Iron deficiency anemia in different trimesters of pregnancy and laboratory diagnosis with hematological parameters and serum ferritin concentration

Jinu Varghese; Atul Khajuria

doi:10.4103/sujhs.sujhs_35_22

A CLINICAL UPDATED REVIEW

Year : 2022 | Volume : 8 | Issue : 2 | Page : 156-168

Iron deficiency anemia in different trimesters of pregnancy and laboratory diagnosis with hematological parameters and serum ferritin concentration

Jinu Varghese, Atul Khajuria
Department of Pathology, Medical Lab Technology, NIMS College of Paramedical Technology, Jaipur, Rajasthan, India

Date of Submission	19-Nov-2022
Date of Decision	23-Nov-2022
Date of Acceptance	24-Nov-2022
Date of Web Publication	11-Jan-2023

Correspondence Address:
Atul Khajuria
Medical Lab Technology, NIMS College of Paramedical Technology, Jaipur, Rajasthan
India

Source of Support: None, Conflict of Interest: None

DOI: 10.4103/sujhs.sujhs_35_22

Abstract

This article aims to provide an overview of the iron deficiency anemia (IDA) in different trimesters of pregnancy and laboratory diagnosis with hematological parameters and serum ferritin concentration. It is a laboratory leadership and quality management-based time bound prospective study that explains about IDA in different trimesters of pregnancy, causes of IDA, response to treatment, preventing IDA during pregnancy, lab leadership, and quality management.

Keywords: Anemia, complete blood count, iron deficiency anemia, peripheral smear, serum ferritin levels, trimesters of pregnancy

How to cite this article:
Varghese J, Khajuria A. Iron deficiency anemia in different trimesters of pregnancy and laboratory diagnosis with hematological parameters and serum ferritin concentration. Santosh Univ J Health Sci 2022;8:156-68

How to cite this URL:
Varghese J, Khajuria A. Iron deficiency anemia in different trimesters of pregnancy and laboratory diagnosis with hematological parameters and serum ferritin concentration. Santosh Univ J Health Sci [serial online] 2022 [cited 2023 May 30];8:156-68. Available from: http://www.sujhs.org/text.asp?2022/8/2/156/367570

Introduction

As per the World Health Organization (WHO), anemia is the most common disease, affecting more than 1.5 billion people worldwide. It occurs in every age group of people. noticeably, women in the childbearing age and in different trimesters of pregnancy. Many women lack the sufficient amount of iron needed for the second and third trimesters. When their body needs more iron than it has available, they can become anemic. Mild anemia is normal during pregnancy due to an increase in blood volume. Normally, body iron stores are maintained by the absorption of a small amount from the diet. Nearly 10% from the total intake of iron from the diet and by the re utilization of iron released from the breakdown of hemoglobin after red cells are removed from the blood circulation at the end of their normal life span by the reticuloendothelial system (Mainly spleen and liver).^{[1],[2],[3],[4],[5]}

Women are more apt to become iron deficient than men because their normal blood loss occurs in a monthly loss of 3570 mL of blood during each menstrual period and also due to this loss of iron is not compensating with the iron intake. In case of different trimesters of pregnancy, the normal cause of iron deficiency is due to the loss of iron to the growing fetus. Other reasons for developing iron deficiency for antenatal mothers are due to decreased iron intake in the diet or decreased absorption of iron or through increased or abnormal loss of iron from the body.

Causes of Iron Deficiency Anemia

Insufficient iron in the diet
Decreased absorption of dietary iron, due to either to the presence in the diet of excessive phytates and other substances which bind iron, or due to abnormalities of absorption of the iron through the intestinal mucosa, as in the various malabsorption syndromes
Diminished iron stores at birth, this happens especially in premature infants
Multiple pregnancies, especially if associated with an already borderline sufficient or insufficient diet or increased blood loss
Chronic blood loss from any condition, particularly gastrointestinal bleeding (peptic ulcer, cancer of the stomach or bowels, chronic bleeding from enlarged veins either in the esophagus, esophageal varices or ano-rectal region, piles, or hemorrhoids), bleeding in the urinary tract (hematuria) or vaginal bleeding (abnormal menstrual bleeding or cancer)
Hookworm infestation can be a chronic blood loss from the gastrointestinal tract. The amount lost depends on the severity of the infestation. Each adult hookworm drinks about 0.010.05 ml per day. (some reports give higher figures, up to 0.67ml worm per day), so that a relatively light infestation of only 100 worms would mean a loss of 25 ml per day, and a gravy infestation of 1000 or more worms would mean a loss of 2050 ml or more each day
Certain chronic infections.

