Challenges in the LaboratoryDiagnosis of Anemia

Dr. Behzad Poopak, DCLS PhD.Associate Professor of Hematology

Islamic Azad University, Tehran Medical Branch

Objectives

I will review following topics in my presentation:

■ Epidemiology of Anemia

■ Erythrocyte Morphology Challenges

■ Reticulocyte parameters & its application in Anemia Diagnosis

■ Review of Functional Iron Deficiency

■ Diagnostic Approach to Auto-Immune Hemolytic Anemia

■ Challenges in Thalassemia / Hbopathies

■ PNH Diagnostic Problems

Introduction

DOI 10.1182/blood-2016-08-672246

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The requirement for iron

increases gradually from 0.8

mg/d in the 1st trimester to

7.5mg/d in the 3rd

Total YLD due to anemia in 2010, by country

Sant-Rayn Pasricha Blood 2014;123:611-612

years lived with disability, or YLD, which captures the averageduration and relative severity of the disease

Iran

■ Despite increasing efforts worldwide, there remains a surprisingly

large global burden of the disease, particularly among young

children (impaired mental or motor development, the burden

among children under age 5 actually increased.) and women,

■ The global prevalence of anemia decreased 1990 - 2010 (from

40.2% to 32.9%),

■ By comparison, this global anemia burden is greater than burden

associated with major depression (63.2 million YLDs), chronic

respiratory diseases (49.3 million YLDs), and general injuries (47.2

million YLDs). years lived with disability

Anemia & Clinical Laboratory

■ When investigating patients with suspected anemia, 4

fundamental aspects have to be considered:

1. Confirm Dx of Anemia & Classify it.

2. What is the cause of the anemia?

3. What is the urgency for correcting the anemia,

i.e. is a blood transfusion or other urgent intervention indicated?

4. Monitoring the treatment response

WHO/CDC Anemia Cut-offsover or under diagnosis

– Men■ Age 12-14 years: <12.5 g/dl (CDC)

■ Age 15-17 years: <13.3 g/dl (CDC)

■ Age >17 years: <13 g/dl (WHO) or

<13.5 g/dl (CDC)

– Women (Non-pregnant, non-

lactating)

■ Age 12-14 years: <11.8 g/dl

■ Age 15-17 years: <12.0 g/dl

■ Age >17 years: <12 g/dl (WHO & CDC)

• Women in Pregnancy (CDC

Guidelines <5th percentile) 1st Trimester: <11.0 g/dl

2nd Trimester: <10.5 g/dl

3rd Trimester: <11.0 g/dl

• Children (CDC Guidelines

<5th percentile) Age 1-2 years: <11.0 g/dl

Age 2-5 years: <11.1 g/dl

Age 5-8 years: <11.5 g/dl

Age 8-11 years: <11.9 g/dl

The WHO defines severe anemia in all persons as a Hb of <7 g/dL

and very severe anemia as a Hb of <4 g/dL

Adjustment of max. Hb concentration and Hctvalues for anemia - over or under diagnosis

Erythrocyte Morphology Challenges

■ Different Nomenclature

■ No correlation bet. Morphology & Pathology or

Interpretation

■ No common grading system

■ Some erythrocyte morphology missed

■ Different report format

■ Role of Automation in morphology assessment

Three Types of Smear Examination

1. Blood Smear Scan (BSS)

■ Synonyms: platelet scan, platelet estimate, blood smear

examination without a DIFF.

2. Blood Smear Examination (BSE)

■ Synonyms: manual DIFF, DIFF

3. Blood Smear Review (BSR)

■ Synonyms: blood smear interpretation, physician review of

blood smear,

Erythrocyte Morphology Challenges, cont.

■ Abnormal morphologic findings are reported in various ways:

(i) A simple description,

(ii)The use of terms such as present or absent,

(iii) A semi-quantitative determination,

mild (+), moderate (++), marked (+++)

(iv) A quantitative percentage of the morphological abnormalities:

normal (<5%), mild (5–25%), moderate (25–50%), marked (>50%)

2015 John Wiley & Sons Ltd,

Int. Jnl. Lab. Hem. 2015, 37, 287–303

Erythrocyte MorphologyICSH important recommendation

■ The 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

■ According to the Rumke table distribution, a minimum of 1000

RBC should be evaluated to provide a precise percentage of the

cells having a particular morphological abnormality.

