C hallenges /New Parameters or Tests - PowerPoint Presentation

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Challenges /New Parameters or Tests in the Laboratory Diagnosis of Anemia

Dr. Behzad Poopak, DCLS PhD.Associate Professor of HematologyIslamic Azad University, Tehran Medical Branch

Payvand Clinical Specialty Lab.

Network Member

of

Molecular Diagnostic Centers of IFCC

With Quality Certificate Awarded By Health Reference Laboratory

Ministry Of Health and Medical Education

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ObjectivesI will review following topics in my presentation:Erythrocyte Morphology ChallengesReticulocyte parameters & its application in Anemia DiagnosisDiagnostic Approach to Auto-Immune Hemolytic AnemiaPNH Diagnostic Problems

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Erythrocyte Morphology ChallengesDifferent NomenclatureNo correlation bet. Morphology & Pathology or InterpretationNo common grading systemSome erythrocyte morphology missed Different report formatRole of Automation in morphology assessment

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2015 John Wiley & Sons Ltd, Int. Jnl. Lab. Hem. 2015, 37, 287–303

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Erythrocyte MorphologyICSH important recommendationThe use of grading some cell morphology using Cell Counter parameters Higher level of accuracy & precision compared with

observer use of the optical light microscope,Example: rbc size abnormalities –MCV for microcytosis and macrocytosis, and MCH

for hypochromia

and hyperchromia. However, it is important that the laboratory establishes policies to review peripheral blood smears

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Morphology Grading TableGrading is not equal for all morphologyFew/+ applied only for schistocyte

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Missed Erythrocyte MorphologyIrregularly Contracted CellsUnstable haemoglobinG6PD deficiency, Hemoglobinopathies

Irregularly contracted cells are smaller & denser

rbc

which lack an area of central pallor but are not as regular in shape as spherocytes.

Spherocyte

Spherocyte

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Neglected Erythrocyte MorphologySchistocytesTTP, HUS, DIC – a wide range of fragmented red cells with polychromatic cells and other damaged cells. Platelets are absent from the film

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Helmet Cells

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Typical ErythrogramsCold Agglutinins: RBCs 4. 41 after 370C

VolumeCell Hb

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Cold Autoagglutination

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Reticulocyte Count & its ParametersPitfallsReticulocyte Definition?Report format: % or Absolute countManual (CV:20%) or automated Retic. Count?Important Retic. Parameters: IRF (Immature Reticulocyte Fraction), CHr (mean hemoglobin content of reticulocytes)

Is Lab responsible for Reticulocyte production index (RPI) calculation? RPI = Relative Retic. Count X X

SAMG, January 2017, Vol. 107, No. 1

Hct of PatientHct of Normal

1

Maturation Time

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LFRLow Fluorescence Retics.MFRMedium Fluorescence Retics.

HFRHigh Fluorescence Retics.

Little RNA

More RNAHigh Level of RNAMature Retics.

Semi-Mature Retics.

Immature Retics.Reference Interval: 86.5-98.5%

Reference Interval: 1.5-11.3%Reference Interval: 0-1.4%

IRF (Immature Reticulocyte Fraction):

HFR (High Fluorescent Reticulocyte)

MFR (Medium Fluorescent Reticulocyte)

LFR (Low Fluorescent Reticulocyte)

Reference Range:

IRF, Female: 1.1-15.9% Male: 1.5-13.7%

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IRF, a sensitive indicator of erythropoietic activity Serial testing after BMT can show successful engraftmentA rise in the IRF occur earlier than any other available

test, including absolute neutrophil count IRF >20% from the post BMT value suggests successful erythroid engraftment.

IRF is a sensitive measure of early hematopoietic recovery following intensive chemotherapy.

An early and reliable indicator of adequacy of response to EPO therapy in patients with anemia of CRF, AIDS and malignancy.It can also be used to monitor response to other treatments for anemia such as iron, folate and vitamin B12

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The percentage of hypochromic red cells (%HRC) is the best-established variable for the identification of FID - Hypochromic red cells are those with Hb <280 g/l. -%HRC≥ 6% was found to

be superior to measurements of sTfR , ZPP, ferritin & TIBC in differentiating between iron-deficient and iron sufficient patients with CRF receiving maintenance doses of ESAs

Reticulocyte hemoglobin content (CHr

) is the next most established option. CHr <29 pg predicts FID in patients receiving ESA therapyBoth tests have limitations in terms

of sample stability or equipment availability.

