Board Review- Part 1:

Benign HemePath

Peripheral Blood

Smear Examination

Elevated MCV = Macrocytosis

MCV > 100um3

• B12/Folate deficiency, aplastic anemia, MDS

• Autoimmune hemolytic anemia

• Liver disease, hypothyroidism, alcoholism

• Cold agglutinin disease

Decreased MCV = Microcytosis

MCV < 80um3

• Iron deficiency

• Thalassemias

• Anemia of chronic

disease

• Hemoglobinopathies

– C, E, S, D

Iron Panel Interpretation

Cause of

anemia

Serum

iron

TIBC Percent

saturation

Iron

deficiency

↓ ↑ ↓

Thalassemias ↑ / N ↓ / N

↑ / N

Sideroblastic

anemia

↑ ↓ / N

↑

Chronic

disease

N/↓ ↓ N

Pathologic Red Blood Cells in

Peripheral Blood Smears

Type of Cell Underlying Change Disease States

Acanthocyte (spur cell) Altered cell membrane lipids Abetalipoproteinemia, liver

disease, postsplenectomy,

McLeod phenotype

Bite Cell (degmacyte) Heinz body pitting by spleen G6PD deficiency, drug-induced

oxidant hemolysis

Ovalocyte (elliptocyte) Abnormal cytoskeletal proteins Hereditary elliptocytosis

Rouleaux Circulating paraprotein Paraproteinemia

Schistocyte (helmet cell) Mechanical destruction in

microvasculature

DIC, TTP, HUS, prosthetic heart

valves

Spherocyte Decreased membrane

redundancy

Hereditary sphereocytosis,

immunohemolytic anemia (warm

Ab)

Stomatocyte Membrane defect with

abnormal cation permeation

Hereditary stomatocytosis, liver

disease

Target Cell (codocyte) Increased redundancy of cell

membrane

Liver disease, beta thalassemia

postsplenectomy, Hgb C/D/E/S

Burr Cell (ecchinocyte) Altered membrane lipids Usually artifactual but maybe

uremia

Tear Drop Cell (dacrocyte) Myelofibrosis

•Irregular, long, sharply pointed and

bent spicules

•Absence of central pallor

•Most commonly seen in liver disease

Acanthocytes (Spur cells)

Abetalipoproteinemia: absence of apolipoprotein B results in inability to transport

triglycerides in the blood-> Altered cell membrane lipids-> Acanthocytes

McLeod phenotype/syndrome

• Absence of erythrocyte surface Kx

antigen (part of Kell antigen group)

• Acanthocytes with chronic but well

compensated hemolytic anemia

• Disease processes include muscular

dystrophy, cardiomyopathy

• May be associated with chronic

granulomatous disease

Tear Drop Cells (Dacrocytes)

Myelofibrosis or bone marrow infiltrate

Bite Cells

• G6PD deficiency

G6PD deficiency

STOMATOCYTES

RBC with slit-like or rectangular area of central pallor, a mouth

Most often seen in liver disease

Burr Cells (Ecchinocytes)

Projections- smaller

more regular than

acanthocytes

Often artifactual but

may be seen in

UREMIA

Spherocytes

-Hereditary sphereocytosis,

-Immunohemolytic anemia (warm Ab)

Hereditary spherocytosis

• Northern European ancestry

• Spectrin, ankyrin, band 3 or band 4.1 deficiency

Hereditary spherocytosis

• Defects in vertical stabilization of the phospholipid bilayer of the RBC membrane cause separation of the spectrin - phospholipid bilayer

• Normal biconcave red cell loses membrane fragments and adopts a spherical shape • Inflexible cells are trapped in the splenic cords, phagocytosed by macrophages

Osmotic Fragility Test

• Equal volumes of blood are placed in a

series of hypotonic solutions

• Allowed to reach equilibrium (24 hr,

37 C); centrifuged

• The RBC swells and lysis occurs

• As the % saline decreases

further, the amount of lysis

increases (determined by

optical density )

Normal

HS

Elliptocytes

Hereditary elliptocytosis

• Autosomal dominant trait

• Spectrin abnormality or deficiency

of protein band 4.1

• Asymptomatic without anemia and

usually with no splenomegaly and

only mild hemolysis

21

Hereditary Pyropoikilocytosis

• Same mutations as in HE

• Present in the neonatal

period with severe

hemolytic anemia, RBC

fragments, poikilocytes,

elliptocytes,

microspherocytes,

hyperbilirubinemia

Hereditary Pyropoikilocytosis

(HPP)

