Rest of the Objectives

What lab procedures can be used to work up Hemoglobinopathies?

• CBC / RBC indicies

• reticulocyte count

• Peripheral blood smear

• Hemoglobin solubility test

• Hemoglobin electrophoresis

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What is seen on a peripheral blood smear for HbSS disease?

• Sickle cells

• target cells

• HJ bodies

• pappenheimer bodies

• basophilic stipplings

• nucleated RBC

• spherocyte

What is seen on a peripheral blood smear for HbC disease?

• Target cells

• microspherocytes

• schistocytes

• Hgb crystals “bar of gold”

What is seen on a peripheral blood smear for HbSC disease?

• sickle cell

• target cell

• SC crystals

What is the Hgb Solubility Test?

• blood is added to phosphate buffer and saponin

• sample is deoxygenated

• HbS becomes insoluble

• produces a turbid suspension

• positive = presence of HbS or other hemoglobinopathies

What does the Hgb Solubility Test NOT do?

• tells you whether Pt has: HbSC, HbAS, HbS

• positive result → follow with electrophoresis

What does hemoglobin electrophoresis do?

• separates hemoglobins based on electrophoretic mobility

• allows you to determine what hemoglobins are present and approx how much of each are present

What occurs when you have an alkaline pH for hemoglobin electrophoresis?

• HbS, HbD, HbG migrate together

What occurs when you have an acidic pH for hemoglobin electrophoresis?

• can separate HbS from HbD/HbG

What lab tests are used to diagnose thalassemias?

• CBC

• Blood smear

• Hemoglobin electrophoresis

• HPLC

• Hemoglobin quantification

• Routine Chemistry

What CBC results can indicate Thalassemia?

• norm / ↑ RBC and MCHC

• ↓ MCV/ MCH

• ↓ HCT / Hgb

• normal RDW

What blood smear results indicate homozygous B thalassemia?

• extreme anisocytosis / poikilocytosis

• target cells, ovalocytes, nRBCs

• marker: hypo/micro

What blood smear results indicate heterozygous B thalassemia?

• hypo/micro

• mod/mild anisocytosis/poikilocytosis

• target cells

• basophilic stipplings

Why is hemoglobin electrophoresis important in diagnosing Thalassemias?

• detect ↑ levels of abnormal/normal hemoglobins

  • Hgb H

  • Hgb Barts

  • Hgb Constant Spring

  • other abnormal hemoglobins

How does High-performance liquid chromatography (HPLC) help with diagnosing thalassemias?

• replaces hemoglobin electophoresis in many labs

• confirmatory method following abnormal hemoglobin electrophoresis results

• separates molecules based on their movement through mobile/stationary phase

Why is hemoglobin quantification used for thalassemia diagnosis?

• needed to establish true diagnosis

What can ↑ billirubin indicate?

• thalassemia major / intermedia

• 1-6 mg/dL

• higher in thalassemia intermedia than major

What can serum, iron, TIBC, and serum ferritin indicate?

• differentiates thalassemia carrier from IDA

• assessment of iron load in a patient with thalassemia major / intermedia

What is Hemoglobin H?

• HbH = B4 ( 4 beta chains)

  • occurs when 3 of the 4 alpha genes are deleted

  • leaves 1 functional alpha gene

What is the hemoglobin concentration in Hemoglobin H disease?

• 5-40% HbH

• rest is mostly HbA

• small amounts: HbA2 and Hb Barts

What lab findings indicate Hbg H?

• Hgb: 7-10 g/dL

• retics: 3-10%

• mod mico/hypo

• target cells, heinz bodies

• splenomegaly

• BM: erythroid hyperplasia

What is Hemoglobin Barts?

• y4 = consists of 4 gamma chains

  • occurs when all 4 alpha chains are deleted

  • no normal alpha chains are produced

What lab findings indicate Hemoglobin Barts disease?

