APPROACH TO ANEMIA IN PREGNANCY Sitalakshmi S Professor and Head Department of Clinical Pathology St John’s medical College, Bangalore

What is anemia? Sign? Clinical finding? Laboratory finding? Disease? Pointer to a systemic disease?

The first step in any diagnosis: physical examination determining its cause This may be difficult in pregnancy

A detailed medical, personal, and dietary history : Any family or personal history of anemia A history jaundice, or enlarged spleen Heavy menstrual bleeding in women Any signs of internal bleeding.

Of all the diagnostic tests available, complete blood count is one of the single most valuable tools in assessing the general health of the body. Provides a snap shot of the hematopoietic system at a specific point in time. It is a valuable indicator of disease, either local or systemic

Complete Blood Count (CBC) a panel of tests that measures RBC,WBC and platelets. For diagnosis of anemia, the CBC provides critical information on: the size, volume, and shape of RBC Hemoglobin Anemia is when Hb is < 11 g/dL for pregnant women < 12 g/dL for non-pregnant women

Complete blood counts should be included in evaluation of: As a part of routine examination every sick patient every patient with vague signs of disease every pre-anesthetic evaluation as a recheck test for patients previously diagnosed with erythrocyte, leukocyte or platelet abnormalities.

Results from the CBC test can help: Provide basic information about one’s health Detect a disease condition even before the patient is symptomatic Identify the causes of the disease process Rule out a disease process Assess response to therapy Establish a baseline that can be used for comparison with future test results

CBC The CBC includes quantitative evaluation of erythrocytes, leukocytes and platelets and microscopic examination of the blood film to detect morphological abnormalities that provide valuable insight to various disease conditions.

Components of CBC RBC parameters Platelet parameters WBC parameters - Scattergram Reticulocyte count

Parameters… RBC parameters WBC parameters Platelet parameters RBC count Hemoglobin Hematocrit MCV MCH MCHC RDW Reticulocyte Nucleated RBC WBC parameters WBC count Differential Platelet parameters Platelet count MPV PDW PCT

Hemogram: Hemogram quantitative data (total cell counts, differential cell counts, hematocrit, hemoglobin, red cell indices ) and Qualitative data (blood film evaluation). Proper interpretation depends upon the integration of the two and a systematic approach. For all cell compartments, interpretation can be guided by answering a series of key questions.

All automated hematology analyzers in addition to enumerating the CBC results generate: red cell histograms platelet histograms and white cell histograms or scattergrams

The RBC and platelet histograms depict the cell sizing data in graphic form by plotting relative cell number versus size (volume) whereas scattergrams are two- dimensional or three- dimensional plots of multiparameter data obtained for the DLC.

T Red cell Indices help us to classify an anemia as: mean cell volume (MCV) mean corpuscular hemoglobin (MCH) and mean cell hemoglobin concentration (MCHC) help us to classify an anemia as: regenerative (blood loss or hemolysis) or nonregenerative (production defect) often giving us insight to the etiology

Erythrocyte (RBC) Histogram RL RU PLT RBC 25-75 fl 200-250 fl RBC size: 80-95 fl RBC detection: between 25 and 250 fl Distribution curves are separated by flexible discriminators: RL & RU

RBC - Histogram Distribution Width RDW-CV 100 % RDW – CV is equivalent to 68,26 % of the distribution curve 68.26% Reference range less than 16 % 200 250 fl . RDW-SD 200 250 fl 100% RDW – SD standerd devation at 20 % of the distrubution curve Reference range: 37 - 46 fl 20%

RDW RDW - coefficient of variation of the red blood cell distribution histogram. Quantitative measure of variation in RBC size (anisocytosis), RDW is elevated in: iron deficiency anemia RDW is normal in microcytic anemia of thalassemia.

The new additions to the CBC profile which have useful clinical application. Red cell distribution width (RDW) – an indicator of the degree of anisocytosis Hemoglobin distribution width ( HDW) - is a red cell parameter that measures anisochromia which along with MCV and RDW are useful in the differential diagnosis of microcytic anemia.

Red cell indices in Anemia Anemia associated with thalassemia minor: low MCV, a normal RDW, and elevated HDW Iron deficiency anemia: Low MCV, an increase in both RDW and HDW Marrow regeneration as in haemolytic anemias : elevated MCV, RDW and HDW.

