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?