• approx 80% Hb barts

• Hb barts can not carry O2 effectively

  • incompatible with life

• death in utero or shortly after birth

Main findings of HbH.

• chains: B4

• alpha genes deleted: ¾

• Timing: after birth

• severity: mod/severe

• Major clue: HbH inclusions

Major findings of Hb Barts.

• chains: y4

• alpha genes: 4/4

• timing: fetal / newborn

• severity: usually fatal

• major clue: hydrops fetalis

What is the etiology of Gaucher’s Disease?

• lack of beta-glucocerbrosidase enzyme

• leads to - accumulation of glucocerebroside

• autosomal recessive

What gene is involved in Gaucher’s Disease?

• GBA 1

  • located on chromosome 1 at 1q21

What clinical triad indicates Gauchers Disease?

• hepatomegaly

• Gaucher’s cells in BM

• ↑ serum phosphatase

What is the etiology of Niemann-Pick Disease?

• deficiency of sphingomyelinase

  • leads to accumulation of sphingomyelin and cholesterol

What can indicate Neimann-Pick Disease?

• lipid laden giant foam cells in BM, tissues, and organs

• cherry red spot in macula of eye

What is Tay Sachs Disease?

• progressive neurodegenerative disorder

• fatal by: 2-3 yrs

What is the etiology of Tay Sachs Disease?

• lesions in cns caused by accumulation of gm2 ganglioside in neurocytes

• deficiency of beta hexosaminidase enzyme

  • mutation in hexa gene

What stain is used for viewing Gaucher cells in Gaucher’s Disease?

• Periodic Acid Schiff Stain

• PAS +

Describe the morphology of a Gaucher Cell.

• macrophage like

• 20-80 micrometers

• abundant blue-grey cytoplasm with wrinkled paper appearance

• PAS +

• cytoplasmic inclusions

  • aggregates of alucocerebroside

  • seen in: BM, liver, spleen

Describe the appearance of a Niemann Pick Cell

• macrophage like

• foamy cytoplasm with lysosomes and sphingomyelin

• 20-90 micrometers

• eccentric nucleus

• seen in BM / spleen

When is the clinical onset of Type 1 Gaucher’s Disease (Neuropathic)

• childhood / adulthood

  • 1/450 - 1/1,000

What do these lab findings indicate?

• Hepatosplenomegaly +

• Hematologic complications secondary to hypersplenism +

• Skeletal deterioration (bone crisis/ fractures) +

• Neurodegenerative course - 

• Life expectancy: 6-80+ yrs

• Type 1 Gaucher’s Disease (adult form, neuropathic)

When is the clinical onset of Type 2 Gaucher’s Disease (Acute Neuropathic)

• infant form

  • 1/100,000

What do these findings indicate?

• Hepatosplenomegaly +

• Hematologic complications secondary to hypersplenism +

• Skeletal deterioration (bone crisis/ fractures) -

• Neurodegenerative course +++

• Life expectancy: 2 yrs 

• Type 2, Acute Neuropathic Infant Form (Gaucher’s Disease)

What is the clinical onset of Type III Subacute Neuropathic Gaucher’s disease?

• Childhood / juvenile

  • 1/100,000

What do these findings indicate?

• Hepatosplenomegaly +

• Hematologic complications secondary to hypersplenism +

• Skeletal deterioration (bone crisis/ fractures) +

• Neurodegenerative course ++

• Life expectancy: 20-40 yrs 

• Type III: Subacute Neuropathic Gaucher’s Disease

What causes type A Niemman-Pick Disease?

• mutation: smpd 1 gene

  • most common

• causes a deficiency in sphingomyelinase

What are characteristics of Type A Niemann-Pick Disease?

• buildup of sphingomyelin in liver, kidney, lungs, brain

• presence of niemann pick cells in BM

• presents in infants

  • failure to thrive

  • hepatosplenomegaly

  • rapid neurodegeneration

  • fatal by 2 yrs old

What causes Type B Niemann-Pick Disease?