The severity of anemia is categorized by: Mild anemia Hb 9.5 - 13.0 g/dL Moderate anemia Hb 7.0 - 9.5 g/dL Severe anemia Hb below 7.0 g/dL

Anemia is a laboratory diagnosis? Men Women Hemoglobin (g/dL) 14-17.4 12.3-15.3 Hematocrit (%) 42-50% 36-44% RBC Count (106/mm3) 4.5-5.9 4.1-5.1 Reticulocytes 1.6 ± 0.5% 1.4 ± 0.5% WBC (cells/mm3) ~4,000-11,000 MCV (fL) 80-96 MCH (pg/RBC) 30.4 ± 2.8 MCHC (g/dL of RBC) 34.4 ± 1.1 RDW (%) 11.7-14.5%

Role of Labs Good history and physical examination CBC With Diff Leukopenia with anemia may suggest aplastic anemia Increased Neutrophils may suggest infection Increased Monocytes may suggest Myelodysplasia Thrombocytopenia may suggest hypersplenism, marrow involvement with malignancy, autoimmune destruction, folate deficiency Reticulocyte Count Peripheral Smear

Red cell indices MCV (mean corpuscular volume) The average volume of RBC Hct =  10 (fl) RBC count (m/µL) e.g. Hct= 40% RBC=5.0 (m /µL) MCV= 40/5.0  10 = 80 fl NR= 80-96 fl

MCH (mean corpuscular hemoglobin) The average content of Hb in average RBC. It is directly proportional to the amount of Hb and RBC size. Hb MCH =  10 (pg) RBC count (m/µL) e.g. Hb = 14 g/dl RBC = 4 (m/µL) MCH= 14/4  10 = 35 pg NR= 27-32 pg

NR= 32-36% MCHC (mean corpuscular hemoglobin concentration) Hb (g/dl) Express the average concentration of hemoglobin per unit volume of RBC. It defined as the ratio of the weight of hemoglobin to volume of RBC. Hb (g/dl) MCHC=  100 (%) Hct (%) e.g. Hb = 14 g/dl Hct = 45 % MCHC 14/45  100 = 31% NR= 32-36%

Peripheral smear Reticulocyte smear

All automated hematology analyzers in addition to enumerating the CBC results generate: red cell histograms platelet histograms and white cell histograms or scattergrams

Reticulocyte Count Reticulocyte count is the percent of immature RBCs (released earlier in anemia from the marrow) Normal levels 0.5-1.5% for non anemic stages <1% means Inadequate Production >/equal to 1 means increased production (hemolysis) Corrected reticulocyte count compares anemic to non-anemic counterparts to assess response as reticulocyte count may overestimate response Corrected Reticulocyte Count = % Retic X HCT/45

Reticulocyte Correction Factor RPI = % reticulocytes X HCT/45 X 1/Correction Factor Normal RPI =1 RPI < 2 Hypoproliferative RPI greater than/equal 2 Hyperproliferative Disorder Hematocrit Correction Factor 40-45 1 35-39 1.5 25-34 2 15-24 2.5

CLASSIFICATION OF ANEMIAS Anemias may also be classified functionally into: Hypoproliferative (when there is a proliferation defect) Ineffective (when there is a maturation defect) Hemolytic (when there is a survival defect)

Kinetic Approach Decreased RBC production Increased RBC destruction Lack of nutrients (B12, folate, iron) Bone Marrow Disorder Bone Marrow Suppression Increased RBC destruction Inherited and Acquired Hemolytic Anemias Blood Loss

FUNCTIONAL CLASSIFICATION OF ANEMIAS

CLASSIFICATION OF ANEMIAS Anemias may be classified morphologically based on the average size of the cells and the hemoglobin concentration into: Macrocytic Normochromic, normocytic Hypochromic, microcytic

Morphological Approach Microcytic (MCV < 80) Reduced iron availability Reduced heme synthesis Reduced globin production Normocytic ( 80 < MCV < 100) Macrocytic (MCV > 100) Liver disease, B12, folate

MORPHOLOGICAL CLASSIFICATION OF ANEMIAS

NORMOCYTIC NORMOCHROMIC ANEMIAS

MICROCYTIC HYPOCHROMIC ANEMIAS

MACROCYTIC ANEMIAS

Hemoglobin/Hematocrit Symptoms of Anemia Normal or High Hemoglobin/Hematocrit Check other Causes of symptoms e.g. Cardiac Pulmonary Low RBC indices MCV < 80 MCHC < 32 MCV=80-96 MCV > 98 History of acute blood loss Auto immune Hemolytic anemia Anemia of chronic Diseases Anemia of infection B12 and folate levels Serum iron and Total Iron binding capacity of Ferritin Low Iron IDA, chronic diseases, Renal diseases Normal Hb electrophoresis for Thala. High Iron BM exam For Sideroblastic anemia Low B12 PA, GI problems Severe malnutrition. Low folate Folate malnutrition GI problems Liver disease High or Normal MPD Liver Disease