■ As a general recommendation, the ICSH group recommends

providing only a qualitative report for those presenting with RBC

abnormalities; however, a schistocyte count may be of clinical

value for the diagnosis and follow-up of thrombotic

thrombocytopenic purpura (TTP) and haemolytic uraemic

syndrome (HUS)

Erythrocyte MorphologyICSH important recommendation, cont.

Morphology Grading Table

■ Grading is not equal

for all morphology

■ Few/+ applied only for

schistocyte

Missed Erythrocyte Morphology

Irregularly Contracted Cells

■ Unstable haemoglobin

■ G6PD 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

Neglected Erythrocyte Morphology

Schistocytes

■ Thrombotic thrombocytopenic purpura – a wide range of

fragmented red cells with polychromatic cells and other

damaged cells. Platelets are absent from the film

Helmet Cells

Anisocytosis & RDW:1. Count the macrocytic and/or microcytic RBC separately in

10 fields of evenly dispersed RBCs.

2. Divide the total of these fields by 10 to establish the mean.

3. Add both for total and use the table below for grading.

NORMAL Slight ANISO Moderate ANISO Marked ANISO

0-5 6-15 15-30 >30

Dimorphic AnemiaRDW, HDW

Typical Erythrograms

Cold Agglutinins:

RBCs 4. 41 after

370C

Vo

lum

e

Cell Hb

Cold Autoagglutination

Reticulocyte Count & its ParametersPitfalls

■ Reticulocyte Definition?

■ Report format: % or Absolute count

■ Manual (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 XHct of Patient

Hct of Normal

1

Maturation Time

LFRLow Fluorescence Retics.

MFRMedium Fluorescence Retics.

HFRHigh Fluorescence Retics.

Little RNA More RNA High Level of RNA

Mature 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%

IRF, a sensitive indicator of erythropoietic activity

■ Serial testing after BMT can show successful engraftment

■ A 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

Rbc & Retic Parameters

Functional Iron Deficiency (FID)

■ FID: insufficient iron incorporation into erythroid precursors with apparently adequate body iron stores (stainable iron in the BM+ normal serum ferritin)

■ Block in iron transport to the erythroid marrow seen in infectious, inflammatory & malignant diseases, and is a major component of the anemia of chronic disease (ACD).

■ FID, found in some subjects treated with erythropoiesis-stimulating agents (ESAs), especially in subjects with chronic kidney disease (CKD).

■ MCV & MCH values are useful at diagnosis and in assessing, trends over periods of weeks or months. They have no use in assessing acute changes in iron availability secondary to therapy with erythropoiesis-stimulating agents (ESAs).

2013 John Wiley & Sons Ltd

British Journal of Haematology, 2013, 161, 639–648

■ 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 therapy

■ Both tests have limitations in terms of sample stability or equipment

availability.

Rbc & Retic Parameters

Functional Iron Deficiency (FID)

• CHr and %HYPO are direct indicators of FID & ID

• Diagnosis 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.

• .

Management of iron-restricted erythropoiesis in patients with CKD on ESA.

*Where IRE (iron-restricted

erythropoiesis) is defined by:

- Percentage of hypochromic red cells

(%HRC) > 6%.

- Retic. Hb content (CHr) < 29 pg.

- Retic. Hb equivalent (Ret-He) < 30.6 pg

Or indicative values from other red cell or

reticulocyte parameter. CKD, chronic kidney disease;

ESA, erythropoiesis stimulating agent;

FID, functional iron deficiency; HD: hemodialysis.

Iron Deficiency Anemia versus Anemia of Chronic Disorders

■ Ferritin is an acute-phase protein and may be falsely normal

or raised in patients with iron deficiency in the presence of

acute inflammation.

■ In this setting, the gold standard for discriminating IDA from

ACD is evaluating BM iron stores microscopically.

■ Measuring the soluble TF receptor (sTFR) levels and calculating

the sTFR index (sTFR:log ferritin ratio) is useful, a ratio >2

suggests IDA+ACD Unfortunately, STFR testing is not universally

available and not standardized yet.