Rbc & Retic ParametersFunctional Iron Deficiency (FID)

CHr and %HYPO are direct indicators of FID & IDDiagnosis of iron deficiency in early

childhood

CHr

can provide evidence of a

response to iron therapy

approx.

4 days after treatment initiation, that is to say much earlier than with other hematological measurements

.

A

reticulocyte

hemoglobin

equivalent

(Ret-He)

value

<25

pg

is suggestive of

classical iron deficiency

and also

predicts FID

in those receiving ESA therapy.

.

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Cytological Assessment of Iron StoresPerls’ Prussian blue reaction or Iron staining of BM, ‘gold standard’ test Assessment can be misleading if insufficient material is available:

seven or more particles should be available for review, Few hematologists

can honestly say they invariably manage this number on their aspirate films.

Inadequate material was a major factor in a study that concluded that > 30% of reports of absence of stainable iron were inaccurateThe presence of stainable iron does not define Iron incorporationFurthermore, BM examination is uncomfortable and not without complications,

such as post-biopsy pain and bleeding.

2013 John Wiley & Sons Ltd 643British Journal of Haematology, 2013, 161, 639–648

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Lab. findings, IDA, ACD & IDA+ACD

F

erritin <20 μ

g/L confirms iron deficiency,Sensitivity is poor (59 %–73%)Ferritin ≤ 30ng/mL has a 92% sensitivity and 98% specificity for diagnosing IDin the absence of inflammation [e.g. CRP; < 0.5 mg/dl]

Being a positive acute phase reactant, ferritin levels as high as 100

μg/L can occur in iron deficient patients.

Clin Chem Lab Med 2012;50(8):1343–1349S Afr Med J 2016;106(1):53-54.ACD+ID: more frequent in patients with inflammatory diseases and chronic blood losses (e.g. inflammatory bowel disease).

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Diagnostic approach to suspected AIHAWhen a patient presents with suspected AIHA, 3 questions should be considered. Is there hemolysis? Typical laboratory findings: • Bilirubin (unconjugated)

– increased • Reticulocyte count - increased • LDH– may be normal or increased • Haptoglobin – reduced • Blood film – spherocytes

, agglutination or polychromasia

• Urinalysis/dipstick test Hb-uria • Urinary haemosiderin 1 week after onset of intravascular haemolysis 2. Is the hemolysis autoimmune?A positive DAT indicates immune etiology (IgG, IgM, IgA or complement (usually C3d) bound to the

rbc membrane)

3. What is the type of AIHA?British Journal of Haematology, 2017, 176, 395–411

A positive DAT is not specific and is also associated with a wide range of non-hemolytic disease states, possibly through passive deposition of IGs or

immune complexes

; examples

include

Liver

disease,

Chronic

infection,

Malignancy

,

Systemic Lupus Erythematosus

(SLE),

Renal disorders and

Drugs

such as intravenous

IVIg

or

antithymocyte

globulin.

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Positive DAT, Evidence of Hemolysis. Before diagnosing AIHA, ask the following 5 questions:1. Is there a history of blood transfusion in the last 3 months?

o Consider a delayed hemolytic transfusion reaction (DHTR)2. Has the patient received a solid organ or allogeneic hematopoietic

stem cell transplant (HSCT)?

o Consider alloimmune hemolysis caused by major ABO mismatch (HSCT) or passenger lymphocyte syndrome (PLS) (solid organ or HSCT).3. In infants, could this be hemolytic disease of the newborn (HDN)?

4. Has the patient received any relevant drugs

? o Consider drug-induced immune haemolytic anemia (DIIHA).5. Is

there another known cause of hemolysis? o Given the high prevalence of an incidental positive DAT within the hospital population, consider whether there is an alternative cause of hemolysis or abnormal laboratory values

Rarely, AIHA patients test

negative with a tube test DAT

, for example due to

a low affinity antibody

,

low levels of red cell bound antibody

or an

immunoglobulin not tested for

(e.g.

IgA-only AIHA

).

A

gel column agglutination method

is a more sensitive method that is less prone to error than a conventional tube test

AIHA can be diagnosed in 3% of patients testing negative with a gel card method or by using a

red cell elution technique

Recommendation:

• In patients with unexplained hemolysis and a negative screening DAT, retest with a column agglutination DAT method that includes monospecific anti-IgG, anti-IgA and anti-C3d.