• Reminiscent of erthrocyte morphology seen

after thermal burns

• Rare cause of severe hemolytic anemia

• 1/3 of family members with HPP have HE

• Many HPP patients have severe hemolytic

anemia in childhood that evolves into typical

HE later in life

Target Cells

Characteristic of: – Liver disease

– Post-splenectomy

– Hemoglobin disorders

• Beta thalassemia

• Hemoglobinopathies

Hb S, C, D and E

Hgb SC disease with C crystals, Taco cells and sickle cells

“Washington Monument crystals”

Schistocytes

Microangiopathic

hemolytic anemia

DIC, TTP, HUS

Rouleoux and Clumping

Rouleaux:

monoclonal gammopathy

vs. polyclonal gammopathy

RBC Clumping:

cold agglutinin

Hypochromic anemia

MCH < 27 pg

• Disorders of globin synthesis – Thalassemic syndromes

• α-Thalassemia

• ß-Thalassemia

• Disorders of heme synthesis – Sideroblastic anemias

• Hereditary (X-linked, auto. dominant)

• Acquired (lead poisoning, alcoholic, medication, MDS)

• Disorders of Fe metabolism – Fe deficiency

– Chronic disease

– Neoplasia

Iron Deficiency

Reticulocytes

• Decreased cell survival

– Blood loss

– Autoimmune hemolysis

– Nonimmune hemolysis

• TTP, HUS, DIC

• H. spherocytosis

• G6PD

• PNH

• Hemoglobinopathy

• Thalassemia

Nucleated RBCs

• Not normally present in

adult patients PS

• Present in:

– Severe hemolysis

– Myelophthisic condition

– Leukoerythroblastic

smear

Basophilic Stippling

Precipitated ribosomes (RNA)

Fine – variety of anemias:

Siderblastic, sickle cell,

megaloblastic

Coarse – Lead intoxication,

thalassemia

Heinz Bodies

• Precipitated denatured Hgb

• Seen in G6PD deficiency

• Seen with supravital staining – Crystal violet

– Brilliant cresyl blue

Howell Jolly Bodies

Dense,usually

single

Nuclear remnant

Seen in:

- Postsplenectomy

- Hemolytic anemia

- Megaloblastic

anemia

Pappenheimer bodies

Small, dense basophilic granules

(mitochondrial remnant that contains Fe)

Seen in:

- Sideroblastic anemia

- Post-splenectomy

The Malignant Mimicker:

Leukemoid Reaction

• Precursor granulocytes in the PBS

• WBC in the range up to 100K

• Response to severe stress or infection

• Other signs of malignancy not present (i.e. CML)

Neutrophil Disorders with

Abnormal Morphology

• Pelger-Huet anomaly

– Bilobed or nonsegmented nucleus

– asymptomatic

• May-Hegglin anomaly

– Cytoplasmic inclusions resembling Dohle bodies

– Many asymptomatic

• Chediak-Higashi syndrome

– Giant cytoplasmic granules in all granulocytes

– Immunodeficiency

• Alder Reilley Anomaly: inclusion is mucopolysaccharide (PAS+)

• Hypersegmentation

– B12/Folate deficiency, myelodysplasia, chemotherapy, or renal failure

Pelger-Huet Anomaly

• Inherited, AD

• Acquired =

“pseudo” Pelger-

Huet as in MDS

Chediak-Higashi Syndrome

• Autosomal

recessive

• Giant granules

• Severe

immunodeficiency

May Hegglin Anomaly

• Thrombocytopenia

• Enlarged platelets

• Variable neutropenia

• Inclusions also seen in

eosinophils, basophils, and

monocytes

• Autosomal dominant

• Many patients are

asymptomatic

• Non-muscle myosin heavy

chain A (MYH9) mutation

• No impairment on PMN

function

Alder Reilley Anomaly

• Resemble the large

primary granules of

promyelocytes

• Large, purple to purplish-

black, coarse azurophilic

granules

• No impaired PMN function

• Inclusion is a

mucopolysaccharide

(PAS+)

Alder Reilley Anomaly

• Autosomal recessive

• Associated with several

different types of genetic

mucopolysaccharide

disorders (Hurler, Hunter,

San Fillipo, Maroteaux-

Lamy)