Cytological Assessment of Iron Stores

■ Perls’ 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 inaccurate

■ The presence of stainable iron does not define Iron incorporation

■ Furthermore, 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

Ferritin frequency distribution in Anemic Patients, n:2084The highest Tf/log(ferr) ratio among the non-iron-deficient group is 1.70

(median: 0.84), while the lowest Tf/log(ferr) ratio among the iron-deficient

patients is 1.74 (median 3.49) (p < 0.01).

Clin Chem Lab Med 2012;50(8):1343–1349

Peak:

5 – 12 µg/L

Peak:

148µg/L

no

n-iro

n-d

efi

cie

nt

iro

n-d

efi

cie

nt

Lab. findings, IDA, ACD & IDA+ACD

Ferritin <20 μg/L confirms

iron deficiency,

Sensitivity is poor (59 %–73%)

Ferritin ≤ 30ng/mL has a 92%

sensitivity and 98% specificity

for diagnosing ID

in 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):1343–1349

ACD+ID: more frequent in patients with

inflammatory diseases and chronic blood losses

(e.g. inflammatory bowel disease).

Diagnostic approach to suspected AIHA

■ When a patient presents with suspected AIHA, 3 questions should be considered.

1. 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?

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.

British Journal of Haematology, 2017, 176, 395–411

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 (HTR)

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 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.

Diagnostic Lab. Challenges in Thalassemia-Hbopathies

■ Different types of techniques: CAE, CE, HPLC, CC,…

■ IQC & EQA for Manual/automated Tech. are not defined or available

■ Using technique without any notice to sensitivity, eg. CAE is not good HbF <30%

■ Report without any comment or Interpretive notes

■ Complementary techniques are not defined, the performance of the HbA2 analysis must be accurate and should be interpreted in association with the red cell indices.

■ CBC and sometimes other tests are mandatory for final conclusion

■ There is no central or referral Lab & training center for these types of diseases

True HbA2 value = HbA2 value + peak in Z1

- The HbA2 accuracy is especially important in the critical area

between 3.0 and 4.0% and the imprecision should be such that

an SD of 0.05 (or CV, or RSD, of 2%) can be obtained (duplicates

within 0.2% in the final numeric result).

- It is important to both detect and quantitate any HbA2 variant

that is present (due to either an α- or δ-globin chain mutation)

and include it in the total HbA2 reported.

■ WHO International Reference Reagent for HbA2 (2015):5.3+/-

0.066% or other internationally accredited reference material

■ Controls made from samples obtained from staff, usually have

stable HbA2 levels as long as iron deficiency does not occur.

■ Blood donors and women of childbearing age should be avoided

unless their blood count and iron status are confirmed as normal.

■ If this material can be validated by a reference laboratory, it can

also be used as a secondary standard.

Diagnostic Lab. Challenges in Thalassemia-Hbopathies

Commentary notes: Yes or No?

Challenges in PNH Diagnosis

■ Different/variable clinical presentation

■ The disease is rare & most labs have limited experience in PNH testing

■ Test order is a major problem?

■ Specimen Type; PB or BMA?

■ Routine tests such as Ham's test or sucrose hemolysis test is not sensitive test

■ High Sensitivity Flow Cytometry is the choice method

■ Standardize protocol not followed?!?!

■ Report format?

■ Proficiency testing?

38

Chronic

Kidney Disease

Acute Renal Failure

Pulmonary Hypertension

Cardiac

Dysfunction

Stroke / TIAIschemic Bowel

DVT

Hepatic Failure

Signs of PNH the Underlying Threat of Catastrophic Consequences

Common Symptoms of Hemolysis

FatigueImpaired QoL

Anemia

HemoglobinuriaDyspnea

Dysphagia

Abdominal Pain

Erectile Dysfunction

39

PNH TestingICCS PNH Guidelines

Guidelines 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

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%

42

High-Sensitivity Flow Cytometry Is Needed for Accurate Diagnosis and Monitoring

■ 40% of PNH+ Samples Show a Clone of <1%1

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

≤1% Clone Size >1%

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+ RBC’s

Thank you, any question?