Iron Deficiency Anemia: Peripheral Smear The arrows show that the RBCs are smaller and paler Microcytosis &, Hypochromic RBCs

What Causes Iron Deficiency? Blood Loss (occult or overt): PUD, Diverticulosis, Colon Cancer Decreased Iron Absorption: achlorhydria, atrophic gastritis, celiac disease Foods and Medications: phytate, calcium, soy protein, polyphenols decrease iron absorption Uncommon causes: intravascular hemolysis, pulmonary hemosiderosis, EPO, gastric bypass Decreased Intake (rare)

Iron Deficiency Anemia Low Retic Count High RDW Low iron level High TIBC Low ferritin

Degrees of Iron Deficiency

Causes of iron deficiency

Causes of Hypochromic Microcytic Anaemias Blood losses Inadequate intake Enterocyte Erythroid precursor Defects in heme synthesis or iron acquisition Malabsorption Breastfeeding with inadequate supplementary food Preterm, low birth weight Growth spurt Inadequate calorie intake Vegetarian diet Polymenorrhea Parasitic infestations Peptic ulcer Inflammatory bowel disease Meckel diverticulum Celiac disease Helicobacter pylori gastritis Autoimmune atrophic gastritis IRIDA (TMPRSS6 mutation) Chronic inflammation Haemoglobinopathies Sideroblastic anaemia Erythropoietic porphyria DMT1 mutations Ferroportin disease Hereditary atransferrinaemia Hereditary aceruloplasminaemia . 53

Tests for Assessing Iron Status Serum iron Total iron binding capacity (TIBC) Transferrin saturation = serum iron/TIBC x 100 Serum ferritin Serum transferrin receptor (sTfR)/serum ferritin [R/F ratio] Reticulocyte haemoglobin content Stainable iron in bone marrow Serum iron exhibits diurnal variations, is affected by meat and oral iron supplements, and is assay methodology dependent. Transferrin saturation, which is the ratio of serum iron to total iron binding capacity, is low in iron deficiency. Serum ferritin, a good measure of tissue iron stores, and low in iron deficiency, exhibits a remarkable diurnal variation and increases in infection and liver disease. An approach that uses the ratio of serum transferrin receptor to serum ferritin (R/F ratio), has been shown to be an excellent test for body iron stores. But, again, this ratio has limited value in cases of inflammation or liver disease. CHr is The strongest predictor (better than hemoglobin) of Fe deficiency and Fe deficiency anemia in infants, toddlers and children 54

Iron Deficiency Stages Prelatent Latent IDA Parameter Reduced iron stores with normal serum iron levels Exhausted iron stores with normal haemoglobin Low haemoglobin Haemoglobin Normal Decreased MCV/MCH Serum iron TIBC Increased Transferrin saturation Serum ferritin Marrow iron Absent Iron deficiency represents a spectrum (Table_3) ranging from iron depletion, which causes no physiological impairments, to iron-deficiency anemia, which affects the functioning of several organ systems. In iron depletion, the amount of stored iron (e.g., as measured by serum ferritin concentration) is reduced but the amount of functional iron may not be affected (30,31). Persons who have iron depletion have no iron stores to mobilize if the body requires more iron. In iron-deficient erythropoiesis, stored iron is depleted and transport iron (e.g., as measured by transferrin saturation) is reduced further; the amount of iron absorbed is not sufficient to replace the amount lost or to provide the amount needed for growth and function. In this stage, the shortage of iron limits red blood cell production and results in increased erthryocyte protoporphyrin concentration. In iron-deficiency anemia, the most severe form of iron deficiency, the shortage of iron leads to underproduction of iron-containing functional compounds, including Hb. The red blood cells of persons who have iron-deficiency anemia are microcytic and hypochromic (30,31). Abbreviations: IDA, iron deficiency anaemia; MCH, mean corpuscular haemoglobin; MCV, mean corpuscular volume; TIBC, total iron binding capacity.

Case Studies

Anemia Case Study #1 A 22 year old lady has the CBC findings shown. Peripheral RBCs are hypochromic & microcytic.