Because of different lifestyles and socioeconomic conditions between cultures, the prevalence of anemia during pregnancy is highly variable. According to a WHO review of nationally representative surveys from 1993 to 2005, anemia affects approximately 42% of pregnant women worldwide (52% and 23% in developing and developed countries, respectively). Although an estimated 1.6 billion individuals worldwide have anemia, it is generally assumed that 50% of cases of anemia are due to iron deficiency and about twice as many individuals are estimated to be affected by iron deficiency.^{[6],[7],[8],[9],[10],[11]}

Iron Deficiency Anemia in Pregnant Women

Iron deficiency anemia (IDA) is a frequent condition during pregnancy. The worldwide prevalence of anemia in pregnancy is estimated to be approximately 41.8%; nevertheless, the percentage of iron deficiency without anemia is unknown. The overall iron requirement during pregnancy is significantly higher than in the nonpregnant state, despite the temporary respite from iron losses incurred during menstruation. This is due to an exponential increase of iron needed to expand the plasma volume, produce a greater quantity of red blood cells (RBCs), support the growth of the fetal-placental unit, and compensate for iron loss at delivery. The physiological iron demand in pregnant women corresponds roughly to 1000–1200 mg for an average weight of 55 kg. This quantity includes almost 350 mg associated with fetal and placental growth,^{[12],[13],[14]} about 500 mg associated with expansion in red cell mass, and around 250 mg associated with blood loss at delivery. In the course of gestation, iron need presents a variation with a growing trend; in fact, there is a lower iron necessity in the first trimester (0.8 mg/day) and a much higher need in the third trimester (3.07.5 mg/day). At the beginning of pregnancy, approximately 40% of women show low or absent iron stores, and up to 90% of women have iron reserves of <500 mg, which represent an insufficient amount to support the increased iron needs. An overt IDA frequently develops in pregnancy even in developed countries, indicating that the physiologic adaptations are often insufficient to meet the increased requirements, and iron intake is often below nutritional needs. IDA in pregnancy, if not diagnosed and treated, can have a significant impact on maternal and fetal health.

Lab Diagnosis of Iron Deficiency Anemia During Pregnancy

Hematological parameters - Peripheral blood report

Indeed, chronic iron deficiency can affect the general well-being of the mother and leads to fatigue and reduced working capacity. Given the significant adverse impact on maternal-fetal outcomes, early recognition and treatment of this clinical condition are fundamental. Therefore, the laboratory assays are recommended from the first trimester to evaluate the iron status. As per the hematology parameters, the primary class characteristics is a decrease in the hemoglobin concentration and associated fall in the packed cell volume and RBC count. In addition to it, IDA shows low MCV, low MCH, and low MCHC. This anemia is also referred to as hypochromic microcytic anemia.

Hypochromia is the most prominent feature of IDA and a small proportion of cells may show more normal hemoglobinization. There is a varying degree of anisocytosis with the majority of cells being normal or small though occasionally the pale empty cells may be so flattened on the slide as to appear normal in size or even enlarged. Moderate poikilocytosis is noted but the variation in size is usually more marked. There will be little or no polychromasia and the reticulocyte count will be low, unless fresh blood loss has occurred, or unless the patient has just recently received some treatment. Nucleated RBCs may occasionally be seen in very severe cases. The white blood cell count and platelet count are usually normal, though the former may be very slightly lowered in number. Both may be increased if there is superimposed acute bleeding.

Bone marrow

The fragments are usually hypercellular, and the portion of the erythroid cells in the marrow is increased above 20% of the nucleated cells (erythroid hyperplasia). The developing erythroid cells show normal nuclear maturation (are normoblastic), although there are morphological changes due to the delayed and decreased hemoglobinization of the cytoplasm because of the lack of iron. The smears will not show any stainable iron.

Response to the treatment

With the correct treatment, there will be a response of a rise in the reticulocyte count and concomitant increase in polychromasia in the peripheral blood starting about 4^th day and reaching the maximum response about the 7^th or 8^th day. With the availability of iron from treatment, the newly formed cells are fully hemoglobinized. However, the old hypochromic cells are still present in the blood. This gives rise to a blood picture described as a “double population” meaning, there are two distinctly different cell populations (this can also be seen after a blood transfusion in such patients). The morphology of the erythroid cells in the bone marrow reverts to normal, although the erythroid hyperplasia will continue until the anemia is corrected.

Serum Ferritin Levels in Iron Deficiency Anemia

Ferritin is a blood protein and is the main storage protein for iron, alongside hemosiderin. Ferritin is, therefore, a good indicator of how much iron is stored within your body with low levels indicating IDA. Approximately, one-quarter of the total iron in the body is stored as ferritin.

The most reliable parameter to reveal iron deficiency is serum ferritin, and screening of serum ferritin concentration at the beginning of pregnancy is recommended. If serum ferritin is <30 g/L, there is a high probability that iron stores are depleted, even in the absence of anemia. A serum ferritin value <30 g/L is associated with an Hb concentration <11 g/dL during the first trimester, <10.5 g/dL during the second trimester, and <11 g/dL during the third trimester are diagnostic for IDA in pregnant women. Iron therapy should be considered in such cases. However, in the presence of inflammatory processes or chronic diseases, ferritin levels can be falsely normal or elevated, despite the presence of anemia. This is because ferritin reacts as an acute-phase protein. The evaluation of C-reactive protein (CRP) levels may assist in obtaining the correct diagnosis, excluding infections or inflammation. If the CRP value is elevated, re-evaluation of the SF level is recommended after the normalization of CRP concentration. Repeating serum ferritin levels measurement afterward during pregnancy is not necessary if the patient does not show symptoms of anemia. Conversely, Hb concentration should be measured in each trimester. When ferritin levels are 30 g/L, apart from measuring CRP levels, it is necessary to carry out other diagnostic investigations such as the determination of transferrin saturation and serum iron.