• Affects connective tissues,

heart, bone, CNS

Hypersegmentation

• More than 5 lobes

• Seen most commonly in B12/folate def

• Uremia

• Chemotherapy

• Also seen in MDS and other MPN/MDS neoplasms

Eosinophilia

• Allergic/hypersensitivity reactions

• Drug allergies

• Parasitic infections

• Connective tissue/collagen vascular disease

• Neoplasms

– T-cell lymphoma

– Hodgkin lymphoma

• Sarcoidosis

• Hypereosinophilic syndrome/Chronic eosinophilic leukemia

• Chronic/acute leukemia a/w PDGFRRA or PDGFRB mutations

Basophilia

• Much more common in

malignancies like CML

vs. reactive

Reactive Lymphocytosis

• Diseases with nonreactive morphology

– Infectious lymphocytosis (Whooping cough)

– Transient stress lymphocytosis

• Diseases with reactive morphology

– EBV,CMV, Toxo, adenovirus, HHV-6, viral

hepatitis

Atypical/reactive lymphocytes

Plasmacytoid

lymphocyte

Ehrlichiosis

• Found in the SE and S. Central US

• Rickettsial organism

• Transmitted by ticks

Histoplasma

<>

Babesiosis

• Protozoa

• Endemic in the NE US

• Transmitted by the Ixodidae tick

• Morphology similar to malaria

• Tetrad form is diagnostic

• Risk Factors: – Post-splenectomy

– Immunocompromised

Malaria

• Ringed stage

(trophozoite)

• Can see other

stages within

RBCs

Gametocytes

Trophozoites (rings)

Plasmodium falciparum

Multiple rings/cell

Appliqué forms

1-2 chromatin dots

Plasmodium vivax

Early Trophozoites (rings)

Enlarged RBCs

Schuffner’s Dots

Ameoboid rings

Mature trophozoite ->schizont

Giant Platelets

• Size of an RBC

• Usually indicates a hyperreactive bone marrow 2o to underlying condition – ITP, TTP, DIC

• Can be inherited in the form of Bernard-Soulier syndrome, May-Heglin anomaly

Platelet Clumping and

Satellitelosis

• Causes artificially low platelet

counts

• 2o to EDTA used in collection tubes

Solution is to use

sodium citrate instead

of EDTA

Aplastic Anemia

Aplastic Anemia

• Reduction of erythroid,

granulocytic/monocytic, and

megakaryocytic cell lines in the

bone marrow and their progeny in

the peripheral blood.

Hypocellular Bone Marrow

Aplastic Anemia- Causes

• Constitutional- including Fanconi’s

• Idiopathic

• Secondary

– Chemical/drug

– Radiation

– Infection

– Immune disorders

Fanconi’s Anemia

• 2/3 of constitutional aplastic

anemia

• Autosomal recessive

– DNA repair defect (diagnosis:

chromosome breakage analysis)

• Phenotypic abnormalities- bone,

skin, mental retardation

• Aplasia develops by mid childhood

• Progression to AML

Chemical/Drug

• Dose-dependant

– Chemotherapy

– Benzene

• Idiosyncratic

– Chloramphenicol, anticonvulsants,

sulfonamides, gold, NSAIDs

Ionizing Radiation

• Aplastic anemia- acute

• Long-term effects are

myelofibrosis and acute leukemia

Infections

• Hepatitis

– Rare, often fatal

– Non-A, non-B, non-C, non-G

– Usually 6 wks after clinical

symptoms

– No relation to severity of illness

Parvovirus B19

• Usually red cell aplasia

– Selective cytotoxic invasion of

erthryoblasts

• Mild reduction in granulocyte and

megakaryocyte production

• Chronic hemolytic anemia pts. at risk

– Sensitive to cessation in RBC production

– Transient aplastic crisis

• Self limited

Parvovirus B19

Treatment

• Treat the underlying conditions:

infection, d/c meds

• For immune disorders: androgens,

immune modulation, cytokine therapy

• Transfusions

• Bone marrow transplant

Deficiencies of Glycolytic Pathway

-Mature RBCs have the capacity for a limited number of enzymatic reactions

-No mitochondria in RBC, so it depends entirely on anaerobic glycolysis to produce

energy.

-The mature RBC is completely dependent on glucose as a source of energy. Glucose

usually (90%) is catabolized to pyruvate and lactate in the major anaerobic

glycolytic pathway (Embden-Myerhof Pathway). In the process, ATP is generated to

play its major role in maintaining a cation gradient in the RBC, thus protecting it

from premature death.