If also negative, consider preparing and investigating a red cell eluate.

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Arch Pathol Lab Med—Vol 141, February 2017AIHA can be diagnosedin 3% of patients testing negative with a gel cardmethod by using a red cell elution technique

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DAT MethodsConventional Test Tube (CTT) methodgel micro-Column

Solid Phase methods- +

- +

- +Am. J. Hematol. 87:707–709, 2012.

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0.1%

of healthy

blood

donors have a positive DAT finding without evidence of hemolysis.A positive DAT can be found in 1 in 1,000–1:14,000 healthy blood donors without hemolysis

About 2/3 are positive with

IgG and 1/3, with complement. The significance of this finding is unclear, but some individuals may go on to develop AIHA or

cancer.The DAT is positive in 7–8% of hospitalized patients and up to 15% of hospitalized patient specimensTherefore, the significance of a DAT requires clinical correlation.

Positive DAT in Normal Individuals

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Paroxysmal Cold HemoglobinuriaThe rarest type of AIHA, occurs more often in children.The autoantibody is a biphasic hemolysin, which most often has anti-P specificity. The

DAT is positive for C3d and negative for IgG. The diagnosis is confirmed by a Donath-Landsteiner test. A test is positive if hemolysis is detected in the ice followed by incubation

at 370C

reaction and not in the melting ice or 370C reactions.Am. J. Hematol. 87:707–709, 2012.

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Hereditary spherocytosis (HS), Hereditary elliptocytosis (HE), ABO and warm AIHA,Clostridium perfringens sepsis, BurnsG6PD deficiencyAIHA– typical round, dense red cells

SpherocytosisHereditary Membrane Defects & Acquired Conditions

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METHODS FOR LABORATORY DIAGNOSIS OF HSScreening (+ family history and typical clinical features)- First line: RBC morphology on blood smear, Hematology parameters, Biochemical hemolysis parameters- Second line (reduced area or surface to volume (S/V) ratio, increased osmotic fragility)

Hypertonic cryohemolysis, acid glycerol lysis test, osmotic fragility test, pink test Eosine-5-maleimide binding, EMADiagnosis

SDS-PAGEEktacytometry

with osmotic resistance measurementMolecular analysis

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CBC / Retic. Parameters in HSMCV is decreased variably in HS with largest decreases noted in severe forms of HS (due to significant decreases in the spectrin content of the rbc memb.).Importantly, the reticulocyte MCV

(MCVr) is also decreased ( to variable extent depending on the severity of anemia). MCVr decrease in HS but not in AIHA or ABO incompatibility (

does not decreases)

An increased % of hyper-dense cells (Hb conc. >41 g/dl) in association with increased MCHC values of >36 g/dl of mature red cells as well as of reticulocytes (CHCMr). In contrast,

CHCMr values are normal in

AIHA.

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HS with high CHCM >36 g/dl, high % of hyper-dense cells >4%

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CBC: Hereditary Spherocytosis

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Red cells under microscopic examination in HSBand 3 defect with the classical mushroom featureβ spectrin (SPTB) defect with a lot of acanthocytes (red arrows) in association with

spherocytic red cellsThe number of spherocytes

is highly variable from patient to patient:

very few in 25 to 35% of mild cases of HS and in 33% of HS neonates to very large numbers in the more severe forms of HS.

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Confirmatory Tests for HSUsed as 1st line of clinical laboratory testsThe Osmotic Fragility Test, OFT (Before & After Incubation) Glycerol Lysis TestPink test

However, the sensitivity of these tests for diagnosis is low 68% for OFT performed on fresh blood,

61% for the glycerol lysis test and

91% for the Pink test. As a result, flow cytometry measurement of the mean red cell fluorescence, associated red cells following labeling with the dye eosin-5′ maleimide (

EMA) to document surface area loss is

being used an alternate test for diagnosis of HS.

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OFTInitiation of Hemolysis RI: 0.44 %Complete Hemolysis RI: 0.34 %

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MCF : Median Corpuscular Fragility

Payvand Clinical Specialty Lab.