Anemia Case Study #1 A-Hemoglobin Electrophoresis B-Retic count What test would you order for this patient? A-Hemoglobin Electrophoresis B-Retic count C-Stool for occult blood D-Serum Iron studies E-Bone marrow biopsy

Anemia Case Study #1 Two questions: What is your diagnosis? What is the next step for this patient?

Anemia Case Study #1 Answers Question 1 Question 2 Likely Iron Deficiency Anemia Question 2 Serum iron studies

Anemia Case Study #2 A 28 year old lady G2P1 16 weeks gestation has become progressively more fatigued at the end of the day. This has been going on for months. In the past month she has noted paresthesias with numbness in the feet. A CBC demonstrates the findings shown.

Anemia Case Study #2 A peripheral blood smear (the slide is representative of this condition) shows red blood cells displaying macro- ovalocytosis and neutrophils with hypersegmentation.

Anemia Case Study #2 Which of the following tests would be most useful to determine the etiology? A. Hemoglobin electrophoresis B. Reticulocyte count C. Stool for occult blood D. Vitamin B12 assay E. Bone marrow biopsy

Anemia Case Study #2 Questions: What is the diagnosis from these findings? How do you explain the neurologic findings?

Anemia Case Study #2 Answers: Question 1 Question 2 This is a macrocytic (megaloblastic) anemia. The neurologic findings suggest vitamin B12 deficiency (pernicious anemia). Question 2 The B12 deficiency leads to degeneration in the spinal cord (posterior and lateral columns).

Case 3 27 year old lady at 18 weeks gestation referred to the Emergency because of sudden onset of generalized weakness, severe pallor and low grade fever During the last week treated with an antibiotic for pharyngitis

Case 3 On examination: pallor, febrile, mild jaundice; spleen mildly enlarged, soft, tender No lymphadenopathy / hepatomegaly

Case 3 Laboratory evaluation - Set I: - Hemoglobin – 7.9 gm/dl - MCV – 130fl - WBC – 20,000/cmm DC N 88% L05% Myelo 03% Meta 02% Band 02% - Platelets – normal Peripheral blood smear:

Approach to Anemia – Case 3 Laboratory evaluation - Set IV: Blood smear - Anisocytosis (high RDW) - Polychromasia (reticulocytosis) -

Case 3 Laboratory evaluation - Set II: - Reticulocytes – 14% - Corrected reticulocyte count: % Retics X (measured to the expected Hb ratio)/ 14% x 7.9/15 = 7.4% - Reticulocyte Production Index (RPI): Corrected reticulocyte count/shift correction factor 7.4 / 2 = 3.7 (>2)

Case 3 Interpretation: Red cell clumping, Polychromasia, nucleated RBCs, spherocytes Diagnosis: - Hemolytic anemia? Yes

Case 4 Laboratory evaluation - Set III: - LDH – high - Bilirubin – high (unconjugated) - Diagnosis confirmed: Hemolytic anemia

Approach to Anemia – Case 4 Hemolytic anemia – differential diagnosis: - Intra-corpuscular hemoglobinopathy enzymopathy Red cell membrane - Extra-corpuscular hypersplenism mechanical hemolysis immune hemolysis toxic/infectious agents

Approach to Anemia – Case 3 Laboratory evaluation – Set V: - Direct anti-globulin (Coombs) test – IgG +3

Approach to Anemia – Case 3 Summary: - Mild splenomegaly, fever, jaundice - Reticulocytosis - Red cell clumping, few spherocytes, evidence of hemolysis - Positive direct anti-globulin test Warm-type immune hemolytic anemia, possibly – drug related

Case 4 24 year old lady presented at 34 weeks of gestation with headache, vomiting and epigastric pain of acute onset. On examination she had anemia and jaundice. Investigations Hb 5.6g/dl TC 22000/cmm DC N 86 L 5 Bd 3 Myelo 2 meta 4 NRBC 7/100 WBC Platelet count 40.000/cmm

Case 4 Peripheral smear:

Case 4 Biochemical investigations: PT 13 sec C 12 sec INR 1 Serum bilirubin 2 .5 mg Conjugated 0.8 AST 786 IU/L ALT 1030 IU/L Alkaline phosphatase: 240 PT 13 sec C 12 sec INR 1