-Hereditary deficiency of some of the glycolytic enzymes have been documented,

and several cause a hereditary nonspherocytic hemolytic anemia (HNSHA), whereas

others cause multisystem disease

-Most are rare

-Pyruvate kinase deficiency is the most common and comprises 90% of affected

patients

-Hexokinase (AR) Mild-severe

-Glucose-6-isomerase (AR) Mod-severe

-Phosphofructokinase (AR) Mild myopathy

-Aldolase (AR) Mild-mod

-Phosphoglucerate Mild-severe

kinase (X-linked) retardation

-***Pyruvate kinase (AR) Mod-severe

Deficiencies of Glycolytic pathway

Pyruvate Kinase Deficiency

-AR, patient must be homozygous for the trait to be

expressed fully

-Detected in infancy or childhood due to anemia, jaundice, splenomegaly , gall

stones. The severity of the condition is widely variable, even among patients with

the same level of deficiency.

Lab studies

-Norchromic normocytic, or macrocytic anemia with reticulocytosis in the absence

of blood loss, is suggestive of hemolysis.

-Enzyme assay and, more recently, DNA analysis by PCR or single-strand

polymorphism are available to confirm the diagnosis and to identify the carrier

state if the need arises.

Fluorescent Screening test for Pyruvate kinase deficiency:

Principle: Reduced pyridine nucleotide (NADH) fluoresces when illuminated with

long-wave UV light.

Phosphoenolpyruvate (PEP) + ADP--------------> Pyruvate + ATP

Pyruvate + NADH + H+ ----------------------------> Lactate + NAD+

LDH is present in excess of PK, NAD production is limited by PK levels. Thus,

there should be a time dependent loss of fluorescence as NADH is oxidized to

NAD when PK is normal.

In PK-deficiency, fluoresence by NADH fails to disappear.

Pyruvate

kinase (PK)

Lactate dehydogenase

(LDH)

RBC enzyme activity assays

-Spectrophotometric techniques that depend on the absorption of light of the

reduced pyridine nucleotide, NADPH, or NADH at 340nm.

-Reduction results in formation of NADPH or NADH, with increase in

absorbance at 340 nm, and oxidation results in formation of NADP or NAD with

a decrease in absorbance.

-The change in absorbance may be used to calculate enzyme activity.

Deficiencies of Hexose Monophosphate

Shunt Pathway

Hexose monophosphate Shunt (HMP)

G6PD deficiency Common

Gamma-Glutamylcysteine synthase Rare

GSH synthetase Rare

Glutathione reductase Rare

G6PD Deficiency:

-G6PD enzyme located on X chromosome

-> 30 different mutations give rise to a variety of clinical diseases

-Normally 2 isotypes of G6PD: A and B. Can be differentiated based on

electrophoretic mobility

-B isoform most common type of enzyme found in all population groups

-A isoform, found in 20% black men in US, migrates more rapidly on

electrophoretic gels than B. It has similar enzyme activity as B, and does not cause

disease

-11% of US black men have G6PD variant (G6PD A-). It has same electrophoretic

mobility as A, but is unstable, resulting in enzyme loss and ultimate enzyme

deficiency. Older RBC have only 5-15% enzyme

-G6PD A- most clinically significant type of abnormal G6PD among US Blacks

-Other G6PD variants predominate in other racial groups: G6PDMED in Sicilians,

Greeks, Sepharic Jews, Arabs. G6PDCANTON or G6PDMAHIDOL in Asian population

-X linked, so sex-linked inheritance pattern.

-Effect fully seen in affected men

-Carrier women are variable affected depending X chromosome inactivation

Clinical findings

-Episode of hemolysis following infection or ingestion of an oxidant drug

-In G6PD A- def, the hemolytic anemia is self-limited as the young RBC produced in

response to hemolysis have nearly normal enzyme.

-Five classes of G-6-PD deficiency exist based on enzyme activity levels, as follows:

1.Enzyme deficiency with chronic hemolytic anemia

2.Severe enzyme deficiency (enzyme level <10%)

3.Moderate-to-mild enzyme deficiency (enzyme level 10-60%)

4.Very mild-to-no enzyme deficiency (enzyme level > 60%)

5.Increased enzyme activity

Laboratory Tests:

-Acute hemolysis is associated with formation of Heinz bodies (denatured Hgb).

-Bite cells (eccentrocytes) may be seen

-Fluorescent Screening test for G6PD Deficiency:

In the presence of NADP+, G6PD is oxidized to form 6-phosphogluconate and

NADPH. Then

6-Phophogluconate + NADP+ ----------------------- > Ribulose-5-P + NADPH + H+

NADPH can be detected by fluorescence

When G6PD is low, less NADPH is formed.

Normal sample shows a bright fluorescence after 5-10 minutes of incubation,

whereas deficient sample shows no fluorescence.