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Performance of Screening TestsISLH 2013, May-Jean King

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Eosin-5-Maleimide, EMA Flow Cytometry

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Eosin-5′ Maleimide (EMA) Flow Cytometric Test forIn HS diagnosis:Sensitivity:92.7% & specificity: 99.1%, Positive Predictive Value,

PPV: 97.8% ; Negative Predictive Value, NPV:96.9%The EMA-binding is not dependent on the phenotype and is positive also in compensated HS.independent of the molecular defect

in the rbc membrane protein but it may be less sensitive for diagnosis if HS is with undefined molecular defects and with

ankyrin defects.The test can also be positive in Pyropoikilocytosis (HPP)Decreased memb. associated fluorescence in patients with congenital dyserythropoietic anemia type II (

CDAII)

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Advantages of EMA Flow cytometric TestEMA test should replace the much lower sensitive & specific tests since: it is easy to perform particularly in neonates, since only 5 μl of peripheral blood is needed Results are

available in 2 to 3 hrs Samples may be analyzed up to 7 days after

the blood samplingthe gating on the abnormal red cells

allows the avoidance of the bias due to presence of transfused red cell enabling the diagnosis of HS in patients with a recent transfusion history.

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EMA Report Format

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Flow chart for Lab. diagnosis of HS

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Diagnostic Tests: SDS-PAGEDetermines the extent of membrane deficiencyLack of sensitivity to very mild ‘carrier’ HSRecommended if: - Clinical phenotype more severe than predicted from RBC morphology - RBC morphology is more severe

than predicted from parental blood film - Equivocal or borderline results of the screening test - Dx

is not clear prior to splenectomy

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Challenges in PNH DiagnosisDifferent/variable clinical presentationThe disease is rare & most labs have limited experience in PNH testingTest order is a major problem?Specimen Type; PB or BMA?Routine tests such as Ham's test or sucrose hemolysis test is not sensitive testHigh Sensitivity Flow Cytometry is the choice methodStandardize protocol not followed?!?!Report format?Proficiency testing?

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41

Chronic

Kidney Disease

Acute

Renal Failure

Pulmonary HypertensionCardiac Dysfunction

Stroke / TIA

Ischemic Bowel

DVT

Hepatic Failure

Signs of PNH

the Underlying Threat of Catastrophic Consequences

Common Symptoms of Hemolysis

Fatigue

Impaired

QoL

Anemia

Hemoglobinuria

Dyspnea

Dysphagia

Abdominal Pain

Erectile Dysfunction

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PNH TestingICCS PNH GuidelinesGuidelines for the diagnosis and monitoring of paroxysmal nocturnal hemoglobinuria and related disorders by flow cytometry. Borowitz MJ, Craig FE, DiGiuseppe JA, Illingworth AJ, Rosse W, Sutherland DR, Wittwer CT, Richards SJ. Cytometry Part B 2010; 00B: 000-000

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Patient Testing for PNH Using High Resolution Flow Cytometry at Dahl-Chase Dec 2007 – Nov 15, 2013 Patient Selection based on Diagnostic Pathway - 9,289 Total screening tests (including follow-up cases) - 8,836 Total patients screened - 572

PNH positive patients 6.5% 337 Patients w/ PNH Clones > 1% in WBC

3.8%

236 Patients w/ minor PNH Clones <1% in WBC 2.7% Using the Diagnostic Pathway, every 26th Patient has shown a PNH Clone greater than 1%

45

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High-Sensitivity Flow Cytometry Is Needed for Accurate Diagnosis and Monitoring 40% of PNH+ Samples Show a Clone of <1%1

46

1. Movalia MK et al. Poster presented at 53rd Annual meeting of the ASH, San Diego, CA. 2011.

≤1%

Clone Size >1%

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Normal Expression of CD59 (Type I) and Abnormal Expression of CD59 (Type II and III) in RBCs

PNH clone with

complete CD59 deficiency

(Type III cells) and

partial CD59 deficiency

(Type II cells)

PNH clone with

complete CD59 deficiency

(Type III cells)

Normal RBC’s with normal CD59 expression (Type I cells)

Gating on GPA+

(CD235a) RBC’s

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PNH Leukocyte Assay

Granulocytes and Monocytes: FLAER - CD24 - CD14* - CD15 - CD45

Monocytes only (Reflex):

FLAER - CD33** - CD14 - CD64** - CD45

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Thank you, any question?