Case 4 HELLP syndrome Acute fatty liver of pregnancy

Classification of Hemolytic Anemias Hereditary 1. Abnormalities of RBC interior a.Enzyme defects: G-6-PD def,PK def b.Hemoglobinopathies 2. RBC membrane abnormalities a. Hereditary spherocytosis etc. b. PNH   Acquired c. Spur cell anemia 3. Extrinsic factors a. Hypersplenism b. Antibody: immune hemolysis c. Mechanical trauma: MAHA d. Infections, toxins, etc Ref : Harrison’s

A Very Simple Classification of Hemolytic Anemias Intracorpuscular 1- Abnormalities of RBC interior a. Enzyme defects b. Hemoglobinopathies & Thalassemia M 2-RBC membrane abnormalities a. Hereditary spherocytosis, elliptocytosis etc b. Paroxysmal nocturnal hemoglobinuria c. Spur cell anemia 3- Extrinsic factors a. Hypersplenism b. Antibody : immune hemolysis c. Traumatic & Microangiopathic hemolysis d. Infections , toxins , etc Hereditary Extracorpuscular Acquired

Laboratory Evaluation of Hemolysis Extravascular Intravascular   HEMATOLOGIC Routine blood film Reticulocyte count Bone marrow examination Polychromatophilia Erythroid hyperplasia PLASMA OR SERUM Bilirubin Haptoglobin Plasma hemoglobin Lactate dehydrogenase Unconjugated , Absent N/ (Variable) Absent URINE Hemosiderin Hemoglobin + + severe cases

Case 5 A 20 year old lady, 32 weeks gestation presented with fever and anemia. She had received multiple blood transfusions in the past H/O having undergone splenectomy 3 years ago On examination: Pallor Hepatomegaly

Peripheral smear shows many microcytes, target cells, nucleated red cells and Howell Jolly bodies

Hb Electrophoresis Hb A: 11.31% Hb F: 88.69%

B thalassemia Major

B thalassemia Post splenectomy blood picture with malarial parasite

Sickle cell anemia Thalassemia

IDA Target cells Stomatocytes Sickle cells Poikiolocytosis Acanthocytes Spherocytes Ovalocyte Nucleated RBC

spherocytes Hypochromic Macrocytic Microcytic

Normal red cell morphology

Anemia of Chronic Disease EPO production inadequate for the degree of anemia observed or erythroid marrow responds inadequately to stimulation Causes: inflammation, infection, tissue injury, cancer Low serum iron, increased red cell porphyrin, transferrin 15-20%, normal to increased ferritin

Anemia of Chronic Disease

Approach to Anemia An approach to anemia is based on asking 3 main questions: 1. MCV? 2. Reticulocyte count? (Bone marrow function) 3. Involvement of additional lineages?

Approach to Anemia MCV: - Low: Iron deficiency Thalassemia trait Chronic disease Sideroblastic anemia - Normal: Iron deficiency (early) Renal failure

Approach to Anemia - High: Megaloblastic anemia Hemolytic anemia MDS / Aplastic anemia Drug-related Hypothyroidism Liver disease

Approach to Anemia Reticulocyte count: - Adequte (RPI>2): Hemolytic anemias (most) Acute blood loss Liver disease - Inadequate (RPI<2): Iron deficiency Megaloblastic anemia Anemia of chronic disease Bone marrow failure

Approach to Anemia Additional lineage(s) involvement: - Leukopenia: Malignancy Chemotherapy Hypersplenism Drug-related Megaloblastic anemia Aplastic anemia

Approach to Anemia - Thrombocytopenia Drug-related Megaloblastic anemia Aplastic anemia HELLP, DIC

Approach to Anemia In addition, blood smear always needs to be carefully assessed: Schistocytes? Spherocytes? Target cells? Normoblasts? Spur cells (acanthocytes)? Burr cells? Sickle forms? abnormal cells (leukemia, lymphoma)? Malaria parasites?

26 year old lady 14 weeks pregnancy presents with weakness and bleeding gums Hb 7g/dl TC 1200/ul DC N 2 L 98 Platelet count 20 thousand /ul

Hypoplastic anemia

Acute promyelocytic leukemia

Hb 8 g/dl TC 1200/ul DC N 40 L 30 Blast 10 Abnomal promyelocytes 20 Platelet count 20,000/ul

Anemia in pregnancy Iron Deficiency Anemia Vit B 12 and Folate deficiency HELLP syndrome Sickle cell anemia B Thalassemia Autoimmune Haemolytic Anemia Acute myeloblastic Leukemia - APML Aplastic anemia

A logical stepwise approach in the context of clinical presentation selection of appropriate laboratory tests to support the clinical suspicion Use of laboratory automation Arrive at the correct diagnosis