6-phosphogluconate

dehydrogenase

Quantitative G6PD assay:

-Formation of NADPH from NADP+ from G6P is measurable by a change in

absorbance created by NADPH at 340 nm in a spectrophotometer

Heinz body test:

Crystal violet or neutral red is added to blood

Paroxysmal Nocturnal

Hemoglobinuria (PNH)

Introduction

• Recurrent, episodic

– Intravascular hemolysis

– Hemoglobinuria

– Venous thrombosis

• May not always be

– Paroxysmal (acute)

– Nocturnal

– hemoglobinuric

Additional Points

• Clonal disorder

– Not a malignant clone

• Deficiency of complement-control proteins

– On all affected hematopoietic cells

• Increased susceptibility to complement lysis

– Erythrocytes not able to endocytose

membrane attack compex (MAC: end product

of complement cascade) as opposed to

nucleated cells

Complement Control Proteins

•CD55 (decay accelerating

factor: DAF)

–Regulates C3 convertase

•CD59 (membrane inhibitor of

reactive lysis: MIRL)

–Modulates complement-

mediated lysis

Complement Control Proteins

Regulation of C3 Convertase

•CD55 (DAF)

–Inhibits association of

C4b and C2

–Promotes dissociation

of C4bC2a complex

(C3 Convertase)

Complement Control Proteins

• CD59 (MIRL) prevents formation of Membrane Attack Complex and lytic action

Clinical

Nocturnal Hemoglobinuria

•Classical

description

–Dark urine in morning

that clears through

the day

–Sleep decreases pH,

increases

complement activity

-Accumulation of

urine

Flow Cytometry •Preferred

confirmatory test

•Decreased

expression of CD55

and/or CD59

PNH

Normal

PNH

AUTOIMMUNE

HEMOLYTIC

ANEMIAS

WARM TYPE

-Abrupt onset

-May be severe

-Slight female preponderance

-IgG antibodies

-Extravascular hemolysis

-Antibodies commonly against broad Rh

antigens

WARM AIHA

COLD TYPE

-Acute or insidious onset

-Mild to severe anemia

-Female preponderance

-IgM antibodies

-Agglutination of red cells at low temperatures

-Complement activation

-Intravascular and extravascular hemolysis

-Antibodies commonly against I or I antigens

COLD TYPE

• Acute: postinfectious, self limited, younger patients

• Chronic: idiopathic, older patients

• Cold agglutinin disease (CAD): insidious, elderly women,

associated with lymphoproliferative disorders

COLD AIHA

•

LAB PARAMETERS

WARM COLD

DAT 2+ TO 4+ 2+ TO 4+

ANTI IgG + -

ANTI C3 RARE +

TREATMENT

WARM AIHA

-Corticosteroid therapy

-Intravenous IVIG

-Immunosuppressants

-Splenectomy

COLD AIHA

-Immunosuppressants and splenectomy: no role

-Keeping patient warm

-Treatment of underlying disease

-Plasmapheresis

Hemophagocytic syndrome

Syndrome Definition • “Hemophagocytosis” – Process by which

macrophages engulf RBCs, WBCs,

platelets and their precursors

• Hemophagocytic syndrome =

Hemophagocytic lymphohistiocytosis

(HLH)

– Two main groups: Genetic and acquired

– Mechanism: highly active yet ineffective

immune response that is life-threatening

Bone marrow aspirate

HLH - Pathophysiology

• Theory: Inappropriate immune reaction caused by proliferating, active T cells and macrophages with inadequate apoptosis of immunogenic cells

• Impaired cytotoxic function of NK cells

– Decreased NK activity with persistent T-cell activation

• Cytokine hypersecretion, inflammatory response

– IFN-gamma, TNF-alpha, IL-6, IL-10, M-CSF

– Responsible for clinical signs, cytopenias, coagulopathy, high triglycerides

– Infiltrate tissues and lead to necrosis and organ failure

HLH – Criteria

• sCD25 (alpha chain of soluble IL-2 receptor)