A serum ferritin threshold of <100 μg/L or TSAT <20% can be considered diagnostic for iron deficiency in CHF, CKD, and IBD. If serum ferritin is 100–300 μg/L, TSAT <20% is required to confirm iron deficiency.

The level of serum ferritin is a reliable indicator of body iron stores. Exceptions include liver disease, malignant diseases, and treatment of iron-deficiency anemia. IDA among pregnant women is a widespread problem in developing countries, though its influence on neonatal iron status was inconsistently reported in literature.

Ferritin is an acute-phase reactant and levels will rise when there is active infection or inflammation. Participants with a history of chronic illness, such as hepatitis, sickle cell disease, renal disorders, and those with obstetric complications such as preterm labor, preeclampsia, gestational diabetes and HIV infection, were therefore excluded.

Preventing Maternal Anemia During Pregnancy

Daily supplementation with oral ferrous sulfate is effective in preventing maternal anemia and iron deficiency during pregnancy, and reducing the risk of low birth weight. Indeed, international guidelines recommend universal iron supplementation for the management of iron deficiency and IDA during pregnancy.

Laboratory Leadership and Quality Management

Blood sampling

Peripheral blood samples were collected from the pregnant women to determine complete blood count and SR levels before delivery. Complete blood cell count was performed by the Sysmex autoanalyzer on the same day of collection, a Leishman-stained blood smear was prepared for the peripheral blood smear and serum ferritin levels were performed from the blood sample in plain bottles after centrifugation in biochemistry analyzer (Cobas 6000). Elecsys Ferritin test is an immunoassay for the in vitro quantitative determination of Ferritin in human serum/plasma using the Cobas 6000 (601) analyzer.

Data analysis and quality control

All data were expressed as mean ± standard deviation. All analyses were performed internal and external quality controls before testing. A probability value of P < 0.05 was considered to indicate statistical significance.

Standard operating procedure - Peripheral blood smear study

Purpose

Peripheral smear study is used in reporting any abnormal morphology of RBC and WBC and in the diagnosis of various anemias, leukemia and infection of blood parasites, for example, malaria.

Responsibility

Pathologists, clinical scientists or laboratory technologists should be responsible to ensure standard procedure has been followed for peripheral smear study.

Definition and abbreviations

DLC: Differential leukocyte count
PBS: Peripheral blood smear
TLC: Total leukocyte count
ANC: Absolute neutrophil count
NRBC: Nucleated RBC.

Procedure

Principle

To establish the absolute value of each type of leukocyte, confirm the electronic differential and describe RBC and platelet morphology. Electronic Differential - The analyzer determines the cell identification based on predetermined criteria set by the manufacturer. When criteria are exceeded or cell population is not defined, the instrument FLAGS the population of cells as positive, indicating that they should be reviewed visually by a tech
Scan – The technologist scans the slide for at least 2030 s and/or 2030 fields to determine presence of abnormal WBCs and/or to confirm the accuracy of the electronic differential. If in agreement and with no abnormalities, the differential is reported as “Electronic diff verified by slide review”. If not, a manual differential is reported. RBCs and platelets are examined, and morphology is reported with every slide review
Manual differential - The first 100 leukocytes on a blood smear are identified and counted by a technologist using the manual counter; the leukocytes are differentiated, and the cell types are reported.

Performance characteristics

Tolerance limits for an acceptable slide:
Minimum of 2.5 cm in length and at least 1 cm from end
Gradual termination in thickness, with feather edge at termination
Acceptable morphology (without artifacts) within the working area
Film should be narrower than the slide and have smooth continuous side margins
Stain should be even and consistent with correct color for each structure (i.e. deep blue nucleus, pink, or blue cytoplasm)
Leukocytes should be well preserved; beware of anticoagulant effects such as excessive vacuolization
There should be less than 10% smudge cells (unless lymphoproliferative disorder).

Type of sample

Whole blood K2 EDTA
Amount of specimen: One drop (≈50 ul) of well mixed EDTA whole blood
Collection and handling requirements: Blood should be mixed immediately upon collection in EDTA to ensure the specimen does not clot
Stability and Storage: Specimen can remain at the room temperature for 4 h, use with caution if over 4 h. Most refrigerated specimens are stable for 48 h though some may show deterioration as early as 6 h after collection. If specimen integrity is suspect, a recollection should be initiated. Specimen stability may be extended to 72 h so long as a slide is examined to rule out a loss of integrity (cellular breakdown, platelet clumping, agglutination, etc.)
If refrigerated, let the tube sit at room temperature for 30 min and invert it by hand, end to end, before preparing the smear.

