histiocytes with emperipolesis
-surrounding infiltrate rich in plasma cells
-self limiting, rarely fatal
-histiocytes express what immunohistochemistry markers?
S100
CD11b
CD14
Lysozyme
HLA-DR
And unexpectedly CD31
Lymphangiogram
Sinus expansion pattern in lymph nodes
Whipple Disease
-Male predominance
-Infiltration of multiple organs by foamy histiocytes
-intestinal, joint, CNS, cardiac valve involvment and common
Lymph nodes contain PAS+,diastase-resistant, AFB-bacilli (Tropheryma whippelii)
Hemophagocytic syndrome
Characterized by histiocytes with internalized degenerated and partially digested lymphoid cells.
Associated with EBV and certain types of lymphoma
Dermatopathic lymphadenitis
Found in pts with benign and malignant skin disorders and characterized by paracortical expansion by histiocytes containing faint pigment.

In mycosis fungoides this is an ominous sign.
suppurative granulomatous lymphadenopathy
a. cat-scratch
b. lymphogranuloma venereum
c. tularemia
necrotizing granulomata without suppuration
a. mycobacterial infection
b. brucellosis
c. fungal
d. yersinial
necrotizing granulomata without suppuration in the thorax or mediastinum
M. tuberculosis
H. capsulatum
C. immitis
necrotizing granulomata without suppuration in cervical nodes
M. scrofulaceum
necrotizing granulomata without suppuration in intraabdominal nodes, ileal peyer patches or appendix
yersinial
diffuse pattern benign lymphadenopathy
viral: CMV, HSV, measles