HLH and Infection

• Viruses (esp. EBV), bacteria, parasites, spirochetes,

mycobacteria, fungi

• Difficulty distinguishing acquired from familial

– Familial cases may be precipitated by infection

– May mimic an infectious illness

HLH and Infection

• Impractical to exclude all possible infectious causes

• If HLH criteria are met, then screen for EBV, CMV,

parvovirus B19, HIV

• History should guide testing

– HIV infection: Pneumococcus, Pneumocystis,

Histoplasma, Penicillium marneffei

– Animal exposure: Brucella, Rickettsia

– Travel exposure: Leishmania, malaria

HLH and other Associations

• Malignancy

– T-cell lymphoma

• Autoimmune diseases

– Juvenile RA, Still’s disease (systemic-onset juvenile

idiopathic arthritis), SLE

HLH - Laboratory

• CBC, AST/ALT, bilirubin, triglycerides, ferritin, fibrinogen

• Bone marrow: Insensitive

– May be negative in 2/3 of initial aspirates

• sCD25 >2400 U/ml

– Highly specific serum parameter

• NK cell perforin expression

• Genetic testing

– PRF1, Munc13-4 (available at Cincinnati Children's Hospital)

HLH - Treatment

• Immediate goal: Immune suppression

– Corticosteroids

– Cyclosporin A: prevents T cell activation

– Etoposide: Critical for EBV-infected pts, inhibits

EBNA synthesis in EBV-infected cells

• Genetic HLH: Bone marrow transplant

CRYOGLOBULINAEMIA

CRYOGLOBULINS

• Cryoglobulins are plasma proteins that

precipitate at low temperature ( 0 to 4 degree

Celsius ) and dissolve at higher temperature

• They were first described in 1933 by Wintrobe

and Buell (at John Hopkins Hosp)

• In 1962, Lospaluto et al. showed that

cryoglobulins contains more than one

immunoglobulin ( Am J Med 1962;32:142-7 )

Peripheral blood smear with clumps of

precipitated cryoglobulin

CLASSIFICATION( Brouet et al.’s )

• TYPE I:- Single Monoclonal Immunoglobulins

– Type I CGB consists of a single monoclonal Ig, usually IgG or IgM,

infrequently IgA, and very rarely monoclonal light chain protein

• TYPE II :- Mixed Monoclonal/ polyclonal IG

– Type II CGB are mixed CGB consist of two or more IG of different

classes. One component of the complex is a monoclonal protein

(with a high proportion being IgM), that has rheumatology factor

activity, in association with polyclonal IG component

• TYPE III :- Mixed polyclonal IG

– Type III CGB are also mixed CGB, consisting of two or more IG of

different classes, however, each component is a polyclonal IG

CRYOGLOBULIN TYPES &

ASSOCIATED DISEASES

Types of CGB Immunochemical

composition Associated diseases Comments

Type I : Single

Monoclonal IG

IgG

IgA

Monoclonal

L-Chain

IgM

B-cell dyscrasis:

Myeloma

W. Macroglobulinemia

CLL

Hairy Cell Leukaemia

Cryoglobulin

concentratio

n is usually

HIGH (>

5mg/ml )

CRYOGLOBULIN TYPES &

ASSOCIATED DISEASES

Type of CGB Immunochemical

composition

Associated Diseases Comments

Type II: Mixed

Monoclonal

/Polyclonal IG

IgM-IgG

IgG-IgG

IgA-IgG

B- Cell dyscasias:

Myeloma

W. Macroglobulinaemia

CLL

HCV

Circulating

immune

complexes

CGB con.

usually(5mg/ml)

CRYOGLOBULIN TYPES & ASSOCIATED DISEASES

Type of CGB Immunochemical

composition

Associated Diseases Comments

Type III : Mixed

Polyclonal IG

IgM-IgG

IgM-IgG-IgA

Autoimmune disease:

Rheumatoid arthritis

SLE

Sjogren’s syndrome

Hepatitis

Vasculitis

Circulating

immune complexes

in response to Ig

challenge in

Rheumatoid disease

and chronic

infections

CGB conc. is

usually < 1mg/ml )

CLINICAL ASSOCIATION

• Type I cryoglobulinemia are more likely to be symtomatic and are usually associated with acrocyanosis, retinal haemorrhage, Raynaud’s phenomenon, and arterial thrombosis

• High levels of type I cryoglobulins are associated with symptoms of hyperviscosity

• Mixed CG, are less commonly symptomatic and are usually associated with vascular purpura and arthritis/ arthalgia; this is secondary to deposition within tissue (immune complexes ) which activate complement and induce localized inflammation

HEPATITIS C VIRUS-ASSOCIATED

CRYOGLOBULINEMIA

• When no underlying disease is present, this is referred

to as essential mixed cryoglobulinaemia ( EMC )

• In EMC, the possible role of hepatotropic viruses has

been suggested by the high frequency of co-existing

liver abnormalities

• The prevalence of anti-hepatitis C virus (HCV )

antibodies and the correlation with clinical and

serological parameters of EMC have been

investigated

ANALYSIS PROCEDURE

• A white precipitate (cryoglobulin) appears in the

serum after 24-72 hours of storage at 37oC

• The cryoglobulins can be quatitated by:

Measuring the cryoglobulin protein

spectrophotometrically by absorbance at 280mm

• Ig component of cryoglobulins can be determine by:

– Immunoelectrophoresis

– Isoelectric focusing in asociation with

immunofaxition

THERAPEUTIC MODALITIES FOR

SYMPTOMATIC PATIENTS

• Corticosteriods

• Subcutaneous interferon alpha ( IFN ), with or without plasma

exchange

• Care must be given to ensure blood components are warmed

before infusion

Bone Marrow Disorders

of Nonhematopoietic

Elements

Lipid Storage Disorders

• Hereditary lysosomal

enzyme deficiency

– Autosomal recessive

• Partially degraded

lipids accumulate in

macrophages

– Liver, spleen, bone

marrow, etc.

• Organomegaly,

cytopenias

Gaucher’s Disease

• Enzyme deficiency: Glucocerebrosidase

• Accumulate Glucocerebroside

• 3 clinical forms, all AR

– Type I- adult

– Type II- acute infantile, neuronopathic

– Type III- subacute juvenile, neuronopathic

Gaucher’s Disease

• Gaucher cell

– “Wrinkled

cigarette paper”

or “crepe paper”

– PAS positive

Pseudo-Gaucher Cell

• Indistinguishable from true Gaucher

cell

• Conditions with high cell membrane

turnover

– CML, hemoglobinopathies, myeloma

– Increased burden of glucocerebroside

Gaucher Cell Look-alike

• MAI infection

• Macrophages packed

with organisms

– Negatively staining

organisms mimics

striations of Gaucher

cells.

Niemann-Pick Disease

• Enzyme deficiency: Sphingomyelinase

• Accumulate Sphingomyelin

• 3 clinical forms, all AR • Type A- early onset

• Type B- adult, no cerebral involvement

• Type C- late onset

Niemann-Pick Disease

• Foamy macrophages

– “Bubbly” appearance

– Weak PAS+

– Oil Red O and Sudan

black positive

• May also present as

Sea blue histiocytes

Sea Blue Histiocytes

• Macrophages contain ceroid

– Lipofuscin-like pigment

– H&E-> yellow-brown

– Wright/Geimsa-> bright blue green

– PAS positive

• High turnover states: CML, ITP

• Lipidoses and hyperlipidemia

Sea Blue Histiocytes

H & E

Geimsa

Sarcoidosis

• Epitheloid granulomas in lung and

other organs

– Bone marrow involvement 17%

– A/w lymphadenopathy, spleenomegaly,

lymphopenia

Sarcoidosis

• Epitheloid histiocytes

– Small, noncaseating

granulomas

Bone Marrow Infarction

• Coagulative necrosis

• Associated with neoplastic

process, vasocculsive disorders

and hemoglobinopathies

Bone Marrow Infarction

• Homogenously

staining

• Ghost cells

Serous Fat Atrophy

• AKA gelatinous

transformation

• Associated with

starvation and

wasting diseases

• Homogenous

extracellular

substance with

“gelantinous”

appearance

Renal Osteodystrophy

• Chronic renal insufficiency

– 2o hyperparathyroidism

• Increased osteoclast activity- irregular

scalloping of bony trabeculae and

peritrabecular fibrosis

Renal Osteodystrophy

• “Scalloping” of

bony trabeculae

• Peritrabecular

fibrosis

Nonmalignant

Lymphadenopathy

Reactive Lymphoid Hyperplasia

Follicular Pattern

• Numerous enlarged, oddly shaped follicles

• Prominent germinal centers

• Tingible body macrophages

• Nonhomogenous lymphoid population

• Frequent mitoses

• Polyclonal surface immunoglobulins

• Germinal centers negative for bcl-2

Reactive Lymphoid Hyperplasia

Sinus Pattern

• Prominent sinuses

• Histiocyte hyperplasia

• Proliferation of plasma cells

• Polyclonal surface immunoglobulins

Progressive Transformation of Germinal Center

Progressive Transformation of Germinal Center: expanded

follicular center infiltrated by mantle cells. A benign process.