Type of container with additive

EDTA
REQUIRED REAGENTS/EQUIPMENT:
Slides
Slide Spreader
Field's Stain (Refer to Procedure Steps)
Sysmex XN 550/XN 1000
Light Microscope
Manual differential counter
Oil.

Environmental and safety control

Laboratory MSDS (Material Safety Data Sheet) should be followed for environment and safety control.

Safety precautions

Handle all reagents with care and avoid contact with eye, mouth, and skin
Eye wash stations should be available in the laboratory
Handle all samples as potentially infectious
Discard used reagents and samples as per disposal procedure.

Procedure steps

Slides are made and stained for peripheral blood film examination, hemograms with flags and abnormal platelets (See slide review criteria).

Manual smear preparation

Mix blood well before the smear is made by inverting end to end 10 times
Prepare a manually made slide by hand. Take a clean dry slide
Place a drop of EDTA whole blood near the edge of the slide
Place a smaller piece of glass with a width slightly less than that of the slide called a spreader at an angle of about 45° to the surface of the slide
Pull back the spreader until it touches the drop of blood. Put the spreader forward to the end of the slide with a smooth movement
Manually label slide with patient name and sample order number, or place patient's ID aliquot sticker on slide assuring that the label does not extend over the edges of the slide
Dry the blood smear at the room temperature.

Staining the slide

(Field's Stain) Fix the slide by dipping five times for 1 s in solution # 1 methanol. Drain the surplus onto a paper towel.

Dip the slide five times for 1 s in solution # 2 eosin. Carefully drain the surplus onto a paper towel
Dip the slide five times for 1 s in solution # 3 methylene blue. Wash the smear using tap water and stand the slide in a draining rack or on the laboratory counter to dry
Keep the washed slides on a paper towel, with the lower edge of slides touching the paper towel
Wipe the back of the slide with a wet gauze pad or paper towel to remove any dried stain from the back of the slide.

Examination of stained smears

After staining, examine the slide on a low power, 1020 X for quality and later under 100X oil immersion objective of a light microscope.

Check the overall staining of the slide for proper staining of RBC s and inner structures of white blood cells
Check the distribution of the WBCs. Is there an increase or decrease in any cell type? Are the WBCs at the feathered edge and edges representative of the rest of the slide? Larger cells and clumps of cells tend to be carried to the feathered edge. Among the cells likely to be found in this region are immature leukocytes, nucleated red cells, megakaryocyte nuclei, platelet clumps, mitotic forms, tumor cells, macrophages, microfilariae and monocytes, and neutrophils containing red cells, bacteria, and parasites
Note: If abnormal cells are not evenly distributed throughout the slide. Consider doing a 200300 cell differential incorporating a portion of the feathered edge or slide edge to ensure that all cell types are adequately represented
Check for any rare immature or abnormal nucleated cells, platelet clumps, giant platelets, or megakaryocyte fragments
Check the RBC distribution for Rouleaux and agglutination. Look for RBC features of: micro, macro, hypo, poly, poikilo, target cells, and nucleated RBCs and RBC inclusions. When there are >5 NRBC/100 WBC, the WBC needs to be corrected if your analyzer does not correct for NRBCs
Look for a good area to do the differential.

Scanning: Select an area just behind the feathered edge where the red cells are barely touching

Scan the slide on 50X or 100X for 2030 s.

The Comment “Electronic diff. verified by slide review can be resulted out with the absolute counts from the automated differential. Manual differential is same as automated
Scan agrees comment can be used provided that the scan agrees with the electronic differential, there are no vote-outs, there are no immature cells (metas, myelos, pros, or blasts), and all results for Basos, Eos, and Monos are within the normal limits. If the manual result values for Basos, Eos, or Monos are abnormal, report the manual differential (check slide review criteria)
Report the absolute values from the manual differential if:
Presence of any immature cells
Manual differential varies from the electronic differential by greater than the 95% confidence limits.

Counting differential

With the 50X oil or 100X oil objective, perform a differential by counting the first 100 WBCs located in sequence, using the manual counter, keyboard counter, or DI. Results are expressed in percent on the keyboard/counter. Absolute values are calculated by calculator
Criteria for performing a 2 tech manual differential - A second tech will perform another differential on slides with the following abnormal parameters. If the two techs do not agree, remake the slides and repeat. If the two techs still do not agree, the sample and slides are reviewed by the technical leader/pathologist.

Types of Cells (Refer to [Appendix 1] for expected normal values for differentials).

Segmented neutrophil- (mature neutrophil)

Medium size (1015 um diameter) with two to five unequal nuclear lobes connected by a filament. Purple chromatin coarsely clumped with a few open areas randomly distributed. Pinkish tan or light lilac cytoplasm-may contain a few red-purple residual primary granules.

Neutrophil band (counted as mature neutrophil)

Similar to segmented neutrophil except for nuclear shape. The connection between the end of the beginning lobe formation of the nucleus is band-like rather than a filament. The connecting band is wide enough to contain nuclear material. If no nuclear material can be seen in the connecting band, the cell is a segmented neutrophil.