• mutation: smpd 1 gene

• deficiency in sphingomyelinase (exhibit 10-20% of normal enzyme activity)

• buildup of sphingomyelin in lungs, liver, kidney

What findings can indicate Type B Niemann-Pick Disease?

• presence of Niemann-pick cells in BM

• presents later in life

  • does not live past adolescence

• scant neurologic symptoms

• progressive hepatosplenomegaly

• heart disease

How does Type C Neumann Pick Disease occur?

• mutations in npc1 or npc 2

• cholesterol, biphosphate, and sphingolipid buildup in lysosomal storage organelles of macrophages

What findings indicate Type C Neumann Pick Disease?

• systemic, neurologic and psychiatric symptoms

• poor prognosis

• affects children’s and adults - longer lifespan

What happens to a cell in Gaucher’s Disease?

• macrophages swallow waste material

• forms a lysosome containing waste material

• no enzyme = majority of waste material remains in the lysosome

How do you interpret a scatterplot from a flow cytometry analysis?

• flow cytometry scatterplot displays individual cells according to measured properties

• each dot = individual cell or particle passing through instrument

• there are two main light scatter measurements

  • forward scatter (FS/FSC)

  • Side Scatter (SS/SSC or RALS)

What does forward scatter do?

• approximate cell sizes

What does side scatter do?

• reflects cellular complexity / granularity

How do you read the axises on a FSC vs. SSC plot?

• X axis: forward scatter (increasing from left →right)

• Y axis: side scatter (increasing bottom → top)

What do Lymphocytes look like on a Flow Cytometry Plot?

• relative FSC: low

• Relative SSC: low

• location: lower left

What do Monocytes look like on a Flow Cytometry Plot?

• relative FSC: intermediate / high

• Relative SSC: intermediate

• typical location: middle

What do Granulocytes look like on a Flow Cytometry Plot?

• relative FSC: intermediate to high

• relative SSC: high

• typical location: upper region often towards the right

What do the different quadrants mean on a plot?

• Upper Left

  • Y marker +

  • X marker -

• Lower Left

  • both markers -

• Upper right

  • both markers +

• Lower right

  • Y marker -

  • X marker +

What is Flow Cytometry?

• flow - moving fluid

• cyto- physical properties of cells

• metry - measurement

• differentiates cell counts / microparticles

• 10,000 cells per second

• uses monoclonal Ab to differentiate cells

How does Flow Cytometry Work?

• Fluids

  • transports particles / cells in single file through a laser

  • pressure and saline sheath fluid

• optics

  • directs light to PTM using filters

• electronics (photodetectors)

  • converts light → digital signals that are plotted as events on a scatterplot or contour plot

What occurs during Forward Scatter?

• approximates cell size

• larger cell = greater signal

What occurs during side or right angle scatter?

• measures cell complexity / granularity

• larger complexity = larger signal

Define gating.

• separation of subpopulation from a heterogenous population to optimize analysis in multiparameter experiments

What is electron gating?

• process of selecting a specific cell population for analysis based on its measured characteristics

What is a Gate?

• region on a scatterplot of fluorescence plot

• events inside region are included in selected population

• events outside are excluded from particular analysis

Why is gating usfeul?

• blood specimen contains many cell types

  • if you want to analyze a particular population, you need to separate it analytically from others

What is hydrodynamic focusing in flow cytometry?

• process in which sheath fluid surrounds and narrows the sample stream so that cells pass through the laser in a controlled nearly single-file arrangement

Why is hydrodynamic focusing useful?

• allows instrument to analyze cells individually and consistently

What CDs are expressed in hematopoietic stem cells?

• CD34

What CDs are expressed in B cells?

• CD34

• CD19

• CD10

• CD21

• CD22

What CDs are expressed in a proerthroblast?

• CD33

• CD72

• GPA

• CD36+