Maybe a/w NLPHL

Infectious Mononucleosis

• EBV virus

• Clinical information: febrile, exudative pharyngitis, cervical lymphadenopathy, splenomegaly, abnormal LFTs, common in adolescence/young adulthood, rare after 40 y/o

• Peripheral blood lymphocytosis with atypical lymphocytes

• Expanded paracortex (T-cell zone) by many immunoblasts & R-S like cells

• Mottled pattern, foci of necrosis

• Different from NHL: polymorphous background of transformed lymphocytes, persistent of reactive follicles, architecture preservation. Immuno for R-S like cells: (+) for CD45, CD30 and CD20; (-) for CD15. Also many large CD8-pos cells

Measles Lymphadenitis

• Measles (rubeola) or history of recent vaccination

• Axillary, cervical, inguinal lymph nodes

• Mottled histologic pattern

• Follicular hyperplasia

• Proliferation of immunoblasts

• Warthin-Finkeldey giant cells (syncytia of lymphocytes)

HIV Lymphadenitis (persistent generalized

lymphadenopathy)

• Pattern A (Acute): Enlarged lymph node with hyperplastic follicles and reactive germinal centers, naked follicular centers, folliculolysis by mantle-zone cells, monocytoid B cells in sinus, Warthin-Finkeldey giant cells

• Pattern B (Chronic): Involution of germinal centers, depletion of lymphocytes, increased plasma cells, vascular hyperplasia

• Pattern C (Burnout): Small or absent follicles with hyalinized germinal centers and collagen-ensheathed arterioles (“lollipop”), plasma cells, more severe lymphocyte depletion-> naked stroma

Toxoplasma Lymphadenitis

• Most common parasitic infection in US

• Cat definitive host; Toxoplasma gondii

• Posterior cervical LN most often affected

• Triad: Reactive germinal centers; perifollicular and intrafollicular clusters of epithelioid histiocytes; patches of monocytoid cells in sinus

• Serologic tests for confirmation

Kikuchi-Fujimoto

(Subacute necrotizing lymphadenitis)

• Affects predominantly young Asians

• Females more often than males

• Benign course, spontaneous remission

• Fever, cervical lymphadenapthy

• Patchy necrosis, marked apoptosis, nuclear debris

• Aggregates of histiocytes

• Neutrophils, eosinophils absent

• Kawasaki’s disease: infancy and childhood. Histology similar to Kikuchi + fibrin thrombi

Systemic Lupus

Lymphadenopathy

• Architecture effaced

• Follicles inconspicuous

• Necrosis focal or confluent

• Nuclear debris and hematoxylin bodies (basophilic masses of

DNA)

• Presence of plasma cells, immunoblasts

• Vasculitis with fibrinoid necrosis

• Granulomas, neutrophils and eosinophils: absent

SHML-Rosai-Dorfmann Disease

Histologic Features

• Effacement of follicles

• Dilatation of sinuses

• Proliferation of sinus histiocytes with emperipolesis (lymphocytes in histiocytes)

• Lipid-laden macrophages

• Absence of necrosis

• Lack of mitoses

Castleman

Lymphadenopathy

• Hyaline-vascular type

• Plasma cell type

• Mixed type

• Multicentric

Castleman Lymphadenopathy:

Hyaline-Vascular Type

• More common; younger patients

• Isolated lymph node mass; often mediastinal

• Frequently asymptomatic

• Hyaline deposits in atrophic germinal centers

• Collagen-ensheathed (“lollipop”) arterioles

• Concentric layering of lymphocytes (“target” follicles)

• Interfollicular vascular hyperplasia

Castleman Lymphadenopathy:

Plasma Cell Type

• Older patients

• Systemic symptoms

• Possible associations: polyneuropathies,

organomegaly, endocrinopathy, monoclonal

gammopathy, skin changes (POEMS

syndrome)

• Hyperplastic lymphoid follicles with active

germinal centers

• Interfollicular sheets of plasma cells

Castleman’s Disease, plasma cell type

Castleman Lymphadenopathy

Multicentric Form

• Atypical lymphoproliferative disorder

• Most are plasma cell type or mixed type (features of both types)

• Older patients

• Severe systemic symptoms

• Multiple lymphoid organs involved

• High levels of IL-6 in lymph nodes & serum

• Kaposi sarcoma/HHV-8 often present

• HIV infection often associated

Dermatopathic Lymphadenopathy

• LNs draining areas of dermatitis, also a/w cutaneous T cell lymphoma

• Pruritis, eosinophilia

• Axillary, inguinal lymphadenopathy

• Architecture preserved; follicular hyperplasia

• Paracortical, palely stained, confluent areas of histiocytes/interdigitating dendritic cells/ Langerhan’s cells. Histiocytes are the major component.

• Melanin pigment-laden macrophages

• Immuno: CD1a+ (LCs); and S100+ (IDCs and LCs)