Eosinophil

Similar to the segmented or band neutrophil except for a two-lobed nucleus and presence of numerous bright orange spherical cytoplasmic granules. Usually 1216 um in size.

Basophil

Similar to the mature neutrophil except for a round or indented light red-purple staining nucleus and presence of large purple-black cytoplasmic granules that may obscure the nucleus. 1014 um in size.

Lymphocytes

Small to large (814 um) in size and mononuclear with a high nuclear cytoplasmic (N/C) ratio. Nucleus: Round, oval or slightly indented with semi-densely blocked red-purple chromatin, the larger the cell, the less dense in the nucleus, nucleolus occasionally visible, especially in larger cells and in thin areas. Cytoplasm: pale or medium blue, translucent and may contain several azurophilic (red-purple) granules, larger lymphocytes have a smaller N/C ratio than small ones. Variant forms including reactive (dark cytoplasm or edges), immature (contains a nucleoli), indented/clefted nucleus, plasmacytoid lymphs, and lymphs with cytoplasmic projections will be categorized at “Atypical”.

Plasma cell

1015 um in size with a round or oval shape and low N/C ratio. Nucleus is usually eccentrically located with semi-coarsely blocked chromatin similar to lymphocytes. The cytoplasm is medium to deep blue with a prominent perinuclear clear area.

Monocyte

Medium to large (1020 um) mononuclear, with a medium N/C ratio. Nucleus is usually convoluted or horseshoe-shaped, can be round, oval, indented, deeply lobulated, or even segmented. It contains evenly distributed chromatin that is pale and delicate staining. Cytoplasm is usually abundant, dull gray-blue with a few coarse red-purple or tiny dust-like granules may be present, causing a “ground glass” like appearance.

Nucleated red blood cell and megakaryocytes

Nucleated RBC s and megakaryocytes are counted during the 100 WBC differential. The keypad is designed to keep them separate from the 100 WBCs. When using a manual cell counter, keep them separate from the 100 WBCs.

If NRBCs or megakaryocyte fragments are present, they are reported along with the manual differential. When there are more than 5 NRBCs per 100 WBCs, the total WBC count must be corrected using this formula: WBC times 100 divided by the number of NRBC plus 100. A WBC corrected for the presence of NRBCs will be lower than the uncorrected value.

**Example: The WBC count from the analyzer is 20,000. Multiply the WBC count by100.(=2,000,000). There are 5 NRBC per 100 WBC per 100 WBC and 10 megakaryocytes fragments per 100 WBC found upon slide review. Add the number of mega frags and NRBC together and add 100 (=115). Divide 2,000,000 by 115 = corrected WBC of 17,391.

**If the WBC is corrected, absolute differential values must be calculated using the corrected WBC.

Atypical lymphocytes

Variant lymph forms including reactive (dark cytoplasm or edges), immature (contains a nucleoli), indented/clefted nucleus, plasmacytoid lymphs, and lymphs with cytoplasmic projections will be categorized as “Atypical”
Up to 5% atypical lymphocytes are considered normal and do not need to be commented on. (5% of the total WBCs, not 5% of the total Lymphs.) Quantitate the number of atypical lymphs with descriptive terms.

Occasional (occ) = >5 to 30%

Moderate (mod) = >30 to 60%
Many = >60%.

Smudge and basket cells

Smudge cells will be counted as WBCs in the manual diff.

If the cell has a dense purple appearance, count as lymph. If characteristics of a neutrophil, eosinophils, or basophils, count, respectively, as those cell types.

If the manual diff agrees with the electronic diff report “Electronic diff verified by slide review”
If the electronic diff is voted out, report the manual diff with smudge cells counted.

White Blood Cell Estimate

Perform WBC estimation to confirm automated WBC. Note abnormal populations. Results should match the instrument within 10%
To estimate WBC:
Perform in an area where RBCs are just starting to overlap
Average the number of WBCs from 1020 fields
If using a 50X oil objective, multiply the average by 3
If using a 40X or “high dry” objective
Using a long disposable pipette, draw off and discard the majority of plasma, avoiding WBC and platelet layer
Draw up a buffy coat area of both tubes, getting just a small portion of the plasma and red cells, place on a glass slide, and mix. Alternatively, the pipette can be dispensed into a plain round bottomed tube and mixed
Using a plain Hematocrit tube, transfer a drop to spread for a manual smear. Refer to Lab-5074 Differential: Counting and Morphology for Manual Smear Preparation
When dry, stain slide for manual diff. Perform a differential on the buffy coat slides. Use the original slide for morphology or estimates.

Red cell morphology

RBC morphology characteristics are measured and reported by scanning 10 fields in different areas of the slide with evenly dispersed red cells using the 100X oil immersion objective. This area is usually where cells are just touching or beginning to overlap. The test code “RBC Morphology” is reported as Normal or Present. The individual components of morphology are reported by using the following grading steps. Variance of one grading step is acceptable tech to tech variation when grading RBC morphology.

Red blood cell grading system

Other cellular abnormalities- the following are not quantitated (Refer to [Appendix 2] for abnormal cells).

