Challenges in the Laboratory Diagnosis of Anemia

Transcription

1 Challenges in the Laboratory Diagnosis of Anemia Dr. Behzad Poopak, DCLS PhD. Associate Professor of Hematology Islamic Azad University, Tehran Medical Branch

2 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

3 Introduction The requirement for iron increases gradually from 0.8 mg/d in the 1 st trimester to 7.5mg/d in the 3 rd DOI /blood

4 Total YLD due to anemia in 2010, by country years lived with disability, or YLD, which captures the average duration and relative severity of the disease 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 Iran (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 Sant-Rayn Pasricha Blood 2014;123:

5 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

6 WHO/CDC Anemia Cut-offs over or under diagnosis Men Age years: <12.5 g/dl (CDC) Age years: <13.3 g/dl (CDC) Age >17 years: <13 g/dl (WHO) or <13.5 g/dl (CDC) Women (Non-pregnant, nonlactating) Age years: <11.8 g/dl Age years: <12.0 g/dl Age >17 years: <12 g/dl (WHO & CDC) Women in Pregnancy (CDC Guidelines <5th percentile) 1 st Trimester: <11.0 g/dl 2 nd Trimester: <10.5 g/dl 3 rd 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

7 Adjustment of max. Hb concentration and Hct values for anemia - over or under diagnosis

8 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

9 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,

10 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%)

11 2015 John Wiley & Sons Ltd, Int. Jnl. Lab. Hem. 2015, 37,

12 Erythrocyte Morphology ICSH 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

13 Erythrocyte Morphology ICSH important recommendation, cont. 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)

14 Morphology Grading Table Grading is not equal for all morphology Few/+ applied only for schistocyte

15 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

16 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

17 Helmet Cells

18 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 >30

19 Dimorphic Anemia RDW, HDW

20 Volume Cold Agglutinins: RBCs after 37 0 C Typical Erythrograms Cell Hb

21 Cold Autoagglutination

22 Reticulocyte Count & its Parameters Pitfalls 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 Hct of Patient Hct of Normal X 1 Maturation Time

23 IRF (Immature Reticulocyte Fraction): HFR (High Fluorescent Reticulocyte) MFR (Medium Fluorescent Reticulocyte) LFR (Low Fluorescent Reticulocyte) Reference Range: IRF, Female: % Male: % LFR Low Fluorescence Retics. MFR Medium Fluorescence Retics. HFR High Fluorescence Retics. Little RNA More RNA High Level of RNA Mature Retics. Semi-Mature Retics. Immature Retics. Reference Interval: % Reference Interval: % Reference Interval: 0-1.4%

24 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 B 12

25 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 erythropoiesisstimulating 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) John Wiley & Sons Ltd British Journal of Haematology, 2013, 161,

26 Rbc CHr& and Retic %HYPO Parameters are direct indicators of FID & ID Functional Diagnosis of iron Iron deficiency Deficiency in early childhood (FID) CHr can provide evidence of a response to iron therapy approx. 4 The days percentage after treatment of hypochromic initiation, that red cells is to say (%HRC) much is earlier the bestestablished other hematological variable for measurements. the identification of FID than with A - Hypochromic reticulocytered hemoglobin cells are those equivalent with Hb <280 (Ret-He) g/l. value <25 pg is suggestive of classical iron deficiency and also predicts FID in those receiving -%HRC 6% ESA was therapy. 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.

27 Management of ironrestricted 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.

28 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.

29 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 John Wiley & Sons Ltd 643 British Journal of Haematology, 2013, 161,

30 non-iron-deficient iron-deficient Ferritin frequency distribution in Anemic Patients, n:2084 The 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). Peak: 148µg/L Peak: 5 12 µg/l Clin Chem Lab Med 2012;50(8):

31 Lab. findings, IDA, ACD & IDA+ACD ACD+ID: more frequent in patients with inflammatory diseases and chronic blood losses (e.g. inflammatory bowel disease). 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):