WBC anomalies resulted as Present: Dohle Bodies, Toxic Granulation, Auer Rods, Vacuolated, hypersegmented (6 or more segments present), bilobed, and agranular neutrophils
RBC anomalies resulted as Present: Rouleaux, Basophilic Stippling, Pappenheimer Bodies, Howell-Jolly Bodies, and Cabot Rings

Basophilic Stippling should only be called when coarse basophilic stippling is present or when there are 2 or more cells with fine stippling present per high power field.

PLT morphology: resulted as Normal, Giant, and/or Agranular, and unable to report

Giant platelets should only be called when the platelets are larger than a normal red cell (larger than 8 μm). Care should be taken when a patient has macrocytic or microcytic RBC s as this can make the determination of giant platelets more difficult
The morphology of “Unable to report” should only be used in very rare cases where not a single platelet is visible upon slide review. If a platelet is found on the slide, the morphology should be based off of that platelet (example, one normal platelet is found upon slide review, resulting in the morphology as normal).

White blood cell/red blood cell parasites or Bacteria

Malaria: Various forms including rings with Schuffner's dot may be seen in the RBCs depending on the stage of the parasite's life cycle.

Platelet estimation and morphology

Estimate whether platelet number appears normal, increased, or decreased and compare to electronic platelet count
Platelet morphology is reported as Normal, Giant, or unable to report

Platelets are 23 μm in diameter with no nucleus and red-purple granules
Look for agranular platelets and giant platelets (platelets >8 μm).

Platelet estimate:

Using 100X oil, find an area where the RBCs touch or just overlap
Count 10 fields and calculate the average
Multiply the average number of platelets per field by a “Factor”. It takes practice to find a factor that works for you.

Example: 10, 9, 10, 8, 5, 4, 9, 11, 9, 7 counted for a total of 82. Divide 82 by number of fields (10) = 8.2 average
8.2 multiplied by a factor of 12 = 98.4 for an estimated platelet count.

If platelet clumps are detected and the platelet count has been verified:

Delete the platelet result in LIS
Enter the appropriate canned comment regarding platelet clumping and adequacy.

Quality control procedures

The quality of each slide is assessed for the correct color of the inner structures. For example, in a neutrophil, the nucleus is deep blue-purple, and cytoplasm is pinkish. If needed, another slide is made and stained
Proficiency testing: Testing of PBS by two pathologist correlation.

k. Critical Value:

Peripheral blood smear with bands (>10.0%), blasts (>1.0%), microangiopathic hemolytic changes or malaria.

l. Clinical interpretation

Reporting results

Verify that your results and estimations agree with electronic results, including:

MCHC/hypochromic
MCV/anisocytosis/macro/micro
WBC/estimate
Plt/estimate
HGB/polychromasia, etc.

LIS: After finishing slide review, the CBC is completed by resulting in a Manual differential or “Electronic diff verified by slide review.” RBC and platelet morphology are also results. The Complete comment should be verified last and will remove the specimen from the worklist.

Interpretation of results

Look under oil immersion for the following features.

Red blood cells

Size of red blood cells

Normal RBC is about 7.2 microns in diameter. Normal cells are called normocyte. When the cells are smaller than normal that are less than 6 μ in diameter, it is called a microcyte and when larger than normal – more than 9 μ in diameter, it is called a macrocyte. When the variation in size is greater than normal, it is called anisocytosis.

Shapes of red blood cells

Normal red cells are biconcave in shape and appear circular on smear. Abnormal cells may assume different shapes. Variation in shapes is known as poikilocytosis. These shapes are described as follows:

Ovalocytes/elliptocytes
Pencil shaped cells
Target cells
Sickle cells
Schizocytes.

Crenated cells
Acanthocytes/Burr cells
Crescent bodies.

Color

Look for chromaticity. Normal cells have a central area of pallor. When this normal pallor is increased, it is called hypochromia. Some cells appear purplish or pinkish and are the polychromatic erythrocytes. Some cells appear to be hyperchromic – without a central area of pallor.

Inclusions of red blood cells

Basophilic stippling: These are small blue or back granules seen in red cells
Howell-jolly bodies-These appear small, round, densely staining, dark blue particles. Usually found near the periphery of the cells
Cabot rings- These are pale staining rings or figures of 8.

White blood cells

Look for number, morphology, nuclear granules, distribution, abnormal cells or immature forms.

Platelets

Look for number, platelet distribution, morphology and aggregation.

Parasites

Look for intra-cellular (malaria) and extracellular parasites.

m. Potential Sources of Variations:

PreAnalytic

Unlabeled specimen, Clotted specimen
Dirty slides do not give an even smear
Heparinized specimen, Sodium citrate tube may be used for the differential and PLT estimate but not for any other estimation or morphology as the dilution factor will affect results. Sodium Citrate may be used for platelet estimation when a patient clumps in EDTA but this result will always be accompanied by a comment denoting that the result is from a Sodium Citrate tube and the result may differ from EDTA
Blood greater than 4 h old should be used with caution as the integrity of the WBCs may be in question
Smear should be made immediately after putting the drop of blood on the slide; a delay will cause uneven distribution of white cells on the film
Jerky movement of the spreader slide and the loss of contact between the spreader and the smear slide will yield poor smears.

Analytic: Sample analysis

Post analytic

Transcription error (Oral and written).

Standard operating procedure - serum ferritin levels.

Purpose

Elecsys Ferritin test is an immunoassay for the in vitro quantitative determination of Ferritin in human serum/plasma using the Cobas 6000 (601) analyzer.

Responsibility

It is mandatory for all Laboratory Technologists to follow this standard procedure on performing Ferritin tests
Laboratory director should ensure and check that this Standard operating procedure is being practiced.

Definition and abbreviations

Plain Tube (Serum)
ECLIA: Electrochemiluminescence Immunoassay
PC VARIA: PreciControl Varia
FERR: Ferritin.

Procedure

Principle

Electrochemiluminescence immunoassay “ECLIA” – Sandwich Principle.

Performance characteristics

Method validation done and passed

Precision Passed
Comparison Passed
Accuracy, Linearity Passed.

Measuring range

0.500 – 100000 ng/mL.

Lower detection limit: 0.500 ng/mL.

Type of sample

Serum is preferred
Plasma is acceptable.

Patient preparation

Fasting and non-fasting is accepted.

Type of container with additives

Plain tube (Red top)
Gel tube (Yellow top)
Sodium or Lithium Heparin (Green top)
K2-EDTA and K3-EDTA (Purple/Lavender top).

Required reagents/equipment

Equipments

COBAS 6000 (e601)
Bench Centrifuge
Calibrated pipettes
Sample racks
Reagent racks.

Reagents

The reagent rackpack is labeled as FERR
M Streptavidin-coated microparticles (transparent cap), 1 bottle, 6.5 mL: Streptavidin-coated microparticles 0.72 mg/mL; preservative
R1 Anti-Ferritin-Ab ~ biotin (gray cap), 1 bottle, 10 mL: Biotinylated monoclonal anti-ferritin antibody (mouse) 3.0 mg/L; phosphate buffer 100 mmol/L, pH 7.2; preservative
R2 Anti-ferritin-Ab ~ Ru (bpy) (black cap), 1 bottle, 10 mL: Monoclonal anti-ferritin antibody (mouse) labeled with ruthenium complex 6.0 mg/L; phosphate buffer 100 mmol/L, pH 7.2;
Ferritin I and II CalSet:
Storage Stability:

Store at 2°C8°C – stable up to stated expiration date
Stability of opened calibrators:

at 2°C8°C: 12 weeks
on the analyzers at 20°C25°C: use only once

PreciControl Varia:
Storage Stability:

Store at 2°C8°C
Stability of reconstituted calibrators:

At −20°C (± 5°C): 31 days (freeze only one)
At 2°C8°C: 72 h
On the analyzers at 20°C25°C: up to 5 h

Diluent Universal
ProCell M and CleanCell M
ProbeWash M and PreClean M
Accessory Materials: assay tips and assay cup, solid waste bag, sysclean adapter, cobas cups.

Environmental and safety control

Refer to Laboratory and Safety Policy and MSDS.

Calibration procedures (metrological traceability)

Traceability: The Ferritin assay (03737551190) has been standardized against the Ferritin assay (11820982122). The Ferritin assay (11820982122) has been standardized against the Enzymun-Test Ferritin method. This in turn has been standardized against the 1^st International Standard (IS) NIBSC (National Institute for Biological Standards and Control) “Reagent for Ferritin (human liver)” 80/602
Calibration frequency: Calibration must be performed once per reagent lot using fresh reagent (i.e. not more than 24 h since the reagent kit was registered on the analyzer)
Calibration intervals may be extended based on acceptable verification of calibration by the laboratory
Renewed calibration is recommended as follows:

After 8 weeks when using the same reagent lot
After 7 days (when using the same reagent kit on the analyzer)
As required: e.g., quality control findings outside the defined limits.

Calibration procedure

Always check the lot number and expiry date of the calibrators. Download the calibrator
Allow the calibrator to come to the room temperature after taking it out from the refrigerator
If running with barcode

4.8.5.4.1. Go to “Calibration” highlight FERR then press save.

Put the calibrator in the calibration rack (Black rack), with the barcode facing the barcode reader

Press the “start” button to start the assay
If running manually
Assign the assay to the calibration rack (black rack)
Transfer calibrator in cobas cup and put it in the assigned rack
Go to “Calibration” highlight FERR then press save
Press the “start” button to start the assay.

Procedure steps

Assay procedure

Centrifuge the clotted sample to separate the serum from cells
Place the sample on the sample rack (gray rack). Barcode facing the barcode reader
Samples are processed using barcodes and with the host interface; however, in certain cases, manual entry of test orders may be required. If in cases there is sample barcode error, enter manually the 12 digit sample episode number or do manual barcoding on the “test selection”. Press “ENTER” then go to “sample barcode error”, assign rack number and position number then press “OK” and “SAVE”
Press “START” to initiate assay
Wait for the test to be done then print the result
Laboratory Technician on duty validates the results and checks for flags. Consult the Chief Laboratory Technician or the Pathologist on Duty for panic values
Results are forwarded to the data entry for entry into LIS.