32 Diagnostic approach to suspected AIHA When a patient presents with suspected AIHA, 3 questions should A be positive considered. DAT is not specific and is also associated with a 1. wide Is there range hemolysis?; of non-hemolytic Typical laboratory disease findings: states, possibly through passive Bilirubin deposition (unconjugated) of IGs or increased immune Reticulocyte complexes; count examples - increased include LDH may be normal or increased Urinalysis/dipstick test Hb-uria onset of intravascular haemolysis 2. Is the hemolysis autoimmune? Haptoglobin reduced Liver Blood disease, film spherocytes, agglutination or polychromasia Chronic infection, Malignancy, Systemic Lupus Erythematosus (SLE), Renal disorders and Drugs such as intravenous IVIg or antithymocyte globulin. Urinary haemosiderin 1 week after 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,

33 Positive DAT, Evidence of Hemolysis. Before diagnosing AIHA, ask the following 5 questions: Rarely, 1. Is there AIHA a patients history of test blood negative transfusion with a tube in the test last DAT, 3 months? for example due to a low affinity antibody, low levels of red cell bound antibody or an immunoglobulin not o Consider a delayed hemolytic transfusion reaction (HTR) tested for (e.g. IgA-only AIHA). 2. Has the patient received a solid organ or allogeneic hematopoietic stem cell A transplant gel column (HSCT)? agglutination method is a more sensitive method that is less prone to error than a conventional tube test o Consider alloimmune hemolysis caused by major ABO mismatch (HSCT) or passenger lymphocyte syndrome (PLS) (solid organ or HSCT). AIHA can be diagnosed in 3% of patients testing negative with a gel card method by using 3. In a infants, red cell could elution this technique be hemolytic disease of the newborn (HDN)? 4. Has the patient received any relevant drugs? Recommendation: o Consider drug-induced immune haemolytic anemia (DIIHA). In patients with unexplained hemolysis and a negative screening DAT, retest with 5. Is there another known cause of hemolysis? a column agglutination DAT method that includes monospecific anti-igg, anti-iga and anti-c3d. 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 If also negative, consider preparing and investigating a red cell eluate.

34 Diagnostic Lab. Challenges in Thalassemia-Hbopathies - The Different HbA2 accuracy types of techniques: is especially CAE, important CE, HPLC, CC, in the critical area between IQC & 3.0 EQA and for Manual/automated 4.0% and the imprecision Tech. are not should defined be or such available that an SD Using of 0.05 technique (or CV, without or RSD, any notice of 2%) to can sensitivity, be obtained eg. CAE is (duplicates not good within HbF 0.2% <30% in the final numeric result). - It is Report important without to any both comment detect or and Interpretive quantitate notesany HbA2 variant that is present (due to either an α- or δ-globin chain mutation) Complementary techniques are not defined, the performance of the and HbA2 include analysis it in must the be total accurate HbA2 and reported. 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

35 Diagnostic Lab. Challenges in Thalassemia- Hbopathies WHO International Reference Reagent for HbA2 (2015):5.3+/ % 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.

36 Commentary notes: Yes or No?

37 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 Common Symptoms of Hemolysis Signs of PNH the Underlying Threat of Catastrophic Consequences Dyspnea Dysphagia Abdominal Pain Impaired QoL Fatigue Hemoglobinuria Erectile Dysfunction Anemia DVT Acute Renal Failure Hepatic Failure Chronic Kidney Disease Cardiac Dysfunction Stroke / TIA Ischemic Bowel 38 Pulmonary Hypertension

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40 PNH Testing ICCS 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:

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42 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 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 26 th Patient has shown a PNH Clone greater than 1% 42

43 High-Sensitivity Flow Cytometry Is Needed for Accurate Diagnosis and Monitoring 40% of PNH+ Samples Show a Clone of <1% 1 1% Clone Size >1% Movalia MK et al. Poster presented at 53rd Annual meeting of the ASH, San Diego, CA

44 Normal Expression of CD59 (Type I) and Abnormal Expression of CD59 (Type II and III) in RBCs Normal RBC s with normal CD59 expression (Type I cells) PNH clone with complete CD59 deficiency (Type III cells) Gating on GPA+ RBC s PNH clone with complete CD59 deficiency (Type III cells) and partial CD59 deficiency (Type II cells)

45 Thank you, any question?