Quality control procedures

Use PreciControl Varia. In addition, other suitable control material can be used
Controls for the various concentration ranges should be run individually at least once every 24 h when the test is in use, once per reagent kit, and following each calibration
Procedure
Always check the lot number and expiry date of the control reagent
Download the PreControl Varia, then activate the tests
Allow the reagent to come to room temperature, let it stand for about 15 min upon removal from the refrigerator
Reconstitute the PreControl Varia with 3ml of deionized water and allow it to stand closed for 30 min to reconstitute. Mix carefully, avoiding foam formation. Aliquots intended for storage at − 20°C (± 5°C) should be frozen immediately.
If running with barcode
Go to “QC '', then “STATUS”, highlight FERR then press “ SAVE”
Put the control reagent in the control rack (white rack)
Press “START” to initiate the assay.

If running manually:

Assign the assay to the control rack (white rack)
Transfer control reagent in cobas cup and put it in the assigned rack
Go to “QC'' then “STATUS'' highlight FERR then press save
Press the “start” button to initiate the assay.

Interference (lipemia, hemolysis, bilirubinemia, drugs)

The assay is unaffected by icterus

(Bilirubin ≤1112 μmol/L or ≤65 mg/dL)
Hemolysis (Hb <0.31 mmol/L or ≤0.5 g/dL)
Lipemia (Intralipid <3300 mg/dL)
Biotin (≤205 nmol/L or ≤50 ng/mL
Samples should not be taken from patients receiving therapy with high biotin doses (i.e. >5 mg/day) until at least 8 h following the last biotin administration
No interference was observed from rheumatoid factors up to a concentration of 2500 IU/mL.

Biological reference

Reportable range

0.5002000 ng/mL
Values below detection limit are reported as <0.500 ng/mL
Values above the measuring range are reported as >2000 ng/mL

1.1. CRITICAL VALUE: N/A

Procedure for calculating results/MOU

Results are automatically calculated by the machine
Measurement of Uncertainty will be computed as: CV% multiplied by 2.

1.2. Instructions for Determining Results When The Result Is Not Within Measurement Interval

1. Samples with ferritin concentrations above the measuring range can be diluted with Diluent Universal. The recommended dilution is 1:50 (either automatically by the analyzers or manually). The concentration of the diluted sample must be >40 μg/L (ng/mL).

Clinical interpretation

a. Under steady-state conditions, the serum ferritin concentration is proportional to the total body iron stores: 1 ng of serum ferritin per mL corresponds to 10 mg of total iron stores. 6, 7, 8 Therefore, in the literature, the measurement of serum ferritin levels is proposed as the best and most convenient laboratory test to estimate iron stores and diagnose iron deficiency or iron related disorders. 6, 8, 5,9 It has substituted the invasive and semiquantitative histochemical examination of bone marrow aspirate or biopsy as the gold standard for diagnosis of IDA

2. Serum ferritin is a good indicator of storage iron in the body; however it does not provide information about the amount of iron actually available for erythropoiesis. Decreased serum ferritin concentrations of 400 μg/L) may have many implications: Ferritin being an acute phase reactant, elevated serum ferritin values can occur in patients with infections, acute or chronic inflammation and malignant tumors, despite acute iron deficiency. Elevated ferritin levels unrelated to iron stores are also seen in case of alcoholic or viral hepatitis and chronic renal failure. Diagnosis should be made looking at the entire clinical situation of the individual patient.

Potential sources of variation

Preanalytical

Using old samples. Patient serum Stable for 7 days at 2°C8°C, 30 days at −20°C
Foam formation in all reagents and sample types (specimens, calibrators, and controls)

Use of reagent kits beyond the expiration date
Un centrifuge samples containing precipitates before performing the assay
Use of heat-inactivated samples
Use of samples and controls stabilized with azide.

Analytical: Interferences, Equipment malfunctioning.

Post-Analytical sources of variability: Oral, hand-written, and electronic transcriptional error.

Conclusion

IDA in pregnancy is a public health problem. Undiagnosed and untreated IDA can have a great impact on maternal and fetal health. According to the WHO, anemia is the most common disease, affecting more than 1.5 billion people worldwide. IDA is a frequent condition during pregnancy. The global prevalence of anemia in pregnancy is estimated to be approximately 41.8%; nevertheless, the percentage of iron deficiency without anemia is unknown. The overall iron requirement during pregnancy is significantly higher than in the nonpregnant state, despite the temporary respite from iron losses incurred during menstruation Moreover, IDA accounts for 50% of cases of anemia. IDA is common during pregnancy and the postpartum period and can lead to serious maternal and fetal complications. There is a need for more studies to establish the cause of anemia in pregnant women in this area. The most reliable parameters to reveal IDA in pregnant women are the assessment of hematological parameters and serum ferritin levels.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

References

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